50% increase in maximum isometric tetanic specific force (sPo) was obtained by taurine supplementation in mdx mice aged 4 and 6 weeks [78,81]. However, no treatment effect (1 g/kg/day) on specific tetanic force was detected in muscle of mice aged 5-7 weeks, whereas fore limb force, assessed by means of a grip strength meter, was ameliorated [96]. Furthermore, a study performed by Barker reported no effects of taurine treatment (2.5% w/v) on peak twitch force, maximum specific force, nor fatigue or recovery in mdx mice treated from week 2 until week 4 [97]. Similarly, no effect on maximum specific force in 6-week-old mdx mice was observed when high doses of taurine (16 g/kg/day) were administered [98].","In 6-month-old mdx mice, taurine could not ameliorate specific maximum isometric force production. However, after a fatigue protocol consisting of recurrent electrical stimulation, the EDL of taurine treated mdx animals was more resistant to fatigue, as shown by a significant higher force production at the end of stimulation relative to force production at the beginning. Furthermore, EDL muscle of taurine treated mdx animals showed a significantly better recovery capacity at 10-60 min after stimulation than untreated mdx animals [79]. Taurine treatment in exercised mdx mice significantly increased in vivo forelimb muscle strength normalized to body weight [96,99], but did not alter locomotor activity of mdx mice. A similar finding was reported in a study that supplemented unexercised mdx mice. Taurine supplementation (±4 g/kg/day) in 6-week-old unexercised mdx mice significantly increased grip strength [81]. Whereas no effect of high taurine treatment (16 g/kg/day) was observed on normalized grip strength [98].","In summary, benefit of taurine supplementation on muscle force differed considerably between published studies. Although multiple studies have been executed, these used different concentrations, ingestion methods, and treatment protocols, which might explain the obtained conflicting results, and which hampers interpretation.","Although different treatment conditions and read-outs were used, multiple studies have shown anti-inflammatory and anti-oxidative effects of taurine treatment in mdx mice [59,80,81,95]. The anti-oxidant and anti-inflammatory actions of taurine have been proposed as a mechanism by which taurine protects dystrophic tissue from damage. In evidence, muscle of taurine-treated mdx mice showed significantly less NF-κβ positive fibers, TNF-α levels, neutrophil elastase, and MPO activity [59,80,81,96]. Accordingly, taurine-treated mdx mice showed lower levels of disulfide and protein thiol oxidation in muscle compared to untreated mdx mice, which could point towards protection against oxidative stress [59,81,98,100]. Similarly, ROS levels were significantly reduced upon taurine treatment in muscles of exercised dystrophic mice, as shown by dihydroethidium (DHE) staining, which reacts with O 2 − [96]. In addition, taurine normalized the resting macroscopic ionic conductance (gm), a measure for ROS production, to WT levels [96].","In mdx mice, the threshold of the membrane potential at which a contraction is elicited is shifted towards a more negative value than in control mice. Thus, contraction is induced upon a lower depolarization state than in WT animals [96,101]. This mechanical threshold is indicative for E-C functioning and suggests alterations in E-C-coupling and/or calcium homeostasis [96,101]. Interestingly, taurine treatment in mdx mice has been shown to partially restore the mechanical threshold [96,99,101]. Taurine treatment in mdx did not alter expression of proteins involved in E-C coupling such as calcium sensitive receptors (RyR), calcium channels (DHPR channels), calcium ATP-ase pump (SERCA) and calcium binding proteins (calsequestrin) [78]. Contrastingly, taurine supplementation (2.5% w/v) in rats significantly increased expression of calsequestrin 1 in the muscle [102]. Although in the mdx mouse and rat study the same dose of taurine was used (2.5% w/v), this discrepancy in outcome might be explained by age-dependent calsequestrin regulation, changes in calsequestrin regulation upon dystrophin deficiency, species-dependent regulation, or differences in treatment protocol. However, a specific explanation cannot be pinpointed with the current literature studies available.","Histopathological characteristics such as necrotic myofibers and fibers with centralized nuclei, indicative for regeneration ensuing myofiber damage, are conspicuous in Hematoxylin-Eosin stained sections of mdx muscles and are used to evaluate therapeutic effectiveness [59]. In the study of Barker, taurine (2.5% w/v) reduced the amount of noncontractile area in young mdx mice, whereas no effect on the percentage regenerative fibers could be observed [78]. Other studies have reported a significant increase in healthy myofibers with peripheral nuclei upon taurine treatment [59], a decrease of histopathological features and myofiber necrosis [80,96]. Thus, taurine seems to alleviate histological features related to dystrophinopathy.","Combined use of taurine (1 g/kg) and α-methylprednisolone (PDN) (1 mg/kg), a synthetic adrenocortical hormone, in the exercised mdx mouse model significantly improved muscle strength in such a way that a synergistic effect was proposed by Cozzoli et al. [103]. The increase in fore limb muscle strength after 4 weeks of treatment was significantly higher in mice that received combined therapy compared to mice treated with PDN. Similarly, the increment in muscle force normalized to body weight was remarkably elevated in mice that received taurine + PDN compared to single-drug treatments. Furthermore, combined treatment normalized the rheobase potential to WT values, however similar results were observed in taurine-treated mice. No synergistic effect of combination treatment compared to PDN-treatment was detected regarding histopathological markers that included the amount of centronucleated fibers, necrosis and non-muscle tissue [103].","Contrastingly, the study of Barker et al. reported no effects of combined treatment on muscle strength. These opposing findings might be explained by differences in experimental set-up since in the latter study therapeutic intervention was initiated more closely to onset of damage, higher taurine concentrations were used for a shorter period of time and analysis occurred at the peak of damage [97]. Besides possible synergistic effects of combined treatment, taurine could also counteract side-effects related to corticosteroids. Dexamethasone causes muscle atrophy and significantly lowers the myotube diameter, which is restored upon taurine treatment [18]. Furthermore, taurine protects against glucocorticoid-induced mitochondrial dysfunction of the bone and might attenuate corticoid-induced osteoporosis and or osteonecrosis, pathological features that are conspicuous in glucocorticoid-treated DMD patients [104][105][106].","In some rat studies, different test regimes of taurine supplementation (3% w/v for 4 weeks [107], 100 mg/kg for 2 weeks [108]; 500 mg/kg for 2 weeks [109]) resulted in increased muscle taurine content, whilst other long-term studies (1% w/v ≈ 50 mg/kg for 22 weeks [110]) could not report increased muscle taurine levels. Similarly, in mouse studies muscle taurine levels were elevated upon taurine supplementation (4% w/v for 3 weeks [59]; 3% w/v for 4 weeks [79]). Interestingly, one study showed that continuous taurine supplementation (2.5% w/v) in mdx mice increased muscle taurine concentration up to the age of 4 weeks, but when treatment was prolonged up to the age of 10 weeks, muscle taurine concentration was significantly decreased in taurine supplemented mdx mice compared to untreated mdx mice [78]. Similarly, high doses of taurine (8% w/v ≈ 16 g/kg/day) added to the drinking water up until the age of 6 weeks, did not increase muscle taurine content [98]. Therefore, it might be hypothesized that the muscle taurine content is strictly regulated. Since chronic treatment might not be able to increase intramuscular taurine levels it is not known if long-term taurine treatment could effectively attenuate muscle damage. Furthermore, taurine intake (≈5 g/day) for a period of 7 days was reported not to alter muscle taurine levels in humans [111].","In general, taurine is well tolerated and safe if used in appropriate concentrations [112]. One study has reported gastro-intestinal complaints; however, taurine was used in combination with other nutritional supplements [112,113]. Another study aimed to investigate taurine supplementation in patients with end-stage renal failure. In healthy subjects, excessive taurine is excreted; however, due to kidney failure the taurine overload was not immediately cleared and these patients suffered from dizziness and vertigo [114] which was the reason to discontinue the study.","High taurine treatment (16 g/kg) in 6 week-old animals significantly reduced the body weight of mdx mice by more than 20% and shortened tibia length by 10%. Of note, the body weight and tibia length of untreated mdx mice were comparable to those of WT animals in this experiment [98]. Similarly, taurine treatment (3% w/v) for 4 weeks in adult mdx mice (5 months old) reduced body weight and muscle mass of mdx mice; however, the weight of these animals still exceeded that of WT animals [79]. Interestingly, no effect of taurine on body weight was observed in WT animals. Obesity is commonly observed in DMD patients, especially in glucocorticoid-treated patients [7,115,116]. Therefore, it remains unclear if taurine-induced decreased body weight would be disadvantageous in these patients. Obviously if growth is hampered, this should be avoided. A study conducted in piglets reported a reduced gain to feed ratio upon higher taurine supplementation (3% taurine diet) compared to untreated piglets whilst supplementation with 0.3% taurine might improve growth [117]. Terrill et al. have hypothesized that high taurine supplementation in young animals interferes with the taurine synthesis pathway, shifting the equilibrium to the left inducing increased cysteine levels in the plasma which could hamper growth [98].","Approximately 50-65% of DMD patients are using vitamins or nutritional supplements [115,116], which indicates that the quest for supportive treatment, including but not limited to taurine, in DMD is relevant. A detailed overview of nutritional supplements that are under investigation for the treatment of DMD is provided in the review of Boccanegra et al. [118].","The current nutritional guidelines for patients are very similar to those used for the general population. If serum 25-hydroxy-vitamin D drops below 30 ng/mL or calcium intake is low, the use of vitamin D and, respectively, calcium intake under the form of calcium citrate or calcium carbonate is advised in order to stimulate bone health, which could be impaired in DMD patients [116,117,119]. In addition, other nutrients are currently under investigation. Coenzyme Q10, a naturally occurring anti-oxidant, significantly improved muscle strength in steroid-treated DMD patients [118,120]. Similarly, beneficial effects of L-arginine, creatine, omega-3 fatty acids have been observed in clinical trials [118].","Taurine is involved in numerous processes such as protein stabilization and osmotic homeostasis and appears to be indispensable for physiological muscle function as evidenced in the TauT KO mouse. We further zoomed in on DMD, a severe muscle disorder that showed altered regulation of taurine and its transporter. Multiple facets of dystrophinopathology and potential mechanisms on how taurine might act on these pathological features have been proposed and discussed in view of the mdx mouse model. Promising results have been obtained in the mdx mouse model in terms of inflammation, muscle strength, oxidative stress, etc. and led to the conclusion that taurine supplementation is relevant for DMD pathology. Former clinical trials conducted to evaluate the anti-aging or mood stabilizing effects of taurine have shown that taurine is well tolerated and considered safe upon appropriate use. However, up until now, no clinical trials have been conducted that evaluated taurine as a treatment for DMD patients. We propose that taurine has the potential to act as supportive therapy in combination with glucocorticoids for the treatment of DMD patients and further studies should be conducted to evaluate the effectiveness of chronic taurine supplementation."],"string":"[\n \"Taurine or 2-aminoethane-sulfonic acid is primarily a free occurring sulfur-containing amino acid. Unlike most other amino acids, it is not a building block for proteins, yet classifies as a conditionally essential amino acid that is abundant in excitable tissues such as brain, retina, heart, and skeletal muscle, where intracellular concentrations range from 20 to 70 mmol/kg. Taurine is either taken up from diet, for example from fish and meat, or can be synthesized from other amino acids such as cysteine or methionine. Taurine has versatile functions: it plays an important role in osmoregulation, acts as a stabilizer of the cell membrane and of proteins, has anti-oxidant and anti-inflammatory functions, regulates mitochondrial tRNA activities, is involved in calcium homeostasis, etc. [1][2][3].\",\n \"In this review, we focused on the role of taurine in muscle disease, especially in Duchenne Muscular Dystrophy (DMD), a progressive muscle wasting disorder affecting approximately 1 per 5000 male births [4]. Muscle weakness is conspicuous in the hip-and pelvic area first, and later spreads to distal regions. Patients become wheelchair-dependent in their early teens and eventually require cardiac and respiratory care since the muscles of the heart and respiratory system are affected in a life-threatening manner. While awaiting curative treatments to enter the clinic, glucocorticoids are the standard of care, and can prolong life-expectancy of DMD patients [5,6]. Although the precise mechanism by which glucocorticoids slow down disease progression in DMD is not completely understood, its anti-inflammatory action might play a crucial role. However, the use of glucocorticoids negatively influences bone health, which is already impaired in DMD patients [7]. A comprehensive overview of emerging genetic therapies in DMD is provided in the review of Sun et al. [8] The genetic cause of DMD is mutations in the dystrophin gene, located on chromosome X, which hampers the production of functional dystrophin protein. The latter is a keycomponent of the dystrophin-associated protein complex (DAPC) that provides stability to muscle fibers during contraction and relaxation by connecting the intracellular actin cytoskeleton to the basal lamina [7]. Besides membrane stabilization, DAPC also fulfils a role in signal transduction. Dystrophic muscles encounter chronic inflammation, oxidative stress, and ischemia. Eventually these detrimental processes lead to loss of muscle mass and muscle fibrosis [7].\",\n \"This review explores the physiological role of taurine in skeletal muscle and focuses on the consequences of its disturbed balance in DMD. The therapeutic potential of taurine as a dietary supplement for DMD will be scrutinized.\",\n \"A first clue towards an important role for taurine in the muscle was its relatively high abundance. Proper insights regarding the function of taurine in physiological muscle function were acquired upon the generation of taurine transporter (TauT) knock-out (KO) mouse models and ablation of muscle taurine content. Lack of taurine impaired the conductance velocity of the muscle without affecting nerve conductance speed [2]. In addition, exercise performance was seriously hampered in TauT KO mice as shown by a significantly lower running speed. In a different experimental set-up, the total running distance was only 20% of the distance travelled by age-matched wild type (WT) mice [2,9,10]. Besides running tests, the reduced exercise capacity of TauT KO mice also became apparent during a weight-loaded swimming test that showed an 80% decrease in swimming time compared to WT [11]. This study reported structural changes in morphology of TauT KO muscle; however, Warskulat et al. hypothesized that hampered exercise performance was likely attributed to muscle dysfunction, resulting from taurine deficiency. In evidence, serum creatine kinase levels are increased in TauT KO and serum lactate levels were raised after exercise [2]. Some of the pathological characteristics of TauT KO models such as necrotic myofibers and reduced exercise capacity resemble the features of the mdx mouse model [12,13]. Another approach to evaluate the effect of taurine depletion is the use of guanodinoethane sulfonate (GES). GES, a taurine transporter antagonist, reduces muscle taurine content by 60% [14]. Previously taurine was shown to enhance calcium uptake and release in myofibers concurrently with an increase in force production, whereas myofibers derived from GES-treated mice showed reduced force production at relevant stimulation frequencies. Interestingly, fatigue was reportedly attenuated upon GES-treatment [14].\",\n \"Furthermore, the lifespan of TauT KO male mice is significantly lower than those of WT mice, 511 and 686 days, respectively. Reduced life expectancy together with increased expression of p16INK4a, an indicator of senescence, in TauT KO mice allowed to hypothesize that taurine might be involved in aging [15,16]. Furthermore, it was suggested that taurine delays muscle-specific senescence including sarcopenia in tumor necrosis factor (TNF)-α stimulated L6 myogenic rat cells. In evidence, differences in the regulation of inflammation, autophagy, and apoptosis have been reported upon taurine treatment [17]. In addition, TNF-α stimulation of L6 myogenic rat cells hampered muscle differentiation which could be restored by taurine. Presumably this effect was mediated through the PI3/AKT signaling pathway since myocyte enhancer factor-2 (MEF-2), a transcription factor involved in myogenic differentiation, was markedly decreased after knockdown of AKT [17]. Furthermore, expression of TauT increased during muscle differentiation and was even further enhanced upon binding of MEF-2 to the promotor region of TauT [18].\",\n \"In conclusion, depletion of muscle taurine levels either through TauT KO or via pharmacological inhibition of the taurine transporter by GES alters force output and exercise performance. Therefore, taurine seems essential for the preservation of physiological muscle function. The sections below provide an overview of the main cellular processes in which taurine plays a role in relation to the muscle.\",\n \"Exposure to hyperosmolar conditions can induce several detrimental cellular effects including interference with transcriptional and translational activity, induction of oxidative stress, DNA damage, and can even elicit apoptosis under certain conditions [19,20]. Thus, safeguarding the osmotic equilibrium is essential for ensuring cellular health. When cells are exposed to an environment high on NaCl, fluid is retracted from the intracellular compartment causing cellular shrinkage, molecular crowding, and increased ionic forces. In order to counteract these deleterious processes, the cell's response is a regulatory volume increase (RVI) that includes activation of inorganic ion transporters (e.g., Na + /K + /2Cl − cotransporter, the Na + /H +/− , and the Cl − /HCO 3 − exchanger), allowing an influx of ions accompanied by osmotic uptake of water [19][20][21][22][23]. However, this condition is unfavorable over time due to increased intracellular ionic forces that could interact with macromolecules. Thus secondarily, accumulation of organic osmolytes (e.g., taurine) that replace inorganic electrolytes results in normalization of ionic strength whilst preserving cellular volume and protein stability [19,20]. Rather than stimulation of de novo synthesis, osmotic stress is most likely to enhance cellular import of taurine [20]. Both the designated TauT as well as the proton-coupled amino-acid transporter (PAT) 1 are capable of accumulating taurine in the cell [24]. However, TauT is considered as the principal transporter of taurine in muscle cells as evidenced by a 98% reduction of taurine content in a TauT KO mouse [10]. Transcription of TauT mRNA is upregulated under hypertonic conditions due to binding of Nuclear Factor of Activated T-cells 5 (NFAT-5) to the 5 flank region of the TauT gene. NFAT-5, also known as tonicity responsive element binding protein (TonEBP) acts as a transcription factor of SLC6A6, the gene encoding TauT, and thus allows cellular accumulation of taurine [25,26].\",\n \"Exercise can affect the osmotic balance in muscle fibers. Muscle subjected to an intensive exercise protocol resulted in increased myofiber volume, cross-sectional area, and water concentration by more than 15%, indicative for muscle fiber swelling [27,28]. The rise in myofiber water content can be partially explained by water production in cellular metabolic processes that take place during exercise [27][28][29]. Additionally, intracellular solute concentrations might be elevated during exercise as a result of phosphocreatine splitting and increased lactate and H + , ensuing water influx in order to retain osmotic balance [28] and could contribute to myofiber swelling as well. This volume increase is followed by a compensatory mechanism named the regulatory volume decrease (RVD) that releases electrolytes (e.g., K + , HCO 3 − , Cl − ) and osmolytes such as taurine concurrently with water in order to normalize cellular volume [19,20,[26][27][28][29][30][31]. Thus, taurine is released in order to counteract myofiber swelling, a phenomenon which occurs during exercise [28,31].\",\n \"The stabilizing effect of taurine is mentioned in many papers. However, the mechanism by which taurine is able to exert stabilization is poorly described. In order to comprehend this characteristic, it is important to understand its interaction with water molecules, considering the chemical properties related to its molecular structure. One of the most popular hypotheses that could explain protein stabilization by osmolytes is based on preferential exclusion [32][33][34][35]. This principle builds on unfavorable interactions between proteins and osmolytes in terms of Gibbs adsorption isotherm [32]. In a denatured state, the area of the peptide backbone by which osmolytes can interact is larger and results in increased Gibbs energy (unfavorable). In order to reduce these interactions, the thermodynamic component drives the folding equilibrium towards its native state, also referred to as the osmophobic effect, which is associated with a much lower Gibbs energy. This simplified explanation implies that in the presence of stabilizing osmolytes, the Gibbs energy of the denatured state is much higher compared to Gibbs energy of the folded state [32]. Therefore, the folded protein conformation is favored and osmolytes act as protein stabilizers [32,33]. In general, the presence of osmolytes results in a specific distribution of water molecules around the proteins in a preferential hydrated state and osmolyte exclusion from the protein backbone [32][33][34][35].\",\n \"Furthermore, stabilizing actions have been attributed to taurine, as well as direct interaction with protein side chains [33][34][35]. The amino group of taurine orients itself preferentially to the protein side chain. This strengthens the hydrogen bonded network of water surrounding the protein and stabilizes its native form. The latter appears to contradict the preferential exclusion theory; however, such interactions between osmolytes and side chains have also been discussed by Bolen et al. [32]. It should be noted that protein side chains are associated with other characteristics than the protein backbone and favorable interactions between osmolyte and side chains might occur. Supposedly, the latter does not substantially alter the protein folding state [32]. In the article by Brudziak et al., the protein was hydrated in the presence of taurine [35]. This might suggest that besides limited interactions between osmolytes and protein side chains, the protein is still preferentially surrounded by water molecules. Taurine was able to increase the thermal stability of both lysozyme and ubiquitin protein [35][36][37][38], although the extent of stabilization was protein specific [35].\",\n \"In addition to protein stabilization, membrane stabilizing properties of taurine were hypothesized by Huxtable and Bressler [39]. Taurine inhibits the activity of phospholipid methyltransferase, which catalyzes the methylation of phosphatidylethanolamine to form phosphatidylcholine and thus taurine could alter the composition and consequently the properties and stability of phospholipid membranes [40][41][42]. In evidence, the presence of taurine decreased the viscosity of erythrocytic membranes, suggesting taurine might increase membrane fluidity [43].\",\n \"Eccentric muscle contraction might induce denaturation of myofibrillar proteins as hypothesized by Paulsen et al. [44]. In addition, the unfolded protein response (UPR) is activated during exercise [45,46] which might indicate that proteins struggle to maintain native folding conformations. Interestingly, prolonged exercise increased the denaturation temperature of albumin, pointing to enhanced thermal stability [47]. The importance of taurine in protein stabilization is illustrated in the TauT KO mouse model [15]. It is assumed that a lack of taurine allows accumulation of unfolded and/or misfolded proteins in skeletal muscle which activates expression of genes involved in the UPR. Thus, taurine plays a key role in protein homeostasis of skeletal muscle [15,48].\",\n \"Under physiological conditions, reactive oxygen species (ROS) are balanced by antioxidant mechanisms that detoxify reactive species. A limited amount of ROS is produced during exercise and exerts advantageous effects on force generation. In addition, low levels of ROS might protect against injury through adaptations in cellular signaling upon regular training exercise [27,47,49], whereas high levels of ROS are associated with muscle dysfunction [50]. Although direct scavenging of the main ROS (e.g., superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH)) by taurine is considered unlikely [51,52], taurine is believed to relieve oxidative stress through neutralization of hypochlorous acid (HOCl) and upregulation of antioxidant enzymes [53][54][55]. Upon inflammation, neutrophils become activated and release myeloperoxidase (MPO). MPO catalyzes the reaction of chloride and H 2 O 2 , a classic ROS molecule, resulting in the formation of HOCl. HOCl has toxic effects and interferes with cellular processes including molecular transport and pump capacity [56]. Recently, it has been hypothesized that HOCl could alter excitation-contraction (E-C) coupling and impairs muscle force production [57]. HOCl is converted to TauCl after interaction with taurine. TauCl possesses anti-inflammatory properties and increases antioxidant enzymes including heme oxygenase 1 in murine microglial cells and muscle cells [58,59]. Taurine supplementation increased activity of antioxidant enzymes such as superoxide dismutase and catalase, measured in the blood of patients with type II diabetes [60]. Similar results were obtained in the liver and kidney of an ethanol-induced oxidative stress mouse model [61].\",\n \"Furthermore, antioxidant effects of taurine on superoxide production and lipid peroxidation were observed in the muscle of eccentric exercised rats [62]. Similarly oxidative lipid damage was reduced by taurine treatment in a mouse model of muscle overuse [63].\",\n \"Taurine participates in the synthesis of mitochondrial proteins, more specifically proteins that require tRNA (Leu) and tRNA (Lys) with uridine on a Wobble position [64]. If the first base of the tRNA anticodon is uridine, then the classic Watson-Crick rules are substituted by Wobble base pairing rules. According to this hypothesis, H-bonds are formed between the first base of the anticodon (tRNA) and the third position of the mRNA codon. Contrastingly to Watson-Crick base pairing, Wobble pairing suggests that uridine on a Wobble position at the anticodon can form H-bonds with A, U, G, and C on the third position of the mRNA codon mRNA. Whereas, taurine-conjugated uridine tRNA will promote the formation of H-bonds between uridine and A or G on the third codon position and is required for appropriate translation to leucine (UUA/UUG) [64]. Taurine modification of uridine is required in some mitochondrial tRNAs to ensure a more specific codon-anticodon interaction and proper mitochondrial protein synthesis. Cytochrome b, ND5, and ND6 are mitochondrial proteins containing UUG codons, and synthesis of these proteins is potentially hampered if taurine conjugation of mitochondrial tRNA Leu(UUR) is deficient. ND5 and ND6 are functional subunits of oxidative phosphorylation complex I, that catalyzes electron transport from NADH to coenzyme Q [64,65]. As the process of oxidative phosphorylation is one of the main sources of ROS [50] in myofibers, preservation of mitochondrial function is considered essential in the safeguarding of oxidative stress.\",\n \"Specific mutations in tRNA (Lys) and tRNA (Leu) are associated with respectively myoclonic epilepsy with ragged red fibers (MERRF) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). MERRF and MELAS are mitochondrial diseases that show disturbed protein synthesis and pathological characteristics such as exercise intolerance, thus of which some aspects resemble the TauT KO phenotype [9,64,66]. Of note, taurine supplementation in MELAS patients improved the occurrence of stroke-like episodes [67].\",\n \"Calcium is an essential cellular building block and a universal carrier of biological information. As a diffusible intracellular second messenger, calcium is involved in signaling transduction pathways and can regulate many different processes ranging from neurotransmitter release, bone formation, and blood coagulation to muscle contraction [68]. More specifically, calcium is essential during the E-C coupling mechanism that transforms the electrical input (action potential) to a mechanical output (contraction). Upon adequate stimulation, an action potential is generated and propagates over the sarcolemma to the transverse tubules that contain L-type calcium channels e.g., dihydropyridine receptors (DHPR). In the skeletal muscle, DHPR are in close contact with the ryanodine receptors (RyR) of the sarcoplasmic reticulum (SR), which releases calcium into the cytosol. Binding of calcium to troponin C induces the translocation of tropomyosin, thereby allowing the formation of cross-bridges between actin and myosin filaments and subsequent contraction [69].\",\n \"Temporal and spatial changes of calcium concentration in cytoplasm or organelles are monitored by a multitude of calcium sensing proteins that determine the character and duration of the cellular response. The SR is an organelle involved in storage of calcium which is released into the cytoplasmic environment upon muscle stimulation. Taurine partially preserved SR function upon exposure to phospholipase C, which is known to hamper calcium transport, in SR derived from rat skeletal muscle. Furthermore, in the presence of 15 mM taurine, uptake of calcium oxalate by the SR was increased by more than 20% [39].\",\n \"Similarly, taurine significantly increased the accumulation of calcium in SR of skinned EDL rat myofibers [70]. In this experiment, the membrane of the myofibers was mechanically removed which allowed to control intracellular taurine concentrations. The enhanced calcium SR load and subsequent release upon stimulation might explain the increment in force response upon depolarization. The authors hypothesized that taurine modulates SR calcium pump activity, allowing increased calcium accumulation [70]. These results are consistent with observations in human skeletal muscle fibers type I and II; interestingly, these different muscle fibers contain a different type of SR calcium transport ATPase (SERCA)-isoform, thus taurine might modulate either activity and/or calcium affinity [71]. Of note, a small decline in calcium sensitivity was observed in the presence of taurine in SR of skinned EDL myofibers [70].\",\n \"Furthermore, voltage clamp experiments carried out in cardiomyocyte derived from guinea pig showed that the effect of taurine on calcium influx is highly dependent of the extracellular calcium concentration. In the presence of taurine (20 mM), calcium influx was slightly increased upon low extracellular calcium concentration (0.8 mM) and vice versa, the calcium influx was slightly yet significant decreased when extracellular calcium concentrations were high (3.6 mM). No effect of taurine on inward calcium current could be observed at an extracellular calcium level of 1.8 mM, suggesting that taurine aims at maintaining intracellular calcium homeostasis [72]. However, taurine modulated resting membrane potential regardless of extracellular calcium channels.\",\n \"Previously, it has been suggested that alterations in taurine and/or its regulation associate with dystrophinopathology [73][74][75]. Lack of dystrophin results in destabilization of DAPC and thereby rendering the sarcolemma more susceptible to contraction-induced damage. Membrane fragility is observed in dystrophin deficient muscle as evidenced by increased permeability to dyes such as Evans Blue and Procion Orange, accumulation of serum proteins such as albumin and IgG, increased levels of creatine kinase in the blood, and sarcolemma rupture in myofibers [7,76,77].\",\n \"Taurine regulation is altered in dystrophic tissues of mouse models. Some studies reported a significant downregulation of the TauT in muscles of the mdx mouse model [73], whereas others reported no significant differences between mdx mice and age-matched control mice [78,79]. Seemingly, TauT is significantly downregulated in young mdx mouse and normalizes to control levels over time.\",\n \"Similar to the expression of TauT, differences in muscle taurine content with age were reported as well, yet with considerable differences between studies. Taurine content of mdx muscle was comparable to controls at age 3-4 weeks, but increased by age 10 weeks [78,80]. A significant downregulation of taurine in mdx mice aged 4 and 6 weeks compared to age-matched control mice was reported [59,73], whereas other studies found no significant difference in taurine content between control and mdx mice at age 6 weeks [73,81]. A decline in taurine content was on the other hand observed in the plantaris muscle of 6-month-old mdx mice compared to wet weight yet did not hold up when compared to dry weight [79]. Contrasting results have been reported regarding the taurine content in mdx mice, however most of these studies conclude that taurine is differentially regulated in mdx mice compared to control mice at a certain time point. The expression of TauT and taurine content in the muscle of mdx mice compared to age-matched control mice is summarized in Table 1. Interestingly, muscle taurine levels increased in glucocorticoid treated mdx mice, pointing to its reestablishment by immunosuppressive therapeutic interventions [75]. As opposed to the observations in the mdx mouse model a significant increase in muscle taurine and its transporter were discerned in 8-month-old Golden Retriever Muscular dystrophy (GRMD) canine model [74]. Similarly, taurine regulation in DMD patients differs from healthy control patients, as shown by increased levels of muscle TauT protein in DMD patients [82,83]. Thus, expression of TauT is differently regulated in the mdx mouse than in the GRMD-model and DMD patients. Interestingly, the phenotype in the mdx mouse model is milder than in GRMD dogs and DMD patients. Myofiber necrosis and muscle weakness become conspicuous approximately 3 weeks after mdx mice are born. Myofiber necrosis in the mdx mouse is most explicit in the young to juvenile period, followed by necrosis at a slower pace in adult mice [74,80]. The course of disease in mdx mice thereby differs from the more progressive pathology in GRMD dogs and DMD patients, that is characterized by fatty replacement and fibrosis at an early age [74]. These differences in pathological features might be linked to regulation of TauT.\",\n \"Mitochondria participate in cellular energy production by synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation [84]. This process includes transfer of electrons, required for ATP-synthesis, and is inevitably linked to production of ROS. Under physiological conditions, basal levels of ROS are generated as a by-product of oxidative phosphorylation. However, ROS production is enhanced upon mitochondrial dysfunction [84]. Upon oxidative phosphorylation, electron transfer is facilitated by mitochondrial protein complexes that resides within the inner mitochondrial membrane [84]. However, Onopiuk found significantly reduced protein levels of complex III, cytochrome-c reductase, and complex V, the ATP synthase, in immortalized myoblasts of mdx mice. Of note, myoblasts of mdx mice (SC-5), and control myoblasts (IMO) were used [85]. Neither of these myoblasts expressed dystrophin protein, only dystrophin mRNA was present in control myoblasts. In addition, Onopiuk et al. showed an increase in mitochondrial membrane potential [85]. Taken together, these results suggest mitochondrial dysfunction and inevitably, increased ROS production in dystrophin deficient cells. Taurine reduces ROS by upregulation of antioxidant enzymes and might preserve electron transport chain activity by safeguarding mitochondrial protein synthesis of subunits involved in the respiratory chain. In evidence, reduced taurine levels hamper expression of ND6, subunit of complex I in the mitochondria of cardiomyocytes derived from rat [64,86].\",\n \"Excessive calcium levels are observed in dystrophic myofibers; however, calcium entry through sarcolemmal tears is not considered as the main contributor to calcium overload in dystrophic myofibers. Apparently, the open probability of calcium leak channels in the proximity of micro-tears are increased, thereby allowing an increased calcium influx [87].\",\n \"Additionally, increased expression and activation of store-operated calcium channels, presumably induced by calcium-independent phospholipase A2, is observed in dystrophin deficient myofibers and could contribute to calcium overload as well [84,[88][89][90]. Moreover, a correlation between the dystrophic phenotype and expression of a stretch-activated channel, transient receptor potential canonical (TRPC) channel 1, was discovered in different muscles of the mdx mouse. The diaphragm of mdx mice was affected the most, as shown by increased Evans Blue permeability, and showed a significant upregulation of TRPC1 expression compared to controls [88]. Thus, the involvement of the TRPC channels might also contribute to the calcium overload as evidenced by increased expression of various TRPC channels in mdx mice. The cytoplasmic calcium concentration is not only determined by calcium channels/exchangers but also by the SR uptake and release of calcium, which plays an essential role during E-C coupling. RyR, responsible for the release of calcium from SR upon depolarization, is hyper nitrosylated in dystrophic muscle and consecutive depletion of calstabin-1 upon RyR-nitrosylation results in calcium leakage [84,[89][90][91][92]. During relaxation, cytoplasmic calcium ions are sequestered by SERCA, thereby lowering its cytoplasmic concentration. In the mdx mouse model, the removal of calcium ions by the SR is hampered, suggesting reduced SERCA functioning. In conclusion, calcium homeostasis is disturbed in dystrophic muscle on many levels. Chronic cytoplasmic calcium overload will induce activation of degrading pathways mediated by phospholipase 2 and proteases which eventually can lead to myofiber death [7,90].\",\n \"In addition, cytoplasmic calcium overload can induce accumulation of calcium in the mitochondria and mitochondrial dysfunction. Multiple pathways have been proposed by which mitochondrial calcium overload can induce ROS production, such as mitochondrial permeability transition (MPT)-mediated release of anti-oxidative enzymes, dislocation of mitochondrial proteins involved in electron transports including cytochrome C, induction of NO, etc. [84,93]. Mitochondrial calcium overload can elicit MPT pore formation. A permeable pore is formed that spans inner and outer mitochondrial membranes and results in mitochondrial swelling [84]. Furthermore, dystrophic muscle cells show an increased susceptibility to calcium and therefore are more prone to MPT pore formation, that eventually cause mitochondrial swelling and death [87,91]. In evidence, mitochondrial swelling, indicative for MPT pore formation, was induced at a lower calcium load in mitochondria compared to WT [84,90,[92][93][94]. Interestingly, taurine is able to attenuate calcium-induced swelling of mitochondria derived from skeletal muscle [95].\",\n \"A beneficial effect of taurine on muscle force remains controversial: supported by some studies yet disproved by others. A significant increase in peak twitch force was obtained in taurine supplemented (2.5% w/v in drinking water) mdx mice compared to untreated mdx mice at 4 weeks. However, this effect was abrogated in juvenile and adult mdx mice, respectively aged 10 weeks (2.5% w/v) and 6 months (3% w/v) [78,79]. Similarly, a >50% increase in maximum isometric tetanic specific force (sPo) was obtained by taurine supplementation in mdx mice aged 4 and 6 weeks [78,81]. However, no treatment effect (1 g/kg/day) on specific tetanic force was detected in muscle of mice aged 5-7 weeks, whereas fore limb force, assessed by means of a grip strength meter, was ameliorated [96]. Furthermore, a study performed by Barker reported no effects of taurine treatment (2.5% w/v) on peak twitch force, maximum specific force, nor fatigue or recovery in mdx mice treated from week 2 until week 4 [97]. Similarly, no effect on maximum specific force in 6-week-old mdx mice was observed when high doses of taurine (16 g/kg/day) were administered [98].\",\n \"In 6-month-old mdx mice, taurine could not ameliorate specific maximum isometric force production. However, after a fatigue protocol consisting of recurrent electrical stimulation, the EDL of taurine treated mdx animals was more resistant to fatigue, as shown by a significant higher force production at the end of stimulation relative to force production at the beginning. Furthermore, EDL muscle of taurine treated mdx animals showed a significantly better recovery capacity at 10-60 min after stimulation than untreated mdx animals [79]. Taurine treatment in exercised mdx mice significantly increased in vivo forelimb muscle strength normalized to body weight [96,99], but did not alter locomotor activity of mdx mice. A similar finding was reported in a study that supplemented unexercised mdx mice. Taurine supplementation (±4 g/kg/day) in 6-week-old unexercised mdx mice significantly increased grip strength [81]. Whereas no effect of high taurine treatment (16 g/kg/day) was observed on normalized grip strength [98].\",\n \"In summary, benefit of taurine supplementation on muscle force differed considerably between published studies. Although multiple studies have been executed, these used different concentrations, ingestion methods, and treatment protocols, which might explain the obtained conflicting results, and which hampers interpretation.\",\n \"Although different treatment conditions and read-outs were used, multiple studies have shown anti-inflammatory and anti-oxidative effects of taurine treatment in mdx mice [59,80,81,95]. The anti-oxidant and anti-inflammatory actions of taurine have been proposed as a mechanism by which taurine protects dystrophic tissue from damage. In evidence, muscle of taurine-treated mdx mice showed significantly less NF-κβ positive fibers, TNF-α levels, neutrophil elastase, and MPO activity [59,80,81,96]. Accordingly, taurine-treated mdx mice showed lower levels of disulfide and protein thiol oxidation in muscle compared to untreated mdx mice, which could point towards protection against oxidative stress [59,81,98,100]. Similarly, ROS levels were significantly reduced upon taurine treatment in muscles of exercised dystrophic mice, as shown by dihydroethidium (DHE) staining, which reacts with O 2 − [96]. In addition, taurine normalized the resting macroscopic ionic conductance (gm), a measure for ROS production, to WT levels [96].\",\n \"In mdx mice, the threshold of the membrane potential at which a contraction is elicited is shifted towards a more negative value than in control mice. Thus, contraction is induced upon a lower depolarization state than in WT animals [96,101]. This mechanical threshold is indicative for E-C functioning and suggests alterations in E-C-coupling and/or calcium homeostasis [96,101]. Interestingly, taurine treatment in mdx mice has been shown to partially restore the mechanical threshold [96,99,101]. Taurine treatment in mdx did not alter expression of proteins involved in E-C coupling such as calcium sensitive receptors (RyR), calcium channels (DHPR channels), calcium ATP-ase pump (SERCA) and calcium binding proteins (calsequestrin) [78]. Contrastingly, taurine supplementation (2.5% w/v) in rats significantly increased expression of calsequestrin 1 in the muscle [102]. Although in the mdx mouse and rat study the same dose of taurine was used (2.5% w/v), this discrepancy in outcome might be explained by age-dependent calsequestrin regulation, changes in calsequestrin regulation upon dystrophin deficiency, species-dependent regulation, or differences in treatment protocol. However, a specific explanation cannot be pinpointed with the current literature studies available.\",\n \"Histopathological characteristics such as necrotic myofibers and fibers with centralized nuclei, indicative for regeneration ensuing myofiber damage, are conspicuous in Hematoxylin-Eosin stained sections of mdx muscles and are used to evaluate therapeutic effectiveness [59]. In the study of Barker, taurine (2.5% w/v) reduced the amount of noncontractile area in young mdx mice, whereas no effect on the percentage regenerative fibers could be observed [78]. Other studies have reported a significant increase in healthy myofibers with peripheral nuclei upon taurine treatment [59], a decrease of histopathological features and myofiber necrosis [80,96]. Thus, taurine seems to alleviate histological features related to dystrophinopathy.\",\n \"Combined use of taurine (1 g/kg) and α-methylprednisolone (PDN) (1 mg/kg), a synthetic adrenocortical hormone, in the exercised mdx mouse model significantly improved muscle strength in such a way that a synergistic effect was proposed by Cozzoli et al. [103]. The increase in fore limb muscle strength after 4 weeks of treatment was significantly higher in mice that received combined therapy compared to mice treated with PDN. Similarly, the increment in muscle force normalized to body weight was remarkably elevated in mice that received taurine + PDN compared to single-drug treatments. Furthermore, combined treatment normalized the rheobase potential to WT values, however similar results were observed in taurine-treated mice. No synergistic effect of combination treatment compared to PDN-treatment was detected regarding histopathological markers that included the amount of centronucleated fibers, necrosis and non-muscle tissue [103].\",\n \"Contrastingly, the study of Barker et al. reported no effects of combined treatment on muscle strength. These opposing findings might be explained by differences in experimental set-up since in the latter study therapeutic intervention was initiated more closely to onset of damage, higher taurine concentrations were used for a shorter period of time and analysis occurred at the peak of damage [97]. Besides possible synergistic effects of combined treatment, taurine could also counteract side-effects related to corticosteroids. Dexamethasone causes muscle atrophy and significantly lowers the myotube diameter, which is restored upon taurine treatment [18]. Furthermore, taurine protects against glucocorticoid-induced mitochondrial dysfunction of the bone and might attenuate corticoid-induced osteoporosis and or osteonecrosis, pathological features that are conspicuous in glucocorticoid-treated DMD patients [104][105][106].\",\n \"In some rat studies, different test regimes of taurine supplementation (3% w/v for 4 weeks [107], 100 mg/kg for 2 weeks [108]; 500 mg/kg for 2 weeks [109]) resulted in increased muscle taurine content, whilst other long-term studies (1% w/v ≈ 50 mg/kg for 22 weeks [110]) could not report increased muscle taurine levels. Similarly, in mouse studies muscle taurine levels were elevated upon taurine supplementation (4% w/v for 3 weeks [59]; 3% w/v for 4 weeks [79]). Interestingly, one study showed that continuous taurine supplementation (2.5% w/v) in mdx mice increased muscle taurine concentration up to the age of 4 weeks, but when treatment was prolonged up to the age of 10 weeks, muscle taurine concentration was significantly decreased in taurine supplemented mdx mice compared to untreated mdx mice [78]. Similarly, high doses of taurine (8% w/v ≈ 16 g/kg/day) added to the drinking water up until the age of 6 weeks, did not increase muscle taurine content [98]. Therefore, it might be hypothesized that the muscle taurine content is strictly regulated. Since chronic treatment might not be able to increase intramuscular taurine levels it is not known if long-term taurine treatment could effectively attenuate muscle damage. Furthermore, taurine intake (≈5 g/day) for a period of 7 days was reported not to alter muscle taurine levels in humans [111].\",\n \"In general, taurine is well tolerated and safe if used in appropriate concentrations [112]. One study has reported gastro-intestinal complaints; however, taurine was used in combination with other nutritional supplements [112,113]. Another study aimed to investigate taurine supplementation in patients with end-stage renal failure. In healthy subjects, excessive taurine is excreted; however, due to kidney failure the taurine overload was not immediately cleared and these patients suffered from dizziness and vertigo [114] which was the reason to discontinue the study.\",\n \"High taurine treatment (16 g/kg) in 6 week-old animals significantly reduced the body weight of mdx mice by more than 20% and shortened tibia length by 10%. Of note, the body weight and tibia length of untreated mdx mice were comparable to those of WT animals in this experiment [98]. Similarly, taurine treatment (3% w/v) for 4 weeks in adult mdx mice (5 months old) reduced body weight and muscle mass of mdx mice; however, the weight of these animals still exceeded that of WT animals [79]. Interestingly, no effect of taurine on body weight was observed in WT animals. Obesity is commonly observed in DMD patients, especially in glucocorticoid-treated patients [7,115,116]. Therefore, it remains unclear if taurine-induced decreased body weight would be disadvantageous in these patients. Obviously if growth is hampered, this should be avoided. A study conducted in piglets reported a reduced gain to feed ratio upon higher taurine supplementation (3% taurine diet) compared to untreated piglets whilst supplementation with 0.3% taurine might improve growth [117]. Terrill et al. have hypothesized that high taurine supplementation in young animals interferes with the taurine synthesis pathway, shifting the equilibrium to the left inducing increased cysteine levels in the plasma which could hamper growth [98].\",\n \"Approximately 50-65% of DMD patients are using vitamins or nutritional supplements [115,116], which indicates that the quest for supportive treatment, including but not limited to taurine, in DMD is relevant. A detailed overview of nutritional supplements that are under investigation for the treatment of DMD is provided in the review of Boccanegra et al. [118].\",\n \"The current nutritional guidelines for patients are very similar to those used for the general population. If serum 25-hydroxy-vitamin D drops below 30 ng/mL or calcium intake is low, the use of vitamin D and, respectively, calcium intake under the form of calcium citrate or calcium carbonate is advised in order to stimulate bone health, which could be impaired in DMD patients [116,117,119]. In addition, other nutrients are currently under investigation. Coenzyme Q10, a naturally occurring anti-oxidant, significantly improved muscle strength in steroid-treated DMD patients [118,120]. Similarly, beneficial effects of L-arginine, creatine, omega-3 fatty acids have been observed in clinical trials [118].\",\n \"Taurine is involved in numerous processes such as protein stabilization and osmotic homeostasis and appears to be indispensable for physiological muscle function as evidenced in the TauT KO mouse. We further zoomed in on DMD, a severe muscle disorder that showed altered regulation of taurine and its transporter. Multiple facets of dystrophinopathology and potential mechanisms on how taurine might act on these pathological features have been proposed and discussed in view of the mdx mouse model. Promising results have been obtained in the mdx mouse model in terms of inflammation, muscle strength, oxidative stress, etc. and led to the conclusion that taurine supplementation is relevant for DMD pathology. Former clinical trials conducted to evaluate the anti-aging or mood stabilizing effects of taurine have shown that taurine is well tolerated and considered safe upon appropriate use. However, up until now, no clinical trials have been conducted that evaluated taurine as a treatment for DMD patients. We propose that taurine has the potential to act as supportive therapy in combination with glucocorticoids for the treatment of DMD patients and further studies should be conducted to evaluate the effectiveness of chronic taurine supplementation.\"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["Introduction","Involvement of Taurine in Physiological Skeletal Muscle Functioning","Knowledge Gained from Knockout Models","Taurine and Its Role in Osmotic Homeostasis","Taurine and Its Role in Protein and Membrane Stabilization","Taurine and Its Role in Oxidative Stress","The Role of Taurine in Mitochondrial Protein Synthesis","Taurine and Its Role in Calcium Homeostasis","Pathophysiological Characteristics of Taurine in DMD","Regulation in Dystrophin Deficiency","Role in Oxidative Stress Management and Mitochondrial Protein Synthesis","Dysregulation of Calcium Homeostasis","Taurine Supplementation as a Therapeutic Strategy for DMD","Effect of Taurine on Muscle Force","Effect of Taurine on Oxidative Stress and Inflammation","Effects of Taurine on E-C Coupling","Effect of Taurine on Histopathological Characteristics of the Mdx Mouse","Combinatory Use of Taurine and Glucocorticoids","Potential Caveats of Taurine Treatment","Other Nutritional Supplements Used in Duchenne Muscular Dystrophy","Conclusions","Table 1 ."],"string":"[\n \"Introduction\",\n \"Involvement of Taurine in Physiological Skeletal Muscle Functioning\",\n \"Knowledge Gained from Knockout Models\",\n \"Taurine and Its Role in Osmotic Homeostasis\",\n \"Taurine and Its Role in Protein and Membrane Stabilization\",\n \"Taurine and Its Role in Oxidative Stress\",\n \"The Role of Taurine in Mitochondrial Protein Synthesis\",\n \"Taurine and Its Role in Calcium Homeostasis\",\n \"Pathophysiological Characteristics of Taurine in DMD\",\n \"Regulation in Dystrophin Deficiency\",\n \"Role in Oxidative Stress Management and Mitochondrial Protein Synthesis\",\n \"Dysregulation of Calcium Homeostasis\",\n \"Taurine Supplementation as a Therapeutic Strategy for DMD\",\n \"Effect of Taurine on Muscle Force\",\n \"Effect of Taurine on Oxidative Stress and Inflammation\",\n \"Effects of Taurine on E-C Coupling\",\n \"Effect of Taurine on Histopathological Characteristics of the Mdx Mouse\",\n \"Combinatory Use of Taurine and Glucocorticoids\",\n \"Potential Caveats of Taurine Treatment\",\n \"Other Nutritional Supplements Used in Duchenne Muscular Dystrophy\",\n \"Conclusions\",\n \"Table 1 .\"\n]"},"annotations_table":{"kind":"list like","value":["Timepoint of Analysis \nMuscle TauT Content \nMuscle Taurine Content \nMuscle Type \nReference \n\n18 days \n↓ in mdx mice \n≈ controls \nquadriceps \n73 \n\n22 days \n/ \n≈ controls \nquadriceps \n80 \n\n28 days \n28 days \n\n≈ controls \n≈ controls \n\n↓ in mdx mice \n≈ controls \n\nquadriceps \ntibialis anterior \n\n73 \n78 \n\n6 weeks \n6 weeks \n6 weeks \n\n↓ in mdx mice \n/ \n/ \n\n≈ controls \n↓ in mdx mice \n≈ controls \n\nquadriceps \nquad/gas \ntibialis anterior \n\n73 \n59 \n81 \n\n10 weeks \n≈ controls \n↑ in mdx \ntibialis anterior \n78 \n\n6 months \n≈ controls \n\n↓ in mdx mice (wet weight) \n≈ controls \n(dry weight) \n\nEDL (TauT); plantaris \n(taurine) \n79 \n\n"],"string":"[\n \"Timepoint of Analysis \\nMuscle TauT Content \\nMuscle Taurine Content \\nMuscle Type \\nReference \\n\\n18 days \\n↓ in mdx mice \\n≈ controls \\nquadriceps \\n73 \\n\\n22 days \\n/ \\n≈ controls \\nquadriceps \\n80 \\n\\n28 days \\n28 days \\n\\n≈ controls \\n≈ controls \\n\\n↓ in mdx mice \\n≈ controls \\n\\nquadriceps \\ntibialis anterior \\n\\n73 \\n78 \\n\\n6 weeks \\n6 weeks \\n6 weeks \\n\\n↓ in mdx mice \\n/ \\n/ \\n\\n≈ controls \\n↓ in mdx mice \\n≈ controls \\n\\nquadriceps \\nquad/gas \\ntibialis anterior \\n\\n73 \\n59 \\n81 \\n\\n10 weeks \\n≈ controls \\n↑ in mdx \\ntibialis anterior \\n78 \\n\\n6 months \\n≈ controls \\n\\n↓ in mdx mice (wet weight) \\n≈ controls \\n(dry weight) \\n\\nEDL (TauT); plantaris \\n(taurine) \\n79 \\n\\n\"\n]"},"annotations_tableref":{"kind":"list like","value":["Table 1"],"string":"[\n \"Table 1\"\n]"},"annotations_title":{"kind":"list like","value":[],"string":"[]"},"annotations_venue":{"kind":"list like","value":[],"string":"[]"}}},{"rowIdx":84739,"cells":{"corpusid":{"kind":"number","value":204943882,"string":"204,943,882"},"updated":{"kind":"string","value":"2022-02-03T17:39:25Z"},"content_source_oainfo_license":{"kind":"string","value":"CCBY"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"https://www.frontiersin.org/articles/10.3389/fendo.2019.00741/pdf"},"content_source_oainfo_status":{"kind":"string","value":"GOLD"},"content_source_pdfsha":{"kind":"string","value":"81bd6c904a1445c00b06b088979b75c39c749582"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.3389/fendo.2019.00741"},"externalids_mag":{"kind":"string","value":"2982633354"},"externalids_pubmed":{"kind":"string","value":"31736878"},"externalids_pubmedcentral":{"kind":"string","value":"6828820"},"content_text":{"kind":"string","value":"\nA Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice\n1 October 2019\n\nVaclav Kasicka \nMartin Hubalek \nYu Zeng zengyu@njmu.edu.cn \nWu Y \nHan M Wang \nY \nGao Y \nCui X \nXu P \nJi C \nZhong T \nYou L \nZeng Y \nYanting Wu \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nAffiliated Maternity and Child Health Care Hospital of Nantong University\nNanTongChina\n\nMei Han \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nYan Wang \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nYao Gao \nDepartment of Endocrinology, Children's Hospital\nNanjing Medical University\nNanjingChina\n\nXianwei Cui \nPengfei Xu \nChenbo Ji \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nTianying Zhong \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nLianghui You y_lianghui@126.com \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nYu Zeng \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\n\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nLianghui You\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nCzechia, Czechia\n\nA Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice\n\nFrontiers in Endocrinology | www.frontiersin.org\n107411 October 201910.3389/fendo.2019.00741Received: 22 July 2019 Accepted: 14 October 2019ORIGINAL RESEARCH Edited by: Honoo Satake, Suntory Foundation for Life Sciences, Japan Reviewed by: *Correspondence: † These authors have contributed equally to this work as joint first authors Specialty section: This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology Citation: (2019) A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice. Front. Endocrinol. 10:741.mass spectrometrysecreted peptideskeletal muscleinsulin resistanceIrs1-Akt signaling pathwayPgc1α\nAs an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.\n\nINTRODUCTION\n\nSkeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis (1). Accordingly, the dysregulation of skeletal muscle metabolism also arise a number of metabolic disorders such as hyperinsulinaemia, excessive hepatic gluconeogenesis (2), abnormal lipid accumulation (3), impaired glucose uptake and metabolic inflexibility (4). Furthermore, disorders in skeletal muscle play a central role in the development of type 2 diabetic mellitus (T2DM), obesity and lead to other related complications (5). Thus, it is of great interest to deeply characterize the pathogenesis of dysregulation on skeletal muscle glucose/lipid homeostasis to whole-body endocrine and metabolic functions.\n\nAs an important secretory organ, skeletal muscle has drawn attention to be a potential target tissue for treating metabolic disorders (6). Thus, analysis of skeletal muscle secretome opens up a novel route for comprehending the communication of this tissue with other tissues such as adipose tissue (7), bone (8), liver (9) and pancreas (10,11). In light of recently reported experimental evidence, a variety of proteins generated by muscles fibers and released into the circulation are classified as myokines (12), most of which have autocrine, paracrine, and endocrine effects not only in muscle fiber growth (13) but also systemic metabolism (14). The first identified myokine IL-6 presented a vital locally muscular effects such as skeletal muscle growth and glucose/lipid metabolism (15, 16). Furthermore, IL-6 also could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues (17)(18)(19). Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM (20). These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. However, knowledge of the skeletal muscle secretome is scarcely reported under pathophysiology of metabolic diseases such as T2DM and obesity. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established.\n\nPeptides in length of 3-50 amino acids residues, which are widely characterized in mouse and human, are termed as a sort of compounds produced or secreted by endocrine gland tissues as well as certain types of cells (21,22). And Abbreviations: T2DM, type 2 diabetic mellitus; GO, gene ontology; KCRM, creatine kinase m-type; Aldo A, fructose-bisphosphate aldolase A; Mr, relative molecular mass; pI, isoelectric point; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FA, formic acid; DDA, data dependent acquisition; FDR, false discovery rate; PSPEP, Proteomics System Performance Evaluation Pipeline; RIPA, Radio Immunoprecipitation Assay; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; TPIS, triosephosphate isomerase; ENOB, beta-enolase; PYGM, glycogen phosphorylase, muscle form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; MLRS, myosin regulatory light chain 2, skeletal muscle isoform; MYH6, myosin heavy chain 6; MYH7, myosin heavy chain 7; MYBPH, myosin-binding protein H; MYL3, myosin light chain 3; MYL1; LDH, myosin light chain 1; lactate dehydrogenase. these endogenous peptides have important physiological action, including neuroregulation (23), cell differentiation (24) and energy metabolism (25) and dysregulation of peptide hormone signaling have been implicated in a wide range of diseases (26,27). In view of that, further insight into identification of novel peptides is of major importance. Benefited from the progresses in peptide extraction method and application of modern analytical methods, (U)HPLC, nano-LC and CE, hyphenated with tandem mass spectrometry (MS/MS) technology (28,29), various techniques used for quantitative peptidomics have been applied to address the challenging question of identifying peptides with potential bioactive under the physiological or disease condition (30). More importantly, the peptidomics is widely used to identify biological markers (31), discover new drug (32) and therapeutic targets (33). Recently, quantative peptidomics has also been conducted in endocrine studies (22,34,35), however, the secreted peptidomics from skeletal muscle under the insulin-resistant condition was not fully characterized.\n\nHerein, we performed liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to help characterize the secretome from cultured skeletal muscle tissues of db/db mice at peptides level and identify putative bioactive peptides. A global secreted peptides were established and bioinformatics analysis of precursor proteins provided a possible relationship of differential peptides with T2DM or insulin resistance. Additionally, the biological effects of these secreted peptides on C2C12 myotubes elucidated a possible regulatory role in insulin signaling-and mitochondrial-related genes expression. Taken together, these observations will encourage us to investigate function of these secreted peptides from cultured skeletal muscle tissues with other tissues under the diabetic state, thus representing a promising strategy for prevention and treatment of insulin resistance as well as the associated metabolic disorders.\n\n\nMATERIALS AND METHODS\n\n\nEthics Statement\n\nAll the studies involving mice acquired approval from the Ethical Committee of Nanjing Medical University. All procedure involving mice were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval Number: IACUC-1812053).\n\n\nAnimal Experiments and Sample Preparation\n\nTwelve-week-old male C57BLKS/J db/db mice (n = 8, db/db group) and age-matched WT controls (n = 8, NC group) were purchased from the Model Animal Research Center of Nanjing Medical University. After adaptive raising for one week, mice were sacrificed by cervical dislocation and skeletal muscle tissues were isolated from the left hind leg (each mice of 100 ∼ 150 mg). Subsequent operations were carried out under a laminar airflow hood to decrease contamination. The visible blood vessels and connective tissue were removed from the tissue. After rinsed with PBS, the skeletal muscle tissues were cut into small pieces (3-4 mm 3 ) with scissors as described by an established protocol (36,37). Tissue cutting will lead to release of damaged cells slowly into the medium. Additionally, a small amount of serum proteins in the tissue pieces will diffuse out during culture period. Therefore, necessary washing procedures during culture were adopted to obtain medium samples (referred to as secretome) containing mainly skeletal muscle tissue-derived secreted components as previously reported (38,39). Tissue fragments were placed in a 10 cm plate (200∼300 mg from two mice as one sample) containing 10 mL serum/phenol red free DMEM/F12 medium (Gibco, Grand Island, CA, USA). After incubation in a humidified incubator at 37 • C under 95% O2 and 5% CO2 for 48 h, the medium was immediately supplemented with protease inhibitors and centrifuged (845 g, 10 min, 4 • C) to wipe off cell debris and dead cell. Supernatant samples from each group (NC or db/db group) were harvested from four individual tissue culture dishes independently (n = 4 per group). Lactate dehydrogenase (LDH) (36,40,41) and IL-6 (42) expression levels were detected to evaluate skeletal muscle vitality and capability during the in vitro culture. Then the supernatant samples obtained were stored at −80 • C until further processing.\n\n\nPeptide Extraction and Desalting\n\nFirst, both supernatant samples were concentrated to 1-2 mL by centrifuges for speed vacuum (LaboGene, Allerød, Denmark). Then equal volume of U2 solution containing 8 M urea and 100 mM tetraethyl-ammonium bromide in pH 8.0 was added to the concentrated supernatant for denaturation. Followed by centrifugation for 30 min (13,000 g, 30 • C), the medium supernatant was transferred to a new centrifuge tube. Subsequently, proteins were reduced by 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide successively. The protein concentrations of supernatant from cultured skeletal muscle samples were detected by Bradford method (43) and integrity of these samples were evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) combined with silver staining. Afterwards the peptides were separated from samples by treatment with Amicon R Ultra Centrifugal Filters in 10-kDa (Merck Millipore, Billerica, MA, USA) according to the manufacturer's instruction as previously described (37). The \"peptidome\" present in the filtrates was desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and the desalted peptide solution was vacuum-dried with centrifuges for speed vacuum (SCAN SPEED 40, LaboGene) as previously described (44) and immediately frozen at −80 • C until the following processing.\n\n\nLiquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)\n\nThe LC-MS/MS analysis was conducted similarly to the previous protocols (45). The peptide samples were redissolved in 2% (v/v) acetonitrile/0.1% formic acid (FA) (v/v) and 5 µL solution was injected into an A Triple TOFTM 5600 mass spectrometer (AB Sciex, Redwood City, CA, USA) coupled to a ekspert TM nanoLC400 liquid chromatography (AB Sciex) via a nanosource electrospray interface equipped with distal coated SilicaTip emitters (New Objective, Woburn, MA, USA). First, load peptide onto a C18 trap column (5 µm, 100 µm × 20 mm, AB Sciex) and elute at 300 nL/min onto a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Shanghai, China) in the gradient as long as 120 min. These two mobile phases included buffer A containing 2% acetonitrile/ 0.1% FA/ 98% H2O (v/v) and buffer B containing 98% acetonitrile/0.1% FA/2% H2O (v/v). Then peptide mixture was eluted at a flow rate of 0.3 µL/min in a gradient generated with Solvent A containing 98% water and 2% acetonitrile containing 0.1% FA (v/v) and Solvent B containing 2% water and 98% acetonitrile containing 0.1% FA (v/v) according to the previously described reports (45,46). The mass spectrometer was operated in positive mode with a spray voltage of 2500 V, 206.84kPa for the curtain gas, 41.37kPa for the nebulizer gas and 150 • C as temperature. A data dependent acquisition (DDA) method was applied and a full scan MS spectrum (300-1500 m/z) with accumulation time of 0.25 s was adopted. Top 30 precursor ions for fragmentation based on the highest intensity were selected. The collections of MS1 spectra were in the range 350-1500 m/z, and MS2 spectra were in the range of 100-1500 m/z. A total of 47,709 MS/MS were collected from all LC-MS/MS analyses.\n\n\nPeptide Identification and Quantitative Analysis\n\nProtein Pilot Software (https://sciex.com.cn/products/software/ proteinpilot-software, version4.5,AB SCIEX) was adopted to analyze the original MS/MS file data (47). For peptides identification, the Paragon algorithm was employed against the Mus_musculus SwissProt sequence database (a total of 85210 items, updated in January 2019). The following parameters were installed: The parameters were set as follows: 1) cysteine modified with iodoacetamide; 2) biological modifications were selected as ID focus. The strategy of the automatic decoy database search was employed to estimate false discovery rate (FDR) calculation using the Proteomics System Performance Evaluation Pipeline (PSPEP) Software integrated in protein pilot-software. Only unique peptides (global FDR values < 1%) were considered for further analysis. Skyline v4.2 software was employed for MS1 filtering and ion chromatogram extractions for peptides label-free quantification (48,49). And the parameters setting for skyline MS1 filtering were according to previous studies (48). Using the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.\n\n\nBioinformatics and Annotations\n\nThe relative molecular mass and isoelectric point of each peptide were calculated by the online tool (http://web.expasy. org/compute.pi/). All the precursors protein of differentially expressed peptides as one group were imported into for GO (http://www.geneontology.org/) (50) and pathway analysis (https://www.kegg.jp/) for predicting potential functions. The online tools UniProt Database (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/) were adopted to explore if the peptides' sequences were positioned in the conserved structural domains or regions of their precursors. The Open Targets Platform database (www.targetvalidation.org/) was adopted to investigate precursors associated with diabetes and obesity as previously reported (24). For visualization, clustergram and volcano plot graphs in this study were drawn with R language (http://www.r-project.org/). For determination of differentially expressed peptides, fold change was computed as the average values of biological duplication (n = 4).\n\n\nSynthetic Peptides\n\nAll the peptides used in this study were custom-synthesized and HPLC-purified by Science Peptide Biological Technology Co., Ltd. (Shanghai, China) through the solid-phase method as described reported (51). The purity in 95% for each peptide was confirmed by HPLC-MS method. All the used peptides were stored in lyophilization at−20 • C until dissolved with sterile water immediately for treatment with cells in vitro.\n\n\nCell Culture and Peptide Treatment\n\nC2C12 cells, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China) at 37 • C with 5% CO2. On the fourth day of cell differentiation, the C2C12 cells were pre-treated with synthesized peptides or solvent for 48 h at the same concentration of 50 µM, and then starved for 24 h with serumfree L-DMEM (Gibco). Subsequently, myotubes were incubated in L-DMEM in the presence or absence of 100 nM of insulin for 30 min. At the indicated time, the cells were collected for the following analysis. \n\n\nWestern Blot Analysis and Antibodies\n\n\nReverse Transcription and Real-Time Quantitative PCR\n\nTotal RNA was isolated using trizol reagent (Life Technologies, Carlsbad, CA, USA). And the cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was determined by real-time quantitative PCR (ABI ViiA7, Applied Biosystems, Foster City, California, USA) using the SYBR Green array. The relative gene expression was analyzed based on the 2 − CT method with normalization of the data to PPIA. The primers used for the real-time quantitative PCR were listed in Table S1.\n\n\nStatistical Analysis\n\nData were analyzed with GraphPad Prism 7 (San Diego, CA, USA), and the results were shown as the mean ± standard deviation (SD). Peptides with a fold change larger than 1.5 or <0.67 with a Student's t-test p-value <0.05 were selected as differently expressed peptides.\n\n\nRESULTS\n\n\nIdentification of Secreted Peptides From Cultured db/db Skeletal Muscle Tissues\n\nConsidering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity. LDH and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period. LDH content of culture medium was assessed as an indicator of cell lysis at 0 h∼24 h, 24 h∼48 h and 48 h∼72 h; no significant increased in LDH occurred from 48 h to 72 h (not shown in the manuscript). Secretion of IL-6 remained stable from 24 h to 48 h in culture by ELISA (not shown in the manuscript). Therefore, we chose the 24 h∼48 h culture as an optimal time point. The protein composition and integrity of the secreted samples were evaluated by SDS-PAGE with silver staining in Figure S1. After validation of skeletal muscle culture system and samples assessment, peptides from four different culture dishes per group (control and db/db mice) were individually extracted from supernatants and analyzed via label-free mass spectrometry based quantification. To gain more insights into biological differences between control and db/db mice skeletal muscle, we employed LC-MS/MS technology and identified 3384 peptides, of which 2664 peptides showed valid quantitative values in the detection. A total of 63 peptides were identified differentially secreted, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 peptides were down-regulated (fold change < 0.67, P < 0.05) in medium supernatants from insulin resistant-skeletal muscle tissues. Visualization methods such as hierarchical clustering and volcano plot showed distinct peptides secretion profiles as shown in Figures 1A,B. The differentially secreted peptides were summarized in Table 1.\n\n\nCharactrization of the Basic Feature of Differentially Secreted Peptides\n\nTo characterize the general feature of differentially secreted peptides, we further analyzed the relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs. pI. The results indicated that the Mr varied from 1011.1 Da to 2670.9 Da with 70% between 1,300 and 2,100 Da (Figure 2A), and pI varied from 3.7 to 9.7, with 51% between 4 and 7 ( Figure 2B). Additionally, distribution of pI vs. Mr could divide these peptides into three groups around pI4, pI6 and pI10 ( Figure 2C).\n\n\nComprehensive Functional Assessment and Intercellular Location of Precursor Proteins of These Differential Peptides\n\nTo make comprehensive functional assessment of these differential peptides, we consider all the precursor proteins as one group and performed GO and Pathways analysis of them. First of all, the precursor proteins of these differential peptides identified from the cultured control and db/db skeletal muscle were classified using Gene Ontology categories, which revealed the majority of proteins were associated with striated muscle contraction and glucose metabolic process ( Figure 3A). The top 20 GO terms were listed in Figure 3A. The precursor proteins annotated involved in these GO terms were presented in Table S2. Subsequent pathway analysis in the KEGG database revealed a significant enrichment in metabolic pathway and glycolysis/gluconeogenesis process ( Figure 3B). The top 14 pathway terms were listed in Figure 3B. The precursor proteins annotated involved in these pathway terms were presented in Table S3. We further discriminated the up-and down-regulated peptides and performed GO and Pathways analysis of these two groups of precursor proteins. This analysis may bring overlapping terms such as phosphorylation, phosphagen metabolic process and phosphorus metabolic process in up-or down-regulated peptides, and a smaller number of Search tool for the retrieval of interacting genes/proteins (STRING) was used to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides. The size of node represents the number of interacting proteins and the color of node represents the bitscore of matching results from STRING database. The thicker the line (edge), the higher the reliability (evaluated by combined score >0.4).\n\nterms in Pathway analysis (deriving up-regulated peptides). These analysis were presented in Figure S2. Additionally, the major intracellular locations of the precursor proteins were considered from literature sources, illustrating that 64% proteins were cytoplasmic and 15% proteins were cytoskeletal seen in Figure 3C. Based on above analysis, we found that a vast of precursor proteins were assigned to glucose metabolic process include ALDO A, triosephosphate isomerase (TPIS), beta-enolase (ENOB), glycogen phosphorylase, muscle form (PYGM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1(PGK1), specifically assigned to glycolysis/gluconeogenesis process. Another kind of proteins enriched in regulation of striated muscle contraction terms (GO) and regulation of actin cytoskeleton (Pathway) were myosin regulatory light chain 2, skeletal muscle isoform(MLRS), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin-binding protein H (MYBPH) and myosin light chain 3 (MYL3),both belonging to sarcomeric proteins. Subsequently, we used search tool for the retrieval of interacting genes/proteins (STRING) to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides in Figure 3D. Based on protein-protein interaction and co-occurrence in KEGG pathways and literature mining, this network was constructed from 25 proteins that matched to the STRING database and the matching results were listed in Table S4. As shown from this network, 22 nodes representing precursor protein constituted the interaction diagram, which could form 93 reliable a-to-b interaction relationship by combined score (more than 0.4) as shown in Table S5. \n\n\nCleavage Pattern of Differentially Secreted Peptides\n\nIt is widely postulated that most peptides can be attributed mainly to the proteolytic enzymes as well as their type and level (cleavage specificity and activity), which can differ during disease (52)(53)(54). Briefly, the N-terminal pre-cleavage site, N terminus, C terminus, and C-terminal post-cleavage site of the identified peptides were commonly used to investigate the nature of proteolytic enzymes within serum (55) or tissues (56). Thus, we analyzed the distribution of peptide cleavage site and found that Lysine (K) was the most frequent cleavage site of N-terminal amino acid of identified peptides and Alanine (A) was the most frequent cleavage site of C-terminal amino acid of identified peptides. Lysine (K) was the most frequent cleavage site of Nterminal amino acid of preceding peptides, while Leucine (L) was the commonest C-terminal amino acid of preceding peptides ( Figure 4A). These data indicate that the pattern of cleavage sites may represent the specificity of cleavage and activity of proteolytic enzymes under the diabetic condition. Additionally, we also tried to align peptide sequences on the same precursor sequence to construct a \"peptide alignment map\". Searched from our samples, the largest number of identified peptides (n = 1 7) (Figure 4B) originated from KCRM and the second largest number of identified peptides (n = 6) came from ALDO A (Figure 4B), elucidating that these peptides were easily cleaved by certain kind of endogenous enzymes.\n\n\nPutative Bioactive Peptides Associated With Diabetes and Obesity\n\nTo check for specific domain structures or patterns in the identified peptides comparison with its precursor proteins, we retrieved domain information from the UniProt and Pfam database. In order to validate that if the peptides exerted important roles in the related metabolic diseases, we adopted the Open Targets Platform database to investigate protein precursors. In our searching results, most peptides (n = 44) from the identified pepetides (n = 63) were located in the functional domains ( Table 2). Additionally, 27 out of 44 peptides located in the functional domain were predicted closely related with both obesity and diabetes ( Table 3). These observations will encourage us to further investigate properties of the putative secreted peptides in skeletal muscle under the diabetic state.\n\n\nThe Effect of Candidated Peptides (P1-P7) on Insulin and Mitochondrial-Related Pathways in Skeletal Muscle Cells\n\nTo explicit the putative function of differentially secreted peptides, 7 differentially secreted peptides, which had already been annotated derived from authentic protein from Uniprot database, located in the functional motifs and showed relatively high abundance from MS detection, were chosen for further analysis. Table S6. Sequentially, we termed these candidated peptides P1-P7 in order. To use C2C12 myotubes as cell models in vitro, the results of changes in inuslin signaling revealed that up-regulated peptides P2-P4 addition could both significantly decrease the phosphorylation of Irs1 (Ser-307) and Akt (Ser-473) under insulin stimulation (100 nM, 30 min) as shown in Figures 5A,B, in spite of bringing a modestly up-regulation of p-Irs1 (Ser-307) at the basal status. Moreover, mitochondrial-related marker protein Pgc1α was significantly attenuated in the insulin-stimulated C2C12 cells by up-regulated peptides P1-P4 as shown in Figures 5E,F. Among these downregulated peptides, only peptide 7 presented a promotion on p-Akt (Ser-473) expression level both under the basal and insulin stimulation status as shown in Figures 5C,D. And these up-regulated peptide 1-4 could decrease Pgc1α protein level and down-regulated peptide 6 and 7 administration enhanced Pgc1α protein level by the stimulation of insulin presented in Figures 5E,F. We further evaluated Gut 4 (a key gene for glucose transport) and Pgc1α mRNA levels by peptides treatment upon insulin stimulation. The results also indicated that Peptide 3 and 4 modestly decreased Pgc1αexpression at mRNA levels seen in Figure 5G, while Peptide 6 and 7 significantly up-regulated both Glut4 and Pgc1α expression at mRNA levels seen in Figure 5H. These observations revealed that these candidated peptides (P1-P7) may affect the expressions of insulin signaling or mitochondrial-related genes in skeletal muscle cells.\n\n\nDISCUSSION\n\nEmerging as a widely known secrete organ, skeletal muscle tissues have been extensively discussed using a wide range of comparative and quantitative proteomic methods (57). Proteomic scannings of the medium supernatant from skeletal muscle cells have already afford approaches to identify a large number of secreted proteins (58)(59)(60)(61)(62)(63). Particularly, one such research reported the alteration of insulin effect on the secretome profile of skeletal muscle cells, revealing the changes of protein levels secreted from skeletal muscle during activation of insulin signaling pathway (58). A recent study also generated a comprehensive secretome analysis of skeletal muscle cells under palmitic acid-induced insulin resistance (61). In addition to these proteins, many other muscle-secreted compounds have been also come to light, including exosome (64,65), metabolites (66), and miRNAs (67). These studies have greatly expanded our knowledge of skeletal muscle secretome and might afford possibilities for exploring novel molecular targets in the maintenance of skeletal muscle physiology and even whole-body metabolism. However, to date few studies has focused on the peptides present in either skeletal muscle tissues or cells. Therefore, we attempted to comprehensively profile peptides that may play roles in regulating insulin sensitivity and offer enormous promise for exploring molecular mechanisms underlying insulin resistance.\n\nIn present study, a total of 63 peptides were differentially secreted in medium supernatants from cultured skeletal muscle tissues of db/db mice, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 were down-regulated (fold change < 0.67, P < 0.05) shown in Figures 1A,B. The differences of dysregulation peptides may indeed reflect the changes between control and insulin-resistant mice in some extent. However, db/db mouse is a well-established leptin receptordeficient animal model (68,69). Despite no studies revealed the association between leptin receptor deficiency and peptide dysregulation/protein degradation so far, the possible effect by this mutation deserves to be taken into account and other nongenetic mice models could be adopted in the future studies. Based on our previous study (70), 10-kDa filters method was used to extract peptides in order to intercept proteins secreted by skeletal muscle to the conditional media. Characterization of the basic feature (Mr and pI) of these identified peptides (Figures 2A,B) only reflected the differences of distribution in the diabetic skeletal muscle tissues, but also proved the reliability of the used peptide extraction method.\n\nSubsequently, GO and Pathway analysis revealed that the precursors protein of these peptides were mostly involved in muscle contraction and metabolism processes (Figures 3A,B). An interesting finding from our study was that most of the differentially identified peptides derived from cytosolic, cytoskeletal and mitochondrial proteins (Figures 3A,B), which differed from the secretory pathway peptides (71). This (34). Similarly, another peptidomic anaylsis of brain slices cultures and media also pointed that vast majority of secreted peptides arose from intracellular proteins (72). There does also exist some evidence that these identified N-or C-terminus protein yielding peptides, rather than internal fragments, raised the possibility that they are produced by selective processing rather than protein degradation (73). Taken together with previous researches, the current results show us meaningful hints that these intracellular peptides may be secreted via nonclassical mechanisms. Actually, another important origin of peptides is proteolytic degradation processes in body fluid under the physiological or pathologically processes. Most regulatory peptides were efficiently degraded by plasma enzymes once secreted into the bloodstream, which exerted anti-diabetic therapeutic function (74,75) or were identified as disease markers (76). Recently, Federico Aletti et al. (77) used protease activity detection and specific enzymes analysis to explain a large presence of circulating peptides under hemorrhagic shock, which gave us a possible way to evaluate peptides origin. Thus, a fundamental validation is whether the proteins-derived peptides are actually secreted from skeletal muscle cells per se or proteolytic degradation and are of biologically active. Among the differentially secreted peptides from cultured skeletal muscle tissues of db/db mice, we found that three peptides derived from GAPDH, four peptides derived from ALDO A and one peptides derived from PYGM were upregulated in the conditional medium from db/db groups as shown in Table 1. As described by Dustin S. Hittel et.al, a global proteomic survey of skeletal muscle revealed a statistically significant up-regulation in glycolytic enzymes GAPDH and ALDO A protein levels in obese/overweight patients (78). Another quantitative protein profile also identified a more abundant levels of GAPDH and PYGM in skeletal muscle from T2DM groups compared with the control groups (79). In addition to pointing the importance of the mitochondrial numbers and impairments under the insulin-resistant states (80)(81)(82), several studies noted that glycolytic capacity is higher in skeletal muscle of patients with T2DM or obesity (83). Notably, stronger changes of peptides derived from sarcomeric proteins such as myosin light chain 1 (MYL1) and MYL3 were also observed in the conditional medium from diabetic muscles as shown in Table 1. MYL1 and MYL3 are representive markers of fast-muscle and slow-muscle respectively, which were both regulated by insulin stimulation (79). And previous studies observed that property of T2DM individuals muscle was shifted to a fast-twitch glycolytic phenotype (84). In fact, deficiency in sarcomeric proteins in skeletal muscle also suggested their importance in skeletal muscle physiologic and pathological processes. On the whole, our results of increased glycolytic enzyme-or sarcomeric proteins-derived peptides secreted from insulin resistant skeletal muscle may support the hypothesis that altered glycolytic capacities or fiber types under the diabetic status contribute to this difference.\n\nTill now, the putative function of these peptides are not clear. Therefore, we evaluated whether these peptides originated from functional domains of the corresponding precursor protein using the UniProt and Pfam database. In our searching results, most peptides (n = 44) from the identified peptides (n = 63) were located in the functional domains as listed in Table 2. Specifically, most peptides derived from functional enzymes were located in the enzymatic activity region, including TPI- TCTP-derived peptide (119-AEQIKHILANF-129) and CAH3-derived peptide . We also found another kind of S-Nitrosylation modifying sites for affecting GAPDH enzymatic activity (90). Therefore, future efforts need to be established for investigating the potential role of peptides on insulin sensitive cells in vitro or whole-body metabolism in vivo.\n\nThe above analyses provided a possibility to evaluate the biological effects of these differentially secreted peptides. As widely reported, insulin resistance in skeletal muscle is tightly connected with the deficit in insulin signaling (91). Consequently, the role of phosphorylation of Irs1 and Akt in signaling pathways is very crucial in anti-hyperglycemia and insulin sensitivity (92). In this result, we found these up-regulated peptides 1-4 both exerted a significantly attenuated insulin action in C2C12 cells, evaluated by a decreased level of p-Irs1 (Ser-307) and p-Akt (Ser-473) seen in Figures 5A,B, whereas only one down-regulated peptide P7 could remarkably promote insulin signaling only via Irs1 signaling pathway seen in Figures 5C,D. Peroxisome proliferator-activated receptor γ coactiva-tor-1(Pgc-1), which displays a dominant role through tight modulation of mitochondrial biogenesis and respiration, has also been demonstrated to participate in skeletal muscle insulin signaling and metabolic homeostasis (93). The up-regulated peptides 1-4 also brought out a decreased protein level of Pgc-1α in C2C12 cells as shown in Figures 5E,F, and down-regulated peptide 6 and 7 administration also gave rise to the increased Pgc1α mRNA and protein level in Figures 5E,F,H. Taken together, these selected peptides secreted from db/db mice skeletal muscle presented a promotive or inhibitory effect on insulin and mitochondrialrelated pathways in skeletal muscle cells by an autocrine manner. Notably, peptide 4 (231-RVPTPNVSVVDLTCRLEKPAK−252) derived from GAPDH displayed a most significant inhibitory effect toward these candidate peptides. However, the relationship between GAPDH-derived peptide and its precursor protein is to be determined. On the other hand, more methods need to be employed for the wider cell signaling screen and further research is required to explore the biologic function of skeletal muscle-secreted peptides on adipocytes or liver cells.\n\nTo our knowledge, no large-scale quantitative peptidomic analysis has been performed on skeletal muscle to elucidate secreted peptides profiles under the diabetic status. The present study identified and quantified changes with a label-free discovery using LC-MS/MS technology to construct a global secreted peptides picture. Further bioinformatics analysis of precursors comprehensively provided an atlas of peptides that may exist roles in regulating insulin sensitivity. This represented a new perspective toward exploring insulin resistance pathogenesis. Additionally, the detailed biological effects of these secreted peptides on skeletal muscle insulin resistance or cross-talk with other tissues remained to be elucidated in the future study.\n\n\nDATA AVAILABILITY STATEMENT\n\nThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.\n\n\nETHICS STATEMENT\n\nThe protocol has been approved by the Institutional Animal Care and Use Committee of Nanjing medical university (Approval Number: IACUC-1812053).\n\n\nAUTHOR CONTRIBUTIONS\n\nYWu and MH performed experiments and interpreted results of experiments. YWa and YG prepared the figures. XC and PX analyzed the data. CJ and TZ participated discussion. YZ helped write the manuscript with providing assistance. LY conceived and designed experiments, provided funding to regents, and approved final version of manuscript. All authors read and approved the final manuscript. \n\n\nSUPPLEMENTARY MATERIAL\n\nThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2019.00741/full#supplementary-material Figure S1 | SDS-PAGE associated with silver staining of the medium supernatants from cultured skeletal muscle tissues (Con vs. db groups). Table S1 | Primers used in this study. Table S2 | GO terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. Table S3 | Pathway terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. \n\n\nAt the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.\n\nFIGURE 1 |\n1Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.\n\nFIGURE 2 |\n2Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.\n\nFIGURE 3 |\n3Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)\n\nFIGURE 4 |\n4Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.\n\nFIGURE 5 |\n5Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.\n\n\nThe up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in\n\n\n413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),\n\nFUNDING\nThis study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science\n\nFigure S2 |\nS2Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.\n\nTABLE 1 |\n1Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nUp-regulated peptides \n\nAVGAVFDISNADRLGSSEVEQ \nP07310 \nKCRM \n2163 \n9.728 \n0.005 \n\nTNGAFTGEISPGMIKDLGATWV \nP17751 \nTPIS \n2264 \n8.019 \n0.035 \n\nIADLVVGLCTGQIK \nP21550 \nENOB \n1486 \n6.77 \n0.000 \n\nNYKPQEEYPDLSKHNNHMA \nP07310 \nKCRM \n2315 \n4.843 \n0.024 \n\nEEIEEDAGLGNGGLGRLAAC \nQ9WUB3 \nPYGM \n2030 \n4.411 \n0.004 \n\nGQCGDVLRALGQNPTQAEV \nP09542 \nMYL3 \n2013 \n4.077 \n0.033 \n\nAGPLCLQEVDEPPQHAL \nP70296 \nPEBP1 \n1873 \n4.038 \n0.004 \n\nEAFTVIDQNRDGIID \nP97457 \nMLRS \n1705 \n3.947 \n0.026 \n\nDLFDPIIQDRHGGY \nP07310 \nKCRM \n1645 \n3.668 \n0.022 \n\nKDLFDPIIQD \nP07310 \nKCRM \n1203 \n3.282 \n0.000 \n\nLDDVIQTGVDNPGHP \nP07310 \nKCRM \n1576 \n3.265 \n0.001 \n\nSEIIDVSSKAEEVK \nA2ASS6 \nTITIN \n1533 \n3.134 \n0.002 \n\nVIACIGEKLDE \nP17751 \nTPIS \n1246 \n2.976 \n0.003 \n\nGTNPTNAEVKKVLGNPSNEEM \nQ545G5 \nMYL1 \n2180 \n2.838 \n0.006 \n\nADRLGSSEVEQVQLV \nP07310 \nKCRM \n1629 \n2.804 \n0.001 \n\nIGQGTPIPDLPEVKRV \nA2AQA9 \nNEB \n1719 \n2.739 \n0.002 \n\nSTTAPPIQSPLPVIPHQK \nE9PYJ9 \nLDB3 \n1910 \n2.685 \n0.023 \n\nKADDGRPFPQVI \nP05064 \nALDOA \n1342 \n2.644 \n0.021 \n\nKSMTEQEQQQLIDD \nP07310 \nKCRM \n1692 \n2.442 \n0.001 \n\nGGGASLELLEGKVLPGVDA \nP09411 \nPGK1 \n1781 \n2.264 \n0.028 \n\nLEGTLLKPNMVTPGHA \nP05064 \nALDOA \n1677 \n2.208 \n0.010 \n\nVMVGMGQKDSYVGDEAQSK \nP68134 \nACTS \n2028 \n2.168 \n0.021 \n\nDLEEATLQHEATAAALR \nQ02566 \nMYH6 \n1838 \n2.07 \n0.038 \n\nAELQEVQITEEKPLLPG \nQ9QYG0 \nNDRG2 \n1935 \n2.043 \n0.048 \n\nSEGKCAELEEELKTVTNNL \nP58771 \nTPM1 \n2163 \n2.038 \n0.049 \n\nRSIKGYTLPPHC \nP07310 \nKCRM \n1428 \n1.967 \n0.001 \n\nNSNSHSSTFDAGAGIALNDNFVK \nP16858 \nG3P \n2365 \n1.935 \n0.038 \n\nTGKATSEASVSTPEETAPEPAKVPT \nP70402 \nMYBPH \n2484 \n1.919 \n0.003 \n\nDNEYGYSNRVVDL \nP16858 \nG3P \n1543 \n1.898 \n0.004 \n\nEELDAMMKEASGPINF \nP97457 \nMLRS \n1781 \n1.853 \n0.012 \n\nSNNKDQGGYEDFVEGLRV \nQ545G5 \nMYL1 \n2026 \n1.816 \n0.016 \n\nAEQIKHILANF \nP63028 \nTCTP \n1283 \n1.787 \n0.044 \n\nGADPEDVITGAFK \nP97457 \nMLRS \n1319 \n1.782 \n0.031 \n\nTSIEDAPITVQSKINQ \nA2AQA9 \nNEB \n1743 \n1.739 \n0.028 \n\nLKGGDDLDPNYVLS \nP07310 \nKCRM \n1505 \n1.732 \n0.040 \n\nIQTGVDNPGHPF \nQ6P8J7 \nKCRS \n1281 \n1.600 \n0.045 \n\nAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n1957 \n1.595 \n0.009 \n\nGQEQWEEGDLYDKEKQQKPF \nE9Q8K5 \nTITIN \n2481 \n1.586 \n0.036 \n\nAGTNGETTTQGLDGLSERCA \nP05064 \nALDOA \n2037 \n1.582 \n0.033 \n\nRVPTPNVSVVDLTCRLEKPAK \nP16858 \nG3P \n2378 \n1.531 \n0.022 \n\nKVPEKPEVVEKVEPAPLK \nF7CR78 \nF7CR78 \n2015 \n1.527 \n0.026 \n\nGETTTQGLDGLSERCAQ \nP05064 \nALDOA \n1822 \n1.526 \n0.042 \n\nDown-regulated peptides \n\nKIEFTPEQIEEF \nP09542 \nMYL3 \n1509 \n0.612 \n0.021 \n\nDLSAIKIEFSKEQQEDF \nP05977 \nMYL1 \n2026 \n0.600 \n0.019 \n\nHELYPIAKGDNQSPI \nP16015 \nCAH3 \n1681 \n0.559 \n0.038 \n\nNSLTGEFKGKY \nP07310 \nKCRM \n1243 \n0.545 \n0.027 \n\nDEPKPYPYPNLDDF \nQ3V1D3 \nAMPD1 \n1709 \n0.543 \n0.034 \n\nASHHPADFTPAVHA \nQ91VB8 \nHBA-A1 \n1457 \n0.537 \n0.016 \n\n(Continued) \n\nFrontiers in Endocrinology | www.frontiersin.org \n\nTABLE 1 |\n1ContinuedPeptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nSQVGDVLRALGTNPTNAE \nQ545G5 \nMYL1 \n1841 \n0.516 \n0.047 \n\nADEIAKAQVAQPGGDTI \nP70349 \nHINT1 \n1725 \n0.515 \n0.038 \n\nDKETPSGFTLDDV \nP07310 \nKCRM \n1423 \n0.478 \n0.037 \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n2654 \n0.467 \n0.038 \n\nPHPYPALTPEQKK \nP05064 \nALDOA \n1505 \n0.407 \n0.028 \n\nQLVVDGVKLM \nP07310 \nKCRM \n1101 \n0.400 \n0.001 \n\nKDLFDPIIQDRHG \nP07310 \nKCRM \n1553 \n0.395 \n0.022 \n\nKGVVPLAGTNGETTTQGLDGLSER \nP05064 \nALDOA \n2399 \n0.367 \n0.015 \n\nRVFDKEGNGTVMGAELR \nQ545G5 \nMYL1 \n1878 \n0.341 \n0.043 \n\nNADEVGGEALGRL \nA8DUK4 \nHBB-BS \n1300 \n0.339 \n0.037 \n\nNEHLGYVLTCPS \nP07310 \nKCRM \n1389 \n0.328 \n0.031 \n\nASHTEEEVSVSVPEVQKKTVTEEK \nE9Q8K5 \nTITIN \n2669 \n0.253 \n0.023 \n\nNEEDHLRV \nP07310 \nKCRM \n1010 \n0.212 \n0.027 \n\nVLTCPSNLGTGLRG \nP07310 \nKCRM \n1444 \n0.201 \n0.018 \n\nGGYKPTDKHKTDL \nP07310 \nKCRM \n1459 \n0.127 \n0.006 \n\nobservation was also demonstrated by other peptidomic studies. \nFrom Steven W. Taylor group' results, several identified peptides \nin the human islet cultures were derived from intracellular and \ncytoskeletal proteins such as microtubule-associated protein 4 \nand ubiquitin, which may result from a greater level of cellular \nstress \n\nTABLE 2 |\n2Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported asPeptide sequence \nProtein \nLocation \nDomain \nDescription \n\nUp-regualted peptides \n\nAVGAVFDISNADRLGSSEVEQ \nKCRM \n329-349 \n125-367 \nPhosphagen kinase C-terminal \n\nTNGAFTGEISPGMIKDLGATWV \nTPIS \n121-142 \n57-295 \nTIM \n\nIADLVVGLCTGQIK \nENOB \n381-394 \n142-432 \nEnolase C \n\nNYKPQEEYPDLSKHNNHMA \nKCRM \n13-31 \n11-98 \nPhosphagen kinase N-terminal \n\nEEIEEDAGLGNGGLGRLAAC \nPYGM \n124-143 \n113-828 \nPhosphorylase \n\nGQCGDVLRALGQNPTQAEV \nMYL3 \n83-101 \n58-95 \nEF-hand 1 \n\nEAFTVIDQNRDGIID \nMLRS \n32-46 \n25-60 \nEF-hand 1 \n\nDLFDPIIQDRHGGY \nKCRM \n87-100 \n11-98 \nPhosphagen kinase N-terminal \n\nKDLFDPIIQD \nKCRM \n86-95 \n11-98 \nPhosphagen kinase N-terminal \n\nLDDVIQTGVDNPGHP \nKCRM \n53-67 \n11-98 \nPhosphagen kinase N-terminal \n\nVIACIGEKLDE \nTPIS \n174-184 \n57-295 \nTIM \n\nGTNPTNAEVKKVLGNPSNEEM \nMYL1 \n39-59 \n6-41 \nEF-hand \n\nADRLGSSEVEQVQLV \nKCRM \n339-353 \n125-367 \nPhosphagen kinase C-terminal \n\nKADDGRPFPQVI \nALDOA \n87-98 \n15-364 \nGlycolytic \n\nKSMTEQEQQQLIDD \nKCRM \n177-190 \n125-367 \nPhosphagen kinase C-terminal \n\nGGGASLELLEGKVLPGVDA \nPGK1 \n395-413 \n9-406 \nPGK \n\nLEGTLLKPNMVTPGHA \nALDOA \n224-239 \n15-364 \nGlycolytic \n\nVMVGMGQKDSYVGDEAQSK \nACTS \n45-63 \n4-377 \nActin \n\nDLEEATLQHEATAAALR \nMYH6 \n1,179-1,195 \n845-1,926 \nMyosin_tail_1 \n\nSEGKCAELEEELKTVTNNL \nTPM1 \n186-204 \n1-284 \nCoiled coili \n\nRSIKGYTLPPHC \nKCRM \n135-146 \n125-367 \nPhosphagen kinase C-terminal \n\nSNNKDQGGYEDFVEGLRV \nMYL1 \n77-94 \n83-118 \nEF-hand \n\nAEQIKHILANF \nTCTP \n119-129 \n1-172 \nTCTP \n\nGADPEDVITGAFK \nMLRS \n93-105 \n95-130 \nEF-hand 2 \n\nLKGGDDLDPNYVLS \nKCRM \n115-128 \n125-367 \nPhosphagen kinase C-terminal \n\nGQEQWEEGDLYDKEKQQKPF \nTITIN \n1,691-1,710 \n1,709-1,799 \nIg-like \n\nAGTNGETTTQGLDGLSERCA \nALDOA \n117-136 \n15-364 \nGlycolytic \n\nRVPTPNVSVVDLTCRLEKPAK \nGAPDH \n232-252 \n243-248 \n[IL]-x-C-x-x-[DE] motif \n\nGETTTQGLDGLSERCAQ \nALDOA \n121-137 \n15-364 \nGlycolytic \n\nDown-regualted peptides \n\nKIEFTPEQIEEF \nMYL3 \n52-63 \n58-95 \nEF-hand 1 \n\nDLSAIKIEFSKEQQEDF \nMYL1 \n33-49 \n44-79 \nEF-hand 1 \n\nHELYPIAKGDNQSPI \nCAH3 \n17-31 \n3-259 \nAlpha-carbonic anhydrase \n\nNSLTGEFKGKY \nKCRM \n163-173 \n125-367 \nPhosphagen kinase C-terminal \n\nASHHPADFTPAVHA \nHBA-A1 \n111-124 \n3-142 \nGLOBIN \n\nDKETPSGFTLDDV \nKCRM \n44-56 \n11-98 \nPhosphagen kinase N-terminal \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nMYL1 \n76-100 \n83-118 \nEF-hand \n\nQLVVDGVKLM \nKCRM \n351-360 \n125-367 \nPhosphagen kinase C-terminal \n\nKDLFDPIIQDRHG \nKCRM \n86-98 \n11-98 \nPhosphagen kinase N-terminal \n\nKGVVPLAGTNGETTTQGLDGLSER \nALDOA \n111-134 \n15-364 \nGlycolytic \n\nRVFDKEGNGTVMGAELR \nMYL1 \n147-163 \n133-150 \nEF-hand \n\nNADEVGGEALGRL \nHBB-BS \n20-32 \n2-147 \nGLOBIN \n\nNEHLGYVLTCPS \nKCRM \n274-285 \n125-367 \nPhosphagen kinase C-terminal \n\nNEEDHLRV \nKCRM \n230-237 \n125-367 \nPhosphagen kinase C-terminal \n\nVLTCPSNLGTGLRG \nKCRM \n280-293 \n125-367 \nPhosphagen kinase C-terminal \n\n\n\nTABLE 3 |\n3Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.Protein name \nDescription \nPeptide number \nAssociation score \nwith obesity # \n\nAssociation score with \ndiabetes mellitus # \n\nTPI1 \nTriosephosphate isomerase \n2 \n0.012 \n0.012 \n\nKCRM \nCreatine kinase M-type \n17 \n0.022 \n0.017 \n\nTTN \nTitin \n1 \n0.026 \n0.063 \n\nGAPDH \nGlyceraldehyde-3-phosphate dehydrogenase \n1 \n0.071 \n0.117 \n\nHBA-A1 \nAlpha globin 1 \n1 \n0.014 \n0.038 \n\nENOB \nBeta-enolase \n1 \n0.040 \n0.048 \n\nCAH3 \nCarbonic anhydrase 3 \n1 \n0.074 \n0.094 \n\nTCTP \nTranslationally-controlled tumor protein \n1 \n0.028 \n0.040 \n\nHBB-BS \nBeta-globin \n1 \n0.008 \n0.020 \n\nACTS \nActin, alpha skeletal muscle \n1 \n0.006 \n0.188 \n\n# \n\nTable S4 |\nS4The matching results of the precursor proteins from STRING database.\n\nTable S5 |\nS5Precursor proteins-to-precursor proteins interaction from STRING database.\n\nTable S6 |\nS6The information of candidate peptides for functional evaluation.\nFrontiers in Endocrinology | www.frontiersin.org\nOctober 2019 | Volume 10 | Article 741\n\nThe effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man. 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Snapshot peptidomics of the regulated secretory pathway. Mol Cell Proteomics. (2009) 8:1638-47. doi: 10.1074/mcp.M900044-MCP200\n\nAnalysis of peptides secreted from cultured mouse brain tissue. J S Gelman, S Dasgupta, I Berezniuk, L D Fricker, 10.1016/j.bbapap.2013.01.043Biochim Biophys Acta. 1834Gelman JS, Dasgupta S, Berezniuk I, Fricker LD. Analysis of peptides secreted from cultured mouse brain tissue. Biochim Biophys Acta. (2013) 1834:2408-17. doi: 10.1016/j.bbapap.2013.01.043\n\nAnalysis of mouse brain peptides using mass spectrometrybased peptidomics: implications for novel functions ranging from nonclassical neuropeptides to microproteins. L D Fricker, 10.1039/c003317kMol Biosyst. 6Fricker LD. Analysis of mouse brain peptides using mass spectrometry- based peptidomics: implications for novel functions ranging from non- classical neuropeptides to microproteins. Mol Biosyst. (2010) 6:1355-65. doi: 10.1039/c003317k\n\nStimulatory effect of xenin-8 on insulin and glucagon secretion in the perfused rat pancreas. R A Silvestre, J Rodríguez-Gallardo, E M Egido, R Hernández, J Marco, 10.1016/S0167-0115(03)00147-2Regul Pept. 115Silvestre RA, Rodríguez-Gallardo J, Egido EM, Hernández R, Marco J. Stimulatory effect of xenin-8 on insulin and glucagon secretion in the perfused rat pancreas. Regul Pept. (2003) 115:25-9. doi: 10.1016/S0167-0115(03)00147-2\n\nCharacterisation of the biological activity of xenin-25 degradation fragment peptides. C M Martin, V Parthsarathy, V Pathak, V A Gault, P R Flatt, N Irwin, 10.1530/JOE-13-0617J Endocrinol. 221Martin CM, Parthsarathy V, Pathak V, Gault VA, Flatt PR, Irwin N. Characterisation of the biological activity of xenin-25 degradation fragment peptides. J Endocrinol. (2014) 221:193-200. doi: 10.1530/JOE-13-0617\n\nCirculating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling. A P Wiita, G W Hsu, C M Lu, J H Esensten, J A Wells, 10.1073/pnas.1405987111Proc Natl Acad Sci. 111Wiita AP, Hsu GW, Lu CM, Esensten JH, Wells JA. Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling. Proc Natl Acad Sci USA. (2014) 111:7594-9. doi: 10.1073/pnas.1405987111\n\nPeptidomic analysis of rat plasma: proteolysis in hemorrhagic shock. F Aletti, E Maffioli, A Negri, M H Santamaria, F A Delano, E B Kistler, 10.1097/SHK.0000000000000532Shock. 45Aletti F, Maffioli E, Negri A, Santamaria MH, DeLano FA, Kistler EB, et al. Peptidomic analysis of rat plasma: proteolysis in hemorrhagic shock. Shock. (2016) 45:540-54. doi: 10.1097/SHK.0000000000 000532\n\nProteome analysis of skeletal muscle from obese and morbidly obese women. D S Hittel, Y Hathout, E P Hoffman, J A Houmard, 10.2337/diabetes.54.5.1283Diabetes. 54Hittel DS, Hathout Y, Hoffman EP, Houmard JA. Proteome analysis of skeletal muscle from obese and morbidly obese women. Diabetes. (2005) 54:1283-8. doi: 10.2337/diabetes.54.5.1283\n\nThe proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes. J Giebelstein, G Poschmann, K Højlund, W Schechinger, J W Dietrich, K Levin, 10.1007/s00125-012-2456-xDiabetologia. 55Giebelstein J, Poschmann G, Højlund K, Schechinger W, Dietrich JW, Levin K, et al. The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes. Diabetologia. (2012) 55:1114-27. doi: 10.1007/s00125-012-2456-x\n\nMitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes. M Mogensen, K Sahlin, M Fernström, D Glintborg, B F Vind, H Beck-Nielsen, 10.2337/db06-0981Diabetes. 56Mogensen M, Sahlin K, Fernström M, Glintborg D, Vind BF, Beck-Nielsen H, et al. Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes. Diabetes. (2007) 56:1592-9. doi: 10.2337/db06-0981\n\nInsulin signaling meets mitochondria in metabolism. Z Cheng, Y Tseng, M F White, 10.1016/j.tem.2010.06.005Trends Endocrinol Metab. 21Cheng Z, Tseng Y, White MF. Insulin signaling meets mitochondria in metabolism. Trends Endocrinol Metab. (2010) 21:589-98. doi: 10.1016/j.tem.2010.06.005\n\nDeficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. V B Ritov, E V Menshikova, K Azuma, R Wood, F G Toledo, B H Goodpaster, 10.1152/ajpendo.00317.2009Am J Physiol Endocrinol Metab. 298Ritov VB, Menshikova EV, Azuma K, Wood R, Toledo FG, Goodpaster BH, et al. Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. Am J Physiol Endocrinol Metab. (2010) 298:E49-58. doi: 10.1152/ajpendo.00317.2009\n\nCharacterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods. T E Thingholm, S Bak, H Beck-Nielsen, O N Jensen, M Gaster, 10.1074/mcp.M110.006650Mol Cell Proteomics. 10Thingholm TE, Bak S, Beck-Nielsen H, Jensen ON, Gaster M. Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods. Mol Cell Proteomics. (2011) 10:M110.006650. doi: 10.1074/mcp.M110.006650\n\nAltered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. A Oberbach, Y Bossenz, S Lehmann, J Niebauer, V Adams, R Paschke, 10.2337/diacare.29.04.06.dc05-1854Diabetes Care. 29Oberbach A, Bossenz Y, Lehmann S, Niebauer J, Adams V, Paschke R, et al. Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. Diabetes Care. (2006) 29:895-900. doi: 10.2337/diacare.29.04.06.dc05-1854\n\nTwo structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors. K Denessiouk, S Permyakov, A Denesyuk, E Permyakov, M S Johnson, 10.1371/journal.pone.0109287PLoS ONE. 9109287Denessiouk K, Permyakov S, Denesyuk A, Permyakov E, Johnson MS. Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors. PLoS ONE. (2014) 9:e109287. doi: 10.1371/journal.pone.0109287\n\nPeptide binding by a fragment of calmodulin composed of EF-hands 2 and 3. Biochemistry-us. T M Lakowski, G M Lee, B Lelj-Garolla, M Okon, R E Reid, L P Mcintosh, 10.1021/bi700265j46Lakowski TM, Lee GM, Lelj-Garolla B, Okon M, Reid RE, McIntosh LP. Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3. Biochemistry-us. (2007) 46:8525-36. doi: 10.1021/bi700265j\n\nCalcium-binding proteins 1: EF-hands. H Kawasaki, R H Kretsinger, Protein Profile. 2Kawasaki H, Kretsinger RH. Calcium-binding proteins 1: EF-hands. Protein Profile. (1995) 2:297-490.\n\nCa2+ and AMPK both mediate stimulation of glucose transport by muscle contractions. D C Wright, K A Hucker, J O Holloszy, D H Han, 10.2337/diabetes.53.2.330Diabetes. 53Wright DC, Hucker KA, Holloszy JO, Han DH. Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions. Diabetes. (2004) 53:330-5. doi: 10.2337/diabetes.53.2.330\n\nExercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles. D R Park, K H Park, B J Kim, C S Yoon, U H Kim, 10.2337/db14-0939Diabetes. 64Park DR, Park KH, Kim BJ, Yoon CS, Kim UH. Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles. Diabetes. (2015) 64:1224-34. doi: 10.2337/db14-0939\n\nTarget-selective protein S-nitrosylation by sequence motif recognition. J Jia, A Arif, F Terenzi, B Willard, E F Plow, S L Hazen, 10.1016/j.cell.2014.09.032Cell. 159Jia J, Arif A, Terenzi F, Willard B, Plow EF, Hazen SL, et al. Target-selective protein S-nitrosylation by sequence motif recognition. Cell. (2014) 159:623- 34. doi: 10.1016/j.cell.2014.09.032\n\nInsulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. S Guo, 10.1530/JOE-13-0327J Endocrinol. 220Guo S. Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. J Endocrinol. (2014) 220:T1-23. doi: 10.1530/JOE-13-0327\n\nBioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice. Z Q Wang, X H Zhang, Y Yu, A Poulev, D Ribnicky, Z E Floyd, 10.1016/j.jnutbio.2010.09.004J Nutr Biochem. 22Wang ZQ, Zhang XH, Yu Y, Poulev A, Ribnicky D, Floyd ZE, et al. Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice. J Nutr Biochem. (2011) 22:1064-73. doi: 10.1016/j.jnutbio.2010.09.004\n\nPGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. I Pagel-Langenickel, Bao J Joseph, J J Schwartz, D R Mantell, B S Xu, X , 10.1074/jbc.M800842200J Biol Chem. 283Pagel-Langenickel I, Bao J, Joseph JJ, Schwartz DR, Mantell BS, Xu X, et al. PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. J Biol Chem. (2008) 283:22464-72. doi: 10.1074/jbc.M800842200\n"},"annotations_abstract":{"kind":"list like","value":["As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders."],"string":"[\n \"As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.\"\n]"},"annotations_author":{"kind":"list like","value":["Vaclav Kasicka ","Martin Hubalek ","Yu Zeng zengyu@njmu.edu.cn ","Wu Y ","Han M Wang ","Y ","Gao Y ","Cui X ","Xu P ","Ji C ","Zhong T ","You L ","Zeng Y ","Yanting Wu \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nAffiliated Maternity and Child Health Care Hospital of Nantong University\nNanTongChina\n","Mei Han \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","Yan Wang \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","Yao Gao \nDepartment of Endocrinology, Children's Hospital\nNanjing Medical University\nNanjingChina\n","Xianwei Cui ","Pengfei Xu ","Chenbo Ji \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","Tianying Zhong \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","Lianghui You y_lianghui@126.com \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","Yu Zeng \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n","\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nLianghui You\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nCzechia, Czechia\n"],"string":"[\n \"Vaclav Kasicka \",\n \"Martin Hubalek \",\n \"Yu Zeng zengyu@njmu.edu.cn \",\n \"Wu Y \",\n \"Han M Wang \",\n \"Y \",\n \"Gao Y \",\n \"Cui X \",\n \"Xu P \",\n \"Ji C \",\n \"Zhong T \",\n \"You L \",\n \"Zeng Y \",\n \"Yanting Wu \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\\nAffiliated Maternity and Child Health Care Hospital of Nantong University\\nNanTongChina\\n\",\n \"Mei Han \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"Yan Wang \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"Yao Gao \\nDepartment of Endocrinology, Children's Hospital\\nNanjing Medical University\\nNanjingChina\\n\",\n \"Xianwei Cui \",\n \"Pengfei Xu \",\n \"Chenbo Ji \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"Tianying Zhong \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"Lianghui You y_lianghui@126.com \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"Yu Zeng \\nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\\n\",\n \"\\nInstitute of Organic Chemistry and Biochemistry (ASCR)\\nLianghui You\\nInstitute of Organic Chemistry and Biochemistry (ASCR)\\nCzechia, Czechia\\n\"\n]"},"annotations_authoraffiliation":{"kind":"list like","value":["Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Affiliated Maternity and Child Health Care Hospital of Nantong University\nNanTongChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Department of Endocrinology, Children's Hospital\nNanjing Medical University\nNanjingChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina","Institute of Organic Chemistry and Biochemistry (ASCR)\nLianghui You\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nCzechia, Czechia"],"string":"[\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Affiliated Maternity and Child Health Care Hospital of Nantong University\\nNanTongChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Department of Endocrinology, Children's Hospital\\nNanjing Medical University\\nNanjingChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\\nNanjingChina\",\n \"Institute of Organic Chemistry and Biochemistry (ASCR)\\nLianghui You\\nInstitute of Organic Chemistry and Biochemistry (ASCR)\\nCzechia, Czechia\"\n]"},"annotations_authorfirstname":{"kind":"list like","value":["Vaclav","Martin","Yu","Wu","Y","Han","M","Y","Gao","Y","Cui","X","Xu","P","Ji","C","Zhong","T","You","L","Zeng","Y","Yanting","Mei","Yan","Yao","Xianwei","Pengfei","Chenbo","Tianying","Lianghui","Yu"],"string":"[\n \"Vaclav\",\n \"Martin\",\n \"Yu\",\n \"Wu\",\n \"Y\",\n \"Han\",\n \"M\",\n \"Y\",\n \"Gao\",\n \"Y\",\n \"Cui\",\n \"X\",\n \"Xu\",\n \"P\",\n \"Ji\",\n \"C\",\n \"Zhong\",\n \"T\",\n \"You\",\n \"L\",\n \"Zeng\",\n \"Y\",\n \"Yanting\",\n \"Mei\",\n \"Yan\",\n \"Yao\",\n \"Xianwei\",\n \"Pengfei\",\n \"Chenbo\",\n \"Tianying\",\n \"Lianghui\",\n \"Yu\"\n]"},"annotations_authorlastname":{"kind":"list like","value":["Kasicka","Hubalek","Zeng","Wang","Wu","Han","Wang","Gao","Cui","Xu","Ji","Zhong","You","Zeng"],"string":"[\n \"Kasicka\",\n \"Hubalek\",\n \"Zeng\",\n \"Wang\",\n \"Wu\",\n \"Han\",\n \"Wang\",\n \"Gao\",\n 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\"Dasari\",\n \"Karakelides\",\n \"Bergen\",\n \"Nair\",\n \"Zhang\",\n \"Liu\",\n \"Zhou\",\n \"Li\",\n \"Alolga\",\n \"Qi\",\n \"Yin\",\n \"Knolhoff\",\n \"Rosenberg\",\n \"Millet\",\n \"Gillette\",\n \"Sweedler\",\n \"Shilov\",\n \"Seymour\",\n \"Patel\",\n \"Loboda\",\n \"Tang\",\n \"Keating\",\n \"Schilling\",\n \"Rardin\",\n \"Maclean\",\n \"Zawadzka\",\n \"Frewen\",\n \"Cusack\",\n \"Rardin\",\n \"Newman\",\n \"Held\",\n \"Cusack\",\n \"Sorensen\",\n \"Li\",\n \"Ashburner\",\n \"Ball\",\n \"Blake\",\n \"Botstein\",\n \"Butler\",\n \"Cherry\",\n \"Shen\",\n \"Li\",\n \"Wang\",\n \"Wang\",\n \"Huang\",\n \"Cao\",\n \"Villanueva\",\n \"Philip\",\n \"Chaparro\",\n \"Li\",\n \"Toledo-Crow\",\n \"Denoyer\",\n \"Koomen\",\n \"Li\",\n \"Xiao\",\n \"Liu\",\n \"Coombes\",\n \"Abbruzzese\",\n \"Villanueva\",\n \"Shaffer\",\n \"Philip\",\n \"Chaparro\",\n \"Erdjument-Bromage\",\n \"Olshen\",\n \"Wang\",\n \"Zhu\",\n \"Hu\",\n \"Qin\",\n \"Ye\",\n \"Zou\",\n \"Zhang\",\n \"Liang\",\n \"Cheng\",\n \"Cao\",\n \"Wu\",\n \"Wang\",\n \"Deshmukh\",\n \"Yoon\",\n \"Yea\",\n \"Kim\",\n \"Choi\",\n \"Park\",\n \"Lee\",\n \"Norheim\",\n \"Raastad\",\n \"Thiede\",\n \"Rustan\",\n \"Drevon\",\n \"Haugen\",\n \"Raschke\",\n \"Eckardt\",\n \"Bjørklund Holven\",\n \"Jensen\",\n \"Eckel\",\n \"Deshmukh\",\n \"Cox\",\n \"Jensen\",\n \"Meissner\",\n \"Mann\",\n \"Yoon\",\n \"Kim\",\n \"Jang\",\n \"Ghim\",\n \"Park\",\n \"Song\",\n \"Furuichi\",\n \"Manabe\",\n \"Takagi\",\n \"Aoki\",\n \"Fujii\",\n \"Forterre\",\n \"Jalabert\",\n \"Chikh\",\n \"Pesenti\",\n \"Euthine\",\n \"Granjon\",\n \"Jalabert\",\n \"Vial\",\n \"Guay\",\n \"Wiklander\",\n \"Nordin\",\n \"Aswad\",\n \"Ibrahim\",\n \"Neinast\",\n \"Arany\",\n \"Nielsen\",\n \"Scheele\",\n \"Yfanti\",\n \"Akerström\",\n \"Nielsen\",\n \"Pedersen\",\n \"Coleman\",\n \"Rees\",\n \"Alcolado\",\n \"Gao\",\n \"Wang\",\n \"Huang\",\n \"Cui\",\n \"Li\",\n \"Wang\",\n \"Sasaki\",\n \"Satomi\",\n \"Takao\",\n \"Minamino\",\n \"Gelman\",\n \"Dasgupta\",\n \"Berezniuk\",\n \"Fricker\",\n \"Fricker\",\n \"Silvestre\",\n \"Rodríguez-Gallardo\",\n \"Egido\",\n \"Hernández\",\n \"Marco\",\n \"Martin\",\n \"Parthsarathy\",\n \"Pathak\",\n \"Gault\",\n \"Flatt\",\n \"Irwin\",\n \"Wiita\",\n \"Hsu\",\n \"Lu\",\n \"Esensten\",\n \"Wells\",\n \"Aletti\",\n \"Maffioli\",\n \"Negri\",\n \"Santamaria\",\n \"Delano\",\n \"Kistler\",\n \"Hittel\",\n \"Hathout\",\n \"Hoffman\",\n \"Houmard\",\n \"Giebelstein\",\n \"Poschmann\",\n \"Højlund\",\n \"Schechinger\",\n \"Dietrich\",\n \"Levin\",\n \"Mogensen\",\n \"Sahlin\",\n \"Fernström\",\n \"Glintborg\",\n \"Vind\",\n \"Beck-Nielsen\",\n \"Cheng\",\n \"Tseng\",\n \"White\",\n \"Ritov\",\n \"Menshikova\",\n \"Azuma\",\n \"Wood\",\n \"Toledo\",\n \"Goodpaster\",\n \"Thingholm\",\n \"Bak\",\n \"Beck-Nielsen\",\n \"Jensen\",\n \"Gaster\",\n \"Oberbach\",\n \"Bossenz\",\n \"Lehmann\",\n \"Niebauer\",\n \"Adams\",\n \"Paschke\",\n \"Denessiouk\",\n \"Permyakov\",\n \"Denesyuk\",\n \"Permyakov\",\n \"Johnson\",\n \"Lakowski\",\n \"Lee\",\n \"Lelj-Garolla\",\n \"Okon\",\n \"Reid\",\n \"Mcintosh\",\n \"Kawasaki\",\n \"Kretsinger\",\n \"Wright\",\n \"Hucker\",\n \"Holloszy\",\n \"Han\",\n \"Park\",\n \"Park\",\n \"Kim\",\n \"Yoon\",\n \"Kim\",\n \"Jia\",\n \"Arif\",\n \"Terenzi\",\n \"Willard\",\n \"Plow\",\n \"Hazen\",\n \"Guo\",\n \"Wang\",\n \"Zhang\",\n \"Yu\",\n \"Poulev\",\n \"Ribnicky\",\n \"Floyd\",\n \"Pagel-Langenickel\",\n \"Joseph\",\n \"Schwartz\",\n \"Mantell\",\n \"Xu\"\n]"},"annotations_bibentry":{"kind":"list like","value":["The effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man. 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(2011) 301:E1013-21. doi: 10.1152/ajpendo.00326.2011\",\n \"Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells. S Raschke, K Eckardt, K Bjørklund Holven, J Jensen, J Eckel, 10.1371/journal.pone.0062008PLoS ONE. 862008Raschke S, Eckardt K, Bjørklund Holven K, Jensen J, Eckel J. Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells. PLoS ONE. (2013) 8:e62008. doi: 10.1371/journal.pone.0062008\",\n \"Secretome analysis of lipid-induced insulin resistance in skeletal muscle cells by a combined experimental and bioinformatics workflow. A S Deshmukh, J Cox, L J Jensen, F Meissner, M Mann, 10.1021/acs.jproteome.5b00720J Proteome Res. 14Deshmukh AS, Cox J, Jensen LJ, Meissner F, Mann M. Secretome analysis of lipid-induced insulin resistance in skeletal muscle cells by a combined experimental and bioinformatics workflow. J Proteome Res. (2015) 14:4885- 95. doi: 10.1021/acs.jproteome.5b00720\",\n \"Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance. J H Yoon, D Kim, J H Jang, J Ghim, S Park, P Song, 10.1074/mcp.M114.039651Mol Cell Proteomics. 14Yoon JH, Kim D, Jang JH, Ghim J, Park S, Song P, et al. Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance. Mol Cell Proteomics. (2015) 14:882-92. doi: 10.1074/mcp.M114. 039651\",\n \"Evidence for acute contraction-induced myokine secretion by C2C12 myotubes. Y Furuichi, Y Manabe, M Takagi, M Aoki, N L Fujii, 10.1371/journal.pone.0206146PLoS ONE. 13206146Furuichi Y, Manabe Y, Takagi M, Aoki M, Fujii NL. Evidence for acute contraction-induced myokine secretion by C2C12 myotubes. PLoS ONE. (2018) 13:e206146. doi: 10.1371/journal.pone.0206146\",\n \"Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation. A Forterre, A Jalabert, K Chikh, S Pesenti, V Euthine, A Granjon, 10.4161/cc.26808Cell Cycle. 13Forterre A, Jalabert A, Chikh K, Pesenti S, Euthine V, Granjon A, et al. Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation. Cell Cycle. (2014) 13:78-89. doi: 10.4161/cc.26808\",\n \"Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice. A Jalabert, G Vial, C Guay, O P Wiklander, J Z Nordin, H Aswad, 10.1007/s00125-016-3882-yDiabetologia. 59Jalabert A, Vial G, Guay C, Wiklander OP, Nordin JZ, Aswad H, et al. Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice. Diabetologia. (2016) 59:1049-58. doi: 10.1007/s00125-016-3882-y\",\n \"Myobolites: muscle-derived metabolites with paracrine and systemic effects. A Ibrahim, M Neinast, Z P Arany, 10.1016/j.coph.2017.03.007Curr Opin Pharmacol. 34Ibrahim A, Neinast M, Arany ZP. Myobolites: muscle-derived metabolites with paracrine and systemic effects. Curr Opin Pharmacol. (2017) 34:15-20. doi: 10.1016/j.coph.2017.03.007\",\n \"Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle. S Nielsen, C Scheele, C Yfanti, T Akerström, A R Nielsen, B K Pedersen, 10.1113/jphysiol.2010.189860J Physiol. 588Nielsen S, Scheele C, Yfanti C, Akerström T, Nielsen AR, Pedersen BK, et al. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle. J Physiol. (2010) 588:4029-37. doi: 10.1113/jphysiol.2010.189860\",\n \"Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. D L Coleman, 10.1007/BF00429772Diabetologia. 14Coleman DL. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia. (1978) 14:141-8. doi: 10.1007/BF00429772\",\n \"Animal models of diabetes mellitus. D A Rees, J C Alcolado, 10.1111/j.1464-5491.2005.01499.xDiabet Med. 22Rees DA, Alcolado JC. Animal models of diabetes mellitus. Diabet Med. (2005) 22:359-70. doi: 10.1111/j.1464-5491.2005.01499.x\",\n \"Identification and characterization of metformin on peptidomic profiling in human visceral adipocytes. Y Gao, X Wang, F Huang, X Cui, Y Li, X Wang, 10.1002/jcb.26347J Cell Biochem. 119Gao Y, Wang X, Huang F, Cui X, Li Y, Wang X, et al. Identification and characterization of metformin on peptidomic profiling in human visceral adipocytes. J Cell Biochem. (2018) 119:1866-78. doi: 10.1002/jcb.26347\",\n \"Snapshot peptidomics of the regulated secretory pathway. K Sasaki, Y Satomi, T Takao, N Minamino, 10.1074/mcp.M900044-MCP200Mol Cell Proteomics. 8Sasaki K, Satomi Y, Takao T, Minamino N. Snapshot peptidomics of the regulated secretory pathway. Mol Cell Proteomics. (2009) 8:1638-47. doi: 10.1074/mcp.M900044-MCP200\",\n \"Analysis of peptides secreted from cultured mouse brain tissue. J S Gelman, S Dasgupta, I Berezniuk, L D Fricker, 10.1016/j.bbapap.2013.01.043Biochim Biophys Acta. 1834Gelman JS, Dasgupta S, Berezniuk I, Fricker LD. Analysis of peptides secreted from cultured mouse brain tissue. Biochim Biophys Acta. (2013) 1834:2408-17. doi: 10.1016/j.bbapap.2013.01.043\",\n \"Analysis of mouse brain peptides using mass spectrometrybased peptidomics: implications for novel functions ranging from nonclassical neuropeptides to microproteins. L D Fricker, 10.1039/c003317kMol Biosyst. 6Fricker LD. Analysis of mouse brain peptides using mass spectrometry- based peptidomics: implications for novel functions ranging from non- classical neuropeptides to microproteins. Mol Biosyst. 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D S Hittel, Y Hathout, E P Hoffman, J A Houmard, 10.2337/diabetes.54.5.1283Diabetes. 54Hittel DS, Hathout Y, Hoffman EP, Houmard JA. Proteome analysis of skeletal muscle from obese and morbidly obese women. Diabetes. (2005) 54:1283-8. doi: 10.2337/diabetes.54.5.1283\",\n \"The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes. J Giebelstein, G Poschmann, K Højlund, W Schechinger, J W Dietrich, K Levin, 10.1007/s00125-012-2456-xDiabetologia. 55Giebelstein J, Poschmann G, Højlund K, Schechinger W, Dietrich JW, Levin K, et al. The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes. Diabetologia. (2012) 55:1114-27. doi: 10.1007/s00125-012-2456-x\",\n \"Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes. M Mogensen, K Sahlin, M Fernström, D Glintborg, B F Vind, H Beck-Nielsen, 10.2337/db06-0981Diabetes. 56Mogensen M, Sahlin K, Fernström M, Glintborg D, Vind BF, Beck-Nielsen H, et al. Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes. Diabetes. (2007) 56:1592-9. doi: 10.2337/db06-0981\",\n \"Insulin signaling meets mitochondria in metabolism. Z Cheng, Y Tseng, M F White, 10.1016/j.tem.2010.06.005Trends Endocrinol Metab. 21Cheng Z, Tseng Y, White MF. Insulin signaling meets mitochondria in metabolism. Trends Endocrinol Metab. (2010) 21:589-98. doi: 10.1016/j.tem.2010.06.005\",\n \"Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. V B Ritov, E V Menshikova, K Azuma, R Wood, F G Toledo, B H Goodpaster, 10.1152/ajpendo.00317.2009Am J Physiol Endocrinol Metab. 298Ritov VB, Menshikova EV, Azuma K, Wood R, Toledo FG, Goodpaster BH, et al. Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. Am J Physiol Endocrinol Metab. (2010) 298:E49-58. doi: 10.1152/ajpendo.00317.2009\",\n \"Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods. T E Thingholm, S Bak, H Beck-Nielsen, O N Jensen, M Gaster, 10.1074/mcp.M110.006650Mol Cell Proteomics. 10Thingholm TE, Bak S, Beck-Nielsen H, Jensen ON, Gaster M. Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods. Mol Cell Proteomics. (2011) 10:M110.006650. doi: 10.1074/mcp.M110.006650\",\n \"Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. A Oberbach, Y Bossenz, S Lehmann, J Niebauer, V Adams, R Paschke, 10.2337/diacare.29.04.06.dc05-1854Diabetes Care. 29Oberbach A, Bossenz Y, Lehmann S, Niebauer J, Adams V, Paschke R, et al. Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. Diabetes Care. (2006) 29:895-900. doi: 10.2337/diacare.29.04.06.dc05-1854\",\n \"Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors. K Denessiouk, S Permyakov, A Denesyuk, E Permyakov, M S Johnson, 10.1371/journal.pone.0109287PLoS ONE. 9109287Denessiouk K, Permyakov S, Denesyuk A, Permyakov E, Johnson MS. Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors. PLoS ONE. (2014) 9:e109287. doi: 10.1371/journal.pone.0109287\",\n \"Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3. 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D R Park, K H Park, B J Kim, C S Yoon, U H Kim, 10.2337/db14-0939Diabetes. 64Park DR, Park KH, Kim BJ, Yoon CS, Kim UH. Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles. Diabetes. (2015) 64:1224-34. doi: 10.2337/db14-0939\",\n \"Target-selective protein S-nitrosylation by sequence motif recognition. J Jia, A Arif, F Terenzi, B Willard, E F Plow, S L Hazen, 10.1016/j.cell.2014.09.032Cell. 159Jia J, Arif A, Terenzi F, Willard B, Plow EF, Hazen SL, et al. Target-selective protein S-nitrosylation by sequence motif recognition. Cell. (2014) 159:623- 34. doi: 10.1016/j.cell.2014.09.032\",\n \"Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. S Guo, 10.1530/JOE-13-0327J Endocrinol. 220Guo S. Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. J Endocrinol. (2014) 220:T1-23. doi: 10.1530/JOE-13-0327\",\n \"Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice. Z Q Wang, X H Zhang, Y Yu, A Poulev, D Ribnicky, Z E Floyd, 10.1016/j.jnutbio.2010.09.004J Nutr Biochem. 22Wang ZQ, Zhang XH, Yu Y, Poulev A, Ribnicky D, Floyd ZE, et al. Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice. J Nutr Biochem. (2011) 22:1064-73. doi: 10.1016/j.jnutbio.2010.09.004\",\n \"PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. I Pagel-Langenickel, Bao J Joseph, J J Schwartz, D R Mantell, B S Xu, X , 10.1074/jbc.M800842200J Biol Chem. 283Pagel-Langenickel I, Bao J, Joseph JJ, Schwartz DR, Mantell BS, Xu X, et al. PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. J Biol Chem. (2008) 283:22464-72. doi: 10.1074/jbc.M800842200\"\n]"},"annotations_bibref":{"kind":"list like","value":["(1)","(2)","(4)","(5)","(6)","(8)","(9)","(10,","11)","(12)","(13)","(14)","16)","(17)","(18)","(19)","(20)","(21,","22)","(23)","(24)","(25)","(26,","27)","(28,","29)","(30)","(31)","(33)","(22,","34,","35)","(36,","37)","(38,","39)","(36,","40,","41)","(43)","(37)","(44)","(45)","(45,","46)","(47)","(48,","49)","(48)","(24)","(51)","(52)","(53)","(54)","(55)","(56)","(57)","(58)","(59)","(60)","(61)","(62)","(63)","(58)","(61)","(64,","65)","(66)","(67)","(68,","69)","(70)","(71)","(34)","(72)","(73)","(74,","75)","(76)","(77)","(78)","(79)","(80)","(81)","(82)","(83)","(79)","(84)","(90)","(91)","(92)","(93)","(85)","(86,","87)","(88)","(89)"],"string":"[\n \"(1)\",\n \"(2)\",\n \"(4)\",\n \"(5)\",\n \"(6)\",\n \"(8)\",\n \"(9)\",\n \"(10,\",\n \"11)\",\n \"(12)\",\n \"(13)\",\n \"(14)\",\n \"16)\",\n \"(17)\",\n \"(18)\",\n \"(19)\",\n \"(20)\",\n \"(21,\",\n \"22)\",\n 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doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man","Compartmentalized acyl-CoA metabolism in skeletal muscle regulates systemic glucose homeostasis","Muscle-specific deletion of Prkaa1 enhances skeletal muscle lipid accumulation in mice fed a high-fat diet","Age-related impairments in skeletal muscle PDH phosphorylation and plasma lactate are indicative of metabolic inflexibility and the effects of exercise training","The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome","Muscle as a paracrine and endocrine organ","A PGC1-alphadependent myokine that drives brown-fat-like development of white fat and thermogenesis","Muscle and bone, two interconnected tissues","Perilipin 5 deletion unmasks an endoplasmic reticulum stressfibroblast growth factor 21 axis in skeletal muscle","Skeletal muscle to pancreatic β-Cell cross-talk: the effect of humoral mediators liberated by muscle contraction and acute exercise on β-cell apoptosis","Adipose-and muscle-derived Wnts trigger pancreatic beta-cell adaptation to systemic insulin resistance","Muscle-to-organ cross talk mediated by myokines","The role of myokines in muscle health and disease","Dual role of interleukin-6 in regulating insulin sensitivity in murine skeletal muscle","Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle","Interleukin-6 stimulates lipolysis and fat oxidation in humans","Differential effects of interleukin-6 and−10 on skeletal muscle and liver insulin action in vivo","Do skeletal muscle-secreted factors influence the function of pancreatic beta-cells?","A peptidomics strategy for discovering endogenous bioactive peptides","Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling","Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release","Profiling analysis reveals the potential contribution of peptides to human adipocyte differentiation","TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity","Natriuretic peptides in cardiometabolic regulation and disease","Neuroendocrine-derived peptides promote prostate cancer cell survival through activation of IGF-1R signaling","Origins, technological development, and applications of peptidomics","Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015-mid 2018)","Quantitative peptidomics: general considerations","Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice","Urinary peptidomics in kidney disease and drug research","Development of a prosaposin-derived therapeutic cyclic peptide that targets ovarian cancer via the tumor microenvironment","Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of fulllength peptide hormones than found using cell lysis procedures","Quantitative mass spectrometry reveals food intake-induced neuropeptide level changes in rat brain: functional assessment of selected neuropeptides as feeding regulators","Testosterone stimulates adipose tissue 11beta-hydroxysteroid dehydrogenase type 1 expression in a depot-specific manner in children","Analysis of secreted peptidome from omental adipose tissue in polycystic ovarian syndrome patients","Characterization of the human visceral adipose tissue secretome","Secretome analysis of rat adipose tissues shows locationspecific roles for each depot type","Gender differences in both spontaneous and stimulated leptin secretion by human omental adipose tissue in vitro: dexamethasone and estradiol stimulate leptin release in women, but not in men","Adiponectin secretion and response to pioglitazone is depot dependent in cultured human adipose tissue","Muscle tissue as an endocrine organ: comparative secretome profiling of slow-oxidative and fast-glycolytic rat muscle explants and its variation with exercise","Protein determination-method matters","Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice","A comparative proteomic characterization and nutritional assessment of naturally-and artificially-cultivated Cordyceps sinensis","Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes","The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra","Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation","Labelfree quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways","Gene ontology: tool for the unification of biology. The Gene Ontology Consortium","A novel peptide suppresses adipogenic differentiation through activation of the AMPK pathway","Correcting common errors in identifying cancer-specific serum peptide signatures","Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery","Differential exoprotease activities confer tumorspecific serum peptidome patterns","Comprehensive analysis of the N and C terminus of endogenous serum peptides reveals a highly conserved cleavage site pattern derived from proteolytic enzymes","Peptidomic analysis of fetal heart tissue for identification of endogenous peptides involved in tetralogy of fallot","Proteomics of skeletal muscle: focus on insulin resistance and exercise biology","Comparative proteomic analysis of the insulin-induced L6 myotube secretome","Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training","Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells","Secretome analysis of lipid-induced insulin resistance in skeletal muscle cells by a combined experimental and bioinformatics workflow","Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance","Evidence for acute contraction-induced myokine secretion by C2C12 myotubes","Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation","Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice","Myobolites: muscle-derived metabolites with paracrine and systemic effects","Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle","Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice","Animal models of diabetes mellitus","Identification and characterization of metformin on peptidomic profiling in human visceral adipocytes","Snapshot peptidomics of the regulated secretory pathway","Analysis of peptides secreted from cultured mouse brain tissue","Analysis of mouse brain peptides using mass spectrometrybased peptidomics: implications for novel functions ranging from nonclassical neuropeptides to microproteins","Stimulatory effect of xenin-8 on insulin and glucagon secretion in the perfused rat pancreas","Characterisation of the biological activity of xenin-25 degradation fragment peptides","Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling","Peptidomic analysis of rat plasma: proteolysis in hemorrhagic shock","Proteome analysis of skeletal muscle from obese and morbidly obese women","The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes","Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes","Insulin signaling meets mitochondria in metabolism","Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity","Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods","Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes","Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors","Calcium-binding proteins 1: EF-hands","Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions","Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles","Target-selective protein S-nitrosylation by sequence motif recognition","Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms","Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice","PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle"],"string":"[\n \"The effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man\",\n \"Compartmentalized acyl-CoA metabolism in skeletal muscle regulates systemic glucose homeostasis\",\n \"Muscle-specific deletion of Prkaa1 enhances skeletal muscle lipid accumulation in mice fed a high-fat diet\",\n \"Age-related impairments in skeletal muscle PDH phosphorylation and plasma lactate are indicative of metabolic inflexibility and the effects of exercise training\",\n \"The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome\",\n \"Muscle as a paracrine and endocrine organ\",\n \"A PGC1-alphadependent myokine that drives brown-fat-like development of white fat and thermogenesis\",\n \"Muscle and bone, two interconnected tissues\",\n \"Perilipin 5 deletion unmasks an endoplasmic reticulum stressfibroblast growth factor 21 axis in skeletal muscle\",\n \"Skeletal muscle to pancreatic β-Cell cross-talk: the effect of humoral mediators liberated by muscle contraction and acute exercise on β-cell apoptosis\",\n \"Adipose-and muscle-derived Wnts trigger pancreatic beta-cell adaptation to systemic insulin resistance\",\n \"Muscle-to-organ cross talk mediated by myokines\",\n \"The role of myokines in muscle health and disease\",\n \"Dual role of interleukin-6 in regulating insulin sensitivity in murine skeletal muscle\",\n \"Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle\",\n \"Interleukin-6 stimulates lipolysis and fat oxidation in humans\",\n \"Differential effects of interleukin-6 and−10 on skeletal muscle and liver insulin action in vivo\",\n \"Do skeletal muscle-secreted factors influence the function of pancreatic beta-cells?\",\n \"A peptidomics strategy for discovering endogenous bioactive peptides\",\n \"Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling\",\n \"Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release\",\n \"Profiling analysis reveals the potential contribution of peptides to human adipocyte differentiation\",\n \"TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity\",\n \"Natriuretic peptides in cardiometabolic regulation and disease\",\n \"Neuroendocrine-derived peptides promote prostate cancer cell survival through activation of IGF-1R signaling\",\n \"Origins, technological development, and applications of peptidomics\",\n \"Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015-mid 2018)\",\n \"Quantitative peptidomics: general considerations\",\n \"Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice\",\n \"Urinary peptidomics in kidney disease and drug research\",\n \"Development of a prosaposin-derived therapeutic cyclic peptide that targets ovarian cancer via the tumor microenvironment\",\n \"Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of fulllength peptide hormones than found using cell lysis procedures\",\n \"Quantitative mass spectrometry reveals food intake-induced neuropeptide level changes in rat brain: functional assessment of selected neuropeptides as feeding regulators\",\n \"Testosterone stimulates adipose tissue 11beta-hydroxysteroid dehydrogenase type 1 expression in a depot-specific manner in children\",\n \"Analysis of secreted peptidome from omental adipose tissue in polycystic ovarian syndrome patients\",\n \"Characterization of the human visceral adipose tissue secretome\",\n \"Secretome analysis of rat adipose tissues shows locationspecific roles for each depot type\",\n \"Gender differences in both spontaneous and stimulated leptin secretion by human omental adipose tissue in vitro: dexamethasone and estradiol stimulate leptin release in women, but not in men\",\n \"Adiponectin secretion and response to pioglitazone is depot dependent in cultured human adipose tissue\",\n \"Muscle tissue as an endocrine organ: comparative secretome profiling of slow-oxidative and fast-glycolytic rat muscle explants and its variation with exercise\",\n \"Protein determination-method matters\",\n \"Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice\",\n \"A comparative proteomic characterization and nutritional assessment of naturally-and artificially-cultivated Cordyceps sinensis\",\n \"Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes\",\n \"The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra\",\n \"Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation\",\n \"Labelfree quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways\",\n \"Gene ontology: tool for the unification of biology. The Gene Ontology Consortium\",\n \"A novel peptide suppresses adipogenic differentiation through activation of the AMPK pathway\",\n \"Correcting common errors in identifying cancer-specific serum peptide signatures\",\n \"Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery\",\n \"Differential exoprotease activities confer tumorspecific serum peptidome patterns\",\n \"Comprehensive analysis of the N and C terminus of endogenous serum peptides reveals a highly conserved cleavage site pattern derived from proteolytic enzymes\",\n \"Peptidomic analysis of fetal heart tissue for identification of endogenous peptides involved in tetralogy of fallot\",\n \"Proteomics of skeletal muscle: focus on insulin resistance and exercise biology\",\n \"Comparative proteomic analysis of the insulin-induced L6 myotube secretome\",\n \"Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training\",\n \"Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells\",\n \"Secretome analysis of lipid-induced insulin resistance in skeletal muscle cells by a combined experimental and bioinformatics workflow\",\n \"Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance\",\n \"Evidence for acute contraction-induced myokine secretion by C2C12 myotubes\",\n \"Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation\",\n \"Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice\",\n \"Myobolites: muscle-derived metabolites with paracrine and systemic effects\",\n \"Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle\",\n \"Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice\",\n \"Animal models of diabetes mellitus\",\n \"Identification and characterization of metformin on peptidomic profiling in human visceral adipocytes\",\n \"Snapshot peptidomics of the regulated secretory pathway\",\n \"Analysis of peptides secreted from cultured mouse brain tissue\",\n \"Analysis of mouse brain peptides using mass spectrometrybased peptidomics: implications for novel functions ranging from nonclassical neuropeptides to microproteins\",\n \"Stimulatory effect of xenin-8 on insulin and glucagon secretion in the perfused rat pancreas\",\n \"Characterisation of the biological activity of xenin-25 degradation fragment peptides\",\n \"Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling\",\n \"Peptidomic analysis of rat plasma: proteolysis in hemorrhagic shock\",\n \"Proteome analysis of skeletal muscle from obese and morbidly obese women\",\n \"The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes\",\n \"Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes\",\n \"Insulin signaling meets mitochondria in metabolism\",\n \"Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity\",\n \"Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods\",\n \"Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes\",\n \"Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors\",\n \"Calcium-binding proteins 1: EF-hands\",\n \"Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions\",\n \"Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles\",\n \"Target-selective protein S-nitrosylation by sequence motif recognition\",\n \"Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms\",\n \"Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice\",\n \"PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["Diabetes","Diabetes","J Physiol Biochem","Am J Physiol Endocrinol Metab","Proc Natl Acad Sci","Curr Opin Pharmacol","Nature","Ageing Res Rev","Diabetes","J Clin Endocrinol Metab","Sci Rep","Adipocyte","Curr Opin Rheumatol","Diabetologia","Mol Endocrinol","J Clin Endocrinol Metab","Diabetes","Am J Physiol Endocrinol Metab","The potential role of contraction-induced myokines in the regulation of metabolic function for the prevention and treatment of type 2 diabetes. Front Endocrinol","J Proteome Res","Diabetes","Br J Pharmacol","Proteomics Clin Appl","Proc Natl Acad Sci","Nat Rev Cardiol","Prostate","Methods Mol Biol","J Sep Sci","Methods Mol Biol","Kidney Int","Expert Opin Drug Discov","Sci Transl Med","J Proteome Res","Mol Cell Proteomics","J Clin Endocrinol Metab","J Cell Physiol","Mol Cell Proteomics","J Proteomics","J Clin Endocrinol Metab","Am J Physiol Endocrinol Metab","J Proteomics","Foods","Am J Physiol Endocrinol Metab","J Proteomics","J Proteome Res","Mol Cell Proteomics","Mol Cell Proteomics","Proc Natl Acad Sci","Nat Genet","Biochem Biophys Res Commun","J Proteome Res","J Proteome Res","J Clin Invest","Protein Cell","DNA Cell Biol","Proteomes","Proteomics","Am J Physiol Endocrinol Metab","PLoS ONE","J Proteome Res","Mol Cell Proteomics","PLoS ONE","Cell Cycle","Diabetologia","Curr Opin Pharmacol","J Physiol","Diabetologia","Diabet Med","J Cell Biochem","Mol Cell Proteomics","Biochim Biophys Acta","Mol Biosyst","Regul Pept","J Endocrinol","Proc Natl Acad Sci","Shock","Diabetes","Diabetologia","Diabetes","Trends Endocrinol Metab","Am J Physiol Endocrinol Metab","Mol Cell Proteomics","Diabetes Care","PLoS ONE","Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3. Biochemistry-us","Protein Profile","Diabetes","Diabetes","Cell","J Endocrinol","J Nutr Biochem","J Biol Chem"],"string":"[\n \"Diabetes\",\n \"Diabetes\",\n \"J Physiol Biochem\",\n \"Am J Physiol Endocrinol Metab\",\n \"Proc Natl Acad Sci\",\n \"Curr Opin Pharmacol\",\n \"Nature\",\n \"Ageing Res Rev\",\n \"Diabetes\",\n \"J Clin Endocrinol Metab\",\n \"Sci Rep\",\n \"Adipocyte\",\n \"Curr Opin Rheumatol\",\n \"Diabetologia\",\n \"Mol Endocrinol\",\n \"J Clin Endocrinol Metab\",\n \"Diabetes\",\n \"Am J Physiol Endocrinol Metab\",\n \"The potential role of contraction-induced myokines in the regulation of metabolic function for the prevention and treatment of type 2 diabetes. Front Endocrinol\",\n \"J Proteome Res\",\n \"Diabetes\",\n \"Br J Pharmacol\",\n \"Proteomics Clin Appl\",\n \"Proc Natl Acad Sci\",\n \"Nat Rev Cardiol\",\n \"Prostate\",\n \"Methods Mol Biol\",\n \"J Sep Sci\",\n \"Methods Mol Biol\",\n \"Kidney Int\",\n \"Expert Opin Drug Discov\",\n \"Sci Transl Med\",\n \"J Proteome Res\",\n \"Mol Cell Proteomics\",\n \"J Clin Endocrinol Metab\",\n \"J Cell Physiol\",\n \"Mol Cell Proteomics\",\n \"J Proteomics\",\n \"J Clin Endocrinol Metab\",\n \"Am J Physiol Endocrinol Metab\",\n \"J Proteomics\",\n \"Foods\",\n \"Am J Physiol Endocrinol Metab\",\n \"J Proteomics\",\n \"J Proteome Res\",\n \"Mol Cell Proteomics\",\n \"Mol Cell Proteomics\",\n \"Proc Natl Acad Sci\",\n \"Nat Genet\",\n \"Biochem Biophys Res Commun\",\n \"J Proteome Res\",\n \"J Proteome Res\",\n \"J Clin Invest\",\n \"Protein Cell\",\n \"DNA Cell Biol\",\n \"Proteomes\",\n \"Proteomics\",\n \"Am J Physiol Endocrinol Metab\",\n \"PLoS ONE\",\n \"J Proteome Res\",\n \"Mol Cell Proteomics\",\n \"PLoS ONE\",\n \"Cell Cycle\",\n \"Diabetologia\",\n \"Curr Opin Pharmacol\",\n \"J Physiol\",\n \"Diabetologia\",\n \"Diabet Med\",\n \"J Cell Biochem\",\n \"Mol Cell Proteomics\",\n \"Biochim Biophys Acta\",\n \"Mol Biosyst\",\n \"Regul Pept\",\n \"J Endocrinol\",\n \"Proc Natl Acad Sci\",\n \"Shock\",\n \"Diabetes\",\n \"Diabetologia\",\n \"Diabetes\",\n \"Trends Endocrinol Metab\",\n \"Am J Physiol Endocrinol Metab\",\n \"Mol Cell Proteomics\",\n \"Diabetes Care\",\n \"PLoS ONE\",\n \"Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3. Biochemistry-us\",\n \"Protein Profile\",\n \"Diabetes\",\n \"Diabetes\",\n \"Cell\",\n \"J Endocrinol\",\n \"J Nutr Biochem\",\n \"J Biol Chem\"\n]"},"annotations_figure":{"kind":"list like","value":["\n\nAt the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.","\nFIGURE 1 |\n1Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.","\nFIGURE 2 |\n2Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.","\nFIGURE 3 |\n3Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)","\nFIGURE 4 |\n4Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.","\nFIGURE 5 |\n5Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.","\n\nThe up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in","\n\n413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),","\nFUNDING\nThis study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science","\nFigure S2 |\nS2Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.","\nTABLE 1 |\n1Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nUp-regulated peptides \n\nAVGAVFDISNADRLGSSEVEQ \nP07310 \nKCRM \n2163 \n9.728 \n0.005 \n\nTNGAFTGEISPGMIKDLGATWV \nP17751 \nTPIS \n2264 \n8.019 \n0.035 \n\nIADLVVGLCTGQIK \nP21550 \nENOB \n1486 \n6.77 \n0.000 \n\nNYKPQEEYPDLSKHNNHMA \nP07310 \nKCRM \n2315 \n4.843 \n0.024 \n\nEEIEEDAGLGNGGLGRLAAC \nQ9WUB3 \nPYGM \n2030 \n4.411 \n0.004 \n\nGQCGDVLRALGQNPTQAEV \nP09542 \nMYL3 \n2013 \n4.077 \n0.033 \n\nAGPLCLQEVDEPPQHAL \nP70296 \nPEBP1 \n1873 \n4.038 \n0.004 \n\nEAFTVIDQNRDGIID \nP97457 \nMLRS \n1705 \n3.947 \n0.026 \n\nDLFDPIIQDRHGGY \nP07310 \nKCRM \n1645 \n3.668 \n0.022 \n\nKDLFDPIIQD \nP07310 \nKCRM \n1203 \n3.282 \n0.000 \n\nLDDVIQTGVDNPGHP \nP07310 \nKCRM \n1576 \n3.265 \n0.001 \n\nSEIIDVSSKAEEVK \nA2ASS6 \nTITIN \n1533 \n3.134 \n0.002 \n\nVIACIGEKLDE \nP17751 \nTPIS \n1246 \n2.976 \n0.003 \n\nGTNPTNAEVKKVLGNPSNEEM \nQ545G5 \nMYL1 \n2180 \n2.838 \n0.006 \n\nADRLGSSEVEQVQLV \nP07310 \nKCRM \n1629 \n2.804 \n0.001 \n\nIGQGTPIPDLPEVKRV \nA2AQA9 \nNEB \n1719 \n2.739 \n0.002 \n\nSTTAPPIQSPLPVIPHQK \nE9PYJ9 \nLDB3 \n1910 \n2.685 \n0.023 \n\nKADDGRPFPQVI \nP05064 \nALDOA \n1342 \n2.644 \n0.021 \n\nKSMTEQEQQQLIDD \nP07310 \nKCRM \n1692 \n2.442 \n0.001 \n\nGGGASLELLEGKVLPGVDA \nP09411 \nPGK1 \n1781 \n2.264 \n0.028 \n\nLEGTLLKPNMVTPGHA \nP05064 \nALDOA \n1677 \n2.208 \n0.010 \n\nVMVGMGQKDSYVGDEAQSK \nP68134 \nACTS \n2028 \n2.168 \n0.021 \n\nDLEEATLQHEATAAALR \nQ02566 \nMYH6 \n1838 \n2.07 \n0.038 \n\nAELQEVQITEEKPLLPG \nQ9QYG0 \nNDRG2 \n1935 \n2.043 \n0.048 \n\nSEGKCAELEEELKTVTNNL \nP58771 \nTPM1 \n2163 \n2.038 \n0.049 \n\nRSIKGYTLPPHC \nP07310 \nKCRM \n1428 \n1.967 \n0.001 \n\nNSNSHSSTFDAGAGIALNDNFVK \nP16858 \nG3P \n2365 \n1.935 \n0.038 \n\nTGKATSEASVSTPEETAPEPAKVPT \nP70402 \nMYBPH \n2484 \n1.919 \n0.003 \n\nDNEYGYSNRVVDL \nP16858 \nG3P \n1543 \n1.898 \n0.004 \n\nEELDAMMKEASGPINF \nP97457 \nMLRS \n1781 \n1.853 \n0.012 \n\nSNNKDQGGYEDFVEGLRV \nQ545G5 \nMYL1 \n2026 \n1.816 \n0.016 \n\nAEQIKHILANF \nP63028 \nTCTP \n1283 \n1.787 \n0.044 \n\nGADPEDVITGAFK \nP97457 \nMLRS \n1319 \n1.782 \n0.031 \n\nTSIEDAPITVQSKINQ \nA2AQA9 \nNEB \n1743 \n1.739 \n0.028 \n\nLKGGDDLDPNYVLS \nP07310 \nKCRM \n1505 \n1.732 \n0.040 \n\nIQTGVDNPGHPF \nQ6P8J7 \nKCRS \n1281 \n1.600 \n0.045 \n\nAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n1957 \n1.595 \n0.009 \n\nGQEQWEEGDLYDKEKQQKPF \nE9Q8K5 \nTITIN \n2481 \n1.586 \n0.036 \n\nAGTNGETTTQGLDGLSERCA \nP05064 \nALDOA \n2037 \n1.582 \n0.033 \n\nRVPTPNVSVVDLTCRLEKPAK \nP16858 \nG3P \n2378 \n1.531 \n0.022 \n\nKVPEKPEVVEKVEPAPLK \nF7CR78 \nF7CR78 \n2015 \n1.527 \n0.026 \n\nGETTTQGLDGLSERCAQ \nP05064 \nALDOA \n1822 \n1.526 \n0.042 \n\nDown-regulated peptides \n\nKIEFTPEQIEEF \nP09542 \nMYL3 \n1509 \n0.612 \n0.021 \n\nDLSAIKIEFSKEQQEDF \nP05977 \nMYL1 \n2026 \n0.600 \n0.019 \n\nHELYPIAKGDNQSPI \nP16015 \nCAH3 \n1681 \n0.559 \n0.038 \n\nNSLTGEFKGKY \nP07310 \nKCRM \n1243 \n0.545 \n0.027 \n\nDEPKPYPYPNLDDF \nQ3V1D3 \nAMPD1 \n1709 \n0.543 \n0.034 \n\nASHHPADFTPAVHA \nQ91VB8 \nHBA-A1 \n1457 \n0.537 \n0.016 \n\n(Continued) \n\nFrontiers in Endocrinology | www.frontiersin.org ","\nTABLE 1 |\n1ContinuedPeptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nSQVGDVLRALGTNPTNAE \nQ545G5 \nMYL1 \n1841 \n0.516 \n0.047 \n\nADEIAKAQVAQPGGDTI \nP70349 \nHINT1 \n1725 \n0.515 \n0.038 \n\nDKETPSGFTLDDV \nP07310 \nKCRM \n1423 \n0.478 \n0.037 \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n2654 \n0.467 \n0.038 \n\nPHPYPALTPEQKK \nP05064 \nALDOA \n1505 \n0.407 \n0.028 \n\nQLVVDGVKLM \nP07310 \nKCRM \n1101 \n0.400 \n0.001 \n\nKDLFDPIIQDRHG \nP07310 \nKCRM \n1553 \n0.395 \n0.022 \n\nKGVVPLAGTNGETTTQGLDGLSER \nP05064 \nALDOA \n2399 \n0.367 \n0.015 \n\nRVFDKEGNGTVMGAELR \nQ545G5 \nMYL1 \n1878 \n0.341 \n0.043 \n\nNADEVGGEALGRL \nA8DUK4 \nHBB-BS \n1300 \n0.339 \n0.037 \n\nNEHLGYVLTCPS \nP07310 \nKCRM \n1389 \n0.328 \n0.031 \n\nASHTEEEVSVSVPEVQKKTVTEEK \nE9Q8K5 \nTITIN \n2669 \n0.253 \n0.023 \n\nNEEDHLRV \nP07310 \nKCRM \n1010 \n0.212 \n0.027 \n\nVLTCPSNLGTGLRG \nP07310 \nKCRM \n1444 \n0.201 \n0.018 \n\nGGYKPTDKHKTDL \nP07310 \nKCRM \n1459 \n0.127 \n0.006 \n\nobservation was also demonstrated by other peptidomic studies. \nFrom Steven W. Taylor group' results, several identified peptides \nin the human islet cultures were derived from intracellular and \ncytoskeletal proteins such as microtubule-associated protein 4 \nand ubiquitin, which may result from a greater level of cellular \nstress ","\nTABLE 2 |\n2Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported asPeptide sequence \nProtein \nLocation \nDomain \nDescription \n\nUp-regualted peptides \n\nAVGAVFDISNADRLGSSEVEQ \nKCRM \n329-349 \n125-367 \nPhosphagen kinase C-terminal \n\nTNGAFTGEISPGMIKDLGATWV \nTPIS \n121-142 \n57-295 \nTIM \n\nIADLVVGLCTGQIK \nENOB \n381-394 \n142-432 \nEnolase C \n\nNYKPQEEYPDLSKHNNHMA \nKCRM \n13-31 \n11-98 \nPhosphagen kinase N-terminal \n\nEEIEEDAGLGNGGLGRLAAC \nPYGM \n124-143 \n113-828 \nPhosphorylase \n\nGQCGDVLRALGQNPTQAEV \nMYL3 \n83-101 \n58-95 \nEF-hand 1 \n\nEAFTVIDQNRDGIID \nMLRS \n32-46 \n25-60 \nEF-hand 1 \n\nDLFDPIIQDRHGGY \nKCRM \n87-100 \n11-98 \nPhosphagen kinase N-terminal \n\nKDLFDPIIQD \nKCRM \n86-95 \n11-98 \nPhosphagen kinase N-terminal \n\nLDDVIQTGVDNPGHP \nKCRM \n53-67 \n11-98 \nPhosphagen kinase N-terminal \n\nVIACIGEKLDE \nTPIS \n174-184 \n57-295 \nTIM \n\nGTNPTNAEVKKVLGNPSNEEM \nMYL1 \n39-59 \n6-41 \nEF-hand \n\nADRLGSSEVEQVQLV \nKCRM \n339-353 \n125-367 \nPhosphagen kinase C-terminal \n\nKADDGRPFPQVI \nALDOA \n87-98 \n15-364 \nGlycolytic \n\nKSMTEQEQQQLIDD \nKCRM \n177-190 \n125-367 \nPhosphagen kinase C-terminal \n\nGGGASLELLEGKVLPGVDA \nPGK1 \n395-413 \n9-406 \nPGK \n\nLEGTLLKPNMVTPGHA \nALDOA \n224-239 \n15-364 \nGlycolytic \n\nVMVGMGQKDSYVGDEAQSK \nACTS \n45-63 \n4-377 \nActin \n\nDLEEATLQHEATAAALR \nMYH6 \n1,179-1,195 \n845-1,926 \nMyosin_tail_1 \n\nSEGKCAELEEELKTVTNNL \nTPM1 \n186-204 \n1-284 \nCoiled coili \n\nRSIKGYTLPPHC \nKCRM \n135-146 \n125-367 \nPhosphagen kinase C-terminal \n\nSNNKDQGGYEDFVEGLRV \nMYL1 \n77-94 \n83-118 \nEF-hand \n\nAEQIKHILANF \nTCTP \n119-129 \n1-172 \nTCTP \n\nGADPEDVITGAFK \nMLRS \n93-105 \n95-130 \nEF-hand 2 \n\nLKGGDDLDPNYVLS \nKCRM \n115-128 \n125-367 \nPhosphagen kinase C-terminal \n\nGQEQWEEGDLYDKEKQQKPF \nTITIN \n1,691-1,710 \n1,709-1,799 \nIg-like \n\nAGTNGETTTQGLDGLSERCA \nALDOA \n117-136 \n15-364 \nGlycolytic \n\nRVPTPNVSVVDLTCRLEKPAK \nGAPDH \n232-252 \n243-248 \n[IL]-x-C-x-x-[DE] motif \n\nGETTTQGLDGLSERCAQ \nALDOA \n121-137 \n15-364 \nGlycolytic \n\nDown-regualted peptides \n\nKIEFTPEQIEEF \nMYL3 \n52-63 \n58-95 \nEF-hand 1 \n\nDLSAIKIEFSKEQQEDF \nMYL1 \n33-49 \n44-79 \nEF-hand 1 \n\nHELYPIAKGDNQSPI \nCAH3 \n17-31 \n3-259 \nAlpha-carbonic anhydrase \n\nNSLTGEFKGKY \nKCRM \n163-173 \n125-367 \nPhosphagen kinase C-terminal \n\nASHHPADFTPAVHA \nHBA-A1 \n111-124 \n3-142 \nGLOBIN \n\nDKETPSGFTLDDV \nKCRM \n44-56 \n11-98 \nPhosphagen kinase N-terminal \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nMYL1 \n76-100 \n83-118 \nEF-hand \n\nQLVVDGVKLM \nKCRM \n351-360 \n125-367 \nPhosphagen kinase C-terminal \n\nKDLFDPIIQDRHG \nKCRM \n86-98 \n11-98 \nPhosphagen kinase N-terminal \n\nKGVVPLAGTNGETTTQGLDGLSER \nALDOA \n111-134 \n15-364 \nGlycolytic \n\nRVFDKEGNGTVMGAELR \nMYL1 \n147-163 \n133-150 \nEF-hand \n\nNADEVGGEALGRL \nHBB-BS \n20-32 \n2-147 \nGLOBIN \n\nNEHLGYVLTCPS \nKCRM \n274-285 \n125-367 \nPhosphagen kinase C-terminal \n\nNEEDHLRV \nKCRM \n230-237 \n125-367 \nPhosphagen kinase C-terminal \n\nVLTCPSNLGTGLRG \nKCRM \n280-293 \n125-367 \nPhosphagen kinase C-terminal \n\n","\nTABLE 3 |\n3Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.Protein name \nDescription \nPeptide number \nAssociation score \nwith obesity # \n\nAssociation score with \ndiabetes mellitus # \n\nTPI1 \nTriosephosphate isomerase \n2 \n0.012 \n0.012 \n\nKCRM \nCreatine kinase M-type \n17 \n0.022 \n0.017 \n\nTTN \nTitin \n1 \n0.026 \n0.063 \n\nGAPDH \nGlyceraldehyde-3-phosphate dehydrogenase \n1 \n0.071 \n0.117 \n\nHBA-A1 \nAlpha globin 1 \n1 \n0.014 \n0.038 \n\nENOB \nBeta-enolase \n1 \n0.040 \n0.048 \n\nCAH3 \nCarbonic anhydrase 3 \n1 \n0.074 \n0.094 \n\nTCTP \nTranslationally-controlled tumor protein \n1 \n0.028 \n0.040 \n\nHBB-BS \nBeta-globin \n1 \n0.008 \n0.020 \n\nACTS \nActin, alpha skeletal muscle \n1 \n0.006 \n0.188 \n\n# ","\nTable S4 |\nS4The matching results of the precursor proteins from STRING database.","\nTable S5 |\nS5Precursor proteins-to-precursor proteins interaction from STRING database.","\nTable S6 |\nS6The information of candidate peptides for functional evaluation."],"string":"[\n \"\\n\\nAt the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.\",\n \"\\nFIGURE 1 |\\n1Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.\",\n \"\\nFIGURE 2 |\\n2Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.\",\n \"\\nFIGURE 3 |\\n3Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)\",\n \"\\nFIGURE 4 |\\n4Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.\",\n \"\\nFIGURE 5 |\\n5Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.\",\n \"\\n\\nThe up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in\",\n \"\\n\\n413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),\",\n \"\\nFUNDING\\nThis study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science\",\n \"\\nFigure S2 |\\nS2Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.\",\n \"\\nTABLE 1 |\\n1Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.Peptide sequence \\nProtein ID \\nProtein name \\nMr(KDa) \\nFold change \\nP-value \\n\\nUp-regulated peptides \\n\\nAVGAVFDISNADRLGSSEVEQ \\nP07310 \\nKCRM \\n2163 \\n9.728 \\n0.005 \\n\\nTNGAFTGEISPGMIKDLGATWV \\nP17751 \\nTPIS \\n2264 \\n8.019 \\n0.035 \\n\\nIADLVVGLCTGQIK \\nP21550 \\nENOB \\n1486 \\n6.77 \\n0.000 \\n\\nNYKPQEEYPDLSKHNNHMA \\nP07310 \\nKCRM \\n2315 \\n4.843 \\n0.024 \\n\\nEEIEEDAGLGNGGLGRLAAC \\nQ9WUB3 \\nPYGM \\n2030 \\n4.411 \\n0.004 \\n\\nGQCGDVLRALGQNPTQAEV \\nP09542 \\nMYL3 \\n2013 \\n4.077 \\n0.033 \\n\\nAGPLCLQEVDEPPQHAL \\nP70296 \\nPEBP1 \\n1873 \\n4.038 \\n0.004 \\n\\nEAFTVIDQNRDGIID \\nP97457 \\nMLRS \\n1705 \\n3.947 \\n0.026 \\n\\nDLFDPIIQDRHGGY \\nP07310 \\nKCRM \\n1645 \\n3.668 \\n0.022 \\n\\nKDLFDPIIQD \\nP07310 \\nKCRM \\n1203 \\n3.282 \\n0.000 \\n\\nLDDVIQTGVDNPGHP \\nP07310 \\nKCRM \\n1576 \\n3.265 \\n0.001 \\n\\nSEIIDVSSKAEEVK \\nA2ASS6 \\nTITIN \\n1533 \\n3.134 \\n0.002 \\n\\nVIACIGEKLDE \\nP17751 \\nTPIS \\n1246 \\n2.976 \\n0.003 \\n\\nGTNPTNAEVKKVLGNPSNEEM \\nQ545G5 \\nMYL1 \\n2180 \\n2.838 \\n0.006 \\n\\nADRLGSSEVEQVQLV \\nP07310 \\nKCRM \\n1629 \\n2.804 \\n0.001 \\n\\nIGQGTPIPDLPEVKRV \\nA2AQA9 \\nNEB \\n1719 \\n2.739 \\n0.002 \\n\\nSTTAPPIQSPLPVIPHQK \\nE9PYJ9 \\nLDB3 \\n1910 \\n2.685 \\n0.023 \\n\\nKADDGRPFPQVI \\nP05064 \\nALDOA \\n1342 \\n2.644 \\n0.021 \\n\\nKSMTEQEQQQLIDD \\nP07310 \\nKCRM \\n1692 \\n2.442 \\n0.001 \\n\\nGGGASLELLEGKVLPGVDA \\nP09411 \\nPGK1 \\n1781 \\n2.264 \\n0.028 \\n\\nLEGTLLKPNMVTPGHA \\nP05064 \\nALDOA \\n1677 \\n2.208 \\n0.010 \\n\\nVMVGMGQKDSYVGDEAQSK \\nP68134 \\nACTS \\n2028 \\n2.168 \\n0.021 \\n\\nDLEEATLQHEATAAALR \\nQ02566 \\nMYH6 \\n1838 \\n2.07 \\n0.038 \\n\\nAELQEVQITEEKPLLPG \\nQ9QYG0 \\nNDRG2 \\n1935 \\n2.043 \\n0.048 \\n\\nSEGKCAELEEELKTVTNNL \\nP58771 \\nTPM1 \\n2163 \\n2.038 \\n0.049 \\n\\nRSIKGYTLPPHC \\nP07310 \\nKCRM \\n1428 \\n1.967 \\n0.001 \\n\\nNSNSHSSTFDAGAGIALNDNFVK \\nP16858 \\nG3P \\n2365 \\n1.935 \\n0.038 \\n\\nTGKATSEASVSTPEETAPEPAKVPT \\nP70402 \\nMYBPH \\n2484 \\n1.919 \\n0.003 \\n\\nDNEYGYSNRVVDL \\nP16858 \\nG3P \\n1543 \\n1.898 \\n0.004 \\n\\nEELDAMMKEASGPINF \\nP97457 \\nMLRS \\n1781 \\n1.853 \\n0.012 \\n\\nSNNKDQGGYEDFVEGLRV \\nQ545G5 \\nMYL1 \\n2026 \\n1.816 \\n0.016 \\n\\nAEQIKHILANF \\nP63028 \\nTCTP \\n1283 \\n1.787 \\n0.044 \\n\\nGADPEDVITGAFK \\nP97457 \\nMLRS \\n1319 \\n1.782 \\n0.031 \\n\\nTSIEDAPITVQSKINQ \\nA2AQA9 \\nNEB \\n1743 \\n1.739 \\n0.028 \\n\\nLKGGDDLDPNYVLS \\nP07310 \\nKCRM \\n1505 \\n1.732 \\n0.040 \\n\\nIQTGVDNPGHPF \\nQ6P8J7 \\nKCRS \\n1281 \\n1.600 \\n0.045 \\n\\nAEVKKVLGNPSNEEMNAK \\nQ545G5 \\nMYL1 \\n1957 \\n1.595 \\n0.009 \\n\\nGQEQWEEGDLYDKEKQQKPF \\nE9Q8K5 \\nTITIN \\n2481 \\n1.586 \\n0.036 \\n\\nAGTNGETTTQGLDGLSERCA \\nP05064 \\nALDOA \\n2037 \\n1.582 \\n0.033 \\n\\nRVPTPNVSVVDLTCRLEKPAK \\nP16858 \\nG3P \\n2378 \\n1.531 \\n0.022 \\n\\nKVPEKPEVVEKVEPAPLK \\nF7CR78 \\nF7CR78 \\n2015 \\n1.527 \\n0.026 \\n\\nGETTTQGLDGLSERCAQ \\nP05064 \\nALDOA \\n1822 \\n1.526 \\n0.042 \\n\\nDown-regulated peptides \\n\\nKIEFTPEQIEEF \\nP09542 \\nMYL3 \\n1509 \\n0.612 \\n0.021 \\n\\nDLSAIKIEFSKEQQEDF \\nP05977 \\nMYL1 \\n2026 \\n0.600 \\n0.019 \\n\\nHELYPIAKGDNQSPI \\nP16015 \\nCAH3 \\n1681 \\n0.559 \\n0.038 \\n\\nNSLTGEFKGKY \\nP07310 \\nKCRM \\n1243 \\n0.545 \\n0.027 \\n\\nDEPKPYPYPNLDDF \\nQ3V1D3 \\nAMPD1 \\n1709 \\n0.543 \\n0.034 \\n\\nASHHPADFTPAVHA \\nQ91VB8 \\nHBA-A1 \\n1457 \\n0.537 \\n0.016 \\n\\n(Continued) \\n\\nFrontiers in Endocrinology | www.frontiersin.org \",\n \"\\nTABLE 1 |\\n1ContinuedPeptide sequence \\nProtein ID \\nProtein name \\nMr(KDa) \\nFold change \\nP-value \\n\\nSQVGDVLRALGTNPTNAE \\nQ545G5 \\nMYL1 \\n1841 \\n0.516 \\n0.047 \\n\\nADEIAKAQVAQPGGDTI \\nP70349 \\nHINT1 \\n1725 \\n0.515 \\n0.038 \\n\\nDKETPSGFTLDDV \\nP07310 \\nKCRM \\n1423 \\n0.478 \\n0.037 \\n\\nLGTNPTNAEVKKVLGNPSNEEMNAK \\nQ545G5 \\nMYL1 \\n2654 \\n0.467 \\n0.038 \\n\\nPHPYPALTPEQKK \\nP05064 \\nALDOA \\n1505 \\n0.407 \\n0.028 \\n\\nQLVVDGVKLM \\nP07310 \\nKCRM \\n1101 \\n0.400 \\n0.001 \\n\\nKDLFDPIIQDRHG \\nP07310 \\nKCRM \\n1553 \\n0.395 \\n0.022 \\n\\nKGVVPLAGTNGETTTQGLDGLSER \\nP05064 \\nALDOA \\n2399 \\n0.367 \\n0.015 \\n\\nRVFDKEGNGTVMGAELR \\nQ545G5 \\nMYL1 \\n1878 \\n0.341 \\n0.043 \\n\\nNADEVGGEALGRL \\nA8DUK4 \\nHBB-BS \\n1300 \\n0.339 \\n0.037 \\n\\nNEHLGYVLTCPS \\nP07310 \\nKCRM \\n1389 \\n0.328 \\n0.031 \\n\\nASHTEEEVSVSVPEVQKKTVTEEK \\nE9Q8K5 \\nTITIN \\n2669 \\n0.253 \\n0.023 \\n\\nNEEDHLRV \\nP07310 \\nKCRM \\n1010 \\n0.212 \\n0.027 \\n\\nVLTCPSNLGTGLRG \\nP07310 \\nKCRM \\n1444 \\n0.201 \\n0.018 \\n\\nGGYKPTDKHKTDL \\nP07310 \\nKCRM \\n1459 \\n0.127 \\n0.006 \\n\\nobservation was also demonstrated by other peptidomic studies. \\nFrom Steven W. Taylor group' results, several identified peptides \\nin the human islet cultures were derived from intracellular and \\ncytoskeletal proteins such as microtubule-associated protein 4 \\nand ubiquitin, which may result from a greater level of cellular \\nstress \",\n \"\\nTABLE 2 |\\n2Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported asPeptide sequence \\nProtein \\nLocation \\nDomain \\nDescription \\n\\nUp-regualted peptides \\n\\nAVGAVFDISNADRLGSSEVEQ \\nKCRM \\n329-349 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nTNGAFTGEISPGMIKDLGATWV \\nTPIS \\n121-142 \\n57-295 \\nTIM \\n\\nIADLVVGLCTGQIK \\nENOB \\n381-394 \\n142-432 \\nEnolase C \\n\\nNYKPQEEYPDLSKHNNHMA \\nKCRM \\n13-31 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nEEIEEDAGLGNGGLGRLAAC \\nPYGM \\n124-143 \\n113-828 \\nPhosphorylase \\n\\nGQCGDVLRALGQNPTQAEV \\nMYL3 \\n83-101 \\n58-95 \\nEF-hand 1 \\n\\nEAFTVIDQNRDGIID \\nMLRS \\n32-46 \\n25-60 \\nEF-hand 1 \\n\\nDLFDPIIQDRHGGY \\nKCRM \\n87-100 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nKDLFDPIIQD \\nKCRM \\n86-95 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nLDDVIQTGVDNPGHP \\nKCRM \\n53-67 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nVIACIGEKLDE \\nTPIS \\n174-184 \\n57-295 \\nTIM \\n\\nGTNPTNAEVKKVLGNPSNEEM \\nMYL1 \\n39-59 \\n6-41 \\nEF-hand \\n\\nADRLGSSEVEQVQLV \\nKCRM \\n339-353 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nKADDGRPFPQVI \\nALDOA \\n87-98 \\n15-364 \\nGlycolytic \\n\\nKSMTEQEQQQLIDD \\nKCRM \\n177-190 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nGGGASLELLEGKVLPGVDA \\nPGK1 \\n395-413 \\n9-406 \\nPGK \\n\\nLEGTLLKPNMVTPGHA \\nALDOA \\n224-239 \\n15-364 \\nGlycolytic \\n\\nVMVGMGQKDSYVGDEAQSK \\nACTS \\n45-63 \\n4-377 \\nActin \\n\\nDLEEATLQHEATAAALR \\nMYH6 \\n1,179-1,195 \\n845-1,926 \\nMyosin_tail_1 \\n\\nSEGKCAELEEELKTVTNNL \\nTPM1 \\n186-204 \\n1-284 \\nCoiled coili \\n\\nRSIKGYTLPPHC \\nKCRM \\n135-146 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nSNNKDQGGYEDFVEGLRV \\nMYL1 \\n77-94 \\n83-118 \\nEF-hand \\n\\nAEQIKHILANF \\nTCTP \\n119-129 \\n1-172 \\nTCTP \\n\\nGADPEDVITGAFK \\nMLRS \\n93-105 \\n95-130 \\nEF-hand 2 \\n\\nLKGGDDLDPNYVLS \\nKCRM \\n115-128 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nGQEQWEEGDLYDKEKQQKPF \\nTITIN \\n1,691-1,710 \\n1,709-1,799 \\nIg-like \\n\\nAGTNGETTTQGLDGLSERCA \\nALDOA \\n117-136 \\n15-364 \\nGlycolytic \\n\\nRVPTPNVSVVDLTCRLEKPAK \\nGAPDH \\n232-252 \\n243-248 \\n[IL]-x-C-x-x-[DE] motif \\n\\nGETTTQGLDGLSERCAQ \\nALDOA \\n121-137 \\n15-364 \\nGlycolytic \\n\\nDown-regualted peptides \\n\\nKIEFTPEQIEEF \\nMYL3 \\n52-63 \\n58-95 \\nEF-hand 1 \\n\\nDLSAIKIEFSKEQQEDF \\nMYL1 \\n33-49 \\n44-79 \\nEF-hand 1 \\n\\nHELYPIAKGDNQSPI \\nCAH3 \\n17-31 \\n3-259 \\nAlpha-carbonic anhydrase \\n\\nNSLTGEFKGKY \\nKCRM \\n163-173 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nASHHPADFTPAVHA \\nHBA-A1 \\n111-124 \\n3-142 \\nGLOBIN \\n\\nDKETPSGFTLDDV \\nKCRM \\n44-56 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nLGTNPTNAEVKKVLGNPSNEEMNAK \\nMYL1 \\n76-100 \\n83-118 \\nEF-hand \\n\\nQLVVDGVKLM \\nKCRM \\n351-360 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nKDLFDPIIQDRHG \\nKCRM \\n86-98 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nKGVVPLAGTNGETTTQGLDGLSER \\nALDOA \\n111-134 \\n15-364 \\nGlycolytic \\n\\nRVFDKEGNGTVMGAELR \\nMYL1 \\n147-163 \\n133-150 \\nEF-hand \\n\\nNADEVGGEALGRL \\nHBB-BS \\n20-32 \\n2-147 \\nGLOBIN \\n\\nNEHLGYVLTCPS \\nKCRM \\n274-285 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nNEEDHLRV \\nKCRM \\n230-237 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nVLTCPSNLGTGLRG \\nKCRM \\n280-293 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\n\",\n \"\\nTABLE 3 |\\n3Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.Protein name \\nDescription \\nPeptide number \\nAssociation score \\nwith obesity # \\n\\nAssociation score with \\ndiabetes mellitus # \\n\\nTPI1 \\nTriosephosphate isomerase \\n2 \\n0.012 \\n0.012 \\n\\nKCRM \\nCreatine kinase M-type \\n17 \\n0.022 \\n0.017 \\n\\nTTN \\nTitin \\n1 \\n0.026 \\n0.063 \\n\\nGAPDH \\nGlyceraldehyde-3-phosphate dehydrogenase \\n1 \\n0.071 \\n0.117 \\n\\nHBA-A1 \\nAlpha globin 1 \\n1 \\n0.014 \\n0.038 \\n\\nENOB \\nBeta-enolase \\n1 \\n0.040 \\n0.048 \\n\\nCAH3 \\nCarbonic anhydrase 3 \\n1 \\n0.074 \\n0.094 \\n\\nTCTP \\nTranslationally-controlled tumor protein \\n1 \\n0.028 \\n0.040 \\n\\nHBB-BS \\nBeta-globin \\n1 \\n0.008 \\n0.020 \\n\\nACTS \\nActin, alpha skeletal muscle \\n1 \\n0.006 \\n0.188 \\n\\n# \",\n \"\\nTable S4 |\\nS4The matching results of the precursor proteins from STRING database.\",\n \"\\nTable S5 |\\nS5Precursor proteins-to-precursor proteins interaction from STRING database.\",\n \"\\nTable S6 |\\nS6The information of candidate peptides for functional evaluation.\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["At the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.","Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.","Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.","Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)","Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.","Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.","The up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in","413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),","This study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science","Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.","Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.","Continued","Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported as","Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.","The matching results of the precursor proteins from STRING database.","Precursor proteins-to-precursor proteins interaction from STRING database.","The information of candidate peptides for functional evaluation."],"string":"[\n \"At the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.\",\n \"Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.\",\n \"Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.\",\n \"Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)\",\n \"Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.\",\n \"Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.\",\n \"The up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in\",\n \"413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),\",\n \"This study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science\",\n \"Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.\",\n \"Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.\",\n \"Continued\",\n \"Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported as\",\n \"Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.\",\n \"The matching results of the precursor proteins from STRING database.\",\n \"Precursor proteins-to-precursor proteins interaction from STRING database.\",\n \"The information of candidate peptides for functional evaluation.\"\n]"},"annotations_figureref":{"kind":"list like","value":["Figure S1","Figures 1A,B","(Figure 2A)","Figure 2B)","Figure 2C","Figure 3A)","Figure 3A","Figure 3B)","Figure 3B","Figure S2","Figure 3C","Figure 3D","Figure 4A)","(Figure 4B","(Figure 4B)","Figures 5A,B,","Figures 5E,F","Figures 5C,D.","Figures 5E,F","Figure 5G","Figure 5H","Figures 1A,B.","(Figures 2A,B)","(Figures 3A,B)","(Figures 3A,B)","Figures 5A,B","Figures 5C,D.","Figures 5E,F","Figures 5E,F,H","Figure S1"],"string":"[\n \"Figure S1\",\n \"Figures 1A,B\",\n \"(Figure 2A)\",\n \"Figure 2B)\",\n \"Figure 2C\",\n \"Figure 3A)\",\n \"Figure 3A\",\n \"Figure 3B)\",\n \"Figure 3B\",\n \"Figure S2\",\n \"Figure 3C\",\n \"Figure 3D\",\n \"Figure 4A)\",\n \"(Figure 4B\",\n \"(Figure 4B)\",\n \"Figures 5A,B,\",\n \"Figures 5E,F\",\n \"Figures 5C,D.\",\n \"Figures 5E,F\",\n \"Figure 5G\",\n \"Figure 5H\",\n \"Figures 1A,B.\",\n \"(Figures 2A,B)\",\n \"(Figures 3A,B)\",\n \"(Figures 3A,B)\",\n \"Figures 5A,B\",\n \"Figures 5C,D.\",\n \"Figures 5E,F\",\n \"Figures 5E,F,H\",\n \"Figure S1\"\n]"},"annotations_formula":{"kind":"list like","value":[],"string":"[]"},"annotations_paragraph":{"kind":"list like","value":["Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis (1). Accordingly, the dysregulation of skeletal muscle metabolism also arise a number of metabolic disorders such as hyperinsulinaemia, excessive hepatic gluconeogenesis (2), abnormal lipid accumulation (3), impaired glucose uptake and metabolic inflexibility (4). Furthermore, disorders in skeletal muscle play a central role in the development of type 2 diabetic mellitus (T2DM), obesity and lead to other related complications (5). Thus, it is of great interest to deeply characterize the pathogenesis of dysregulation on skeletal muscle glucose/lipid homeostasis to whole-body endocrine and metabolic functions.","As an important secretory organ, skeletal muscle has drawn attention to be a potential target tissue for treating metabolic disorders (6). Thus, analysis of skeletal muscle secretome opens up a novel route for comprehending the communication of this tissue with other tissues such as adipose tissue (7), bone (8), liver (9) and pancreas (10,11). In light of recently reported experimental evidence, a variety of proteins generated by muscles fibers and released into the circulation are classified as myokines (12), most of which have autocrine, paracrine, and endocrine effects not only in muscle fiber growth (13) but also systemic metabolism (14). The first identified myokine IL-6 presented a vital locally muscular effects such as skeletal muscle growth and glucose/lipid metabolism (15, 16). Furthermore, IL-6 also could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues (17)(18)(19). Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM (20). These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. However, knowledge of the skeletal muscle secretome is scarcely reported under pathophysiology of metabolic diseases such as T2DM and obesity. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established.","Peptides in length of 3-50 amino acids residues, which are widely characterized in mouse and human, are termed as a sort of compounds produced or secreted by endocrine gland tissues as well as certain types of cells (21,22). And Abbreviations: T2DM, type 2 diabetic mellitus; GO, gene ontology; KCRM, creatine kinase m-type; Aldo A, fructose-bisphosphate aldolase A; Mr, relative molecular mass; pI, isoelectric point; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FA, formic acid; DDA, data dependent acquisition; FDR, false discovery rate; PSPEP, Proteomics System Performance Evaluation Pipeline; RIPA, Radio Immunoprecipitation Assay; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; TPIS, triosephosphate isomerase; ENOB, beta-enolase; PYGM, glycogen phosphorylase, muscle form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; MLRS, myosin regulatory light chain 2, skeletal muscle isoform; MYH6, myosin heavy chain 6; MYH7, myosin heavy chain 7; MYBPH, myosin-binding protein H; MYL3, myosin light chain 3; MYL1; LDH, myosin light chain 1; lactate dehydrogenase. these endogenous peptides have important physiological action, including neuroregulation (23), cell differentiation (24) and energy metabolism (25) and dysregulation of peptide hormone signaling have been implicated in a wide range of diseases (26,27). In view of that, further insight into identification of novel peptides is of major importance. Benefited from the progresses in peptide extraction method and application of modern analytical methods, (U)HPLC, nano-LC and CE, hyphenated with tandem mass spectrometry (MS/MS) technology (28,29), various techniques used for quantitative peptidomics have been applied to address the challenging question of identifying peptides with potential bioactive under the physiological or disease condition (30). More importantly, the peptidomics is widely used to identify biological markers (31), discover new drug (32) and therapeutic targets (33). Recently, quantative peptidomics has also been conducted in endocrine studies (22,34,35), however, the secreted peptidomics from skeletal muscle under the insulin-resistant condition was not fully characterized.","Herein, we performed liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to help characterize the secretome from cultured skeletal muscle tissues of db/db mice at peptides level and identify putative bioactive peptides. A global secreted peptides were established and bioinformatics analysis of precursor proteins provided a possible relationship of differential peptides with T2DM or insulin resistance. Additionally, the biological effects of these secreted peptides on C2C12 myotubes elucidated a possible regulatory role in insulin signaling-and mitochondrial-related genes expression. Taken together, these observations will encourage us to investigate function of these secreted peptides from cultured skeletal muscle tissues with other tissues under the diabetic state, thus representing a promising strategy for prevention and treatment of insulin resistance as well as the associated metabolic disorders.","All the studies involving mice acquired approval from the Ethical Committee of Nanjing Medical University. All procedure involving mice were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval Number: IACUC-1812053).","Twelve-week-old male C57BLKS/J db/db mice (n = 8, db/db group) and age-matched WT controls (n = 8, NC group) were purchased from the Model Animal Research Center of Nanjing Medical University. After adaptive raising for one week, mice were sacrificed by cervical dislocation and skeletal muscle tissues were isolated from the left hind leg (each mice of 100 ∼ 150 mg). Subsequent operations were carried out under a laminar airflow hood to decrease contamination. The visible blood vessels and connective tissue were removed from the tissue. After rinsed with PBS, the skeletal muscle tissues were cut into small pieces (3-4 mm 3 ) with scissors as described by an established protocol (36,37). Tissue cutting will lead to release of damaged cells slowly into the medium. Additionally, a small amount of serum proteins in the tissue pieces will diffuse out during culture period. Therefore, necessary washing procedures during culture were adopted to obtain medium samples (referred to as secretome) containing mainly skeletal muscle tissue-derived secreted components as previously reported (38,39). Tissue fragments were placed in a 10 cm plate (200∼300 mg from two mice as one sample) containing 10 mL serum/phenol red free DMEM/F12 medium (Gibco, Grand Island, CA, USA). After incubation in a humidified incubator at 37 • C under 95% O2 and 5% CO2 for 48 h, the medium was immediately supplemented with protease inhibitors and centrifuged (845 g, 10 min, 4 • C) to wipe off cell debris and dead cell. Supernatant samples from each group (NC or db/db group) were harvested from four individual tissue culture dishes independently (n = 4 per group). Lactate dehydrogenase (LDH) (36,40,41) and IL-6 (42) expression levels were detected to evaluate skeletal muscle vitality and capability during the in vitro culture. Then the supernatant samples obtained were stored at −80 • C until further processing.","First, both supernatant samples were concentrated to 1-2 mL by centrifuges for speed vacuum (LaboGene, Allerød, Denmark). Then equal volume of U2 solution containing 8 M urea and 100 mM tetraethyl-ammonium bromide in pH 8.0 was added to the concentrated supernatant for denaturation. Followed by centrifugation for 30 min (13,000 g, 30 • C), the medium supernatant was transferred to a new centrifuge tube. Subsequently, proteins were reduced by 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide successively. The protein concentrations of supernatant from cultured skeletal muscle samples were detected by Bradford method (43) and integrity of these samples were evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) combined with silver staining. Afterwards the peptides were separated from samples by treatment with Amicon R Ultra Centrifugal Filters in 10-kDa (Merck Millipore, Billerica, MA, USA) according to the manufacturer's instruction as previously described (37). The \"peptidome\" present in the filtrates was desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and the desalted peptide solution was vacuum-dried with centrifuges for speed vacuum (SCAN SPEED 40, LaboGene) as previously described (44) and immediately frozen at −80 • C until the following processing.","The LC-MS/MS analysis was conducted similarly to the previous protocols (45). The peptide samples were redissolved in 2% (v/v) acetonitrile/0.1% formic acid (FA) (v/v) and 5 µL solution was injected into an A Triple TOFTM 5600 mass spectrometer (AB Sciex, Redwood City, CA, USA) coupled to a ekspert TM nanoLC400 liquid chromatography (AB Sciex) via a nanosource electrospray interface equipped with distal coated SilicaTip emitters (New Objective, Woburn, MA, USA). First, load peptide onto a C18 trap column (5 µm, 100 µm × 20 mm, AB Sciex) and elute at 300 nL/min onto a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Shanghai, China) in the gradient as long as 120 min. These two mobile phases included buffer A containing 2% acetonitrile/ 0.1% FA/ 98% H2O (v/v) and buffer B containing 98% acetonitrile/0.1% FA/2% H2O (v/v). Then peptide mixture was eluted at a flow rate of 0.3 µL/min in a gradient generated with Solvent A containing 98% water and 2% acetonitrile containing 0.1% FA (v/v) and Solvent B containing 2% water and 98% acetonitrile containing 0.1% FA (v/v) according to the previously described reports (45,46). The mass spectrometer was operated in positive mode with a spray voltage of 2500 V, 206.84kPa for the curtain gas, 41.37kPa for the nebulizer gas and 150 • C as temperature. A data dependent acquisition (DDA) method was applied and a full scan MS spectrum (300-1500 m/z) with accumulation time of 0.25 s was adopted. Top 30 precursor ions for fragmentation based on the highest intensity were selected. The collections of MS1 spectra were in the range 350-1500 m/z, and MS2 spectra were in the range of 100-1500 m/z. A total of 47,709 MS/MS were collected from all LC-MS/MS analyses.","Protein Pilot Software (https://sciex.com.cn/products/software/ proteinpilot-software, version4.5,AB SCIEX) was adopted to analyze the original MS/MS file data (47). For peptides identification, the Paragon algorithm was employed against the Mus_musculus SwissProt sequence database (a total of 85210 items, updated in January 2019). The following parameters were installed: The parameters were set as follows: 1) cysteine modified with iodoacetamide; 2) biological modifications were selected as ID focus. The strategy of the automatic decoy database search was employed to estimate false discovery rate (FDR) calculation using the Proteomics System Performance Evaluation Pipeline (PSPEP) Software integrated in protein pilot-software. Only unique peptides (global FDR values < 1%) were considered for further analysis. Skyline v4.2 software was employed for MS1 filtering and ion chromatogram extractions for peptides label-free quantification (48,49). And the parameters setting for skyline MS1 filtering were according to previous studies (48). Using the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.","The relative molecular mass and isoelectric point of each peptide were calculated by the online tool (http://web.expasy. org/compute.pi/). All the precursors protein of differentially expressed peptides as one group were imported into for GO (http://www.geneontology.org/) (50) and pathway analysis (https://www.kegg.jp/) for predicting potential functions. The online tools UniProt Database (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/) were adopted to explore if the peptides' sequences were positioned in the conserved structural domains or regions of their precursors. The Open Targets Platform database (www.targetvalidation.org/) was adopted to investigate precursors associated with diabetes and obesity as previously reported (24). For visualization, clustergram and volcano plot graphs in this study were drawn with R language (http://www.r-project.org/). For determination of differentially expressed peptides, fold change was computed as the average values of biological duplication (n = 4).","All the peptides used in this study were custom-synthesized and HPLC-purified by Science Peptide Biological Technology Co., Ltd. (Shanghai, China) through the solid-phase method as described reported (51). The purity in 95% for each peptide was confirmed by HPLC-MS method. All the used peptides were stored in lyophilization at−20 • C until dissolved with sterile water immediately for treatment with cells in vitro.","C2C12 cells, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China) at 37 • C with 5% CO2. On the fourth day of cell differentiation, the C2C12 cells were pre-treated with synthesized peptides or solvent for 48 h at the same concentration of 50 µM, and then starved for 24 h with serumfree L-DMEM (Gibco). Subsequently, myotubes were incubated in L-DMEM in the presence or absence of 100 nM of insulin for 30 min. At the indicated time, the cells were collected for the following analysis. ","Total RNA was isolated using trizol reagent (Life Technologies, Carlsbad, CA, USA). And the cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was determined by real-time quantitative PCR (ABI ViiA7, Applied Biosystems, Foster City, California, USA) using the SYBR Green array. The relative gene expression was analyzed based on the 2 − CT method with normalization of the data to PPIA. The primers used for the real-time quantitative PCR were listed in Table S1.","Data were analyzed with GraphPad Prism 7 (San Diego, CA, USA), and the results were shown as the mean ± standard deviation (SD). Peptides with a fold change larger than 1.5 or <0.67 with a Student's t-test p-value <0.05 were selected as differently expressed peptides.","Considering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity. LDH and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period. LDH content of culture medium was assessed as an indicator of cell lysis at 0 h∼24 h, 24 h∼48 h and 48 h∼72 h; no significant increased in LDH occurred from 48 h to 72 h (not shown in the manuscript). Secretion of IL-6 remained stable from 24 h to 48 h in culture by ELISA (not shown in the manuscript). Therefore, we chose the 24 h∼48 h culture as an optimal time point. The protein composition and integrity of the secreted samples were evaluated by SDS-PAGE with silver staining in Figure S1. After validation of skeletal muscle culture system and samples assessment, peptides from four different culture dishes per group (control and db/db mice) were individually extracted from supernatants and analyzed via label-free mass spectrometry based quantification. To gain more insights into biological differences between control and db/db mice skeletal muscle, we employed LC-MS/MS technology and identified 3384 peptides, of which 2664 peptides showed valid quantitative values in the detection. A total of 63 peptides were identified differentially secreted, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 peptides were down-regulated (fold change < 0.67, P < 0.05) in medium supernatants from insulin resistant-skeletal muscle tissues. Visualization methods such as hierarchical clustering and volcano plot showed distinct peptides secretion profiles as shown in Figures 1A,B. The differentially secreted peptides were summarized in Table 1.","To characterize the general feature of differentially secreted peptides, we further analyzed the relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs. pI. The results indicated that the Mr varied from 1011.1 Da to 2670.9 Da with 70% between 1,300 and 2,100 Da (Figure 2A), and pI varied from 3.7 to 9.7, with 51% between 4 and 7 ( Figure 2B). Additionally, distribution of pI vs. Mr could divide these peptides into three groups around pI4, pI6 and pI10 ( Figure 2C).","To make comprehensive functional assessment of these differential peptides, we consider all the precursor proteins as one group and performed GO and Pathways analysis of them. First of all, the precursor proteins of these differential peptides identified from the cultured control and db/db skeletal muscle were classified using Gene Ontology categories, which revealed the majority of proteins were associated with striated muscle contraction and glucose metabolic process ( Figure 3A). The top 20 GO terms were listed in Figure 3A. The precursor proteins annotated involved in these GO terms were presented in Table S2. Subsequent pathway analysis in the KEGG database revealed a significant enrichment in metabolic pathway and glycolysis/gluconeogenesis process ( Figure 3B). The top 14 pathway terms were listed in Figure 3B. The precursor proteins annotated involved in these pathway terms were presented in Table S3. We further discriminated the up-and down-regulated peptides and performed GO and Pathways analysis of these two groups of precursor proteins. This analysis may bring overlapping terms such as phosphorylation, phosphagen metabolic process and phosphorus metabolic process in up-or down-regulated peptides, and a smaller number of Search tool for the retrieval of interacting genes/proteins (STRING) was used to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides. The size of node represents the number of interacting proteins and the color of node represents the bitscore of matching results from STRING database. The thicker the line (edge), the higher the reliability (evaluated by combined score >0.4).","terms in Pathway analysis (deriving up-regulated peptides). These analysis were presented in Figure S2. Additionally, the major intracellular locations of the precursor proteins were considered from literature sources, illustrating that 64% proteins were cytoplasmic and 15% proteins were cytoskeletal seen in Figure 3C. Based on above analysis, we found that a vast of precursor proteins were assigned to glucose metabolic process include ALDO A, triosephosphate isomerase (TPIS), beta-enolase (ENOB), glycogen phosphorylase, muscle form (PYGM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1(PGK1), specifically assigned to glycolysis/gluconeogenesis process. Another kind of proteins enriched in regulation of striated muscle contraction terms (GO) and regulation of actin cytoskeleton (Pathway) were myosin regulatory light chain 2, skeletal muscle isoform(MLRS), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin-binding protein H (MYBPH) and myosin light chain 3 (MYL3),both belonging to sarcomeric proteins. Subsequently, we used search tool for the retrieval of interacting genes/proteins (STRING) to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides in Figure 3D. Based on protein-protein interaction and co-occurrence in KEGG pathways and literature mining, this network was constructed from 25 proteins that matched to the STRING database and the matching results were listed in Table S4. As shown from this network, 22 nodes representing precursor protein constituted the interaction diagram, which could form 93 reliable a-to-b interaction relationship by combined score (more than 0.4) as shown in Table S5. ","It is widely postulated that most peptides can be attributed mainly to the proteolytic enzymes as well as their type and level (cleavage specificity and activity), which can differ during disease (52)(53)(54). Briefly, the N-terminal pre-cleavage site, N terminus, C terminus, and C-terminal post-cleavage site of the identified peptides were commonly used to investigate the nature of proteolytic enzymes within serum (55) or tissues (56). Thus, we analyzed the distribution of peptide cleavage site and found that Lysine (K) was the most frequent cleavage site of N-terminal amino acid of identified peptides and Alanine (A) was the most frequent cleavage site of C-terminal amino acid of identified peptides. Lysine (K) was the most frequent cleavage site of Nterminal amino acid of preceding peptides, while Leucine (L) was the commonest C-terminal amino acid of preceding peptides ( Figure 4A). These data indicate that the pattern of cleavage sites may represent the specificity of cleavage and activity of proteolytic enzymes under the diabetic condition. Additionally, we also tried to align peptide sequences on the same precursor sequence to construct a \"peptide alignment map\". Searched from our samples, the largest number of identified peptides (n = 1 7) (Figure 4B) originated from KCRM and the second largest number of identified peptides (n = 6) came from ALDO A (Figure 4B), elucidating that these peptides were easily cleaved by certain kind of endogenous enzymes.","To check for specific domain structures or patterns in the identified peptides comparison with its precursor proteins, we retrieved domain information from the UniProt and Pfam database. In order to validate that if the peptides exerted important roles in the related metabolic diseases, we adopted the Open Targets Platform database to investigate protein precursors. In our searching results, most peptides (n = 44) from the identified pepetides (n = 63) were located in the functional domains ( Table 2). Additionally, 27 out of 44 peptides located in the functional domain were predicted closely related with both obesity and diabetes ( Table 3). These observations will encourage us to further investigate properties of the putative secreted peptides in skeletal muscle under the diabetic state.","To explicit the putative function of differentially secreted peptides, 7 differentially secreted peptides, which had already been annotated derived from authentic protein from Uniprot database, located in the functional motifs and showed relatively high abundance from MS detection, were chosen for further analysis. Table S6. Sequentially, we termed these candidated peptides P1-P7 in order. To use C2C12 myotubes as cell models in vitro, the results of changes in inuslin signaling revealed that up-regulated peptides P2-P4 addition could both significantly decrease the phosphorylation of Irs1 (Ser-307) and Akt (Ser-473) under insulin stimulation (100 nM, 30 min) as shown in Figures 5A,B, in spite of bringing a modestly up-regulation of p-Irs1 (Ser-307) at the basal status. Moreover, mitochondrial-related marker protein Pgc1α was significantly attenuated in the insulin-stimulated C2C12 cells by up-regulated peptides P1-P4 as shown in Figures 5E,F. Among these downregulated peptides, only peptide 7 presented a promotion on p-Akt (Ser-473) expression level both under the basal and insulin stimulation status as shown in Figures 5C,D. And these up-regulated peptide 1-4 could decrease Pgc1α protein level and down-regulated peptide 6 and 7 administration enhanced Pgc1α protein level by the stimulation of insulin presented in Figures 5E,F. We further evaluated Gut 4 (a key gene for glucose transport) and Pgc1α mRNA levels by peptides treatment upon insulin stimulation. The results also indicated that Peptide 3 and 4 modestly decreased Pgc1αexpression at mRNA levels seen in Figure 5G, while Peptide 6 and 7 significantly up-regulated both Glut4 and Pgc1α expression at mRNA levels seen in Figure 5H. These observations revealed that these candidated peptides (P1-P7) may affect the expressions of insulin signaling or mitochondrial-related genes in skeletal muscle cells.","Emerging as a widely known secrete organ, skeletal muscle tissues have been extensively discussed using a wide range of comparative and quantitative proteomic methods (57). Proteomic scannings of the medium supernatant from skeletal muscle cells have already afford approaches to identify a large number of secreted proteins (58)(59)(60)(61)(62)(63). Particularly, one such research reported the alteration of insulin effect on the secretome profile of skeletal muscle cells, revealing the changes of protein levels secreted from skeletal muscle during activation of insulin signaling pathway (58). A recent study also generated a comprehensive secretome analysis of skeletal muscle cells under palmitic acid-induced insulin resistance (61). In addition to these proteins, many other muscle-secreted compounds have been also come to light, including exosome (64,65), metabolites (66), and miRNAs (67). These studies have greatly expanded our knowledge of skeletal muscle secretome and might afford possibilities for exploring novel molecular targets in the maintenance of skeletal muscle physiology and even whole-body metabolism. However, to date few studies has focused on the peptides present in either skeletal muscle tissues or cells. Therefore, we attempted to comprehensively profile peptides that may play roles in regulating insulin sensitivity and offer enormous promise for exploring molecular mechanisms underlying insulin resistance.","In present study, a total of 63 peptides were differentially secreted in medium supernatants from cultured skeletal muscle tissues of db/db mice, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 were down-regulated (fold change < 0.67, P < 0.05) shown in Figures 1A,B. The differences of dysregulation peptides may indeed reflect the changes between control and insulin-resistant mice in some extent. However, db/db mouse is a well-established leptin receptordeficient animal model (68,69). Despite no studies revealed the association between leptin receptor deficiency and peptide dysregulation/protein degradation so far, the possible effect by this mutation deserves to be taken into account and other nongenetic mice models could be adopted in the future studies. Based on our previous study (70), 10-kDa filters method was used to extract peptides in order to intercept proteins secreted by skeletal muscle to the conditional media. Characterization of the basic feature (Mr and pI) of these identified peptides (Figures 2A,B) only reflected the differences of distribution in the diabetic skeletal muscle tissues, but also proved the reliability of the used peptide extraction method.","Subsequently, GO and Pathway analysis revealed that the precursors protein of these peptides were mostly involved in muscle contraction and metabolism processes (Figures 3A,B). An interesting finding from our study was that most of the differentially identified peptides derived from cytosolic, cytoskeletal and mitochondrial proteins (Figures 3A,B), which differed from the secretory pathway peptides (71). This (34). Similarly, another peptidomic anaylsis of brain slices cultures and media also pointed that vast majority of secreted peptides arose from intracellular proteins (72). There does also exist some evidence that these identified N-or C-terminus protein yielding peptides, rather than internal fragments, raised the possibility that they are produced by selective processing rather than protein degradation (73). Taken together with previous researches, the current results show us meaningful hints that these intracellular peptides may be secreted via nonclassical mechanisms. Actually, another important origin of peptides is proteolytic degradation processes in body fluid under the physiological or pathologically processes. Most regulatory peptides were efficiently degraded by plasma enzymes once secreted into the bloodstream, which exerted anti-diabetic therapeutic function (74,75) or were identified as disease markers (76). Recently, Federico Aletti et al. (77) used protease activity detection and specific enzymes analysis to explain a large presence of circulating peptides under hemorrhagic shock, which gave us a possible way to evaluate peptides origin. Thus, a fundamental validation is whether the proteins-derived peptides are actually secreted from skeletal muscle cells per se or proteolytic degradation and are of biologically active. Among the differentially secreted peptides from cultured skeletal muscle tissues of db/db mice, we found that three peptides derived from GAPDH, four peptides derived from ALDO A and one peptides derived from PYGM were upregulated in the conditional medium from db/db groups as shown in Table 1. As described by Dustin S. Hittel et.al, a global proteomic survey of skeletal muscle revealed a statistically significant up-regulation in glycolytic enzymes GAPDH and ALDO A protein levels in obese/overweight patients (78). Another quantitative protein profile also identified a more abundant levels of GAPDH and PYGM in skeletal muscle from T2DM groups compared with the control groups (79). In addition to pointing the importance of the mitochondrial numbers and impairments under the insulin-resistant states (80)(81)(82), several studies noted that glycolytic capacity is higher in skeletal muscle of patients with T2DM or obesity (83). Notably, stronger changes of peptides derived from sarcomeric proteins such as myosin light chain 1 (MYL1) and MYL3 were also observed in the conditional medium from diabetic muscles as shown in Table 1. MYL1 and MYL3 are representive markers of fast-muscle and slow-muscle respectively, which were both regulated by insulin stimulation (79). And previous studies observed that property of T2DM individuals muscle was shifted to a fast-twitch glycolytic phenotype (84). In fact, deficiency in sarcomeric proteins in skeletal muscle also suggested their importance in skeletal muscle physiologic and pathological processes. On the whole, our results of increased glycolytic enzyme-or sarcomeric proteins-derived peptides secreted from insulin resistant skeletal muscle may support the hypothesis that altered glycolytic capacities or fiber types under the diabetic status contribute to this difference.","Till now, the putative function of these peptides are not clear. Therefore, we evaluated whether these peptides originated from functional domains of the corresponding precursor protein using the UniProt and Pfam database. In our searching results, most peptides (n = 44) from the identified peptides (n = 63) were located in the functional domains as listed in Table 2. Specifically, most peptides derived from functional enzymes were located in the enzymatic activity region, including TPI- TCTP-derived peptide (119-AEQIKHILANF-129) and CAH3-derived peptide . We also found another kind of S-Nitrosylation modifying sites for affecting GAPDH enzymatic activity (90). Therefore, future efforts need to be established for investigating the potential role of peptides on insulin sensitive cells in vitro or whole-body metabolism in vivo.","The above analyses provided a possibility to evaluate the biological effects of these differentially secreted peptides. As widely reported, insulin resistance in skeletal muscle is tightly connected with the deficit in insulin signaling (91). Consequently, the role of phosphorylation of Irs1 and Akt in signaling pathways is very crucial in anti-hyperglycemia and insulin sensitivity (92). In this result, we found these up-regulated peptides 1-4 both exerted a significantly attenuated insulin action in C2C12 cells, evaluated by a decreased level of p-Irs1 (Ser-307) and p-Akt (Ser-473) seen in Figures 5A,B, whereas only one down-regulated peptide P7 could remarkably promote insulin signaling only via Irs1 signaling pathway seen in Figures 5C,D. Peroxisome proliferator-activated receptor γ coactiva-tor-1(Pgc-1), which displays a dominant role through tight modulation of mitochondrial biogenesis and respiration, has also been demonstrated to participate in skeletal muscle insulin signaling and metabolic homeostasis (93). The up-regulated peptides 1-4 also brought out a decreased protein level of Pgc-1α in C2C12 cells as shown in Figures 5E,F, and down-regulated peptide 6 and 7 administration also gave rise to the increased Pgc1α mRNA and protein level in Figures 5E,F,H. Taken together, these selected peptides secreted from db/db mice skeletal muscle presented a promotive or inhibitory effect on insulin and mitochondrialrelated pathways in skeletal muscle cells by an autocrine manner. Notably, peptide 4 (231-RVPTPNVSVVDLTCRLEKPAK−252) derived from GAPDH displayed a most significant inhibitory effect toward these candidate peptides. However, the relationship between GAPDH-derived peptide and its precursor protein is to be determined. On the other hand, more methods need to be employed for the wider cell signaling screen and further research is required to explore the biologic function of skeletal muscle-secreted peptides on adipocytes or liver cells.","To our knowledge, no large-scale quantitative peptidomic analysis has been performed on skeletal muscle to elucidate secreted peptides profiles under the diabetic status. The present study identified and quantified changes with a label-free discovery using LC-MS/MS technology to construct a global secreted peptides picture. Further bioinformatics analysis of precursors comprehensively provided an atlas of peptides that may exist roles in regulating insulin sensitivity. This represented a new perspective toward exploring insulin resistance pathogenesis. Additionally, the detailed biological effects of these secreted peptides on skeletal muscle insulin resistance or cross-talk with other tissues remained to be elucidated in the future study.","The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.","The protocol has been approved by the Institutional Animal Care and Use Committee of Nanjing medical university (Approval Number: IACUC-1812053).","YWu and MH performed experiments and interpreted results of experiments. YWa and YG prepared the figures. XC and PX analyzed the data. CJ and TZ participated discussion. YZ helped write the manuscript with providing assistance. LY conceived and designed experiments, provided funding to regents, and approved final version of manuscript. All authors read and approved the final manuscript. ","The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2019.00741/full#supplementary-material Figure S1 | SDS-PAGE associated with silver staining of the medium supernatants from cultured skeletal muscle tissues (Con vs. db groups). Table S1 | Primers used in this study. Table S2 | GO terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. Table S3 | Pathway terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. "],"string":"[\n \"Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis (1). Accordingly, the dysregulation of skeletal muscle metabolism also arise a number of metabolic disorders such as hyperinsulinaemia, excessive hepatic gluconeogenesis (2), abnormal lipid accumulation (3), impaired glucose uptake and metabolic inflexibility (4). Furthermore, disorders in skeletal muscle play a central role in the development of type 2 diabetic mellitus (T2DM), obesity and lead to other related complications (5). Thus, it is of great interest to deeply characterize the pathogenesis of dysregulation on skeletal muscle glucose/lipid homeostasis to whole-body endocrine and metabolic functions.\",\n \"As an important secretory organ, skeletal muscle has drawn attention to be a potential target tissue for treating metabolic disorders (6). Thus, analysis of skeletal muscle secretome opens up a novel route for comprehending the communication of this tissue with other tissues such as adipose tissue (7), bone (8), liver (9) and pancreas (10,11). In light of recently reported experimental evidence, a variety of proteins generated by muscles fibers and released into the circulation are classified as myokines (12), most of which have autocrine, paracrine, and endocrine effects not only in muscle fiber growth (13) but also systemic metabolism (14). The first identified myokine IL-6 presented a vital locally muscular effects such as skeletal muscle growth and glucose/lipid metabolism (15, 16). Furthermore, IL-6 also could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues (17)(18)(19). Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM (20). These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. However, knowledge of the skeletal muscle secretome is scarcely reported under pathophysiology of metabolic diseases such as T2DM and obesity. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established.\",\n \"Peptides in length of 3-50 amino acids residues, which are widely characterized in mouse and human, are termed as a sort of compounds produced or secreted by endocrine gland tissues as well as certain types of cells (21,22). And Abbreviations: T2DM, type 2 diabetic mellitus; GO, gene ontology; KCRM, creatine kinase m-type; Aldo A, fructose-bisphosphate aldolase A; Mr, relative molecular mass; pI, isoelectric point; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FA, formic acid; DDA, data dependent acquisition; FDR, false discovery rate; PSPEP, Proteomics System Performance Evaluation Pipeline; RIPA, Radio Immunoprecipitation Assay; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; TPIS, triosephosphate isomerase; ENOB, beta-enolase; PYGM, glycogen phosphorylase, muscle form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; MLRS, myosin regulatory light chain 2, skeletal muscle isoform; MYH6, myosin heavy chain 6; MYH7, myosin heavy chain 7; MYBPH, myosin-binding protein H; MYL3, myosin light chain 3; MYL1; LDH, myosin light chain 1; lactate dehydrogenase. these endogenous peptides have important physiological action, including neuroregulation (23), cell differentiation (24) and energy metabolism (25) and dysregulation of peptide hormone signaling have been implicated in a wide range of diseases (26,27). In view of that, further insight into identification of novel peptides is of major importance. Benefited from the progresses in peptide extraction method and application of modern analytical methods, (U)HPLC, nano-LC and CE, hyphenated with tandem mass spectrometry (MS/MS) technology (28,29), various techniques used for quantitative peptidomics have been applied to address the challenging question of identifying peptides with potential bioactive under the physiological or disease condition (30). More importantly, the peptidomics is widely used to identify biological markers (31), discover new drug (32) and therapeutic targets (33). Recently, quantative peptidomics has also been conducted in endocrine studies (22,34,35), however, the secreted peptidomics from skeletal muscle under the insulin-resistant condition was not fully characterized.\",\n \"Herein, we performed liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to help characterize the secretome from cultured skeletal muscle tissues of db/db mice at peptides level and identify putative bioactive peptides. A global secreted peptides were established and bioinformatics analysis of precursor proteins provided a possible relationship of differential peptides with T2DM or insulin resistance. Additionally, the biological effects of these secreted peptides on C2C12 myotubes elucidated a possible regulatory role in insulin signaling-and mitochondrial-related genes expression. Taken together, these observations will encourage us to investigate function of these secreted peptides from cultured skeletal muscle tissues with other tissues under the diabetic state, thus representing a promising strategy for prevention and treatment of insulin resistance as well as the associated metabolic disorders.\",\n \"All the studies involving mice acquired approval from the Ethical Committee of Nanjing Medical University. All procedure involving mice were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval Number: IACUC-1812053).\",\n \"Twelve-week-old male C57BLKS/J db/db mice (n = 8, db/db group) and age-matched WT controls (n = 8, NC group) were purchased from the Model Animal Research Center of Nanjing Medical University. After adaptive raising for one week, mice were sacrificed by cervical dislocation and skeletal muscle tissues were isolated from the left hind leg (each mice of 100 ∼ 150 mg). Subsequent operations were carried out under a laminar airflow hood to decrease contamination. The visible blood vessels and connective tissue were removed from the tissue. After rinsed with PBS, the skeletal muscle tissues were cut into small pieces (3-4 mm 3 ) with scissors as described by an established protocol (36,37). Tissue cutting will lead to release of damaged cells slowly into the medium. Additionally, a small amount of serum proteins in the tissue pieces will diffuse out during culture period. Therefore, necessary washing procedures during culture were adopted to obtain medium samples (referred to as secretome) containing mainly skeletal muscle tissue-derived secreted components as previously reported (38,39). Tissue fragments were placed in a 10 cm plate (200∼300 mg from two mice as one sample) containing 10 mL serum/phenol red free DMEM/F12 medium (Gibco, Grand Island, CA, USA). After incubation in a humidified incubator at 37 • C under 95% O2 and 5% CO2 for 48 h, the medium was immediately supplemented with protease inhibitors and centrifuged (845 g, 10 min, 4 • C) to wipe off cell debris and dead cell. Supernatant samples from each group (NC or db/db group) were harvested from four individual tissue culture dishes independently (n = 4 per group). Lactate dehydrogenase (LDH) (36,40,41) and IL-6 (42) expression levels were detected to evaluate skeletal muscle vitality and capability during the in vitro culture. Then the supernatant samples obtained were stored at −80 • C until further processing.\",\n \"First, both supernatant samples were concentrated to 1-2 mL by centrifuges for speed vacuum (LaboGene, Allerød, Denmark). Then equal volume of U2 solution containing 8 M urea and 100 mM tetraethyl-ammonium bromide in pH 8.0 was added to the concentrated supernatant for denaturation. Followed by centrifugation for 30 min (13,000 g, 30 • C), the medium supernatant was transferred to a new centrifuge tube. Subsequently, proteins were reduced by 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide successively. The protein concentrations of supernatant from cultured skeletal muscle samples were detected by Bradford method (43) and integrity of these samples were evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) combined with silver staining. Afterwards the peptides were separated from samples by treatment with Amicon R Ultra Centrifugal Filters in 10-kDa (Merck Millipore, Billerica, MA, USA) according to the manufacturer's instruction as previously described (37). The \\\"peptidome\\\" present in the filtrates was desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and the desalted peptide solution was vacuum-dried with centrifuges for speed vacuum (SCAN SPEED 40, LaboGene) as previously described (44) and immediately frozen at −80 • C until the following processing.\",\n \"The LC-MS/MS analysis was conducted similarly to the previous protocols (45). The peptide samples were redissolved in 2% (v/v) acetonitrile/0.1% formic acid (FA) (v/v) and 5 µL solution was injected into an A Triple TOFTM 5600 mass spectrometer (AB Sciex, Redwood City, CA, USA) coupled to a ekspert TM nanoLC400 liquid chromatography (AB Sciex) via a nanosource electrospray interface equipped with distal coated SilicaTip emitters (New Objective, Woburn, MA, USA). First, load peptide onto a C18 trap column (5 µm, 100 µm × 20 mm, AB Sciex) and elute at 300 nL/min onto a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Shanghai, China) in the gradient as long as 120 min. These two mobile phases included buffer A containing 2% acetonitrile/ 0.1% FA/ 98% H2O (v/v) and buffer B containing 98% acetonitrile/0.1% FA/2% H2O (v/v). Then peptide mixture was eluted at a flow rate of 0.3 µL/min in a gradient generated with Solvent A containing 98% water and 2% acetonitrile containing 0.1% FA (v/v) and Solvent B containing 2% water and 98% acetonitrile containing 0.1% FA (v/v) according to the previously described reports (45,46). The mass spectrometer was operated in positive mode with a spray voltage of 2500 V, 206.84kPa for the curtain gas, 41.37kPa for the nebulizer gas and 150 • C as temperature. A data dependent acquisition (DDA) method was applied and a full scan MS spectrum (300-1500 m/z) with accumulation time of 0.25 s was adopted. Top 30 precursor ions for fragmentation based on the highest intensity were selected. The collections of MS1 spectra were in the range 350-1500 m/z, and MS2 spectra were in the range of 100-1500 m/z. A total of 47,709 MS/MS were collected from all LC-MS/MS analyses.\",\n \"Protein Pilot Software (https://sciex.com.cn/products/software/ proteinpilot-software, version4.5,AB SCIEX) was adopted to analyze the original MS/MS file data (47). For peptides identification, the Paragon algorithm was employed against the Mus_musculus SwissProt sequence database (a total of 85210 items, updated in January 2019). The following parameters were installed: The parameters were set as follows: 1) cysteine modified with iodoacetamide; 2) biological modifications were selected as ID focus. The strategy of the automatic decoy database search was employed to estimate false discovery rate (FDR) calculation using the Proteomics System Performance Evaluation Pipeline (PSPEP) Software integrated in protein pilot-software. Only unique peptides (global FDR values < 1%) were considered for further analysis. Skyline v4.2 software was employed for MS1 filtering and ion chromatogram extractions for peptides label-free quantification (48,49). And the parameters setting for skyline MS1 filtering were according to previous studies (48). Using the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.\",\n \"The relative molecular mass and isoelectric point of each peptide were calculated by the online tool (http://web.expasy. org/compute.pi/). All the precursors protein of differentially expressed peptides as one group were imported into for GO (http://www.geneontology.org/) (50) and pathway analysis (https://www.kegg.jp/) for predicting potential functions. The online tools UniProt Database (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/) were adopted to explore if the peptides' sequences were positioned in the conserved structural domains or regions of their precursors. The Open Targets Platform database (www.targetvalidation.org/) was adopted to investigate precursors associated with diabetes and obesity as previously reported (24). For visualization, clustergram and volcano plot graphs in this study were drawn with R language (http://www.r-project.org/). For determination of differentially expressed peptides, fold change was computed as the average values of biological duplication (n = 4).\",\n \"All the peptides used in this study were custom-synthesized and HPLC-purified by Science Peptide Biological Technology Co., Ltd. (Shanghai, China) through the solid-phase method as described reported (51). The purity in 95% for each peptide was confirmed by HPLC-MS method. All the used peptides were stored in lyophilization at−20 • C until dissolved with sterile water immediately for treatment with cells in vitro.\",\n \"C2C12 cells, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China) at 37 • C with 5% CO2. On the fourth day of cell differentiation, the C2C12 cells were pre-treated with synthesized peptides or solvent for 48 h at the same concentration of 50 µM, and then starved for 24 h with serumfree L-DMEM (Gibco). Subsequently, myotubes were incubated in L-DMEM in the presence or absence of 100 nM of insulin for 30 min. At the indicated time, the cells were collected for the following analysis. \",\n \"Total RNA was isolated using trizol reagent (Life Technologies, Carlsbad, CA, USA). And the cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was determined by real-time quantitative PCR (ABI ViiA7, Applied Biosystems, Foster City, California, USA) using the SYBR Green array. The relative gene expression was analyzed based on the 2 − CT method with normalization of the data to PPIA. The primers used for the real-time quantitative PCR were listed in Table S1.\",\n \"Data were analyzed with GraphPad Prism 7 (San Diego, CA, USA), and the results were shown as the mean ± standard deviation (SD). Peptides with a fold change larger than 1.5 or <0.67 with a Student's t-test p-value <0.05 were selected as differently expressed peptides.\",\n \"Considering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity. LDH and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period. LDH content of culture medium was assessed as an indicator of cell lysis at 0 h∼24 h, 24 h∼48 h and 48 h∼72 h; no significant increased in LDH occurred from 48 h to 72 h (not shown in the manuscript). Secretion of IL-6 remained stable from 24 h to 48 h in culture by ELISA (not shown in the manuscript). Therefore, we chose the 24 h∼48 h culture as an optimal time point. The protein composition and integrity of the secreted samples were evaluated by SDS-PAGE with silver staining in Figure S1. After validation of skeletal muscle culture system and samples assessment, peptides from four different culture dishes per group (control and db/db mice) were individually extracted from supernatants and analyzed via label-free mass spectrometry based quantification. To gain more insights into biological differences between control and db/db mice skeletal muscle, we employed LC-MS/MS technology and identified 3384 peptides, of which 2664 peptides showed valid quantitative values in the detection. A total of 63 peptides were identified differentially secreted, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 peptides were down-regulated (fold change < 0.67, P < 0.05) in medium supernatants from insulin resistant-skeletal muscle tissues. Visualization methods such as hierarchical clustering and volcano plot showed distinct peptides secretion profiles as shown in Figures 1A,B. The differentially secreted peptides were summarized in Table 1.\",\n \"To characterize the general feature of differentially secreted peptides, we further analyzed the relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs. pI. The results indicated that the Mr varied from 1011.1 Da to 2670.9 Da with 70% between 1,300 and 2,100 Da (Figure 2A), and pI varied from 3.7 to 9.7, with 51% between 4 and 7 ( Figure 2B). Additionally, distribution of pI vs. Mr could divide these peptides into three groups around pI4, pI6 and pI10 ( Figure 2C).\",\n \"To make comprehensive functional assessment of these differential peptides, we consider all the precursor proteins as one group and performed GO and Pathways analysis of them. First of all, the precursor proteins of these differential peptides identified from the cultured control and db/db skeletal muscle were classified using Gene Ontology categories, which revealed the majority of proteins were associated with striated muscle contraction and glucose metabolic process ( Figure 3A). The top 20 GO terms were listed in Figure 3A. The precursor proteins annotated involved in these GO terms were presented in Table S2. Subsequent pathway analysis in the KEGG database revealed a significant enrichment in metabolic pathway and glycolysis/gluconeogenesis process ( Figure 3B). The top 14 pathway terms were listed in Figure 3B. The precursor proteins annotated involved in these pathway terms were presented in Table S3. We further discriminated the up-and down-regulated peptides and performed GO and Pathways analysis of these two groups of precursor proteins. This analysis may bring overlapping terms such as phosphorylation, phosphagen metabolic process and phosphorus metabolic process in up-or down-regulated peptides, and a smaller number of Search tool for the retrieval of interacting genes/proteins (STRING) was used to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides. The size of node represents the number of interacting proteins and the color of node represents the bitscore of matching results from STRING database. The thicker the line (edge), the higher the reliability (evaluated by combined score >0.4).\",\n \"terms in Pathway analysis (deriving up-regulated peptides). These analysis were presented in Figure S2. Additionally, the major intracellular locations of the precursor proteins were considered from literature sources, illustrating that 64% proteins were cytoplasmic and 15% proteins were cytoskeletal seen in Figure 3C. Based on above analysis, we found that a vast of precursor proteins were assigned to glucose metabolic process include ALDO A, triosephosphate isomerase (TPIS), beta-enolase (ENOB), glycogen phosphorylase, muscle form (PYGM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1(PGK1), specifically assigned to glycolysis/gluconeogenesis process. Another kind of proteins enriched in regulation of striated muscle contraction terms (GO) and regulation of actin cytoskeleton (Pathway) were myosin regulatory light chain 2, skeletal muscle isoform(MLRS), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin-binding protein H (MYBPH) and myosin light chain 3 (MYL3),both belonging to sarcomeric proteins. Subsequently, we used search tool for the retrieval of interacting genes/proteins (STRING) to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides in Figure 3D. Based on protein-protein interaction and co-occurrence in KEGG pathways and literature mining, this network was constructed from 25 proteins that matched to the STRING database and the matching results were listed in Table S4. As shown from this network, 22 nodes representing precursor protein constituted the interaction diagram, which could form 93 reliable a-to-b interaction relationship by combined score (more than 0.4) as shown in Table S5. \",\n \"It is widely postulated that most peptides can be attributed mainly to the proteolytic enzymes as well as their type and level (cleavage specificity and activity), which can differ during disease (52)(53)(54). Briefly, the N-terminal pre-cleavage site, N terminus, C terminus, and C-terminal post-cleavage site of the identified peptides were commonly used to investigate the nature of proteolytic enzymes within serum (55) or tissues (56). Thus, we analyzed the distribution of peptide cleavage site and found that Lysine (K) was the most frequent cleavage site of N-terminal amino acid of identified peptides and Alanine (A) was the most frequent cleavage site of C-terminal amino acid of identified peptides. Lysine (K) was the most frequent cleavage site of Nterminal amino acid of preceding peptides, while Leucine (L) was the commonest C-terminal amino acid of preceding peptides ( Figure 4A). These data indicate that the pattern of cleavage sites may represent the specificity of cleavage and activity of proteolytic enzymes under the diabetic condition. Additionally, we also tried to align peptide sequences on the same precursor sequence to construct a \\\"peptide alignment map\\\". Searched from our samples, the largest number of identified peptides (n = 1 7) (Figure 4B) originated from KCRM and the second largest number of identified peptides (n = 6) came from ALDO A (Figure 4B), elucidating that these peptides were easily cleaved by certain kind of endogenous enzymes.\",\n \"To check for specific domain structures or patterns in the identified peptides comparison with its precursor proteins, we retrieved domain information from the UniProt and Pfam database. In order to validate that if the peptides exerted important roles in the related metabolic diseases, we adopted the Open Targets Platform database to investigate protein precursors. In our searching results, most peptides (n = 44) from the identified pepetides (n = 63) were located in the functional domains ( Table 2). Additionally, 27 out of 44 peptides located in the functional domain were predicted closely related with both obesity and diabetes ( Table 3). These observations will encourage us to further investigate properties of the putative secreted peptides in skeletal muscle under the diabetic state.\",\n \"To explicit the putative function of differentially secreted peptides, 7 differentially secreted peptides, which had already been annotated derived from authentic protein from Uniprot database, located in the functional motifs and showed relatively high abundance from MS detection, were chosen for further analysis. Table S6. Sequentially, we termed these candidated peptides P1-P7 in order. To use C2C12 myotubes as cell models in vitro, the results of changes in inuslin signaling revealed that up-regulated peptides P2-P4 addition could both significantly decrease the phosphorylation of Irs1 (Ser-307) and Akt (Ser-473) under insulin stimulation (100 nM, 30 min) as shown in Figures 5A,B, in spite of bringing a modestly up-regulation of p-Irs1 (Ser-307) at the basal status. Moreover, mitochondrial-related marker protein Pgc1α was significantly attenuated in the insulin-stimulated C2C12 cells by up-regulated peptides P1-P4 as shown in Figures 5E,F. Among these downregulated peptides, only peptide 7 presented a promotion on p-Akt (Ser-473) expression level both under the basal and insulin stimulation status as shown in Figures 5C,D. And these up-regulated peptide 1-4 could decrease Pgc1α protein level and down-regulated peptide 6 and 7 administration enhanced Pgc1α protein level by the stimulation of insulin presented in Figures 5E,F. We further evaluated Gut 4 (a key gene for glucose transport) and Pgc1α mRNA levels by peptides treatment upon insulin stimulation. The results also indicated that Peptide 3 and 4 modestly decreased Pgc1αexpression at mRNA levels seen in Figure 5G, while Peptide 6 and 7 significantly up-regulated both Glut4 and Pgc1α expression at mRNA levels seen in Figure 5H. These observations revealed that these candidated peptides (P1-P7) may affect the expressions of insulin signaling or mitochondrial-related genes in skeletal muscle cells.\",\n \"Emerging as a widely known secrete organ, skeletal muscle tissues have been extensively discussed using a wide range of comparative and quantitative proteomic methods (57). Proteomic scannings of the medium supernatant from skeletal muscle cells have already afford approaches to identify a large number of secreted proteins (58)(59)(60)(61)(62)(63). Particularly, one such research reported the alteration of insulin effect on the secretome profile of skeletal muscle cells, revealing the changes of protein levels secreted from skeletal muscle during activation of insulin signaling pathway (58). A recent study also generated a comprehensive secretome analysis of skeletal muscle cells under palmitic acid-induced insulin resistance (61). In addition to these proteins, many other muscle-secreted compounds have been also come to light, including exosome (64,65), metabolites (66), and miRNAs (67). These studies have greatly expanded our knowledge of skeletal muscle secretome and might afford possibilities for exploring novel molecular targets in the maintenance of skeletal muscle physiology and even whole-body metabolism. However, to date few studies has focused on the peptides present in either skeletal muscle tissues or cells. Therefore, we attempted to comprehensively profile peptides that may play roles in regulating insulin sensitivity and offer enormous promise for exploring molecular mechanisms underlying insulin resistance.\",\n \"In present study, a total of 63 peptides were differentially secreted in medium supernatants from cultured skeletal muscle tissues of db/db mice, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 were down-regulated (fold change < 0.67, P < 0.05) shown in Figures 1A,B. The differences of dysregulation peptides may indeed reflect the changes between control and insulin-resistant mice in some extent. However, db/db mouse is a well-established leptin receptordeficient animal model (68,69). Despite no studies revealed the association between leptin receptor deficiency and peptide dysregulation/protein degradation so far, the possible effect by this mutation deserves to be taken into account and other nongenetic mice models could be adopted in the future studies. Based on our previous study (70), 10-kDa filters method was used to extract peptides in order to intercept proteins secreted by skeletal muscle to the conditional media. Characterization of the basic feature (Mr and pI) of these identified peptides (Figures 2A,B) only reflected the differences of distribution in the diabetic skeletal muscle tissues, but also proved the reliability of the used peptide extraction method.\",\n \"Subsequently, GO and Pathway analysis revealed that the precursors protein of these peptides were mostly involved in muscle contraction and metabolism processes (Figures 3A,B). An interesting finding from our study was that most of the differentially identified peptides derived from cytosolic, cytoskeletal and mitochondrial proteins (Figures 3A,B), which differed from the secretory pathway peptides (71). This (34). Similarly, another peptidomic anaylsis of brain slices cultures and media also pointed that vast majority of secreted peptides arose from intracellular proteins (72). There does also exist some evidence that these identified N-or C-terminus protein yielding peptides, rather than internal fragments, raised the possibility that they are produced by selective processing rather than protein degradation (73). Taken together with previous researches, the current results show us meaningful hints that these intracellular peptides may be secreted via nonclassical mechanisms. Actually, another important origin of peptides is proteolytic degradation processes in body fluid under the physiological or pathologically processes. Most regulatory peptides were efficiently degraded by plasma enzymes once secreted into the bloodstream, which exerted anti-diabetic therapeutic function (74,75) or were identified as disease markers (76). Recently, Federico Aletti et al. (77) used protease activity detection and specific enzymes analysis to explain a large presence of circulating peptides under hemorrhagic shock, which gave us a possible way to evaluate peptides origin. Thus, a fundamental validation is whether the proteins-derived peptides are actually secreted from skeletal muscle cells per se or proteolytic degradation and are of biologically active. Among the differentially secreted peptides from cultured skeletal muscle tissues of db/db mice, we found that three peptides derived from GAPDH, four peptides derived from ALDO A and one peptides derived from PYGM were upregulated in the conditional medium from db/db groups as shown in Table 1. As described by Dustin S. Hittel et.al, a global proteomic survey of skeletal muscle revealed a statistically significant up-regulation in glycolytic enzymes GAPDH and ALDO A protein levels in obese/overweight patients (78). Another quantitative protein profile also identified a more abundant levels of GAPDH and PYGM in skeletal muscle from T2DM groups compared with the control groups (79). In addition to pointing the importance of the mitochondrial numbers and impairments under the insulin-resistant states (80)(81)(82), several studies noted that glycolytic capacity is higher in skeletal muscle of patients with T2DM or obesity (83). Notably, stronger changes of peptides derived from sarcomeric proteins such as myosin light chain 1 (MYL1) and MYL3 were also observed in the conditional medium from diabetic muscles as shown in Table 1. MYL1 and MYL3 are representive markers of fast-muscle and slow-muscle respectively, which were both regulated by insulin stimulation (79). And previous studies observed that property of T2DM individuals muscle was shifted to a fast-twitch glycolytic phenotype (84). In fact, deficiency in sarcomeric proteins in skeletal muscle also suggested their importance in skeletal muscle physiologic and pathological processes. On the whole, our results of increased glycolytic enzyme-or sarcomeric proteins-derived peptides secreted from insulin resistant skeletal muscle may support the hypothesis that altered glycolytic capacities or fiber types under the diabetic status contribute to this difference.\",\n \"Till now, the putative function of these peptides are not clear. Therefore, we evaluated whether these peptides originated from functional domains of the corresponding precursor protein using the UniProt and Pfam database. In our searching results, most peptides (n = 44) from the identified peptides (n = 63) were located in the functional domains as listed in Table 2. Specifically, most peptides derived from functional enzymes were located in the enzymatic activity region, including TPI- TCTP-derived peptide (119-AEQIKHILANF-129) and CAH3-derived peptide . We also found another kind of S-Nitrosylation modifying sites for affecting GAPDH enzymatic activity (90). Therefore, future efforts need to be established for investigating the potential role of peptides on insulin sensitive cells in vitro or whole-body metabolism in vivo.\",\n \"The above analyses provided a possibility to evaluate the biological effects of these differentially secreted peptides. As widely reported, insulin resistance in skeletal muscle is tightly connected with the deficit in insulin signaling (91). Consequently, the role of phosphorylation of Irs1 and Akt in signaling pathways is very crucial in anti-hyperglycemia and insulin sensitivity (92). In this result, we found these up-regulated peptides 1-4 both exerted a significantly attenuated insulin action in C2C12 cells, evaluated by a decreased level of p-Irs1 (Ser-307) and p-Akt (Ser-473) seen in Figures 5A,B, whereas only one down-regulated peptide P7 could remarkably promote insulin signaling only via Irs1 signaling pathway seen in Figures 5C,D. Peroxisome proliferator-activated receptor γ coactiva-tor-1(Pgc-1), which displays a dominant role through tight modulation of mitochondrial biogenesis and respiration, has also been demonstrated to participate in skeletal muscle insulin signaling and metabolic homeostasis (93). The up-regulated peptides 1-4 also brought out a decreased protein level of Pgc-1α in C2C12 cells as shown in Figures 5E,F, and down-regulated peptide 6 and 7 administration also gave rise to the increased Pgc1α mRNA and protein level in Figures 5E,F,H. Taken together, these selected peptides secreted from db/db mice skeletal muscle presented a promotive or inhibitory effect on insulin and mitochondrialrelated pathways in skeletal muscle cells by an autocrine manner. Notably, peptide 4 (231-RVPTPNVSVVDLTCRLEKPAK−252) derived from GAPDH displayed a most significant inhibitory effect toward these candidate peptides. However, the relationship between GAPDH-derived peptide and its precursor protein is to be determined. On the other hand, more methods need to be employed for the wider cell signaling screen and further research is required to explore the biologic function of skeletal muscle-secreted peptides on adipocytes or liver cells.\",\n \"To our knowledge, no large-scale quantitative peptidomic analysis has been performed on skeletal muscle to elucidate secreted peptides profiles under the diabetic status. The present study identified and quantified changes with a label-free discovery using LC-MS/MS technology to construct a global secreted peptides picture. Further bioinformatics analysis of precursors comprehensively provided an atlas of peptides that may exist roles in regulating insulin sensitivity. This represented a new perspective toward exploring insulin resistance pathogenesis. Additionally, the detailed biological effects of these secreted peptides on skeletal muscle insulin resistance or cross-talk with other tissues remained to be elucidated in the future study.\",\n \"The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.\",\n \"The protocol has been approved by the Institutional Animal Care and Use Committee of Nanjing medical university (Approval Number: IACUC-1812053).\",\n \"YWu and MH performed experiments and interpreted results of experiments. YWa and YG prepared the figures. XC and PX analyzed the data. CJ and TZ participated discussion. YZ helped write the manuscript with providing assistance. LY conceived and designed experiments, provided funding to regents, and approved final version of manuscript. All authors read and approved the final manuscript. \",\n \"The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2019.00741/full#supplementary-material Figure S1 | SDS-PAGE associated with silver staining of the medium supernatants from cultured skeletal muscle tissues (Con vs. db groups). Table S1 | Primers used in this study. Table S2 | GO terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. Table S3 | Pathway terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. \"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Ethics Statement","Animal Experiments and Sample Preparation","Peptide Extraction and Desalting","Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)","Peptide Identification and Quantitative Analysis","Bioinformatics and Annotations","Synthetic Peptides","Cell Culture and Peptide Treatment","Western Blot Analysis and Antibodies","Reverse Transcription and Real-Time Quantitative PCR","Statistical Analysis","RESULTS","Identification of Secreted Peptides From Cultured db/db Skeletal Muscle Tissues","Charactrization of the Basic Feature of Differentially Secreted Peptides","Comprehensive Functional Assessment and Intercellular Location of Precursor Proteins of These Differential Peptides","Cleavage Pattern of Differentially Secreted Peptides","Putative Bioactive Peptides Associated With Diabetes and Obesity","The Effect of Candidated Peptides (P1-P7) on Insulin and Mitochondrial-Related Pathways in Skeletal Muscle Cells","DISCUSSION","DATA AVAILABILITY STATEMENT","ETHICS STATEMENT","AUTHOR CONTRIBUTIONS","SUPPLEMENTARY MATERIAL","FIGURE 1 |","FIGURE 2 |","FIGURE 3 |","FIGURE 4 |","FIGURE 5 |","FUNDING","Figure S2 |","TABLE 1 |","TABLE 1 |","TABLE 2 |","TABLE 3 |","Table S4 |","Table S5 |","Table S6 |"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Ethics Statement\",\n \"Animal Experiments and Sample Preparation\",\n \"Peptide Extraction and Desalting\",\n \"Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)\",\n \"Peptide Identification and Quantitative Analysis\",\n \"Bioinformatics and Annotations\",\n \"Synthetic Peptides\",\n \"Cell Culture and Peptide Treatment\",\n \"Western Blot Analysis and Antibodies\",\n \"Reverse Transcription and Real-Time Quantitative PCR\",\n \"Statistical Analysis\",\n \"RESULTS\",\n \"Identification of Secreted Peptides From Cultured db/db Skeletal Muscle Tissues\",\n \"Charactrization of the Basic Feature of Differentially Secreted Peptides\",\n \"Comprehensive Functional Assessment and Intercellular Location of Precursor Proteins of These Differential Peptides\",\n \"Cleavage Pattern of Differentially Secreted Peptides\",\n \"Putative Bioactive Peptides Associated With Diabetes and Obesity\",\n \"The Effect of Candidated Peptides (P1-P7) on Insulin and Mitochondrial-Related Pathways in Skeletal Muscle Cells\",\n \"DISCUSSION\",\n \"DATA AVAILABILITY STATEMENT\",\n \"ETHICS STATEMENT\",\n \"AUTHOR CONTRIBUTIONS\",\n \"SUPPLEMENTARY MATERIAL\",\n \"FIGURE 1 |\",\n \"FIGURE 2 |\",\n \"FIGURE 3 |\",\n \"FIGURE 4 |\",\n \"FIGURE 5 |\",\n \"FUNDING\",\n \"Figure S2 |\",\n \"TABLE 1 |\",\n \"TABLE 1 |\",\n \"TABLE 2 |\",\n \"TABLE 3 |\",\n \"Table S4 |\",\n \"Table S5 |\",\n \"Table S6 |\"\n]"},"annotations_table":{"kind":"list like","value":["Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nUp-regulated peptides \n\nAVGAVFDISNADRLGSSEVEQ \nP07310 \nKCRM \n2163 \n9.728 \n0.005 \n\nTNGAFTGEISPGMIKDLGATWV \nP17751 \nTPIS \n2264 \n8.019 \n0.035 \n\nIADLVVGLCTGQIK \nP21550 \nENOB \n1486 \n6.77 \n0.000 \n\nNYKPQEEYPDLSKHNNHMA \nP07310 \nKCRM \n2315 \n4.843 \n0.024 \n\nEEIEEDAGLGNGGLGRLAAC \nQ9WUB3 \nPYGM \n2030 \n4.411 \n0.004 \n\nGQCGDVLRALGQNPTQAEV \nP09542 \nMYL3 \n2013 \n4.077 \n0.033 \n\nAGPLCLQEVDEPPQHAL \nP70296 \nPEBP1 \n1873 \n4.038 \n0.004 \n\nEAFTVIDQNRDGIID \nP97457 \nMLRS \n1705 \n3.947 \n0.026 \n\nDLFDPIIQDRHGGY \nP07310 \nKCRM \n1645 \n3.668 \n0.022 \n\nKDLFDPIIQD \nP07310 \nKCRM \n1203 \n3.282 \n0.000 \n\nLDDVIQTGVDNPGHP \nP07310 \nKCRM \n1576 \n3.265 \n0.001 \n\nSEIIDVSSKAEEVK \nA2ASS6 \nTITIN \n1533 \n3.134 \n0.002 \n\nVIACIGEKLDE \nP17751 \nTPIS \n1246 \n2.976 \n0.003 \n\nGTNPTNAEVKKVLGNPSNEEM \nQ545G5 \nMYL1 \n2180 \n2.838 \n0.006 \n\nADRLGSSEVEQVQLV \nP07310 \nKCRM \n1629 \n2.804 \n0.001 \n\nIGQGTPIPDLPEVKRV \nA2AQA9 \nNEB \n1719 \n2.739 \n0.002 \n\nSTTAPPIQSPLPVIPHQK \nE9PYJ9 \nLDB3 \n1910 \n2.685 \n0.023 \n\nKADDGRPFPQVI \nP05064 \nALDOA \n1342 \n2.644 \n0.021 \n\nKSMTEQEQQQLIDD \nP07310 \nKCRM \n1692 \n2.442 \n0.001 \n\nGGGASLELLEGKVLPGVDA \nP09411 \nPGK1 \n1781 \n2.264 \n0.028 \n\nLEGTLLKPNMVTPGHA \nP05064 \nALDOA \n1677 \n2.208 \n0.010 \n\nVMVGMGQKDSYVGDEAQSK \nP68134 \nACTS \n2028 \n2.168 \n0.021 \n\nDLEEATLQHEATAAALR \nQ02566 \nMYH6 \n1838 \n2.07 \n0.038 \n\nAELQEVQITEEKPLLPG \nQ9QYG0 \nNDRG2 \n1935 \n2.043 \n0.048 \n\nSEGKCAELEEELKTVTNNL \nP58771 \nTPM1 \n2163 \n2.038 \n0.049 \n\nRSIKGYTLPPHC \nP07310 \nKCRM \n1428 \n1.967 \n0.001 \n\nNSNSHSSTFDAGAGIALNDNFVK \nP16858 \nG3P \n2365 \n1.935 \n0.038 \n\nTGKATSEASVSTPEETAPEPAKVPT \nP70402 \nMYBPH \n2484 \n1.919 \n0.003 \n\nDNEYGYSNRVVDL \nP16858 \nG3P \n1543 \n1.898 \n0.004 \n\nEELDAMMKEASGPINF \nP97457 \nMLRS \n1781 \n1.853 \n0.012 \n\nSNNKDQGGYEDFVEGLRV \nQ545G5 \nMYL1 \n2026 \n1.816 \n0.016 \n\nAEQIKHILANF \nP63028 \nTCTP \n1283 \n1.787 \n0.044 \n\nGADPEDVITGAFK \nP97457 \nMLRS \n1319 \n1.782 \n0.031 \n\nTSIEDAPITVQSKINQ \nA2AQA9 \nNEB \n1743 \n1.739 \n0.028 \n\nLKGGDDLDPNYVLS \nP07310 \nKCRM \n1505 \n1.732 \n0.040 \n\nIQTGVDNPGHPF \nQ6P8J7 \nKCRS \n1281 \n1.600 \n0.045 \n\nAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n1957 \n1.595 \n0.009 \n\nGQEQWEEGDLYDKEKQQKPF \nE9Q8K5 \nTITIN \n2481 \n1.586 \n0.036 \n\nAGTNGETTTQGLDGLSERCA \nP05064 \nALDOA \n2037 \n1.582 \n0.033 \n\nRVPTPNVSVVDLTCRLEKPAK \nP16858 \nG3P \n2378 \n1.531 \n0.022 \n\nKVPEKPEVVEKVEPAPLK \nF7CR78 \nF7CR78 \n2015 \n1.527 \n0.026 \n\nGETTTQGLDGLSERCAQ \nP05064 \nALDOA \n1822 \n1.526 \n0.042 \n\nDown-regulated peptides \n\nKIEFTPEQIEEF \nP09542 \nMYL3 \n1509 \n0.612 \n0.021 \n\nDLSAIKIEFSKEQQEDF \nP05977 \nMYL1 \n2026 \n0.600 \n0.019 \n\nHELYPIAKGDNQSPI \nP16015 \nCAH3 \n1681 \n0.559 \n0.038 \n\nNSLTGEFKGKY \nP07310 \nKCRM \n1243 \n0.545 \n0.027 \n\nDEPKPYPYPNLDDF \nQ3V1D3 \nAMPD1 \n1709 \n0.543 \n0.034 \n\nASHHPADFTPAVHA \nQ91VB8 \nHBA-A1 \n1457 \n0.537 \n0.016 \n\n(Continued) \n\nFrontiers in Endocrinology | www.frontiersin.org ","Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nSQVGDVLRALGTNPTNAE \nQ545G5 \nMYL1 \n1841 \n0.516 \n0.047 \n\nADEIAKAQVAQPGGDTI \nP70349 \nHINT1 \n1725 \n0.515 \n0.038 \n\nDKETPSGFTLDDV \nP07310 \nKCRM \n1423 \n0.478 \n0.037 \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n2654 \n0.467 \n0.038 \n\nPHPYPALTPEQKK \nP05064 \nALDOA \n1505 \n0.407 \n0.028 \n\nQLVVDGVKLM \nP07310 \nKCRM \n1101 \n0.400 \n0.001 \n\nKDLFDPIIQDRHG \nP07310 \nKCRM \n1553 \n0.395 \n0.022 \n\nKGVVPLAGTNGETTTQGLDGLSER \nP05064 \nALDOA \n2399 \n0.367 \n0.015 \n\nRVFDKEGNGTVMGAELR \nQ545G5 \nMYL1 \n1878 \n0.341 \n0.043 \n\nNADEVGGEALGRL \nA8DUK4 \nHBB-BS \n1300 \n0.339 \n0.037 \n\nNEHLGYVLTCPS \nP07310 \nKCRM \n1389 \n0.328 \n0.031 \n\nASHTEEEVSVSVPEVQKKTVTEEK \nE9Q8K5 \nTITIN \n2669 \n0.253 \n0.023 \n\nNEEDHLRV \nP07310 \nKCRM \n1010 \n0.212 \n0.027 \n\nVLTCPSNLGTGLRG \nP07310 \nKCRM \n1444 \n0.201 \n0.018 \n\nGGYKPTDKHKTDL \nP07310 \nKCRM \n1459 \n0.127 \n0.006 \n\nobservation was also demonstrated by other peptidomic studies. \nFrom Steven W. Taylor group' results, several identified peptides \nin the human islet cultures were derived from intracellular and \ncytoskeletal proteins such as microtubule-associated protein 4 \nand ubiquitin, which may result from a greater level of cellular \nstress ","Peptide sequence \nProtein \nLocation \nDomain \nDescription \n\nUp-regualted peptides \n\nAVGAVFDISNADRLGSSEVEQ \nKCRM \n329-349 \n125-367 \nPhosphagen kinase C-terminal \n\nTNGAFTGEISPGMIKDLGATWV \nTPIS \n121-142 \n57-295 \nTIM \n\nIADLVVGLCTGQIK \nENOB \n381-394 \n142-432 \nEnolase C \n\nNYKPQEEYPDLSKHNNHMA \nKCRM \n13-31 \n11-98 \nPhosphagen kinase N-terminal \n\nEEIEEDAGLGNGGLGRLAAC \nPYGM \n124-143 \n113-828 \nPhosphorylase \n\nGQCGDVLRALGQNPTQAEV \nMYL3 \n83-101 \n58-95 \nEF-hand 1 \n\nEAFTVIDQNRDGIID \nMLRS \n32-46 \n25-60 \nEF-hand 1 \n\nDLFDPIIQDRHGGY \nKCRM \n87-100 \n11-98 \nPhosphagen kinase N-terminal \n\nKDLFDPIIQD \nKCRM \n86-95 \n11-98 \nPhosphagen kinase N-terminal \n\nLDDVIQTGVDNPGHP \nKCRM \n53-67 \n11-98 \nPhosphagen kinase N-terminal \n\nVIACIGEKLDE \nTPIS \n174-184 \n57-295 \nTIM \n\nGTNPTNAEVKKVLGNPSNEEM \nMYL1 \n39-59 \n6-41 \nEF-hand \n\nADRLGSSEVEQVQLV \nKCRM \n339-353 \n125-367 \nPhosphagen kinase C-terminal \n\nKADDGRPFPQVI \nALDOA \n87-98 \n15-364 \nGlycolytic \n\nKSMTEQEQQQLIDD \nKCRM \n177-190 \n125-367 \nPhosphagen kinase C-terminal \n\nGGGASLELLEGKVLPGVDA \nPGK1 \n395-413 \n9-406 \nPGK \n\nLEGTLLKPNMVTPGHA \nALDOA \n224-239 \n15-364 \nGlycolytic \n\nVMVGMGQKDSYVGDEAQSK \nACTS \n45-63 \n4-377 \nActin \n\nDLEEATLQHEATAAALR \nMYH6 \n1,179-1,195 \n845-1,926 \nMyosin_tail_1 \n\nSEGKCAELEEELKTVTNNL \nTPM1 \n186-204 \n1-284 \nCoiled coili \n\nRSIKGYTLPPHC \nKCRM \n135-146 \n125-367 \nPhosphagen kinase C-terminal \n\nSNNKDQGGYEDFVEGLRV \nMYL1 \n77-94 \n83-118 \nEF-hand \n\nAEQIKHILANF \nTCTP \n119-129 \n1-172 \nTCTP \n\nGADPEDVITGAFK \nMLRS \n93-105 \n95-130 \nEF-hand 2 \n\nLKGGDDLDPNYVLS \nKCRM \n115-128 \n125-367 \nPhosphagen kinase C-terminal \n\nGQEQWEEGDLYDKEKQQKPF \nTITIN \n1,691-1,710 \n1,709-1,799 \nIg-like \n\nAGTNGETTTQGLDGLSERCA \nALDOA \n117-136 \n15-364 \nGlycolytic \n\nRVPTPNVSVVDLTCRLEKPAK \nGAPDH \n232-252 \n243-248 \n[IL]-x-C-x-x-[DE] motif \n\nGETTTQGLDGLSERCAQ \nALDOA \n121-137 \n15-364 \nGlycolytic \n\nDown-regualted peptides \n\nKIEFTPEQIEEF \nMYL3 \n52-63 \n58-95 \nEF-hand 1 \n\nDLSAIKIEFSKEQQEDF \nMYL1 \n33-49 \n44-79 \nEF-hand 1 \n\nHELYPIAKGDNQSPI \nCAH3 \n17-31 \n3-259 \nAlpha-carbonic anhydrase \n\nNSLTGEFKGKY \nKCRM \n163-173 \n125-367 \nPhosphagen kinase C-terminal \n\nASHHPADFTPAVHA \nHBA-A1 \n111-124 \n3-142 \nGLOBIN \n\nDKETPSGFTLDDV \nKCRM \n44-56 \n11-98 \nPhosphagen kinase N-terminal \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nMYL1 \n76-100 \n83-118 \nEF-hand \n\nQLVVDGVKLM \nKCRM \n351-360 \n125-367 \nPhosphagen kinase C-terminal \n\nKDLFDPIIQDRHG \nKCRM \n86-98 \n11-98 \nPhosphagen kinase N-terminal \n\nKGVVPLAGTNGETTTQGLDGLSER \nALDOA \n111-134 \n15-364 \nGlycolytic \n\nRVFDKEGNGTVMGAELR \nMYL1 \n147-163 \n133-150 \nEF-hand \n\nNADEVGGEALGRL \nHBB-BS \n20-32 \n2-147 \nGLOBIN \n\nNEHLGYVLTCPS \nKCRM \n274-285 \n125-367 \nPhosphagen kinase C-terminal \n\nNEEDHLRV \nKCRM \n230-237 \n125-367 \nPhosphagen kinase C-terminal \n\nVLTCPSNLGTGLRG \nKCRM \n280-293 \n125-367 \nPhosphagen kinase C-terminal \n\n","Protein name \nDescription \nPeptide number \nAssociation score \nwith obesity # \n\nAssociation score with \ndiabetes mellitus # \n\nTPI1 \nTriosephosphate isomerase \n2 \n0.012 \n0.012 \n\nKCRM \nCreatine kinase M-type \n17 \n0.022 \n0.017 \n\nTTN \nTitin \n1 \n0.026 \n0.063 \n\nGAPDH \nGlyceraldehyde-3-phosphate dehydrogenase \n1 \n0.071 \n0.117 \n\nHBA-A1 \nAlpha globin 1 \n1 \n0.014 \n0.038 \n\nENOB \nBeta-enolase \n1 \n0.040 \n0.048 \n\nCAH3 \nCarbonic anhydrase 3 \n1 \n0.074 \n0.094 \n\nTCTP \nTranslationally-controlled tumor protein \n1 \n0.028 \n0.040 \n\nHBB-BS \nBeta-globin \n1 \n0.008 \n0.020 \n\nACTS \nActin, alpha skeletal muscle \n1 \n0.006 \n0.188 \n\n# "],"string":"[\n \"Peptide sequence \\nProtein ID \\nProtein name \\nMr(KDa) \\nFold change \\nP-value \\n\\nUp-regulated peptides \\n\\nAVGAVFDISNADRLGSSEVEQ \\nP07310 \\nKCRM \\n2163 \\n9.728 \\n0.005 \\n\\nTNGAFTGEISPGMIKDLGATWV \\nP17751 \\nTPIS \\n2264 \\n8.019 \\n0.035 \\n\\nIADLVVGLCTGQIK \\nP21550 \\nENOB \\n1486 \\n6.77 \\n0.000 \\n\\nNYKPQEEYPDLSKHNNHMA \\nP07310 \\nKCRM \\n2315 \\n4.843 \\n0.024 \\n\\nEEIEEDAGLGNGGLGRLAAC \\nQ9WUB3 \\nPYGM \\n2030 \\n4.411 \\n0.004 \\n\\nGQCGDVLRALGQNPTQAEV \\nP09542 \\nMYL3 \\n2013 \\n4.077 \\n0.033 \\n\\nAGPLCLQEVDEPPQHAL \\nP70296 \\nPEBP1 \\n1873 \\n4.038 \\n0.004 \\n\\nEAFTVIDQNRDGIID \\nP97457 \\nMLRS \\n1705 \\n3.947 \\n0.026 \\n\\nDLFDPIIQDRHGGY \\nP07310 \\nKCRM \\n1645 \\n3.668 \\n0.022 \\n\\nKDLFDPIIQD \\nP07310 \\nKCRM \\n1203 \\n3.282 \\n0.000 \\n\\nLDDVIQTGVDNPGHP \\nP07310 \\nKCRM \\n1576 \\n3.265 \\n0.001 \\n\\nSEIIDVSSKAEEVK \\nA2ASS6 \\nTITIN \\n1533 \\n3.134 \\n0.002 \\n\\nVIACIGEKLDE \\nP17751 \\nTPIS \\n1246 \\n2.976 \\n0.003 \\n\\nGTNPTNAEVKKVLGNPSNEEM \\nQ545G5 \\nMYL1 \\n2180 \\n2.838 \\n0.006 \\n\\nADRLGSSEVEQVQLV \\nP07310 \\nKCRM \\n1629 \\n2.804 \\n0.001 \\n\\nIGQGTPIPDLPEVKRV \\nA2AQA9 \\nNEB \\n1719 \\n2.739 \\n0.002 \\n\\nSTTAPPIQSPLPVIPHQK \\nE9PYJ9 \\nLDB3 \\n1910 \\n2.685 \\n0.023 \\n\\nKADDGRPFPQVI \\nP05064 \\nALDOA \\n1342 \\n2.644 \\n0.021 \\n\\nKSMTEQEQQQLIDD \\nP07310 \\nKCRM \\n1692 \\n2.442 \\n0.001 \\n\\nGGGASLELLEGKVLPGVDA \\nP09411 \\nPGK1 \\n1781 \\n2.264 \\n0.028 \\n\\nLEGTLLKPNMVTPGHA \\nP05064 \\nALDOA \\n1677 \\n2.208 \\n0.010 \\n\\nVMVGMGQKDSYVGDEAQSK \\nP68134 \\nACTS \\n2028 \\n2.168 \\n0.021 \\n\\nDLEEATLQHEATAAALR \\nQ02566 \\nMYH6 \\n1838 \\n2.07 \\n0.038 \\n\\nAELQEVQITEEKPLLPG \\nQ9QYG0 \\nNDRG2 \\n1935 \\n2.043 \\n0.048 \\n\\nSEGKCAELEEELKTVTNNL \\nP58771 \\nTPM1 \\n2163 \\n2.038 \\n0.049 \\n\\nRSIKGYTLPPHC \\nP07310 \\nKCRM \\n1428 \\n1.967 \\n0.001 \\n\\nNSNSHSSTFDAGAGIALNDNFVK \\nP16858 \\nG3P \\n2365 \\n1.935 \\n0.038 \\n\\nTGKATSEASVSTPEETAPEPAKVPT \\nP70402 \\nMYBPH \\n2484 \\n1.919 \\n0.003 \\n\\nDNEYGYSNRVVDL \\nP16858 \\nG3P \\n1543 \\n1.898 \\n0.004 \\n\\nEELDAMMKEASGPINF \\nP97457 \\nMLRS \\n1781 \\n1.853 \\n0.012 \\n\\nSNNKDQGGYEDFVEGLRV \\nQ545G5 \\nMYL1 \\n2026 \\n1.816 \\n0.016 \\n\\nAEQIKHILANF \\nP63028 \\nTCTP \\n1283 \\n1.787 \\n0.044 \\n\\nGADPEDVITGAFK \\nP97457 \\nMLRS \\n1319 \\n1.782 \\n0.031 \\n\\nTSIEDAPITVQSKINQ \\nA2AQA9 \\nNEB \\n1743 \\n1.739 \\n0.028 \\n\\nLKGGDDLDPNYVLS \\nP07310 \\nKCRM \\n1505 \\n1.732 \\n0.040 \\n\\nIQTGVDNPGHPF \\nQ6P8J7 \\nKCRS \\n1281 \\n1.600 \\n0.045 \\n\\nAEVKKVLGNPSNEEMNAK \\nQ545G5 \\nMYL1 \\n1957 \\n1.595 \\n0.009 \\n\\nGQEQWEEGDLYDKEKQQKPF \\nE9Q8K5 \\nTITIN \\n2481 \\n1.586 \\n0.036 \\n\\nAGTNGETTTQGLDGLSERCA \\nP05064 \\nALDOA \\n2037 \\n1.582 \\n0.033 \\n\\nRVPTPNVSVVDLTCRLEKPAK \\nP16858 \\nG3P \\n2378 \\n1.531 \\n0.022 \\n\\nKVPEKPEVVEKVEPAPLK \\nF7CR78 \\nF7CR78 \\n2015 \\n1.527 \\n0.026 \\n\\nGETTTQGLDGLSERCAQ \\nP05064 \\nALDOA \\n1822 \\n1.526 \\n0.042 \\n\\nDown-regulated peptides \\n\\nKIEFTPEQIEEF \\nP09542 \\nMYL3 \\n1509 \\n0.612 \\n0.021 \\n\\nDLSAIKIEFSKEQQEDF \\nP05977 \\nMYL1 \\n2026 \\n0.600 \\n0.019 \\n\\nHELYPIAKGDNQSPI \\nP16015 \\nCAH3 \\n1681 \\n0.559 \\n0.038 \\n\\nNSLTGEFKGKY \\nP07310 \\nKCRM \\n1243 \\n0.545 \\n0.027 \\n\\nDEPKPYPYPNLDDF \\nQ3V1D3 \\nAMPD1 \\n1709 \\n0.543 \\n0.034 \\n\\nASHHPADFTPAVHA \\nQ91VB8 \\nHBA-A1 \\n1457 \\n0.537 \\n0.016 \\n\\n(Continued) \\n\\nFrontiers in Endocrinology | www.frontiersin.org \",\n \"Peptide sequence \\nProtein ID \\nProtein name \\nMr(KDa) \\nFold change \\nP-value \\n\\nSQVGDVLRALGTNPTNAE \\nQ545G5 \\nMYL1 \\n1841 \\n0.516 \\n0.047 \\n\\nADEIAKAQVAQPGGDTI \\nP70349 \\nHINT1 \\n1725 \\n0.515 \\n0.038 \\n\\nDKETPSGFTLDDV \\nP07310 \\nKCRM \\n1423 \\n0.478 \\n0.037 \\n\\nLGTNPTNAEVKKVLGNPSNEEMNAK \\nQ545G5 \\nMYL1 \\n2654 \\n0.467 \\n0.038 \\n\\nPHPYPALTPEQKK \\nP05064 \\nALDOA \\n1505 \\n0.407 \\n0.028 \\n\\nQLVVDGVKLM \\nP07310 \\nKCRM \\n1101 \\n0.400 \\n0.001 \\n\\nKDLFDPIIQDRHG \\nP07310 \\nKCRM \\n1553 \\n0.395 \\n0.022 \\n\\nKGVVPLAGTNGETTTQGLDGLSER \\nP05064 \\nALDOA \\n2399 \\n0.367 \\n0.015 \\n\\nRVFDKEGNGTVMGAELR \\nQ545G5 \\nMYL1 \\n1878 \\n0.341 \\n0.043 \\n\\nNADEVGGEALGRL \\nA8DUK4 \\nHBB-BS \\n1300 \\n0.339 \\n0.037 \\n\\nNEHLGYVLTCPS \\nP07310 \\nKCRM \\n1389 \\n0.328 \\n0.031 \\n\\nASHTEEEVSVSVPEVQKKTVTEEK \\nE9Q8K5 \\nTITIN \\n2669 \\n0.253 \\n0.023 \\n\\nNEEDHLRV \\nP07310 \\nKCRM \\n1010 \\n0.212 \\n0.027 \\n\\nVLTCPSNLGTGLRG \\nP07310 \\nKCRM \\n1444 \\n0.201 \\n0.018 \\n\\nGGYKPTDKHKTDL \\nP07310 \\nKCRM \\n1459 \\n0.127 \\n0.006 \\n\\nobservation was also demonstrated by other peptidomic studies. \\nFrom Steven W. Taylor group' results, several identified peptides \\nin the human islet cultures were derived from intracellular and \\ncytoskeletal proteins such as microtubule-associated protein 4 \\nand ubiquitin, which may result from a greater level of cellular \\nstress \",\n \"Peptide sequence \\nProtein \\nLocation \\nDomain \\nDescription \\n\\nUp-regualted peptides \\n\\nAVGAVFDISNADRLGSSEVEQ \\nKCRM \\n329-349 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nTNGAFTGEISPGMIKDLGATWV \\nTPIS \\n121-142 \\n57-295 \\nTIM \\n\\nIADLVVGLCTGQIK \\nENOB \\n381-394 \\n142-432 \\nEnolase C \\n\\nNYKPQEEYPDLSKHNNHMA \\nKCRM \\n13-31 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nEEIEEDAGLGNGGLGRLAAC \\nPYGM \\n124-143 \\n113-828 \\nPhosphorylase \\n\\nGQCGDVLRALGQNPTQAEV \\nMYL3 \\n83-101 \\n58-95 \\nEF-hand 1 \\n\\nEAFTVIDQNRDGIID \\nMLRS \\n32-46 \\n25-60 \\nEF-hand 1 \\n\\nDLFDPIIQDRHGGY \\nKCRM \\n87-100 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nKDLFDPIIQD \\nKCRM \\n86-95 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nLDDVIQTGVDNPGHP \\nKCRM \\n53-67 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nVIACIGEKLDE \\nTPIS \\n174-184 \\n57-295 \\nTIM \\n\\nGTNPTNAEVKKVLGNPSNEEM \\nMYL1 \\n39-59 \\n6-41 \\nEF-hand \\n\\nADRLGSSEVEQVQLV \\nKCRM \\n339-353 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nKADDGRPFPQVI \\nALDOA \\n87-98 \\n15-364 \\nGlycolytic \\n\\nKSMTEQEQQQLIDD \\nKCRM \\n177-190 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nGGGASLELLEGKVLPGVDA \\nPGK1 \\n395-413 \\n9-406 \\nPGK \\n\\nLEGTLLKPNMVTPGHA \\nALDOA \\n224-239 \\n15-364 \\nGlycolytic \\n\\nVMVGMGQKDSYVGDEAQSK \\nACTS \\n45-63 \\n4-377 \\nActin \\n\\nDLEEATLQHEATAAALR \\nMYH6 \\n1,179-1,195 \\n845-1,926 \\nMyosin_tail_1 \\n\\nSEGKCAELEEELKTVTNNL \\nTPM1 \\n186-204 \\n1-284 \\nCoiled coili \\n\\nRSIKGYTLPPHC \\nKCRM \\n135-146 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nSNNKDQGGYEDFVEGLRV \\nMYL1 \\n77-94 \\n83-118 \\nEF-hand \\n\\nAEQIKHILANF \\nTCTP \\n119-129 \\n1-172 \\nTCTP \\n\\nGADPEDVITGAFK \\nMLRS \\n93-105 \\n95-130 \\nEF-hand 2 \\n\\nLKGGDDLDPNYVLS \\nKCRM \\n115-128 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nGQEQWEEGDLYDKEKQQKPF \\nTITIN \\n1,691-1,710 \\n1,709-1,799 \\nIg-like \\n\\nAGTNGETTTQGLDGLSERCA \\nALDOA \\n117-136 \\n15-364 \\nGlycolytic \\n\\nRVPTPNVSVVDLTCRLEKPAK \\nGAPDH \\n232-252 \\n243-248 \\n[IL]-x-C-x-x-[DE] motif \\n\\nGETTTQGLDGLSERCAQ \\nALDOA \\n121-137 \\n15-364 \\nGlycolytic \\n\\nDown-regualted peptides \\n\\nKIEFTPEQIEEF \\nMYL3 \\n52-63 \\n58-95 \\nEF-hand 1 \\n\\nDLSAIKIEFSKEQQEDF \\nMYL1 \\n33-49 \\n44-79 \\nEF-hand 1 \\n\\nHELYPIAKGDNQSPI \\nCAH3 \\n17-31 \\n3-259 \\nAlpha-carbonic anhydrase \\n\\nNSLTGEFKGKY \\nKCRM \\n163-173 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nASHHPADFTPAVHA \\nHBA-A1 \\n111-124 \\n3-142 \\nGLOBIN \\n\\nDKETPSGFTLDDV \\nKCRM \\n44-56 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nLGTNPTNAEVKKVLGNPSNEEMNAK \\nMYL1 \\n76-100 \\n83-118 \\nEF-hand \\n\\nQLVVDGVKLM \\nKCRM \\n351-360 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nKDLFDPIIQDRHG \\nKCRM \\n86-98 \\n11-98 \\nPhosphagen kinase N-terminal \\n\\nKGVVPLAGTNGETTTQGLDGLSER \\nALDOA \\n111-134 \\n15-364 \\nGlycolytic \\n\\nRVFDKEGNGTVMGAELR \\nMYL1 \\n147-163 \\n133-150 \\nEF-hand \\n\\nNADEVGGEALGRL \\nHBB-BS \\n20-32 \\n2-147 \\nGLOBIN \\n\\nNEHLGYVLTCPS \\nKCRM \\n274-285 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nNEEDHLRV \\nKCRM \\n230-237 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\nVLTCPSNLGTGLRG \\nKCRM \\n280-293 \\n125-367 \\nPhosphagen kinase C-terminal \\n\\n\",\n \"Protein name \\nDescription \\nPeptide number \\nAssociation score \\nwith obesity # \\n\\nAssociation score with \\ndiabetes mellitus # \\n\\nTPI1 \\nTriosephosphate isomerase \\n2 \\n0.012 \\n0.012 \\n\\nKCRM \\nCreatine kinase M-type \\n17 \\n0.022 \\n0.017 \\n\\nTTN \\nTitin \\n1 \\n0.026 \\n0.063 \\n\\nGAPDH \\nGlyceraldehyde-3-phosphate dehydrogenase \\n1 \\n0.071 \\n0.117 \\n\\nHBA-A1 \\nAlpha globin 1 \\n1 \\n0.014 \\n0.038 \\n\\nENOB \\nBeta-enolase \\n1 \\n0.040 \\n0.048 \\n\\nCAH3 \\nCarbonic anhydrase 3 \\n1 \\n0.074 \\n0.094 \\n\\nTCTP \\nTranslationally-controlled tumor protein \\n1 \\n0.028 \\n0.040 \\n\\nHBB-BS \\nBeta-globin \\n1 \\n0.008 \\n0.020 \\n\\nACTS \\nActin, alpha skeletal muscle \\n1 \\n0.006 \\n0.188 \\n\\n# \"\n]"},"annotations_tableref":{"kind":"list like","value":["Table S1","Table 1","Table S2","Table S3","Table S4","Table S5","Table 2)","Table 3)","Table S6","Table 1","Table 1","Table 2","Table S2","Table S3"],"string":"[\n \"Table S1\",\n \"Table 1\",\n \"Table S2\",\n \"Table S3\",\n \"Table S4\",\n \"Table S5\",\n \"Table 2)\",\n \"Table 3)\",\n \"Table S6\",\n \"Table 1\",\n \"Table 1\",\n \"Table 2\",\n \"Table S2\",\n \"Table S3\"\n]"},"annotations_title":{"kind":"list like","value":["A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice","A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice"],"string":"[\n \"A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice\",\n \"A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice\"\n]"},"annotations_venue":{"kind":"list like","value":["Frontiers in Endocrinology | www.frontiersin.org"],"string":"[\n \"Frontiers in Endocrinology | www.frontiersin.org\"\n]"}}},{"rowIdx":84740,"cells":{"corpusid":{"kind":"number","value":13635771,"string":"13,635,771"},"updated":{"kind":"string","value":"2022-03-19T02:41:50Z"},"content_source_oainfo_license":{"kind":"string","value":"CCBY"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"https://www.mdpi.com/1422-0067/13/7/7902/pdf"},"content_source_oainfo_status":{"kind":"string","value":"GOLD"},"content_source_pdfsha":{"kind":"string","value":"25cf22b87121018681c2d8cd096ca798727de852"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.3390/ijms13077902"},"externalids_mag":{"kind":"string","value":"2085991986"},"externalids_pubmed":{"kind":"string","value":"22942680"},"externalids_pubmedcentral":{"kind":"string","value":"3430211"},"content_text":{"kind":"string","value":"\nA Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways\n2012\n\nXu Hou \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nYuqin Shen \nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nChao Zhang \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nLiru Zhang lrzhang10@mails.jlu.edu.cnl.z. \nYanyan Qin \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nYongli Yu ylyu@mail.jlu.edu.cn \nDepartment of Immunology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n\nLiying Wang \nDepartment of Molecular Biology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n\nXinhua Sun \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nA Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways\n\nInt. J. Mol. Sci\n13201210.3390/ijms13077902Received: 10 May 2012; in revised form: 11 May 2012 / Accepted: 15 June 2012 /International Journal of Molecular Sciences * Author to whom correspondence should be addressed; E-Mail: sunxinh@jlu.edu.cn;oligodeoxynucleotideosteoblastdifferentiationERK1/2 MAPKp38 MAPK\nA specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.OPEN ACCESSInt. J. Mol. Sci. 2012, 13 7903\n\nIntroduction\n\nOsteoblasts play an extremely important role in bone formation and remodeling processes. Osteoblasts are derived from mesenchymal stem cells, and can differentiate into mature and functional osteoblasts that produce extracellular matrix proteins and regulators of matrix mineralization [1]. Runx2 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Runx2 −/− mice show a complete lack of both intramembranous and endochondral ossification owing to maturation arrest of osteoblasts [2,3]. However, Runx2 can regulate the expression of osteoblast-specific genes including ALP, type I collagen and osteocalcin. To date, numerous reports have shown that signaling pathways and transcription factors could participate in osteoblastogenesis by regulating the production or activity of Runx2 [4]. Furthermore, Runx2 is necessary throughout life to promote the differentiation of new osteoblasts during bone remodeling [5].\n\nOligodeoxynucleotides (ODNs) containing unmethylated nucleotide motifs are immunostimulatory in vertebrates, and some ODNs containing CpG motifs are used for treating cancer, virus-associated diseases, and infections [6][7][8][9]. Recently, specific ODNs were found to have an effect on modulating osteoclast-and osteoblast-lineage cells [10]. In addition, CpG-ODN with a phosphorothioate backbone inhibits the BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines [11]. Chang et al. demonstrated that a novel CpG-ODN enhances the inhibitory effects on osteoclast differentiation by downregulation of TREM-2 [12]. In our previous studies, we have shown that ODN MT01, a synthetic 27-mer single stranded ODN with a design based on human mitochondrial DNA, promotes the proliferation and activation of osteoblasts [13], and stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts [14]. However, the detailed signaling pathway in which ODN MT01 modulates the differentiation of osteoblasts has not been fully elucidated.\n\nMitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific protein kinases, including p38, JNK and ERK1/2 (p44/p42). These molecules play important roles in cell differentiation, proliferation and death [15], and are activated by phosphorylation of tyrosine/threonine residues in signal transduction cascades. Previous studies have shown that the p38 MAPK and ERK1/2 pathways are involved in the proliferation and differentiation of osteoblasts [16][17][18]. Therefore, we hypothesized that ODN MT01 might regulate the differentiation of osteoblastic cells via the MAPKs pathway.\n\nThis study aimed to investigate the regulation of MAPKs in response to ODN MT01, and to elucidate the involvement of the MAPK signaling pathway in regulation of Runx2, osteocalcin, type I collagen and ALP expression in ODN MT01-treated MG 63 cells.\n\n\nResults and Discussion\n\n\nInternalization Analysis of ODNs\n\nThe design of ODN MT01, a 27-mer ODN, was based on the motif sequence 5'-ACCCCCTCTACCCCCTCT-3' in human mitochondrial DNA. MT01 is a cytosine-rich ODN and contains five successive cytosines in each of its 9-mer motifs (5'-ACCCCCTCT-3') [19]. To investigate whether the effects of an ODN required internalization into cells, Cy5-labeled ODN MT01 was synthesized to trace the sites of ODN actions. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, and is temperature-and energy-dependent [20]. This result was consistent with our observations, as shown in Figure 1, ODN MT01 entered cells by endocytosis after incubation with MG 63 cells for 3 h. The fluorescence intensity was enhanced over time, and the intensity at 6 h was significantly higher than that at 3 h, and the intensity at 12 h was higher than that at 3 and 6 h. It was apparent that ODN MT01 stimulation of osteoblasts required internalization. \n\n\nEffects of ODN MT01 on MAPK Signaling Proteins ERK1/2 and p38\n\nTo investigate the effect of ODN MT01 on MAPKs, both total and phosphorylated levels of MAPKs were investigated by Western blotting after 0, 30, 60 min, 24 h and 48 h of treatment with 1 µg/mL ODN MT01. To better demonstrate that the effects of MT01 in MG 63 cells were in response to the specific sequence of MT01 we tested another ODN sequence, ODN FC003, a 24-mer ODN(5'-TCTCTCTCTCTCTCTCTCTCTCTC-3'). The design of ODN FC003 was also based on human mitochondrial DNA, and contained successive thymidine-cytosine nucleotides. In our previous studies [13,14], we found that ODN FC003 is inactive in the regulation of osteoblasts and bone marrow mesenchymal stem cells. Therefore, we chose this sequence as a negative control. As shown in Figure 2A, phosphorylation of ERK1/2 was observed after 30 min of treatment with ODN MT01, which increased by 13.1-fold after 60 min compared with that at 0 min. Furthermore, the active effect lasted up to 48 h (8.9-fold increase), while the total level of ERK1/2 protein remained unchanged. For p38, there was no obvious change in the phosphorylation level before 30 min. However, p38 phosphorylation increased after 60 min of treatment with MT01 ( Figure 2B), and showed an increase of 4.7 fold at 24 h, which lasted up to 48 h. As shown in Figure 2 A and B, the FC003 sequence had no active effect on MG 63 cells via ERK1/2 or p38 MAPKs. Thus, these findings indicated that ODN MT01 induced the phosphorylation of ERK1/2 and p38 MAPK in MG 63 cells.\n\nTo investigate the mechanisms by which ODNs regulate the differentiation of osteoblasts, it is important to identify the functional role of signaling pathways activated by ODNs. At present, several signaling pathways have been shown to be involved in osteogenesis, such as MAPKs, BMP-2-Smad and NF-κB [21][22][23]. Among them, p38 and ERK1/2 MAPKs have been reported to be important for early osteoblast differentiation in various cell lines [24]. In this study, we found that MT01 was capable of activating ERK1/2 and p38 MAPK pathways indicating that MT01 induced the phosphorylation of MAPKs at the early stage of osteoblast differentiation. Previous reports have shown that ERK and p38 are phosphorylated in response to CpG-ODN, and p38 is phosphorylated to a lesser extent [25]. Thus, our findings were consistent with previous reports. ERK1/2 reacted more rapidly than p38 MAPK, and activation of both lasted up to 48 h after treatment with MT01. Once MAPK is activated by ODNs, transcription factors and other kinases may be phosphorylated to initiate events such as gene expression and post-translational protein modifications. \n\n\nEffects of ERK and p38 Inhibitors on Up-Regulation of ALP Activity Induced by ODN MT01\n\nTo investigate the involvement of ERK and p38 MAPK in modulation of ALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38 (U0126 and SB203580, respectively) were used. Cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. As shown in Figure 2, compared with the control (phosphate-buffered saline (PBS) treatment), ALP activity was significantly up-regulated after ODN MT01 treatment at 48 and 72 h. Notably, U0126 and SB203580 significantly inhibited the up-regulation induced by ODN MT01.\n\nThe activity of osteogenic differentiation marker ALP is an important index to indicate osteogenesis at an early stage. As shown in Figure 3, after 24 h of treatment with ODN MT01, the activity of ALP was increased, compared with that in the control, but the difference was not significant. The density of cells increased after 48 and 72 h, and ODN MT01 exhibited an obvious promoting effect on osteogenic differentiation. In addition, inhibitors significantly downregulated ALP activity induced by ODN MT01 after 48 and 72 h. These results indicated that the ERK and p38 MAPK pathways were probably involved in ODN MT01-induced promotion of ALP activity. \n\n\nEffect of ERK and p38 Inhibitors on Runx2 Expression in Response to ODN MT01\n\nThe above results confirmed that ODN MT01 induced the phosphorylation of ERK and p38 MAPK and further activated these MAPK signaling pathways. To investigate the role of Runx2, which is one of the most important factors for osteoblast differentiation, we used the ERK inhibitor U0126 and p38 inhibitor SB203580 to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 for 72 h. The total and phosphorylated protein levels of Runx2 were detected using Western blotting. As shown in Figure 4, there were obvious differences in the phosphorylation levels of ERK and p38 between the MT01-treatment and control groups; p-ERK and p-p38 were significantly inhibited in the presence of U0126 and SB203580, which were induced by MT01. For Runx2, there was no obvious change in protein levels in these groups, but MT01 induced notable phosphorylation of Runx2 (1.8-fold increase compared with the control). However, the up-regulated phosphorylation of Runx2 induced by MT01 was inhibited in the presence of U0126 and SB203580.\n\nRunx2 is an essential transcription factor for osteoblast differentiation and bone formation, and inactivation of one Runx2 allele will cause cleidocranial dysplasia syndrome, a disease characterized by delayed osteoblast differentiation for bone formation through intramembranous ossification in mice and humans [3,26]. Previous reports have shown that extracellular matrix production can induce osteoblast differentiation by increasing Runx2 transcriptional activity, but not the mRNA or protein levels [27][28][29], which concurs with our results. Runx2 activity is controlled by various extracellular signaling pathways, and post-translational modifications (phosphorylation, acetylation and ubiquitination) can affect its stability and activity [30,31]. Among the post-translational modifications, the role of Runx2 phosphorylation has been well characterized. Ge et al. found that Runx2 is regulated by ERK1/2 and p38 MAPK-mediated phosphorylation [32], and ERK is more important for Runx2 phosphorylation than p38 [33]. Interestingly, in the present study, we found the same results in which inhibition of ERK showed a stronger effect on the expression of p-Runx2 (1-fold increase compared with the control) than that of p38 inhibition (1.2-fold increase compared with the control). These results suggested that Runx2 is phosphorylated on a complex set of sites that partially overlap between ERK and p38. Another study found that phosphorylation of p38 MAPK induces activation of Runx2 via TAK1 and MEK3 signaling pathways during early osteoblastic differentiation [21]. Here, we showed that MT01 greatly increased Runx2 phosphorylation, which was potently repressed by inhibitors of ERK and p38. Taken together, our results were consistent with other findings and demonstrated that MT01-induced phosphorylation of Runx2 was mediated by activation of both ERK1/2 and p38 MAPK signaling pathways, and ERK MAPK may be more important. \n\n\nEffects of ERK and p38 Inhibitors on the Expression of Osteocalcin and Type I Collagen\n\nRunx2 has been shown to regulate the expression of osteoblast-specific genes, such as ALP, osteocalcin and type I collagen [34]. To further confirm our results, we analyzed the expression of subsets of osteogenic marker genes including osteocalcin and type I collagen. We used ERK and p38 inhibitors to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 or ODN FC003 for 15 days. Then, the mRNA and protein expression of osteocalcin and type I collagen were analyzed by real-time PCR and Western blot, respectively. As shown in Figure 5A, compared with the control g, the protein expression of osteocalcin was increased by ODN MT01 treatment. Compared with ODN MT01 treatment, the protein level of osteocalcin was significantly decreased when cells were pretreated with the ERK and p38 inhibitors. The expression level of type I collagen exhibited a similar tendency with osteocalcin. As shown in Figure 5B,C, the mRNA levels were concurrent with the protein expression. Osteocalcin protein expression marks the late phase of osteogenic differentiation, and is only produced by mineralizing cells types. In addition, osteocalcin is considered as a specific marker of osteoblast differentiation and maturity. Type I collagen is the major product of osteoblasts, and accounts for 90% of the matrix protein content [35]. Type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life. Osteocalcin and type I collagen protein expression induced by ODN MT01 was increased at late stages of osteoblast differentiation, suggesting that ODN MT01 controll of the expression of osteocalcin and type I collagen in these cells could also control osteoblast differentiation and function. Furthermore, ERK and p38 MAPK inhibitors obviously decreased protein expression induced by MT01. Therefore, ODN MT01 induced the expression of osteocalcin and type I collagen via ERK and p38 MAPK signaling pathways.\n\n\nExperimental Section\n\n\nMaterials\n\nODNs MT01 (5'-ACCCCCTCTACCCCCTCTACCCCCTCT-3') and FC003 (5'-TCTCTCTCTCTC TCTCTCTCTCTC-3') were synthesized by TaKaRa (Dalian, China), and were dissolved in axenic PBS. The human osteoblastic cell line MG 63 was obtained from the American Type Culture Collection (CRL-1427). An Alkaline phosphatase kit and micro-BCA assay kit were obtained from Jiancheng Biological Reagent Co. (Nanjing, China). A real-time PCR kit was purchased from TaKaRa (Tokyo, Japan). Monoclonal antibodies against p38, p-p38, p44/p42 and p-p44/42, were purchased from Santa Cruz Biotech. and those against Runx2, osteocalcin and type I collagen were purchased from Abcam (UK). The anti-phospho-Runx2 antibody was purchased from Abcam Co (UK). The phosphorylation site was Ser533.\n\n\nCell Culture\n\nMG 63 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO 2 . Cells were seeded at an initial density of 2 × 10 5 cells/mL. To investigate the effect of ODN MT01 on MAPKs, cells were treated with 1 µg/mL ODN MT01 for 0, 30, 60 min, 24 and 48 h. To investigate activation of Runx2, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 72 h. To investigate the effects of ERK and p38 inhibitors on the mRNA and protein levels of osteocalcin and type I collagen, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 15 days. Medium was changed every 3 days, and ODNs and inhibitors were added at the same time.\n\n\nFluorescent Labeling of MT01\n\nCy5 was conjugated to the 5'-end of MT01, and then dissolved with PBS. Cells were cultured on 24 × 24 mm coverslips at a density of 1 × 10 4 cells/well overnight. Then, cells were treated with 1 µg/mL Cy5-labeled MT01 for 3, 6 and 12 h. Cells were washed three times with PBS according to the time points. While protected from light, Hoechst 33342 was added at 1 µg/mL for 5 min to stain nuclei. A laser scanning confocal microscope (Olympus IX81, Japan) was used to observe cell climb-chips. Stained cells were imaged with Olympus Fluoview FV1000 Viewer (Version1.7.c; Olympus Corporation: Tokyo, Japan, 2007).\n\n\nWestern Blot Analysis\n\nWestern blotting was used to evaluate total and phosphorylated protein levels of p38 and p44/42. MG 63 cells were cultured in 10-cm dishes and treated with 1 µg/mL ODN MT01 for the indicated times. To evaluate the protein levels of Runx2 and p-Runx2, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without ODN MT01 for 72 h. To investigate the levels of osteocalcin and type I collagen, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without ODN MT01 for 15 days. Then cells were collected, washed twice with cold Tris-buffered saline and resuspended in lysis buffer (50 mM Tris, pH 7.6, 0.01% EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/mL leupeptin). The protein concentration was measured using a BCA Protein Assay Reagent kit. Protein samples (40 μg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Then membranes were incubated with secondary antibodies at 20 °C for 2 h, and developed using an ECL chemiluminescent system. Loading differences were normalized using a GAPDH antibody.\n\n\nALP Activity Assay\n\nCells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. Then, the cells were collected and lysed to measure ALP activity. The assay was conducted using an Alkaline Phosphatase Kit, according to the manufacturer's instructions. The protein concentration of cell lysates was measured using a micro-BCA assay kit, and ALP activity was normalized to the total protein concentration. Values were the averages of triplicate measurements.\n\n\nReal-Time PCR\n\nTo quantify mRNA expression levels, real-time PCR was performed with cDNA samples. Primers were designed using qPrimerDepot(Nucleic Acids Res. 2007), a primer database for quantitative real-time PCR. Real-time PCR was performed using an ABI Steponeplus (ABI PRISM, Carlsbad, NM, USA), which allowed real-time monitoring of increases in PCR product concentrations after each cycle based on the fluorescence of the double-stranded DNA specific dye SYBR green. The number of cycles required to produce a detectable product above background was measured for each sample. These cycle numbers were then used to calculate fold differences in the initial mRNA level for each sample using the following method. First, the cycle number difference for GAPDH, a housekeeping gene, was determined in the control sample and appropriate ODN MT01-treated sample. Values were the averages of triplicate measurements. PCR primers used were as follows: GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'; Type I collagen forward, 5'-AGGGCCAAGACGAAGACATC-3' and reverse, 5'-AGATCACGTCATCGCACAACA-3'; OC forward, 5'-TGAGAGCCCTCACACTCCTC-3' and reverse, 5'-GCCGTAGAAGCGCCGATAGGC-3'.\n\n\nStatistical Analysis\n\nAll experiments were performed with triplicate independent samples and repeated at least twice. Results were expressed as the mean ± SD. ANOVA and the Bonferroni post-hoc test were used to compare the differences between the ODN MT01-treatment group and other groups. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed with SAS software (version 8.0, SAS: Cary, NC, USA).\n\n\nConclusions\n\nIn this study, a specific ODN, ODN MT01, was endocytosed by osteoblasts and found to up-regulate the expression level of osteocalcin, type I collagen and ALP in MG 63 cells via the ERK1/2 and p38 MAPK pathways. This study provides further insight into the use of ODN MT01 for in vitro experimentation, and supports the potential use of ODN MT01 to regulate the rebuilding of bone.\n\nFigure 1 .\n1Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.\n\nFigure 1 .\n1Cont\n\nFigure 2 .\n2Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.\n\nFigure 3 .\n3Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72\n\nFigure 4 .\n4Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.\n\nFigure 5 .\n5MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD (\n© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).\nAcknowledgmentsThis research was supported by the Natural Science Funds of Jilin Province, China (No. 201015203). Quanshun Li is gratefully acknowledged for his correction and proofreading of the manuscript.\nRegulation of gene expression in osteoblasts. E D Jensen, R Gopalakrishnan, J J Westendorf, Biofactors. 36Jensen, E.D.; Gopalakrishnan, R.; Westendorf, J.J. Regulation of gene expression in osteoblasts. Biofactors 2010, 36, 25-32.\n\nCbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. 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Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2012, 27, 538-551.\n\nRegulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. A Yamaguchi, T Komori, T Suda, Endocr. Rev. 21Yamaguchi, A.; Komori, T.; Suda, T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 2000, 21, 393-411.\n\nCbfa1 contributes to the osteoblast-specific expression of type I collagen genes. B Kern, J Shen, M Starbuck, G Karsenty, J. Biol. Chem. 276Kern, B.; Shen, J.; Starbuck, M.; Karsenty, G. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J. Biol. Chem. 2001, 276, 7101-7107.\n"},"annotations_abstract":{"kind":"list like","value":["A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.OPEN ACCESSInt. J. Mol. Sci. 2012, 13 7903"],"string":"[\n \"A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.OPEN ACCESSInt. J. Mol. Sci. 2012, 13 7903\"\n]"},"annotations_author":{"kind":"list like","value":["Xu Hou \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n","Yuqin Shen \nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n","Chao Zhang \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n","Liru Zhang lrzhang10@mails.jlu.edu.cnl.z. ","Yanyan Qin \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n","Yongli Yu ylyu@mail.jlu.edu.cn \nDepartment of Immunology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n","Liying Wang \nDepartment of Molecular Biology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n","Xinhua Sun \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n"],"string":"[\n \"Xu Hou \\nDepartment of Orthodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\",\n \"Yuqin Shen \\nDepartment of Periodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\",\n \"Chao Zhang \\nDepartment of Orthodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\",\n \"Liru Zhang lrzhang10@mails.jlu.edu.cnl.z. \",\n \"Yanyan Qin \\nDepartment of Orthodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\\nDepartment of Periodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\",\n \"Yongli Yu ylyu@mail.jlu.edu.cn \\nDepartment of Immunology, Medical College of Norman Bethune\\nJilin University\\n130021ChangchunChina\\n\",\n \"Liying Wang \\nDepartment of Molecular Biology, Medical College of Norman Bethune\\nJilin University\\n130021ChangchunChina\\n\",\n \"Xinhua Sun \\nDepartment of Orthodontics\\nSchool of Stomatology\\nJilin University\\n1500 Qinghua Road130021ChangchunChina\\n\"\n]"},"annotations_authoraffiliation":{"kind":"list like","value":["Department of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina","Department of Periodontics\nSchool of Stomatology\nJilin University\n1500 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\"Li\",\n \"Liu\",\n \"Pan\",\n \"Suzuki\",\n \"Guicheux\",\n \"Palmer\",\n \"Miura\",\n \"Oiso\",\n \"Bonjour\",\n \"Caverzasio\",\n \"Zou\",\n \"Amcheslavsky\",\n \"Wagner\",\n \"Karsenty\",\n \"Xiao\",\n \"Cui\",\n \"Ducy\",\n \"Karsenty\",\n \"Franceschi\",\n \"Xiao\",\n \"Jiang\",\n \"Thomas\",\n \"Benson\",\n \"Guan\",\n \"Karsenty\",\n \"Franceschi\",\n \"Franceschi\",\n \"Ge\",\n \"Xiao\",\n \"Roca\",\n \"Jiang\",\n \"Bae\",\n \"Lee\",\n \"Jeon\",\n \"Lee\",\n \"Choi\",\n \"Lee\",\n \"Kim\",\n \"Jin\",\n \"Ryoo\",\n \"Choi\",\n \"Yoshida\",\n \"Nishino\",\n \"Ge\",\n \"Xiao\",\n \"Jiang\",\n \"Yang\",\n \"Hatch\",\n \"Roca\",\n \"Franceschi\",\n \"Ge\",\n \"Yang\",\n \"Zhao\",\n \"Yu\",\n \"Kirkwood\",\n \"Franceschi\",\n \"Yamaguchi\",\n \"Komori\",\n \"Suda\",\n \"Kern\",\n \"Shen\",\n \"Starbuck\",\n \"Karsenty\"\n]"},"annotations_bibentry":{"kind":"list like","value":["Regulation of gene expression in osteoblasts. E D Jensen, R Gopalakrishnan, J J Westendorf, Biofactors. 36Jensen, E.D.; Gopalakrishnan, R.; Westendorf, J.J. Regulation of gene expression in osteoblasts. Biofactors 2010, 36, 25-32.","Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. F Otto, A P Thornell, T Crompton, A Denzel, K C Gilmour, I R Rosewell, G W Stamp, R S Beddington, S Mundlos, B R Olsen, Cell. 89Otto, F.; Thornell, A.P.; Crompton, T.; Denzel, A.; Gilmour, K.C.; Rosewell, I.R.; Stamp, G.W.; Beddington, R.S.; Mundlos, S.; Olsen, B.R.; et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997, 89, 765-771.","Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. S Mundlos, F Otto, C Mundlos, J B Mulliken, A S Aylsworth, S Albright, D Lindhout, W G Cole, W Henn, J H Knoll, Cell. 89Mundlos, S.; Otto, F.; Mundlos, C.; Mulliken, J.B.; Aylsworth, A.S.; Albright, S.; Lindhout, D.; Cole, W.G.; Henn, W.; Knoll, J.H.; et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997, 89, 773-779.","Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. G S Stein, J B Lian, A J Van Wijnen, J L Stein, M Montecino, A Javed, S K Zaidi, D W Young, J Y Choi, S M Pockwinse, Oncogene. 23Stein, G.S.; Lian, J.B.; van Wijnen, A.J.; Stein, J.L.; Montecino, M.; Javed, A.; Zaidi, S.K.; Young, D.W.; Choi, J.Y.; Pockwinse, S.M. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 2004, 23, 4315-4329.","A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. P Ducy, M Starbuck, M Priemel, J Shen, G Pinero, V Geoffroy, M Amling, G Karsenty, Ducy, P.; Starbuck, M.; Priemel, M.; Shen, J.; Pinero, G.; Geoffroy, V.; Amling, M.; Karsenty, G. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.",". Genes Dev. 13Genes Dev. 1999, 13, 1025-1036.","Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. P Cerkovnik, B Jezersek Novakovic, V Stegel, S Novakovic, Int. J. Oncol. 38Cerkovnik, P.; Jezersek Novakovic, B.; Stegel, V.; Novakovic, S. Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. Int. J. Oncol. 2011, 38, 1749-1758.","Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. C S Liu, Y Sun, Y H Hu, L Sun, Vaccine. 28Liu, C.S.; Sun, Y.; Hu, Y.H.; Sun, L. Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. Vaccine 2010, 28, 4153-4161.","Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. M Bao, Y Zhang, M Wan, L Dai, X Hu, X Wu, L Wang, P Deng, J Wang, J Chen, Clin. Immunol. 118Bao, M.; Zhang, Y.; Wan, M.; Dai, L.; Hu, X.; Wu, X.; Wang, L.; Deng, P.; Wang, J.; Chen, J.; et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. Clin. Immunol. 2006, 118, 180-187.","Synthetic oligonucleotides as modulators of inflammation. D Klinman, H Shirota, D Tross, T Sato, S Klaschik, J. Leukoc Biol. 84Klinman, D.; Shirota, H.; Tross, D.; Sato, T.; Klaschik, S. Synthetic oligonucleotides as modulators of inflammation. J. Leukoc Biol. 2008, 84, 958-964.","Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. A Amcheslavsky, H Hemmi, S Akira, J. Bone Miner. Res. 20Amcheslavsky, A.; Hemmi, H.; Akira, S.; Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. J. Bone Miner. Res. 2005, 20, 1692-1699.","CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. N N Norgaard, T Holien, S Jonsson, H Hella, T Espevik, A Sundan, T Standal, J. Immunol. 185Norgaard, N.N.; Holien, T.; Jonsson, S.; Hella, H.; Espevik, T.; Sundan, A.; Standal, T. CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. J. Immunol. 2010, 185, 3131-3139.","Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. J H Chang, E J Chang, H H Kim, S K Kim, Biochem. Biophys. Res. Commun. 389Chang, J.H.; Chang, E.J.; Kim, H.H.; Kim, S.K. Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. Biochem. Biophys. Res. Commun. 2009, 389, 28-33.","An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Z Feng, Y Shen, L Wang, L Cheng, J Wang, Q Li, W Shi, X Sun, Int. J. Mol. Sci. 12Feng, Z.; Shen, Y.; Wang, L.; Cheng, L.; Wang, J.; Li, Q.; Shi, W.; Sun, X. An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Int. J. Mol. Sci. 2011, 12, 2543-2555.","An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Y Shen, Z Feng, C Lin, X Hou, X Wang, J Wang, Y Yu, L Wang, X Sun, Int. J. Mol. Sci. 13Shen, Y.; Feng, Z.; Lin, C.; Hou, X.; Wang, X.; Wang, J.; Yu, Y.; Wang, L.; Sun, X. An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Int. J. Mol. Sci. 2012, 13, 2877-2892.","Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. A M Bennett, N K Tonks, Science. 278Bennett, A.M.; Tonks, N.K. Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science 1997, 278, 1288-1291.","Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, 10.1002/jbmr.561J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2011, 27, doi: 10.1002/jbmr.561.","Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. H M Jeong, E H Han, Y H Jin, Y P Hwang, H G Kim, B H Park, J Y Kim, Y C Chung, K Y Lee, H G Jeong, Food Chem. Toxicol. 48Jeong, H.M.; Han, E.H.; Jin, Y.H.; Hwang, Y.P.; Kim, H.G.; Park, B.H.; Kim, J.Y.; Chung, Y.C.; Lee, K.Y.; Jeong, H.G. Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. Food Chem. Toxicol. 2010, 48, 3362-3368.","Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. D Reddi, S J Brown, G N Belibasakis, Microb. Pathog. 51Reddi, D.; Brown, S.J.; Belibasakis, G.N. Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. Microb. Pathog. 2011, 51, 415-420.","Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice. G Yang, M Wan, Y Zhang, L Sun, R Sun, D Hu, X Zhou, L Wang, X Wu, Y Yu, Immunology. 131Yang, G.; Wan, M.; Zhang, Y.; Sun, L.; Sun, R.; Hu, D.; Zhou, X.; Wang, L.; Wu, X.; Yu, Y. Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice. Immunology 2010, 131, 501-512","Therapeutic potential of Toll-like receptor 9 activation. A M Krieg, Nat. Rev. Drug. Discov. 5Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug. Discov. 2006, 5, 471-484.","The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. M B Greenblatt, J H Shim, W Zou, D Sitara, M Schweitzer, D Hu, S Lotinun, Y Sano, R Baron, J M Park, J. Clin. Invest. 120Greenblatt, M.B.; Shim, J.H.; Zou, W.; Sitara, D.; Schweitzer, M.; Hu, D.; Lotinun, S.; Sano, Y.; Baron, R.; Park, J.M.; et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J. Clin. Invest. 2010, 120, 2457-2473.","Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. M Kawano, W Ariyoshi, K Iwanaga, T Okinaga, M Habu, I Yoshioka, K Tominaga, T Nishihara, Biochem. Biophys. Res. Commun. 405Kawano, M.; Ariyoshi, W.; Iwanaga, K.; Okinaga, T.; Habu, M.; Yoshioka, I.; Tominaga, K.; Nishihara, T. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. Biochem. Biophys. Res. Commun. 2011, 405, 575-580.","Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. D Zhang, H Zheng, J Zhao, L Lin, C Li, J Liu, Y Pan, J. Periodontal. Res. 46Zhang, D.; Zheng, H.; Zhao, J.; Lin, L.; Li, C.; Liu, J.; Pan, Y. Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. J. Periodontal. Res. 2011, 46, 31-38.","Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. A Suzuki, J Guicheux, G Palmer, Y Miura, Y Oiso, J P Bonjour, J Caverzasio, 30BoneSuzuki, A.; Guicheux, J.; Palmer, G.; Miura, Y.; Oiso, Y.; Bonjour, J.P.; Caverzasio, J. Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. Bone 2002, 30, 91-98.","Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. W Zou, A Amcheslavsky, J. Biol. Chem. 278Zou, W.; Amcheslavsky, A.; Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. J. Biol. Chem. 2003, 278, 16732-16740.","Genetic control of skeletal development. E F Wagner, G Karsenty, Curr. Opin. Genet. Dev. 11Wagner, E.F.; Karsenty, G. Genetic control of skeletal development. Curr. Opin. Genet. Dev. 2001, 11, 527-532.","Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. G Xiao, Y Cui, P Ducy, G Karsenty, R T Franceschi, Mol. Endocrinol. 11Xiao, G.; Cui, Y.; Ducy, P.; Karsenty, G.; Franceschi, R.T. Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. Mol. Endocrinol. 1997, 11, 1103-1113.","MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. G Xiao, D Jiang, P Thomas, M D Benson, K Guan, G Karsenty, R T Franceschi, J. Biol. Chem. 275Xiao, G.; Jiang, D.; Thomas, P.; Benson, M.D.; Guan, K.; Karsenty, G.; Franceschi, R.T. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J. Biol. Chem. 2000, 275, 4453-4459.","Transcriptional regulation of osteoblasts. R T Franceschi, C Ge, G Xiao, H Roca, D Jiang, Cells Tissues Organs. 189Franceschi, R.T.; Ge, C.; Xiao, G.; Roca, H.; Jiang, D. Transcriptional regulation of osteoblasts. Cells Tissues Organs 2009, 189, 144-152.","Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. S C Bae, Y H Lee, Gene. 366Bae, S.C.; Lee, Y.H. Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. Gene 2006, 366, 58-66.",". E J Jeon, K Y Lee, N S Choi, M H Lee, H N Kim, Y H Jin, H M Ryoo, J Y Choi, M Yoshida, N Nishino, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylationJeon, E.J.; Lee, K.Y.; Choi, N.S.; Lee, M.H.; Kim, H.N.; Jin, Y.H.; Ryoo, H.M.; Choi, J.Y.; Yoshida, M.; Nishino, N.; et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation.",". J. Biol. Chem. 281J. Biol. Chem. 2006, 281, 16502-16511.","Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor. C Ge, G Xiao, D Jiang, Q Yang, N E Hatch, H Roca, R T Franceschi, Ge, C.; Xiao, G.; Jiang, D.; Yang, Q.; Hatch, N.E.; Roca, H.; Franceschi, R.T. Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor.",". J. Biol. Chem. 284J. Biol. Chem. 2009, 284, 32533-32543.","Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2012, 27, 538-551.","Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. A Yamaguchi, T Komori, T Suda, Endocr. Rev. 21Yamaguchi, A.; Komori, T.; Suda, T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 2000, 21, 393-411.","Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. B Kern, J Shen, M Starbuck, G Karsenty, J. Biol. Chem. 276Kern, B.; Shen, J.; Starbuck, M.; Karsenty, G. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J. Biol. Chem. 2001, 276, 7101-7107."],"string":"[\n \"Regulation of gene expression in osteoblasts. E D Jensen, R Gopalakrishnan, J J Westendorf, Biofactors. 36Jensen, E.D.; Gopalakrishnan, R.; Westendorf, J.J. Regulation of gene expression in osteoblasts. Biofactors 2010, 36, 25-32.\",\n \"Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. F Otto, A P Thornell, T Crompton, A Denzel, K C Gilmour, I R Rosewell, G W Stamp, R S Beddington, S Mundlos, B R Olsen, Cell. 89Otto, F.; Thornell, A.P.; Crompton, T.; Denzel, A.; Gilmour, K.C.; Rosewell, I.R.; Stamp, G.W.; Beddington, R.S.; Mundlos, S.; Olsen, B.R.; et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997, 89, 765-771.\",\n \"Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. S Mundlos, F Otto, C Mundlos, J B Mulliken, A S Aylsworth, S Albright, D Lindhout, W G Cole, W Henn, J H Knoll, Cell. 89Mundlos, S.; Otto, F.; Mundlos, C.; Mulliken, J.B.; Aylsworth, A.S.; Albright, S.; Lindhout, D.; Cole, W.G.; Henn, W.; Knoll, J.H.; et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997, 89, 773-779.\",\n \"Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. G S Stein, J B Lian, A J Van Wijnen, J L Stein, M Montecino, A Javed, S K Zaidi, D W Young, J Y Choi, S M Pockwinse, Oncogene. 23Stein, G.S.; Lian, J.B.; van Wijnen, A.J.; Stein, J.L.; Montecino, M.; Javed, A.; Zaidi, S.K.; Young, D.W.; Choi, J.Y.; Pockwinse, S.M. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 2004, 23, 4315-4329.\",\n \"A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. P Ducy, M Starbuck, M Priemel, J Shen, G Pinero, V Geoffroy, M Amling, G Karsenty, Ducy, P.; Starbuck, M.; Priemel, M.; Shen, J.; Pinero, G.; Geoffroy, V.; Amling, M.; Karsenty, G. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.\",\n \". Genes Dev. 13Genes Dev. 1999, 13, 1025-1036.\",\n \"Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. P Cerkovnik, B Jezersek Novakovic, V Stegel, S Novakovic, Int. J. Oncol. 38Cerkovnik, P.; Jezersek Novakovic, B.; Stegel, V.; Novakovic, S. Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. Int. J. Oncol. 2011, 38, 1749-1758.\",\n \"Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. C S Liu, Y Sun, Y H Hu, L Sun, Vaccine. 28Liu, C.S.; Sun, Y.; Hu, Y.H.; Sun, L. Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. Vaccine 2010, 28, 4153-4161.\",\n \"Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. M Bao, Y Zhang, M Wan, L Dai, X Hu, X Wu, L Wang, P Deng, J Wang, J Chen, Clin. Immunol. 118Bao, M.; Zhang, Y.; Wan, M.; Dai, L.; Hu, X.; Wu, X.; Wang, L.; Deng, P.; Wang, J.; Chen, J.; et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. Clin. Immunol. 2006, 118, 180-187.\",\n \"Synthetic oligonucleotides as modulators of inflammation. D Klinman, H Shirota, D Tross, T Sato, S Klaschik, J. Leukoc Biol. 84Klinman, D.; Shirota, H.; Tross, D.; Sato, T.; Klaschik, S. Synthetic oligonucleotides as modulators of inflammation. J. Leukoc Biol. 2008, 84, 958-964.\",\n \"Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. A Amcheslavsky, H Hemmi, S Akira, J. Bone Miner. Res. 20Amcheslavsky, A.; Hemmi, H.; Akira, S.; Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. J. Bone Miner. Res. 2005, 20, 1692-1699.\",\n \"CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. N N Norgaard, T Holien, S Jonsson, H Hella, T Espevik, A Sundan, T Standal, J. Immunol. 185Norgaard, N.N.; Holien, T.; Jonsson, S.; Hella, H.; Espevik, T.; Sundan, A.; Standal, T. CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. J. Immunol. 2010, 185, 3131-3139.\",\n \"Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. J H Chang, E J Chang, H H Kim, S K Kim, Biochem. Biophys. Res. Commun. 389Chang, J.H.; Chang, E.J.; Kim, H.H.; Kim, S.K. Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. Biochem. Biophys. Res. Commun. 2009, 389, 28-33.\",\n \"An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Z Feng, Y Shen, L Wang, L Cheng, J Wang, Q Li, W Shi, X Sun, Int. J. Mol. Sci. 12Feng, Z.; Shen, Y.; Wang, L.; Cheng, L.; Wang, J.; Li, Q.; Shi, W.; Sun, X. An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Int. J. Mol. Sci. 2011, 12, 2543-2555.\",\n \"An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Y Shen, Z Feng, C Lin, X Hou, X Wang, J Wang, Y Yu, L Wang, X Sun, Int. J. Mol. Sci. 13Shen, Y.; Feng, Z.; Lin, C.; Hou, X.; Wang, X.; Wang, J.; Yu, Y.; Wang, L.; Sun, X. An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Int. J. Mol. Sci. 2012, 13, 2877-2892.\",\n \"Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. A M Bennett, N K Tonks, Science. 278Bennett, A.M.; Tonks, N.K. Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science 1997, 278, 1288-1291.\",\n \"Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, 10.1002/jbmr.561J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2011, 27, doi: 10.1002/jbmr.561.\",\n \"Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. H M Jeong, E H Han, Y H Jin, Y P Hwang, H G Kim, B H Park, J Y Kim, Y C Chung, K Y Lee, H G Jeong, Food Chem. Toxicol. 48Jeong, H.M.; Han, E.H.; Jin, Y.H.; Hwang, Y.P.; Kim, H.G.; Park, B.H.; Kim, J.Y.; Chung, Y.C.; Lee, K.Y.; Jeong, H.G. Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. Food Chem. Toxicol. 2010, 48, 3362-3368.\",\n \"Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. D Reddi, S J Brown, G N Belibasakis, Microb. Pathog. 51Reddi, D.; Brown, S.J.; Belibasakis, G.N. Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. Microb. Pathog. 2011, 51, 415-420.\",\n \"Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice. G Yang, M Wan, Y Zhang, L Sun, R Sun, D Hu, X Zhou, L Wang, X Wu, Y Yu, Immunology. 131Yang, G.; Wan, M.; Zhang, Y.; Sun, L.; Sun, R.; Hu, D.; Zhou, X.; Wang, L.; Wu, X.; Yu, Y. Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice. Immunology 2010, 131, 501-512\",\n \"Therapeutic potential of Toll-like receptor 9 activation. A M Krieg, Nat. Rev. Drug. Discov. 5Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug. Discov. 2006, 5, 471-484.\",\n \"The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. M B Greenblatt, J H Shim, W Zou, D Sitara, M Schweitzer, D Hu, S Lotinun, Y Sano, R Baron, J M Park, J. Clin. Invest. 120Greenblatt, M.B.; Shim, J.H.; Zou, W.; Sitara, D.; Schweitzer, M.; Hu, D.; Lotinun, S.; Sano, Y.; Baron, R.; Park, J.M.; et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J. Clin. Invest. 2010, 120, 2457-2473.\",\n \"Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. M Kawano, W Ariyoshi, K Iwanaga, T Okinaga, M Habu, I Yoshioka, K Tominaga, T Nishihara, Biochem. Biophys. Res. Commun. 405Kawano, M.; Ariyoshi, W.; Iwanaga, K.; Okinaga, T.; Habu, M.; Yoshioka, I.; Tominaga, K.; Nishihara, T. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. Biochem. Biophys. Res. Commun. 2011, 405, 575-580.\",\n \"Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. D Zhang, H Zheng, J Zhao, L Lin, C Li, J Liu, Y Pan, J. Periodontal. Res. 46Zhang, D.; Zheng, H.; Zhao, J.; Lin, L.; Li, C.; Liu, J.; Pan, Y. Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. J. Periodontal. Res. 2011, 46, 31-38.\",\n \"Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. A Suzuki, J Guicheux, G Palmer, Y Miura, Y Oiso, J P Bonjour, J Caverzasio, 30BoneSuzuki, A.; Guicheux, J.; Palmer, G.; Miura, Y.; Oiso, Y.; Bonjour, J.P.; Caverzasio, J. Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. Bone 2002, 30, 91-98.\",\n \"Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. W Zou, A Amcheslavsky, J. Biol. Chem. 278Zou, W.; Amcheslavsky, A.; Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. J. Biol. Chem. 2003, 278, 16732-16740.\",\n \"Genetic control of skeletal development. E F Wagner, G Karsenty, Curr. Opin. Genet. Dev. 11Wagner, E.F.; Karsenty, G. Genetic control of skeletal development. Curr. Opin. Genet. Dev. 2001, 11, 527-532.\",\n \"Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. G Xiao, Y Cui, P Ducy, G Karsenty, R T Franceschi, Mol. Endocrinol. 11Xiao, G.; Cui, Y.; Ducy, P.; Karsenty, G.; Franceschi, R.T. Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. Mol. Endocrinol. 1997, 11, 1103-1113.\",\n \"MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. G Xiao, D Jiang, P Thomas, M D Benson, K Guan, G Karsenty, R T Franceschi, J. Biol. Chem. 275Xiao, G.; Jiang, D.; Thomas, P.; Benson, M.D.; Guan, K.; Karsenty, G.; Franceschi, R.T. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J. Biol. Chem. 2000, 275, 4453-4459.\",\n \"Transcriptional regulation of osteoblasts. R T Franceschi, C Ge, G Xiao, H Roca, D Jiang, Cells Tissues Organs. 189Franceschi, R.T.; Ge, C.; Xiao, G.; Roca, H.; Jiang, D. Transcriptional regulation of osteoblasts. Cells Tissues Organs 2009, 189, 144-152.\",\n \"Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. S C Bae, Y H Lee, Gene. 366Bae, S.C.; Lee, Y.H. Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. Gene 2006, 366, 58-66.\",\n \". E J Jeon, K Y Lee, N S Choi, M H Lee, H N Kim, Y H Jin, H M Ryoo, J Y Choi, M Yoshida, N Nishino, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylationJeon, E.J.; Lee, K.Y.; Choi, N.S.; Lee, M.H.; Kim, H.N.; Jin, Y.H.; Ryoo, H.M.; Choi, J.Y.; Yoshida, M.; Nishino, N.; et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation.\",\n \". J. Biol. Chem. 281J. Biol. Chem. 2006, 281, 16502-16511.\",\n \"Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor. C Ge, G Xiao, D Jiang, Q Yang, N E Hatch, H Roca, R T Franceschi, Ge, C.; Xiao, G.; Jiang, D.; Yang, Q.; Hatch, N.E.; Roca, H.; Franceschi, R.T. Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor.\",\n \". J. Biol. Chem. 284J. Biol. Chem. 2009, 284, 32533-32543.\",\n \"Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2012, 27, 538-551.\",\n \"Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. A Yamaguchi, T Komori, T Suda, Endocr. Rev. 21Yamaguchi, A.; Komori, T.; Suda, T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 2000, 21, 393-411.\",\n \"Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. B Kern, J Shen, M Starbuck, G Karsenty, J. Biol. Chem. 276Kern, B.; Shen, J.; Starbuck, M.; Karsenty, G. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J. Biol. Chem. 2001, 276, 7101-7107.\"\n]"},"annotations_bibref":{"kind":"list like","value":["[1]","[2,","3]","[4]","[5]","[6]","[7]","[8]","[9]","[10]","[11]","[12]","[13]","[14]","[15]","[16]","[17]","[18]","[19]","[20]","[13,","14]","[21]","[22]","[23]","[24]","[25]","[3,","26]","[27]","[28]","[29]","[30,","31]","[32]","[33]","[21]","[34]","[35]"],"string":"[\n \"[1]\",\n \"[2,\",\n \"3]\",\n \"[4]\",\n \"[5]\",\n \"[6]\",\n \"[7]\",\n \"[8]\",\n \"[9]\",\n \"[10]\",\n \"[11]\",\n \"[12]\",\n \"[13]\",\n \"[14]\",\n \"[15]\",\n \"[16]\",\n \"[17]\",\n \"[18]\",\n \"[19]\",\n \"[20]\",\n \"[13,\",\n \"14]\",\n \"[21]\",\n \"[22]\",\n \"[23]\",\n \"[24]\",\n \"[25]\",\n \"[3,\",\n \"26]\",\n \"[27]\",\n \"[28]\",\n \"[29]\",\n \"[30,\",\n \"31]\",\n \"[32]\",\n \"[33]\",\n \"[21]\",\n \"[34]\",\n \"[35]\"\n]"},"annotations_bibtitle":{"kind":"list like","value":["Regulation of gene expression in osteoblasts","Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development","Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia","Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression","Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells","Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity","Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide","Synthetic oligonucleotides as modulators of inflammation","Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis","CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis","Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation","An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast","An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis","Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases","Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity","Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation","Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK","Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice","Therapeutic potential of Toll-like receptor 9 activation","The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice","Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid","Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway","Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9","Genetic control of skeletal development","Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence","MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1","Transcriptional regulation of osteoblasts","Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation","Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity","Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1","Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes"],"string":"[\n \"Regulation of gene expression in osteoblasts\",\n \"Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development\",\n \"Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia\",\n \"Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression\",\n \"Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells\",\n \"Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity\",\n \"Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide\",\n \"Synthetic oligonucleotides as modulators of inflammation\",\n \"Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis\",\n \"CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis\",\n \"Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation\",\n \"An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast\",\n \"An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis\",\n \"Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases\",\n \"Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity\",\n \"Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation\",\n \"Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK\",\n \"Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice\",\n \"Therapeutic potential of Toll-like receptor 9 activation\",\n \"The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice\",\n \"Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid\",\n \"Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway\",\n \"Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9\",\n \"Genetic control of skeletal development\",\n \"Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence\",\n \"MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1\",\n \"Transcriptional regulation of osteoblasts\",\n \"Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation\",\n \"Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity\",\n \"Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1\",\n \"Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["Biofactors","Cell","Cell","Oncogene","A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development","Genes Dev","Int. J. Oncol","Vaccine","Clin. Immunol","J. Leukoc Biol","J. Bone Miner. Res","J. Immunol","Biochem. Biophys. Res. Commun","Int. J. Mol. Sci","Int. J. Mol. Sci","Science","J. Bone Miner. Res","Food Chem. Toxicol","Microb. Pathog","Immunology","Nat. Rev. Drug. Discov","J. Clin. Invest","Biochem. Biophys. Res. Commun","J. Periodontal. Res","Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation","J. Biol. Chem","Curr. Opin. Genet. Dev","Mol. Endocrinol","J. Biol. Chem","Cells Tissues Organs","Gene","J. Biol. Chem","Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor","J. Biol. Chem","J. Bone Miner. Res","Endocr. Rev","J. Biol. Chem"],"string":"[\n \"Biofactors\",\n \"Cell\",\n \"Cell\",\n \"Oncogene\",\n \"A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development\",\n \"Genes Dev\",\n \"Int. J. Oncol\",\n \"Vaccine\",\n \"Clin. Immunol\",\n \"J. Leukoc Biol\",\n \"J. Bone Miner. Res\",\n \"J. Immunol\",\n \"Biochem. Biophys. Res. Commun\",\n \"Int. J. Mol. Sci\",\n \"Int. J. Mol. Sci\",\n \"Science\",\n \"J. Bone Miner. Res\",\n \"Food Chem. Toxicol\",\n \"Microb. Pathog\",\n \"Immunology\",\n \"Nat. Rev. Drug. Discov\",\n \"J. Clin. Invest\",\n \"Biochem. Biophys. Res. Commun\",\n \"J. Periodontal. Res\",\n \"Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation\",\n \"J. Biol. Chem\",\n \"Curr. Opin. Genet. Dev\",\n \"Mol. Endocrinol\",\n \"J. Biol. Chem\",\n \"Cells Tissues Organs\",\n \"Gene\",\n \"J. Biol. Chem\",\n \"Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor\",\n \"J. Biol. Chem\",\n \"J. Bone Miner. Res\",\n \"Endocr. Rev\",\n \"J. Biol. Chem\"\n]"},"annotations_figure":{"kind":"list like","value":["\nFigure 1 .\n1Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.","\nFigure 1 .\n1Cont","\nFigure 2 .\n2Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.","\nFigure 3 .\n3Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72","\nFigure 4 .\n4Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.","\nFigure 5 .\n5MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD ("],"string":"[\n \"\\nFigure 1 .\\n1Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.\",\n \"\\nFigure 1 .\\n1Cont\",\n \"\\nFigure 2 .\\n2Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.\",\n \"\\nFigure 3 .\\n3Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72\",\n \"\\nFigure 4 .\\n4Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.\",\n \"\\nFigure 5 .\\n5MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD (\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.","Cont","Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.","Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72","Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.","MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD ("],"string":"[\n \"Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.\",\n \"Cont\",\n \"Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.\",\n \"Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72\",\n \"Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.\",\n \"MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD (\"\n]"},"annotations_figureref":{"kind":"list like","value":["Figure 1","Figure 2A","Figure 2B","Figure 2","Figure 2","Figure 3","Figure 4","Figure 5A","Figure 5B","(Nanjing, China)"],"string":"[\n \"Figure 1\",\n \"Figure 2A\",\n \"Figure 2B\",\n \"Figure 2\",\n \"Figure 2\",\n \"Figure 3\",\n \"Figure 4\",\n \"Figure 5A\",\n \"Figure 5B\",\n \"(Nanjing, China)\"\n]"},"annotations_formula":{"kind":"list like","value":[],"string":"[]"},"annotations_paragraph":{"kind":"list like","value":["Osteoblasts play an extremely important role in bone formation and remodeling processes. Osteoblasts are derived from mesenchymal stem cells, and can differentiate into mature and functional osteoblasts that produce extracellular matrix proteins and regulators of matrix mineralization [1]. Runx2 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Runx2 −/− mice show a complete lack of both intramembranous and endochondral ossification owing to maturation arrest of osteoblasts [2,3]. However, Runx2 can regulate the expression of osteoblast-specific genes including ALP, type I collagen and osteocalcin. To date, numerous reports have shown that signaling pathways and transcription factors could participate in osteoblastogenesis by regulating the production or activity of Runx2 [4]. Furthermore, Runx2 is necessary throughout life to promote the differentiation of new osteoblasts during bone remodeling [5].","Oligodeoxynucleotides (ODNs) containing unmethylated nucleotide motifs are immunostimulatory in vertebrates, and some ODNs containing CpG motifs are used for treating cancer, virus-associated diseases, and infections [6][7][8][9]. Recently, specific ODNs were found to have an effect on modulating osteoclast-and osteoblast-lineage cells [10]. In addition, CpG-ODN with a phosphorothioate backbone inhibits the BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines [11]. Chang et al. demonstrated that a novel CpG-ODN enhances the inhibitory effects on osteoclast differentiation by downregulation of TREM-2 [12]. In our previous studies, we have shown that ODN MT01, a synthetic 27-mer single stranded ODN with a design based on human mitochondrial DNA, promotes the proliferation and activation of osteoblasts [13], and stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts [14]. However, the detailed signaling pathway in which ODN MT01 modulates the differentiation of osteoblasts has not been fully elucidated.","Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific protein kinases, including p38, JNK and ERK1/2 (p44/p42). These molecules play important roles in cell differentiation, proliferation and death [15], and are activated by phosphorylation of tyrosine/threonine residues in signal transduction cascades. Previous studies have shown that the p38 MAPK and ERK1/2 pathways are involved in the proliferation and differentiation of osteoblasts [16][17][18]. Therefore, we hypothesized that ODN MT01 might regulate the differentiation of osteoblastic cells via the MAPKs pathway.","This study aimed to investigate the regulation of MAPKs in response to ODN MT01, and to elucidate the involvement of the MAPK signaling pathway in regulation of Runx2, osteocalcin, type I collagen and ALP expression in ODN MT01-treated MG 63 cells.","The design of ODN MT01, a 27-mer ODN, was based on the motif sequence 5'-ACCCCCTCTACCCCCTCT-3' in human mitochondrial DNA. MT01 is a cytosine-rich ODN and contains five successive cytosines in each of its 9-mer motifs (5'-ACCCCCTCT-3') [19]. To investigate whether the effects of an ODN required internalization into cells, Cy5-labeled ODN MT01 was synthesized to trace the sites of ODN actions. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, and is temperature-and energy-dependent [20]. This result was consistent with our observations, as shown in Figure 1, ODN MT01 entered cells by endocytosis after incubation with MG 63 cells for 3 h. The fluorescence intensity was enhanced over time, and the intensity at 6 h was significantly higher than that at 3 h, and the intensity at 12 h was higher than that at 3 and 6 h. It was apparent that ODN MT01 stimulation of osteoblasts required internalization. ","To investigate the effect of ODN MT01 on MAPKs, both total and phosphorylated levels of MAPKs were investigated by Western blotting after 0, 30, 60 min, 24 h and 48 h of treatment with 1 µg/mL ODN MT01. To better demonstrate that the effects of MT01 in MG 63 cells were in response to the specific sequence of MT01 we tested another ODN sequence, ODN FC003, a 24-mer ODN(5'-TCTCTCTCTCTCTCTCTCTCTCTC-3'). The design of ODN FC003 was also based on human mitochondrial DNA, and contained successive thymidine-cytosine nucleotides. In our previous studies [13,14], we found that ODN FC003 is inactive in the regulation of osteoblasts and bone marrow mesenchymal stem cells. Therefore, we chose this sequence as a negative control. As shown in Figure 2A, phosphorylation of ERK1/2 was observed after 30 min of treatment with ODN MT01, which increased by 13.1-fold after 60 min compared with that at 0 min. Furthermore, the active effect lasted up to 48 h (8.9-fold increase), while the total level of ERK1/2 protein remained unchanged. For p38, there was no obvious change in the phosphorylation level before 30 min. However, p38 phosphorylation increased after 60 min of treatment with MT01 ( Figure 2B), and showed an increase of 4.7 fold at 24 h, which lasted up to 48 h. As shown in Figure 2 A and B, the FC003 sequence had no active effect on MG 63 cells via ERK1/2 or p38 MAPKs. Thus, these findings indicated that ODN MT01 induced the phosphorylation of ERK1/2 and p38 MAPK in MG 63 cells.","To investigate the mechanisms by which ODNs regulate the differentiation of osteoblasts, it is important to identify the functional role of signaling pathways activated by ODNs. At present, several signaling pathways have been shown to be involved in osteogenesis, such as MAPKs, BMP-2-Smad and NF-κB [21][22][23]. Among them, p38 and ERK1/2 MAPKs have been reported to be important for early osteoblast differentiation in various cell lines [24]. In this study, we found that MT01 was capable of activating ERK1/2 and p38 MAPK pathways indicating that MT01 induced the phosphorylation of MAPKs at the early stage of osteoblast differentiation. Previous reports have shown that ERK and p38 are phosphorylated in response to CpG-ODN, and p38 is phosphorylated to a lesser extent [25]. Thus, our findings were consistent with previous reports. ERK1/2 reacted more rapidly than p38 MAPK, and activation of both lasted up to 48 h after treatment with MT01. Once MAPK is activated by ODNs, transcription factors and other kinases may be phosphorylated to initiate events such as gene expression and post-translational protein modifications. ","To investigate the involvement of ERK and p38 MAPK in modulation of ALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38 (U0126 and SB203580, respectively) were used. Cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. As shown in Figure 2, compared with the control (phosphate-buffered saline (PBS) treatment), ALP activity was significantly up-regulated after ODN MT01 treatment at 48 and 72 h. Notably, U0126 and SB203580 significantly inhibited the up-regulation induced by ODN MT01.","The activity of osteogenic differentiation marker ALP is an important index to indicate osteogenesis at an early stage. As shown in Figure 3, after 24 h of treatment with ODN MT01, the activity of ALP was increased, compared with that in the control, but the difference was not significant. The density of cells increased after 48 and 72 h, and ODN MT01 exhibited an obvious promoting effect on osteogenic differentiation. In addition, inhibitors significantly downregulated ALP activity induced by ODN MT01 after 48 and 72 h. These results indicated that the ERK and p38 MAPK pathways were probably involved in ODN MT01-induced promotion of ALP activity. ","The above results confirmed that ODN MT01 induced the phosphorylation of ERK and p38 MAPK and further activated these MAPK signaling pathways. To investigate the role of Runx2, which is one of the most important factors for osteoblast differentiation, we used the ERK inhibitor U0126 and p38 inhibitor SB203580 to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 for 72 h. The total and phosphorylated protein levels of Runx2 were detected using Western blotting. As shown in Figure 4, there were obvious differences in the phosphorylation levels of ERK and p38 between the MT01-treatment and control groups; p-ERK and p-p38 were significantly inhibited in the presence of U0126 and SB203580, which were induced by MT01. For Runx2, there was no obvious change in protein levels in these groups, but MT01 induced notable phosphorylation of Runx2 (1.8-fold increase compared with the control). However, the up-regulated phosphorylation of Runx2 induced by MT01 was inhibited in the presence of U0126 and SB203580.","Runx2 is an essential transcription factor for osteoblast differentiation and bone formation, and inactivation of one Runx2 allele will cause cleidocranial dysplasia syndrome, a disease characterized by delayed osteoblast differentiation for bone formation through intramembranous ossification in mice and humans [3,26]. Previous reports have shown that extracellular matrix production can induce osteoblast differentiation by increasing Runx2 transcriptional activity, but not the mRNA or protein levels [27][28][29], which concurs with our results. Runx2 activity is controlled by various extracellular signaling pathways, and post-translational modifications (phosphorylation, acetylation and ubiquitination) can affect its stability and activity [30,31]. Among the post-translational modifications, the role of Runx2 phosphorylation has been well characterized. Ge et al. found that Runx2 is regulated by ERK1/2 and p38 MAPK-mediated phosphorylation [32], and ERK is more important for Runx2 phosphorylation than p38 [33]. Interestingly, in the present study, we found the same results in which inhibition of ERK showed a stronger effect on the expression of p-Runx2 (1-fold increase compared with the control) than that of p38 inhibition (1.2-fold increase compared with the control). These results suggested that Runx2 is phosphorylated on a complex set of sites that partially overlap between ERK and p38. Another study found that phosphorylation of p38 MAPK induces activation of Runx2 via TAK1 and MEK3 signaling pathways during early osteoblastic differentiation [21]. Here, we showed that MT01 greatly increased Runx2 phosphorylation, which was potently repressed by inhibitors of ERK and p38. Taken together, our results were consistent with other findings and demonstrated that MT01-induced phosphorylation of Runx2 was mediated by activation of both ERK1/2 and p38 MAPK signaling pathways, and ERK MAPK may be more important. ","Runx2 has been shown to regulate the expression of osteoblast-specific genes, such as ALP, osteocalcin and type I collagen [34]. To further confirm our results, we analyzed the expression of subsets of osteogenic marker genes including osteocalcin and type I collagen. We used ERK and p38 inhibitors to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 or ODN FC003 for 15 days. Then, the mRNA and protein expression of osteocalcin and type I collagen were analyzed by real-time PCR and Western blot, respectively. As shown in Figure 5A, compared with the control g, the protein expression of osteocalcin was increased by ODN MT01 treatment. Compared with ODN MT01 treatment, the protein level of osteocalcin was significantly decreased when cells were pretreated with the ERK and p38 inhibitors. The expression level of type I collagen exhibited a similar tendency with osteocalcin. As shown in Figure 5B,C, the mRNA levels were concurrent with the protein expression. Osteocalcin protein expression marks the late phase of osteogenic differentiation, and is only produced by mineralizing cells types. In addition, osteocalcin is considered as a specific marker of osteoblast differentiation and maturity. Type I collagen is the major product of osteoblasts, and accounts for 90% of the matrix protein content [35]. Type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life. Osteocalcin and type I collagen protein expression induced by ODN MT01 was increased at late stages of osteoblast differentiation, suggesting that ODN MT01 controll of the expression of osteocalcin and type I collagen in these cells could also control osteoblast differentiation and function. Furthermore, ERK and p38 MAPK inhibitors obviously decreased protein expression induced by MT01. Therefore, ODN MT01 induced the expression of osteocalcin and type I collagen via ERK and p38 MAPK signaling pathways.","ODNs MT01 (5'-ACCCCCTCTACCCCCTCTACCCCCTCT-3') and FC003 (5'-TCTCTCTCTCTC TCTCTCTCTCTC-3') were synthesized by TaKaRa (Dalian, China), and were dissolved in axenic PBS. The human osteoblastic cell line MG 63 was obtained from the American Type Culture Collection (CRL-1427). An Alkaline phosphatase kit and micro-BCA assay kit were obtained from Jiancheng Biological Reagent Co. (Nanjing, China). A real-time PCR kit was purchased from TaKaRa (Tokyo, Japan). Monoclonal antibodies against p38, p-p38, p44/p42 and p-p44/42, were purchased from Santa Cruz Biotech. and those against Runx2, osteocalcin and type I collagen were purchased from Abcam (UK). The anti-phospho-Runx2 antibody was purchased from Abcam Co (UK). The phosphorylation site was Ser533.","MG 63 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO 2 . Cells were seeded at an initial density of 2 × 10 5 cells/mL. To investigate the effect of ODN MT01 on MAPKs, cells were treated with 1 µg/mL ODN MT01 for 0, 30, 60 min, 24 and 48 h. To investigate activation of Runx2, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 72 h. To investigate the effects of ERK and p38 inhibitors on the mRNA and protein levels of osteocalcin and type I collagen, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 15 days. Medium was changed every 3 days, and ODNs and inhibitors were added at the same time.","Cy5 was conjugated to the 5'-end of MT01, and then dissolved with PBS. Cells were cultured on 24 × 24 mm coverslips at a density of 1 × 10 4 cells/well overnight. Then, cells were treated with 1 µg/mL Cy5-labeled MT01 for 3, 6 and 12 h. Cells were washed three times with PBS according to the time points. While protected from light, Hoechst 33342 was added at 1 µg/mL for 5 min to stain nuclei. A laser scanning confocal microscope (Olympus IX81, Japan) was used to observe cell climb-chips. Stained cells were imaged with Olympus Fluoview FV1000 Viewer (Version1.7.c; Olympus Corporation: Tokyo, Japan, 2007).","Western blotting was used to evaluate total and phosphorylated protein levels of p38 and p44/42. MG 63 cells were cultured in 10-cm dishes and treated with 1 µg/mL ODN MT01 for the indicated times. To evaluate the protein levels of Runx2 and p-Runx2, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without ODN MT01 for 72 h. To investigate the levels of osteocalcin and type I collagen, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without ODN MT01 for 15 days. Then cells were collected, washed twice with cold Tris-buffered saline and resuspended in lysis buffer (50 mM Tris, pH 7.6, 0.01% EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/mL leupeptin). The protein concentration was measured using a BCA Protein Assay Reagent kit. Protein samples (40 μg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Then membranes were incubated with secondary antibodies at 20 °C for 2 h, and developed using an ECL chemiluminescent system. Loading differences were normalized using a GAPDH antibody.","Cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. Then, the cells were collected and lysed to measure ALP activity. The assay was conducted using an Alkaline Phosphatase Kit, according to the manufacturer's instructions. The protein concentration of cell lysates was measured using a micro-BCA assay kit, and ALP activity was normalized to the total protein concentration. Values were the averages of triplicate measurements.","To quantify mRNA expression levels, real-time PCR was performed with cDNA samples. Primers were designed using qPrimerDepot(Nucleic Acids Res. 2007), a primer database for quantitative real-time PCR. Real-time PCR was performed using an ABI Steponeplus (ABI PRISM, Carlsbad, NM, USA), which allowed real-time monitoring of increases in PCR product concentrations after each cycle based on the fluorescence of the double-stranded DNA specific dye SYBR green. The number of cycles required to produce a detectable product above background was measured for each sample. These cycle numbers were then used to calculate fold differences in the initial mRNA level for each sample using the following method. First, the cycle number difference for GAPDH, a housekeeping gene, was determined in the control sample and appropriate ODN MT01-treated sample. Values were the averages of triplicate measurements. PCR primers used were as follows: GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'; Type I collagen forward, 5'-AGGGCCAAGACGAAGACATC-3' and reverse, 5'-AGATCACGTCATCGCACAACA-3'; OC forward, 5'-TGAGAGCCCTCACACTCCTC-3' and reverse, 5'-GCCGTAGAAGCGCCGATAGGC-3'.","All experiments were performed with triplicate independent samples and repeated at least twice. Results were expressed as the mean ± SD. ANOVA and the Bonferroni post-hoc test were used to compare the differences between the ODN MT01-treatment group and other groups. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed with SAS software (version 8.0, SAS: Cary, NC, USA).","In this study, a specific ODN, ODN MT01, was endocytosed by osteoblasts and found to up-regulate the expression level of osteocalcin, type I collagen and ALP in MG 63 cells via the ERK1/2 and p38 MAPK pathways. This study provides further insight into the use of ODN MT01 for in vitro experimentation, and supports the potential use of ODN MT01 to regulate the rebuilding of bone."],"string":"[\n \"Osteoblasts play an extremely important role in bone formation and remodeling processes. Osteoblasts are derived from mesenchymal stem cells, and can differentiate into mature and functional osteoblasts that produce extracellular matrix proteins and regulators of matrix mineralization [1]. Runx2 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Runx2 −/− mice show a complete lack of both intramembranous and endochondral ossification owing to maturation arrest of osteoblasts [2,3]. However, Runx2 can regulate the expression of osteoblast-specific genes including ALP, type I collagen and osteocalcin. To date, numerous reports have shown that signaling pathways and transcription factors could participate in osteoblastogenesis by regulating the production or activity of Runx2 [4]. Furthermore, Runx2 is necessary throughout life to promote the differentiation of new osteoblasts during bone remodeling [5].\",\n \"Oligodeoxynucleotides (ODNs) containing unmethylated nucleotide motifs are immunostimulatory in vertebrates, and some ODNs containing CpG motifs are used for treating cancer, virus-associated diseases, and infections [6][7][8][9]. Recently, specific ODNs were found to have an effect on modulating osteoclast-and osteoblast-lineage cells [10]. In addition, CpG-ODN with a phosphorothioate backbone inhibits the BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines [11]. Chang et al. demonstrated that a novel CpG-ODN enhances the inhibitory effects on osteoclast differentiation by downregulation of TREM-2 [12]. In our previous studies, we have shown that ODN MT01, a synthetic 27-mer single stranded ODN with a design based on human mitochondrial DNA, promotes the proliferation and activation of osteoblasts [13], and stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts [14]. However, the detailed signaling pathway in which ODN MT01 modulates the differentiation of osteoblasts has not been fully elucidated.\",\n \"Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific protein kinases, including p38, JNK and ERK1/2 (p44/p42). These molecules play important roles in cell differentiation, proliferation and death [15], and are activated by phosphorylation of tyrosine/threonine residues in signal transduction cascades. Previous studies have shown that the p38 MAPK and ERK1/2 pathways are involved in the proliferation and differentiation of osteoblasts [16][17][18]. Therefore, we hypothesized that ODN MT01 might regulate the differentiation of osteoblastic cells via the MAPKs pathway.\",\n \"This study aimed to investigate the regulation of MAPKs in response to ODN MT01, and to elucidate the involvement of the MAPK signaling pathway in regulation of Runx2, osteocalcin, type I collagen and ALP expression in ODN MT01-treated MG 63 cells.\",\n \"The design of ODN MT01, a 27-mer ODN, was based on the motif sequence 5'-ACCCCCTCTACCCCCTCT-3' in human mitochondrial DNA. MT01 is a cytosine-rich ODN and contains five successive cytosines in each of its 9-mer motifs (5'-ACCCCCTCT-3') [19]. To investigate whether the effects of an ODN required internalization into cells, Cy5-labeled ODN MT01 was synthesized to trace the sites of ODN actions. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, and is temperature-and energy-dependent [20]. This result was consistent with our observations, as shown in Figure 1, ODN MT01 entered cells by endocytosis after incubation with MG 63 cells for 3 h. The fluorescence intensity was enhanced over time, and the intensity at 6 h was significantly higher than that at 3 h, and the intensity at 12 h was higher than that at 3 and 6 h. It was apparent that ODN MT01 stimulation of osteoblasts required internalization. \",\n \"To investigate the effect of ODN MT01 on MAPKs, both total and phosphorylated levels of MAPKs were investigated by Western blotting after 0, 30, 60 min, 24 h and 48 h of treatment with 1 µg/mL ODN MT01. To better demonstrate that the effects of MT01 in MG 63 cells were in response to the specific sequence of MT01 we tested another ODN sequence, ODN FC003, a 24-mer ODN(5'-TCTCTCTCTCTCTCTCTCTCTCTC-3'). The design of ODN FC003 was also based on human mitochondrial DNA, and contained successive thymidine-cytosine nucleotides. In our previous studies [13,14], we found that ODN FC003 is inactive in the regulation of osteoblasts and bone marrow mesenchymal stem cells. Therefore, we chose this sequence as a negative control. As shown in Figure 2A, phosphorylation of ERK1/2 was observed after 30 min of treatment with ODN MT01, which increased by 13.1-fold after 60 min compared with that at 0 min. Furthermore, the active effect lasted up to 48 h (8.9-fold increase), while the total level of ERK1/2 protein remained unchanged. For p38, there was no obvious change in the phosphorylation level before 30 min. However, p38 phosphorylation increased after 60 min of treatment with MT01 ( Figure 2B), and showed an increase of 4.7 fold at 24 h, which lasted up to 48 h. As shown in Figure 2 A and B, the FC003 sequence had no active effect on MG 63 cells via ERK1/2 or p38 MAPKs. Thus, these findings indicated that ODN MT01 induced the phosphorylation of ERK1/2 and p38 MAPK in MG 63 cells.\",\n \"To investigate the mechanisms by which ODNs regulate the differentiation of osteoblasts, it is important to identify the functional role of signaling pathways activated by ODNs. At present, several signaling pathways have been shown to be involved in osteogenesis, such as MAPKs, BMP-2-Smad and NF-κB [21][22][23]. Among them, p38 and ERK1/2 MAPKs have been reported to be important for early osteoblast differentiation in various cell lines [24]. In this study, we found that MT01 was capable of activating ERK1/2 and p38 MAPK pathways indicating that MT01 induced the phosphorylation of MAPKs at the early stage of osteoblast differentiation. Previous reports have shown that ERK and p38 are phosphorylated in response to CpG-ODN, and p38 is phosphorylated to a lesser extent [25]. Thus, our findings were consistent with previous reports. ERK1/2 reacted more rapidly than p38 MAPK, and activation of both lasted up to 48 h after treatment with MT01. Once MAPK is activated by ODNs, transcription factors and other kinases may be phosphorylated to initiate events such as gene expression and post-translational protein modifications. \",\n \"To investigate the involvement of ERK and p38 MAPK in modulation of ALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38 (U0126 and SB203580, respectively) were used. Cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. As shown in Figure 2, compared with the control (phosphate-buffered saline (PBS) treatment), ALP activity was significantly up-regulated after ODN MT01 treatment at 48 and 72 h. Notably, U0126 and SB203580 significantly inhibited the up-regulation induced by ODN MT01.\",\n \"The activity of osteogenic differentiation marker ALP is an important index to indicate osteogenesis at an early stage. As shown in Figure 3, after 24 h of treatment with ODN MT01, the activity of ALP was increased, compared with that in the control, but the difference was not significant. The density of cells increased after 48 and 72 h, and ODN MT01 exhibited an obvious promoting effect on osteogenic differentiation. In addition, inhibitors significantly downregulated ALP activity induced by ODN MT01 after 48 and 72 h. These results indicated that the ERK and p38 MAPK pathways were probably involved in ODN MT01-induced promotion of ALP activity. \",\n \"The above results confirmed that ODN MT01 induced the phosphorylation of ERK and p38 MAPK and further activated these MAPK signaling pathways. To investigate the role of Runx2, which is one of the most important factors for osteoblast differentiation, we used the ERK inhibitor U0126 and p38 inhibitor SB203580 to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 for 72 h. The total and phosphorylated protein levels of Runx2 were detected using Western blotting. As shown in Figure 4, there were obvious differences in the phosphorylation levels of ERK and p38 between the MT01-treatment and control groups; p-ERK and p-p38 were significantly inhibited in the presence of U0126 and SB203580, which were induced by MT01. For Runx2, there was no obvious change in protein levels in these groups, but MT01 induced notable phosphorylation of Runx2 (1.8-fold increase compared with the control). However, the up-regulated phosphorylation of Runx2 induced by MT01 was inhibited in the presence of U0126 and SB203580.\",\n \"Runx2 is an essential transcription factor for osteoblast differentiation and bone formation, and inactivation of one Runx2 allele will cause cleidocranial dysplasia syndrome, a disease characterized by delayed osteoblast differentiation for bone formation through intramembranous ossification in mice and humans [3,26]. Previous reports have shown that extracellular matrix production can induce osteoblast differentiation by increasing Runx2 transcriptional activity, but not the mRNA or protein levels [27][28][29], which concurs with our results. Runx2 activity is controlled by various extracellular signaling pathways, and post-translational modifications (phosphorylation, acetylation and ubiquitination) can affect its stability and activity [30,31]. Among the post-translational modifications, the role of Runx2 phosphorylation has been well characterized. Ge et al. found that Runx2 is regulated by ERK1/2 and p38 MAPK-mediated phosphorylation [32], and ERK is more important for Runx2 phosphorylation than p38 [33]. Interestingly, in the present study, we found the same results in which inhibition of ERK showed a stronger effect on the expression of p-Runx2 (1-fold increase compared with the control) than that of p38 inhibition (1.2-fold increase compared with the control). These results suggested that Runx2 is phosphorylated on a complex set of sites that partially overlap between ERK and p38. Another study found that phosphorylation of p38 MAPK induces activation of Runx2 via TAK1 and MEK3 signaling pathways during early osteoblastic differentiation [21]. Here, we showed that MT01 greatly increased Runx2 phosphorylation, which was potently repressed by inhibitors of ERK and p38. Taken together, our results were consistent with other findings and demonstrated that MT01-induced phosphorylation of Runx2 was mediated by activation of both ERK1/2 and p38 MAPK signaling pathways, and ERK MAPK may be more important. \",\n \"Runx2 has been shown to regulate the expression of osteoblast-specific genes, such as ALP, osteocalcin and type I collagen [34]. To further confirm our results, we analyzed the expression of subsets of osteogenic marker genes including osteocalcin and type I collagen. We used ERK and p38 inhibitors to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 or ODN FC003 for 15 days. Then, the mRNA and protein expression of osteocalcin and type I collagen were analyzed by real-time PCR and Western blot, respectively. As shown in Figure 5A, compared with the control g, the protein expression of osteocalcin was increased by ODN MT01 treatment. Compared with ODN MT01 treatment, the protein level of osteocalcin was significantly decreased when cells were pretreated with the ERK and p38 inhibitors. The expression level of type I collagen exhibited a similar tendency with osteocalcin. As shown in Figure 5B,C, the mRNA levels were concurrent with the protein expression. Osteocalcin protein expression marks the late phase of osteogenic differentiation, and is only produced by mineralizing cells types. In addition, osteocalcin is considered as a specific marker of osteoblast differentiation and maturity. Type I collagen is the major product of osteoblasts, and accounts for 90% of the matrix protein content [35]. Type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life. Osteocalcin and type I collagen protein expression induced by ODN MT01 was increased at late stages of osteoblast differentiation, suggesting that ODN MT01 controll of the expression of osteocalcin and type I collagen in these cells could also control osteoblast differentiation and function. Furthermore, ERK and p38 MAPK inhibitors obviously decreased protein expression induced by MT01. Therefore, ODN MT01 induced the expression of osteocalcin and type I collagen via ERK and p38 MAPK signaling pathways.\",\n \"ODNs MT01 (5'-ACCCCCTCTACCCCCTCTACCCCCTCT-3') and FC003 (5'-TCTCTCTCTCTC TCTCTCTCTCTC-3') were synthesized by TaKaRa (Dalian, China), and were dissolved in axenic PBS. The human osteoblastic cell line MG 63 was obtained from the American Type Culture Collection (CRL-1427). An Alkaline phosphatase kit and micro-BCA assay kit were obtained from Jiancheng Biological Reagent Co. (Nanjing, China). A real-time PCR kit was purchased from TaKaRa (Tokyo, Japan). Monoclonal antibodies against p38, p-p38, p44/p42 and p-p44/42, were purchased from Santa Cruz Biotech. and those against Runx2, osteocalcin and type I collagen were purchased from Abcam (UK). The anti-phospho-Runx2 antibody was purchased from Abcam Co (UK). The phosphorylation site was Ser533.\",\n \"MG 63 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO 2 . Cells were seeded at an initial density of 2 × 10 5 cells/mL. To investigate the effect of ODN MT01 on MAPKs, cells were treated with 1 µg/mL ODN MT01 for 0, 30, 60 min, 24 and 48 h. To investigate activation of Runx2, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 72 h. To investigate the effects of ERK and p38 inhibitors on the mRNA and protein levels of osteocalcin and type I collagen, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 15 days. Medium was changed every 3 days, and ODNs and inhibitors were added at the same time.\",\n \"Cy5 was conjugated to the 5'-end of MT01, and then dissolved with PBS. Cells were cultured on 24 × 24 mm coverslips at a density of 1 × 10 4 cells/well overnight. Then, cells were treated with 1 µg/mL Cy5-labeled MT01 for 3, 6 and 12 h. Cells were washed three times with PBS according to the time points. While protected from light, Hoechst 33342 was added at 1 µg/mL for 5 min to stain nuclei. A laser scanning confocal microscope (Olympus IX81, Japan) was used to observe cell climb-chips. Stained cells were imaged with Olympus Fluoview FV1000 Viewer (Version1.7.c; Olympus Corporation: Tokyo, Japan, 2007).\",\n \"Western blotting was used to evaluate total and phosphorylated protein levels of p38 and p44/42. MG 63 cells were cultured in 10-cm dishes and treated with 1 µg/mL ODN MT01 for the indicated times. To evaluate the protein levels of Runx2 and p-Runx2, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without ODN MT01 for 72 h. To investigate the levels of osteocalcin and type I collagen, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without ODN MT01 for 15 days. Then cells were collected, washed twice with cold Tris-buffered saline and resuspended in lysis buffer (50 mM Tris, pH 7.6, 0.01% EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/mL leupeptin). The protein concentration was measured using a BCA Protein Assay Reagent kit. Protein samples (40 μg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Then membranes were incubated with secondary antibodies at 20 °C for 2 h, and developed using an ECL chemiluminescent system. Loading differences were normalized using a GAPDH antibody.\",\n \"Cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. Then, the cells were collected and lysed to measure ALP activity. The assay was conducted using an Alkaline Phosphatase Kit, according to the manufacturer's instructions. The protein concentration of cell lysates was measured using a micro-BCA assay kit, and ALP activity was normalized to the total protein concentration. Values were the averages of triplicate measurements.\",\n \"To quantify mRNA expression levels, real-time PCR was performed with cDNA samples. Primers were designed using qPrimerDepot(Nucleic Acids Res. 2007), a primer database for quantitative real-time PCR. Real-time PCR was performed using an ABI Steponeplus (ABI PRISM, Carlsbad, NM, USA), which allowed real-time monitoring of increases in PCR product concentrations after each cycle based on the fluorescence of the double-stranded DNA specific dye SYBR green. The number of cycles required to produce a detectable product above background was measured for each sample. These cycle numbers were then used to calculate fold differences in the initial mRNA level for each sample using the following method. First, the cycle number difference for GAPDH, a housekeeping gene, was determined in the control sample and appropriate ODN MT01-treated sample. Values were the averages of triplicate measurements. PCR primers used were as follows: GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'; Type I collagen forward, 5'-AGGGCCAAGACGAAGACATC-3' and reverse, 5'-AGATCACGTCATCGCACAACA-3'; OC forward, 5'-TGAGAGCCCTCACACTCCTC-3' and reverse, 5'-GCCGTAGAAGCGCCGATAGGC-3'.\",\n \"All experiments were performed with triplicate independent samples and repeated at least twice. Results were expressed as the mean ± SD. ANOVA and the Bonferroni post-hoc test were used to compare the differences between the ODN MT01-treatment group and other groups. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed with SAS software (version 8.0, SAS: Cary, NC, USA).\",\n \"In this study, a specific ODN, ODN MT01, was endocytosed by osteoblasts and found to up-regulate the expression level of osteocalcin, type I collagen and ALP in MG 63 cells via the ERK1/2 and p38 MAPK pathways. This study provides further insight into the use of ODN MT01 for in vitro experimentation, and supports the potential use of ODN MT01 to regulate the rebuilding of bone.\"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["Introduction","Results and Discussion","Internalization Analysis of ODNs","Effects of ODN MT01 on MAPK Signaling Proteins ERK1/2 and p38","Effects of ERK and p38 Inhibitors on Up-Regulation of ALP Activity Induced by ODN MT01","Effect of ERK and p38 Inhibitors on Runx2 Expression in Response to ODN MT01","Effects of ERK and p38 Inhibitors on the Expression of Osteocalcin and Type I Collagen","Experimental Section","Materials","Cell Culture","Fluorescent Labeling of MT01","Western Blot Analysis","ALP Activity Assay","Real-Time PCR","Statistical Analysis","Conclusions","Figure 1 .","Figure 1 .","Figure 2 .","Figure 3 .","Figure 4 .","Figure 5 ."],"string":"[\n \"Introduction\",\n \"Results and Discussion\",\n \"Internalization Analysis of ODNs\",\n \"Effects of ODN MT01 on MAPK Signaling Proteins ERK1/2 and p38\",\n \"Effects of ERK and p38 Inhibitors on Up-Regulation of ALP Activity Induced by ODN MT01\",\n \"Effect of ERK and p38 Inhibitors on Runx2 Expression in Response to ODN MT01\",\n \"Effects of ERK and p38 Inhibitors on the Expression of Osteocalcin and Type I Collagen\",\n \"Experimental Section\",\n \"Materials\",\n \"Cell Culture\",\n \"Fluorescent Labeling of MT01\",\n \"Western Blot Analysis\",\n \"ALP Activity Assay\",\n \"Real-Time PCR\",\n \"Statistical Analysis\",\n \"Conclusions\",\n \"Figure 1 .\",\n \"Figure 1 .\",\n \"Figure 2 .\",\n \"Figure 3 .\",\n \"Figure 4 .\",\n \"Figure 5 .\"\n]"},"annotations_table":{"kind":"list like","value":[],"string":"[]"},"annotations_tableref":{"kind":"list like","value":[],"string":"[]"},"annotations_title":{"kind":"list like","value":["A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways","A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways"],"string":"[\n \"A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways\",\n \"A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways\"\n]"},"annotations_venue":{"kind":"list like","value":["Int. J. Mol. Sci"],"string":"[\n \"Int. J. Mol. Sci\"\n]"}}},{"rowIdx":84741,"cells":{"corpusid":{"kind":"number","value":195064701,"string":"195,064,701"},"updated":{"kind":"string","value":"2022-02-06T16:46:01Z"},"content_source_oainfo_license":{"kind":"null"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"https://doi.org/10.1172/jci.insight.127748"},"content_source_oainfo_status":{"kind":"string","value":"GOLD"},"content_source_pdfsha":{"kind":"string","value":"7763ded8126fd66d292080ffad0b7de65f640443"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.1172/jci.insight.127748"},"externalids_mag":{"kind":"string","value":"2950028168"},"externalids_pubmed":{"kind":"string","value":"31211696"},"externalids_pubmedcentral":{"kind":"null"},"content_text":{"kind":"string","value":"\n\n6-18-2019\n\n\nSchool of Medicine\nWashington University\nWashington University School of Medicine Digital Commons@Becker Digital Commons@Becker\n\n\n\nDepartment of Cell Biology and Physiology\n\n\n\nDivision of Nephrology\nDepartment of Pediatrics\nSchool of Medicine\nWashington University\nSt. LouisMissouriUSA\n\n\nDepartment of Molecular and Cellular Biology\nHenry M. Goldman School of Dental Medicine\nBoston University\nBostonMassachusettsUSA\n\n\nCardiovascular Division\nDepartment of Medicine\nSchool of Medicine\nWashington University\nSt. LouisMissouriUSA\n\n\nSchool of Medicine\nDepartment of Cell Biology and Physiology\nWashington University\nCampus, 660 South Euclid AvenueBox 822863110St. LouisMissouriUSA\n6-18-201910.1172/jci.insight.127748\n\n\nIntracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress\n\nHeterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.\n\n\nIntroduction\n\nThoracic aortic aneurysm and dissection (TAAD) is a major cardiovascular health problem that arises from mutations that alter vascular extracellular matrix (ECM), smooth muscle cell function, or growth factor signaling pathways (1). In contrast to abdominal aortic aneurysms, TAAD has a strong genetic component, with pathogenic variants typically inherited in an autosomal dominant manner (2). A single highly penetrant, pathogenic mutation is responsible for disease in only one-fourth of individuals with TAAD, indicating that other undiscovered genetic factors influence disease onset and progression. In addition to the known pathogenic gene mutations, low-penetrant risk variants interact with other genetic or environmental factors to trigger disease or to increase the risk for developing disease but are not themselves pathogenic (2). Significant risk factors for TAAD, primarily when occurring in the background of low-penetrant disease-predisposing variants, are hypertension and bicuspid aortic valve (1).\n\nA common histopathological feature of TAAD in humans is fragmented elastin in the aortic medial region (3). Elastin is the most abundant ECM protein in the aorta, where it plays a central role in the vessel's ability to expand and recoil in response to pulsatile blood flow. Smooth muscle cells (SMCs) synthesize and organize the protein into fenestrated sheets (lamellae) that separate smooth muscle cell layers. The secreted form of elastin (tropoelastin) undergoes extensive crosslinking outside the cell to form an elastic polymer, which is the functional form of the protein. The enzyme responsible for initiating crosslink formation is lysyl oxidase (LOX), a copper-binding amine oxidase that oxidizes lysine ε-amino groups in elastin and fibrillar collagens to aldehydes, which then spontaneously react to form covalent linkages between and within proteins (4,5). Approximately 90% of the lysine residues in elastin undergo crosslink formation, which makes it one of the most highly crosslinked and most stable proteins in the body (4). Inactivation of the Lox gene Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.\n\nin mice results in early postnatal death due to ruptured aortic aneurysms, emphasizing the critical role of fully crosslinked, functional elastin in maintaining aorta integrity and function (6,7). The gene inactivation studies also show that other LOX isoforms cannot substitute for the loss of LOX in large, elastin-rich vessels.\n\nLOX in vertebrates belongs to a family of 5 copper-dependent enzymes (LOX, LOXL1-4) that show a high degree of homology in their carboxy-terminal catalytic domain but differ in their amino-terminal sequences. LOXL2-4 form a subgroup that shares 4 amino-terminal scavenger receptor cysteine-rich domains that influence substrate binding but appear to have little role in regulating catalytic activity (8,9). LOX and LOXL1, in contrast, are synthesized without scavenger receptor sequences but have a propeptide domain that keeps the enzymes in a latent state until proteolytically processed outside the cell by BMP1/Tolloid-like metalloproteinases (10,11). Substrate specificity for each of the lysyl oxidases is still under investigation, but gene inactivation studies and phylogenetic analysis suggest that LOX and LOXL1 contribute to crosslinking of elastin and fibrillar collagens, whereas LOXL2-4 have different substrate specificities (12,13). All 5 LOX isoforms are synthesized in the ER and traffic to the Golgi, where the inactive apoenzyme binds copper and forms the cofactor lysyl tyrosine quinone (LTQ); both copper and LTQ are essential active site components required for catalytic activity (14).\n\nMutations within LOX in families with TAAD were recently identified by whole exome sequencing (3,15). Aortic disease in these families followed autosomal dominant inheritance, suggesting that 1 mutant LOX allele is sufficient to influence disease initiation or progression. Previously, we identified a LOX catalytic site mutation (p.Met298Arg) in a family with a history of TAAD (3). By introducing this human mutation into the homologous position in the mouse genome using CRISPR/Cas9 genome editing technology (c.857T>G encoding p.M292R; hereafter referred to as Mu), we confirmed that this amino acid change caused ruptured TAAD and perinatal death when both alleles were mutated (Lox Mu/Mu ) (3). In contrast to humans who developed TAAD when heterozygous for this LOX variant, aneurysms or other arterial disease were not observed in mice with a similar genotype (Lox +/Mu ) unless they were challenged with hypertension. In this report, we show that the nature of the functional loss associated with the methionine-to-arginine change in LOX results from a secretion defect, where the protein is retained in the ER and degraded through an autophagy/proteasome pathway. We also show that the vessel wall alterations associated with LOX functional haploinsufficiency increase the likelihood of developing vascular disease in association with environmental factors that increase wall stress.\n\n\nResults\n\nLox +/Mu adult animals are predisposed to aortic dilation in response to hypertension. The generation and characterization of mice expressing the M292R mutation were described previously (3). Mice heterozygous for this Lox missense variant appear grossly normal, have normal blood pressure, and show no increased mortality through 2 years of age. Aortic diameter and circumferential wall stiffness at physiological blood pressure in Lox +/Mu animals are normal, but ascending aortic length measured from the aortic root to the brachiocephalic artery is 10% longer than in WT mice (3).\n\nHypertension is a known risk factor associated with aortic dilation (16). To test whether mice expressing mutant LOX are predisposed to aneurysm formation as a consequence of increased wall stress, Lox +/Mu mice were made hypertensive by subcutaneous delivery of angiotensin II (Ang II) via Alzet osmotic pumps. Pumps containing saline served as the control. Ang II infusion is a well-established model for inducing vessel wall changes in mice and other animals (17,18). Blood pressure measurements using radiotelemetry in awake, freely moving Lox +/+ and Lox +/Mu mice showed an increase of approximately 30 mmHg in systolic pressure and 25-30 mmHg in mean blood pressure in both genotypes 7-10 days after pump implantation (data not shown), consistent with what others have found using this model (19). After 4 weeks of treatment, all animals receiving Ang II had cardiac hypertrophy, indicating altered cardiac physiology consistent with Ang II-induced hypertension ( Figure 1A). However, only Lox +/Mu mice showed substantial dilation and elongation of the ascending aorta in response to Ang II; no change in aortic diameter was observed in Ang II-treated Lox +/+ mice or in Lox +/Mu mice infused with saline alone ( Figure 1B). Biaxial pressure-diameter testing showed the ascending aorta in Ang II-treated Lox +/Mu animals to be approximately 25% larger than the aorta from WT or saline-treated controls at mean physiological blood pressure ( Figure 1C). The shape of the pressure-diameter curves suggests that Ang II treatment did not affect aortic mechanical properties in either mutant or WT animals. Other than in the ascending aorta, no dilation or change in vessel mechanics occurred in the left common carotid artery for either Lox +/+ or Lox +/Mu animals with saline or Ang II treatment ( Figure 1D), nor were dilations or areas of stenosis found in the aortic arch, descending aorta, or abdominal aorta.\n\nAng II treatment leads to vessel wall thickening and changes in vascular cellularity. On a structural level, Ang II treatment resulted in ascending and descending aortic wall thickening in Lox +/Mu animals, but there were no changes in elastic lamellar number or lamellar organization, quantified by measurement of medial area ( Figure 2A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.127748DS1). Elastic lamellar fragmentation, already elevated in Lox +/Mu compared with Lox +/+ animals (3), did not increase in either genotype following Ang II treatment (data not shown). We analyzed elastin crosslinking using an ELISA assay for desmosine, a major crosslink in elastin. We found no difference in desmosine levels in aortic tissue from Lox +/+ and Lox +/Mu mice, confirming normal elastin levels and that elastin crosslinking is not affected in animals heterozygous for the Lox mutation (Supplemental Figure 1C). Hydroxyproline levels (as a measure of total collagen) trended higher in vessels from the mutant animals but were not significantly different from WT when normalized to the change in total protein (Supplemental Figure 1D). Higher protein quantities are consistent with the increase in wall thickness that occurred with Ang II treatment (Supplemental Figure 1B).\n\nChanges in the number and types of cells were observed in the thickened aortic wall of the hypertensive animals. The most substantial change occurred in the adventitia of both the ascending and descending aorta, which underwent considerable expansion in the Lox +/+ and Lox +/Mu animals as a result of Ang II exposure ( Figure 2A). In the medial layer, Ang II infusion led to a decrease in the number of SMCs in Lox +/+ animals but an increase in SMCs in Lox +/Mu mice ( Figure 2B). There was also an increase in intralamellar area in both genotypes, although the change was most notable in Lox +/Mu mice. The nuclei of the intralamellar cells in the mutant media were rounder than cells in Lox +/+ vessels, suggesting a less polarized Lox +/+ and Lox +/Mu animals developed significant cardiac hypertrophy following 4 weeks of treatment with 2 μg/kg/ min Ang II compared with saline-treated controls. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. ****P < 0.0001. (B) The ascending aorta in Lox +/Mu animals is longer than in Lox +/+ animals and undergoes substantial dilation following Ang II treatment. (C) Pressure-diameter measurements at intraluminal pressures ranging from 0 to 175 mmHg. The ascending aorta from Ang II-treated Lox +/Mu animals has a larger diameter over the entire range of pressures compared with Lox +/+ animals treated with Ang II or with saline controls. (D) There was no difference in the diameter or mechanical properties of the left common carotid artery in either genotype with or without Ang II treatment. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each pressure level. Data are presented as mean ± SD. # P < 0.05, Lox +/+ with Ang II vs. Lox +/Mu with Ang II; *P < 0.05, **P < 0.01, Lox +/Mu with saline vs. Lox +/Mu with Ang II.\n\nphenotype and noncircumferential orientation. Immunostaining for smooth muscle α-actin demonstrated uniform reactivity throughout the medial intralamellar spaces, whereas cell staining in the adventitia was heterogeneous (not shown).\n\nStaining for the monocyte/macrophage marker CD68 in descending aorta showed an increased number of CD68 + cells in the media and adventitia of both Lox +/+ and Lox +/Mu descending aorta following Ang II treatment ( Figure 2C). These results are consistent with previous studies showing that infusion of Ang II promotes macrophage accumulation within the aortic wall (20). While the adventitia showed the highest level of immune cell infiltration, macrophage accretion in the medial layer was also observed, but the spatial localization of the CD68 + cells was different for the 2 genotypes. In Ang II-treated Lox +/+ vessels, medial CD68 + cells were associated with the first 2 elastic layers nearest to the intima. Medial CD68 + cells in the aorta of Lox +/Mu animals treated with Ang II, however, were in the outer layers of the media closest to the adventitia ( Figure 2C).\n\nEvaluation of factors that adversely affect vessel wall integrity and SMC function. An explanation for why Lox +/Mu mice are more sensitive than WT animals to Ang II infusion was explored by first checking for possible differences in Ang II receptor expression using quantitative PCR (qPCR). In ascending aorta from Lox +/Mu mice, all 3 Ang II receptors (Agtr1a, Agtr1b, and Agtr2) were detected, and there was no difference in their expression levels . Macrophages (stained green, arrowheads) were detected with an antibody to CD68, and cell nuclei were visualized with DAPI. Red is elastin autofluorescence. The vessel wall was thicker in both Lox +/+ and Lox +/Mu animals following Ang II treatment, with more DAPI + and CD68 + cells in the adventitia of both genotypes. (B) In the medial layer, there were fewer SMCs in the aorta of WT animals treated with Ang II but more cells in Lox +/Mu aorta. Shown are the mean ± SD for cell number counts between the internal and external elastic lamina on 7 tissue sections from each group. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.01. (C) Medial CD68 + cells were mostly located on the luminal side of the media in the Lox +/+ aorta but nearer the adventitia in Lox +/Mu aorta after Ang II treatment.\n\ncompared with WT controls (Supplemental Figure 2A). Thus, the potential for Ang II receptor-mediated signaling is similar in both genotypes.\n\nMany Ang II-induced responses on SMCs are attributable to stimulation of NADPH oxidase with the subsequent generation of ROS (21,22). We used dihydroethidium, a cell-permeable compound that interacts with superoxide, to assess oxidant levels in sections of WT and mutant ascending aorta and found no difference between the 2 genotypes (Supplemental Figure 2B). Thus, differences in levels of oxidative stress do not appear to be a factor in the Ang II susceptibility of Lox +/Mu mice.\n\nIn many models of aortic disease, the presence of proteases that degrade elastin can initiate and propagate disease by degrading the elastic fiber system in the vessel wall. We therefore investigated whether 3 elastolytic MMPs commonly linked to vascular remodeling -MMP2, MMP9, and MMP12 -were differentially expressed in mutant versus WT aorta. There were no differences in Mmp2 and Mmp9 mRNA expression levels, while expression of Mmp12 was diminished in the mutant aorta (Supplemental Figure 2C).\n\nActive LOX is absent in Lox Mu/Mu mouse embryonic fibroblast conditioned medium. To elucidate how the M292R modification affects LOX function, we used mouse embryonic fibroblasts (MEFs) from WT, KO, and mutant animals to characterize LOX secretion and activity. Mutant cells were indistinguishable from WT cells in morphology and in their rate of proliferation (data not shown). LOX activity ( Figure 3A), assessed using an Amplex Red assay, was not detected in cell layer extracts of any MEF genotype (Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-), which was expected, since the full-length intracellular form of LOX (pro-LOX) is an inactive zymogen. LOX enzymatic activity was readily detected in the conditioned medium from Lox +/+ MEFs and showed linear enzyme kinetics over the period of the Amplex Red assay. Conditioned medium from Lox Mu/Mu MEFs, however, had no detectable activity, similar to conditioned medium from cells with inactivated Lox alleles (Lox -/-) ( Figure 3B). Activity was detected in conditioned medium from Lox +/Mu cells, where the linearity and rate of substrate oxidation were comparable to those seen in Lox +/+ cells, indicating that the mutant form of the protein does not adversely affect the enzymatic activity of WT LOX and hence does not function in a dominant negative fashion.\n\nM292R LOX is poorly secreted and is retained in the ER. LOX is maintained as an inactive 50-kDa proenzyme through every stage of the synthetic and secretory pathway. Activation requires secretion into the extracellular space, where proteolytic removal of the N-terminal propeptide portion of the protein generates the 30-kDa active catalytic domain. To determine whether LOX activity is absent in M292R MEF conditioned medium because mutant LOX is not secreted, we performed Western blots using an anti-LOX antibody on Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF cell layers and conditioned medium. Immunoblotting showed increased levels of 50-kDa intracellular pro-LOX protein in cell extracts from Lox Mu/Mu MEFs compared with Lox +/+ cells ( Figure 4A). Conversely, there was abundant 30-kDa mature (processed) LOX in conditioned medium from WT cells but barely detectable levels in medium from Lox Mu/Mu MEFs ( Figure 4B). These findings indicate that the mutant protein is poorly secreted and is retained intracellularly as the proenzyme. Immunofluorescence imaging with an antibody to LOX showed minimal intracellular staining in Lox +/+ cells, confirming that WT LOX is secreted efficiently ( Figure 5A). Staining of Lox Mu/Mu cells, however, showed abundant intracellular staining, consistent with the immunoblot results described above. The staining pattern for intracellular LOX was typical of accumulation within the ER, which was confirmed by colocalization of LOX with calnexin, an ER-resident protein ( Figure 5, B and C).\n\nA direct interaction between LOX and calnexin was shown using coimmunoprecipitation from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF lysates. Lox -/cells served as the negative control. Cell lysates were immunoprecipitated with an anti-calnexin antibody, followed by immunoblotting using an antibody to LOX. LOX protein was detected in calnexin immunoprecipitates from all genotypes except Lox -/-, confirming an interaction between the 2 proteins ( Figure 5D).\n\nM292R LOX has an altered protein conformation. The high level of LOX detected in Lox Mu/Mu MEF calnexin immunoprecipitates suggests that M292R LOX is misfolded and trapped in the ER bound to calnexin. To determine whether M292R LOX is conformationally different from the WT enzyme, we explored possible structural changes by assessing differential sensitivity to proteolytic degradation. Changes in protein structure frequently expose or mask protease binding sites, leading to altered degradation products and a different degradation fingerprint. Immunoblotting of cell lysate from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs incubated at 37°C for 6 hours to exploit protein degradation by endogenous enzymes within the cell lysate showed time-dependent degradation of WT but not M292R LOX ( Figure 6). These results suggest that WT and M292R pro-LOX have different susceptibilities to degradation arising from cell-associated proteases. Differential degradation was also observed when cell lysates were incubated with pancreatic elastase. In digests of Lox +/+ cell lysate, there was a 25-kDa fragment evident with all doses of elastase that was absent in Lox Mu/Mu samples (Supplemental Figure 3, arrow). When treated with the highest dose of elastase, pro-LOX from Lox +/+ and Lox +/Mu samples degraded predominantly to a 30-kDa fragment. LOX from Lox Mu/Mu samples, in contrast, degraded further to mainly a 10-kDa fragment. It is important to note that pancreatic elastase does not cleave after methionine or arginine, so the Met-to-Arg mutation does not create or negate a protease cleavage site for this enzyme. Together, the conformational difference between the WT and mutant proteins suggests that M292R LOX is misfolded, which is consistent with its protracted interaction with calnexin in the ER.\n\nM292R LOX does not elicit an ER stress response. Accumulation of misfolded proteins in the ER generally leads to ER stress, which subsequently activates the unfolded protein response (UPR) (23). UPR triggers 3 primary stress sensors, PERK, IRE1, and ATF6. Under resting conditions, BIP/Grp78 maintains these sensors in an inactive state. Upon ER stress, BIP binds to misfolded proteins, thereby allowing for sensor activation. To determine whether the continuous presence of misfolded mutant LOX induces ER stress, we assessed Bip transcript levels by qPCR. We also measured mRNA levels of 20% change was modest compared with the ~2-fold and ~4-fold increase in Atf4 and Chop, respectively, when ER stress was stimulated by treatment of MEFs with lopinavir/ritonavir, which served as a positive control (data not shown). Electron microscopic analysis of WT and mutant cells showed that ER volume was not markedly different in the 2 cell types (Supplemental Figure 4), suggesting that mutant protein does not accumulate to levels that would produce dilated saccules. Together, these results suggest that LOX protein containing the M292R mutation does not induce substantial ER stress.\n\nIncreased activation of autophagy in Lox Mu/Mu cells. The absence of ER stress in Lox Mu/Mu cells was surprising given that M292R LOX does not leave the ER. To determine whether mutant LOX is being removed from the ER for degradation through the lysosomal pathway, we costained cells for LOX and the lysosomal marker LAMP-2. No colocalization of the 2 proteins was observed, suggesting that the mutant protein was not trafficked through lysosomes (Supplemental Figure 5).\n\nAutophagy is a general mechanism for clearing proteins accumulating in cells. Clearing of misfolded proteins from the ER, a process termed ER-phagy, plays a critical role in ER homeostasis (24). To determine whether mutant LOX directly activates autophagy-mediated protein clearance from the ER, we measured 2 autophagy activation markers, p62 and microtubule-associated protein 1A/1B, light chain 3 (LC3), in WT and Lox Mu/Mu MEFs. Following pretreatment with bafilomycin A1 (BafA1) to inhibit organelle acidification-induced degradation of p62 and LC3, only Lox Mu/Mu cells with and without BafA1 pretreatment had significantly increased p62 expression compared with Lox +/+ cells without BafA1 pretreatment ( Figure 8, A and B). The autophagy activation markers LC3-I and -II were also elevated in mutant compared with WT cells (Figure 8, A, C, and D), providing more evidence for increased autophagy.\n\nWe also used electron microscopy to screen for the presence of autophagosome-like vesicles in WT and mutant cells (Supplemental Figure 4). We observed that multi-and single-membrane vesicles typical of autophagosomes were particularly abundant in the perinuclear region but were also present throughout the cytoplasm of Lox Mu/Mu cells. Such structures were rare in WT cells. Together, the existence of autophagic vesicles along with increased expression of autophagy activation markers in mutant cells is consistent with the clearance of misfolded mutant LOX from the ER through an autophagy/proteasome pathway.\n\nMutant LOX does not influence the secretion of the WT protein.\n\nTo assess whether the secretion of WT LOX is affected by the presence of M292R mutant protein, we transiently expressed plasmids containing WT LOX, and M292R LOX fused to the fluorescent protein mApple, in Lox -/-MEFs. Successful transfection was confirmed by intracellular mApple expression using fluorescence imaging (data not shown). In the conditioned medium of Lox -/cells transfected with empty plasmid, no LOX was observed by Western blot analysis ( Figure 9A). In Lox -/-MEFs transfected with 2.5 μg WT Lox-mApple plasmid, an immunoreactive protein band at approximately 60 kDa was detected in the conditioned media, corresponding to the size of active LOX (30 kDa) fused to mApple (27 kDa). In Lox -/-MEFs transfected with 2.5 μg of M292R Lox-mApple plasmid, LOX protein was observed inside the cell by fluorescence microscopy, but no LOX protein was detected in conditioned medium by Western blotting, consistent with the inability of M292R LOX to be secreted. To determine whether mutant LOX alters the secretion of the WT protein, or if the WT protein acts as a chaperone to facilitate the secretion of the mutant protein, we cotransfected cells with 1.25 μg each of WT and mutant plasmid, which is half as much as was used for single plasmid transfection. If mutant LOX does not affect the secretion of the WT protein, then LOX should be detected in conditioned medium, but at half the level of the single plasmid transfection. If the mutant protein has an adverse effect on WT LOX secretion, then levels will be lower than 50%. If WT LOX acts as a chaperone to facilitate secretion of the mutant protein, then the combination of both secreted proteins would be higher than 50% and possibly equivalent to the WT transfection (1.25 + 1.25 = 2.5 μg). Figure 9 shows that LOX protein was detected in conditioned medium of doubly transfected cells at half the level of singly transfected cells, confirming that the mutant protein does not affect the secretion of WT LOX.\n\nM292R propeptide processing. Our experimental results are consistent with the M292R mutation being a loss-of-function phenotype due to a secretion defect. The final activation step of the proenzyme occurs outside the cell through proteolytic separation of the propeptide and catalytic domains. To determine whether M292R pro-LOX could be processed to the 30-kDa mature form if it were secreted, we incubated cell lysates from WT and mutant MEFs with recombinant BMP1 (rBMP1), the enzyme responsible for propeptide cleavage. Western blot analysis showed that BMP1 appropriately processed both WT and M292R pro-LOX to the 30-kDa mature form (Supplemental Figure 6). Thus, the mutation did not inhibit propeptide processing. The processed mutant protein was not assayed for oxidase activity because intracellular LOX in M292R MEFs does not have bound copper and cannot be enzymatically active.\n\n\nDiscussion\n\nLOX plays a critical role in vascular ECM maturation by catalyzing the crosslinking of elastin and fibrillar collagens, the 2 major ECM protein classes in the aortic wall. Phylogenic studies suggest that the current form of vertebrate LOX coevolved with elastin and helped facilitate the appearance of a closed circulatory system (12,25). Therefore, it is no surprise that mutations in LOX give rise to vascular insufficiency and disease. All LOX mutations associated with TAAD in humans show autosomal dominant inheritance, and most result in loss of function. We know from mouse studies that inactivation of both Lox alleles is lethal and that other members of the LOX family cannot assume LOX's critical role in vascular development and maintenance of the vessel wall. In this report, we utilize a mouse model of a well-characterized human mutation to explore how LOX haploinsufficiency leads to vascular dilation and aneurysm formation. Mice heterozygous for the Lox M292R variant (Lox +/Mu ) have increased elastic lamellar fragmentation in the wall of the ascending aorta (3), but do not develop aortic dilation under normal conditions. The absence of aneurysmal disease was surprising given that humans heterozygous for this same mutation are prone to aneurysm formation and vessel wall changes (3). Variability in the age of disease onset and disease severity in humans, however, suggested that a secondary challenge is a factor in disease initiation and progression. Here we show that hypertension induced by Ang II infusion resulted in vessel wall changes in both Lox +/+ and Lox +/Mu mice, but only Lox +/Mu animals showed aortic dilation localized to the ascending aorta. Several groups have reported that the effects of Ang II are most pronounced in the ascending aorta region in this model (26)(27)(28), but the underlying factors governing this vessel specificity are unknown. Differences in the embryonic origin of the cells that populate the aortic wall are one explanation (29,30), but the composition and maturation of the aortic ECM are also factors. In Lox +/Mu mice, changes in ECM crosslinking associated with LOX insufficiency could have more negative consequences for this vessel segment because of high collagen and elastin levels and the high mechanical stress experienced by the aortic wall during the cardiac cycle.\n\nWhile several Ang II-induced vascular changes were expected (19,27) and common to WT and mutant animals, there were also significant differences. For instance, medial expansion was evident in both genotypes, but the effect was most notable in mutant mice, in which there was an increase in the number of smooth muscle cells between the elastic lamellae. This response is particularly unusual, since the intralamellar space is typically populated by a single smooth muscle cell layer, as can be seen in vessels from saline-treated Lox +/+ and Lox +/Mu animals ( Figure 2). The opposite change occurred in WT vessels, where there were fewer smooth muscle cells in response to Ang II, suggesting Ang II-related apoptosis or growth suppression (26), which we did not assess. mice. Oneway ANOVA with Tukey's multiple-comparisons test was used to assess differences, which were minimal between the 3 genotypes. Data are presented as mean ± SD. ***P < 0.001.\n\nAnother cellular change in the ascending and descending aorta following Ang II treatment was the accumulation of macrophages in the aortic wall of both WT and Lox +/Mu mice. CD68 + cells were found in the adventitia of both genotypes, with fewer cells in the medial layer. In Ang II-treated WT mice, CD68 + cells accumulated in the intima and inner elastic layers of the media and did not localize to breaks in elastic lamella, as has been reported in other models (18,20). Their location within the wall, however, shows that some cells were able to penetrate past the internal elastic lamina into the first or second layer of smooth muscle cells. Medial CD68 + cells in Lox +/Mu mice exposed to Ang II, in contrast, were localized to the outer aspect of the media and occurred at higher numbers than in WT vessels. These results suggest that the cellular origin of the CD68 + cells or the route of inflammatory cell infiltration is different in Lox +/+ and Lox +/Mu vessels as a response to wall stress.\n\nThe location of macrophages in the outer region of the wall provides a mechanistic explanation for why Lox +/Mu , but not WT, aorta dilates following Ang II treatment. ECM degradation is a primary factor leading to vessel wall dilation. Elastases and other matrix-degrading enzymes secreted by macrophages destabilize the vessel wall, particularly when matrix degradation occurs in or near the adventitia -the vessel compartment essential for maintaining the structural integrity of the artery (31). The medial cell hypertrophy and inflammatory cell accumulation in the Lox +/Mu aorta in response to hemodynamic stress are similar in several ways to vascular changes in mouse models of Marfan syndrome with mutations in fibrillin-1, a key elastic fiber protein. In Marfan mice, inflammatory cell accumulation at the medial-adventitial interface correlates with increased elastin degradation and a loss C and D) Also, LC3 was elevated in Lox Mu/Mu cells compared with Lox +/+ cells. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.001.\n\nof structural integrity that leads to vessel wall dilation (32). As in the Ang II-treated Lox +/Mu animals, only the ascending aorta is affected in Marfan mice.\n\nOur previous studies showed that mice with the Lox M292R mutation share numerous phenotypic traits with Lox +/animals (3), suggesting that M292R is a loss-of-function mutation in mice and humans. Even though the amino acid change is in the catalytic domain of LOX close to the copper binding site, it was not clear whether the mutation results in a catalytically dead enzyme or if some other aspect of LOX processing is negatively affected by the mutation. Our data show that the answer fits both possibilities: the mutant protein is retained in the ER and not secreted, and the enzyme is catalytically dead because of its inability to traffic to the Golgi, where it needs to bind copper and form the redox quinone cofactor LTQ required for catalytic activity (33). Even though we found that the propeptide of mutant pro-LOX could be cleaved by rBMP1 similarly to WT LOX, full-length LOX in the ER cannot be catalytically active even if propeptide processing does occur.\n\nWhy mutant LOX is retained in the ER is not clear, but our data point to protein misfolding as a probable cause. ER retention is characteristic of incompletely folded proteins. Folding intermediates of most secreted proteins interact with the calnexin/calreticulin protein complex in the ER to achieve the proper native conformation required for secretion (34). The calnexin/calreticulin protein complex recognizes N-linked glycan structures (LOX has 3 N-linked glycosylation sites in the propeptide region; ref. 11) that undergo cycles of sugar residue removal and addition. Once folded correctly, calnexin and calreticulin dissociate, and the folded proteins are transported from the ER to the Golgi apparatus. Mutant LOX colocalized intracellularly with calnexin and was isolated bound to calnexin in cellular extracts, suggesting that the protein remains misfolded despite repeated folding interactions with calnexin. Furthermore, the differential susceptibility to proteolytic degradation of mutant compared with WT LOX is consistent with structural differences between the 2 proteins.\n\nIt was interesting that mutant LOX accumulation in the ER did not induce an appreciable UPR. We saw no upregulation in mutant cells of the ER stress-related markers Bip (an ER luminal chaperone protein and critical ER stress sensor), Atf4 (a transcription factor downstream of PERK that plays a crucial role in the adaptation to ER stress), or Chop (a transcription factor regulated by ATF4 that drives proapoptotic gene transcription). Nor could we detect in mutant cells an increase in ER volume frequently associated with ER stress. One pathway whereby ER stress is mitigated is removal from the ER of mutant protein and degradation at the lysosome (ERAD-II) or proteasome (ERAD-I) (24). We were unable to localize LOX within lysosomes but did find increased expression of the autophagy markers p62 and LC3. Electron microscopy also identified an increased number of autophagic vesicles in Lox Mu/Mu cells. These results suggest that misfolded mutant LOX is being cleared from the ER through an autophagy/proteasome pathway and would explain why mutant protein levels never reached a critical threshold to sustain an ER stress response. Furthermore, there is mounting evidence that the autophagy and secretory pathways are coregulated and intimately linked (35), which may represent a mechanistic connection into the LOX trafficking defect that we identified. It should be noted that all the experiments probing the fate of the mutant protein were done in cultured cells. It is possible, but unlikely, that in vivo, in tissues, and at developmental stages relevant to the TAAD phenotype, LOX folding, secretion, ER retention, and stress occur differently than in MEFs.\n\nGuo et al. (15) described 2 other LOX active site variants (S280R and S348R) that segregate with disease in families with TAAD. When expressed from transgenes in HeLa cells and examined in cell lysates, a portion of the pro-LOX protein was processed to its active form and was able to oxidize substrate at rates approximately equivalent to the WT enzyme (S348R) or with approximately 50% lower activity (S280R) (2,15). Enzymatic activity in conditioned medium from the transfected cells was not evaluated, so it is not known whether these LOX variants are efficiently secreted. However, the fact that the proteins are catalytically active implies trafficking through the Golgi and proteolytic removal of the propeptide domain. Thus, unlike the M292R mutation, which is retained in the ER, the S348R and S280R variants appear capable of traversing the secretory pathway. How these proteins, which have full (or somewhat reduced) catalytic activity, cause thoracic aortic disease is an interesting question. An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.\n\n\nMethods\n\nAntibodies. Antibodies used in this study are described in Supplemental Table 1.\n\nMouse models and cell isolation. A knockin mouse strain bearing the Lox M292R substitution was generated in the C57BL/6 background using CRISPR/Cas9 genome editing technology as previously described (3). Lox +/mice in the same background are described in ref. 6. Primary MEFs were isolated from the various mouse lines by digestion of E14.5 mouse embryos with trypsin/EDTA at 37°C for 30 minutes in DMEM (38). Cells were collected by centrifugation and grown in DMEM, 10% FBS, 1% penicillin/streptomycin, and 1 mM l-glutamine.\n\nAng II treatment and blood pressure measurements. Telemetry transmitters (Data Sciences International, model PA-C10) were implanted surgically in 3-to 4-month-old mice under isoflurane anesthesia. The catheter was inserted in the left common carotid artery and advanced to the aortic arch, and the transmitter was placed subcutaneously along the abdomen. Animals were allowed to recover for 7 days prior to activation of transmitters, and continuous recording of heart rate and arterial pressure was obtained, with sampling every 5 minutes for 15-second intervals for 3 days. Data were collected and stored using Dataquest ART (Data Sciences International).\n\nAfter baseline blood pressure measurements were obtained, mice were implanted with osmotic pumps (Alzet model 1004, DURECT Corp.) that delivered Ang II (MilliporeSigma) subcutaneously at a dose of 2 μg/kg/min in the interscapular region, according to published procedures (39,40). Mice infused with saline were used as controls. After a week of Ang II treatment, arterial pressure and heart rate were again continuously recorded using the telemeters, with sampling every 5 minutes for 15-second intervals for 3 days. Animals were fed a standard laboratory chow diet. After 28 days, animals were sacrificed under isoflurane anesthesia, and vessels were collected for compliance studies and histological characterization.\n\nVessel compliance measurement. For vessel compliance measurements, the ascending aorta and left common carotid artery were excised and placed in a physiological saline solution. Vessels were then cleaned of surrounding fat and connective tissue and mounted onto a pressure arteriograph (Danish Myo Technology). Experiments were done at 37°C in an organ bath containing physiological saline. The mounted vessels were visualized using a computerized imaging system, allowing continuous recording of vessel diameters. Vessel outer diameters were recorded while intravascular pressure was increased from 0 to 175 mmHg by increments of 25 mmHg (12 seconds per step). The average of 3 outer diameter measurements was taken at each pressure (40,41).\n\nProtein quantification, hydroxyproline, and desmosine assays. Each ascending aorta was hydrolyzed at 105°C overnight with 20 μL of 6 N hydrochloric acid (Thermo Fisher Scientific) and dried under vacuum in a SpeedVac. The dried pellet was dissolved in 400 μL water and filtered with a 0.45-μm filter. Total protein, hydroxyproline, and desmosine levels were determined as described previously (42).\n\nDihydroethidium staining for superoxide detection in aortic sections. Superoxide in aortic sections was detected using dihydroethidum (DHE) staining. In the presence of superoxide, DHE is oxidized to 2-hydroxyethidium, which is highly fluorescent and intercalates within the DNA, staining the nucleus a fluorescent red (43,44). After euthanasia and thoracotomy, the right atrium was clipped, and 5 mL cold (4°C) 1× PBS was flushed through the left ventricle to clear the blood. Ascending aorta was then dissected, placed in Tissue-Tek O.C.T. Compound (Sakura Finetek), and flash frozen in liquid nitrogen. Four-micrometer cryosections were obtained using a Leica CM1850 cryostat and placed on charged microscope slides (Thermo Fisher Scientific). A 10-mM stock solution of DHE (Life Technologies) in DMSO was diluted to 10 μM using 1× PBS. The 10-μM DHE solution was applied to unfixed aortic sections and incubated at 37°C for 30 minutes in the dark. After staining with DHE, sections were then rinsed with 1× PBS for 5 minutes at room temperature (RT) and mounted with a coverslip using ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were obtained on the Zeiss Axioscope fluorescence microscope with a QImaging MicroPublisher 3.3 RTV camera using QCapture software (QImaging). The entire procedure (from mouse euthanasia to image acquisition) occurred within 8 hours.\n\nImmunofluorescence on aortic sections and area measurements. Ascending and descending aortas were dissected as above and placed in O.C.T. compound, and the blocks were frozen on dry ice. Four-micrometer-thick cryosections were placed on glass slides and stored at -80°C. The sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS at 4°C for 10 minutes, washed twice with 1× PBS (5 minutes each) at RT, and blocked with a solution containing 1% BSA (MilliporeSigma), 1% fish gelatin (MilliporeSigma), and 0.05% Triton-X (Acros Organics) in 1× PBS for 1 hour at RT. The aortic sections were then incubated with rat anti-mouse CD68 antibody (Bio-Rad) at a 1:1000 dilution in blocking solution at 4°C overnight. The next day, sections were washed 3 times with 1× PBS (5 minutes each) and incubated in goat anti-rat IgG Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) diluted 1:2000 and DAPI diluted 1:3000 in blocking solution at RT for 1 hour. The sections were washed twice with 1× PBS (5 minutes each) at RT, mounted with a coverslip using ProLong Diamond Antifade Mountant, and imaged using the Axioscope fluorescence microscope equipped with the QImaging MicroPublisher 3.3 RTV camera using QCapture software as above. For area measurements, frozen sections from the descending aorta from 3-month-old mice were prepared as described above. The area between the internal and external elastic laminae was traced in each ×40 image using ImageJ software (NIH) and measured in pixels 2 .\n\nLOX activity assay. MEFs were grown in 10-cm dishes and maintained as above until fully confluent. Twenty-four hours before collection, culture medium was replaced with DMEM lacking phenol red and FBS, supplemented with 50 μg/mL ascorbic acid and 0.1% BSA. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. Cell lysates were collected by scraping in 1× PBS containing protease inhibitors. LOX activity in cell lysate and concentrated culture medium, incubated with and without the LOX inhibitor β-aminopropionitrile (BAPN), was quantified using the Amplex Red assay as previously described (3,45). Resorufin fluorescence, the product of Amplex Red oxidation, was measured at excitation and emission wavelengths (540 and 600 nm, respectively) every 30 minutes for 150 minutes using a BioTek H4 Hybrid Reader. LOX activity was calculated as the difference between total activity and activity in the presence of BAPN. Differences in relative fluorescent units were tested using 2-way ANOVA with Tukey's multiple-comparisons test.\n\nImmunofluorescence imaging of primary cells. Primary MEFs were seeded in 4-chamber glass slides at 3 × 10 5 cells per/well. After 3 days in culture, the cells were fixed with cold methanol (2 washes, 5 seconds each) followed by 3 washes with blocking solution containing 1× PBS, 1% BSA, 1% fish gelatin (Milli-poreSigma), and 0.05% Triton-X100 at pH 7.4, then incubated in blocking solution for 30 minutes at RT. The cells were then incubated with a primary antibody to LOX at 1:1000 in blocking solution overnight at 4°C. After washing the cells with blocking solution to remove the primary antibody, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor 568 secondary antibody at 1:2000 (Thermo Fisher Scientific) and anti-calnexin primary antibody directly labeled with Alexa Fluor 488 at 1:4000 in blocking solution for 30 minutes at RT. The cells were washed with blocking solution 3 times, then mounted using ProLong Diamond Antifade Mountant (Invitrogen) and coverslipped. The samples were imaged using the Axioscope fluorescence microscope and QCapture Pro software.\n\nProtein extraction from cell culture and aortic tissue. Cultured MEFs were switched to serum-free medium 24 hours before protein extraction. MEFs in 10-cm dishes were incubated in 1 mL lysis (RIPA) buffer (MilliporeSigma) containing protease inhibitors for 30 minutes at 4°C. The lysates were scraped from the dish into microfuge tubes and centrifuged at 17,500 g for 5 minutes, and the supernatant was collected. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. The protein concentration in cell lysates and concentrated media was measured using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Laemmli buffer with and without DTT was added to samples for SDS-PAGE and native gel electrophoresis, respectively. Only the samples with DTT were boiled at 100°C for 5 minutes.\n\nRNA extraction and qPCR. RNA was extracted from confluent cell cultures using TRIzol (Thermo Fisher Scientific) following the manufacturer's protocol. For RNA isolation from aortic tissue, ascending aorta from adult Lox +/+ and Lox +/Mu mice were each homogenized in 500 μl TRIzol using QIAGEN TissueLyser. The manufacturer's protocol was followed for RNA isolation. At the time of RNA precipitation, 10 μg glycogen (Mil-liporeSigma) was added as a carrier to each sample. Each RNA pellet was dissolved in 10 μL molecular-grade water. The RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). One microgram of the isolated RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Reverse transcription was then carried out using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). One microliter of cDNA was used for qPCR using TaqMan Fast Universal PCR Master Mix and TaqMan assay primer/probes (Life Technologies) for Mmp12 (Mm00500554_m1), and Gapdh (Mm9999999915_ g1), which was used as an internal control. Ten-microliter reactions were performed in duplicate using a ViiA 7 Real-Time PCR System for Bip, Atf4, and Chop and QuantStudio 3 Real-Time PCR system (Applied Biosystems) for Agtr1a, Agtr1b, Agtr2, Mmp2, Mmp9, and Mmp12. All mRNA levels were normalized to Gapdh expression. rBMP1 treatment. Protein was extracted from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs as described above. Fifty microliters of cell lysates from cultured MEFs containing 50 μg total protein were incubated with 30, 60, and 90 ng rBMP1 (R&D Systems) for 45 minutes at 37°C in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5). The enzyme reaction was stopped by adding Laemmli buffer with DTT and incubated at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.\nBip (Mm00517691_m1), Atf4 (Mm00515325_g1), Chop (Mm01135937_g1), Agtr1a (Mm01957722_s1), Agtr1b (Mm02620758_s1), Agtr2 (Mm00431727_91), Mmp2 (Mm00439498_m1), Mmp9 (Mm00442991_m1),\nWestern blotting. Protein extracts from cells, concentrated condition medium, or tissues (generally 50 μg) were analyzed using 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) run at 100 mV for 1 hour. The protein was then transferred to ProBlott Membrane (Applied Biosystems). The blots were incubated in 5% nonfat dried milk in 0.1% 1× PBS-Tween for 1 hour at RT, then incubated with a primary antibody overnight at 4°C. The next day, the blots were washed with 0.1% 1× PBS-Tween and incubated in ECL anti-rabbit or anti-mouse IgG HRP-Linked Secondary Antibody (GE Healthcare) for 1 hour at RT. The blots were then washed and the immunoreactive bands detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The blots were stripped by incubating in 2% SDS, 75 mM Tris-HCl, and 100 mM β-mercaptoethanol for 1 hour at 65°C while shaking. The blot was then incubated in 0.1% 1× PBS-Tween wash buffer for 1 hour at RT before immunodetection of β-actin (MilliporeSigma) at 1:20,000 in wash buffer, which was used as a loading control. For protein samples from concentrated conditioned media, membranes were stained with Ponceau S solution (MilliporeSigma) before blocking and used as a loading control. Alternatively, stain-free images of Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) taken following the manufacturer's protocol served as a loading control.\n\nCoimmunoprecipitation. Cell lysates were prepared from MEFs 5 days after confluency as described above. Immobilized protein A anti-rabbit IgG beads (50 μL), preequilibrated by washing with lysis buffer, were then added to 250 μL cell extract. After incubation on a rocking platform for 60 minutes at 4°C, the sample was centrifuged at 2500 g for 3 minutes, and the supernatant was incubated with 8 μg immunoprecipitation antibody for 1 hour at 4°C. Fifty microliters of precleared protein A beads was then added, and the samples incubated overnight at 4°C with rocking. Immune complexes bound to the protein A beads were collected by centrifugation and washed with lysis buffer prior to boiling at 100°C for 10 minutes with Laemmli buffer containing DTT. The supernatant was collected by centrifugation, and proteins were separated by SDS-PAGE and immunoblotted following the same protocol described above.\n\nProtein degradation analysis. Confluent cultures of WT and mutant MEFs were treated with 5 μg/mL Brefeldin A (MilliporeSigma) to inhibit protein transport from ER to the Golgi apparatus. This ensured that WT LOX remained in the cells so that the starting amount of protein was the same across genotypes. After 4 hours, the cell lysate was extracted using lysis buffer placed at 37°C to initiate protein degradation. Every hour, 50 μL aliquot of cell lysate was collected, then immediately boiled with Laemmli sample buffer at 100°C for 5 minutes prior to SDS-PAGE analysis. For exogenous protease-mediated degradation, human pancreatic elastase in increasing concentrations (0.625, 1.25, 2.5, 5 ng/μL) was added to cell lysates containing 1 mg/ mL protein at 4°C for 30 minutes. After treatment, elastase activity was inhibited with protease inhibitor cocktail, and samples were boiled with Laemmli sample buffer at 100°C for 5 minutes for SDS-PAGE analysis.\n\nAutophagy markers. Confluent MEFs in 6-well dishes were pretreated with 100 mM BafA1 (MilliporeSigma) for 3 hours before cell lysates were collected. The protein samples were separated on 4%-15% gradient gels for SDS-PAGE analysis. Immunoblotting was performed following the same procedure as above.\n\nLox-mApple plasmid cloning and expression. The mouse Lox cDNA in a mammalian expression vector driven by a pCMV promoter was purchased from GE Healthcare. The Lox open reading frame was PCR amplified using a 5′ primer bearing an XhoI restriction enzyme site (5′-CAGATCTCGAGAGCGTTTCGCCTGGGCTGTGC-3′) and a 3′ primer designed to remove the Lox stop codon bearing a BamHI restriction enzyme site (5′-GGATCCA-CATACGGTGAAATTGTGCAGCCTGAG-3′). The PCR product and the pGEMT shuttle vector (Promega) were digested with XhoI and BamHI, then ligated together using T4 ligase. The mutant Lox plasmid bearing the 893 T>G base pair change was generated using the Lox-pGEMT plasmid as the template, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific), following the manufacturer's protocol (5′-TTACCA-CAGCAGGGACGAATTCAGCCACTATG-3′ and 5′-TGTTGGTGACAGCTGTGCCACTCCC-3′). The WT and mutant Lox-pGEMT plasmid sequences were confirmed using Sanger sequencing performed at the Washington University Protein and Nucleic Acid Chemistry Laboratory. To generate the Lox-mApple plasmids, WT and mutant Lox pGEMT plasmids and the mApple plasmid (46), provided by David W. Piston (Washington University School of Medicine in St. Louis), were digested with XhoI and BamHI restriction enzymes. The WT and mutant mLox cDNA inserts were then ligated into mApple vectors to generate the mLox-mApple fusion plasmids and sequenced to confirm. To express the WT and mutant Lox-mApple plasmids in MEFs, Lipofectamine 3000 reagents (Thermo Fisher Scientific) were used following the manufacturer's protocol.\n\nTransmission electron microscopy. MEFs from Lox +/+ , Lox +/Mu , and Lox Mu/Mu mice, cultured on glass coverslips, were washed in cacodylate buffer (0.15 M cacodylate in 2 mM CaCl 2 , pH 7.4) and fixed for 5 minutes with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer prewarmed to 37°C. The samples were then incubated at RT for an additional hour, washed with cacodylate buffer, and treated with 1% OsO 4 /1.5% potassium ferrocyanide for 1 hour in the dark. After washing with ultrapure water, en bloc staining was performed in 2% uranyl acetate for 1 hour in the dark. The samples were washed again in ultrapure water before dehydration in a graded acetone series. Finally, the samples were embedded in Epon resin and cured in a 60°C oven for 48 hours. The processed samples were imaged using a JEOL 1400 electron microscope.\n\nStatistics. One-way ANOVA with Tukey's multiple-comparisons test was used to determine the significance, if any, between different groups, particularly genotypes. When 2 variables were present, e.g., genotype and treatment or genotype and gene, 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences. If the same sample was measured repeatedly (e.g., at different pressures in the pressure-diameter curves in Figure 1, C and D, or at different time points in Figure 3), then repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was performed. Prism 8 for Mac OS X (GraphPad Software) was used to run statistical analyses. In all numerical figures, data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical test used and significant differences are noted in each figure legend.\n\nStudy approval. All animal experiments were carried out following protocols approved by Washington University School of Medicine Institutional Animal Care and Use Committee.\n\n\nAuthor contributions\n\nVSL, CMH, TJB, and PCT participated in experimental design and acquired and analyzed primary data. NOS helped with data analysis, and RPM designed the research study and analyzed primary data. VSL wrote the initial draft of the manuscript, to which all authors contributed edits.\n\n\nVivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.\n\nFigure 1 .\n1The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both\n\nFigure 2 .\n2Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)\n\nFigure 3 .\n3LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .\n\n\nAtf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the\n\nFigure 4 .\n4Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.\n\nFigure 5 .\n5Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.\n\nFigure 6 .\n6M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.\n\nFigure 7 .\n7M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu\n\nFigure 8 .\n8Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (\n\nFigure 9 .\n9Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A.\ninsight.jci.org https://doi.org/10.1172/jci.insight.127748\nAcknowledgmentsThis work was supported by NIH grants R01HL53325, HL105314 (RPM), R21AR072748 (PCT), and R01HL131961 (NOS). Funds were also provided to RPM by the Ines Mandl Research Foundation. VSL was supported by training grant T32 HL125241 and a Predoctoral Individual National Research Service Award (F31HL136073). CMH was supported by NIH grant K08 HL135400. We thank Robyn Roth for assistance with electron microscopy and gratefully acknowledge assistance from James Fitzpatrick and staff at the Washington University Center for Cellular Imaging (WUCCI), which is supported by Washington University School of Medicine, the Children's Discovery Institute of Washington University and St. Louis Children's Hospital (CDI-CORE-2015-505), and the Foundation for Barnes-Jewish Hospital (grant no. 3770). Additionally, we thank Michael Wallendorf of the Washington University Division of Biostatistics for assistance with statistical data analysis.\nTherapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. D M Milewicz, S K Prakash, F Ramirez, 10.1146/annurev-med-100415-022956Annu Rev Med. 68Milewicz DM, Prakash SK, Ramirez F. Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. 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Methods Cell Biol. 2018;143:147-156.\n\nPhotoconversion in orange and red fluorescent proteins. G J Kremers, K L Hazelwood, C S Murphy, M W Davidson, D W Piston, 10.1038/nmeth.1319Nat Methods. 65Kremers GJ, Hazelwood KL, Murphy CS, Davidson MW, Piston DW. Photoconversion in orange and red fluorescent proteins. Nat Methods. 2009;6(5):355-358.\n"},"annotations_abstract":{"kind":"list like","value":[],"string":"[]"},"annotations_author":{"kind":"list like","value":["\nSchool of Medicine\nWashington University\nWashington University School of Medicine Digital Commons@Becker Digital Commons@Becker\n\n","\nDepartment of Cell Biology and Physiology\n\n","\nDivision of Nephrology\nDepartment of Pediatrics\nSchool of Medicine\nWashington University\nSt. LouisMissouriUSA\n","\nDepartment of Molecular and Cellular Biology\nHenry M. 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D M Milewicz, S K Prakash, F Ramirez, 10.1146/annurev-med-100415-022956Annu Rev Med. 68Milewicz DM, Prakash SK, Ramirez F. Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. Annu Rev Med. 2017;68:51-67.","Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \"risk variants. C S Kwartler, 10.1016/j.ajhg.2018.05.012Am J Hum Genet. 1031Kwartler CS, et al. Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low pene- trant \"risk variants.\" Am J Hum Genet. 2018;103(1):138-143.","Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. V S Lee, 10.1073/pnas.1601442113Proc Natl Acad Sci U S A. 11331Lee VS, et al. Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. Proc Natl Acad Sci U S A. 2016;113(31):8759-8764.","B A Kozel, R P Mecham, Rosenbloom J Elastin, Biology of Extracellular Matrix: An Overview. Berlin-Heidelberg. Mecham RPGermanySpringer-VerlagKozel BA, Mecham RP, Rosenbloom J. Elastin. In: Mecham RP, ed. Biology of Extracellular Matrix: An Overview. Berlin-Heidel- berg, Germany: Springer-Verlag; 2011:267-301.","Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. P C Trackman, 10.1016/j.matbio.2016.01.001Matrix Biol. Trackman PC. Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. Matrix Biol. 2016;52-54:7-18.","Lysyl oxidase is required for vascular and diaphragmatic development in mice. I K Hornstra, S Birge, B Starcher, A J Bailey, R P Mecham, S D Shapiro, 10.1074/jbc.M210144200J Biol Chem. 27816Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem. 2003;278(16):14387-14393.","Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. J M Mäki, 10.1161/01.CIR.0000038109.84500.1ECirculation. 10619Mäki JM, et al. Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation. 2002;106(19):2503-2509.","Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. A J López-Jiménez, T Basak, R M Vanacore, 10.1074/jbc.M117.798603J Biol Chem. 29241López-Jiménez AJ, Basak T, Vanacore RM. Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. J Biol Chem. 2017;292(41):16970-16982.","Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. J M Mäki, H Tikkanen, K I Kivirikko, 10.1016/S0945-053X(01)00157-3Matrix Biol. 207Mäki JM, Tikkanen H, Kivirikko KI. Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. Matrix Biol. 2001;20(7):493-496.","Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. A Borel, 10.1074/jbc.M109499200J Biol Chem. 27652Borel A, et al. Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. J Biol Chem. 2001;276(52):48944-48949.","Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor. P C Trackman, D Bedell-Hogan, J Tang, H M Kagan, J Biol Chem. 26712Trackman PC, Bedell-Hogan D, Tang J, Kagan HM. Post-translational glycosylation and proteolytic processing of a lysyl oxi- dase precursor. J Biol Chem. 1992;267(12):8666-8671.","Origin and evolution of lysyl oxidases. X Grau-Bové, I Ruiz-Trillo, F Rodriguez-Pascual, Sci Rep. 510568Grau-Bové X, Ruiz-Trillo I, Rodriguez-Pascual F. Origin and evolution of lysyl oxidases. Sci Rep. 2015;5:10568.","Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. M Bignon, 10.1182/blood-2010-10-313296Blood. 11814Bignon M, et al. Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood. 2011;118(14):3979-3989.","Lysyl oxidase and lysyl oxidase-like enzymes. H M Kagan, F Ryvkin, Mecham RPSpringer-VerlagBerlin Heidelberg, GermanyThe Extracellular Matrix: An OverviewKagan HM, Ryvkin F. Lysyl oxidase and lysyl oxidase-like enzymes. In: Mecham RP, ed. The Extracellular Matrix: An Overview. Berlin Heidelberg, Germany: Springer-Verlag; 2011:303-335.","LOX mutations predispose to thoracic aortic aneurysms and dissections. D C Guo, 10.1161/CIRCRESAHA.115.307130Circ Res. 1186Guo DC, et al. LOX mutations predispose to thoracic aortic aneurysms and dissections. Circ Res. 2016;118(6):928-934.","Central artery stiffness and thoracic aortopathy. J D Humphrey, G Tellides, 10.1152/ajpheart.00205.2018Am J Physiol Heart Circ Physiol. 3161Humphrey JD, Tellides G. Central artery stiffness and thoracic aortopathy. Am J Physiol Heart Circ Physiol. 2019;316(1):H169-H182.","Aortic aneurysms. H Lu, A Daugherty, Arterioscler Thromb Vasc Biol. 376Lu H, Daugherty A. Aortic aneurysms. Arterioscler Thromb Vasc Biol. 2017;37(6):e59-e65.","Angiotensin II-mediated development of vascular diseases. A Daugherty, L Cassis, 10.1016/j.tcm.2004.01.002Trends Cardiovasc Med. 143Daugherty A, Cassis L. Angiotensin II-mediated development of vascular diseases. Trends Cardiovasc Med. 2004;14(3):117-120.","Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension. J Wu, 10.1161/HYPERTENSIONAHA.115.06123Hypertension. 672Wu J, et al. Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension. Hypertension. 2016;67(2):461-468.","Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. K Saraff, F Babamusta, L A Cassis, A Daugherty, 10.1161/01.ATV.0000085631.76095.64Arterioscler Thromb Vasc Biol. 239Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23(9):1621-1626.","Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. A E Dikalova, M C Góngora, D G Harrison, J D Lambeth, S Dikalov, K K Griendling, 10.1152/ajpheart.00242.2010Am J Physiol Heart Circ Physiol. 2993Dikalova AE, Góngora MC, Harrison DG, Lambeth JD, Dikalov S, Griendling KK. Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. Am J Physiol Heart Circ Physiol. 2010;299(3):H673-H679.","Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm. F Jiménez-Altayó, 10.1016/j.freeradbiomed.2018.02.023Free Radic Biol Med. 118Jiménez-Altayó F, et al. Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneu- rysm. Free Radic Biol Med. 2018;118:44-58.","The unfolded protein response and cell fate control. C Hetz, F R Papa, 10.1016/j.molcel.2017.06.017Mol Cell. 692Hetz C, Papa FR. The unfolded protein response and cell fate control. Mol Cell. 2018;69(2):169-181.",". M Smith, S Wilkinson, Er, 10.1042/EBC20170092Essays Biochem. 616Smith M, Wilkinson S. ER homeostasis and autophagy. Essays Biochem. 2017;61(6):625-635.","Vascular extracellular matrix and arterial mechanics. J E Wagenseil, R P Mecham, 10.1152/physrev.00041.2008Physiol Rev. 893Wagenseil JE, Mecham RP. Vascular extracellular matrix and arterial mechanics. Physiol Rev. 2009;89(3):957-989.","Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. A P Owens, 10.1161/CIRCRESAHA.109.212837Circ Res. 1063Owens AP, et al. Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. Circ Res. 2010;106(3):611-619.","Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice. A Daugherty, D L Rateri, I F Charo, A P Owens, D A Howatt, L A Cassis, 10.1042/CS20090372Clin Sci. 11811Daugherty A, Rateri DL, Charo IF, Owens AP, Howatt DA, Cassis LA. Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice. Clin Sci. 2010;118(11):681-689.","Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissections. B Trachet, 10.1161/ATVBAHA.116.307211Arterioscler Thromb Vasc Biol. 364Trachet B, et al. Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissec- tions. Arterioscler Thromb Vasc Biol. 2016;36(4):673-681.","Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J E Hungerford, C D Little, 10.1159/000025622J Vasc Res. 361Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999;36(1):2-27.","Developmental basis of vascular smooth muscle diversity. M W Majesky, 10.1161/ATVBAHA.107.141069Arterioscler Thromb Vasc Biol. 276Majesky MW. Developmental basis of vascular smooth muscle diversity. Arterioscler Thromb Vasc Biol. 2007;27(6):1248-1258.","Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. R Pyo, 10.1172/JCI8931J Clin Invest. 10511Pyo R, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest. 2000;105(11):1641-1649.","Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. L Pereira, 10.1073/pnas.96.7.3819Proc Natl Acad Sci U S A. 967Pereira L, et al. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci U S A. 1999;96(7):3819-3823.","Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors. J P Klinman, F ; Bonnot, Pqq, Ttq, Tpq Ctq, Ltq , 10.1021/cr400475gChem Rev. 1148Klinman JP, Bonnot F. Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ. Chem Rev. 2014;114(8):4343-4365.","Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. J X Zhang, I Braakman, K E Matlack, A Helenius, 10.1091/mbc.8.10.1943Mol Biol Cell. 810Zhang JX, Braakman I, Matlack KE, Helenius A. Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell. 1997;8(10):1943-1954.","The link between autophagy and secretion: a story of multitasking proteins. H Farhan, M Kundu, S Ferro-Novick, 10.1091/mbc.e16-11-0762Mol Biol Cell. 289Farhan H, Kundu M, Ferro-Novick S. The link between autophagy and secretion: a story of multitasking proteins. Mol Biol Cell. 2017;28(9):1161-1164.","Functional importance of lysyl oxidase family propeptide regions. P C Trackman, 10.1007/s12079-017-0424-4J Cell Commun Signal. 121Trackman PC. Functional importance of lysyl oxidase family propeptide regions. J Cell Commun Signal. 2018;12(1):45-53.","Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. S D Vallet, 10.1038/s41598-018-30190-6Sci Rep. 8111768Vallet SD, et al. Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. Sci Rep. 2018;8(1):11768.","Isolation and handling of mouse embryonic fibroblasts. K Jain, P J Verma, J Liu, 10.1007/978-1-4939-1215-5_13Methods Mol Biol. 1194Jain K, Verma PJ, Liu J. Isolation and handling of mouse embryonic fibroblasts. Methods Mol Biol. 2014;1194:247-252.","Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. A Daugherty, M W Manning, L A Cassis, 10.1172/JCI7818J Clin Invest. 10511Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000;105(11):1605-1612.","Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. C M Halabi, T J Broekelmann, R H Knutsen, L Ye, R P Mecham, B A Kozel, Am J Physiol Heart Circ Physiol. 3095Halabi CM, Broekelmann TJ, Knutsen RH, Ye L, Mecham RP, Kozel BA. Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. Am J Physiol Heart Circ Physiol. 2015;309(5):H1008-H1016.","Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. G Faury, 10.1172/JCI19028J Clin Invest. 1129Faury G, et al. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. J Clin Invest. 2003;112(9):1419-1428.","Measurement of elastin, collagen, and total protein levels in tissues. I Stoilov, B C Starcher, R P Mecham, T J Broekelmann, 10.1016/bs.mcb.2017.08.008Methods Cell Biol. 143Stoilov I, Starcher BC, Mecham RP, Broekelmann TJ. Measurement of elastin, collagen, and total protein levels in tissues. Methods Cell Biol. 2018;143:133-146.","Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity. H Cai, S Dikalov, K K Griendling, D G Harrison, 10.1007/978-1-59745-571-8_20Methods Mol Med. 139Cai H, Dikalov S, Griendling KK, Harrison DG. Detection of reactive oxygen species and nitric oxide in vascular cells and tis- sues: comparison of sensitivity and specificity. Methods Mol Med. 2007;139:293-311.","Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. B Fink, K Laude, L Mccann, A Doughan, D G Harrison, S Dikalov, 10.1152/ajpcell.00028.2004Am J Physiol Cell Physiol. 2874Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S. Detection of intracellular superoxide formation in endothe- lial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol. 2004;287(4):C895-C902.","Measurement of lysyl oxidase activity from small tissue samples and cell cultures. P C Trackman, M V Bais, 10.1016/bs.mcb.2017.08.009Methods Cell Biol. 143Trackman PC, Bais MV. Measurement of lysyl oxidase activity from small tissue samples and cell cultures. Methods Cell Biol. 2018;143:147-156.","Photoconversion in orange and red fluorescent proteins. G J Kremers, K L Hazelwood, C S Murphy, M W Davidson, D W Piston, 10.1038/nmeth.1319Nat Methods. 65Kremers GJ, Hazelwood KL, Murphy CS, Davidson MW, Piston DW. Photoconversion in orange and red fluorescent proteins. Nat Methods. 2009;6(5):355-358."],"string":"[\n \"Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. D M Milewicz, S K Prakash, F Ramirez, 10.1146/annurev-med-100415-022956Annu Rev Med. 68Milewicz DM, Prakash SK, Ramirez F. Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. Annu Rev Med. 2017;68:51-67.\",\n \"Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \\\"risk variants. C S Kwartler, 10.1016/j.ajhg.2018.05.012Am J Hum Genet. 1031Kwartler CS, et al. Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low pene- trant \\\"risk variants.\\\" Am J Hum Genet. 2018;103(1):138-143.\",\n \"Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. V S Lee, 10.1073/pnas.1601442113Proc Natl Acad Sci U S A. 11331Lee VS, et al. Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. Proc Natl Acad Sci U S A. 2016;113(31):8759-8764.\",\n \"B A Kozel, R P Mecham, Rosenbloom J Elastin, Biology of Extracellular Matrix: An Overview. Berlin-Heidelberg. Mecham RPGermanySpringer-VerlagKozel BA, Mecham RP, Rosenbloom J. Elastin. In: Mecham RP, ed. Biology of Extracellular Matrix: An Overview. Berlin-Heidel- berg, Germany: Springer-Verlag; 2011:267-301.\",\n \"Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. P C Trackman, 10.1016/j.matbio.2016.01.001Matrix Biol. Trackman PC. Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. Matrix Biol. 2016;52-54:7-18.\",\n \"Lysyl oxidase is required for vascular and diaphragmatic development in mice. I K Hornstra, S Birge, B Starcher, A J Bailey, R P Mecham, S D Shapiro, 10.1074/jbc.M210144200J Biol Chem. 27816Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem. 2003;278(16):14387-14393.\",\n \"Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. J M Mäki, 10.1161/01.CIR.0000038109.84500.1ECirculation. 10619Mäki JM, et al. Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation. 2002;106(19):2503-2509.\",\n \"Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. A J López-Jiménez, T Basak, R M Vanacore, 10.1074/jbc.M117.798603J Biol Chem. 29241López-Jiménez AJ, Basak T, Vanacore RM. Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. J Biol Chem. 2017;292(41):16970-16982.\",\n \"Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. J M Mäki, H Tikkanen, K I Kivirikko, 10.1016/S0945-053X(01)00157-3Matrix Biol. 207Mäki JM, Tikkanen H, Kivirikko KI. Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. Matrix Biol. 2001;20(7):493-496.\",\n \"Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. A Borel, 10.1074/jbc.M109499200J Biol Chem. 27652Borel A, et al. Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. J Biol Chem. 2001;276(52):48944-48949.\",\n \"Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor. P C Trackman, D Bedell-Hogan, J Tang, H M Kagan, J Biol Chem. 26712Trackman PC, Bedell-Hogan D, Tang J, Kagan HM. Post-translational glycosylation and proteolytic processing of a lysyl oxi- dase precursor. J Biol Chem. 1992;267(12):8666-8671.\",\n \"Origin and evolution of lysyl oxidases. X Grau-Bové, I Ruiz-Trillo, F Rodriguez-Pascual, Sci Rep. 510568Grau-Bové X, Ruiz-Trillo I, Rodriguez-Pascual F. Origin and evolution of lysyl oxidases. Sci Rep. 2015;5:10568.\",\n \"Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. M Bignon, 10.1182/blood-2010-10-313296Blood. 11814Bignon M, et al. Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood. 2011;118(14):3979-3989.\",\n \"Lysyl oxidase and lysyl oxidase-like enzymes. H M Kagan, F Ryvkin, Mecham RPSpringer-VerlagBerlin Heidelberg, GermanyThe Extracellular Matrix: An OverviewKagan HM, Ryvkin F. Lysyl oxidase and lysyl oxidase-like enzymes. In: Mecham RP, ed. The Extracellular Matrix: An Overview. Berlin Heidelberg, Germany: Springer-Verlag; 2011:303-335.\",\n \"LOX mutations predispose to thoracic aortic aneurysms and dissections. D C Guo, 10.1161/CIRCRESAHA.115.307130Circ Res. 1186Guo DC, et al. LOX mutations predispose to thoracic aortic aneurysms and dissections. Circ Res. 2016;118(6):928-934.\",\n \"Central artery stiffness and thoracic aortopathy. J D Humphrey, G Tellides, 10.1152/ajpheart.00205.2018Am J Physiol Heart Circ Physiol. 3161Humphrey JD, Tellides G. Central artery stiffness and thoracic aortopathy. Am J Physiol Heart Circ Physiol. 2019;316(1):H169-H182.\",\n \"Aortic aneurysms. H Lu, A Daugherty, Arterioscler Thromb Vasc Biol. 376Lu H, Daugherty A. Aortic aneurysms. Arterioscler Thromb Vasc Biol. 2017;37(6):e59-e65.\",\n \"Angiotensin II-mediated development of vascular diseases. A Daugherty, L Cassis, 10.1016/j.tcm.2004.01.002Trends Cardiovasc Med. 143Daugherty A, Cassis L. Angiotensin II-mediated development of vascular diseases. Trends Cardiovasc Med. 2004;14(3):117-120.\",\n \"Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension. J Wu, 10.1161/HYPERTENSIONAHA.115.06123Hypertension. 672Wu J, et al. Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension. Hypertension. 2016;67(2):461-468.\",\n \"Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. K Saraff, F Babamusta, L A Cassis, A Daugherty, 10.1161/01.ATV.0000085631.76095.64Arterioscler Thromb Vasc Biol. 239Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23(9):1621-1626.\",\n \"Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. A E Dikalova, M C Góngora, D G Harrison, J D Lambeth, S Dikalov, K K Griendling, 10.1152/ajpheart.00242.2010Am J Physiol Heart Circ Physiol. 2993Dikalova AE, Góngora MC, Harrison DG, Lambeth JD, Dikalov S, Griendling KK. Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. Am J Physiol Heart Circ Physiol. 2010;299(3):H673-H679.\",\n \"Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm. F Jiménez-Altayó, 10.1016/j.freeradbiomed.2018.02.023Free Radic Biol Med. 118Jiménez-Altayó F, et al. Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneu- rysm. Free Radic Biol Med. 2018;118:44-58.\",\n \"The unfolded protein response and cell fate control. C Hetz, F R Papa, 10.1016/j.molcel.2017.06.017Mol Cell. 692Hetz C, Papa FR. The unfolded protein response and cell fate control. Mol Cell. 2018;69(2):169-181.\",\n \". M Smith, S Wilkinson, Er, 10.1042/EBC20170092Essays Biochem. 616Smith M, Wilkinson S. ER homeostasis and autophagy. Essays Biochem. 2017;61(6):625-635.\",\n \"Vascular extracellular matrix and arterial mechanics. J E Wagenseil, R P Mecham, 10.1152/physrev.00041.2008Physiol Rev. 893Wagenseil JE, Mecham RP. Vascular extracellular matrix and arterial mechanics. Physiol Rev. 2009;89(3):957-989.\",\n \"Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. A P Owens, 10.1161/CIRCRESAHA.109.212837Circ Res. 1063Owens AP, et al. Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. Circ Res. 2010;106(3):611-619.\",\n \"Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice. A Daugherty, D L Rateri, I F Charo, A P Owens, D A Howatt, L A Cassis, 10.1042/CS20090372Clin Sci. 11811Daugherty A, Rateri DL, Charo IF, Owens AP, Howatt DA, Cassis LA. Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice. Clin Sci. 2010;118(11):681-689.\",\n \"Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissections. B Trachet, 10.1161/ATVBAHA.116.307211Arterioscler Thromb Vasc Biol. 364Trachet B, et al. Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissec- tions. Arterioscler Thromb Vasc Biol. 2016;36(4):673-681.\",\n \"Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J E Hungerford, C D Little, 10.1159/000025622J Vasc Res. 361Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999;36(1):2-27.\",\n \"Developmental basis of vascular smooth muscle diversity. M W Majesky, 10.1161/ATVBAHA.107.141069Arterioscler Thromb Vasc Biol. 276Majesky MW. Developmental basis of vascular smooth muscle diversity. Arterioscler Thromb Vasc Biol. 2007;27(6):1248-1258.\",\n \"Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. R Pyo, 10.1172/JCI8931J Clin Invest. 10511Pyo R, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest. 2000;105(11):1641-1649.\",\n \"Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. L Pereira, 10.1073/pnas.96.7.3819Proc Natl Acad Sci U S A. 967Pereira L, et al. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci U S A. 1999;96(7):3819-3823.\",\n \"Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors. J P Klinman, F ; Bonnot, Pqq, Ttq, Tpq Ctq, Ltq , 10.1021/cr400475gChem Rev. 1148Klinman JP, Bonnot F. Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ. Chem Rev. 2014;114(8):4343-4365.\",\n \"Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. J X Zhang, I Braakman, K E Matlack, A Helenius, 10.1091/mbc.8.10.1943Mol Biol Cell. 810Zhang JX, Braakman I, Matlack KE, Helenius A. Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell. 1997;8(10):1943-1954.\",\n \"The link between autophagy and secretion: a story of multitasking proteins. H Farhan, M Kundu, S Ferro-Novick, 10.1091/mbc.e16-11-0762Mol Biol Cell. 289Farhan H, Kundu M, Ferro-Novick S. The link between autophagy and secretion: a story of multitasking proteins. Mol Biol Cell. 2017;28(9):1161-1164.\",\n \"Functional importance of lysyl oxidase family propeptide regions. P C Trackman, 10.1007/s12079-017-0424-4J Cell Commun Signal. 121Trackman PC. Functional importance of lysyl oxidase family propeptide regions. J Cell Commun Signal. 2018;12(1):45-53.\",\n \"Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. S D Vallet, 10.1038/s41598-018-30190-6Sci Rep. 8111768Vallet SD, et al. Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. Sci Rep. 2018;8(1):11768.\",\n \"Isolation and handling of mouse embryonic fibroblasts. K Jain, P J Verma, J Liu, 10.1007/978-1-4939-1215-5_13Methods Mol Biol. 1194Jain K, Verma PJ, Liu J. Isolation and handling of mouse embryonic fibroblasts. Methods Mol Biol. 2014;1194:247-252.\",\n \"Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. A Daugherty, M W Manning, L A Cassis, 10.1172/JCI7818J Clin Invest. 10511Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000;105(11):1605-1612.\",\n \"Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. C M Halabi, T J Broekelmann, R H Knutsen, L Ye, R P Mecham, B A Kozel, Am J Physiol Heart Circ Physiol. 3095Halabi CM, Broekelmann TJ, Knutsen RH, Ye L, Mecham RP, Kozel BA. Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. Am J Physiol Heart Circ Physiol. 2015;309(5):H1008-H1016.\",\n \"Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. G Faury, 10.1172/JCI19028J Clin Invest. 1129Faury G, et al. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. J Clin Invest. 2003;112(9):1419-1428.\",\n \"Measurement of elastin, collagen, and total protein levels in tissues. I Stoilov, B C Starcher, R P Mecham, T J Broekelmann, 10.1016/bs.mcb.2017.08.008Methods Cell Biol. 143Stoilov I, Starcher BC, Mecham RP, Broekelmann TJ. Measurement of elastin, collagen, and total protein levels in tissues. Methods Cell Biol. 2018;143:133-146.\",\n \"Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity. H Cai, S Dikalov, K K Griendling, D G Harrison, 10.1007/978-1-59745-571-8_20Methods Mol Med. 139Cai H, Dikalov S, Griendling KK, Harrison DG. Detection of reactive oxygen species and nitric oxide in vascular cells and tis- sues: comparison of sensitivity and specificity. Methods Mol Med. 2007;139:293-311.\",\n \"Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. B Fink, K Laude, L Mccann, A Doughan, D G Harrison, S Dikalov, 10.1152/ajpcell.00028.2004Am J Physiol Cell Physiol. 2874Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S. Detection of intracellular superoxide formation in endothe- lial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol. 2004;287(4):C895-C902.\",\n \"Measurement of lysyl oxidase activity from small tissue samples and cell cultures. P C Trackman, M V Bais, 10.1016/bs.mcb.2017.08.009Methods Cell Biol. 143Trackman PC, Bais MV. Measurement of lysyl oxidase activity from small tissue samples and cell cultures. Methods Cell Biol. 2018;143:147-156.\",\n \"Photoconversion in orange and red fluorescent proteins. G J Kremers, K L Hazelwood, C S Murphy, M W Davidson, D W Piston, 10.1038/nmeth.1319Nat Methods. 65Kremers GJ, Hazelwood KL, Murphy CS, Davidson MW, Piston DW. Photoconversion in orange and red fluorescent proteins. Nat Methods. 2009;6(5):355-358.\"\n]"},"annotations_bibref":{"kind":"list like","value":["(1)","(2)","(2)","(1)","(3)","(4,","5)","(4)","(6,","7)","(8,","9)","(10,","11)","(12,","13)","(14)","(3,","15)","(3)","(3)","(16)","(17,","18)","(19)","(20)","(21,","22)","(23)","(24)","(12,","25)","(26)","(27)","(28)","(29,","30)","(19,","27)","(18,","20)","(31)","(32)","(33)","(34)","(24)","(35)","(15)","(2,","15)","(5,","36,","37)","(3)","6","(38)","(39,","40)","(40,","41)","(42)","(43,","44)","(3,","45)","(46)"],"string":"[\n \"(1)\",\n \"(2)\",\n \"(2)\",\n \"(1)\",\n \"(3)\",\n \"(4,\",\n \"5)\",\n \"(4)\",\n \"(6,\",\n \"7)\",\n \"(8,\",\n \"9)\",\n \"(10,\",\n \"11)\",\n \"(12,\",\n \"13)\",\n \"(14)\",\n \"(3,\",\n \"15)\",\n \"(3)\",\n \"(3)\",\n \"(16)\",\n \"(17,\",\n \"18)\",\n \"(19)\",\n \"(20)\",\n \"(21,\",\n \"22)\",\n \"(23)\",\n \"(24)\",\n \"(12,\",\n \"25)\",\n \"(26)\",\n \"(27)\",\n \"(28)\",\n \"(29,\",\n \"30)\",\n \"(19,\",\n \"27)\",\n \"(18,\",\n \"20)\",\n \"(31)\",\n \"(32)\",\n \"(33)\",\n \"(34)\",\n \"(24)\",\n \"(35)\",\n \"(15)\",\n \"(2,\",\n \"15)\",\n \"(5,\",\n \"36,\",\n \"37)\",\n \"(3)\",\n \"6\",\n \"(38)\",\n \"(39,\",\n \"40)\",\n \"(40,\",\n \"41)\",\n \"(42)\",\n \"(43,\",\n \"44)\",\n \"(3,\",\n \"45)\",\n \"(46)\"\n]"},"annotations_bibtitle":{"kind":"list like","value":["Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models","Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \"risk variants","Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans","Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone","Lysyl oxidase is required for vascular and diaphragmatic development in mice","Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice","Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV","Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains","Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1","Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor","Origin and evolution of lysyl oxidases","Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane","LOX mutations predispose to thoracic aortic aneurysms and dissections","Central artery stiffness and thoracic aortopathy","Aortic aneurysms","Angiotensin II-mediated development of vascular diseases","Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension","Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice","Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling","Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm","The unfolded protein response and cell fate control","Vascular extracellular matrix and arterial mechanics","Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3","Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice","Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissections","Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall","Developmental basis of vascular smooth muscle diversity","Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms","Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1","Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors","Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations","The link between autophagy and secretion: a story of multitasking proteins","Functional importance of lysyl oxidase family propeptide regions","Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners","Isolation and handling of mouse embryonic fibroblasts","Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice","Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness","Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency","Measurement of elastin, collagen, and total protein levels in tissues","Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity","Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay","Measurement of lysyl oxidase activity from small tissue samples and cell cultures","Photoconversion in orange and red fluorescent proteins"],"string":"[\n \"Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models\",\n \"Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \\\"risk variants\",\n \"Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans\",\n \"Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone\",\n \"Lysyl oxidase is required for vascular and diaphragmatic development in mice\",\n \"Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice\",\n \"Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV\",\n \"Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains\",\n \"Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1\",\n \"Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor\",\n \"Origin and evolution of lysyl oxidases\",\n \"Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane\",\n \"LOX mutations predispose to thoracic aortic aneurysms and dissections\",\n \"Central artery stiffness and thoracic aortopathy\",\n \"Aortic aneurysms\",\n \"Angiotensin II-mediated development of vascular diseases\",\n \"Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension\",\n \"Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice\",\n \"Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling\",\n \"Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm\",\n \"The unfolded protein response and cell fate control\",\n \"Vascular extracellular matrix and arterial mechanics\",\n \"Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3\",\n \"Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice\",\n \"Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissections\",\n \"Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall\",\n \"Developmental basis of vascular smooth muscle diversity\",\n \"Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms\",\n \"Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1\",\n \"Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors\",\n \"Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations\",\n \"The link between autophagy and secretion: a story of multitasking proteins\",\n \"Functional importance of lysyl oxidase family propeptide regions\",\n \"Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners\",\n \"Isolation and handling of mouse embryonic fibroblasts\",\n \"Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice\",\n \"Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness\",\n \"Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency\",\n \"Measurement of elastin, collagen, and total protein levels in tissues\",\n \"Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity\",\n \"Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay\",\n \"Measurement of lysyl oxidase activity from small tissue samples and cell cultures\",\n \"Photoconversion in orange and red fluorescent proteins\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["Annu Rev Med","Am J Hum Genet","Proc Natl Acad Sci U S A","Biology of Extracellular Matrix: An Overview. Berlin-Heidelberg","Matrix Biol","J Biol Chem","Circulation","J Biol Chem","Matrix Biol","J Biol Chem","J Biol Chem","Sci Rep","Blood","Lysyl oxidase and lysyl oxidase-like enzymes","Circ Res","Am J Physiol Heart Circ Physiol","Arterioscler Thromb Vasc Biol","Trends Cardiovasc Med","Hypertension","Arterioscler Thromb Vasc Biol","Am J Physiol Heart Circ Physiol","Free Radic Biol Med","Mol Cell","Essays Biochem","Physiol Rev","Circ Res","Clin Sci","Arterioscler Thromb Vasc Biol","J Vasc Res","Arterioscler Thromb Vasc Biol","J Clin Invest","Proc Natl Acad Sci U S A","Chem Rev","Mol Biol Cell","Mol Biol Cell","J Cell Commun Signal","Sci Rep","Methods Mol Biol","J Clin Invest","Am J Physiol Heart Circ Physiol","J Clin Invest","Methods Cell Biol","Methods Mol Med","Am J Physiol Cell Physiol","Methods Cell Biol","Nat Methods","Germany"],"string":"[\n \"Annu Rev Med\",\n \"Am J Hum Genet\",\n \"Proc Natl Acad Sci U S A\",\n \"Biology of Extracellular Matrix: An Overview. Berlin-Heidelberg\",\n \"Matrix Biol\",\n \"J Biol Chem\",\n \"Circulation\",\n \"J Biol Chem\",\n \"Matrix Biol\",\n \"J Biol Chem\",\n \"J Biol Chem\",\n \"Sci Rep\",\n \"Blood\",\n \"Lysyl oxidase and lysyl oxidase-like enzymes\",\n \"Circ Res\",\n \"Am J Physiol Heart Circ Physiol\",\n \"Arterioscler Thromb Vasc Biol\",\n \"Trends Cardiovasc Med\",\n \"Hypertension\",\n \"Arterioscler Thromb Vasc Biol\",\n \"Am J Physiol Heart Circ Physiol\",\n \"Free Radic Biol Med\",\n \"Mol Cell\",\n \"Essays Biochem\",\n \"Physiol Rev\",\n \"Circ Res\",\n \"Clin Sci\",\n \"Arterioscler Thromb Vasc Biol\",\n \"J Vasc Res\",\n \"Arterioscler Thromb Vasc Biol\",\n \"J Clin Invest\",\n \"Proc Natl Acad Sci U S A\",\n \"Chem Rev\",\n \"Mol Biol Cell\",\n \"Mol Biol Cell\",\n \"J Cell Commun Signal\",\n \"Sci Rep\",\n \"Methods Mol Biol\",\n \"J Clin Invest\",\n \"Am J Physiol Heart Circ Physiol\",\n \"J Clin Invest\",\n \"Methods Cell Biol\",\n \"Methods Mol Med\",\n \"Am J Physiol Cell Physiol\",\n \"Methods Cell Biol\",\n \"Nat Methods\",\n \"Germany\"\n]"},"annotations_figure":{"kind":"list like","value":["\n\nVivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.","\nFigure 1 .\n1The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both","\nFigure 2 .\n2Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)","\nFigure 3 .\n3LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .","\n\nAtf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the","\nFigure 4 .\n4Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.","\nFigure 5 .\n5Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.","\nFigure 6 .\n6M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.","\nFigure 7 .\n7M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu","\nFigure 8 .\n8Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (","\nFigure 9 .\n9Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A."],"string":"[\n \"\\n\\nVivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.\",\n \"\\nFigure 1 .\\n1The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both\",\n \"\\nFigure 2 .\\n2Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)\",\n \"\\nFigure 3 .\\n3LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .\",\n \"\\n\\nAtf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the\",\n \"\\nFigure 4 .\\n4Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.\",\n \"\\nFigure 5 .\\n5Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.\",\n \"\\nFigure 6 .\\n6M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.\",\n \"\\nFigure 7 .\\n7M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu\",\n \"\\nFigure 8 .\\n8Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (\",\n \"\\nFigure 9 .\\n9Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A.\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["Vivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.","The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both","Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)","LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .","Atf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the","Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.","Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.","M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.","M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu","Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (","Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A."],"string":"[\n \"Vivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.\",\n \"The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both\",\n \"Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)\",\n \"LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .\",\n \"Atf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the\",\n \"Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.\",\n \"Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.\",\n \"M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.\",\n \"M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu\",\n \"Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (\",\n \"Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A.\"\n]"},"annotations_figureref":{"kind":"list like","value":["Figure 1A)","Figure 1B)","Figure 1C","Figure 1D","Figure 2A","Figure 1A","Figure 1C","Figure 1D","Figure 1B)","Figure 2A","Figure 2B","Figure 2C","Figure 2C)","Figure 2A)","Figure 2B","Figure 2C","Figure 3A","Figure 3B","Figure 4A","Figure 4B","Figure 5A","Figure 5","Figure 5D","Figure 6","Figure 3","Figure 4)","Figure 5","Figure 8, A and B)","(Figure 8","Figure 4)","Figure 9A","Figure 9","Figure 6)","Figure 2","C and D)","Mmp12 (Mm00500554_m1)","Figure 1","Figure 3"],"string":"[\n \"Figure 1A)\",\n \"Figure 1B)\",\n \"Figure 1C\",\n \"Figure 1D\",\n \"Figure 2A\",\n \"Figure 1A\",\n \"Figure 1C\",\n \"Figure 1D\",\n \"Figure 1B)\",\n \"Figure 2A\",\n \"Figure 2B\",\n \"Figure 2C\",\n \"Figure 2C)\",\n \"Figure 2A)\",\n \"Figure 2B\",\n \"Figure 2C\",\n \"Figure 3A\",\n \"Figure 3B\",\n \"Figure 4A\",\n \"Figure 4B\",\n \"Figure 5A\",\n \"Figure 5\",\n \"Figure 5D\",\n \"Figure 6\",\n \"Figure 3\",\n \"Figure 4)\",\n \"Figure 5\",\n \"Figure 8, A and B)\",\n \"(Figure 8\",\n \"Figure 4)\",\n \"Figure 9A\",\n \"Figure 9\",\n \"Figure 6)\",\n \"Figure 2\",\n \"C and D)\",\n \"Mmp12 (Mm00500554_m1)\",\n \"Figure 1\",\n \"Figure 3\"\n]"},"annotations_formula":{"kind":"list like","value":["Bip (Mm00517691_m1), Atf4 (Mm00515325_g1), Chop (Mm01135937_g1), Agtr1a (Mm01957722_s1), Agtr1b (Mm02620758_s1), Agtr2 (Mm00431727_91), Mmp2 (Mm00439498_m1), Mmp9 (Mm00442991_m1),"],"string":"[\n \"Bip (Mm00517691_m1), Atf4 (Mm00515325_g1), Chop (Mm01135937_g1), Agtr1a (Mm01957722_s1), Agtr1b (Mm02620758_s1), Agtr2 (Mm00431727_91), Mmp2 (Mm00439498_m1), Mmp9 (Mm00442991_m1),\"\n]"},"annotations_paragraph":{"kind":"list like","value":["Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.","Thoracic aortic aneurysm and dissection (TAAD) is a major cardiovascular health problem that arises from mutations that alter vascular extracellular matrix (ECM), smooth muscle cell function, or growth factor signaling pathways (1). In contrast to abdominal aortic aneurysms, TAAD has a strong genetic component, with pathogenic variants typically inherited in an autosomal dominant manner (2). A single highly penetrant, pathogenic mutation is responsible for disease in only one-fourth of individuals with TAAD, indicating that other undiscovered genetic factors influence disease onset and progression. In addition to the known pathogenic gene mutations, low-penetrant risk variants interact with other genetic or environmental factors to trigger disease or to increase the risk for developing disease but are not themselves pathogenic (2). Significant risk factors for TAAD, primarily when occurring in the background of low-penetrant disease-predisposing variants, are hypertension and bicuspid aortic valve (1).","A common histopathological feature of TAAD in humans is fragmented elastin in the aortic medial region (3). Elastin is the most abundant ECM protein in the aorta, where it plays a central role in the vessel's ability to expand and recoil in response to pulsatile blood flow. Smooth muscle cells (SMCs) synthesize and organize the protein into fenestrated sheets (lamellae) that separate smooth muscle cell layers. The secreted form of elastin (tropoelastin) undergoes extensive crosslinking outside the cell to form an elastic polymer, which is the functional form of the protein. The enzyme responsible for initiating crosslink formation is lysyl oxidase (LOX), a copper-binding amine oxidase that oxidizes lysine ε-amino groups in elastin and fibrillar collagens to aldehydes, which then spontaneously react to form covalent linkages between and within proteins (4,5). Approximately 90% of the lysine residues in elastin undergo crosslink formation, which makes it one of the most highly crosslinked and most stable proteins in the body (4). Inactivation of the Lox gene Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.","in mice results in early postnatal death due to ruptured aortic aneurysms, emphasizing the critical role of fully crosslinked, functional elastin in maintaining aorta integrity and function (6,7). The gene inactivation studies also show that other LOX isoforms cannot substitute for the loss of LOX in large, elastin-rich vessels.","LOX in vertebrates belongs to a family of 5 copper-dependent enzymes (LOX, LOXL1-4) that show a high degree of homology in their carboxy-terminal catalytic domain but differ in their amino-terminal sequences. LOXL2-4 form a subgroup that shares 4 amino-terminal scavenger receptor cysteine-rich domains that influence substrate binding but appear to have little role in regulating catalytic activity (8,9). LOX and LOXL1, in contrast, are synthesized without scavenger receptor sequences but have a propeptide domain that keeps the enzymes in a latent state until proteolytically processed outside the cell by BMP1/Tolloid-like metalloproteinases (10,11). Substrate specificity for each of the lysyl oxidases is still under investigation, but gene inactivation studies and phylogenetic analysis suggest that LOX and LOXL1 contribute to crosslinking of elastin and fibrillar collagens, whereas LOXL2-4 have different substrate specificities (12,13). All 5 LOX isoforms are synthesized in the ER and traffic to the Golgi, where the inactive apoenzyme binds copper and forms the cofactor lysyl tyrosine quinone (LTQ); both copper and LTQ are essential active site components required for catalytic activity (14).","Mutations within LOX in families with TAAD were recently identified by whole exome sequencing (3,15). Aortic disease in these families followed autosomal dominant inheritance, suggesting that 1 mutant LOX allele is sufficient to influence disease initiation or progression. Previously, we identified a LOX catalytic site mutation (p.Met298Arg) in a family with a history of TAAD (3). By introducing this human mutation into the homologous position in the mouse genome using CRISPR/Cas9 genome editing technology (c.857T>G encoding p.M292R; hereafter referred to as Mu), we confirmed that this amino acid change caused ruptured TAAD and perinatal death when both alleles were mutated (Lox Mu/Mu ) (3). In contrast to humans who developed TAAD when heterozygous for this LOX variant, aneurysms or other arterial disease were not observed in mice with a similar genotype (Lox +/Mu ) unless they were challenged with hypertension. In this report, we show that the nature of the functional loss associated with the methionine-to-arginine change in LOX results from a secretion defect, where the protein is retained in the ER and degraded through an autophagy/proteasome pathway. We also show that the vessel wall alterations associated with LOX functional haploinsufficiency increase the likelihood of developing vascular disease in association with environmental factors that increase wall stress.","Lox +/Mu adult animals are predisposed to aortic dilation in response to hypertension. The generation and characterization of mice expressing the M292R mutation were described previously (3). Mice heterozygous for this Lox missense variant appear grossly normal, have normal blood pressure, and show no increased mortality through 2 years of age. Aortic diameter and circumferential wall stiffness at physiological blood pressure in Lox +/Mu animals are normal, but ascending aortic length measured from the aortic root to the brachiocephalic artery is 10% longer than in WT mice (3).","Hypertension is a known risk factor associated with aortic dilation (16). To test whether mice expressing mutant LOX are predisposed to aneurysm formation as a consequence of increased wall stress, Lox +/Mu mice were made hypertensive by subcutaneous delivery of angiotensin II (Ang II) via Alzet osmotic pumps. Pumps containing saline served as the control. Ang II infusion is a well-established model for inducing vessel wall changes in mice and other animals (17,18). Blood pressure measurements using radiotelemetry in awake, freely moving Lox +/+ and Lox +/Mu mice showed an increase of approximately 30 mmHg in systolic pressure and 25-30 mmHg in mean blood pressure in both genotypes 7-10 days after pump implantation (data not shown), consistent with what others have found using this model (19). After 4 weeks of treatment, all animals receiving Ang II had cardiac hypertrophy, indicating altered cardiac physiology consistent with Ang II-induced hypertension ( Figure 1A). However, only Lox +/Mu mice showed substantial dilation and elongation of the ascending aorta in response to Ang II; no change in aortic diameter was observed in Ang II-treated Lox +/+ mice or in Lox +/Mu mice infused with saline alone ( Figure 1B). Biaxial pressure-diameter testing showed the ascending aorta in Ang II-treated Lox +/Mu animals to be approximately 25% larger than the aorta from WT or saline-treated controls at mean physiological blood pressure ( Figure 1C). The shape of the pressure-diameter curves suggests that Ang II treatment did not affect aortic mechanical properties in either mutant or WT animals. Other than in the ascending aorta, no dilation or change in vessel mechanics occurred in the left common carotid artery for either Lox +/+ or Lox +/Mu animals with saline or Ang II treatment ( Figure 1D), nor were dilations or areas of stenosis found in the aortic arch, descending aorta, or abdominal aorta.","Ang II treatment leads to vessel wall thickening and changes in vascular cellularity. On a structural level, Ang II treatment resulted in ascending and descending aortic wall thickening in Lox +/Mu animals, but there were no changes in elastic lamellar number or lamellar organization, quantified by measurement of medial area ( Figure 2A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.127748DS1). Elastic lamellar fragmentation, already elevated in Lox +/Mu compared with Lox +/+ animals (3), did not increase in either genotype following Ang II treatment (data not shown). We analyzed elastin crosslinking using an ELISA assay for desmosine, a major crosslink in elastin. We found no difference in desmosine levels in aortic tissue from Lox +/+ and Lox +/Mu mice, confirming normal elastin levels and that elastin crosslinking is not affected in animals heterozygous for the Lox mutation (Supplemental Figure 1C). Hydroxyproline levels (as a measure of total collagen) trended higher in vessels from the mutant animals but were not significantly different from WT when normalized to the change in total protein (Supplemental Figure 1D). Higher protein quantities are consistent with the increase in wall thickness that occurred with Ang II treatment (Supplemental Figure 1B).","Changes in the number and types of cells were observed in the thickened aortic wall of the hypertensive animals. The most substantial change occurred in the adventitia of both the ascending and descending aorta, which underwent considerable expansion in the Lox +/+ and Lox +/Mu animals as a result of Ang II exposure ( Figure 2A). In the medial layer, Ang II infusion led to a decrease in the number of SMCs in Lox +/+ animals but an increase in SMCs in Lox +/Mu mice ( Figure 2B). There was also an increase in intralamellar area in both genotypes, although the change was most notable in Lox +/Mu mice. The nuclei of the intralamellar cells in the mutant media were rounder than cells in Lox +/+ vessels, suggesting a less polarized Lox +/+ and Lox +/Mu animals developed significant cardiac hypertrophy following 4 weeks of treatment with 2 μg/kg/ min Ang II compared with saline-treated controls. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. ****P < 0.0001. (B) The ascending aorta in Lox +/Mu animals is longer than in Lox +/+ animals and undergoes substantial dilation following Ang II treatment. (C) Pressure-diameter measurements at intraluminal pressures ranging from 0 to 175 mmHg. The ascending aorta from Ang II-treated Lox +/Mu animals has a larger diameter over the entire range of pressures compared with Lox +/+ animals treated with Ang II or with saline controls. (D) There was no difference in the diameter or mechanical properties of the left common carotid artery in either genotype with or without Ang II treatment. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each pressure level. Data are presented as mean ± SD. # P < 0.05, Lox +/+ with Ang II vs. Lox +/Mu with Ang II; *P < 0.05, **P < 0.01, Lox +/Mu with saline vs. Lox +/Mu with Ang II.","phenotype and noncircumferential orientation. Immunostaining for smooth muscle α-actin demonstrated uniform reactivity throughout the medial intralamellar spaces, whereas cell staining in the adventitia was heterogeneous (not shown).","Staining for the monocyte/macrophage marker CD68 in descending aorta showed an increased number of CD68 + cells in the media and adventitia of both Lox +/+ and Lox +/Mu descending aorta following Ang II treatment ( Figure 2C). These results are consistent with previous studies showing that infusion of Ang II promotes macrophage accumulation within the aortic wall (20). While the adventitia showed the highest level of immune cell infiltration, macrophage accretion in the medial layer was also observed, but the spatial localization of the CD68 + cells was different for the 2 genotypes. In Ang II-treated Lox +/+ vessels, medial CD68 + cells were associated with the first 2 elastic layers nearest to the intima. Medial CD68 + cells in the aorta of Lox +/Mu animals treated with Ang II, however, were in the outer layers of the media closest to the adventitia ( Figure 2C).","Evaluation of factors that adversely affect vessel wall integrity and SMC function. An explanation for why Lox +/Mu mice are more sensitive than WT animals to Ang II infusion was explored by first checking for possible differences in Ang II receptor expression using quantitative PCR (qPCR). In ascending aorta from Lox +/Mu mice, all 3 Ang II receptors (Agtr1a, Agtr1b, and Agtr2) were detected, and there was no difference in their expression levels . Macrophages (stained green, arrowheads) were detected with an antibody to CD68, and cell nuclei were visualized with DAPI. Red is elastin autofluorescence. The vessel wall was thicker in both Lox +/+ and Lox +/Mu animals following Ang II treatment, with more DAPI + and CD68 + cells in the adventitia of both genotypes. (B) In the medial layer, there were fewer SMCs in the aorta of WT animals treated with Ang II but more cells in Lox +/Mu aorta. Shown are the mean ± SD for cell number counts between the internal and external elastic lamina on 7 tissue sections from each group. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.01. (C) Medial CD68 + cells were mostly located on the luminal side of the media in the Lox +/+ aorta but nearer the adventitia in Lox +/Mu aorta after Ang II treatment.","compared with WT controls (Supplemental Figure 2A). Thus, the potential for Ang II receptor-mediated signaling is similar in both genotypes.","Many Ang II-induced responses on SMCs are attributable to stimulation of NADPH oxidase with the subsequent generation of ROS (21,22). We used dihydroethidium, a cell-permeable compound that interacts with superoxide, to assess oxidant levels in sections of WT and mutant ascending aorta and found no difference between the 2 genotypes (Supplemental Figure 2B). Thus, differences in levels of oxidative stress do not appear to be a factor in the Ang II susceptibility of Lox +/Mu mice.","In many models of aortic disease, the presence of proteases that degrade elastin can initiate and propagate disease by degrading the elastic fiber system in the vessel wall. We therefore investigated whether 3 elastolytic MMPs commonly linked to vascular remodeling -MMP2, MMP9, and MMP12 -were differentially expressed in mutant versus WT aorta. There were no differences in Mmp2 and Mmp9 mRNA expression levels, while expression of Mmp12 was diminished in the mutant aorta (Supplemental Figure 2C).","Active LOX is absent in Lox Mu/Mu mouse embryonic fibroblast conditioned medium. To elucidate how the M292R modification affects LOX function, we used mouse embryonic fibroblasts (MEFs) from WT, KO, and mutant animals to characterize LOX secretion and activity. Mutant cells were indistinguishable from WT cells in morphology and in their rate of proliferation (data not shown). LOX activity ( Figure 3A), assessed using an Amplex Red assay, was not detected in cell layer extracts of any MEF genotype (Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-), which was expected, since the full-length intracellular form of LOX (pro-LOX) is an inactive zymogen. LOX enzymatic activity was readily detected in the conditioned medium from Lox +/+ MEFs and showed linear enzyme kinetics over the period of the Amplex Red assay. Conditioned medium from Lox Mu/Mu MEFs, however, had no detectable activity, similar to conditioned medium from cells with inactivated Lox alleles (Lox -/-) ( Figure 3B). Activity was detected in conditioned medium from Lox +/Mu cells, where the linearity and rate of substrate oxidation were comparable to those seen in Lox +/+ cells, indicating that the mutant form of the protein does not adversely affect the enzymatic activity of WT LOX and hence does not function in a dominant negative fashion.","M292R LOX is poorly secreted and is retained in the ER. LOX is maintained as an inactive 50-kDa proenzyme through every stage of the synthetic and secretory pathway. Activation requires secretion into the extracellular space, where proteolytic removal of the N-terminal propeptide portion of the protein generates the 30-kDa active catalytic domain. To determine whether LOX activity is absent in M292R MEF conditioned medium because mutant LOX is not secreted, we performed Western blots using an anti-LOX antibody on Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF cell layers and conditioned medium. Immunoblotting showed increased levels of 50-kDa intracellular pro-LOX protein in cell extracts from Lox Mu/Mu MEFs compared with Lox +/+ cells ( Figure 4A). Conversely, there was abundant 30-kDa mature (processed) LOX in conditioned medium from WT cells but barely detectable levels in medium from Lox Mu/Mu MEFs ( Figure 4B). These findings indicate that the mutant protein is poorly secreted and is retained intracellularly as the proenzyme. Immunofluorescence imaging with an antibody to LOX showed minimal intracellular staining in Lox +/+ cells, confirming that WT LOX is secreted efficiently ( Figure 5A). Staining of Lox Mu/Mu cells, however, showed abundant intracellular staining, consistent with the immunoblot results described above. The staining pattern for intracellular LOX was typical of accumulation within the ER, which was confirmed by colocalization of LOX with calnexin, an ER-resident protein ( Figure 5, B and C).","A direct interaction between LOX and calnexin was shown using coimmunoprecipitation from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF lysates. Lox -/cells served as the negative control. Cell lysates were immunoprecipitated with an anti-calnexin antibody, followed by immunoblotting using an antibody to LOX. LOX protein was detected in calnexin immunoprecipitates from all genotypes except Lox -/-, confirming an interaction between the 2 proteins ( Figure 5D).","M292R LOX has an altered protein conformation. The high level of LOX detected in Lox Mu/Mu MEF calnexin immunoprecipitates suggests that M292R LOX is misfolded and trapped in the ER bound to calnexin. To determine whether M292R LOX is conformationally different from the WT enzyme, we explored possible structural changes by assessing differential sensitivity to proteolytic degradation. Changes in protein structure frequently expose or mask protease binding sites, leading to altered degradation products and a different degradation fingerprint. Immunoblotting of cell lysate from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs incubated at 37°C for 6 hours to exploit protein degradation by endogenous enzymes within the cell lysate showed time-dependent degradation of WT but not M292R LOX ( Figure 6). These results suggest that WT and M292R pro-LOX have different susceptibilities to degradation arising from cell-associated proteases. Differential degradation was also observed when cell lysates were incubated with pancreatic elastase. In digests of Lox +/+ cell lysate, there was a 25-kDa fragment evident with all doses of elastase that was absent in Lox Mu/Mu samples (Supplemental Figure 3, arrow). When treated with the highest dose of elastase, pro-LOX from Lox +/+ and Lox +/Mu samples degraded predominantly to a 30-kDa fragment. LOX from Lox Mu/Mu samples, in contrast, degraded further to mainly a 10-kDa fragment. It is important to note that pancreatic elastase does not cleave after methionine or arginine, so the Met-to-Arg mutation does not create or negate a protease cleavage site for this enzyme. Together, the conformational difference between the WT and mutant proteins suggests that M292R LOX is misfolded, which is consistent with its protracted interaction with calnexin in the ER.","M292R LOX does not elicit an ER stress response. Accumulation of misfolded proteins in the ER generally leads to ER stress, which subsequently activates the unfolded protein response (UPR) (23). UPR triggers 3 primary stress sensors, PERK, IRE1, and ATF6. Under resting conditions, BIP/Grp78 maintains these sensors in an inactive state. Upon ER stress, BIP binds to misfolded proteins, thereby allowing for sensor activation. To determine whether the continuous presence of misfolded mutant LOX induces ER stress, we assessed Bip transcript levels by qPCR. We also measured mRNA levels of 20% change was modest compared with the ~2-fold and ~4-fold increase in Atf4 and Chop, respectively, when ER stress was stimulated by treatment of MEFs with lopinavir/ritonavir, which served as a positive control (data not shown). Electron microscopic analysis of WT and mutant cells showed that ER volume was not markedly different in the 2 cell types (Supplemental Figure 4), suggesting that mutant protein does not accumulate to levels that would produce dilated saccules. Together, these results suggest that LOX protein containing the M292R mutation does not induce substantial ER stress.","Increased activation of autophagy in Lox Mu/Mu cells. The absence of ER stress in Lox Mu/Mu cells was surprising given that M292R LOX does not leave the ER. To determine whether mutant LOX is being removed from the ER for degradation through the lysosomal pathway, we costained cells for LOX and the lysosomal marker LAMP-2. No colocalization of the 2 proteins was observed, suggesting that the mutant protein was not trafficked through lysosomes (Supplemental Figure 5).","Autophagy is a general mechanism for clearing proteins accumulating in cells. Clearing of misfolded proteins from the ER, a process termed ER-phagy, plays a critical role in ER homeostasis (24). To determine whether mutant LOX directly activates autophagy-mediated protein clearance from the ER, we measured 2 autophagy activation markers, p62 and microtubule-associated protein 1A/1B, light chain 3 (LC3), in WT and Lox Mu/Mu MEFs. Following pretreatment with bafilomycin A1 (BafA1) to inhibit organelle acidification-induced degradation of p62 and LC3, only Lox Mu/Mu cells with and without BafA1 pretreatment had significantly increased p62 expression compared with Lox +/+ cells without BafA1 pretreatment ( Figure 8, A and B). The autophagy activation markers LC3-I and -II were also elevated in mutant compared with WT cells (Figure 8, A, C, and D), providing more evidence for increased autophagy.","We also used electron microscopy to screen for the presence of autophagosome-like vesicles in WT and mutant cells (Supplemental Figure 4). We observed that multi-and single-membrane vesicles typical of autophagosomes were particularly abundant in the perinuclear region but were also present throughout the cytoplasm of Lox Mu/Mu cells. Such structures were rare in WT cells. Together, the existence of autophagic vesicles along with increased expression of autophagy activation markers in mutant cells is consistent with the clearance of misfolded mutant LOX from the ER through an autophagy/proteasome pathway.","Mutant LOX does not influence the secretion of the WT protein.","To assess whether the secretion of WT LOX is affected by the presence of M292R mutant protein, we transiently expressed plasmids containing WT LOX, and M292R LOX fused to the fluorescent protein mApple, in Lox -/-MEFs. Successful transfection was confirmed by intracellular mApple expression using fluorescence imaging (data not shown). In the conditioned medium of Lox -/cells transfected with empty plasmid, no LOX was observed by Western blot analysis ( Figure 9A). In Lox -/-MEFs transfected with 2.5 μg WT Lox-mApple plasmid, an immunoreactive protein band at approximately 60 kDa was detected in the conditioned media, corresponding to the size of active LOX (30 kDa) fused to mApple (27 kDa). In Lox -/-MEFs transfected with 2.5 μg of M292R Lox-mApple plasmid, LOX protein was observed inside the cell by fluorescence microscopy, but no LOX protein was detected in conditioned medium by Western blotting, consistent with the inability of M292R LOX to be secreted. To determine whether mutant LOX alters the secretion of the WT protein, or if the WT protein acts as a chaperone to facilitate the secretion of the mutant protein, we cotransfected cells with 1.25 μg each of WT and mutant plasmid, which is half as much as was used for single plasmid transfection. If mutant LOX does not affect the secretion of the WT protein, then LOX should be detected in conditioned medium, but at half the level of the single plasmid transfection. If the mutant protein has an adverse effect on WT LOX secretion, then levels will be lower than 50%. If WT LOX acts as a chaperone to facilitate secretion of the mutant protein, then the combination of both secreted proteins would be higher than 50% and possibly equivalent to the WT transfection (1.25 + 1.25 = 2.5 μg). Figure 9 shows that LOX protein was detected in conditioned medium of doubly transfected cells at half the level of singly transfected cells, confirming that the mutant protein does not affect the secretion of WT LOX.","M292R propeptide processing. Our experimental results are consistent with the M292R mutation being a loss-of-function phenotype due to a secretion defect. The final activation step of the proenzyme occurs outside the cell through proteolytic separation of the propeptide and catalytic domains. To determine whether M292R pro-LOX could be processed to the 30-kDa mature form if it were secreted, we incubated cell lysates from WT and mutant MEFs with recombinant BMP1 (rBMP1), the enzyme responsible for propeptide cleavage. Western blot analysis showed that BMP1 appropriately processed both WT and M292R pro-LOX to the 30-kDa mature form (Supplemental Figure 6). Thus, the mutation did not inhibit propeptide processing. The processed mutant protein was not assayed for oxidase activity because intracellular LOX in M292R MEFs does not have bound copper and cannot be enzymatically active.","LOX plays a critical role in vascular ECM maturation by catalyzing the crosslinking of elastin and fibrillar collagens, the 2 major ECM protein classes in the aortic wall. Phylogenic studies suggest that the current form of vertebrate LOX coevolved with elastin and helped facilitate the appearance of a closed circulatory system (12,25). Therefore, it is no surprise that mutations in LOX give rise to vascular insufficiency and disease. All LOX mutations associated with TAAD in humans show autosomal dominant inheritance, and most result in loss of function. We know from mouse studies that inactivation of both Lox alleles is lethal and that other members of the LOX family cannot assume LOX's critical role in vascular development and maintenance of the vessel wall. In this report, we utilize a mouse model of a well-characterized human mutation to explore how LOX haploinsufficiency leads to vascular dilation and aneurysm formation. Mice heterozygous for the Lox M292R variant (Lox +/Mu ) have increased elastic lamellar fragmentation in the wall of the ascending aorta (3), but do not develop aortic dilation under normal conditions. The absence of aneurysmal disease was surprising given that humans heterozygous for this same mutation are prone to aneurysm formation and vessel wall changes (3). Variability in the age of disease onset and disease severity in humans, however, suggested that a secondary challenge is a factor in disease initiation and progression. Here we show that hypertension induced by Ang II infusion resulted in vessel wall changes in both Lox +/+ and Lox +/Mu mice, but only Lox +/Mu animals showed aortic dilation localized to the ascending aorta. Several groups have reported that the effects of Ang II are most pronounced in the ascending aorta region in this model (26)(27)(28), but the underlying factors governing this vessel specificity are unknown. Differences in the embryonic origin of the cells that populate the aortic wall are one explanation (29,30), but the composition and maturation of the aortic ECM are also factors. In Lox +/Mu mice, changes in ECM crosslinking associated with LOX insufficiency could have more negative consequences for this vessel segment because of high collagen and elastin levels and the high mechanical stress experienced by the aortic wall during the cardiac cycle.","While several Ang II-induced vascular changes were expected (19,27) and common to WT and mutant animals, there were also significant differences. For instance, medial expansion was evident in both genotypes, but the effect was most notable in mutant mice, in which there was an increase in the number of smooth muscle cells between the elastic lamellae. This response is particularly unusual, since the intralamellar space is typically populated by a single smooth muscle cell layer, as can be seen in vessels from saline-treated Lox +/+ and Lox +/Mu animals ( Figure 2). The opposite change occurred in WT vessels, where there were fewer smooth muscle cells in response to Ang II, suggesting Ang II-related apoptosis or growth suppression (26), which we did not assess. mice. Oneway ANOVA with Tukey's multiple-comparisons test was used to assess differences, which were minimal between the 3 genotypes. Data are presented as mean ± SD. ***P < 0.001.","Another cellular change in the ascending and descending aorta following Ang II treatment was the accumulation of macrophages in the aortic wall of both WT and Lox +/Mu mice. CD68 + cells were found in the adventitia of both genotypes, with fewer cells in the medial layer. In Ang II-treated WT mice, CD68 + cells accumulated in the intima and inner elastic layers of the media and did not localize to breaks in elastic lamella, as has been reported in other models (18,20). Their location within the wall, however, shows that some cells were able to penetrate past the internal elastic lamina into the first or second layer of smooth muscle cells. Medial CD68 + cells in Lox +/Mu mice exposed to Ang II, in contrast, were localized to the outer aspect of the media and occurred at higher numbers than in WT vessels. These results suggest that the cellular origin of the CD68 + cells or the route of inflammatory cell infiltration is different in Lox +/+ and Lox +/Mu vessels as a response to wall stress.","The location of macrophages in the outer region of the wall provides a mechanistic explanation for why Lox +/Mu , but not WT, aorta dilates following Ang II treatment. ECM degradation is a primary factor leading to vessel wall dilation. Elastases and other matrix-degrading enzymes secreted by macrophages destabilize the vessel wall, particularly when matrix degradation occurs in or near the adventitia -the vessel compartment essential for maintaining the structural integrity of the artery (31). The medial cell hypertrophy and inflammatory cell accumulation in the Lox +/Mu aorta in response to hemodynamic stress are similar in several ways to vascular changes in mouse models of Marfan syndrome with mutations in fibrillin-1, a key elastic fiber protein. In Marfan mice, inflammatory cell accumulation at the medial-adventitial interface correlates with increased elastin degradation and a loss C and D) Also, LC3 was elevated in Lox Mu/Mu cells compared with Lox +/+ cells. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.001.","of structural integrity that leads to vessel wall dilation (32). As in the Ang II-treated Lox +/Mu animals, only the ascending aorta is affected in Marfan mice.","Our previous studies showed that mice with the Lox M292R mutation share numerous phenotypic traits with Lox +/animals (3), suggesting that M292R is a loss-of-function mutation in mice and humans. Even though the amino acid change is in the catalytic domain of LOX close to the copper binding site, it was not clear whether the mutation results in a catalytically dead enzyme or if some other aspect of LOX processing is negatively affected by the mutation. Our data show that the answer fits both possibilities: the mutant protein is retained in the ER and not secreted, and the enzyme is catalytically dead because of its inability to traffic to the Golgi, where it needs to bind copper and form the redox quinone cofactor LTQ required for catalytic activity (33). Even though we found that the propeptide of mutant pro-LOX could be cleaved by rBMP1 similarly to WT LOX, full-length LOX in the ER cannot be catalytically active even if propeptide processing does occur.","Why mutant LOX is retained in the ER is not clear, but our data point to protein misfolding as a probable cause. ER retention is characteristic of incompletely folded proteins. Folding intermediates of most secreted proteins interact with the calnexin/calreticulin protein complex in the ER to achieve the proper native conformation required for secretion (34). The calnexin/calreticulin protein complex recognizes N-linked glycan structures (LOX has 3 N-linked glycosylation sites in the propeptide region; ref. 11) that undergo cycles of sugar residue removal and addition. Once folded correctly, calnexin and calreticulin dissociate, and the folded proteins are transported from the ER to the Golgi apparatus. Mutant LOX colocalized intracellularly with calnexin and was isolated bound to calnexin in cellular extracts, suggesting that the protein remains misfolded despite repeated folding interactions with calnexin. Furthermore, the differential susceptibility to proteolytic degradation of mutant compared with WT LOX is consistent with structural differences between the 2 proteins.","It was interesting that mutant LOX accumulation in the ER did not induce an appreciable UPR. We saw no upregulation in mutant cells of the ER stress-related markers Bip (an ER luminal chaperone protein and critical ER stress sensor), Atf4 (a transcription factor downstream of PERK that plays a crucial role in the adaptation to ER stress), or Chop (a transcription factor regulated by ATF4 that drives proapoptotic gene transcription). Nor could we detect in mutant cells an increase in ER volume frequently associated with ER stress. One pathway whereby ER stress is mitigated is removal from the ER of mutant protein and degradation at the lysosome (ERAD-II) or proteasome (ERAD-I) (24). We were unable to localize LOX within lysosomes but did find increased expression of the autophagy markers p62 and LC3. Electron microscopy also identified an increased number of autophagic vesicles in Lox Mu/Mu cells. These results suggest that misfolded mutant LOX is being cleared from the ER through an autophagy/proteasome pathway and would explain why mutant protein levels never reached a critical threshold to sustain an ER stress response. Furthermore, there is mounting evidence that the autophagy and secretory pathways are coregulated and intimately linked (35), which may represent a mechanistic connection into the LOX trafficking defect that we identified. It should be noted that all the experiments probing the fate of the mutant protein were done in cultured cells. It is possible, but unlikely, that in vivo, in tissues, and at developmental stages relevant to the TAAD phenotype, LOX folding, secretion, ER retention, and stress occur differently than in MEFs.","Guo et al. (15) described 2 other LOX active site variants (S280R and S348R) that segregate with disease in families with TAAD. When expressed from transgenes in HeLa cells and examined in cell lysates, a portion of the pro-LOX protein was processed to its active form and was able to oxidize substrate at rates approximately equivalent to the WT enzyme (S348R) or with approximately 50% lower activity (S280R) (2,15). Enzymatic activity in conditioned medium from the transfected cells was not evaluated, so it is not known whether these LOX variants are efficiently secreted. However, the fact that the proteins are catalytically active implies trafficking through the Golgi and proteolytic removal of the propeptide domain. Thus, unlike the M292R mutation, which is retained in the ER, the S348R and S280R variants appear capable of traversing the secretory pathway. How these proteins, which have full (or somewhat reduced) catalytic activity, cause thoracic aortic disease is an interesting question. An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.","Antibodies. Antibodies used in this study are described in Supplemental Table 1.","Mouse models and cell isolation. A knockin mouse strain bearing the Lox M292R substitution was generated in the C57BL/6 background using CRISPR/Cas9 genome editing technology as previously described (3). Lox +/mice in the same background are described in ref. 6. Primary MEFs were isolated from the various mouse lines by digestion of E14.5 mouse embryos with trypsin/EDTA at 37°C for 30 minutes in DMEM (38). Cells were collected by centrifugation and grown in DMEM, 10% FBS, 1% penicillin/streptomycin, and 1 mM l-glutamine.","Ang II treatment and blood pressure measurements. Telemetry transmitters (Data Sciences International, model PA-C10) were implanted surgically in 3-to 4-month-old mice under isoflurane anesthesia. The catheter was inserted in the left common carotid artery and advanced to the aortic arch, and the transmitter was placed subcutaneously along the abdomen. Animals were allowed to recover for 7 days prior to activation of transmitters, and continuous recording of heart rate and arterial pressure was obtained, with sampling every 5 minutes for 15-second intervals for 3 days. Data were collected and stored using Dataquest ART (Data Sciences International).","After baseline blood pressure measurements were obtained, mice were implanted with osmotic pumps (Alzet model 1004, DURECT Corp.) that delivered Ang II (MilliporeSigma) subcutaneously at a dose of 2 μg/kg/min in the interscapular region, according to published procedures (39,40). Mice infused with saline were used as controls. After a week of Ang II treatment, arterial pressure and heart rate were again continuously recorded using the telemeters, with sampling every 5 minutes for 15-second intervals for 3 days. Animals were fed a standard laboratory chow diet. After 28 days, animals were sacrificed under isoflurane anesthesia, and vessels were collected for compliance studies and histological characterization.","Vessel compliance measurement. For vessel compliance measurements, the ascending aorta and left common carotid artery were excised and placed in a physiological saline solution. Vessels were then cleaned of surrounding fat and connective tissue and mounted onto a pressure arteriograph (Danish Myo Technology). Experiments were done at 37°C in an organ bath containing physiological saline. The mounted vessels were visualized using a computerized imaging system, allowing continuous recording of vessel diameters. Vessel outer diameters were recorded while intravascular pressure was increased from 0 to 175 mmHg by increments of 25 mmHg (12 seconds per step). The average of 3 outer diameter measurements was taken at each pressure (40,41).","Protein quantification, hydroxyproline, and desmosine assays. Each ascending aorta was hydrolyzed at 105°C overnight with 20 μL of 6 N hydrochloric acid (Thermo Fisher Scientific) and dried under vacuum in a SpeedVac. The dried pellet was dissolved in 400 μL water and filtered with a 0.45-μm filter. Total protein, hydroxyproline, and desmosine levels were determined as described previously (42).","Dihydroethidium staining for superoxide detection in aortic sections. Superoxide in aortic sections was detected using dihydroethidum (DHE) staining. In the presence of superoxide, DHE is oxidized to 2-hydroxyethidium, which is highly fluorescent and intercalates within the DNA, staining the nucleus a fluorescent red (43,44). After euthanasia and thoracotomy, the right atrium was clipped, and 5 mL cold (4°C) 1× PBS was flushed through the left ventricle to clear the blood. Ascending aorta was then dissected, placed in Tissue-Tek O.C.T. Compound (Sakura Finetek), and flash frozen in liquid nitrogen. Four-micrometer cryosections were obtained using a Leica CM1850 cryostat and placed on charged microscope slides (Thermo Fisher Scientific). A 10-mM stock solution of DHE (Life Technologies) in DMSO was diluted to 10 μM using 1× PBS. The 10-μM DHE solution was applied to unfixed aortic sections and incubated at 37°C for 30 minutes in the dark. After staining with DHE, sections were then rinsed with 1× PBS for 5 minutes at room temperature (RT) and mounted with a coverslip using ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were obtained on the Zeiss Axioscope fluorescence microscope with a QImaging MicroPublisher 3.3 RTV camera using QCapture software (QImaging). The entire procedure (from mouse euthanasia to image acquisition) occurred within 8 hours.","Immunofluorescence on aortic sections and area measurements. Ascending and descending aortas were dissected as above and placed in O.C.T. compound, and the blocks were frozen on dry ice. Four-micrometer-thick cryosections were placed on glass slides and stored at -80°C. The sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS at 4°C for 10 minutes, washed twice with 1× PBS (5 minutes each) at RT, and blocked with a solution containing 1% BSA (MilliporeSigma), 1% fish gelatin (MilliporeSigma), and 0.05% Triton-X (Acros Organics) in 1× PBS for 1 hour at RT. The aortic sections were then incubated with rat anti-mouse CD68 antibody (Bio-Rad) at a 1:1000 dilution in blocking solution at 4°C overnight. The next day, sections were washed 3 times with 1× PBS (5 minutes each) and incubated in goat anti-rat IgG Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) diluted 1:2000 and DAPI diluted 1:3000 in blocking solution at RT for 1 hour. The sections were washed twice with 1× PBS (5 minutes each) at RT, mounted with a coverslip using ProLong Diamond Antifade Mountant, and imaged using the Axioscope fluorescence microscope equipped with the QImaging MicroPublisher 3.3 RTV camera using QCapture software as above. For area measurements, frozen sections from the descending aorta from 3-month-old mice were prepared as described above. The area between the internal and external elastic laminae was traced in each ×40 image using ImageJ software (NIH) and measured in pixels 2 .","LOX activity assay. MEFs were grown in 10-cm dishes and maintained as above until fully confluent. Twenty-four hours before collection, culture medium was replaced with DMEM lacking phenol red and FBS, supplemented with 50 μg/mL ascorbic acid and 0.1% BSA. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. Cell lysates were collected by scraping in 1× PBS containing protease inhibitors. LOX activity in cell lysate and concentrated culture medium, incubated with and without the LOX inhibitor β-aminopropionitrile (BAPN), was quantified using the Amplex Red assay as previously described (3,45). Resorufin fluorescence, the product of Amplex Red oxidation, was measured at excitation and emission wavelengths (540 and 600 nm, respectively) every 30 minutes for 150 minutes using a BioTek H4 Hybrid Reader. LOX activity was calculated as the difference between total activity and activity in the presence of BAPN. Differences in relative fluorescent units were tested using 2-way ANOVA with Tukey's multiple-comparisons test.","Immunofluorescence imaging of primary cells. Primary MEFs were seeded in 4-chamber glass slides at 3 × 10 5 cells per/well. After 3 days in culture, the cells were fixed with cold methanol (2 washes, 5 seconds each) followed by 3 washes with blocking solution containing 1× PBS, 1% BSA, 1% fish gelatin (Milli-poreSigma), and 0.05% Triton-X100 at pH 7.4, then incubated in blocking solution for 30 minutes at RT. The cells were then incubated with a primary antibody to LOX at 1:1000 in blocking solution overnight at 4°C. After washing the cells with blocking solution to remove the primary antibody, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor 568 secondary antibody at 1:2000 (Thermo Fisher Scientific) and anti-calnexin primary antibody directly labeled with Alexa Fluor 488 at 1:4000 in blocking solution for 30 minutes at RT. The cells were washed with blocking solution 3 times, then mounted using ProLong Diamond Antifade Mountant (Invitrogen) and coverslipped. The samples were imaged using the Axioscope fluorescence microscope and QCapture Pro software.","Protein extraction from cell culture and aortic tissue. Cultured MEFs were switched to serum-free medium 24 hours before protein extraction. MEFs in 10-cm dishes were incubated in 1 mL lysis (RIPA) buffer (MilliporeSigma) containing protease inhibitors for 30 minutes at 4°C. The lysates were scraped from the dish into microfuge tubes and centrifuged at 17,500 g for 5 minutes, and the supernatant was collected. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. The protein concentration in cell lysates and concentrated media was measured using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Laemmli buffer with and without DTT was added to samples for SDS-PAGE and native gel electrophoresis, respectively. Only the samples with DTT were boiled at 100°C for 5 minutes.","RNA extraction and qPCR. RNA was extracted from confluent cell cultures using TRIzol (Thermo Fisher Scientific) following the manufacturer's protocol. For RNA isolation from aortic tissue, ascending aorta from adult Lox +/+ and Lox +/Mu mice were each homogenized in 500 μl TRIzol using QIAGEN TissueLyser. The manufacturer's protocol was followed for RNA isolation. At the time of RNA precipitation, 10 μg glycogen (Mil-liporeSigma) was added as a carrier to each sample. Each RNA pellet was dissolved in 10 μL molecular-grade water. The RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). One microgram of the isolated RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Reverse transcription was then carried out using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). One microliter of cDNA was used for qPCR using TaqMan Fast Universal PCR Master Mix and TaqMan assay primer/probes (Life Technologies) for Mmp12 (Mm00500554_m1), and Gapdh (Mm9999999915_ g1), which was used as an internal control. Ten-microliter reactions were performed in duplicate using a ViiA 7 Real-Time PCR System for Bip, Atf4, and Chop and QuantStudio 3 Real-Time PCR system (Applied Biosystems) for Agtr1a, Agtr1b, Agtr2, Mmp2, Mmp9, and Mmp12. All mRNA levels were normalized to Gapdh expression. rBMP1 treatment. Protein was extracted from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs as described above. Fifty microliters of cell lysates from cultured MEFs containing 50 μg total protein were incubated with 30, 60, and 90 ng rBMP1 (R&D Systems) for 45 minutes at 37°C in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5). The enzyme reaction was stopped by adding Laemmli buffer with DTT and incubated at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.","Western blotting. Protein extracts from cells, concentrated condition medium, or tissues (generally 50 μg) were analyzed using 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) run at 100 mV for 1 hour. The protein was then transferred to ProBlott Membrane (Applied Biosystems). The blots were incubated in 5% nonfat dried milk in 0.1% 1× PBS-Tween for 1 hour at RT, then incubated with a primary antibody overnight at 4°C. The next day, the blots were washed with 0.1% 1× PBS-Tween and incubated in ECL anti-rabbit or anti-mouse IgG HRP-Linked Secondary Antibody (GE Healthcare) for 1 hour at RT. The blots were then washed and the immunoreactive bands detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The blots were stripped by incubating in 2% SDS, 75 mM Tris-HCl, and 100 mM β-mercaptoethanol for 1 hour at 65°C while shaking. The blot was then incubated in 0.1% 1× PBS-Tween wash buffer for 1 hour at RT before immunodetection of β-actin (MilliporeSigma) at 1:20,000 in wash buffer, which was used as a loading control. For protein samples from concentrated conditioned media, membranes were stained with Ponceau S solution (MilliporeSigma) before blocking and used as a loading control. Alternatively, stain-free images of Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) taken following the manufacturer's protocol served as a loading control.","Coimmunoprecipitation. Cell lysates were prepared from MEFs 5 days after confluency as described above. Immobilized protein A anti-rabbit IgG beads (50 μL), preequilibrated by washing with lysis buffer, were then added to 250 μL cell extract. After incubation on a rocking platform for 60 minutes at 4°C, the sample was centrifuged at 2500 g for 3 minutes, and the supernatant was incubated with 8 μg immunoprecipitation antibody for 1 hour at 4°C. Fifty microliters of precleared protein A beads was then added, and the samples incubated overnight at 4°C with rocking. Immune complexes bound to the protein A beads were collected by centrifugation and washed with lysis buffer prior to boiling at 100°C for 10 minutes with Laemmli buffer containing DTT. The supernatant was collected by centrifugation, and proteins were separated by SDS-PAGE and immunoblotted following the same protocol described above.","Protein degradation analysis. Confluent cultures of WT and mutant MEFs were treated with 5 μg/mL Brefeldin A (MilliporeSigma) to inhibit protein transport from ER to the Golgi apparatus. This ensured that WT LOX remained in the cells so that the starting amount of protein was the same across genotypes. After 4 hours, the cell lysate was extracted using lysis buffer placed at 37°C to initiate protein degradation. Every hour, 50 μL aliquot of cell lysate was collected, then immediately boiled with Laemmli sample buffer at 100°C for 5 minutes prior to SDS-PAGE analysis. For exogenous protease-mediated degradation, human pancreatic elastase in increasing concentrations (0.625, 1.25, 2.5, 5 ng/μL) was added to cell lysates containing 1 mg/ mL protein at 4°C for 30 minutes. After treatment, elastase activity was inhibited with protease inhibitor cocktail, and samples were boiled with Laemmli sample buffer at 100°C for 5 minutes for SDS-PAGE analysis.","Autophagy markers. Confluent MEFs in 6-well dishes were pretreated with 100 mM BafA1 (MilliporeSigma) for 3 hours before cell lysates were collected. The protein samples were separated on 4%-15% gradient gels for SDS-PAGE analysis. Immunoblotting was performed following the same procedure as above.","Lox-mApple plasmid cloning and expression. The mouse Lox cDNA in a mammalian expression vector driven by a pCMV promoter was purchased from GE Healthcare. The Lox open reading frame was PCR amplified using a 5′ primer bearing an XhoI restriction enzyme site (5′-CAGATCTCGAGAGCGTTTCGCCTGGGCTGTGC-3′) and a 3′ primer designed to remove the Lox stop codon bearing a BamHI restriction enzyme site (5′-GGATCCA-CATACGGTGAAATTGTGCAGCCTGAG-3′). The PCR product and the pGEMT shuttle vector (Promega) were digested with XhoI and BamHI, then ligated together using T4 ligase. The mutant Lox plasmid bearing the 893 T>G base pair change was generated using the Lox-pGEMT plasmid as the template, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific), following the manufacturer's protocol (5′-TTACCA-CAGCAGGGACGAATTCAGCCACTATG-3′ and 5′-TGTTGGTGACAGCTGTGCCACTCCC-3′). The WT and mutant Lox-pGEMT plasmid sequences were confirmed using Sanger sequencing performed at the Washington University Protein and Nucleic Acid Chemistry Laboratory. To generate the Lox-mApple plasmids, WT and mutant Lox pGEMT plasmids and the mApple plasmid (46), provided by David W. Piston (Washington University School of Medicine in St. Louis), were digested with XhoI and BamHI restriction enzymes. The WT and mutant mLox cDNA inserts were then ligated into mApple vectors to generate the mLox-mApple fusion plasmids and sequenced to confirm. To express the WT and mutant Lox-mApple plasmids in MEFs, Lipofectamine 3000 reagents (Thermo Fisher Scientific) were used following the manufacturer's protocol.","Transmission electron microscopy. MEFs from Lox +/+ , Lox +/Mu , and Lox Mu/Mu mice, cultured on glass coverslips, were washed in cacodylate buffer (0.15 M cacodylate in 2 mM CaCl 2 , pH 7.4) and fixed for 5 minutes with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer prewarmed to 37°C. The samples were then incubated at RT for an additional hour, washed with cacodylate buffer, and treated with 1% OsO 4 /1.5% potassium ferrocyanide for 1 hour in the dark. After washing with ultrapure water, en bloc staining was performed in 2% uranyl acetate for 1 hour in the dark. The samples were washed again in ultrapure water before dehydration in a graded acetone series. Finally, the samples were embedded in Epon resin and cured in a 60°C oven for 48 hours. The processed samples were imaged using a JEOL 1400 electron microscope.","Statistics. One-way ANOVA with Tukey's multiple-comparisons test was used to determine the significance, if any, between different groups, particularly genotypes. When 2 variables were present, e.g., genotype and treatment or genotype and gene, 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences. If the same sample was measured repeatedly (e.g., at different pressures in the pressure-diameter curves in Figure 1, C and D, or at different time points in Figure 3), then repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was performed. Prism 8 for Mac OS X (GraphPad Software) was used to run statistical analyses. In all numerical figures, data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical test used and significant differences are noted in each figure legend.","Study approval. All animal experiments were carried out following protocols approved by Washington University School of Medicine Institutional Animal Care and Use Committee.","VSL, CMH, TJB, and PCT participated in experimental design and acquired and analyzed primary data. NOS helped with data analysis, and RPM designed the research study and analyzed primary data. VSL wrote the initial draft of the manuscript, to which all authors contributed edits."],"string":"[\n \"Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.\",\n \"Thoracic aortic aneurysm and dissection (TAAD) is a major cardiovascular health problem that arises from mutations that alter vascular extracellular matrix (ECM), smooth muscle cell function, or growth factor signaling pathways (1). In contrast to abdominal aortic aneurysms, TAAD has a strong genetic component, with pathogenic variants typically inherited in an autosomal dominant manner (2). A single highly penetrant, pathogenic mutation is responsible for disease in only one-fourth of individuals with TAAD, indicating that other undiscovered genetic factors influence disease onset and progression. In addition to the known pathogenic gene mutations, low-penetrant risk variants interact with other genetic or environmental factors to trigger disease or to increase the risk for developing disease but are not themselves pathogenic (2). Significant risk factors for TAAD, primarily when occurring in the background of low-penetrant disease-predisposing variants, are hypertension and bicuspid aortic valve (1).\",\n \"A common histopathological feature of TAAD in humans is fragmented elastin in the aortic medial region (3). Elastin is the most abundant ECM protein in the aorta, where it plays a central role in the vessel's ability to expand and recoil in response to pulsatile blood flow. Smooth muscle cells (SMCs) synthesize and organize the protein into fenestrated sheets (lamellae) that separate smooth muscle cell layers. The secreted form of elastin (tropoelastin) undergoes extensive crosslinking outside the cell to form an elastic polymer, which is the functional form of the protein. The enzyme responsible for initiating crosslink formation is lysyl oxidase (LOX), a copper-binding amine oxidase that oxidizes lysine ε-amino groups in elastin and fibrillar collagens to aldehydes, which then spontaneously react to form covalent linkages between and within proteins (4,5). Approximately 90% of the lysine residues in elastin undergo crosslink formation, which makes it one of the most highly crosslinked and most stable proteins in the body (4). Inactivation of the Lox gene Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.\",\n \"in mice results in early postnatal death due to ruptured aortic aneurysms, emphasizing the critical role of fully crosslinked, functional elastin in maintaining aorta integrity and function (6,7). The gene inactivation studies also show that other LOX isoforms cannot substitute for the loss of LOX in large, elastin-rich vessels.\",\n \"LOX in vertebrates belongs to a family of 5 copper-dependent enzymes (LOX, LOXL1-4) that show a high degree of homology in their carboxy-terminal catalytic domain but differ in their amino-terminal sequences. LOXL2-4 form a subgroup that shares 4 amino-terminal scavenger receptor cysteine-rich domains that influence substrate binding but appear to have little role in regulating catalytic activity (8,9). LOX and LOXL1, in contrast, are synthesized without scavenger receptor sequences but have a propeptide domain that keeps the enzymes in a latent state until proteolytically processed outside the cell by BMP1/Tolloid-like metalloproteinases (10,11). Substrate specificity for each of the lysyl oxidases is still under investigation, but gene inactivation studies and phylogenetic analysis suggest that LOX and LOXL1 contribute to crosslinking of elastin and fibrillar collagens, whereas LOXL2-4 have different substrate specificities (12,13). All 5 LOX isoforms are synthesized in the ER and traffic to the Golgi, where the inactive apoenzyme binds copper and forms the cofactor lysyl tyrosine quinone (LTQ); both copper and LTQ are essential active site components required for catalytic activity (14).\",\n \"Mutations within LOX in families with TAAD were recently identified by whole exome sequencing (3,15). Aortic disease in these families followed autosomal dominant inheritance, suggesting that 1 mutant LOX allele is sufficient to influence disease initiation or progression. Previously, we identified a LOX catalytic site mutation (p.Met298Arg) in a family with a history of TAAD (3). By introducing this human mutation into the homologous position in the mouse genome using CRISPR/Cas9 genome editing technology (c.857T>G encoding p.M292R; hereafter referred to as Mu), we confirmed that this amino acid change caused ruptured TAAD and perinatal death when both alleles were mutated (Lox Mu/Mu ) (3). In contrast to humans who developed TAAD when heterozygous for this LOX variant, aneurysms or other arterial disease were not observed in mice with a similar genotype (Lox +/Mu ) unless they were challenged with hypertension. In this report, we show that the nature of the functional loss associated with the methionine-to-arginine change in LOX results from a secretion defect, where the protein is retained in the ER and degraded through an autophagy/proteasome pathway. We also show that the vessel wall alterations associated with LOX functional haploinsufficiency increase the likelihood of developing vascular disease in association with environmental factors that increase wall stress.\",\n \"Lox +/Mu adult animals are predisposed to aortic dilation in response to hypertension. The generation and characterization of mice expressing the M292R mutation were described previously (3). Mice heterozygous for this Lox missense variant appear grossly normal, have normal blood pressure, and show no increased mortality through 2 years of age. Aortic diameter and circumferential wall stiffness at physiological blood pressure in Lox +/Mu animals are normal, but ascending aortic length measured from the aortic root to the brachiocephalic artery is 10% longer than in WT mice (3).\",\n \"Hypertension is a known risk factor associated with aortic dilation (16). To test whether mice expressing mutant LOX are predisposed to aneurysm formation as a consequence of increased wall stress, Lox +/Mu mice were made hypertensive by subcutaneous delivery of angiotensin II (Ang II) via Alzet osmotic pumps. Pumps containing saline served as the control. Ang II infusion is a well-established model for inducing vessel wall changes in mice and other animals (17,18). Blood pressure measurements using radiotelemetry in awake, freely moving Lox +/+ and Lox +/Mu mice showed an increase of approximately 30 mmHg in systolic pressure and 25-30 mmHg in mean blood pressure in both genotypes 7-10 days after pump implantation (data not shown), consistent with what others have found using this model (19). After 4 weeks of treatment, all animals receiving Ang II had cardiac hypertrophy, indicating altered cardiac physiology consistent with Ang II-induced hypertension ( Figure 1A). However, only Lox +/Mu mice showed substantial dilation and elongation of the ascending aorta in response to Ang II; no change in aortic diameter was observed in Ang II-treated Lox +/+ mice or in Lox +/Mu mice infused with saline alone ( Figure 1B). Biaxial pressure-diameter testing showed the ascending aorta in Ang II-treated Lox +/Mu animals to be approximately 25% larger than the aorta from WT or saline-treated controls at mean physiological blood pressure ( Figure 1C). The shape of the pressure-diameter curves suggests that Ang II treatment did not affect aortic mechanical properties in either mutant or WT animals. Other than in the ascending aorta, no dilation or change in vessel mechanics occurred in the left common carotid artery for either Lox +/+ or Lox +/Mu animals with saline or Ang II treatment ( Figure 1D), nor were dilations or areas of stenosis found in the aortic arch, descending aorta, or abdominal aorta.\",\n \"Ang II treatment leads to vessel wall thickening and changes in vascular cellularity. On a structural level, Ang II treatment resulted in ascending and descending aortic wall thickening in Lox +/Mu animals, but there were no changes in elastic lamellar number or lamellar organization, quantified by measurement of medial area ( Figure 2A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.127748DS1). Elastic lamellar fragmentation, already elevated in Lox +/Mu compared with Lox +/+ animals (3), did not increase in either genotype following Ang II treatment (data not shown). We analyzed elastin crosslinking using an ELISA assay for desmosine, a major crosslink in elastin. We found no difference in desmosine levels in aortic tissue from Lox +/+ and Lox +/Mu mice, confirming normal elastin levels and that elastin crosslinking is not affected in animals heterozygous for the Lox mutation (Supplemental Figure 1C). Hydroxyproline levels (as a measure of total collagen) trended higher in vessels from the mutant animals but were not significantly different from WT when normalized to the change in total protein (Supplemental Figure 1D). Higher protein quantities are consistent with the increase in wall thickness that occurred with Ang II treatment (Supplemental Figure 1B).\",\n \"Changes in the number and types of cells were observed in the thickened aortic wall of the hypertensive animals. The most substantial change occurred in the adventitia of both the ascending and descending aorta, which underwent considerable expansion in the Lox +/+ and Lox +/Mu animals as a result of Ang II exposure ( Figure 2A). In the medial layer, Ang II infusion led to a decrease in the number of SMCs in Lox +/+ animals but an increase in SMCs in Lox +/Mu mice ( Figure 2B). There was also an increase in intralamellar area in both genotypes, although the change was most notable in Lox +/Mu mice. The nuclei of the intralamellar cells in the mutant media were rounder than cells in Lox +/+ vessels, suggesting a less polarized Lox +/+ and Lox +/Mu animals developed significant cardiac hypertrophy following 4 weeks of treatment with 2 μg/kg/ min Ang II compared with saline-treated controls. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. ****P < 0.0001. (B) The ascending aorta in Lox +/Mu animals is longer than in Lox +/+ animals and undergoes substantial dilation following Ang II treatment. (C) Pressure-diameter measurements at intraluminal pressures ranging from 0 to 175 mmHg. The ascending aorta from Ang II-treated Lox +/Mu animals has a larger diameter over the entire range of pressures compared with Lox +/+ animals treated with Ang II or with saline controls. (D) There was no difference in the diameter or mechanical properties of the left common carotid artery in either genotype with or without Ang II treatment. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each pressure level. Data are presented as mean ± SD. # P < 0.05, Lox +/+ with Ang II vs. Lox +/Mu with Ang II; *P < 0.05, **P < 0.01, Lox +/Mu with saline vs. Lox +/Mu with Ang II.\",\n \"phenotype and noncircumferential orientation. Immunostaining for smooth muscle α-actin demonstrated uniform reactivity throughout the medial intralamellar spaces, whereas cell staining in the adventitia was heterogeneous (not shown).\",\n \"Staining for the monocyte/macrophage marker CD68 in descending aorta showed an increased number of CD68 + cells in the media and adventitia of both Lox +/+ and Lox +/Mu descending aorta following Ang II treatment ( Figure 2C). These results are consistent with previous studies showing that infusion of Ang II promotes macrophage accumulation within the aortic wall (20). While the adventitia showed the highest level of immune cell infiltration, macrophage accretion in the medial layer was also observed, but the spatial localization of the CD68 + cells was different for the 2 genotypes. In Ang II-treated Lox +/+ vessels, medial CD68 + cells were associated with the first 2 elastic layers nearest to the intima. Medial CD68 + cells in the aorta of Lox +/Mu animals treated with Ang II, however, were in the outer layers of the media closest to the adventitia ( Figure 2C).\",\n \"Evaluation of factors that adversely affect vessel wall integrity and SMC function. An explanation for why Lox +/Mu mice are more sensitive than WT animals to Ang II infusion was explored by first checking for possible differences in Ang II receptor expression using quantitative PCR (qPCR). In ascending aorta from Lox +/Mu mice, all 3 Ang II receptors (Agtr1a, Agtr1b, and Agtr2) were detected, and there was no difference in their expression levels . Macrophages (stained green, arrowheads) were detected with an antibody to CD68, and cell nuclei were visualized with DAPI. Red is elastin autofluorescence. The vessel wall was thicker in both Lox +/+ and Lox +/Mu animals following Ang II treatment, with more DAPI + and CD68 + cells in the adventitia of both genotypes. (B) In the medial layer, there were fewer SMCs in the aorta of WT animals treated with Ang II but more cells in Lox +/Mu aorta. Shown are the mean ± SD for cell number counts between the internal and external elastic lamina on 7 tissue sections from each group. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.01. (C) Medial CD68 + cells were mostly located on the luminal side of the media in the Lox +/+ aorta but nearer the adventitia in Lox +/Mu aorta after Ang II treatment.\",\n \"compared with WT controls (Supplemental Figure 2A). Thus, the potential for Ang II receptor-mediated signaling is similar in both genotypes.\",\n \"Many Ang II-induced responses on SMCs are attributable to stimulation of NADPH oxidase with the subsequent generation of ROS (21,22). We used dihydroethidium, a cell-permeable compound that interacts with superoxide, to assess oxidant levels in sections of WT and mutant ascending aorta and found no difference between the 2 genotypes (Supplemental Figure 2B). Thus, differences in levels of oxidative stress do not appear to be a factor in the Ang II susceptibility of Lox +/Mu mice.\",\n \"In many models of aortic disease, the presence of proteases that degrade elastin can initiate and propagate disease by degrading the elastic fiber system in the vessel wall. We therefore investigated whether 3 elastolytic MMPs commonly linked to vascular remodeling -MMP2, MMP9, and MMP12 -were differentially expressed in mutant versus WT aorta. There were no differences in Mmp2 and Mmp9 mRNA expression levels, while expression of Mmp12 was diminished in the mutant aorta (Supplemental Figure 2C).\",\n \"Active LOX is absent in Lox Mu/Mu mouse embryonic fibroblast conditioned medium. To elucidate how the M292R modification affects LOX function, we used mouse embryonic fibroblasts (MEFs) from WT, KO, and mutant animals to characterize LOX secretion and activity. Mutant cells were indistinguishable from WT cells in morphology and in their rate of proliferation (data not shown). LOX activity ( Figure 3A), assessed using an Amplex Red assay, was not detected in cell layer extracts of any MEF genotype (Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-), which was expected, since the full-length intracellular form of LOX (pro-LOX) is an inactive zymogen. LOX enzymatic activity was readily detected in the conditioned medium from Lox +/+ MEFs and showed linear enzyme kinetics over the period of the Amplex Red assay. Conditioned medium from Lox Mu/Mu MEFs, however, had no detectable activity, similar to conditioned medium from cells with inactivated Lox alleles (Lox -/-) ( Figure 3B). Activity was detected in conditioned medium from Lox +/Mu cells, where the linearity and rate of substrate oxidation were comparable to those seen in Lox +/+ cells, indicating that the mutant form of the protein does not adversely affect the enzymatic activity of WT LOX and hence does not function in a dominant negative fashion.\",\n \"M292R LOX is poorly secreted and is retained in the ER. LOX is maintained as an inactive 50-kDa proenzyme through every stage of the synthetic and secretory pathway. Activation requires secretion into the extracellular space, where proteolytic removal of the N-terminal propeptide portion of the protein generates the 30-kDa active catalytic domain. To determine whether LOX activity is absent in M292R MEF conditioned medium because mutant LOX is not secreted, we performed Western blots using an anti-LOX antibody on Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF cell layers and conditioned medium. Immunoblotting showed increased levels of 50-kDa intracellular pro-LOX protein in cell extracts from Lox Mu/Mu MEFs compared with Lox +/+ cells ( Figure 4A). Conversely, there was abundant 30-kDa mature (processed) LOX in conditioned medium from WT cells but barely detectable levels in medium from Lox Mu/Mu MEFs ( Figure 4B). These findings indicate that the mutant protein is poorly secreted and is retained intracellularly as the proenzyme. Immunofluorescence imaging with an antibody to LOX showed minimal intracellular staining in Lox +/+ cells, confirming that WT LOX is secreted efficiently ( Figure 5A). Staining of Lox Mu/Mu cells, however, showed abundant intracellular staining, consistent with the immunoblot results described above. The staining pattern for intracellular LOX was typical of accumulation within the ER, which was confirmed by colocalization of LOX with calnexin, an ER-resident protein ( Figure 5, B and C).\",\n \"A direct interaction between LOX and calnexin was shown using coimmunoprecipitation from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF lysates. Lox -/cells served as the negative control. Cell lysates were immunoprecipitated with an anti-calnexin antibody, followed by immunoblotting using an antibody to LOX. LOX protein was detected in calnexin immunoprecipitates from all genotypes except Lox -/-, confirming an interaction between the 2 proteins ( Figure 5D).\",\n \"M292R LOX has an altered protein conformation. The high level of LOX detected in Lox Mu/Mu MEF calnexin immunoprecipitates suggests that M292R LOX is misfolded and trapped in the ER bound to calnexin. To determine whether M292R LOX is conformationally different from the WT enzyme, we explored possible structural changes by assessing differential sensitivity to proteolytic degradation. Changes in protein structure frequently expose or mask protease binding sites, leading to altered degradation products and a different degradation fingerprint. Immunoblotting of cell lysate from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs incubated at 37°C for 6 hours to exploit protein degradation by endogenous enzymes within the cell lysate showed time-dependent degradation of WT but not M292R LOX ( Figure 6). These results suggest that WT and M292R pro-LOX have different susceptibilities to degradation arising from cell-associated proteases. Differential degradation was also observed when cell lysates were incubated with pancreatic elastase. In digests of Lox +/+ cell lysate, there was a 25-kDa fragment evident with all doses of elastase that was absent in Lox Mu/Mu samples (Supplemental Figure 3, arrow). When treated with the highest dose of elastase, pro-LOX from Lox +/+ and Lox +/Mu samples degraded predominantly to a 30-kDa fragment. LOX from Lox Mu/Mu samples, in contrast, degraded further to mainly a 10-kDa fragment. It is important to note that pancreatic elastase does not cleave after methionine or arginine, so the Met-to-Arg mutation does not create or negate a protease cleavage site for this enzyme. Together, the conformational difference between the WT and mutant proteins suggests that M292R LOX is misfolded, which is consistent with its protracted interaction with calnexin in the ER.\",\n \"M292R LOX does not elicit an ER stress response. Accumulation of misfolded proteins in the ER generally leads to ER stress, which subsequently activates the unfolded protein response (UPR) (23). UPR triggers 3 primary stress sensors, PERK, IRE1, and ATF6. Under resting conditions, BIP/Grp78 maintains these sensors in an inactive state. Upon ER stress, BIP binds to misfolded proteins, thereby allowing for sensor activation. To determine whether the continuous presence of misfolded mutant LOX induces ER stress, we assessed Bip transcript levels by qPCR. We also measured mRNA levels of 20% change was modest compared with the ~2-fold and ~4-fold increase in Atf4 and Chop, respectively, when ER stress was stimulated by treatment of MEFs with lopinavir/ritonavir, which served as a positive control (data not shown). Electron microscopic analysis of WT and mutant cells showed that ER volume was not markedly different in the 2 cell types (Supplemental Figure 4), suggesting that mutant protein does not accumulate to levels that would produce dilated saccules. Together, these results suggest that LOX protein containing the M292R mutation does not induce substantial ER stress.\",\n \"Increased activation of autophagy in Lox Mu/Mu cells. The absence of ER stress in Lox Mu/Mu cells was surprising given that M292R LOX does not leave the ER. To determine whether mutant LOX is being removed from the ER for degradation through the lysosomal pathway, we costained cells for LOX and the lysosomal marker LAMP-2. No colocalization of the 2 proteins was observed, suggesting that the mutant protein was not trafficked through lysosomes (Supplemental Figure 5).\",\n \"Autophagy is a general mechanism for clearing proteins accumulating in cells. Clearing of misfolded proteins from the ER, a process termed ER-phagy, plays a critical role in ER homeostasis (24). To determine whether mutant LOX directly activates autophagy-mediated protein clearance from the ER, we measured 2 autophagy activation markers, p62 and microtubule-associated protein 1A/1B, light chain 3 (LC3), in WT and Lox Mu/Mu MEFs. Following pretreatment with bafilomycin A1 (BafA1) to inhibit organelle acidification-induced degradation of p62 and LC3, only Lox Mu/Mu cells with and without BafA1 pretreatment had significantly increased p62 expression compared with Lox +/+ cells without BafA1 pretreatment ( Figure 8, A and B). The autophagy activation markers LC3-I and -II were also elevated in mutant compared with WT cells (Figure 8, A, C, and D), providing more evidence for increased autophagy.\",\n \"We also used electron microscopy to screen for the presence of autophagosome-like vesicles in WT and mutant cells (Supplemental Figure 4). We observed that multi-and single-membrane vesicles typical of autophagosomes were particularly abundant in the perinuclear region but were also present throughout the cytoplasm of Lox Mu/Mu cells. Such structures were rare in WT cells. Together, the existence of autophagic vesicles along with increased expression of autophagy activation markers in mutant cells is consistent with the clearance of misfolded mutant LOX from the ER through an autophagy/proteasome pathway.\",\n \"Mutant LOX does not influence the secretion of the WT protein.\",\n \"To assess whether the secretion of WT LOX is affected by the presence of M292R mutant protein, we transiently expressed plasmids containing WT LOX, and M292R LOX fused to the fluorescent protein mApple, in Lox -/-MEFs. Successful transfection was confirmed by intracellular mApple expression using fluorescence imaging (data not shown). In the conditioned medium of Lox -/cells transfected with empty plasmid, no LOX was observed by Western blot analysis ( Figure 9A). In Lox -/-MEFs transfected with 2.5 μg WT Lox-mApple plasmid, an immunoreactive protein band at approximately 60 kDa was detected in the conditioned media, corresponding to the size of active LOX (30 kDa) fused to mApple (27 kDa). In Lox -/-MEFs transfected with 2.5 μg of M292R Lox-mApple plasmid, LOX protein was observed inside the cell by fluorescence microscopy, but no LOX protein was detected in conditioned medium by Western blotting, consistent with the inability of M292R LOX to be secreted. To determine whether mutant LOX alters the secretion of the WT protein, or if the WT protein acts as a chaperone to facilitate the secretion of the mutant protein, we cotransfected cells with 1.25 μg each of WT and mutant plasmid, which is half as much as was used for single plasmid transfection. If mutant LOX does not affect the secretion of the WT protein, then LOX should be detected in conditioned medium, but at half the level of the single plasmid transfection. If the mutant protein has an adverse effect on WT LOX secretion, then levels will be lower than 50%. If WT LOX acts as a chaperone to facilitate secretion of the mutant protein, then the combination of both secreted proteins would be higher than 50% and possibly equivalent to the WT transfection (1.25 + 1.25 = 2.5 μg). Figure 9 shows that LOX protein was detected in conditioned medium of doubly transfected cells at half the level of singly transfected cells, confirming that the mutant protein does not affect the secretion of WT LOX.\",\n \"M292R propeptide processing. Our experimental results are consistent with the M292R mutation being a loss-of-function phenotype due to a secretion defect. The final activation step of the proenzyme occurs outside the cell through proteolytic separation of the propeptide and catalytic domains. To determine whether M292R pro-LOX could be processed to the 30-kDa mature form if it were secreted, we incubated cell lysates from WT and mutant MEFs with recombinant BMP1 (rBMP1), the enzyme responsible for propeptide cleavage. Western blot analysis showed that BMP1 appropriately processed both WT and M292R pro-LOX to the 30-kDa mature form (Supplemental Figure 6). Thus, the mutation did not inhibit propeptide processing. The processed mutant protein was not assayed for oxidase activity because intracellular LOX in M292R MEFs does not have bound copper and cannot be enzymatically active.\",\n \"LOX plays a critical role in vascular ECM maturation by catalyzing the crosslinking of elastin and fibrillar collagens, the 2 major ECM protein classes in the aortic wall. Phylogenic studies suggest that the current form of vertebrate LOX coevolved with elastin and helped facilitate the appearance of a closed circulatory system (12,25). Therefore, it is no surprise that mutations in LOX give rise to vascular insufficiency and disease. All LOX mutations associated with TAAD in humans show autosomal dominant inheritance, and most result in loss of function. We know from mouse studies that inactivation of both Lox alleles is lethal and that other members of the LOX family cannot assume LOX's critical role in vascular development and maintenance of the vessel wall. In this report, we utilize a mouse model of a well-characterized human mutation to explore how LOX haploinsufficiency leads to vascular dilation and aneurysm formation. Mice heterozygous for the Lox M292R variant (Lox +/Mu ) have increased elastic lamellar fragmentation in the wall of the ascending aorta (3), but do not develop aortic dilation under normal conditions. The absence of aneurysmal disease was surprising given that humans heterozygous for this same mutation are prone to aneurysm formation and vessel wall changes (3). Variability in the age of disease onset and disease severity in humans, however, suggested that a secondary challenge is a factor in disease initiation and progression. Here we show that hypertension induced by Ang II infusion resulted in vessel wall changes in both Lox +/+ and Lox +/Mu mice, but only Lox +/Mu animals showed aortic dilation localized to the ascending aorta. Several groups have reported that the effects of Ang II are most pronounced in the ascending aorta region in this model (26)(27)(28), but the underlying factors governing this vessel specificity are unknown. Differences in the embryonic origin of the cells that populate the aortic wall are one explanation (29,30), but the composition and maturation of the aortic ECM are also factors. In Lox +/Mu mice, changes in ECM crosslinking associated with LOX insufficiency could have more negative consequences for this vessel segment because of high collagen and elastin levels and the high mechanical stress experienced by the aortic wall during the cardiac cycle.\",\n \"While several Ang II-induced vascular changes were expected (19,27) and common to WT and mutant animals, there were also significant differences. For instance, medial expansion was evident in both genotypes, but the effect was most notable in mutant mice, in which there was an increase in the number of smooth muscle cells between the elastic lamellae. This response is particularly unusual, since the intralamellar space is typically populated by a single smooth muscle cell layer, as can be seen in vessels from saline-treated Lox +/+ and Lox +/Mu animals ( Figure 2). The opposite change occurred in WT vessels, where there were fewer smooth muscle cells in response to Ang II, suggesting Ang II-related apoptosis or growth suppression (26), which we did not assess. mice. Oneway ANOVA with Tukey's multiple-comparisons test was used to assess differences, which were minimal between the 3 genotypes. Data are presented as mean ± SD. ***P < 0.001.\",\n \"Another cellular change in the ascending and descending aorta following Ang II treatment was the accumulation of macrophages in the aortic wall of both WT and Lox +/Mu mice. CD68 + cells were found in the adventitia of both genotypes, with fewer cells in the medial layer. In Ang II-treated WT mice, CD68 + cells accumulated in the intima and inner elastic layers of the media and did not localize to breaks in elastic lamella, as has been reported in other models (18,20). Their location within the wall, however, shows that some cells were able to penetrate past the internal elastic lamina into the first or second layer of smooth muscle cells. Medial CD68 + cells in Lox +/Mu mice exposed to Ang II, in contrast, were localized to the outer aspect of the media and occurred at higher numbers than in WT vessels. These results suggest that the cellular origin of the CD68 + cells or the route of inflammatory cell infiltration is different in Lox +/+ and Lox +/Mu vessels as a response to wall stress.\",\n \"The location of macrophages in the outer region of the wall provides a mechanistic explanation for why Lox +/Mu , but not WT, aorta dilates following Ang II treatment. ECM degradation is a primary factor leading to vessel wall dilation. Elastases and other matrix-degrading enzymes secreted by macrophages destabilize the vessel wall, particularly when matrix degradation occurs in or near the adventitia -the vessel compartment essential for maintaining the structural integrity of the artery (31). The medial cell hypertrophy and inflammatory cell accumulation in the Lox +/Mu aorta in response to hemodynamic stress are similar in several ways to vascular changes in mouse models of Marfan syndrome with mutations in fibrillin-1, a key elastic fiber protein. In Marfan mice, inflammatory cell accumulation at the medial-adventitial interface correlates with increased elastin degradation and a loss C and D) Also, LC3 was elevated in Lox Mu/Mu cells compared with Lox +/+ cells. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.001.\",\n \"of structural integrity that leads to vessel wall dilation (32). As in the Ang II-treated Lox +/Mu animals, only the ascending aorta is affected in Marfan mice.\",\n \"Our previous studies showed that mice with the Lox M292R mutation share numerous phenotypic traits with Lox +/animals (3), suggesting that M292R is a loss-of-function mutation in mice and humans. Even though the amino acid change is in the catalytic domain of LOX close to the copper binding site, it was not clear whether the mutation results in a catalytically dead enzyme or if some other aspect of LOX processing is negatively affected by the mutation. Our data show that the answer fits both possibilities: the mutant protein is retained in the ER and not secreted, and the enzyme is catalytically dead because of its inability to traffic to the Golgi, where it needs to bind copper and form the redox quinone cofactor LTQ required for catalytic activity (33). Even though we found that the propeptide of mutant pro-LOX could be cleaved by rBMP1 similarly to WT LOX, full-length LOX in the ER cannot be catalytically active even if propeptide processing does occur.\",\n \"Why mutant LOX is retained in the ER is not clear, but our data point to protein misfolding as a probable cause. ER retention is characteristic of incompletely folded proteins. Folding intermediates of most secreted proteins interact with the calnexin/calreticulin protein complex in the ER to achieve the proper native conformation required for secretion (34). The calnexin/calreticulin protein complex recognizes N-linked glycan structures (LOX has 3 N-linked glycosylation sites in the propeptide region; ref. 11) that undergo cycles of sugar residue removal and addition. Once folded correctly, calnexin and calreticulin dissociate, and the folded proteins are transported from the ER to the Golgi apparatus. Mutant LOX colocalized intracellularly with calnexin and was isolated bound to calnexin in cellular extracts, suggesting that the protein remains misfolded despite repeated folding interactions with calnexin. Furthermore, the differential susceptibility to proteolytic degradation of mutant compared with WT LOX is consistent with structural differences between the 2 proteins.\",\n \"It was interesting that mutant LOX accumulation in the ER did not induce an appreciable UPR. We saw no upregulation in mutant cells of the ER stress-related markers Bip (an ER luminal chaperone protein and critical ER stress sensor), Atf4 (a transcription factor downstream of PERK that plays a crucial role in the adaptation to ER stress), or Chop (a transcription factor regulated by ATF4 that drives proapoptotic gene transcription). Nor could we detect in mutant cells an increase in ER volume frequently associated with ER stress. One pathway whereby ER stress is mitigated is removal from the ER of mutant protein and degradation at the lysosome (ERAD-II) or proteasome (ERAD-I) (24). We were unable to localize LOX within lysosomes but did find increased expression of the autophagy markers p62 and LC3. Electron microscopy also identified an increased number of autophagic vesicles in Lox Mu/Mu cells. These results suggest that misfolded mutant LOX is being cleared from the ER through an autophagy/proteasome pathway and would explain why mutant protein levels never reached a critical threshold to sustain an ER stress response. Furthermore, there is mounting evidence that the autophagy and secretory pathways are coregulated and intimately linked (35), which may represent a mechanistic connection into the LOX trafficking defect that we identified. It should be noted that all the experiments probing the fate of the mutant protein were done in cultured cells. It is possible, but unlikely, that in vivo, in tissues, and at developmental stages relevant to the TAAD phenotype, LOX folding, secretion, ER retention, and stress occur differently than in MEFs.\",\n \"Guo et al. (15) described 2 other LOX active site variants (S280R and S348R) that segregate with disease in families with TAAD. When expressed from transgenes in HeLa cells and examined in cell lysates, a portion of the pro-LOX protein was processed to its active form and was able to oxidize substrate at rates approximately equivalent to the WT enzyme (S348R) or with approximately 50% lower activity (S280R) (2,15). Enzymatic activity in conditioned medium from the transfected cells was not evaluated, so it is not known whether these LOX variants are efficiently secreted. However, the fact that the proteins are catalytically active implies trafficking through the Golgi and proteolytic removal of the propeptide domain. Thus, unlike the M292R mutation, which is retained in the ER, the S348R and S280R variants appear capable of traversing the secretory pathway. How these proteins, which have full (or somewhat reduced) catalytic activity, cause thoracic aortic disease is an interesting question. An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.\",\n \"Antibodies. Antibodies used in this study are described in Supplemental Table 1.\",\n \"Mouse models and cell isolation. A knockin mouse strain bearing the Lox M292R substitution was generated in the C57BL/6 background using CRISPR/Cas9 genome editing technology as previously described (3). Lox +/mice in the same background are described in ref. 6. Primary MEFs were isolated from the various mouse lines by digestion of E14.5 mouse embryos with trypsin/EDTA at 37°C for 30 minutes in DMEM (38). Cells were collected by centrifugation and grown in DMEM, 10% FBS, 1% penicillin/streptomycin, and 1 mM l-glutamine.\",\n \"Ang II treatment and blood pressure measurements. Telemetry transmitters (Data Sciences International, model PA-C10) were implanted surgically in 3-to 4-month-old mice under isoflurane anesthesia. The catheter was inserted in the left common carotid artery and advanced to the aortic arch, and the transmitter was placed subcutaneously along the abdomen. Animals were allowed to recover for 7 days prior to activation of transmitters, and continuous recording of heart rate and arterial pressure was obtained, with sampling every 5 minutes for 15-second intervals for 3 days. Data were collected and stored using Dataquest ART (Data Sciences International).\",\n \"After baseline blood pressure measurements were obtained, mice were implanted with osmotic pumps (Alzet model 1004, DURECT Corp.) that delivered Ang II (MilliporeSigma) subcutaneously at a dose of 2 μg/kg/min in the interscapular region, according to published procedures (39,40). Mice infused with saline were used as controls. After a week of Ang II treatment, arterial pressure and heart rate were again continuously recorded using the telemeters, with sampling every 5 minutes for 15-second intervals for 3 days. Animals were fed a standard laboratory chow diet. After 28 days, animals were sacrificed under isoflurane anesthesia, and vessels were collected for compliance studies and histological characterization.\",\n \"Vessel compliance measurement. For vessel compliance measurements, the ascending aorta and left common carotid artery were excised and placed in a physiological saline solution. Vessels were then cleaned of surrounding fat and connective tissue and mounted onto a pressure arteriograph (Danish Myo Technology). Experiments were done at 37°C in an organ bath containing physiological saline. The mounted vessels were visualized using a computerized imaging system, allowing continuous recording of vessel diameters. Vessel outer diameters were recorded while intravascular pressure was increased from 0 to 175 mmHg by increments of 25 mmHg (12 seconds per step). The average of 3 outer diameter measurements was taken at each pressure (40,41).\",\n \"Protein quantification, hydroxyproline, and desmosine assays. Each ascending aorta was hydrolyzed at 105°C overnight with 20 μL of 6 N hydrochloric acid (Thermo Fisher Scientific) and dried under vacuum in a SpeedVac. The dried pellet was dissolved in 400 μL water and filtered with a 0.45-μm filter. Total protein, hydroxyproline, and desmosine levels were determined as described previously (42).\",\n \"Dihydroethidium staining for superoxide detection in aortic sections. Superoxide in aortic sections was detected using dihydroethidum (DHE) staining. In the presence of superoxide, DHE is oxidized to 2-hydroxyethidium, which is highly fluorescent and intercalates within the DNA, staining the nucleus a fluorescent red (43,44). After euthanasia and thoracotomy, the right atrium was clipped, and 5 mL cold (4°C) 1× PBS was flushed through the left ventricle to clear the blood. Ascending aorta was then dissected, placed in Tissue-Tek O.C.T. Compound (Sakura Finetek), and flash frozen in liquid nitrogen. Four-micrometer cryosections were obtained using a Leica CM1850 cryostat and placed on charged microscope slides (Thermo Fisher Scientific). A 10-mM stock solution of DHE (Life Technologies) in DMSO was diluted to 10 μM using 1× PBS. The 10-μM DHE solution was applied to unfixed aortic sections and incubated at 37°C for 30 minutes in the dark. After staining with DHE, sections were then rinsed with 1× PBS for 5 minutes at room temperature (RT) and mounted with a coverslip using ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were obtained on the Zeiss Axioscope fluorescence microscope with a QImaging MicroPublisher 3.3 RTV camera using QCapture software (QImaging). The entire procedure (from mouse euthanasia to image acquisition) occurred within 8 hours.\",\n \"Immunofluorescence on aortic sections and area measurements. Ascending and descending aortas were dissected as above and placed in O.C.T. compound, and the blocks were frozen on dry ice. Four-micrometer-thick cryosections were placed on glass slides and stored at -80°C. The sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS at 4°C for 10 minutes, washed twice with 1× PBS (5 minutes each) at RT, and blocked with a solution containing 1% BSA (MilliporeSigma), 1% fish gelatin (MilliporeSigma), and 0.05% Triton-X (Acros Organics) in 1× PBS for 1 hour at RT. The aortic sections were then incubated with rat anti-mouse CD68 antibody (Bio-Rad) at a 1:1000 dilution in blocking solution at 4°C overnight. The next day, sections were washed 3 times with 1× PBS (5 minutes each) and incubated in goat anti-rat IgG Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) diluted 1:2000 and DAPI diluted 1:3000 in blocking solution at RT for 1 hour. The sections were washed twice with 1× PBS (5 minutes each) at RT, mounted with a coverslip using ProLong Diamond Antifade Mountant, and imaged using the Axioscope fluorescence microscope equipped with the QImaging MicroPublisher 3.3 RTV camera using QCapture software as above. For area measurements, frozen sections from the descending aorta from 3-month-old mice were prepared as described above. The area between the internal and external elastic laminae was traced in each ×40 image using ImageJ software (NIH) and measured in pixels 2 .\",\n \"LOX activity assay. MEFs were grown in 10-cm dishes and maintained as above until fully confluent. Twenty-four hours before collection, culture medium was replaced with DMEM lacking phenol red and FBS, supplemented with 50 μg/mL ascorbic acid and 0.1% BSA. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. Cell lysates were collected by scraping in 1× PBS containing protease inhibitors. LOX activity in cell lysate and concentrated culture medium, incubated with and without the LOX inhibitor β-aminopropionitrile (BAPN), was quantified using the Amplex Red assay as previously described (3,45). Resorufin fluorescence, the product of Amplex Red oxidation, was measured at excitation and emission wavelengths (540 and 600 nm, respectively) every 30 minutes for 150 minutes using a BioTek H4 Hybrid Reader. LOX activity was calculated as the difference between total activity and activity in the presence of BAPN. Differences in relative fluorescent units were tested using 2-way ANOVA with Tukey's multiple-comparisons test.\",\n \"Immunofluorescence imaging of primary cells. Primary MEFs were seeded in 4-chamber glass slides at 3 × 10 5 cells per/well. After 3 days in culture, the cells were fixed with cold methanol (2 washes, 5 seconds each) followed by 3 washes with blocking solution containing 1× PBS, 1% BSA, 1% fish gelatin (Milli-poreSigma), and 0.05% Triton-X100 at pH 7.4, then incubated in blocking solution for 30 minutes at RT. The cells were then incubated with a primary antibody to LOX at 1:1000 in blocking solution overnight at 4°C. After washing the cells with blocking solution to remove the primary antibody, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor 568 secondary antibody at 1:2000 (Thermo Fisher Scientific) and anti-calnexin primary antibody directly labeled with Alexa Fluor 488 at 1:4000 in blocking solution for 30 minutes at RT. The cells were washed with blocking solution 3 times, then mounted using ProLong Diamond Antifade Mountant (Invitrogen) and coverslipped. The samples were imaged using the Axioscope fluorescence microscope and QCapture Pro software.\",\n \"Protein extraction from cell culture and aortic tissue. Cultured MEFs were switched to serum-free medium 24 hours before protein extraction. MEFs in 10-cm dishes were incubated in 1 mL lysis (RIPA) buffer (MilliporeSigma) containing protease inhibitors for 30 minutes at 4°C. The lysates were scraped from the dish into microfuge tubes and centrifuged at 17,500 g for 5 minutes, and the supernatant was collected. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. The protein concentration in cell lysates and concentrated media was measured using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Laemmli buffer with and without DTT was added to samples for SDS-PAGE and native gel electrophoresis, respectively. Only the samples with DTT were boiled at 100°C for 5 minutes.\",\n \"RNA extraction and qPCR. RNA was extracted from confluent cell cultures using TRIzol (Thermo Fisher Scientific) following the manufacturer's protocol. For RNA isolation from aortic tissue, ascending aorta from adult Lox +/+ and Lox +/Mu mice were each homogenized in 500 μl TRIzol using QIAGEN TissueLyser. The manufacturer's protocol was followed for RNA isolation. At the time of RNA precipitation, 10 μg glycogen (Mil-liporeSigma) was added as a carrier to each sample. Each RNA pellet was dissolved in 10 μL molecular-grade water. The RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). One microgram of the isolated RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Reverse transcription was then carried out using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). One microliter of cDNA was used for qPCR using TaqMan Fast Universal PCR Master Mix and TaqMan assay primer/probes (Life Technologies) for Mmp12 (Mm00500554_m1), and Gapdh (Mm9999999915_ g1), which was used as an internal control. Ten-microliter reactions were performed in duplicate using a ViiA 7 Real-Time PCR System for Bip, Atf4, and Chop and QuantStudio 3 Real-Time PCR system (Applied Biosystems) for Agtr1a, Agtr1b, Agtr2, Mmp2, Mmp9, and Mmp12. All mRNA levels were normalized to Gapdh expression. rBMP1 treatment. Protein was extracted from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs as described above. Fifty microliters of cell lysates from cultured MEFs containing 50 μg total protein were incubated with 30, 60, and 90 ng rBMP1 (R&D Systems) for 45 minutes at 37°C in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5). The enzyme reaction was stopped by adding Laemmli buffer with DTT and incubated at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.\",\n \"Western blotting. Protein extracts from cells, concentrated condition medium, or tissues (generally 50 μg) were analyzed using 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) run at 100 mV for 1 hour. The protein was then transferred to ProBlott Membrane (Applied Biosystems). The blots were incubated in 5% nonfat dried milk in 0.1% 1× PBS-Tween for 1 hour at RT, then incubated with a primary antibody overnight at 4°C. The next day, the blots were washed with 0.1% 1× PBS-Tween and incubated in ECL anti-rabbit or anti-mouse IgG HRP-Linked Secondary Antibody (GE Healthcare) for 1 hour at RT. The blots were then washed and the immunoreactive bands detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The blots were stripped by incubating in 2% SDS, 75 mM Tris-HCl, and 100 mM β-mercaptoethanol for 1 hour at 65°C while shaking. The blot was then incubated in 0.1% 1× PBS-Tween wash buffer for 1 hour at RT before immunodetection of β-actin (MilliporeSigma) at 1:20,000 in wash buffer, which was used as a loading control. For protein samples from concentrated conditioned media, membranes were stained with Ponceau S solution (MilliporeSigma) before blocking and used as a loading control. Alternatively, stain-free images of Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) taken following the manufacturer's protocol served as a loading control.\",\n \"Coimmunoprecipitation. Cell lysates were prepared from MEFs 5 days after confluency as described above. Immobilized protein A anti-rabbit IgG beads (50 μL), preequilibrated by washing with lysis buffer, were then added to 250 μL cell extract. After incubation on a rocking platform for 60 minutes at 4°C, the sample was centrifuged at 2500 g for 3 minutes, and the supernatant was incubated with 8 μg immunoprecipitation antibody for 1 hour at 4°C. Fifty microliters of precleared protein A beads was then added, and the samples incubated overnight at 4°C with rocking. Immune complexes bound to the protein A beads were collected by centrifugation and washed with lysis buffer prior to boiling at 100°C for 10 minutes with Laemmli buffer containing DTT. The supernatant was collected by centrifugation, and proteins were separated by SDS-PAGE and immunoblotted following the same protocol described above.\",\n \"Protein degradation analysis. Confluent cultures of WT and mutant MEFs were treated with 5 μg/mL Brefeldin A (MilliporeSigma) to inhibit protein transport from ER to the Golgi apparatus. This ensured that WT LOX remained in the cells so that the starting amount of protein was the same across genotypes. After 4 hours, the cell lysate was extracted using lysis buffer placed at 37°C to initiate protein degradation. Every hour, 50 μL aliquot of cell lysate was collected, then immediately boiled with Laemmli sample buffer at 100°C for 5 minutes prior to SDS-PAGE analysis. For exogenous protease-mediated degradation, human pancreatic elastase in increasing concentrations (0.625, 1.25, 2.5, 5 ng/μL) was added to cell lysates containing 1 mg/ mL protein at 4°C for 30 minutes. After treatment, elastase activity was inhibited with protease inhibitor cocktail, and samples were boiled with Laemmli sample buffer at 100°C for 5 minutes for SDS-PAGE analysis.\",\n \"Autophagy markers. Confluent MEFs in 6-well dishes were pretreated with 100 mM BafA1 (MilliporeSigma) for 3 hours before cell lysates were collected. The protein samples were separated on 4%-15% gradient gels for SDS-PAGE analysis. Immunoblotting was performed following the same procedure as above.\",\n \"Lox-mApple plasmid cloning and expression. The mouse Lox cDNA in a mammalian expression vector driven by a pCMV promoter was purchased from GE Healthcare. The Lox open reading frame was PCR amplified using a 5′ primer bearing an XhoI restriction enzyme site (5′-CAGATCTCGAGAGCGTTTCGCCTGGGCTGTGC-3′) and a 3′ primer designed to remove the Lox stop codon bearing a BamHI restriction enzyme site (5′-GGATCCA-CATACGGTGAAATTGTGCAGCCTGAG-3′). The PCR product and the pGEMT shuttle vector (Promega) were digested with XhoI and BamHI, then ligated together using T4 ligase. The mutant Lox plasmid bearing the 893 T>G base pair change was generated using the Lox-pGEMT plasmid as the template, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific), following the manufacturer's protocol (5′-TTACCA-CAGCAGGGACGAATTCAGCCACTATG-3′ and 5′-TGTTGGTGACAGCTGTGCCACTCCC-3′). The WT and mutant Lox-pGEMT plasmid sequences were confirmed using Sanger sequencing performed at the Washington University Protein and Nucleic Acid Chemistry Laboratory. To generate the Lox-mApple plasmids, WT and mutant Lox pGEMT plasmids and the mApple plasmid (46), provided by David W. Piston (Washington University School of Medicine in St. Louis), were digested with XhoI and BamHI restriction enzymes. The WT and mutant mLox cDNA inserts were then ligated into mApple vectors to generate the mLox-mApple fusion plasmids and sequenced to confirm. To express the WT and mutant Lox-mApple plasmids in MEFs, Lipofectamine 3000 reagents (Thermo Fisher Scientific) were used following the manufacturer's protocol.\",\n \"Transmission electron microscopy. MEFs from Lox +/+ , Lox +/Mu , and Lox Mu/Mu mice, cultured on glass coverslips, were washed in cacodylate buffer (0.15 M cacodylate in 2 mM CaCl 2 , pH 7.4) and fixed for 5 minutes with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer prewarmed to 37°C. The samples were then incubated at RT for an additional hour, washed with cacodylate buffer, and treated with 1% OsO 4 /1.5% potassium ferrocyanide for 1 hour in the dark. After washing with ultrapure water, en bloc staining was performed in 2% uranyl acetate for 1 hour in the dark. The samples were washed again in ultrapure water before dehydration in a graded acetone series. Finally, the samples were embedded in Epon resin and cured in a 60°C oven for 48 hours. The processed samples were imaged using a JEOL 1400 electron microscope.\",\n \"Statistics. One-way ANOVA with Tukey's multiple-comparisons test was used to determine the significance, if any, between different groups, particularly genotypes. When 2 variables were present, e.g., genotype and treatment or genotype and gene, 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences. If the same sample was measured repeatedly (e.g., at different pressures in the pressure-diameter curves in Figure 1, C and D, or at different time points in Figure 3), then repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was performed. Prism 8 for Mac OS X (GraphPad Software) was used to run statistical analyses. In all numerical figures, data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical test used and significant differences are noted in each figure legend.\",\n \"Study approval. All animal experiments were carried out following protocols approved by Washington University School of Medicine Institutional Animal Care and Use Committee.\",\n \"VSL, CMH, TJB, and PCT participated in experimental design and acquired and analyzed primary data. NOS helped with data analysis, and RPM designed the research study and analyzed primary data. VSL wrote the initial draft of the manuscript, to which all authors contributed edits.\"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["Intracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress","Introduction","Results","Discussion","Methods","Author contributions","Figure 1 .","Figure 2 .","Figure 3 .","Figure 4 .","Figure 5 .","Figure 6 .","Figure 7 .","Figure 8 .","Figure 9 ."],"string":"[\n \"Intracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress\",\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Methods\",\n \"Author contributions\",\n \"Figure 1 .\",\n \"Figure 2 .\",\n \"Figure 3 .\",\n \"Figure 4 .\",\n \"Figure 5 .\",\n \"Figure 6 .\",\n \"Figure 7 .\",\n \"Figure 8 .\",\n \"Figure 9 .\"\n]"},"annotations_table":{"kind":"list like","value":[],"string":"[]"},"annotations_tableref":{"kind":"list like","value":["Table 1"],"string":"[\n \"Table 1\"\n]"},"annotations_title":{"kind":"list like","value":[],"string":"[]"},"annotations_venue":{"kind":"list like","value":[],"string":"[]"}}},{"rowIdx":84742,"cells":{"corpusid":{"kind":"number","value":201836719,"string":"201,836,719"},"updated":{"kind":"string","value":"2022-02-04T21:02:50Z"},"content_source_oainfo_license":{"kind":"string","value":"CCBY"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"http://www.jlr.org/content/60/11/1807.full.pdf"},"content_source_oainfo_status":{"kind":"string","value":"HYBRID"},"content_source_pdfsha":{"kind":"string","value":"ad5e3dbde1c06e288b88d8a128bb0cb660702320"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.1194/jlr.m092379"},"externalids_mag":{"kind":"string","value":"2971567338"},"externalids_pubmed":{"kind":"string","value":"31484694"},"externalids_pubmedcentral":{"kind":"null"},"content_text":{"kind":"string","value":"\nMetabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling\n2019\n\nSoke Chee Kwong \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n\nAmira Hajirah \nAbd Jamil \nAnthony Rhodes \nNur Aishah Taib \n§ \nIvy Chung ivychung@ummc.edu.my \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n\n\nFaculty of Medicine\nDepartments of Pharmacology\nthe Translational Core Laboratory\nUniversity of Malaya\n\n\nMetabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling\n\nJournal of Lipid Research\n601807201910.1194/jlr.M092379Manuscript received 7 January 2019 and in revised form 6 August 2019.This article is available online at This work was supported by University of Malaya High Impact Research Grant UM.C/HIR/MOHE/06; University of Malaya Research Grant RP019-b; and The authors declare no conflicts of interest. Published, JLR Papers in Press, September 4, 2019 DOI https://doi.org/10.1194/jlr.M092379 1 To whom correspondence should be addressed.Abbreviations: CPT1A, carnitine palmitoyltransferase 1AER, estro- gen receptorFABP, fatty acid binding proteinFAO, FA oxidationGEO, Gene Expression OmnibusGLS1, glutaminase 1GLUT1, glu- cose transporter 1HER2, human epidermal growth factor receptor 2MCAD, medium-chain acyl-CoA dehydrogenaseMTT, methyl thiazolyl tetrazoliumOCR, oxygen consumption rateOD, optical densityPR, progesterone receptorTNBC, triple-negative breast cancer\nERs) and progesterone receptors (PRs), as well as human epidermal growth factor receptor 2 (HER2). It is the most aggressive subtype of breast cancer, affecting about 15-20% of total breast cancer cases. Compared with other subtypes, TNBC tumors are associated with higher histologic grade and larger tumor size (1, 2). The current standard regimen for TNBC patients is a combination of surgery and chemotherapy (3), which often fails to effectively slow down the tumor progression. Hence, TNBC patients have lower overall survival (81% vs. 91%) and disease-free survival (72% vs. 86%) compared with non-TNBC patients (4). Given the lack of ER and HER2 in this subtype, there is no molecular characterization that allows this subtype to be targeted.Uncontrolled proliferation of cancer cells is metabolically demanding. The metabolic pathways utilized by cancer cells to sustain high-energy demands and biosynthesis differ from those employed by healthy cells (5). Challenged by hostile environments such as hypoxia and acute interruptions in nutrient availability, cancer cells typically develop metabolic plasticity, which enables the utilization of available nutrients as bioenergetic substrates. This metabolic flexibility allows maintained ATP production under varying physiological and pathological conditions and is primarily regulated by substrate concentration, hormone levels, blood flow, oxygen supply, and workload(6).Cancer-associated changes in cellular metabolism may also be a direct consequence of oncogenic signal transduction.Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, partly due to the lack of targeted therapy available. Cancer cells heavily reprogram their metabolism and acquire metabolic plasticity to satisfy the high-energy demand due to uncontrolled proliferation. Accumulating evidence shows that deregulated lipid metabolism affects cancer cell survival, and therefore we sought to understand the function of fatty acid binding protein 7 (FABP7), which is expressed predominantly in TNBC tissues. As FABP7 was not detected in the TNBC cell lines tested, Hs578T and MDA-MB-231 cells were transduced with lentiviral particles containing either FABP7 open reading frame or red fluorescent protein. During serum starvation, when lipids were significantly reduced, FABP7 decreased the viability of Hs578T, but not of MDA-MB-231, cells. FABP7overexpressing Hs578T (Hs-FABP7) cells failed to efficiently utilize other available bioenergetic substrates such as glucose to sustain ATP production, which led to S/G2 phase arrest and cell death. We further showed that this metabolic phenotype was mediated by PPAR- signaling, despite the lack of fatty acids in culture media, as Hs-FABP7 cells attempted to survive. This study provides imperative evidence of metabolic vulnerabilities driven by FABP7 via PPAR- signaling.-Kwong, S. C., A. H. A. Jamil, A. Rhodes, N. A. Taib, and I. Chung. Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling. J. Lipid Res. 2019. 60: 1807-1817.Supplementary key words fatty acid binding protein • peroxisome proliferator-activated receptor alpha • cancer • nutrient deprivation • fatty acid metabolism • metabolic adaptation Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptors\n\nAKT activation augments the Warburg effect and renders the cancer cells addicted to glucose (7). Myc protein promotes mitochondrial glutaminolysis and glutamine addiction by shunting glucose away from mitochondrial metabolism (8). Cancer cells that fail to demonstrate metabolic flexibility during nutrient deprivation could become vulnerable in their survival (9). Hence, metabolic vulnerabilities exhibited by cancer cells can be targeted for cancer management (6,10).\n\nAlterations in lipid-and cholesterol-associated pathways encountered in tumors are now increasingly recognized and more frequently described (11,12), but not completely understood. Many cancer types show a strong lipid avidity, in which increasing uptake of exogenous lipid sources (13) or overactivation of endogenous lipid synthesis (14) is observed. Fatty acid binding protein 7 (FABP7), a member of the FABP intracellular lipid chaperone family, has been shown to be upregulated in TNBC compared with other breast cancer subtypes (15,16). FABPs regulates lipid metabolism by increasing fatty acid uptake (17), fatty acid oxidation (FAO) (18), and lipolysis (19). Yet, in TNBC, correlation of FABP7 expression and disease prognosis is debatable. While one study showed that FABP7 expression correlates with lower overall and recurrence-free survival (20), several have shown that FABP7-positive basal tumors (synonymous with TNBC) are associated with better prognosis (21,22). It is unclear, at this point, how FABP7-governed pathways impact the survival of TNBC.\n\nIn this study, we explored the roles of FABP7 in adaptation to nutrient depletion in TNBC cell lines. We showed that overexpression of FABP7 decreased the viability of Hs578T cells during serum starvation. This led to cell-cycle arrest and a significant increase in cell death. We further showed that this phenotype was mediated by PPAR-regulated genes.\n\n\nMATERIALS AND METHODS\n\n\nCell culture\n\nAll cell lines used in this study were purchased from American Type Culture Collection (Gaithersburg, MD). They were cultured in medium according to the manufacturer's protocol and supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), unless specified otherwise. Hs578T, MCF7, MDA-MB-231, and MDA-MB-435S were maintained in DMEM high glucose while BT474 was cultured in MEM. The cell lines were incubated at 37°C and 5% CO 2 atmosphere.\n\nFor nutrient-deprivation experiments, the cells were first seeded in complete medium (10% FBS). The next day (hereafter referred to as 0 h), the cells were washed once with PBS and replaced with nutrient-deprived medium. For the glucose-and glutamine-starvation experiments, 10% FBS were added into the basal medium. Basal medium used for glucose starvation was glucose-free DMEM (Life Technologies, catalog no. 11966-025) that contained 4 mM l-glutamine. For the glutamine-starvation experiment, glutamine-free DMEM (Life Technologies, catalog no. 11960-044) containing 25 mM glucose was used. The serumstarvation experiment mimicked a culture condition with deprived lipids. The cells were challenged with serum-free DMEM (Life Technologies, catalog no. 11965-092) that contains high glucose (25 mM) with l-glutamine (4 mM), but without sodium pyruvate, HEPES, lipids, and growth factors.\n\nThe PPAR- antagonist (GW6471) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a nonlethal concentration (2 M).\n\n\nData mining at GEO database\n\nA microarray profile of selected TNBC was obtained from the publicly available Gene Expression Omnibus (GEO) database. The search keyword used was \"triple negative breast cancer,\" and the results were filtered with \"Homo sapiens.\" The search was conducted from September 1 to 30, 2018.\n\n\nGeneration of FABP7-transduced TNBC cell lines\n\nTNBC cell lines Hs578T and MDA-MB-231 were transduced with FABP7 gene using Thermo Scientific Precision LentiORF constructs at MOI 10 (Clone ID: PLOHS_100004067). For their control counterparts, the cells were transduced with Precision LentiORF RFP (catalog no. OHS5833). The transduced cells were selected with blasticidin for 30 days to generate stable cell line.\n\n\nMTT assay\n\nCells were seeded in 96-well plates and subjected to different growth conditions. At time of termination, 10 l of methyl thiazolyl tetrazolium (MTT) solution (5 mg/ml) (Sigma, St. Louis, MO) was added into each well for 4 h, before the addition of 100 l of 10% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals overnight. Absorbance was measured at 575 nm with reference to 650 nm. All experiments were performed in triplicate and repeated three times.\n\n\nRNA extraction and qRT-PCR\n\nTotal RNA extraction was carried out using Trizol (Invitrogen, Carlsbad, CA) following the recommended protocol. About 500 ng of RNA was converted into cDNA using DyNAmo cDNA synthesis kit (Finnzymes, Vantaa, Finland). For quantitative RT-PCR (qRT-PCR), 5 ng/l cDNA template was added into pool solution containing 5× HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Tartu, Estonia), 10 pmol/l forward and reverse primers, and UltraPure distilled water. ABI StepOne Plus (Applied Biosystem, Foster City, CA) was used, and 40 cycles of amplification were performed. The expression of metabolic genes was normalized to housekeeping genes 18S rRNA and ribosomal protein L13a. Sequence of the primers used is described in supplemental Table S1.\n\n\nProtein extraction and Western blotting\n\nProtein lysate was harvested by scraping the cells in cold PBS. After centrifugation, PBS was discarded, and the cells were incubated in lysis buffer (0.1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1× phosphatase, and 1× protease inhibitors) for 30 min on ice. The mixture was centrifuged for 20 min, and supernatant was collected as protein lysate. Protein concentration was quantified using Bradford assay (Bio-Rad, Hercules, CA). A total of 30 g of protein was resolved on 15% SDS-PAGE prior to transferring on PVDF membrane. Target proteins were detected using primary Abs FABP7 (Cell Signaling Technology) and ECL prime Western blotting detection reagent (Amersham, GE Healthcare Lifesciences, Sweden) before being visualized with gel-documentation system (Biospectrum 410, UVP).\n\n\nBrdU cell-proliferation assay\n\nThe BrdU cell-proliferation assay kit (Cell Signaling Technology, Beverly, MA) was used to measure the cell-proliferation rate at 24, 48, and 72 h after serum starvation. The data were normalized to their counterparts cultured in complete medium. The cells were incubated with 10X BrdU solution for three h, prior to fixation for 30 min and staining with 1× BrdU Detection Antibody for 1 h. The stained cells were washed and incubated with 1× HRPconjugated secondary antibody solution for 30 min, before TMB solution was added. STOP solution was added after 15 min, and absorbance was read at 450 nm.\n\n\nCell-cycle analysis with propidium iodide\n\nCell-cycle analysis was conducted on the cells at 0, 24, 48, and 72 h after serum starvation. The cells were fixed with 70% ethanol at 4°C for 30 min, before two washes with PBS. Enzymatic removal of RNA was achieved by incubating the cells with 100 g/ml RNase for 15 min before DNA staining with 50 g/ml propidium iodide. The cells were analyzed using a BD FACSCanto II flow cytometer, and the cell-cycle distribution of the cell was analyzed with Modfit LT software. The samples were run in triplicates, and the mean value was calculated.\n\n\nAnnexin V apoptosis assay\n\nCells were harvested for apoptosis assay at 0, 24, 48, and 72 h after serum starvation. Floating cells in the culture medium and the trypsinized cells were collected and washed once with ice-cold PBS, before they were resuspended in 100 l of assay buffer (1 part buffer: 9 parts distilled water) with 5 l of annexin V-PE and 5 l of 7AAD (BD Bioscience, Franklin Lakes, NJ). The cells were vortexed gently and kept in the dark for 15 min, before adding 400 l of buffer. The staining was analyzed using a BD FACSCanto II flow cytometer and viewed using FACS DiVa Software (BD Bioscience, San Jose, CA). All samples were run in triplicates, and mean value was calculated.\n\n\nMetabolic activity assays\n\nGAPDH, glutaminase (GSL), and FAO enzyme activities were measured using calorimetric assay kits from Biomedical Research Service Center, State University of New York (Buffalo, NY). All samples were harvested using 1× Cell Lysis Solution. Protein concentration of the samples was assessed with a Bradford assay and normalized to 1 mg/ml. GAPDH activity was measured using GAPDH enzyme assay kit (catalog no. E-101). Briefly, 10 l of sample or water (as blank) was incubated in 50 l of GAPDH assay solution. After gentle agitation, the plate was kept in a non-CO 2 incubator at 37°C for 60 min. For GSL assay (catalog no. E-133), 10 l of sample was incubated in either 40 l of glutamine solution or water. Following a 2 h incubation period in a non-CO 2 incubator at 37°C, 50 l of TA assay solution was added, and the plate was further incubated for another 1 h. For FAO enzyme assay (catalog no. E-141), 50 l of FAO Assay Solution or control solution was added to 10 l of the protein sample. The plate was kept in a non-CO 2 incubator at 37°C for 60 min. All experiments were terminated by adding 50 l of 3% acetic acid, and the plate was read at optical density of 492 nm (OD 492) with a spectrophotometer. Blank reading was subtracted from the sample reading. GAPDH activity in IU/l unit was determined by multiplying OD by 16.98. Glutaminase (GLS) activity in IU/l unit = mol/ (l·min) = (OD × 1,000 × 150 l) ÷ (120 min × 0.6 cm × 18 × 10 l) = OD × 11.58. For FAO assay, the subtracted OD represents the FAO activity of the sample.\n\n\nMeasurement of OCR\n\nBasal oxygen consumption rate (OCR) was carried out with a Mito Fuel Flex Test Kit on XFe96 Bioanalyzer (Agilent). Briefly, 4,000 cells were seeded on a Seahorse XF96-well assay plate in complete medium. The cells grown in complete medium were terminated at 24 h after seeding. For the serum-starvation experiment, the cells were left incubated overnight before they were washed with PBS and incubated in serum-free medium for 24 h. Upon termination, all wells were replaced with assay medium, and baseline OCR was immediately measured for 18 min. The basal OCR was normalized to protein concentration of the cells.\n\n\nImmunofluorescence staining\n\nThe cells were seeded on a coverslip in complete medium. After overnight incubation, the cells were either harvested or challenged with serum-free medium for 24 h. The cells were fixed with 4% formaldehyde for 30 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 1 h. The cells were incubated with FABP7 primary antibody (1:2,000; Cell Signaling catalog no. 13347S) and PPAR- primary antibody (1:400; Santa Cruz Biotechnology, catalog no. sc-398394), followed by Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific). The incubation time was 90 min for primary antibody and 60 min for secondary antibody. The cells were washed three times with PBS after staining with each antibody. All the procedures were carried out on ice. The coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories Cat#H-1200) and analyzed with a Leica TCS SP5 II laser microscope (Leica, Heidelberg, Germany) using 60× objective.\n\n\nStatistical analysis\n\nStatistical analysis assessing the difference between the means of two groups was performed using Student's t-test with Graph-Pad-Prism (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.\n\n\nRESULTS\n\n\nFABP7 overexpression decreased viability of Hs578T, but not MDA-MB-231, cells in serum starvation\n\nThe expression of FABP7 was determined in human breast cancer tissues and TNBC cell lines. Analysis from the GEO database (dataset GSE65194) revealed that FABP7 expression was significantly higher in TNBC (n = 55) compared with non-TNBC (n = 98) subtypes (Fig. 1A). When examined in a panel of breast cancer cell lines, FABP7 showed low expression in all cells including MCF7 (luminal A), BT474 (luminal B), MDA-MB-231, and Hs578T (TNBC) cells, when compared with MDA-MB-435S, a melanoma cell line reported to express high FABP7 (Fig. 1B). Correspondingly, FABP7 protein was not detected in these breast cancer cell lines (Fig. 1C).\n\nTo understand the functional role of FABP7 in TNBC cells, we established FABP7-overexpressing Hs578T and MDA-MB-231 TNBC cells by transduction using lentiviral particles containing FABP7 open reading frame (Hs-FABP7 and 231-FABP7, respectively) and their control counterparts using lentiviral particles containing red fluorescent protein (Hs-RFP and 231-RFP, respectively) (Fig. 1D). When the cells were cultured in complete medium for 72 h, there was no difference in cell growth between cells expressing FABP7 and their respective RFP controls (Fig. 2). However, when cultured in glucose-or glutamine-deprived condition, both FABP7-overexpressing cells and RFP controls demonstrated substantial reduction in cell viability with greater effect observed in glucose-deprived than glutamine-deprived medium ( Fig. 2A,B).\n\nInterestingly, when lipids were significantly reduced during serum starvation, overexpression of FABP7 resulted in decreased viability of Hs578T cells (Fig. 2C). At 24 h after serum starvation, the cell viability of Hs-FABP7 cells decreased by 10%, which progressively decreased to approximately 70% at 72 h after starvation (P < 0.0001 vs. Hs-RFP cells). Cell viability of Hs-RFP cells was not affected by serum starvation, indicating that the decreased viability in Hs-FABP7 cells may be related to FABP7 expression. This phenotype appeared to be specific to Hs578T cells, as FABP7 expression did not affect the viability of MDA-MB-231 cells in serum-free medium (Fig. 2C).\n\n\nDecreased viability of Hs-FABP7 cells in serum starvation was due to reduced proliferation, cell-cycle arrest, and cell death\n\nThe reduction in cell viability of Hs-FABP7 cells during serum starvation resulted from a decrease in cell proliferation. Hs-FABP7 cells demonstrated a 70% reduction in BrdU uptake as early as 24 h after serum starvation, whereas the control Hs-RFP cells only showed a decrease by 20% (Fig. 3A). The nonresponsive MDA-MB-231 cells maintained their proliferation rate in the range of 85-89% during serum starvation, regardless of FABP7 expression.\n\nFlow-cytometry analysis showed that FABP7 induced cellcycle arrest in Hs578T cells during serum starvation ( Fig. 3B and supplemental Fig. S1). Hs-RFP cells showed a higher distribution of cells (80%) in G1 phase throughout serum starvation, indicating that the cells were able to complete the cell cycle and proliferate, parallel to increased cell viability over time. In contrast, Hs-FABP7 cells demonstrated a consistently higher percentage of cells in S and G2 phase upon serum starvation. For instance, at 48 h after serum starvation, 24% of Hs-FABP7 cells were accumulated in S phase and 8.23% in G2 phase, whereas only 5.15% and 1.99% of Hs-RFP cells were detected at S and G2 phase (P = 0.0052 for S phase and P = 0.0241 for G2 phase). The percentage of cells in S/G2 phase increased to 40.15% in Hs-FABP7 cells compared with 14.29% in Hs-RFP cells at 72 h after serum starvation. Cell arrest in S/G2 phase in Hs-FABP7 cells may partly contribute to the decreased cell viability during serum starvation. This effect was not observed in MDA-MB-231 cells (Fig. 3B).\n\nThe decrease in Hs-FABP7 cell viability during serum starvation was potentially attributed to apoptosis and necrosis (Fig. 3C). More necrotic cell death and late apoptosis was observed in Hs-FABP7 cells (34.26% and 21.3%) than in Hs-RFP cells (18.83% and 4.63%) at 72 h after serum starvation (P < 0.0001). Upon serum starvation, increasing cell death was observed in MDA-MB-231 cells, but with no significant difference observed between 231-RFP and 231-FABP7 cells. Taken together, the decreased cell viability in Hs-FABP7 cells during serum starvation was due to reduced proliferation rate, cell cycle arrest at S and G2 phase, and cell death.\n\n\nPerturbations in the metabolism and mitochondrial oxygen consumption in Hs-FABP7 cells during serum starvation\n\nGiven the role of FABP7 in fatty acid metabolism, we investigated the mechanism of FABP7-induced cell death by exploring a panel of genes and enzyme activities involved in the metabolism of glucose, glutamine, and fatty acid ( Fig. 4 and supplemental Figs. S2, S3). In complete medium, despite the increase in glucose transporter 1 (GLUT1) mRNA expression, glycolysis-related genes phosphofructokinase 1 (PFK1) and GAPDH remained unchanged in Hs-FABP7 cells, whereas the genes regulating FAO, carnitine palmitoyltransferase 1A (CPT1A), and medium-chain acyl-CoA dehydrogenase (MCAD) were markedly upregulated (Fig. 4A). In Hs-FABP7 cells, the expression of GLS1, encoding the rate-limiting enzyme for glutaminolysis, was not significantly different than in Hs-RFP cells. When cultured in complete medium, Hs-FABP7 cells did not show any differences in GAPDH, GLS, and FAO activities when compared with Hs-RFP cells 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001. ( Fig. 4B-D). Subsequently, mitochondrial OCR was similar in Hs-FABP7 cells and in the control Hs-RFP cells (Fig. 4E).\n\nHowever, when challenged with serum starvation, a widespread downregulation of glucose and glutamine metabolism-related genes were observed in Hs-FABP7 cells when compared with Hs-RFP cells. Interestingly, despite the absence of serum (lipids) during serum starvation, the gene expression of CPT1A and MCAD was about 2-fold higher in Hs-FABP7 cells than Hs-RFP cells (Fig. 4A). Serum starvation generally decreased glycolytic enzyme activity in Hs578T cells, but FABP7-overexpressing cells had a further 30% reduction (P = 0.0121) in GAPDH activity compared with Hs-RFP cells (Fig. 4B). Although GLS activity remained not significantly different in both cells during serum starvation, GLS activity was significantly lower (P = 0.0296) in Hs-FABP7 cells cultured in serum starvation than in complete medium (Fig. 4C). Interestingly, FAO activity in Hs-FABP7 remained similar with the control Hs-RFP cells (Fig. 4D), despite upregulation of FAO-related genes. This may be explained by the lack of fatty acid in the culture medium to activate the metabolic pathway. Consequently, mitochondrial oxidative capacity of both Hs-RFP and Hs-FABP7 cells was generally lower during serum starvation (P = 0.0001 for both cells); however, Hs-FABP7 cells had much lower OCR (16.69 pmol/min/µg) compared with Hs-RFP cells (21.7 pmol/min/µg) (P < 0.05) (Fig. 4E). These data indicate a lower mitochondrial oxidative capacity particularly in Hs-FABP7 cells, probably due to limited bioenergetic substrates during serum starvation.\n\nIt is worthwhile to note that, unlike Hs578T, MDA-MB-231 cells showed a rather different metabolic profile, as indicated by the expression of metabolic genes ( Fig. 4A and supplemental Fig. S2B). When cultured in complete medium, although FABP7 was not affecting the cell viability, glycolysis was upregulated in 231-FABP7 cells, with about a 1.7-fold increase in GAPDH expression. Meanwhile, a 4-fold decrease in GLS1 expression was observed, indicative of a downregulation of the glutaminolysis pathway. Upon serum starvation, 231-FABP7 cells demonstrated a marked increase in glucose and glutamine metabolismrelated genes, with more than 4-fold increase in GLUT1 and GLS1 expression.\n\nTaken together, our data show that Hs-FABP7 cells, when challenged with a serum-starved condition, were not able to metabolically adapt and effectively utilize other available energetic substrates, especially glucose, and, as a consequence, could not survive.\n\n\nPPAR- signaling mediated FABP7-induced cell death in Hs578T cells under serum starvation\n\nThe upregulation of CPT1A and MCAD mRNA, accompanied with a downregulation of Glut1 mRNA in Hs-FABP7 cells, indicated a possible role of PPAR- signaling, as these genes are direct targets of PPAR- transcription factor. Immunofluorescence staining showed that the expression of PPAR- protein was increased in the nucleus during serum starvation ( Fig. 5A and supplemental Fig. S4A). Similarly, FABP7 protein was observed to translocate into the nucleus (Fig. 5A and supplemental Fig. S4B) in Hs-FABP7 cells upon serum starvation, potentially acting as fatty acid chaperones to deliver ligands to PPAR- protein (23). This suggests that nuclear localization of FABP7 enhanced the action of PPAR- transcriptional activity during serum starvation in Hs-FABP7 cells.\n\nTo investigate whether PPAR- activation was responsible for FABP7-mediated inhibition of cell viability in Hs-FABP7 cells, we treated the cells with a PPAR--specific inhibitor (GW6471). In both Hs578T and MDA-MB-231 cells, treatment of GW6471 at a concentration of 2 M in complete medium had no significant effect on cell viability (Fig. 5B). Yet, the same concentration of GW6471 reversed the cell-growth inhibition observed in Hs-FABP7 cells when exposed to serum-starved conditions (Fig. 5B and supplemental Fig. S5). Subsequent gene-expression analysis revealed that PPAR- target genes were downregulated (CPT1A: 2.43-fold; MCAD: 1.54-fold), when compared with vehicle-treated cells (Fig. 5C). A significant 2-fold increase of GLUT1, PFK1, glucose-6-phosphate dehydrogenase (G6PD), and isocitrate dehydrogenase 1 (IDH1) mRNA expression was observed in GW6471-treated Hs-FABP7 cells (Fig. 5C), indicating that the rescue of cell viability may be due to the switch from lipid to glucose as bioenergetic substrates in these cells. Taken together, FABP7-induced cell death in Hs578T cells during serum starvation is due to activated PPAR- signaling.\n\n\nDISCUSSION\n\nIn this study, we showed that overexpression of FABP7 disabled TNBC cell line Hs578T to metabolically adapt during serum starvation. FABP7-overexpressing cells failed to efficiently utilize glucose as bioenergetics substrates, when challenged with serum starvation. This was accompanied by increased expression of genes controlling FAO as it attempts to utilize free fatty acids liberated by lipolysis from the lipid storage that resulted from preexposure to complete medium (24). However, as free fatty acid storage was depleted, serum-starved FABP7-overexpressing cells were not able to further metabolically adapt and sustain ATP production. The failure of the cells to efficiently utilize glucose for ATP generation leads to cell death. We further showed that the metabolic impact of FABP7 in Hs578T cells during serum starvation is mediated by PPAR- signaling.\n\nFABP7 is predominantly expressed in the nervous system and mammary gland. In the mammary gland, FABP7 inhibits proliferation and promotes differentiation of mammary cells (25). Similarly, in the nervous system, FABP7 is involved in the differentiation of neurons and development of the cortex (26,27). The expression of FABP7 at the edge of astrocytes also suggested that FABP7 may regulate the motility of astrocytes by increasing the uptake of polyunsaturated fatty acids (28). Yet, the evidence of FABP7 expression in TNBC cell lines was rather limited (20,29). A previous study demonstrating the function of endogenous FABP7 in TNBC was reported using MDA-MB-435S, a melanoma cell line formerly misidentified as a TNBC cell line (30). Our study, however, provides evidence on the role of FABP7 as a mediator for tumor cell death during nutrient-deprived conditions. This finding indicates an anti-tumorigenic role of FABP7 in TNBC, which is mediated by specific nutrient availability.\n\nThe metabolic vulnerabilities of FABP7-overexpressing Hs578T cells resulted in S/G2-phase arrest, which subsequently led to cell death. Serum starvation typically induces cell-cycle arrest at G1 phase (31,32), which is observed in Hs-RFP and MDA-MB-231 cells. These cells were able to proliferate, despite a lower proliferation rate, indicating that the lower mitochondrial activity in these cells during serum starvation was sufficient to overcome the metabolic checkpoint and complete the cell cycle (33). Hs-FABP7 cells, however, arrested at the S/G2 phase. G2 arrest has been described as another growth-restricting mechanism in mitogen-starved conditions, likely due to elevated E2F activity (34). Cell arrest could potentially lead to cell death, in which both apoptosis and necrosis were observed in Hs-FABP7 cells upon serum starvation. Apoptosis is a cellular suicide program triggered by stress signals such as nutrient deprivation, which results in the execution of caspasemediated cell death (35). Serum starvation has been shown to induce apoptosis in hepatocellular carcinoma cells (36) and MCF7 breast cancer cells (37), in which the latter was associated with an altered ratio of apoptosis regulating proteins Bax and Bcl-2. Necrosis, on the other hand, may occur when the minimal bioenergetic demands are not met (38), a condition similar to our observations where Hs-FABP7 cells may have limited bioenergetic substrates during serum starvation. In fact, there are common mediators of apoptosis and necrosis, which could potentially contribute to the two modes of cell death observed in our model (39). It is worth noting that FABP7 did not induce cell death in MDA-MB-231 cells during serum starvation, indicating that this cell line is probably more resilient to nutrient deprivation than Hs578T cells. The disparity in the response of Hs578T and MDA-MB-231 cells to serum starvation may also be attributed to differences in their metabolic profiles (40). Hence, it would be worthwhile to examine other TNBC cells with differing metabolic profiles, in delineating the molecular heterogeneity involved in cell death induced by metabolic vulnerabilities.\n\nDuring serum starvation, cancer cells sense and adapt to nutrient deprivation by upregulating glucose metabolism Fig. 5. FABP7-induced cell death in Hs578T cells during serum starvation was mediated by the action of PPAR- signaling. A: Confocal microscopy of immunofluorescence staining of FABP7 (green) and PPAR- (red) proteins in Hs578T and MDA-MB-231 cells after culture in either complete medium or serum-free medium for 24 h. Blue, DAPI. Scale bar, 50 m. Data shown are representative of two independent experiments. B: Cells were treated with PPAR- inhibitor GW6471 (2 M) in complete or serum-starved medium for 72 h, before analyzing for cell viability using an MTT assay. Data shown are mean ± SEM of triplicates; this is a representative of three independent experiments. *** P < 0.0001. C: mRNA expression of key metabolic genes after treatment with 2 M GW6471 in Hs-FABP7 cells for 24 h in serum starvation when compared with vehicle-treated cells. The genes were normalized to housekeeping gene 18S rRNA. CM, complete medium; SFM, serum-free medium. (41,42), besides releasing fatty acids from the lipid droplet through lipolysis for oxidation (24). Central to such adaption, PPAR- is known to be a lipid sensor that maintains cellular energy homeostasis. It is a ligand-activated class of nuclear hormone receptor that binds to peroxisome proliferator response elements (PPREs) to induce transcription of genes such as lipid processing. Its activation aims at increasing lipid combustion in order to yield more energy production, especially in a challenged environment (43). It was shown in rat hepatocytes that PPAR- expression is increased by fasting-and starvation-induced glucocorticoids (44), suggesting the role of PPAR- to maintain cell survival.\n\nIn our model, increased PPAR- expression was observed when the cells were serum-starved, possibly as an adaptation strategy of the cells. The activation of PPAR- was observed in Hs-FABP7 cells during serum starvation, as reflected in the increase of PPAR- target genes, CPT1A and MCAD, which are involved in fatty acid mitochondrial uptake and oxidation (45). Our observation may indicate the usage of fatty acids liberated from lipid droplets as the substrates for FAO. However, the attempt of the cells to survive serum starvation failed as the lipid storage was limited and fatty acids were absent in the serum-free media. In addition, PPAR- activation also potentially inhibited glucose metabolism by targeting the consensus PPRE motif in the promoter region of Glut1, hence repressing its transcription (46). As a result, Hs-FABP7 cells could not efficiently utilize glucose as bioenergetic substrates when serumstarved, which eventually led to cell death.\n\nOur data suggest that PPAR--induced changes are FABP7-dependent. Literature evidence shows that in Cos-7 cells where FABPs and PPAR- are not detected, transfection of the proteins showed that PPAR- activity is amplified by FABP1 and FABP2 (47). In another study, PPAR- transactivation is proportional to levels of L-FABP in human hepatoma HepG2 cells line (48). In our model, upon serum starvation, increased nuclear FABP7 expression was observed, suggesting the role of FABP7 as fatty acids chaperones to transport PPAR- ligands into the nucleus for PPAR- activation. In murine L cells, transduction with FABP1 had dramatically enhanced the uptake of fatty acid into the nucleus (49). Incubation of rat liver nuclei with fluorescence-tagged FABP1 together with fatty acids demonstrated a strong fluorescence response in the nuclei, which otherwise did not happen when fatty acids were absent (50). There has been evidence indicating physical interaction between FABP1 and PPAR- in primary hepatocytes (51). Ablation of L-FABP gene expression significantly impaired fatty acid distribution in the nucleus and affected PPAR- activation in these cells (52). Hence, whether FABP7 and PPAR- require direct contact to induce cell death in TNBC needs to be further investigated. In short, our data suggest that FABP7 facilitates PPAR- activation in serum-starved condition by transporting its ligand (fatty acids) into the nucleus, before enhancing its transcriptional activity.\n\nOur study provides an understanding on the role of FABP7 in regulating the metabolic adaptation of TNBC cell lines. Transcriptional activation of PPAR- in FABP7overexpressing cell can limit their metabolic plasticity when challenged with serum starvation and resulted in reduced survival of these cells. Hence, manipulating metabolic stress in cells expressing FABP7 protein may potentially be an effective targeted therapeutic strategy in FABP7-positive TNBC patients.\n\nFig. 1 .\n1Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.\n\nFig. 2 .\n2The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\n\nFig.\nFig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.\n\nFig. 4 .\n4FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\n\nTriplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. S K Kanapathy Pillai, A Tay, S Nair, C-O Leong, BMC Clin. 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Real time confocal and multiphoton fluorescence imaging in living cells. H Huang, O Starodub, A Mcintosh, A B Kier, F Schroeder, J. Biol. Chem. 277Huang, H., O. Starodub, A. McIntosh, A. B. Kier, and F. Schroeder. 2002. Liver fatty acid-binding protein targets fatty acids to the nu- cleus. Real time confocal and multiphoton fluorescence imaging in living cells. J. Biol. Chem. 277: 29139-29151.\n\nLigand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J W Lawrence, D J Kroll, P I Eacho, J. Lipid Res. 41Lawrence, J. W., D. J. Kroll, and P. I. Eacho. 2000. Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J. Lipid Res. 41: 1390-1401.\n\nL-FABP directly interacts with PPAR in cultured primary hepatocytes. H A Hostetler, A L Mcintosh, B P Atshaves, S M Storey, H R Payne, A B Kier, F Schroeder, J. Lipid Res. 50Hostetler, H. A., A. L. McIntosh, B. P. Atshaves, S. M. Storey, H. R. Payne, A. B. Kier, and F. Schroeder. 2009. L-FABP directly inter- acts with PPAR in cultured primary hepatocytes. J. Lipid Res. 50: 1663-1675.\n\nLiver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes. A L Mcintosh, B P Atshaves, H A Hostetler, H Huang, J Davis, O I Lyuksyutova, D Landrock, A B Kier, F Schroeder, Arch. Biochem. Biophys. 485McIntosh, A. L., B. P. Atshaves, H. A. Hostetler, H. Huang, J. Davis, O. I. Lyuksyutova, D. Landrock, A. B. Kier, and F. Schroeder. 2009. Liver type fatty acid binding protein (L-FABP) gene ablation re- duces nuclear ligand distribution and peroxisome proliferator- activated receptor- activity in cultured primary hepatocytes. Arch. Biochem. Biophys. 485: 160-173.\n"},"annotations_abstract":{"kind":"list like","value":["ERs) and progesterone receptors (PRs), as well as human epidermal growth factor receptor 2 (HER2). It is the most aggressive subtype of breast cancer, affecting about 15-20% of total breast cancer cases. Compared with other subtypes, TNBC tumors are associated with higher histologic grade and larger tumor size (1, 2). The current standard regimen for TNBC patients is a combination of surgery and chemotherapy (3), which often fails to effectively slow down the tumor progression. Hence, TNBC patients have lower overall survival (81% vs. 91%) and disease-free survival (72% vs. 86%) compared with non-TNBC patients (4). Given the lack of ER and HER2 in this subtype, there is no molecular characterization that allows this subtype to be targeted.Uncontrolled proliferation of cancer cells is metabolically demanding. The metabolic pathways utilized by cancer cells to sustain high-energy demands and biosynthesis differ from those employed by healthy cells (5). Challenged by hostile environments such as hypoxia and acute interruptions in nutrient availability, cancer cells typically develop metabolic plasticity, which enables the utilization of available nutrients as bioenergetic substrates. This metabolic flexibility allows maintained ATP production under varying physiological and pathological conditions and is primarily regulated by substrate concentration, hormone levels, blood flow, oxygen supply, and workload(6).Cancer-associated changes in cellular metabolism may also be a direct consequence of oncogenic signal transduction.Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, partly due to the lack of targeted therapy available. Cancer cells heavily reprogram their metabolism and acquire metabolic plasticity to satisfy the high-energy demand due to uncontrolled proliferation. Accumulating evidence shows that deregulated lipid metabolism affects cancer cell survival, and therefore we sought to understand the function of fatty acid binding protein 7 (FABP7), which is expressed predominantly in TNBC tissues. As FABP7 was not detected in the TNBC cell lines tested, Hs578T and MDA-MB-231 cells were transduced with lentiviral particles containing either FABP7 open reading frame or red fluorescent protein. During serum starvation, when lipids were significantly reduced, FABP7 decreased the viability of Hs578T, but not of MDA-MB-231, cells. FABP7overexpressing Hs578T (Hs-FABP7) cells failed to efficiently utilize other available bioenergetic substrates such as glucose to sustain ATP production, which led to S/G2 phase arrest and cell death. We further showed that this metabolic phenotype was mediated by PPAR- signaling, despite the lack of fatty acids in culture media, as Hs-FABP7 cells attempted to survive. This study provides imperative evidence of metabolic vulnerabilities driven by FABP7 via PPAR- signaling.-Kwong, S. C., A. H. A. Jamil, A. Rhodes, N. A. Taib, and I. Chung. Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling. J. Lipid Res. 2019. 60: 1807-1817.Supplementary key words fatty acid binding protein • peroxisome proliferator-activated receptor alpha • cancer • nutrient deprivation • fatty acid metabolism • metabolic adaptation Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptors"],"string":"[\n \"ERs) and progesterone receptors (PRs), as well as human epidermal growth factor receptor 2 (HER2). It is the most aggressive subtype of breast cancer, affecting about 15-20% of total breast cancer cases. Compared with other subtypes, TNBC tumors are associated with higher histologic grade and larger tumor size (1, 2). The current standard regimen for TNBC patients is a combination of surgery and chemotherapy (3), which often fails to effectively slow down the tumor progression. Hence, TNBC patients have lower overall survival (81% vs. 91%) and disease-free survival (72% vs. 86%) compared with non-TNBC patients (4). Given the lack of ER and HER2 in this subtype, there is no molecular characterization that allows this subtype to be targeted.Uncontrolled proliferation of cancer cells is metabolically demanding. The metabolic pathways utilized by cancer cells to sustain high-energy demands and biosynthesis differ from those employed by healthy cells (5). Challenged by hostile environments such as hypoxia and acute interruptions in nutrient availability, cancer cells typically develop metabolic plasticity, which enables the utilization of available nutrients as bioenergetic substrates. This metabolic flexibility allows maintained ATP production under varying physiological and pathological conditions and is primarily regulated by substrate concentration, hormone levels, blood flow, oxygen supply, and workload(6).Cancer-associated changes in cellular metabolism may also be a direct consequence of oncogenic signal transduction.Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, partly due to the lack of targeted therapy available. Cancer cells heavily reprogram their metabolism and acquire metabolic plasticity to satisfy the high-energy demand due to uncontrolled proliferation. Accumulating evidence shows that deregulated lipid metabolism affects cancer cell survival, and therefore we sought to understand the function of fatty acid binding protein 7 (FABP7), which is expressed predominantly in TNBC tissues. As FABP7 was not detected in the TNBC cell lines tested, Hs578T and MDA-MB-231 cells were transduced with lentiviral particles containing either FABP7 open reading frame or red fluorescent protein. During serum starvation, when lipids were significantly reduced, FABP7 decreased the viability of Hs578T, but not of MDA-MB-231, cells. FABP7overexpressing Hs578T (Hs-FABP7) cells failed to efficiently utilize other available bioenergetic substrates such as glucose to sustain ATP production, which led to S/G2 phase arrest and cell death. We further showed that this metabolic phenotype was mediated by PPAR- signaling, despite the lack of fatty acids in culture media, as Hs-FABP7 cells attempted to survive. This study provides imperative evidence of metabolic vulnerabilities driven by FABP7 via PPAR- signaling.-Kwong, S. C., A. H. A. Jamil, A. Rhodes, N. A. Taib, and I. Chung. Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling. J. Lipid Res. 2019. 60: 1807-1817.Supplementary key words fatty acid binding protein • peroxisome proliferator-activated receptor alpha • cancer • nutrient deprivation • fatty acid metabolism • metabolic adaptation Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptors\"\n]"},"annotations_author":{"kind":"list like","value":["Soke Chee Kwong \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n","Amira Hajirah ","Abd Jamil ","Anthony Rhodes ","Nur Aishah Taib ","§ ","Ivy Chung ivychung@ummc.edu.my \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n","\nFaculty of Medicine\nDepartments of Pharmacology\nthe Translational Core Laboratory\nUniversity of Malaya\n\n"],"string":"[\n \"Soke Chee Kwong \\nFaculty of Medicine\\nSchool of Medicine\\nFaculty of Health and Medical Sciences\\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\\nUniversity of Malaya\\n50603Kuala LumpurMalaysia;\\n\\n§ Taylor's University\\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\\n\",\n \"Amira Hajirah \",\n \"Abd Jamil \",\n \"Anthony Rhodes \",\n \"Nur Aishah Taib \",\n \"§ \",\n \"Ivy Chung ivychung@ummc.edu.my \\nFaculty of Medicine\\nSchool of Medicine\\nFaculty of Health and Medical Sciences\\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\\nUniversity of Malaya\\n50603Kuala LumpurMalaysia;\\n\\n§ Taylor's University\\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\\n\",\n \"\\nFaculty of Medicine\\nDepartments of Pharmacology\\nthe Translational Core Laboratory\\nUniversity of Malaya\\n\\n\"\n]"},"annotations_authoraffiliation":{"kind":"list like","value":["Faculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;","§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia","Faculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;","§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia","Faculty of Medicine\nDepartments of Pharmacology\nthe Translational Core Laboratory\nUniversity of Malaya\n"],"string":"[\n \"Faculty of Medicine\\nSchool of Medicine\\nFaculty of Health and Medical Sciences\\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\\nUniversity of Malaya\\n50603Kuala LumpurMalaysia;\",\n \"§ Taylor's University\\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\",\n \"Faculty of Medicine\\nSchool of Medicine\\nFaculty of Health and Medical Sciences\\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\\nUniversity of Malaya\\n50603Kuala LumpurMalaysia;\",\n \"§ Taylor's University\\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\",\n \"Faculty of Medicine\\nDepartments of Pharmacology\\nthe Translational Core Laboratory\\nUniversity of Malaya\\n\"\n]"},"annotations_authorfirstname":{"kind":"list like","value":["Soke","Amira","Abd","Anthony","Nur","Aishah","§","Ivy"],"string":"[\n \"Soke\",\n \"Amira\",\n \"Abd\",\n \"Anthony\",\n \"Nur\",\n \"Aishah\",\n \"§\",\n \"Ivy\"\n]"},"annotations_authorlastname":{"kind":"list like","value":["Chee Kwong","Hajirah","Jamil","Rhodes","Taib","Chung"],"string":"[\n \"Chee Kwong\",\n \"Hajirah\",\n \"Jamil\",\n \"Rhodes\",\n \"Taib\",\n \"Chung\"\n]"},"annotations_bibauthor":{"kind":"list like","value":["S K Kanapathy Pillai, ","A Tay, ","S Nair, ","C-O Leong, ","R Dent, ","M Trudeau, ","K I Pritchard, ","W M Hanna, ","H K Kahn, ","C A Sawka, ","L A Lickley, ","E Rawlinson, ","P Sun, ","S A Narod, ","E Rodler, ","L Korde, ","J Gralow, ","H G Kaplan, ","J A Malmgren, ","Y Zhang, ","J-M Yang, ","E Obre, ","R Rossignol, ","R L Elstrom, ","D E Bauer, ","M Buzzai, ","R Karnauskas, ","M H Harris, ","D R Plas, ","H Zhuang, ","R M Cinalli, ","A Alavi, ","C M Rudin, ","D R Wise, ","R J Deberardinis, ","A Mancuso, ","N Sayed, ","X-Y. 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S K Kanapathy Pillai, A Tay, S Nair, C-O Leong, BMC Clin. Pathol. 1218Kanapathy Pillai, S. K., A. Tay, S. Nair, and C-O. Leong. 2012. Triple- negative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. BMC Clin. Pathol. 12: 18.","Triple-negative breast cancer: clinical features and patterns of recurrence. R Dent, M Trudeau, K I Pritchard, W M Hanna, H K Kahn, C A Sawka, L A Lickley, E Rawlinson, P Sun, S A Narod, Clin. Cancer Res. 13Dent, R., M. Trudeau, K. I. Pritchard, W. M. Hanna, H. K. Kahn, C. A. Sawka, L. A. Lickley, E. Rawlinson, P. Sun, and S. A. Narod. 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res. 13: 4429-4434.","Current treatment options in triple negative breast cancer. E Rodler, L Korde, J Gralow, Breast Dis. 32Rodler, E., L. Korde, and J. Gralow. 2010. Current treatment op- tions in triple negative breast cancer. Breast Dis. 32: 99-122.","Impact of triple negative phenotype on breast cancer prognosis. H G Kaplan, J A Malmgren, Breast J. 14Kaplan, H. G., and J. A. Malmgren. 2008. Impact of triple negative phenotype on breast cancer prognosis. Breast J. 14: 456-463.","Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention. Y Zhang, J-M Yang, Cancer Biol. Ther. 14Zhang, Y., and J-M. Yang. 2013. Altered energy metabolism in can- cer: a unique opportunity for therapeutic intervention. Cancer Biol. Ther. 14: 81-89.","Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy. E Obre, R Rossignol, Int. J. Biochem. Cell Biol. 59Obre, E., and R. Rossignol. 2015. Emerging concepts in bioenerget- ics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bio- energetic therapy. Int. J. Biochem. Cell Biol. 59: 167-181.","Akt stimulates aerobic glycolysis in cancer cells. R L Elstrom, D E Bauer, M Buzzai, R Karnauskas, M H Harris, D R Plas, H Zhuang, R M Cinalli, A Alavi, C M Rudin, Cancer Res. 64Elstrom, R. L., D. E. Bauer, M. Buzzai, R. Karnauskas, M. H. Harris, D. R. Plas, H. Zhuang, R. M. Cinalli, A. Alavi, C. M. Rudin, et al. 2004. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 64: 3892-3899.","Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. D R Wise, R J Deberardinis, A Mancuso, N Sayed, X-Y. Zhang, H K Pfeiffer, I Nissim, E Daikhin, M Yudkoff, S B Mcmahon, Proc. Natl. Acad. Sci. USA. 105Wise, D. R., R. J. DeBerardinis, A. Mancuso, N. Sayed, X-Y. Zhang, H. K. Pfeiffer, I. Nissim, E. Daikhin, M. Yudkoff, S. B. McMahon, et al. 2008. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc. Natl. Acad. Sci. USA. 105: 18782-18787.","The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation. M Buzzai, D E Bauer, R G Jones, R J Deberardinis, G Hatzivassiliou, R L Elstrom, C B Thompson, Oncogene. 24Buzzai, M., D. E. Bauer, R. G. Jones, R. J. DeBerardinis, G. Hatzivassiliou, R. L. Elstrom, and C. B. Thompson. 2005. The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation. Oncogene. 24: 4165-4173.","Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies. K A Olson, J C Schell, J Rutter, Trends Biochem. Sci. 41Olson, K. A., J. C. Schell, and J. Rutter. 2016. Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies. Trends Biochem. Sci. 41: 219-230.","Lipid metabolic reprogramming in cancer cells. S Beloribi-Djefaflia, S Vasseur, F Guillaumond, Oncogenesis. 5189Beloribi-Djefaflia, S., S. Vasseur, and F. Guillaumond. 2016. Lipid metabolic reprogramming in cancer cells. Oncogenesis. 5: e189.","Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. F Baenke, B Peck, H Miess, A Schulze, Dis. Model. Mech. 6Baenke, F., B. Peck, H. Miess, and A. Schulze. 2013. Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour devel- opment. Dis. Model. Mech. 6: 1353-1363.","Exogenous lipids promote the growth of breast cancer cells via CD36. J Zhao, Z Zhi, C Wang, H Xing, G Song, X Yu, Y Zhu, X Wang, X Zhang, Y Di, Oncol. Rep. 38Zhao, J., Z. Zhi, C. Wang, H. Xing, G. Song, X. Yu, Y. Zhu, X. Wang, X. Zhang, and Y. Di. 2017. Exogenous lipids promote the growth of breast cancer cells via CD36. Oncol. Rep. 38: 2105-2115.","De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy. T Mashima, H Seimiya, T Tsuruo, Br. J. Cancer. 100Mashima, T., H. Seimiya, and T. Tsuruo. 2009. De novo fatty-acid synthesis and related pathways as molecular targets for cancer ther- apy. Br. J. Cancer. 100: 1369-1372.","Overexpression of fatty acid binding protein-7 correlates with basal-like subtype of breast cancer. X Y Tang, S Umemura, H Tsukamoto, N Kumaki, Y Tokuda, R Y Osamura, Pathol. Res. Pract. 206Tang, X. Y., S. Umemura, H. Tsukamoto, N. Kumaki, Y. Tokuda, and R. Y. Osamura. 2010. Overexpression of fatty acid binding pro- tein-7 correlates with basal-like subtype of breast cancer. Pathol. Res. Pract. 206: 98-101.","Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene. Y E Shi, J Ni, G Xiao, Y E Liu, A Fuchs, G Yu, J Su, J M Cosgrove, L Xing, M Zhang, MRG. Cancer Res. 57Shi, Y. E., J. Ni, G. Xiao, Y. E. Liu, A. Fuchs, G. Yu, J. Su, J. M. Cosgrove, L. Xing, M. Zhang, et al. 1997. Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene, MRG. Cancer Res. 57: 3084-3091.","Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. V L Spitsberg, E Matitashvili, R C Gorewit, Eur. J. Biochem. 230Spitsberg, V. L., E. Matitashvili, and R. C. Gorewit. 1995. Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. Eur. J. Biochem. 230: 872-878.","Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis. R E Burrier, C R Manson, P Brecher, Biochim. Biophys. Acta. 919Burrier, R. E., C. R. Manson, and P. Brecher. 1987. Binding of acyl- CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis. Biochim. Biophys. Acta. 919: 221-230.","Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. N R Coe, M A Simpson, D A Bernlohr, J. Lipid Res. 40Coe, N. R., M. A. Simpson, and D. A. Bernlohr. 1999. Targeted dis- ruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. J. Lipid Res. 40: 967-972.","A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer. R Z Liu, K Graham, D D Glubrecht, R Lai, J R Mackey, R Godbout, J. Pathol. 228Liu, R. Z., K. Graham, D. D. Glubrecht, R. Lai, J. R. Mackey, and R. Godbout. 2012. A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer. J. Pathol. 228: 310-321.","Fatty acid binding protein 7 expression and its subcellular localization in breast cancer. A T Alshareeda, E A Rakha, C C Nolan, I O Ellis, A R Green, Breast Cancer Res. Treat. 134Alshareeda, A. T., E. A. Rakha, C. C. Nolan, I. O. Ellis, and A. R. Green. 2012. Fatty acid binding protein 7 expression and its sub- cellular localization in breast cancer. Breast Cancer Res. Treat. 134: 519-529.","The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer. H Zhang, E A Rakha, G Ball, I Spiteri, M Aleskandarany, E Paish, D G Powe, R D Macmillan, C Caldas, I O Ellis, Breast Cancer Res. Treat. 121Zhang, H., E. A. Rakha, G. Ball, I. Spiteri, M. Aleskandarany, E. Paish, D. G. Powe, R. D. Macmillan, C. Caldas, I. O. Ellis, et al. 2010. The proteins FABP7 and OATP2 are associated with the basal phe- notype and patient outcome in human breast cancer. Breast Cancer Res. Treat. 121: 41-51.","Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration. R Mita, M J Beaulieu, C Field, R Godbout, J. Biol. Chem. 285Mita, R., M. J. Beaulieu, C. Field, and R. Godbout. 2010. Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration. J. Biol. Chem. 285: 37005-37015.","Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. A S Rambold, S Cohen, J Lippincott-Schwartz, Dev. Cell. 32Rambold, A. S., S. Cohen, and J. Lippincott-Schwartz. 2015. Fatty acid trafficking in starved cells: regulation by lipid droplet lipoly- sis, autophagy, and mitochondrial fusion dynamics. Dev. Cell. 32: 678-692.","Members of the fatty acid binding protein family are differentiation factors for the mammary gland. Y Yang, E Spitzer, N Kenney, W Zschiesche, M Li, A Kromminga, T Müller, F Spener, A Lezius, J H Veerkamp, J. Cell Biol. 127Yang, Y., E. Spitzer, N. Kenney, W. Zschiesche, M. Li, A. Kromminga, T. Müller, F. Spener, A. Lezius, J. H. Veerkamp, et al. 1994. Members of the fatty acid binding protein family are differentiation factors for the mammary gland. J. Cell Biol. 127: 1097-1109.","Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. S G Kuhar, L Feng, S Vidan, M E Ross, M E Hatten, N Heintz, Development. 117Kuhar, S. G., L. Feng, S. Vidan, M. E. Ross, M. E. Hatten, and N. Heintz. 1993. Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. Development. 117: 97-104.","Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex. Y Arai, N Funatsu, K Numayama-Tsuruta, T Nomura, S Nakamura, N Osumi, J. Neurosci. 25Arai, Y., N. Funatsu, K. Numayama-Tsuruta, T. Nomura, S. Nakamura, and N. Osumi. 2005. Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex. J. Neurosci. 25: 9752-9761.","Brain lipid binding protein (FABP7) as modulator of astrocyte function. M Kipp, T Clarner, S Gingele, F Pott, S Amor, P Van Der, C Valk, Beyer, Physiol. Res. 60Suppl. 1Kipp, M., T. Clarner, S. Gingele, F. Pott, S. Amor, P. van der Valk, and C. Beyer. 2011. Brain lipid binding protein (FABP7) as modula- tor of astrocyte function. Physiol. Res. 60 (Suppl. 1): S49-S60.","Fatty acid uptake and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation. K Bensaad, E Favaro, C A Lewis, B Peck, S Lord, J M Collins, K E Pinnick, S Wigfield, F M Buffa, J L Li, Cell Reports. 9Bensaad, K., E. Favaro, C. A. Lewis, B. Peck, S. Lord, J. M. Collins, K. E. Pinnick, S. Wigfield, F. M. Buffa, J. L. Li, et al. 2014. Fatty acid up- take and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation. Cell Reports. 9: 349-365.","MDA-MB-435 cells are from melanoma, not from breast cancer. M Lacroix, Cancer Chemother. Pharmacol. 63567Lacroix, M. 2009. MDA-MB-435 cells are from melanoma, not from breast cancer. Cancer Chemother. Pharmacol. 63: 567.","Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. J-S Shin, S-W Hong, S-L O Lee, T-H Kim, I-C Park, S -K. An, W K Lee, J S Lim, K I Kim, Y Yang, Int. J. Oncol. 32Shin, J-S., S-W. Hong, S-L. O. Lee, T-H. Kim, I-C. Park, S-K. An, W. K. Lee, J. S. Lim, K. I. Kim, Y. Yang, et al. 2008. Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. Int. J. Oncol. 32: 435-439.","Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro. Y Huang, Z Fu, W Dong, Z Zhang, J Mu, J Zhang, Mol. Med. Rep. 17Huang, Y., Z. Fu, W. Dong, Z. Zhang, J. Mu, and J. Zhang. 2018. Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro. Mol. Med. Rep. 17: 5057-5064.","Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression. S M Schieke, J P Mccoy, Jr , T Finkel, Cell Cycle. 7Schieke, S. M., J. P. McCoy, Jr., and T. Finkel. 2008. Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression. Cell Cycle. 7: 1782-1787.","Restriction beyond the restriction point: mitogen requirement for G2 passage. F Foijer, H Te Riele, Cell Div. 18Foijer, F., and H. te Riele. 2006. Restriction beyond the restriction point: mitogen requirement for G2 passage. Cell Div. 1: 8.","Metabolic regulation of apoptosis in cancer. K Matsuura, K Canfield, W Feng, M Kurokawa, Int. Rev. Cell. Mol. Biol. 327Matsuura, K., K. Canfield, W. Feng, and M. Kurokawa. 2016. Metabolic regulation of apoptosis in cancer. Int. Rev. Cell. Mol. Biol. 327: 43-87.","Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells. X Kou, Y Jing, W Deng, K Sun, Z Han, F Ye, G Yu, Q Fan, L Gao, Q Zhao, BMC Cancer. 13438Kou, X., Y. Jing, W. Deng, K. Sun, Z. Han, F. Ye, G. Yu, Q. Fan, L. Gao, Q. Zhao, et al. 2013. Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells. BMC Cancer. 13: 438.","Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells. Tovar Sepulveda, V A , X Shen, M Falzon, Endocrinology. 143Tovar Sepulveda, V. A., X. Shen, and M. Falzon. 2002. Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells. Endocrinology. 143: 596-606.","Metabolic catastrophe as a means to cancer cell death. S Jin, R S Dipaola, R Mathew, E White, J. Cell Sci. 120Jin, S., R. S. DiPaola, R. Mathew, and E. White. 2007. Metabolic ca- tastrophe as a means to cancer cell death. J. Cell Sci. 120: 379-383.","Crosstalk between apoptosis, necrosis and autophagy. V Nikoletopoulou, M Markaki, K Palikaras, N Tavernarakis, Biochim. Biophys. Acta. 1833Nikoletopoulou, V., M. Markaki, K. Palikaras, and N. Tavernarakis. 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta. 1833: 3448-3459.","Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities. N J Lanning, J P Castle, S J Singh, A N Leon, E A Tovar, A Sanghera, J P Mackeigan, F V Filipp, C R Graveel, Cancer Metab. 56Lanning, N. J., J. P. Castle, S. J. Singh, A. N. Leon, E. A. Tovar, A. Sanghera, J. P. MacKeigan, F. V. Filipp, and C. R. Graveel. 2017. Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities. Cancer Metab. 5: 6.","Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells. N Zheng, K Wang, J He, Y Qiu, G Xie, M Su, W Jia, H Li, Sci. Rep. 625892Zheng, N., K. Wang, J. He, Y. Qiu, G. Xie, M. Su, W. Jia, and H. Li. 2016. Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells. Sci. Rep. 6: 25892.","Serum starvation induces a rapid increase of Akt phosphorylation in ovarian cancer cells. S Dai, A Gocher, L Euscher, A Edelman, FASEB J. 30Suppl. 1Dai, S., A. Gocher, L. Euscher, and A. Edelman. 2016. Serum star- vation induces a rapid increase of Akt phosphorylation in ovarian cancer cells. FASEB J. 30 (Suppl. 1): 714.9.","PPAR: energy combustion, hypolipidemia, inflammation and cancer. S R Pyper, N Viswakarma, S Yu, J K Reddy, Nucl. Recept. Signal. 82Pyper, S. R., N. Viswakarma, S. Yu, and J. K. Reddy. 2010. PPAR: energy combustion, hypolipidemia, inflammation and cancer. Nucl. Recept. Signal. 8: e002.","Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus. M Viana Abranches, F C Esteves De Oliveira, J Bressan, Nutr. Hosp. 26Viana Abranches, M., F. C. Esteves de Oliveira, and J. Bressan. 2011. Peroxisome proliferator-activated receptor: effects on nu- tritional homeostasis, obesity and diabetes mellitus. Nutr. Hosp. 26: 271-279.","Peroxisome proliferator-activated receptor target genes. S Mandard, M Müller, S Kersten, Cell. Mol. Life Sci. 61Mandard, S., M. Müller, and S. Kersten. 2004. Peroxisome pro- liferator-activated receptor target genes. Cell. Mol. Life Sci. 61: 393-416.","PPAR promotes cancer cell Glut1 transcription repression. M You, J Jin, Q Liu, Q Xu, J Shi, Y Hou, J. Cell. Biochem. 118You, M., J. Jin, Q. Liu, Q. Xu, J. Shi, and Y. Hou. 2017. PPAR pro- motes cancer cell Glut1 transcription repression. J. Cell. Biochem. 118: 1556-1562.","Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner. M L Hughes, B Liu, M L Halls, K M Wagstaff, R Patil, T Velkov, D A Jans, N W Bunnett, M J Scanlon, C J Porter, J. Biol. Chem. 290Hughes, M. L., B. Liu, M. L. Halls, K. M. Wagstaff, R. Patil, T. Velkov, D. A. Jans, N. W. Bunnett, M. J. Scanlon, and C. J. Porter. 2015. Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner. J. Biol. Chem. 290: 13895-13906.","Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. C Wolfrum, C M Borrmann, T Börchers, F Spener, Proc. Natl. Acad. Sci. USA. 98Wolfrum, C., C. M. Borrmann, T. Börchers, and F. Spener. 2001. Fatty acids and hypolipidemic drugs regulate peroxisome prolifer- ator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc. Natl. Acad. Sci. USA. 98: 2323-2328.","Liver fatty acid-binding protein targets fatty acids to the nucleus. Real time confocal and multiphoton fluorescence imaging in living cells. H Huang, O Starodub, A Mcintosh, A B Kier, F Schroeder, J. Biol. Chem. 277Huang, H., O. Starodub, A. McIntosh, A. B. Kier, and F. Schroeder. 2002. Liver fatty acid-binding protein targets fatty acids to the nu- cleus. Real time confocal and multiphoton fluorescence imaging in living cells. J. Biol. Chem. 277: 29139-29151.","Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J W Lawrence, D J Kroll, P I Eacho, J. Lipid Res. 41Lawrence, J. W., D. J. Kroll, and P. I. Eacho. 2000. Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J. Lipid Res. 41: 1390-1401.","L-FABP directly interacts with PPAR in cultured primary hepatocytes. H A Hostetler, A L Mcintosh, B P Atshaves, S M Storey, H R Payne, A B Kier, F Schroeder, J. Lipid Res. 50Hostetler, H. A., A. L. McIntosh, B. P. Atshaves, S. M. Storey, H. R. Payne, A. B. Kier, and F. Schroeder. 2009. L-FABP directly inter- acts with PPAR in cultured primary hepatocytes. J. Lipid Res. 50: 1663-1675.","Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes. A L Mcintosh, B P Atshaves, H A Hostetler, H Huang, J Davis, O I Lyuksyutova, D Landrock, A B Kier, F Schroeder, Arch. Biochem. Biophys. 485McIntosh, A. L., B. P. Atshaves, H. A. Hostetler, H. Huang, J. Davis, O. I. Lyuksyutova, D. Landrock, A. B. Kier, and F. Schroeder. 2009. Liver type fatty acid binding protein (L-FABP) gene ablation re- duces nuclear ligand distribution and peroxisome proliferator- activated receptor- activity in cultured primary hepatocytes. Arch. Biochem. Biophys. 485: 160-173."],"string":"[\n \"Triplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. S K Kanapathy Pillai, A Tay, S Nair, C-O Leong, BMC Clin. Pathol. 1218Kanapathy Pillai, S. K., A. Tay, S. Nair, and C-O. Leong. 2012. Triple- negative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. BMC Clin. Pathol. 12: 18.\",\n \"Triple-negative breast cancer: clinical features and patterns of recurrence. R Dent, M Trudeau, K I Pritchard, W M Hanna, H K Kahn, C A Sawka, L A Lickley, E Rawlinson, P Sun, S A Narod, Clin. Cancer Res. 13Dent, R., M. Trudeau, K. I. Pritchard, W. M. Hanna, H. K. Kahn, C. A. Sawka, L. A. Lickley, E. Rawlinson, P. Sun, and S. A. Narod. 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res. 13: 4429-4434.\",\n \"Current treatment options in triple negative breast cancer. E Rodler, L Korde, J Gralow, Breast Dis. 32Rodler, E., L. Korde, and J. Gralow. 2010. Current treatment op- tions in triple negative breast cancer. Breast Dis. 32: 99-122.\",\n \"Impact of triple negative phenotype on breast cancer prognosis. H G Kaplan, J A Malmgren, Breast J. 14Kaplan, H. G., and J. A. Malmgren. 2008. Impact of triple negative phenotype on breast cancer prognosis. Breast J. 14: 456-463.\",\n \"Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention. Y Zhang, J-M Yang, Cancer Biol. Ther. 14Zhang, Y., and J-M. Yang. 2013. Altered energy metabolism in can- cer: a unique opportunity for therapeutic intervention. Cancer Biol. Ther. 14: 81-89.\",\n \"Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy. E Obre, R Rossignol, Int. J. Biochem. Cell Biol. 59Obre, E., and R. Rossignol. 2015. Emerging concepts in bioenerget- ics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bio- energetic therapy. Int. J. Biochem. Cell Biol. 59: 167-181.\",\n \"Akt stimulates aerobic glycolysis in cancer cells. R L Elstrom, D E Bauer, M Buzzai, R Karnauskas, M H Harris, D R Plas, H Zhuang, R M Cinalli, A Alavi, C M Rudin, Cancer Res. 64Elstrom, R. L., D. E. Bauer, M. Buzzai, R. Karnauskas, M. H. Harris, D. R. Plas, H. Zhuang, R. M. Cinalli, A. Alavi, C. M. Rudin, et al. 2004. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 64: 3892-3899.\",\n \"Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. D R Wise, R J Deberardinis, A Mancuso, N Sayed, X-Y. Zhang, H K Pfeiffer, I Nissim, E Daikhin, M Yudkoff, S B Mcmahon, Proc. Natl. Acad. Sci. USA. 105Wise, D. R., R. J. DeBerardinis, A. Mancuso, N. Sayed, X-Y. Zhang, H. K. Pfeiffer, I. Nissim, E. Daikhin, M. Yudkoff, S. B. McMahon, et al. 2008. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc. Natl. Acad. Sci. USA. 105: 18782-18787.\",\n \"The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation. M Buzzai, D E Bauer, R G Jones, R J Deberardinis, G Hatzivassiliou, R L Elstrom, C B Thompson, Oncogene. 24Buzzai, M., D. E. Bauer, R. G. Jones, R. J. DeBerardinis, G. Hatzivassiliou, R. L. Elstrom, and C. B. Thompson. 2005. The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation. Oncogene. 24: 4165-4173.\",\n \"Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies. K A Olson, J C Schell, J Rutter, Trends Biochem. Sci. 41Olson, K. A., J. C. Schell, and J. Rutter. 2016. Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies. Trends Biochem. Sci. 41: 219-230.\",\n \"Lipid metabolic reprogramming in cancer cells. S Beloribi-Djefaflia, S Vasseur, F Guillaumond, Oncogenesis. 5189Beloribi-Djefaflia, S., S. Vasseur, and F. Guillaumond. 2016. Lipid metabolic reprogramming in cancer cells. Oncogenesis. 5: e189.\",\n \"Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. F Baenke, B Peck, H Miess, A Schulze, Dis. Model. Mech. 6Baenke, F., B. Peck, H. Miess, and A. Schulze. 2013. Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour devel- opment. Dis. Model. Mech. 6: 1353-1363.\",\n \"Exogenous lipids promote the growth of breast cancer cells via CD36. J Zhao, Z Zhi, C Wang, H Xing, G Song, X Yu, Y Zhu, X Wang, X Zhang, Y Di, Oncol. Rep. 38Zhao, J., Z. Zhi, C. Wang, H. Xing, G. Song, X. Yu, Y. Zhu, X. Wang, X. Zhang, and Y. Di. 2017. Exogenous lipids promote the growth of breast cancer cells via CD36. Oncol. Rep. 38: 2105-2115.\",\n \"De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy. T Mashima, H Seimiya, T Tsuruo, Br. J. Cancer. 100Mashima, T., H. Seimiya, and T. Tsuruo. 2009. De novo fatty-acid synthesis and related pathways as molecular targets for cancer ther- apy. Br. J. Cancer. 100: 1369-1372.\",\n \"Overexpression of fatty acid binding protein-7 correlates with basal-like subtype of breast cancer. X Y Tang, S Umemura, H Tsukamoto, N Kumaki, Y Tokuda, R Y Osamura, Pathol. Res. Pract. 206Tang, X. Y., S. Umemura, H. Tsukamoto, N. Kumaki, Y. Tokuda, and R. Y. Osamura. 2010. Overexpression of fatty acid binding pro- tein-7 correlates with basal-like subtype of breast cancer. Pathol. Res. Pract. 206: 98-101.\",\n \"Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene. Y E Shi, J Ni, G Xiao, Y E Liu, A Fuchs, G Yu, J Su, J M Cosgrove, L Xing, M Zhang, MRG. Cancer Res. 57Shi, Y. E., J. Ni, G. Xiao, Y. E. Liu, A. Fuchs, G. Yu, J. Su, J. M. Cosgrove, L. Xing, M. Zhang, et al. 1997. Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene, MRG. Cancer Res. 57: 3084-3091.\",\n \"Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. V L Spitsberg, E Matitashvili, R C Gorewit, Eur. J. Biochem. 230Spitsberg, V. L., E. Matitashvili, and R. C. Gorewit. 1995. Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland. Eur. J. Biochem. 230: 872-878.\",\n \"Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis. R E Burrier, C R Manson, P Brecher, Biochim. Biophys. Acta. 919Burrier, R. E., C. R. Manson, and P. Brecher. 1987. Binding of acyl- CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis. Biochim. Biophys. Acta. 919: 221-230.\",\n \"Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. N R Coe, M A Simpson, D A Bernlohr, J. Lipid Res. 40Coe, N. R., M. A. Simpson, and D. A. Bernlohr. 1999. Targeted dis- ruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. J. Lipid Res. 40: 967-972.\",\n \"A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer. R Z Liu, K Graham, D D Glubrecht, R Lai, J R Mackey, R Godbout, J. Pathol. 228Liu, R. Z., K. Graham, D. D. Glubrecht, R. Lai, J. R. Mackey, and R. Godbout. 2012. A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer. J. Pathol. 228: 310-321.\",\n \"Fatty acid binding protein 7 expression and its subcellular localization in breast cancer. A T Alshareeda, E A Rakha, C C Nolan, I O Ellis, A R Green, Breast Cancer Res. Treat. 134Alshareeda, A. T., E. A. Rakha, C. C. Nolan, I. O. Ellis, and A. R. Green. 2012. Fatty acid binding protein 7 expression and its sub- cellular localization in breast cancer. Breast Cancer Res. Treat. 134: 519-529.\",\n \"The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer. H Zhang, E A Rakha, G Ball, I Spiteri, M Aleskandarany, E Paish, D G Powe, R D Macmillan, C Caldas, I O Ellis, Breast Cancer Res. Treat. 121Zhang, H., E. A. Rakha, G. Ball, I. Spiteri, M. Aleskandarany, E. Paish, D. G. Powe, R. D. Macmillan, C. Caldas, I. O. Ellis, et al. 2010. The proteins FABP7 and OATP2 are associated with the basal phe- notype and patient outcome in human breast cancer. Breast Cancer Res. Treat. 121: 41-51.\",\n \"Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration. R Mita, M J Beaulieu, C Field, R Godbout, J. Biol. Chem. 285Mita, R., M. J. Beaulieu, C. Field, and R. Godbout. 2010. Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration. J. Biol. Chem. 285: 37005-37015.\",\n \"Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. A S Rambold, S Cohen, J Lippincott-Schwartz, Dev. Cell. 32Rambold, A. S., S. Cohen, and J. Lippincott-Schwartz. 2015. Fatty acid trafficking in starved cells: regulation by lipid droplet lipoly- sis, autophagy, and mitochondrial fusion dynamics. Dev. Cell. 32: 678-692.\",\n \"Members of the fatty acid binding protein family are differentiation factors for the mammary gland. Y Yang, E Spitzer, N Kenney, W Zschiesche, M Li, A Kromminga, T Müller, F Spener, A Lezius, J H Veerkamp, J. Cell Biol. 127Yang, Y., E. Spitzer, N. Kenney, W. Zschiesche, M. Li, A. Kromminga, T. Müller, F. Spener, A. Lezius, J. H. Veerkamp, et al. 1994. Members of the fatty acid binding protein family are differentiation factors for the mammary gland. J. Cell Biol. 127: 1097-1109.\",\n \"Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. S G Kuhar, L Feng, S Vidan, M E Ross, M E Hatten, N Heintz, Development. 117Kuhar, S. G., L. Feng, S. Vidan, M. E. Ross, M. E. Hatten, and N. Heintz. 1993. Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. Development. 117: 97-104.\",\n \"Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex. Y Arai, N Funatsu, K Numayama-Tsuruta, T Nomura, S Nakamura, N Osumi, J. Neurosci. 25Arai, Y., N. Funatsu, K. Numayama-Tsuruta, T. Nomura, S. Nakamura, and N. Osumi. 2005. Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex. J. Neurosci. 25: 9752-9761.\",\n \"Brain lipid binding protein (FABP7) as modulator of astrocyte function. M Kipp, T Clarner, S Gingele, F Pott, S Amor, P Van Der, C Valk, Beyer, Physiol. Res. 60Suppl. 1Kipp, M., T. Clarner, S. Gingele, F. Pott, S. Amor, P. van der Valk, and C. Beyer. 2011. Brain lipid binding protein (FABP7) as modula- tor of astrocyte function. Physiol. Res. 60 (Suppl. 1): S49-S60.\",\n \"Fatty acid uptake and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation. K Bensaad, E Favaro, C A Lewis, B Peck, S Lord, J M Collins, K E Pinnick, S Wigfield, F M Buffa, J L Li, Cell Reports. 9Bensaad, K., E. Favaro, C. A. Lewis, B. Peck, S. Lord, J. M. Collins, K. E. Pinnick, S. Wigfield, F. M. Buffa, J. L. Li, et al. 2014. Fatty acid up- take and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation. Cell Reports. 9: 349-365.\",\n \"MDA-MB-435 cells are from melanoma, not from breast cancer. M Lacroix, Cancer Chemother. Pharmacol. 63567Lacroix, M. 2009. MDA-MB-435 cells are from melanoma, not from breast cancer. Cancer Chemother. Pharmacol. 63: 567.\",\n \"Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. J-S Shin, S-W Hong, S-L O Lee, T-H Kim, I-C Park, S -K. An, W K Lee, J S Lim, K I Kim, Y Yang, Int. J. Oncol. 32Shin, J-S., S-W. Hong, S-L. O. Lee, T-H. Kim, I-C. Park, S-K. An, W. K. Lee, J. S. Lim, K. I. Kim, Y. Yang, et al. 2008. Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. Int. J. Oncol. 32: 435-439.\",\n \"Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro. Y Huang, Z Fu, W Dong, Z Zhang, J Mu, J Zhang, Mol. Med. Rep. 17Huang, Y., Z. Fu, W. Dong, Z. Zhang, J. Mu, and J. Zhang. 2018. Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro. Mol. Med. Rep. 17: 5057-5064.\",\n \"Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression. S M Schieke, J P Mccoy, Jr , T Finkel, Cell Cycle. 7Schieke, S. M., J. P. McCoy, Jr., and T. Finkel. 2008. Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression. Cell Cycle. 7: 1782-1787.\",\n \"Restriction beyond the restriction point: mitogen requirement for G2 passage. F Foijer, H Te Riele, Cell Div. 18Foijer, F., and H. te Riele. 2006. Restriction beyond the restriction point: mitogen requirement for G2 passage. Cell Div. 1: 8.\",\n \"Metabolic regulation of apoptosis in cancer. K Matsuura, K Canfield, W Feng, M Kurokawa, Int. Rev. Cell. Mol. Biol. 327Matsuura, K., K. Canfield, W. Feng, and M. Kurokawa. 2016. Metabolic regulation of apoptosis in cancer. Int. Rev. Cell. Mol. Biol. 327: 43-87.\",\n \"Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells. X Kou, Y Jing, W Deng, K Sun, Z Han, F Ye, G Yu, Q Fan, L Gao, Q Zhao, BMC Cancer. 13438Kou, X., Y. Jing, W. Deng, K. Sun, Z. Han, F. Ye, G. Yu, Q. Fan, L. Gao, Q. Zhao, et al. 2013. Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells. BMC Cancer. 13: 438.\",\n \"Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells. Tovar Sepulveda, V A , X Shen, M Falzon, Endocrinology. 143Tovar Sepulveda, V. A., X. Shen, and M. Falzon. 2002. Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells. Endocrinology. 143: 596-606.\",\n \"Metabolic catastrophe as a means to cancer cell death. S Jin, R S Dipaola, R Mathew, E White, J. Cell Sci. 120Jin, S., R. S. DiPaola, R. Mathew, and E. White. 2007. Metabolic ca- tastrophe as a means to cancer cell death. J. Cell Sci. 120: 379-383.\",\n \"Crosstalk between apoptosis, necrosis and autophagy. V Nikoletopoulou, M Markaki, K Palikaras, N Tavernarakis, Biochim. Biophys. Acta. 1833Nikoletopoulou, V., M. Markaki, K. Palikaras, and N. Tavernarakis. 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta. 1833: 3448-3459.\",\n \"Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities. N J Lanning, J P Castle, S J Singh, A N Leon, E A Tovar, A Sanghera, J P Mackeigan, F V Filipp, C R Graveel, Cancer Metab. 56Lanning, N. J., J. P. Castle, S. J. Singh, A. N. Leon, E. A. Tovar, A. Sanghera, J. P. MacKeigan, F. V. Filipp, and C. R. Graveel. 2017. Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities. Cancer Metab. 5: 6.\",\n \"Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells. N Zheng, K Wang, J He, Y Qiu, G Xie, M Su, W Jia, H Li, Sci. Rep. 625892Zheng, N., K. Wang, J. He, Y. Qiu, G. Xie, M. Su, W. Jia, and H. Li. 2016. Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells. Sci. Rep. 6: 25892.\",\n \"Serum starvation induces a rapid increase of Akt phosphorylation in ovarian cancer cells. S Dai, A Gocher, L Euscher, A Edelman, FASEB J. 30Suppl. 1Dai, S., A. Gocher, L. Euscher, and A. Edelman. 2016. Serum star- vation induces a rapid increase of Akt phosphorylation in ovarian cancer cells. FASEB J. 30 (Suppl. 1): 714.9.\",\n \"PPAR: energy combustion, hypolipidemia, inflammation and cancer. S R Pyper, N Viswakarma, S Yu, J K Reddy, Nucl. Recept. Signal. 82Pyper, S. R., N. Viswakarma, S. Yu, and J. K. Reddy. 2010. PPAR: energy combustion, hypolipidemia, inflammation and cancer. Nucl. Recept. Signal. 8: e002.\",\n \"Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus. M Viana Abranches, F C Esteves De Oliveira, J Bressan, Nutr. Hosp. 26Viana Abranches, M., F. C. Esteves de Oliveira, and J. Bressan. 2011. Peroxisome proliferator-activated receptor: effects on nu- tritional homeostasis, obesity and diabetes mellitus. Nutr. Hosp. 26: 271-279.\",\n \"Peroxisome proliferator-activated receptor target genes. S Mandard, M Müller, S Kersten, Cell. Mol. Life Sci. 61Mandard, S., M. Müller, and S. Kersten. 2004. Peroxisome pro- liferator-activated receptor target genes. Cell. Mol. Life Sci. 61: 393-416.\",\n \"PPAR promotes cancer cell Glut1 transcription repression. M You, J Jin, Q Liu, Q Xu, J Shi, Y Hou, J. Cell. Biochem. 118You, M., J. Jin, Q. Liu, Q. Xu, J. Shi, and Y. Hou. 2017. PPAR pro- motes cancer cell Glut1 transcription repression. J. Cell. Biochem. 118: 1556-1562.\",\n \"Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner. M L Hughes, B Liu, M L Halls, K M Wagstaff, R Patil, T Velkov, D A Jans, N W Bunnett, M J Scanlon, C J Porter, J. Biol. Chem. 290Hughes, M. L., B. Liu, M. L. Halls, K. M. Wagstaff, R. Patil, T. Velkov, D. A. Jans, N. W. Bunnett, M. J. Scanlon, and C. J. Porter. 2015. Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner. J. Biol. Chem. 290: 13895-13906.\",\n \"Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. C Wolfrum, C M Borrmann, T Börchers, F Spener, Proc. Natl. Acad. Sci. USA. 98Wolfrum, C., C. M. Borrmann, T. Börchers, and F. Spener. 2001. Fatty acids and hypolipidemic drugs regulate peroxisome prolifer- ator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc. Natl. Acad. Sci. USA. 98: 2323-2328.\",\n \"Liver fatty acid-binding protein targets fatty acids to the nucleus. Real time confocal and multiphoton fluorescence imaging in living cells. H Huang, O Starodub, A Mcintosh, A B Kier, F Schroeder, J. Biol. Chem. 277Huang, H., O. Starodub, A. McIntosh, A. B. Kier, and F. Schroeder. 2002. Liver fatty acid-binding protein targets fatty acids to the nu- cleus. Real time confocal and multiphoton fluorescence imaging in living cells. J. Biol. Chem. 277: 29139-29151.\",\n \"Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J W Lawrence, D J Kroll, P I Eacho, J. Lipid Res. 41Lawrence, J. W., D. J. Kroll, and P. I. Eacho. 2000. Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus. J. Lipid Res. 41: 1390-1401.\",\n \"L-FABP directly interacts with PPAR in cultured primary hepatocytes. H A Hostetler, A L Mcintosh, B P Atshaves, S M Storey, H R Payne, A B Kier, F Schroeder, J. Lipid Res. 50Hostetler, H. A., A. L. McIntosh, B. P. Atshaves, S. M. Storey, H. R. Payne, A. B. Kier, and F. Schroeder. 2009. L-FABP directly inter- acts with PPAR in cultured primary hepatocytes. J. Lipid Res. 50: 1663-1675.\",\n \"Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes. A L Mcintosh, B P Atshaves, H A Hostetler, H Huang, J Davis, O I Lyuksyutova, D Landrock, A B Kier, F Schroeder, Arch. Biochem. Biophys. 485McIntosh, A. L., B. P. Atshaves, H. A. Hostetler, H. Huang, J. Davis, O. I. Lyuksyutova, D. Landrock, A. B. Kier, and F. Schroeder. 2009. Liver type fatty acid binding protein (L-FABP) gene ablation re- duces nuclear ligand distribution and peroxisome proliferator- activated receptor- activity in cultured primary hepatocytes. Arch. Biochem. Biophys. 485: 160-173.\"\n]"},"annotations_bibref":{"kind":"list like","value":["(7)","(8)","(9)","(6,","10)","(11,","12)","(13)","(14)","(15,","16)","(17)","(18)","(19)","(20)","(21,","22)","(23)","(24)","(25)","(26,","27)","(28)","(20,","29)","(30)","(31,","32)","(33)","(34)","(35)","(36)","(37)","(38)","(39)","(40)","(41,","42)","(24)","(43)","(44)","(45)","(46)","(47)","(48)","(49)","(50)","(51)","(52)"],"string":"[\n \"(7)\",\n \"(8)\",\n \"(9)\",\n \"(6,\",\n \"10)\",\n \"(11,\",\n \"12)\",\n \"(13)\",\n \"(14)\",\n \"(15,\",\n \"16)\",\n \"(17)\",\n \"(18)\",\n \"(19)\",\n \"(20)\",\n \"(21,\",\n \"22)\",\n \"(23)\",\n \"(24)\",\n \"(25)\",\n \"(26,\",\n \"27)\",\n \"(28)\",\n \"(20,\",\n \"29)\",\n \"(30)\",\n \"(31,\",\n \"32)\",\n \"(33)\",\n \"(34)\",\n \"(35)\",\n \"(36)\",\n \"(37)\",\n \"(38)\",\n \"(39)\",\n \"(40)\",\n \"(41,\",\n \"42)\",\n \"(24)\",\n \"(43)\",\n \"(44)\",\n \"(45)\",\n \"(46)\",\n \"(47)\",\n \"(48)\",\n \"(49)\",\n \"(50)\",\n \"(51)\",\n \"(52)\"\n]"},"annotations_bibtitle":{"kind":"list like","value":["Triplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women","Triple-negative breast cancer: clinical features and patterns of recurrence","Current treatment options in triple negative breast cancer","Impact of triple negative phenotype on breast cancer prognosis","Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention","Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy","Akt stimulates aerobic glycolysis in cancer cells","Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction","The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation","Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies","Lipid metabolic reprogramming in cancer cells","Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development","Exogenous lipids promote the growth of breast cancer cells via CD36","De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy","Overexpression of fatty acid binding protein-7 correlates with basal-like subtype of breast cancer","Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene","Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland","Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis","Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels","A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer","Fatty acid binding protein 7 expression and its subcellular localization in breast cancer","The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer","Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration","Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics","Members of the fatty acid binding protein family are differentiation factors for the mammary gland","Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation","Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex","Brain lipid binding protein (FABP7) as modulator of astrocyte function","Fatty acid uptake and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation","MDA-MB-435 cells are from melanoma, not from breast cancer","Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells","Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro","Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression","Restriction beyond the restriction point: mitogen requirement for G2 passage","Metabolic regulation of apoptosis in cancer","Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells","Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells","Metabolic catastrophe as a means to cancer cell death","Crosstalk between apoptosis, necrosis and autophagy","Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities","Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells","Serum starvation induces a rapid increase of Akt phosphorylation in ovarian cancer cells","PPAR: energy combustion, hypolipidemia, inflammation and cancer","Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus","Peroxisome proliferator-activated receptor target genes","PPAR promotes cancer cell Glut1 transcription repression","Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner","Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus","Liver fatty acid-binding protein targets fatty acids to the nucleus. Real time confocal and multiphoton fluorescence imaging in living cells","Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus","L-FABP directly interacts with PPAR in cultured primary hepatocytes","Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes"],"string":"[\n \"Triplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women\",\n \"Triple-negative breast cancer: clinical features and patterns of recurrence\",\n \"Current treatment options in triple negative breast cancer\",\n \"Impact of triple negative phenotype on breast cancer prognosis\",\n \"Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention\",\n \"Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy\",\n \"Akt stimulates aerobic glycolysis in cancer cells\",\n \"Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction\",\n \"The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation\",\n \"Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies\",\n \"Lipid metabolic reprogramming in cancer cells\",\n \"Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development\",\n \"Exogenous lipids promote the growth of breast cancer cells via CD36\",\n \"De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy\",\n \"Overexpression of fatty acid binding protein-7 correlates with basal-like subtype of breast cancer\",\n \"Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene\",\n \"Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland\",\n \"Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis\",\n \"Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels\",\n \"A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer\",\n \"Fatty acid binding protein 7 expression and its subcellular localization in breast cancer\",\n \"The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer\",\n \"Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration\",\n \"Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics\",\n \"Members of the fatty acid binding protein family are differentiation factors for the mammary gland\",\n \"Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation\",\n \"Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex\",\n \"Brain lipid binding protein (FABP7) as modulator of astrocyte function\",\n \"Fatty acid uptake and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation\",\n \"MDA-MB-435 cells are from melanoma, not from breast cancer\",\n \"Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells\",\n \"Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro\",\n \"Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression\",\n \"Restriction beyond the restriction point: mitogen requirement for G2 passage\",\n \"Metabolic regulation of apoptosis in cancer\",\n \"Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells\",\n \"Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells\",\n \"Metabolic catastrophe as a means to cancer cell death\",\n \"Crosstalk between apoptosis, necrosis and autophagy\",\n \"Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities\",\n \"Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells\",\n \"Serum starvation induces a rapid increase of Akt phosphorylation in ovarian cancer cells\",\n \"PPAR: energy combustion, hypolipidemia, inflammation and cancer\",\n \"Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus\",\n \"Peroxisome proliferator-activated receptor target genes\",\n \"PPAR promotes cancer cell Glut1 transcription repression\",\n \"Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner\",\n \"Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus\",\n \"Liver fatty acid-binding protein targets fatty acids to the nucleus. Real time confocal and multiphoton fluorescence imaging in living cells\",\n \"Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus\",\n \"L-FABP directly interacts with PPAR in cultured primary hepatocytes\",\n \"Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["BMC Clin. Pathol","Clin. Cancer Res","Breast Dis","Breast J","Cancer Biol. Ther","Int. J. Biochem. Cell Biol","Cancer Res","Proc. Natl. Acad. Sci. USA","Oncogene","Trends Biochem. Sci","Oncogenesis","Dis. Model. Mech","Oncol. Rep","Br. J. Cancer","Pathol. Res. Pract","MRG. Cancer Res","Eur. J. Biochem","Biochim. Biophys. Acta","J. Lipid Res","J. Pathol","Breast Cancer Res. Treat","Breast Cancer Res. Treat","J. Biol. Chem","Dev. Cell","J. Cell Biol","Development","J. Neurosci","Physiol. Res","Cell Reports","Cancer Chemother. Pharmacol","Int. J. Oncol","Mol. Med. Rep","Cell Cycle","Cell Div","Int. Rev. Cell. Mol. Biol","BMC Cancer","Endocrinology","J. Cell Sci","Biochim. Biophys. Acta","Cancer Metab","Sci. Rep","FASEB J","Nucl. Recept. Signal","Nutr. Hosp","Cell. Mol. Life Sci","J. Cell. Biochem","J. Biol. Chem","Proc. Natl. Acad. Sci. USA","J. Biol. Chem","J. Lipid Res","J. Lipid Res","Arch. Biochem. Biophys"],"string":"[\n \"BMC Clin. Pathol\",\n \"Clin. Cancer Res\",\n \"Breast Dis\",\n \"Breast J\",\n \"Cancer Biol. Ther\",\n \"Int. J. Biochem. Cell Biol\",\n \"Cancer Res\",\n \"Proc. Natl. Acad. Sci. USA\",\n \"Oncogene\",\n \"Trends Biochem. Sci\",\n \"Oncogenesis\",\n \"Dis. Model. Mech\",\n \"Oncol. Rep\",\n \"Br. J. Cancer\",\n \"Pathol. Res. Pract\",\n \"MRG. Cancer Res\",\n \"Eur. J. Biochem\",\n \"Biochim. Biophys. Acta\",\n \"J. Lipid Res\",\n \"J. Pathol\",\n \"Breast Cancer Res. Treat\",\n \"Breast Cancer Res. Treat\",\n \"J. Biol. Chem\",\n \"Dev. 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FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.","\nFig. 2 .\n2The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.","\nFig.\nFig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.","\nFig. 4 .\n4FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium."],"string":"[\n \"\\nFig. 1 .\\n1Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.\",\n \"\\nFig. 2 .\\n2The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\",\n \"\\nFig.\\nFig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.\",\n \"\\nFig. 4 .\\n4FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.","The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.","Fig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.","FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium."],"string":"[\n \"Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.\",\n \"The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\",\n \"Fig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.\",\n \"FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.\"\n]"},"annotations_figureref":{"kind":"list like","value":["(Fig. 1A)","(Fig. 1B)","(Fig. 1C)","(Fig. 1D)","(Fig. 2)","Fig. 2A,B)","(Fig. 2C","(Fig. 2C","(Fig. 3A)","Fig. 3B and supplemental Fig. S1","(Fig. 3B)","(Fig. 3C","Fig. 4 and supplemental Figs. S2, S3)","(Fig. 4A)","3","Fig. 4B-D)","(Fig. 4E)","(Fig. 4A)","(Fig. 4B)","(Fig. 4C)","(Fig. 4D)","(Fig. 4E)","Fig. 4A and supplemental Fig. S2B","Fig. 5A and supplemental Fig. S4A)","(Fig. 5A and supplemental Fig. S4B","(Fig. 5B","(Fig. 5B","Fig. S5","(Fig. 5C)","(Fig. 5C)","Fig. 5"],"string":"[\n \"(Fig. 1A)\",\n \"(Fig. 1B)\",\n \"(Fig. 1C)\",\n \"(Fig. 1D)\",\n \"(Fig. 2)\",\n \"Fig. 2A,B)\",\n \"(Fig. 2C\",\n \"(Fig. 2C\",\n \"(Fig. 3A)\",\n \"Fig. 3B and supplemental Fig. S1\",\n \"(Fig. 3B)\",\n \"(Fig. 3C\",\n \"Fig. 4 and supplemental Figs. S2, S3)\",\n \"(Fig. 4A)\",\n \"3\",\n \"Fig. 4B-D)\",\n \"(Fig. 4E)\",\n \"(Fig. 4A)\",\n \"(Fig. 4B)\",\n \"(Fig. 4C)\",\n \"(Fig. 4D)\",\n \"(Fig. 4E)\",\n \"Fig. 4A and supplemental Fig. S2B\",\n \"Fig. 5A and supplemental Fig. S4A)\",\n \"(Fig. 5A and supplemental Fig. S4B\",\n \"(Fig. 5B\",\n \"(Fig. 5B\",\n \"Fig. S5\",\n \"(Fig. 5C)\",\n \"(Fig. 5C)\",\n \"Fig. 5\"\n]"},"annotations_formula":{"kind":"list like","value":[],"string":"[]"},"annotations_paragraph":{"kind":"list like","value":["AKT activation augments the Warburg effect and renders the cancer cells addicted to glucose (7). Myc protein promotes mitochondrial glutaminolysis and glutamine addiction by shunting glucose away from mitochondrial metabolism (8). Cancer cells that fail to demonstrate metabolic flexibility during nutrient deprivation could become vulnerable in their survival (9). Hence, metabolic vulnerabilities exhibited by cancer cells can be targeted for cancer management (6,10).","Alterations in lipid-and cholesterol-associated pathways encountered in tumors are now increasingly recognized and more frequently described (11,12), but not completely understood. Many cancer types show a strong lipid avidity, in which increasing uptake of exogenous lipid sources (13) or overactivation of endogenous lipid synthesis (14) is observed. Fatty acid binding protein 7 (FABP7), a member of the FABP intracellular lipid chaperone family, has been shown to be upregulated in TNBC compared with other breast cancer subtypes (15,16). FABPs regulates lipid metabolism by increasing fatty acid uptake (17), fatty acid oxidation (FAO) (18), and lipolysis (19). Yet, in TNBC, correlation of FABP7 expression and disease prognosis is debatable. While one study showed that FABP7 expression correlates with lower overall and recurrence-free survival (20), several have shown that FABP7-positive basal tumors (synonymous with TNBC) are associated with better prognosis (21,22). It is unclear, at this point, how FABP7-governed pathways impact the survival of TNBC.","In this study, we explored the roles of FABP7 in adaptation to nutrient depletion in TNBC cell lines. We showed that overexpression of FABP7 decreased the viability of Hs578T cells during serum starvation. This led to cell-cycle arrest and a significant increase in cell death. We further showed that this phenotype was mediated by PPAR-regulated genes.","All cell lines used in this study were purchased from American Type Culture Collection (Gaithersburg, MD). They were cultured in medium according to the manufacturer's protocol and supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), unless specified otherwise. Hs578T, MCF7, MDA-MB-231, and MDA-MB-435S were maintained in DMEM high glucose while BT474 was cultured in MEM. The cell lines were incubated at 37°C and 5% CO 2 atmosphere.","For nutrient-deprivation experiments, the cells were first seeded in complete medium (10% FBS). The next day (hereafter referred to as 0 h), the cells were washed once with PBS and replaced with nutrient-deprived medium. For the glucose-and glutamine-starvation experiments, 10% FBS were added into the basal medium. Basal medium used for glucose starvation was glucose-free DMEM (Life Technologies, catalog no. 11966-025) that contained 4 mM l-glutamine. For the glutamine-starvation experiment, glutamine-free DMEM (Life Technologies, catalog no. 11960-044) containing 25 mM glucose was used. The serumstarvation experiment mimicked a culture condition with deprived lipids. The cells were challenged with serum-free DMEM (Life Technologies, catalog no. 11965-092) that contains high glucose (25 mM) with l-glutamine (4 mM), but without sodium pyruvate, HEPES, lipids, and growth factors.","The PPAR- antagonist (GW6471) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a nonlethal concentration (2 M).","A microarray profile of selected TNBC was obtained from the publicly available Gene Expression Omnibus (GEO) database. The search keyword used was \"triple negative breast cancer,\" and the results were filtered with \"Homo sapiens.\" The search was conducted from September 1 to 30, 2018.","TNBC cell lines Hs578T and MDA-MB-231 were transduced with FABP7 gene using Thermo Scientific Precision LentiORF constructs at MOI 10 (Clone ID: PLOHS_100004067). For their control counterparts, the cells were transduced with Precision LentiORF RFP (catalog no. OHS5833). The transduced cells were selected with blasticidin for 30 days to generate stable cell line.","Cells were seeded in 96-well plates and subjected to different growth conditions. At time of termination, 10 l of methyl thiazolyl tetrazolium (MTT) solution (5 mg/ml) (Sigma, St. Louis, MO) was added into each well for 4 h, before the addition of 100 l of 10% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals overnight. Absorbance was measured at 575 nm with reference to 650 nm. All experiments were performed in triplicate and repeated three times.","Total RNA extraction was carried out using Trizol (Invitrogen, Carlsbad, CA) following the recommended protocol. About 500 ng of RNA was converted into cDNA using DyNAmo cDNA synthesis kit (Finnzymes, Vantaa, Finland). For quantitative RT-PCR (qRT-PCR), 5 ng/l cDNA template was added into pool solution containing 5× HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Tartu, Estonia), 10 pmol/l forward and reverse primers, and UltraPure distilled water. ABI StepOne Plus (Applied Biosystem, Foster City, CA) was used, and 40 cycles of amplification were performed. The expression of metabolic genes was normalized to housekeeping genes 18S rRNA and ribosomal protein L13a. Sequence of the primers used is described in supplemental Table S1.","Protein lysate was harvested by scraping the cells in cold PBS. After centrifugation, PBS was discarded, and the cells were incubated in lysis buffer (0.1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1× phosphatase, and 1× protease inhibitors) for 30 min on ice. The mixture was centrifuged for 20 min, and supernatant was collected as protein lysate. Protein concentration was quantified using Bradford assay (Bio-Rad, Hercules, CA). A total of 30 g of protein was resolved on 15% SDS-PAGE prior to transferring on PVDF membrane. Target proteins were detected using primary Abs FABP7 (Cell Signaling Technology) and ECL prime Western blotting detection reagent (Amersham, GE Healthcare Lifesciences, Sweden) before being visualized with gel-documentation system (Biospectrum 410, UVP).","The BrdU cell-proliferation assay kit (Cell Signaling Technology, Beverly, MA) was used to measure the cell-proliferation rate at 24, 48, and 72 h after serum starvation. The data were normalized to their counterparts cultured in complete medium. The cells were incubated with 10X BrdU solution for three h, prior to fixation for 30 min and staining with 1× BrdU Detection Antibody for 1 h. The stained cells were washed and incubated with 1× HRPconjugated secondary antibody solution for 30 min, before TMB solution was added. STOP solution was added after 15 min, and absorbance was read at 450 nm.","Cell-cycle analysis was conducted on the cells at 0, 24, 48, and 72 h after serum starvation. The cells were fixed with 70% ethanol at 4°C for 30 min, before two washes with PBS. Enzymatic removal of RNA was achieved by incubating the cells with 100 g/ml RNase for 15 min before DNA staining with 50 g/ml propidium iodide. The cells were analyzed using a BD FACSCanto II flow cytometer, and the cell-cycle distribution of the cell was analyzed with Modfit LT software. The samples were run in triplicates, and the mean value was calculated.","Cells were harvested for apoptosis assay at 0, 24, 48, and 72 h after serum starvation. Floating cells in the culture medium and the trypsinized cells were collected and washed once with ice-cold PBS, before they were resuspended in 100 l of assay buffer (1 part buffer: 9 parts distilled water) with 5 l of annexin V-PE and 5 l of 7AAD (BD Bioscience, Franklin Lakes, NJ). The cells were vortexed gently and kept in the dark for 15 min, before adding 400 l of buffer. The staining was analyzed using a BD FACSCanto II flow cytometer and viewed using FACS DiVa Software (BD Bioscience, San Jose, CA). All samples were run in triplicates, and mean value was calculated.","GAPDH, glutaminase (GSL), and FAO enzyme activities were measured using calorimetric assay kits from Biomedical Research Service Center, State University of New York (Buffalo, NY). All samples were harvested using 1× Cell Lysis Solution. Protein concentration of the samples was assessed with a Bradford assay and normalized to 1 mg/ml. GAPDH activity was measured using GAPDH enzyme assay kit (catalog no. E-101). Briefly, 10 l of sample or water (as blank) was incubated in 50 l of GAPDH assay solution. After gentle agitation, the plate was kept in a non-CO 2 incubator at 37°C for 60 min. For GSL assay (catalog no. E-133), 10 l of sample was incubated in either 40 l of glutamine solution or water. Following a 2 h incubation period in a non-CO 2 incubator at 37°C, 50 l of TA assay solution was added, and the plate was further incubated for another 1 h. For FAO enzyme assay (catalog no. E-141), 50 l of FAO Assay Solution or control solution was added to 10 l of the protein sample. The plate was kept in a non-CO 2 incubator at 37°C for 60 min. All experiments were terminated by adding 50 l of 3% acetic acid, and the plate was read at optical density of 492 nm (OD 492) with a spectrophotometer. Blank reading was subtracted from the sample reading. GAPDH activity in IU/l unit was determined by multiplying OD by 16.98. Glutaminase (GLS) activity in IU/l unit = mol/ (l·min) = (OD × 1,000 × 150 l) ÷ (120 min × 0.6 cm × 18 × 10 l) = OD × 11.58. For FAO assay, the subtracted OD represents the FAO activity of the sample.","Basal oxygen consumption rate (OCR) was carried out with a Mito Fuel Flex Test Kit on XFe96 Bioanalyzer (Agilent). Briefly, 4,000 cells were seeded on a Seahorse XF96-well assay plate in complete medium. The cells grown in complete medium were terminated at 24 h after seeding. For the serum-starvation experiment, the cells were left incubated overnight before they were washed with PBS and incubated in serum-free medium for 24 h. Upon termination, all wells were replaced with assay medium, and baseline OCR was immediately measured for 18 min. The basal OCR was normalized to protein concentration of the cells.","The cells were seeded on a coverslip in complete medium. After overnight incubation, the cells were either harvested or challenged with serum-free medium for 24 h. The cells were fixed with 4% formaldehyde for 30 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 1 h. The cells were incubated with FABP7 primary antibody (1:2,000; Cell Signaling catalog no. 13347S) and PPAR- primary antibody (1:400; Santa Cruz Biotechnology, catalog no. sc-398394), followed by Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific). The incubation time was 90 min for primary antibody and 60 min for secondary antibody. The cells were washed three times with PBS after staining with each antibody. All the procedures were carried out on ice. The coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories Cat#H-1200) and analyzed with a Leica TCS SP5 II laser microscope (Leica, Heidelberg, Germany) using 60× objective.","Statistical analysis assessing the difference between the means of two groups was performed using Student's t-test with Graph-Pad-Prism (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.","The expression of FABP7 was determined in human breast cancer tissues and TNBC cell lines. Analysis from the GEO database (dataset GSE65194) revealed that FABP7 expression was significantly higher in TNBC (n = 55) compared with non-TNBC (n = 98) subtypes (Fig. 1A). When examined in a panel of breast cancer cell lines, FABP7 showed low expression in all cells including MCF7 (luminal A), BT474 (luminal B), MDA-MB-231, and Hs578T (TNBC) cells, when compared with MDA-MB-435S, a melanoma cell line reported to express high FABP7 (Fig. 1B). Correspondingly, FABP7 protein was not detected in these breast cancer cell lines (Fig. 1C).","To understand the functional role of FABP7 in TNBC cells, we established FABP7-overexpressing Hs578T and MDA-MB-231 TNBC cells by transduction using lentiviral particles containing FABP7 open reading frame (Hs-FABP7 and 231-FABP7, respectively) and their control counterparts using lentiviral particles containing red fluorescent protein (Hs-RFP and 231-RFP, respectively) (Fig. 1D). When the cells were cultured in complete medium for 72 h, there was no difference in cell growth between cells expressing FABP7 and their respective RFP controls (Fig. 2). However, when cultured in glucose-or glutamine-deprived condition, both FABP7-overexpressing cells and RFP controls demonstrated substantial reduction in cell viability with greater effect observed in glucose-deprived than glutamine-deprived medium ( Fig. 2A,B).","Interestingly, when lipids were significantly reduced during serum starvation, overexpression of FABP7 resulted in decreased viability of Hs578T cells (Fig. 2C). At 24 h after serum starvation, the cell viability of Hs-FABP7 cells decreased by 10%, which progressively decreased to approximately 70% at 72 h after starvation (P < 0.0001 vs. Hs-RFP cells). Cell viability of Hs-RFP cells was not affected by serum starvation, indicating that the decreased viability in Hs-FABP7 cells may be related to FABP7 expression. This phenotype appeared to be specific to Hs578T cells, as FABP7 expression did not affect the viability of MDA-MB-231 cells in serum-free medium (Fig. 2C).","The reduction in cell viability of Hs-FABP7 cells during serum starvation resulted from a decrease in cell proliferation. Hs-FABP7 cells demonstrated a 70% reduction in BrdU uptake as early as 24 h after serum starvation, whereas the control Hs-RFP cells only showed a decrease by 20% (Fig. 3A). The nonresponsive MDA-MB-231 cells maintained their proliferation rate in the range of 85-89% during serum starvation, regardless of FABP7 expression.","Flow-cytometry analysis showed that FABP7 induced cellcycle arrest in Hs578T cells during serum starvation ( Fig. 3B and supplemental Fig. S1). Hs-RFP cells showed a higher distribution of cells (80%) in G1 phase throughout serum starvation, indicating that the cells were able to complete the cell cycle and proliferate, parallel to increased cell viability over time. In contrast, Hs-FABP7 cells demonstrated a consistently higher percentage of cells in S and G2 phase upon serum starvation. For instance, at 48 h after serum starvation, 24% of Hs-FABP7 cells were accumulated in S phase and 8.23% in G2 phase, whereas only 5.15% and 1.99% of Hs-RFP cells were detected at S and G2 phase (P = 0.0052 for S phase and P = 0.0241 for G2 phase). The percentage of cells in S/G2 phase increased to 40.15% in Hs-FABP7 cells compared with 14.29% in Hs-RFP cells at 72 h after serum starvation. Cell arrest in S/G2 phase in Hs-FABP7 cells may partly contribute to the decreased cell viability during serum starvation. This effect was not observed in MDA-MB-231 cells (Fig. 3B).","The decrease in Hs-FABP7 cell viability during serum starvation was potentially attributed to apoptosis and necrosis (Fig. 3C). More necrotic cell death and late apoptosis was observed in Hs-FABP7 cells (34.26% and 21.3%) than in Hs-RFP cells (18.83% and 4.63%) at 72 h after serum starvation (P < 0.0001). Upon serum starvation, increasing cell death was observed in MDA-MB-231 cells, but with no significant difference observed between 231-RFP and 231-FABP7 cells. Taken together, the decreased cell viability in Hs-FABP7 cells during serum starvation was due to reduced proliferation rate, cell cycle arrest at S and G2 phase, and cell death.","Given the role of FABP7 in fatty acid metabolism, we investigated the mechanism of FABP7-induced cell death by exploring a panel of genes and enzyme activities involved in the metabolism of glucose, glutamine, and fatty acid ( Fig. 4 and supplemental Figs. S2, S3). In complete medium, despite the increase in glucose transporter 1 (GLUT1) mRNA expression, glycolysis-related genes phosphofructokinase 1 (PFK1) and GAPDH remained unchanged in Hs-FABP7 cells, whereas the genes regulating FAO, carnitine palmitoyltransferase 1A (CPT1A), and medium-chain acyl-CoA dehydrogenase (MCAD) were markedly upregulated (Fig. 4A). In Hs-FABP7 cells, the expression of GLS1, encoding the rate-limiting enzyme for glutaminolysis, was not significantly different than in Hs-RFP cells. When cultured in complete medium, Hs-FABP7 cells did not show any differences in GAPDH, GLS, and FAO activities when compared with Hs-RFP cells 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001. ( Fig. 4B-D). Subsequently, mitochondrial OCR was similar in Hs-FABP7 cells and in the control Hs-RFP cells (Fig. 4E).","However, when challenged with serum starvation, a widespread downregulation of glucose and glutamine metabolism-related genes were observed in Hs-FABP7 cells when compared with Hs-RFP cells. Interestingly, despite the absence of serum (lipids) during serum starvation, the gene expression of CPT1A and MCAD was about 2-fold higher in Hs-FABP7 cells than Hs-RFP cells (Fig. 4A). Serum starvation generally decreased glycolytic enzyme activity in Hs578T cells, but FABP7-overexpressing cells had a further 30% reduction (P = 0.0121) in GAPDH activity compared with Hs-RFP cells (Fig. 4B). Although GLS activity remained not significantly different in both cells during serum starvation, GLS activity was significantly lower (P = 0.0296) in Hs-FABP7 cells cultured in serum starvation than in complete medium (Fig. 4C). Interestingly, FAO activity in Hs-FABP7 remained similar with the control Hs-RFP cells (Fig. 4D), despite upregulation of FAO-related genes. This may be explained by the lack of fatty acid in the culture medium to activate the metabolic pathway. Consequently, mitochondrial oxidative capacity of both Hs-RFP and Hs-FABP7 cells was generally lower during serum starvation (P = 0.0001 for both cells); however, Hs-FABP7 cells had much lower OCR (16.69 pmol/min/µg) compared with Hs-RFP cells (21.7 pmol/min/µg) (P < 0.05) (Fig. 4E). These data indicate a lower mitochondrial oxidative capacity particularly in Hs-FABP7 cells, probably due to limited bioenergetic substrates during serum starvation.","It is worthwhile to note that, unlike Hs578T, MDA-MB-231 cells showed a rather different metabolic profile, as indicated by the expression of metabolic genes ( Fig. 4A and supplemental Fig. S2B). When cultured in complete medium, although FABP7 was not affecting the cell viability, glycolysis was upregulated in 231-FABP7 cells, with about a 1.7-fold increase in GAPDH expression. Meanwhile, a 4-fold decrease in GLS1 expression was observed, indicative of a downregulation of the glutaminolysis pathway. Upon serum starvation, 231-FABP7 cells demonstrated a marked increase in glucose and glutamine metabolismrelated genes, with more than 4-fold increase in GLUT1 and GLS1 expression.","Taken together, our data show that Hs-FABP7 cells, when challenged with a serum-starved condition, were not able to metabolically adapt and effectively utilize other available energetic substrates, especially glucose, and, as a consequence, could not survive.","The upregulation of CPT1A and MCAD mRNA, accompanied with a downregulation of Glut1 mRNA in Hs-FABP7 cells, indicated a possible role of PPAR- signaling, as these genes are direct targets of PPAR- transcription factor. Immunofluorescence staining showed that the expression of PPAR- protein was increased in the nucleus during serum starvation ( Fig. 5A and supplemental Fig. S4A). Similarly, FABP7 protein was observed to translocate into the nucleus (Fig. 5A and supplemental Fig. S4B) in Hs-FABP7 cells upon serum starvation, potentially acting as fatty acid chaperones to deliver ligands to PPAR- protein (23). This suggests that nuclear localization of FABP7 enhanced the action of PPAR- transcriptional activity during serum starvation in Hs-FABP7 cells.","To investigate whether PPAR- activation was responsible for FABP7-mediated inhibition of cell viability in Hs-FABP7 cells, we treated the cells with a PPAR--specific inhibitor (GW6471). In both Hs578T and MDA-MB-231 cells, treatment of GW6471 at a concentration of 2 M in complete medium had no significant effect on cell viability (Fig. 5B). Yet, the same concentration of GW6471 reversed the cell-growth inhibition observed in Hs-FABP7 cells when exposed to serum-starved conditions (Fig. 5B and supplemental Fig. S5). Subsequent gene-expression analysis revealed that PPAR- target genes were downregulated (CPT1A: 2.43-fold; MCAD: 1.54-fold), when compared with vehicle-treated cells (Fig. 5C). A significant 2-fold increase of GLUT1, PFK1, glucose-6-phosphate dehydrogenase (G6PD), and isocitrate dehydrogenase 1 (IDH1) mRNA expression was observed in GW6471-treated Hs-FABP7 cells (Fig. 5C), indicating that the rescue of cell viability may be due to the switch from lipid to glucose as bioenergetic substrates in these cells. Taken together, FABP7-induced cell death in Hs578T cells during serum starvation is due to activated PPAR- signaling.","In this study, we showed that overexpression of FABP7 disabled TNBC cell line Hs578T to metabolically adapt during serum starvation. FABP7-overexpressing cells failed to efficiently utilize glucose as bioenergetics substrates, when challenged with serum starvation. This was accompanied by increased expression of genes controlling FAO as it attempts to utilize free fatty acids liberated by lipolysis from the lipid storage that resulted from preexposure to complete medium (24). However, as free fatty acid storage was depleted, serum-starved FABP7-overexpressing cells were not able to further metabolically adapt and sustain ATP production. The failure of the cells to efficiently utilize glucose for ATP generation leads to cell death. We further showed that the metabolic impact of FABP7 in Hs578T cells during serum starvation is mediated by PPAR- signaling.","FABP7 is predominantly expressed in the nervous system and mammary gland. In the mammary gland, FABP7 inhibits proliferation and promotes differentiation of mammary cells (25). Similarly, in the nervous system, FABP7 is involved in the differentiation of neurons and development of the cortex (26,27). The expression of FABP7 at the edge of astrocytes also suggested that FABP7 may regulate the motility of astrocytes by increasing the uptake of polyunsaturated fatty acids (28). Yet, the evidence of FABP7 expression in TNBC cell lines was rather limited (20,29). A previous study demonstrating the function of endogenous FABP7 in TNBC was reported using MDA-MB-435S, a melanoma cell line formerly misidentified as a TNBC cell line (30). Our study, however, provides evidence on the role of FABP7 as a mediator for tumor cell death during nutrient-deprived conditions. This finding indicates an anti-tumorigenic role of FABP7 in TNBC, which is mediated by specific nutrient availability.","The metabolic vulnerabilities of FABP7-overexpressing Hs578T cells resulted in S/G2-phase arrest, which subsequently led to cell death. Serum starvation typically induces cell-cycle arrest at G1 phase (31,32), which is observed in Hs-RFP and MDA-MB-231 cells. These cells were able to proliferate, despite a lower proliferation rate, indicating that the lower mitochondrial activity in these cells during serum starvation was sufficient to overcome the metabolic checkpoint and complete the cell cycle (33). Hs-FABP7 cells, however, arrested at the S/G2 phase. G2 arrest has been described as another growth-restricting mechanism in mitogen-starved conditions, likely due to elevated E2F activity (34). Cell arrest could potentially lead to cell death, in which both apoptosis and necrosis were observed in Hs-FABP7 cells upon serum starvation. Apoptosis is a cellular suicide program triggered by stress signals such as nutrient deprivation, which results in the execution of caspasemediated cell death (35). Serum starvation has been shown to induce apoptosis in hepatocellular carcinoma cells (36) and MCF7 breast cancer cells (37), in which the latter was associated with an altered ratio of apoptosis regulating proteins Bax and Bcl-2. Necrosis, on the other hand, may occur when the minimal bioenergetic demands are not met (38), a condition similar to our observations where Hs-FABP7 cells may have limited bioenergetic substrates during serum starvation. In fact, there are common mediators of apoptosis and necrosis, which could potentially contribute to the two modes of cell death observed in our model (39). It is worth noting that FABP7 did not induce cell death in MDA-MB-231 cells during serum starvation, indicating that this cell line is probably more resilient to nutrient deprivation than Hs578T cells. The disparity in the response of Hs578T and MDA-MB-231 cells to serum starvation may also be attributed to differences in their metabolic profiles (40). Hence, it would be worthwhile to examine other TNBC cells with differing metabolic profiles, in delineating the molecular heterogeneity involved in cell death induced by metabolic vulnerabilities.","During serum starvation, cancer cells sense and adapt to nutrient deprivation by upregulating glucose metabolism Fig. 5. FABP7-induced cell death in Hs578T cells during serum starvation was mediated by the action of PPAR- signaling. A: Confocal microscopy of immunofluorescence staining of FABP7 (green) and PPAR- (red) proteins in Hs578T and MDA-MB-231 cells after culture in either complete medium or serum-free medium for 24 h. Blue, DAPI. Scale bar, 50 m. Data shown are representative of two independent experiments. B: Cells were treated with PPAR- inhibitor GW6471 (2 M) in complete or serum-starved medium for 72 h, before analyzing for cell viability using an MTT assay. Data shown are mean ± SEM of triplicates; this is a representative of three independent experiments. *** P < 0.0001. C: mRNA expression of key metabolic genes after treatment with 2 M GW6471 in Hs-FABP7 cells for 24 h in serum starvation when compared with vehicle-treated cells. The genes were normalized to housekeeping gene 18S rRNA. CM, complete medium; SFM, serum-free medium. (41,42), besides releasing fatty acids from the lipid droplet through lipolysis for oxidation (24). Central to such adaption, PPAR- is known to be a lipid sensor that maintains cellular energy homeostasis. It is a ligand-activated class of nuclear hormone receptor that binds to peroxisome proliferator response elements (PPREs) to induce transcription of genes such as lipid processing. Its activation aims at increasing lipid combustion in order to yield more energy production, especially in a challenged environment (43). It was shown in rat hepatocytes that PPAR- expression is increased by fasting-and starvation-induced glucocorticoids (44), suggesting the role of PPAR- to maintain cell survival.","In our model, increased PPAR- expression was observed when the cells were serum-starved, possibly as an adaptation strategy of the cells. The activation of PPAR- was observed in Hs-FABP7 cells during serum starvation, as reflected in the increase of PPAR- target genes, CPT1A and MCAD, which are involved in fatty acid mitochondrial uptake and oxidation (45). Our observation may indicate the usage of fatty acids liberated from lipid droplets as the substrates for FAO. However, the attempt of the cells to survive serum starvation failed as the lipid storage was limited and fatty acids were absent in the serum-free media. In addition, PPAR- activation also potentially inhibited glucose metabolism by targeting the consensus PPRE motif in the promoter region of Glut1, hence repressing its transcription (46). As a result, Hs-FABP7 cells could not efficiently utilize glucose as bioenergetic substrates when serumstarved, which eventually led to cell death.","Our data suggest that PPAR--induced changes are FABP7-dependent. Literature evidence shows that in Cos-7 cells where FABPs and PPAR- are not detected, transfection of the proteins showed that PPAR- activity is amplified by FABP1 and FABP2 (47). In another study, PPAR- transactivation is proportional to levels of L-FABP in human hepatoma HepG2 cells line (48). In our model, upon serum starvation, increased nuclear FABP7 expression was observed, suggesting the role of FABP7 as fatty acids chaperones to transport PPAR- ligands into the nucleus for PPAR- activation. In murine L cells, transduction with FABP1 had dramatically enhanced the uptake of fatty acid into the nucleus (49). Incubation of rat liver nuclei with fluorescence-tagged FABP1 together with fatty acids demonstrated a strong fluorescence response in the nuclei, which otherwise did not happen when fatty acids were absent (50). There has been evidence indicating physical interaction between FABP1 and PPAR- in primary hepatocytes (51). Ablation of L-FABP gene expression significantly impaired fatty acid distribution in the nucleus and affected PPAR- activation in these cells (52). Hence, whether FABP7 and PPAR- require direct contact to induce cell death in TNBC needs to be further investigated. In short, our data suggest that FABP7 facilitates PPAR- activation in serum-starved condition by transporting its ligand (fatty acids) into the nucleus, before enhancing its transcriptional activity.","Our study provides an understanding on the role of FABP7 in regulating the metabolic adaptation of TNBC cell lines. Transcriptional activation of PPAR- in FABP7overexpressing cell can limit their metabolic plasticity when challenged with serum starvation and resulted in reduced survival of these cells. Hence, manipulating metabolic stress in cells expressing FABP7 protein may potentially be an effective targeted therapeutic strategy in FABP7-positive TNBC patients."],"string":"[\n \"AKT activation augments the Warburg effect and renders the cancer cells addicted to glucose (7). Myc protein promotes mitochondrial glutaminolysis and glutamine addiction by shunting glucose away from mitochondrial metabolism (8). Cancer cells that fail to demonstrate metabolic flexibility during nutrient deprivation could become vulnerable in their survival (9). Hence, metabolic vulnerabilities exhibited by cancer cells can be targeted for cancer management (6,10).\",\n \"Alterations in lipid-and cholesterol-associated pathways encountered in tumors are now increasingly recognized and more frequently described (11,12), but not completely understood. Many cancer types show a strong lipid avidity, in which increasing uptake of exogenous lipid sources (13) or overactivation of endogenous lipid synthesis (14) is observed. Fatty acid binding protein 7 (FABP7), a member of the FABP intracellular lipid chaperone family, has been shown to be upregulated in TNBC compared with other breast cancer subtypes (15,16). FABPs regulates lipid metabolism by increasing fatty acid uptake (17), fatty acid oxidation (FAO) (18), and lipolysis (19). Yet, in TNBC, correlation of FABP7 expression and disease prognosis is debatable. While one study showed that FABP7 expression correlates with lower overall and recurrence-free survival (20), several have shown that FABP7-positive basal tumors (synonymous with TNBC) are associated with better prognosis (21,22). It is unclear, at this point, how FABP7-governed pathways impact the survival of TNBC.\",\n \"In this study, we explored the roles of FABP7 in adaptation to nutrient depletion in TNBC cell lines. We showed that overexpression of FABP7 decreased the viability of Hs578T cells during serum starvation. This led to cell-cycle arrest and a significant increase in cell death. We further showed that this phenotype was mediated by PPAR-regulated genes.\",\n \"All cell lines used in this study were purchased from American Type Culture Collection (Gaithersburg, MD). They were cultured in medium according to the manufacturer's protocol and supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), unless specified otherwise. Hs578T, MCF7, MDA-MB-231, and MDA-MB-435S were maintained in DMEM high glucose while BT474 was cultured in MEM. The cell lines were incubated at 37°C and 5% CO 2 atmosphere.\",\n \"For nutrient-deprivation experiments, the cells were first seeded in complete medium (10% FBS). The next day (hereafter referred to as 0 h), the cells were washed once with PBS and replaced with nutrient-deprived medium. For the glucose-and glutamine-starvation experiments, 10% FBS were added into the basal medium. Basal medium used for glucose starvation was glucose-free DMEM (Life Technologies, catalog no. 11966-025) that contained 4 mM l-glutamine. For the glutamine-starvation experiment, glutamine-free DMEM (Life Technologies, catalog no. 11960-044) containing 25 mM glucose was used. The serumstarvation experiment mimicked a culture condition with deprived lipids. The cells were challenged with serum-free DMEM (Life Technologies, catalog no. 11965-092) that contains high glucose (25 mM) with l-glutamine (4 mM), but without sodium pyruvate, HEPES, lipids, and growth factors.\",\n \"The PPAR- antagonist (GW6471) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a nonlethal concentration (2 M).\",\n \"A microarray profile of selected TNBC was obtained from the publicly available Gene Expression Omnibus (GEO) database. The search keyword used was \\\"triple negative breast cancer,\\\" and the results were filtered with \\\"Homo sapiens.\\\" The search was conducted from September 1 to 30, 2018.\",\n \"TNBC cell lines Hs578T and MDA-MB-231 were transduced with FABP7 gene using Thermo Scientific Precision LentiORF constructs at MOI 10 (Clone ID: PLOHS_100004067). For their control counterparts, the cells were transduced with Precision LentiORF RFP (catalog no. OHS5833). The transduced cells were selected with blasticidin for 30 days to generate stable cell line.\",\n \"Cells were seeded in 96-well plates and subjected to different growth conditions. At time of termination, 10 l of methyl thiazolyl tetrazolium (MTT) solution (5 mg/ml) (Sigma, St. Louis, MO) was added into each well for 4 h, before the addition of 100 l of 10% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals overnight. Absorbance was measured at 575 nm with reference to 650 nm. All experiments were performed in triplicate and repeated three times.\",\n \"Total RNA extraction was carried out using Trizol (Invitrogen, Carlsbad, CA) following the recommended protocol. About 500 ng of RNA was converted into cDNA using DyNAmo cDNA synthesis kit (Finnzymes, Vantaa, Finland). For quantitative RT-PCR (qRT-PCR), 5 ng/l cDNA template was added into pool solution containing 5× HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Tartu, Estonia), 10 pmol/l forward and reverse primers, and UltraPure distilled water. ABI StepOne Plus (Applied Biosystem, Foster City, CA) was used, and 40 cycles of amplification were performed. The expression of metabolic genes was normalized to housekeeping genes 18S rRNA and ribosomal protein L13a. Sequence of the primers used is described in supplemental Table S1.\",\n \"Protein lysate was harvested by scraping the cells in cold PBS. After centrifugation, PBS was discarded, and the cells were incubated in lysis buffer (0.1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1× phosphatase, and 1× protease inhibitors) for 30 min on ice. The mixture was centrifuged for 20 min, and supernatant was collected as protein lysate. Protein concentration was quantified using Bradford assay (Bio-Rad, Hercules, CA). A total of 30 g of protein was resolved on 15% SDS-PAGE prior to transferring on PVDF membrane. Target proteins were detected using primary Abs FABP7 (Cell Signaling Technology) and ECL prime Western blotting detection reagent (Amersham, GE Healthcare Lifesciences, Sweden) before being visualized with gel-documentation system (Biospectrum 410, UVP).\",\n \"The BrdU cell-proliferation assay kit (Cell Signaling Technology, Beverly, MA) was used to measure the cell-proliferation rate at 24, 48, and 72 h after serum starvation. The data were normalized to their counterparts cultured in complete medium. The cells were incubated with 10X BrdU solution for three h, prior to fixation for 30 min and staining with 1× BrdU Detection Antibody for 1 h. The stained cells were washed and incubated with 1× HRPconjugated secondary antibody solution for 30 min, before TMB solution was added. STOP solution was added after 15 min, and absorbance was read at 450 nm.\",\n \"Cell-cycle analysis was conducted on the cells at 0, 24, 48, and 72 h after serum starvation. The cells were fixed with 70% ethanol at 4°C for 30 min, before two washes with PBS. Enzymatic removal of RNA was achieved by incubating the cells with 100 g/ml RNase for 15 min before DNA staining with 50 g/ml propidium iodide. The cells were analyzed using a BD FACSCanto II flow cytometer, and the cell-cycle distribution of the cell was analyzed with Modfit LT software. The samples were run in triplicates, and the mean value was calculated.\",\n \"Cells were harvested for apoptosis assay at 0, 24, 48, and 72 h after serum starvation. Floating cells in the culture medium and the trypsinized cells were collected and washed once with ice-cold PBS, before they were resuspended in 100 l of assay buffer (1 part buffer: 9 parts distilled water) with 5 l of annexin V-PE and 5 l of 7AAD (BD Bioscience, Franklin Lakes, NJ). The cells were vortexed gently and kept in the dark for 15 min, before adding 400 l of buffer. The staining was analyzed using a BD FACSCanto II flow cytometer and viewed using FACS DiVa Software (BD Bioscience, San Jose, CA). All samples were run in triplicates, and mean value was calculated.\",\n \"GAPDH, glutaminase (GSL), and FAO enzyme activities were measured using calorimetric assay kits from Biomedical Research Service Center, State University of New York (Buffalo, NY). All samples were harvested using 1× Cell Lysis Solution. Protein concentration of the samples was assessed with a Bradford assay and normalized to 1 mg/ml. GAPDH activity was measured using GAPDH enzyme assay kit (catalog no. E-101). Briefly, 10 l of sample or water (as blank) was incubated in 50 l of GAPDH assay solution. After gentle agitation, the plate was kept in a non-CO 2 incubator at 37°C for 60 min. For GSL assay (catalog no. E-133), 10 l of sample was incubated in either 40 l of glutamine solution or water. Following a 2 h incubation period in a non-CO 2 incubator at 37°C, 50 l of TA assay solution was added, and the plate was further incubated for another 1 h. For FAO enzyme assay (catalog no. E-141), 50 l of FAO Assay Solution or control solution was added to 10 l of the protein sample. The plate was kept in a non-CO 2 incubator at 37°C for 60 min. All experiments were terminated by adding 50 l of 3% acetic acid, and the plate was read at optical density of 492 nm (OD 492) with a spectrophotometer. Blank reading was subtracted from the sample reading. GAPDH activity in IU/l unit was determined by multiplying OD by 16.98. Glutaminase (GLS) activity in IU/l unit = mol/ (l·min) = (OD × 1,000 × 150 l) ÷ (120 min × 0.6 cm × 18 × 10 l) = OD × 11.58. For FAO assay, the subtracted OD represents the FAO activity of the sample.\",\n \"Basal oxygen consumption rate (OCR) was carried out with a Mito Fuel Flex Test Kit on XFe96 Bioanalyzer (Agilent). Briefly, 4,000 cells were seeded on a Seahorse XF96-well assay plate in complete medium. The cells grown in complete medium were terminated at 24 h after seeding. For the serum-starvation experiment, the cells were left incubated overnight before they were washed with PBS and incubated in serum-free medium for 24 h. Upon termination, all wells were replaced with assay medium, and baseline OCR was immediately measured for 18 min. The basal OCR was normalized to protein concentration of the cells.\",\n \"The cells were seeded on a coverslip in complete medium. After overnight incubation, the cells were either harvested or challenged with serum-free medium for 24 h. The cells were fixed with 4% formaldehyde for 30 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 1 h. The cells were incubated with FABP7 primary antibody (1:2,000; Cell Signaling catalog no. 13347S) and PPAR- primary antibody (1:400; Santa Cruz Biotechnology, catalog no. sc-398394), followed by Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific). The incubation time was 90 min for primary antibody and 60 min for secondary antibody. The cells were washed three times with PBS after staining with each antibody. All the procedures were carried out on ice. The coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories Cat#H-1200) and analyzed with a Leica TCS SP5 II laser microscope (Leica, Heidelberg, Germany) using 60× objective.\",\n \"Statistical analysis assessing the difference between the means of two groups was performed using Student's t-test with Graph-Pad-Prism (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.\",\n \"The expression of FABP7 was determined in human breast cancer tissues and TNBC cell lines. Analysis from the GEO database (dataset GSE65194) revealed that FABP7 expression was significantly higher in TNBC (n = 55) compared with non-TNBC (n = 98) subtypes (Fig. 1A). When examined in a panel of breast cancer cell lines, FABP7 showed low expression in all cells including MCF7 (luminal A), BT474 (luminal B), MDA-MB-231, and Hs578T (TNBC) cells, when compared with MDA-MB-435S, a melanoma cell line reported to express high FABP7 (Fig. 1B). Correspondingly, FABP7 protein was not detected in these breast cancer cell lines (Fig. 1C).\",\n \"To understand the functional role of FABP7 in TNBC cells, we established FABP7-overexpressing Hs578T and MDA-MB-231 TNBC cells by transduction using lentiviral particles containing FABP7 open reading frame (Hs-FABP7 and 231-FABP7, respectively) and their control counterparts using lentiviral particles containing red fluorescent protein (Hs-RFP and 231-RFP, respectively) (Fig. 1D). When the cells were cultured in complete medium for 72 h, there was no difference in cell growth between cells expressing FABP7 and their respective RFP controls (Fig. 2). However, when cultured in glucose-or glutamine-deprived condition, both FABP7-overexpressing cells and RFP controls demonstrated substantial reduction in cell viability with greater effect observed in glucose-deprived than glutamine-deprived medium ( Fig. 2A,B).\",\n \"Interestingly, when lipids were significantly reduced during serum starvation, overexpression of FABP7 resulted in decreased viability of Hs578T cells (Fig. 2C). At 24 h after serum starvation, the cell viability of Hs-FABP7 cells decreased by 10%, which progressively decreased to approximately 70% at 72 h after starvation (P < 0.0001 vs. Hs-RFP cells). Cell viability of Hs-RFP cells was not affected by serum starvation, indicating that the decreased viability in Hs-FABP7 cells may be related to FABP7 expression. This phenotype appeared to be specific to Hs578T cells, as FABP7 expression did not affect the viability of MDA-MB-231 cells in serum-free medium (Fig. 2C).\",\n \"The reduction in cell viability of Hs-FABP7 cells during serum starvation resulted from a decrease in cell proliferation. Hs-FABP7 cells demonstrated a 70% reduction in BrdU uptake as early as 24 h after serum starvation, whereas the control Hs-RFP cells only showed a decrease by 20% (Fig. 3A). The nonresponsive MDA-MB-231 cells maintained their proliferation rate in the range of 85-89% during serum starvation, regardless of FABP7 expression.\",\n \"Flow-cytometry analysis showed that FABP7 induced cellcycle arrest in Hs578T cells during serum starvation ( Fig. 3B and supplemental Fig. S1). Hs-RFP cells showed a higher distribution of cells (80%) in G1 phase throughout serum starvation, indicating that the cells were able to complete the cell cycle and proliferate, parallel to increased cell viability over time. In contrast, Hs-FABP7 cells demonstrated a consistently higher percentage of cells in S and G2 phase upon serum starvation. For instance, at 48 h after serum starvation, 24% of Hs-FABP7 cells were accumulated in S phase and 8.23% in G2 phase, whereas only 5.15% and 1.99% of Hs-RFP cells were detected at S and G2 phase (P = 0.0052 for S phase and P = 0.0241 for G2 phase). The percentage of cells in S/G2 phase increased to 40.15% in Hs-FABP7 cells compared with 14.29% in Hs-RFP cells at 72 h after serum starvation. Cell arrest in S/G2 phase in Hs-FABP7 cells may partly contribute to the decreased cell viability during serum starvation. This effect was not observed in MDA-MB-231 cells (Fig. 3B).\",\n \"The decrease in Hs-FABP7 cell viability during serum starvation was potentially attributed to apoptosis and necrosis (Fig. 3C). More necrotic cell death and late apoptosis was observed in Hs-FABP7 cells (34.26% and 21.3%) than in Hs-RFP cells (18.83% and 4.63%) at 72 h after serum starvation (P < 0.0001). Upon serum starvation, increasing cell death was observed in MDA-MB-231 cells, but with no significant difference observed between 231-RFP and 231-FABP7 cells. Taken together, the decreased cell viability in Hs-FABP7 cells during serum starvation was due to reduced proliferation rate, cell cycle arrest at S and G2 phase, and cell death.\",\n \"Given the role of FABP7 in fatty acid metabolism, we investigated the mechanism of FABP7-induced cell death by exploring a panel of genes and enzyme activities involved in the metabolism of glucose, glutamine, and fatty acid ( Fig. 4 and supplemental Figs. S2, S3). In complete medium, despite the increase in glucose transporter 1 (GLUT1) mRNA expression, glycolysis-related genes phosphofructokinase 1 (PFK1) and GAPDH remained unchanged in Hs-FABP7 cells, whereas the genes regulating FAO, carnitine palmitoyltransferase 1A (CPT1A), and medium-chain acyl-CoA dehydrogenase (MCAD) were markedly upregulated (Fig. 4A). In Hs-FABP7 cells, the expression of GLS1, encoding the rate-limiting enzyme for glutaminolysis, was not significantly different than in Hs-RFP cells. When cultured in complete medium, Hs-FABP7 cells did not show any differences in GAPDH, GLS, and FAO activities when compared with Hs-RFP cells 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001. ( Fig. 4B-D). Subsequently, mitochondrial OCR was similar in Hs-FABP7 cells and in the control Hs-RFP cells (Fig. 4E).\",\n \"However, when challenged with serum starvation, a widespread downregulation of glucose and glutamine metabolism-related genes were observed in Hs-FABP7 cells when compared with Hs-RFP cells. Interestingly, despite the absence of serum (lipids) during serum starvation, the gene expression of CPT1A and MCAD was about 2-fold higher in Hs-FABP7 cells than Hs-RFP cells (Fig. 4A). Serum starvation generally decreased glycolytic enzyme activity in Hs578T cells, but FABP7-overexpressing cells had a further 30% reduction (P = 0.0121) in GAPDH activity compared with Hs-RFP cells (Fig. 4B). Although GLS activity remained not significantly different in both cells during serum starvation, GLS activity was significantly lower (P = 0.0296) in Hs-FABP7 cells cultured in serum starvation than in complete medium (Fig. 4C). Interestingly, FAO activity in Hs-FABP7 remained similar with the control Hs-RFP cells (Fig. 4D), despite upregulation of FAO-related genes. This may be explained by the lack of fatty acid in the culture medium to activate the metabolic pathway. Consequently, mitochondrial oxidative capacity of both Hs-RFP and Hs-FABP7 cells was generally lower during serum starvation (P = 0.0001 for both cells); however, Hs-FABP7 cells had much lower OCR (16.69 pmol/min/µg) compared with Hs-RFP cells (21.7 pmol/min/µg) (P < 0.05) (Fig. 4E). These data indicate a lower mitochondrial oxidative capacity particularly in Hs-FABP7 cells, probably due to limited bioenergetic substrates during serum starvation.\",\n \"It is worthwhile to note that, unlike Hs578T, MDA-MB-231 cells showed a rather different metabolic profile, as indicated by the expression of metabolic genes ( Fig. 4A and supplemental Fig. S2B). When cultured in complete medium, although FABP7 was not affecting the cell viability, glycolysis was upregulated in 231-FABP7 cells, with about a 1.7-fold increase in GAPDH expression. Meanwhile, a 4-fold decrease in GLS1 expression was observed, indicative of a downregulation of the glutaminolysis pathway. Upon serum starvation, 231-FABP7 cells demonstrated a marked increase in glucose and glutamine metabolismrelated genes, with more than 4-fold increase in GLUT1 and GLS1 expression.\",\n \"Taken together, our data show that Hs-FABP7 cells, when challenged with a serum-starved condition, were not able to metabolically adapt and effectively utilize other available energetic substrates, especially glucose, and, as a consequence, could not survive.\",\n \"The upregulation of CPT1A and MCAD mRNA, accompanied with a downregulation of Glut1 mRNA in Hs-FABP7 cells, indicated a possible role of PPAR- signaling, as these genes are direct targets of PPAR- transcription factor. Immunofluorescence staining showed that the expression of PPAR- protein was increased in the nucleus during serum starvation ( Fig. 5A and supplemental Fig. S4A). Similarly, FABP7 protein was observed to translocate into the nucleus (Fig. 5A and supplemental Fig. S4B) in Hs-FABP7 cells upon serum starvation, potentially acting as fatty acid chaperones to deliver ligands to PPAR- protein (23). This suggests that nuclear localization of FABP7 enhanced the action of PPAR- transcriptional activity during serum starvation in Hs-FABP7 cells.\",\n \"To investigate whether PPAR- activation was responsible for FABP7-mediated inhibition of cell viability in Hs-FABP7 cells, we treated the cells with a PPAR--specific inhibitor (GW6471). In both Hs578T and MDA-MB-231 cells, treatment of GW6471 at a concentration of 2 M in complete medium had no significant effect on cell viability (Fig. 5B). Yet, the same concentration of GW6471 reversed the cell-growth inhibition observed in Hs-FABP7 cells when exposed to serum-starved conditions (Fig. 5B and supplemental Fig. S5). Subsequent gene-expression analysis revealed that PPAR- target genes were downregulated (CPT1A: 2.43-fold; MCAD: 1.54-fold), when compared with vehicle-treated cells (Fig. 5C). A significant 2-fold increase of GLUT1, PFK1, glucose-6-phosphate dehydrogenase (G6PD), and isocitrate dehydrogenase 1 (IDH1) mRNA expression was observed in GW6471-treated Hs-FABP7 cells (Fig. 5C), indicating that the rescue of cell viability may be due to the switch from lipid to glucose as bioenergetic substrates in these cells. Taken together, FABP7-induced cell death in Hs578T cells during serum starvation is due to activated PPAR- signaling.\",\n \"In this study, we showed that overexpression of FABP7 disabled TNBC cell line Hs578T to metabolically adapt during serum starvation. FABP7-overexpressing cells failed to efficiently utilize glucose as bioenergetics substrates, when challenged with serum starvation. This was accompanied by increased expression of genes controlling FAO as it attempts to utilize free fatty acids liberated by lipolysis from the lipid storage that resulted from preexposure to complete medium (24). However, as free fatty acid storage was depleted, serum-starved FABP7-overexpressing cells were not able to further metabolically adapt and sustain ATP production. The failure of the cells to efficiently utilize glucose for ATP generation leads to cell death. We further showed that the metabolic impact of FABP7 in Hs578T cells during serum starvation is mediated by PPAR- signaling.\",\n \"FABP7 is predominantly expressed in the nervous system and mammary gland. In the mammary gland, FABP7 inhibits proliferation and promotes differentiation of mammary cells (25). Similarly, in the nervous system, FABP7 is involved in the differentiation of neurons and development of the cortex (26,27). The expression of FABP7 at the edge of astrocytes also suggested that FABP7 may regulate the motility of astrocytes by increasing the uptake of polyunsaturated fatty acids (28). Yet, the evidence of FABP7 expression in TNBC cell lines was rather limited (20,29). A previous study demonstrating the function of endogenous FABP7 in TNBC was reported using MDA-MB-435S, a melanoma cell line formerly misidentified as a TNBC cell line (30). Our study, however, provides evidence on the role of FABP7 as a mediator for tumor cell death during nutrient-deprived conditions. This finding indicates an anti-tumorigenic role of FABP7 in TNBC, which is mediated by specific nutrient availability.\",\n \"The metabolic vulnerabilities of FABP7-overexpressing Hs578T cells resulted in S/G2-phase arrest, which subsequently led to cell death. Serum starvation typically induces cell-cycle arrest at G1 phase (31,32), which is observed in Hs-RFP and MDA-MB-231 cells. These cells were able to proliferate, despite a lower proliferation rate, indicating that the lower mitochondrial activity in these cells during serum starvation was sufficient to overcome the metabolic checkpoint and complete the cell cycle (33). Hs-FABP7 cells, however, arrested at the S/G2 phase. G2 arrest has been described as another growth-restricting mechanism in mitogen-starved conditions, likely due to elevated E2F activity (34). Cell arrest could potentially lead to cell death, in which both apoptosis and necrosis were observed in Hs-FABP7 cells upon serum starvation. Apoptosis is a cellular suicide program triggered by stress signals such as nutrient deprivation, which results in the execution of caspasemediated cell death (35). Serum starvation has been shown to induce apoptosis in hepatocellular carcinoma cells (36) and MCF7 breast cancer cells (37), in which the latter was associated with an altered ratio of apoptosis regulating proteins Bax and Bcl-2. Necrosis, on the other hand, may occur when the minimal bioenergetic demands are not met (38), a condition similar to our observations where Hs-FABP7 cells may have limited bioenergetic substrates during serum starvation. In fact, there are common mediators of apoptosis and necrosis, which could potentially contribute to the two modes of cell death observed in our model (39). It is worth noting that FABP7 did not induce cell death in MDA-MB-231 cells during serum starvation, indicating that this cell line is probably more resilient to nutrient deprivation than Hs578T cells. The disparity in the response of Hs578T and MDA-MB-231 cells to serum starvation may also be attributed to differences in their metabolic profiles (40). Hence, it would be worthwhile to examine other TNBC cells with differing metabolic profiles, in delineating the molecular heterogeneity involved in cell death induced by metabolic vulnerabilities.\",\n \"During serum starvation, cancer cells sense and adapt to nutrient deprivation by upregulating glucose metabolism Fig. 5. FABP7-induced cell death in Hs578T cells during serum starvation was mediated by the action of PPAR- signaling. A: Confocal microscopy of immunofluorescence staining of FABP7 (green) and PPAR- (red) proteins in Hs578T and MDA-MB-231 cells after culture in either complete medium or serum-free medium for 24 h. Blue, DAPI. Scale bar, 50 m. Data shown are representative of two independent experiments. B: Cells were treated with PPAR- inhibitor GW6471 (2 M) in complete or serum-starved medium for 72 h, before analyzing for cell viability using an MTT assay. Data shown are mean ± SEM of triplicates; this is a representative of three independent experiments. *** P < 0.0001. C: mRNA expression of key metabolic genes after treatment with 2 M GW6471 in Hs-FABP7 cells for 24 h in serum starvation when compared with vehicle-treated cells. The genes were normalized to housekeeping gene 18S rRNA. CM, complete medium; SFM, serum-free medium. (41,42), besides releasing fatty acids from the lipid droplet through lipolysis for oxidation (24). Central to such adaption, PPAR- is known to be a lipid sensor that maintains cellular energy homeostasis. It is a ligand-activated class of nuclear hormone receptor that binds to peroxisome proliferator response elements (PPREs) to induce transcription of genes such as lipid processing. Its activation aims at increasing lipid combustion in order to yield more energy production, especially in a challenged environment (43). It was shown in rat hepatocytes that PPAR- expression is increased by fasting-and starvation-induced glucocorticoids (44), suggesting the role of PPAR- to maintain cell survival.\",\n \"In our model, increased PPAR- expression was observed when the cells were serum-starved, possibly as an adaptation strategy of the cells. The activation of PPAR- was observed in Hs-FABP7 cells during serum starvation, as reflected in the increase of PPAR- target genes, CPT1A and MCAD, which are involved in fatty acid mitochondrial uptake and oxidation (45). Our observation may indicate the usage of fatty acids liberated from lipid droplets as the substrates for FAO. However, the attempt of the cells to survive serum starvation failed as the lipid storage was limited and fatty acids were absent in the serum-free media. In addition, PPAR- activation also potentially inhibited glucose metabolism by targeting the consensus PPRE motif in the promoter region of Glut1, hence repressing its transcription (46). As a result, Hs-FABP7 cells could not efficiently utilize glucose as bioenergetic substrates when serumstarved, which eventually led to cell death.\",\n \"Our data suggest that PPAR--induced changes are FABP7-dependent. Literature evidence shows that in Cos-7 cells where FABPs and PPAR- are not detected, transfection of the proteins showed that PPAR- activity is amplified by FABP1 and FABP2 (47). In another study, PPAR- transactivation is proportional to levels of L-FABP in human hepatoma HepG2 cells line (48). In our model, upon serum starvation, increased nuclear FABP7 expression was observed, suggesting the role of FABP7 as fatty acids chaperones to transport PPAR- ligands into the nucleus for PPAR- activation. In murine L cells, transduction with FABP1 had dramatically enhanced the uptake of fatty acid into the nucleus (49). Incubation of rat liver nuclei with fluorescence-tagged FABP1 together with fatty acids demonstrated a strong fluorescence response in the nuclei, which otherwise did not happen when fatty acids were absent (50). There has been evidence indicating physical interaction between FABP1 and PPAR- in primary hepatocytes (51). Ablation of L-FABP gene expression significantly impaired fatty acid distribution in the nucleus and affected PPAR- activation in these cells (52). Hence, whether FABP7 and PPAR- require direct contact to induce cell death in TNBC needs to be further investigated. In short, our data suggest that FABP7 facilitates PPAR- activation in serum-starved condition by transporting its ligand (fatty acids) into the nucleus, before enhancing its transcriptional activity.\",\n \"Our study provides an understanding on the role of FABP7 in regulating the metabolic adaptation of TNBC cell lines. Transcriptional activation of PPAR- in FABP7overexpressing cell can limit their metabolic plasticity when challenged with serum starvation and resulted in reduced survival of these cells. Hence, manipulating metabolic stress in cells expressing FABP7 protein may potentially be an effective targeted therapeutic strategy in FABP7-positive TNBC patients.\"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["MATERIALS AND METHODS","Cell culture","Data mining at GEO database","Generation of FABP7-transduced TNBC cell lines","MTT assay","RNA extraction and qRT-PCR","Protein extraction and Western blotting","BrdU cell-proliferation assay","Cell-cycle analysis with propidium iodide","Annexin V apoptosis assay","Metabolic activity assays","Measurement of OCR","Immunofluorescence staining","Statistical analysis","RESULTS","FABP7 overexpression decreased viability of Hs578T, but not MDA-MB-231, cells in serum starvation","Decreased viability of Hs-FABP7 cells in serum starvation was due to reduced proliferation, cell-cycle arrest, and cell death","Perturbations in the metabolism and mitochondrial oxygen consumption in Hs-FABP7 cells during serum starvation","PPAR- signaling mediated FABP7-induced cell death in Hs578T cells under serum starvation","DISCUSSION","Fig. 1 .","Fig. 2 .","Fig.","Fig. 4 ."],"string":"[\n \"MATERIALS AND METHODS\",\n \"Cell culture\",\n \"Data mining at GEO database\",\n \"Generation of FABP7-transduced TNBC cell lines\",\n \"MTT assay\",\n \"RNA extraction and qRT-PCR\",\n \"Protein extraction and Western blotting\",\n \"BrdU cell-proliferation assay\",\n \"Cell-cycle analysis with propidium iodide\",\n \"Annexin V apoptosis assay\",\n \"Metabolic activity assays\",\n \"Measurement of OCR\",\n \"Immunofluorescence staining\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"FABP7 overexpression decreased viability of Hs578T, but not MDA-MB-231, cells in serum starvation\",\n \"Decreased viability of Hs-FABP7 cells in serum starvation was due to reduced proliferation, cell-cycle arrest, and cell death\",\n \"Perturbations in the metabolism and mitochondrial oxygen consumption in Hs-FABP7 cells during serum starvation\",\n \"PPAR- signaling mediated FABP7-induced cell death in Hs578T cells under serum starvation\",\n \"DISCUSSION\",\n \"Fig. 1 .\",\n \"Fig. 2 .\",\n \"Fig.\",\n \"Fig. 4 .\"\n]"},"annotations_table":{"kind":"list like","value":[],"string":"[]"},"annotations_tableref":{"kind":"list like","value":["Table S1"],"string":"[\n \"Table S1\"\n]"},"annotations_title":{"kind":"list like","value":["Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling","Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling"],"string":"[\n \"Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling\",\n \"Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling\"\n]"},"annotations_venue":{"kind":"list like","value":["Journal of Lipid Research"],"string":"[\n \"Journal of Lipid Research\"\n]"}}},{"rowIdx":84743,"cells":{"corpusid":{"kind":"number","value":236209571,"string":"236,209,571"},"updated":{"kind":"string","value":"2022-01-11T02:58:22Z"},"content_source_oainfo_license":{"kind":"string","value":"CCBY"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/jcmm.16803"},"content_source_oainfo_status":{"kind":"string","value":"GOLD"},"content_source_pdfsha":{"kind":"string","value":"ba785ecf67234bc1c401929c79e39a7028e5082e"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.1111/jcmm.16803"},"externalids_mag":{"kind":"null"},"externalids_pubmed":{"kind":"string","value":"34302427"},"externalids_pubmedcentral":{"kind":"string","value":"8419198"},"content_text":{"kind":"string","value":"\nO R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis\n2021\n\nShanshan Dong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nQi Tian \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nTengfei Zhu \nDepartment of Critical Care Medicine\nShenzhen Third People's Hospital\nShenzhenGuangdongChina\n\n| Kangli Wang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nGanting Lei \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nYanling Liu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nHaofeng Xiong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\n| Lu Shen \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nMeng Wang \nRongjuan Zhao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nHuidan Wu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\n| Bin Li \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nNational Clinical Research Center for Geriatric Disorders\nDepartment of Geriatrics\nXiangya Hospital\nCentral South University\nChangshaHunanChina\n\nQiumeng Zhang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nYujun Yao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\n| Hui Guo \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\n| Kun Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nHunan Key Laboratory of Molecular Precisional Medicine\nCentral South University\nChangshaHunanChina\n\n| Lu Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\n| Zhengmao Hu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nCenter for Medical Genetics\nHunan Key Laboratory of Animal Models for Human Diseases\nCentral South University\nCorrespondence Zhengmao Hu and Lu XiaChangshaHunanChina\n\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\n410078ChangshaHunanChina\n\nO R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis\n\nJ Cell Mol Med\n202110.1111/jcmm.16803Received: 10 February 2021 | Revised: 13 May 2021 | Accepted: 6 July 20218432 |\nHigh myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGFβ pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGFβ signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGFβ signalling and ECM synthesis in the KO cells. The fact that TGFβ signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGFβ signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGFβ signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGFβ signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.K E Y W O R D SECM, high myopia, SLC39A5, Smad, TGFβ signalling, zinc\n\n| INTRODUC TI ON\n\nMyopia is a condition of refractive error causing light to focus in front of the retina, with axial elongation as its most common pathology. High myopia, clinically defined as a refractive error of at least −6.00 diopters (D) or an axial length >26 mm, 1 is one of the leading causes of visual impairment worldwide 2,3 with a significantly increased prevalence. 3,4 Thus, the prevention of high myopia has become a global public health problem. Pedigree analyses and twin studies identified high myopia highly heritable. 5,6 Even though numerous loci (1q41, 4q25, 8p23, etc.) and causative genes (ZNF644, CCDC111, SCO2, etc.) have been identified, 7 the pathogenic mechanism of high myopia remains unclear.\n\nRecently, scleral extracellular matrix (ECM) synthesis or remodelling has been proposed as an underlying cause of high myopia. [8][9][10] According to this hypothesis, scleral collagen synthesis is regulated during myopia progression, 9 while scleral ECM is remodelled in myopic animal. 10 ECM synthesis and remodelling are targets of the TGFβ signalling pathway, one of the most reproducibly dysregulated pathways in myopia development. [10][11][12][13] Total or isoform-specific scleral TGFβ is frequently altered during myopia progression. 9,10,14 In the previous study, we first identified SLC39A5 as a high myopia-associated gene, and mutations of SLC39A5 dysregulated the BMP/TGFβ pathway. 15 Further research confirmed SLC39A5 as one of the top three genes contributing to nonsyndromic high myopia. 16 This study aimed to investigate the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia. We found that SLC39A5 played a key role in maintaining intracellular zinc homeostasis. SLC39A5 depletion-induced zinc deficiency directly destabilized Smad proteins, and Smads instability further impaired TGFβ signalling-mediated ECM synthesis, thus contributing to the pathogenesis of high myopia. \n\n\n| MATERIAL S AND ME THODS\n\n\n| Cell culture\n\n\n| SLC39A5 knockout cell line construction\n\nAccording to the protocols from Zhang laboratory (http://www. genom e-engin eering.org/gecko/), target-specific sgRNA was cloned into the lentiCRISPRv2 vector (sgRNAs are listed in Table S1). Then, lentiviruses were packaged in HEK293T cells by co-transfection of CRISPR-sgRNA/pVSVG/pspAX2. The supernatants containing viral particles were collected 48 h after transfection and filtered (0.22 μm pore size). Then, HEK293 cells were transduced with viral supernatant supplemented and selected with 1 μg/ml puromycin.\n\nAfter transfection, cells were detached and seeded in 10 ml Petri dishes at a density of 200 cells per plate. Puromycin (2 µg/ml) was added to allow the selection of positive clones during the following two weeks. Positive clones were isolated and transferred to sixwell plates. The SLC39A5 knockout clones were validated by direct Sanger sequencing (primers are listed in Table S2). The negative control cell line was generated from HEK293 transformed with the identical lentiviral vector lacking specific sgRNAs.\n\n\n| Zinc quantification\n\nZinc quantification assay (Abcam Cat# ab102507) was performed for accurate measurement of zinc levels according to the manufacturer's instructions. In brief, cells were harvested with NP-40 lysis buffer (EDTA-free), and proteins were deproteinized by an equal volume of 7% TCA Solution Buffer. The supernatants were transferred to new clean tubes after centrifugation. 200 μl Zinc Reaction Buffer was applied to 50 μl tested samples in a 96-well plate. After a 10-min incubation at room temperature, the OD 560 was measured in a microplate reader. The zinc standard curve was plotted after subtracting the background value, and the tested sample concentration was calculated from the standard curve.\n\n\n| RNA-seq and analysis\n\nTotal RNA for RNA-seq library was extracted using TRIzol (Invitrogen Cat# AM9738), validated on a NanoDrop 1000 Spectrophotometer (Thermo) and quantitated using Qubit (Thermo). RNA-seq libraries were ing data were filtered with SOAPnuke (v1.5.2), then mapped to the reference genome using HISAT2 (v2.0.4) and aligned to the reference coding gene set using Bowtie2 (v2.2.5). Differential gene expression analysis between the target and reference sets of treatments was determined using DESeq2 (https://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq.html). Enrichment analysis of annotated different expressed genes was performed with the Phyper (https://en.wikip edia.org/wiki/ Hyper geome tric_distr ibution) based on the hypergeometric test. Table S3). All samples were run in triplicate. qRT-PCR data were analysed by LightCycler 96 software. Relative candidate gene mRNA levels were normalized to those of β-actin or GAPDH. A value of p < 0.05 was considered to be statistically significant.\n\n\n| qRT-PCR validation\n\n\n| Plasmid construction and transient transfection\n\nThe Smad1 zinc-binding site substitutions were generated to assess the correlation between zinc-binding capacity and protein stability. Smad1 WT plasmid was brought from OriGene (OriGene Cat# RC200299), and the four zinc-binding site substitutions (C64A, C109A, C121A and H126A) were separately generated using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in Table S4). Table S5).\n\nFinally, the rescue sequence was cloned into the pLVX-IRES-puro vector to construct the KO rescue plasmid.\n\nSLC30A1 shRNA interference plasmids were also constructed to rescue the poor intracellular zinc level of SLC39A5 KO cells by suppressing the zinc efflux baseline. The three targeting sequences were obtained from the siRNA sequence database (Thermo) and then generated into the pFUGW-lentiviral vector between the XbaI and BamHI restriction sites, respectively (primers are listed in Table S6). \n\n\n| Immunoblotting\n\n\n| Immunofluorescence\n\nCells were seeded on specific coverslips in 12-well plates.\n\nThe cell slides were fixed in 4% paraformaldehyde for 10 min.\n\nPermeabilization was performed with 0.1% PBST (phosphatebuffered saline and 0.1% Triton) for 15 min. After blocking with 5% BSA for 1 h, the cell slides were incubated with primary antibody at recommended dilutions overnight in 4°C and stained with Alex Fluor 488-conjugated second antibody (Jackson Cat# 111-545-144) for 1 h protected from light. DAPI was applied for 1 min as a nuclear marker. Images were acquired with an Eclipse TCS-SP5 inverted confocal microscope (Leica).\n\n\n| Wound healing capacity test\n\nHEK293 cells were seeded into the 2-well culture insert (Ibidi Cat# 81176) pre-coated by Matrigel. After cell attachment for approximately 24 h to form an optically confluent monolayer, the culture insert was removed to create the wound gap and the cells were then cultured with serum-free medium. The wound gap closure was monitored by taking pictures with an Eclipse inverted microscope (Leica) at different time points.\n\n\n| Statistical analysis\n\nGraphPad Prism was used to calculate and plot the mean ± SEM of measured quantities. Significances were assessed by two-way analysis of variance (ANOVA)/Mann-Whitney U test (nonparametric)/Student's t test (parametric), p-value < 0.05 was considered to indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.\n\n\n| RE SULTS\n\n\n| SLC39A5 depletion impairs ECM synthesis\n\nTwo SLC39A5 knockout (KO) cell lines were developed using the CRISPR-Cas9 system in the human embryonic kidney cell line (HEK293) ( Figure 1A). Cell morphology anomalies were markedly observed in the SLC39A5-depleted cells. After regular growth for 2 days, the KO cells revealed a tendency to form small cell aggregates instead of a typical optically confluent monolayer ( Figure 1B).\n\nThis phenotype suggested a potential impairment or abnormality of ECM generation. Considering the influence of ECM dynamics on cell migration, invasion and morphogenesis, we immediately investigated the cellular motility and migratory capacity of the SLC39A5depleted HEK293 cells. Scratch assay was performed in both groups.\n\nA 2-well culture insert generated a uniform gap in the confluent monolayer, and wound healing was imaged at different time points.\n\nThe results showed that the gap in the control group was completely healed after 24 h, while the wound gap in the KO group was not able to close ( Figure S1; Figure 1C).\n\nTo investigate the overall alteration caused by SLC39A5 depletion, RNA-sequencing (RNA-seq) was performed using a Immunofluorescence also verified the decrease in these ECM proteins ( Figures S3 and S4).\n\n\n| SLC39A5 depletion suppresses TGFβ signalling\n\nThe transcription of collagens, FN and LN is closely related to TGFβ signalling, and we have already determined that mutations of SLC39A5 dysregulated TGFβ signalling previously. 15 Considering the transcriptional downregulation of collagens, FN and LN in SLC39A5 KO cells, we hypothesized that reduced SLC39A5 expression would inhibit the TGFβ signalling pathway. Hence, we examined the expression of Smad proteins, which lie on the centre of the TGFβ signalling pathway. The results showed an evident upregulation of total Receptor-Smads (R-Smads) (Smad1, Smad2/3) expression along with a significant decrease in phosphorylated Smad expression (pSmad1/5/9, pSmad2/3) ( Figure S5). Additionally, Co-Smad (Smad4) expression was also increased, while inhibitor-Smad (Smad7) was not affected (Figure S5), suggesting a suppressed activation of the TGFβ signalling, which was confirmed through immunofluorescence ( Figures S6 and S7). The qRT-PCR performed to assess the transcriptional levels of Smad1, Smad2 and Smad4 in KO cells showed increased mRNA expression of the tested proteins ( Figure S8). To validate the phenotype of inhibited TGFβ signalling activation and insufficient ECM synthesis resulting from SLC39A5 depletion, rescue experiments were carefully carried out. As shown in Figure 3, re-expression of SLC39A5 restored intracellular zinc level ( Figure 3A). TGFβ signalling activation and ECM synthesis were also rescued ( Figure \n\n\n| SLC39A5 depletion induces zinc deficiency and destabilizes Smad protein\n\nThe TGFβ pathway is a zinc-regulated pathway ( Figure S9), while SLC39A5 is a zinc transporter. 17,18 These facts prompted us to confirm the involvement of zinc homeostasis in TGFβ signalling alteration in SLC39A5 KO cells. Thus, zinc quantification was performed to detect intracellular zinc levels. As shown in Thus, we selected the most widely expressed zinc efflux protein SLC30A1 (20), as a target. Surprisingly, silencing SLC30A1 counterbalanced the influence of SLC39A5 depletion, which significantly reverted intracellular zinc level close to normal ( Figure S10) and normalized the TGFβ signalling pathway and ECM synthesis ( Figure S11).\n\n\n| DISCUSS ION\n\nSLC39A5 was first identified as a high myopia pathogenic gene in our previous study 15 and was later confirmed as one of the top three genes contributing to nonsyndromic high myopia. 16,[20][21][22][23] However, the mechanisms by which SLC39A5 contributes to high myopia are not well understood. Here, we demonstrated that SLC39A5 may play a role in the pathogenesis of high myopia by modulating TGFβ signalling-mediated ECM synthesis through the regulation of intracellular zinc homeostasis. SLC39A5 depletion significantly lowered the intracellular zinc level, which destabilized Smad proteins, and further inhibited TGFβ signalling-mediated ECM synthesis, which ultimately contributed to the pathogenesis of high myopia.\n\nZinc homeostasis is crucial for all organisms as it serves as a catalytic or structural cofactor for multiple types of proteins. Zinc homeostasis depends on the transport of zinc in both directions across the plasma membrane and both in and out of various vesicular compartments. 24 The SLC39 family facilitates zinc transport into the cytoplasm in the opposite direction to SLC30 family members. 25 Within this network, SLC39A5 is a specific zinc transporter. 17,18,26 Our work confirmed the zinc uptake characteristic of SLC39A5, in which SLC39A5 depletion caused an intracellular decline in zinc levels in HEK293 or lymphocyte cells. Once zinc homeostasis was disrupted, the catalytic or structural stability of proteins was impaired, suggesting that SLC39A5 depletion, indirectly, caused this impairment.\n\nZinc has a profound effect on stabilization of zinc-binding proteins, 27-31 since the proteins responsible for cellular zinc buffering will bind zinc transiently; therefore, their function will strongly depend on the cellular zinc status. 32 It has been proposed that zinc chelation affects protein stability through various mechanisms. The human SOD1 protein requires zinc binding to facilitate the restructuring of apoproteins, 33 while the putative protease of Bacteroides thetaiotaomicron (ppBat) requires zinc chelation to link the β-strands to α-helices in protein structures. 34 In our study, the R-Smads and Co-Smad, which are pivotal to the BMP/TGFβ signalling pathway, 35 F I G U R E 2 SLC39A5 depletion impairs extracellular matrix (ECM) synthesis. (A) shows the pathway enrichment analysis for the differentially expressed genes between the control and KO subpopulations of HEK293 cells. Pathway analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics Resources, with an enrichment p-value cut-off of 0.01. (B) shows the qRT-PCR validation of the differentially expressed genes of the ECM members indicated by RNA-seq. (C-E) shows the expression of several ECM components (COL1, COL2, COL4, FN and LN) in the supernatant, whole-cell lysate or intracellular lysate (trypsin digested) of wild-type (WT) and SLC39A5 knockout (KO) HEK293 cells. All tested ECM components were decreased in both the whole lysate and intracellular lysate. (B) and (C) show statistical analyses of the ECM components in the whole lysate and intracellular lysate, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are zinc-binding proteins. We found that Smad proteins were rapidly degraded during SLC39A5 deletion-induced zinc deficiency.\n\nFurther results suggested that loose zinc coupling, facilitated by a mutant ion-binding site, directly destabilized the exogenous Smad1 proteins, which clearly confirms the necessity of zinc chelation in maintaining the protein stability of Smads, while disturbances in zinc homeostasis induce protein instability.\n\nZinc binding has also been implicated in protein phosphorylation and subcellular translocation. 36 Non-zinc-bound protein kinase C delta (PKCδ) showed a significantly different structure near its phosphorylation site, compared with when it was zinc-bound. Subcellular translocation of PKCδ was also influenced by intracellular zinc. These findings expand our understanding of the phosphorylation decrease in R-Smads during SLC39A5-depleted conditions.\n\nSince R-Smads are directly phosphorylated by upstream receptor kinases, which then form heteromeric complexes with Smad4 to translocate into the nucleus, 37 the zinc deficiency induced by SLC39A5 depletion may barely maintain the Smad protein in a favourable structure for phosphorylation, and may lead to nucleus translocation retention, further impairing the TGFβ signalling.\n\nIt is well recognized that TGFβ signalling directly affects ECM dynamics or triggers it when mediating other biological processes. [38][39][40][41] Herein, suppressed TGFβ signalling in the zinc-deficient conditions restrained ECM synthesis in HEK293. Recently, ECM synthesis and remodelling have attracted significant attention in high myopia aetiology studies. [8][9][10]42 We found that the transcription of all ECM proteins tested (COL1, COL2, COL4, FN and LN) was decreased under zinc-deficient conditions. ECM-induced cell morphogenesis According to this hypothesis, the SLC39A5-induced zinc deficiency suppresses TGFβ signalling, leading to insufficient ECM synthesis, which may ultimately contribute to high myopia development. We further validated this hypothesis by performing rescue experiments.\n\nAs expected, of SLC39A5 re-expression, which recovers zinc influx, and SLC30A1 silencing, which blocks zinc efflux, could identically rescue the alteration of R-Smad phosphorylation and ECM synthesis in SLC39A5-depleted HEK293 cells, by re-establishing zinc homeostasis.\n\nUpon reviewing the relationship between zinc and high myopia, we found plenty of indications of zinc involvement in high myopia development. 43 First, zinc is present abundantly in the ocular system, including the retina and choroid; thus, zinc deficiency produces a variety of ocular manifestations. Furthermore, a number of high myopia gene products are zinc-related proteins.\n\nAmong them, ZNF644, ZFHX1B, ZC3H11B and SCO2 are zinc finger proteins, and HGF and IGF1 are positively regulated by intracellular zinc concentration. Additionally, certain pathogenic genes (LAMA1 and TGFB1) may even be regulated by intracellular zinc, according to the zinc-TGFβ signalling-ECM synthesis flow.\n\nFinally, multiple small molecules that serve as chaperones for disease-related genes or pathways are zinc-related proteins. Smad proteins, together with their anchors (SARA and HGS), are either zinc-binding or zinc finger proteins. These findings strongly highlight the correlation between zinc and high myopia.\n\nIn summary, our data suggest that depletion of SLC39A5 induces zinc deficiency, which restrains TGFβ signalling-mediated ECM synthesis, thus possibly contributing to high myopia pathogenesis. Zinc homeostasis appears to be a dominant aetiological factor of high myopia development. \n\n\nACK N OWLED G EM ENTS\n\n\nCO N FLI C T O F I NTE R E S T\n\nThe authors confirm that there are no conflicts of interest. \n\n\nAUTH O R CO NTR I B UTI O N\n\n\nDATA AVA I L A B I L I T Y S TAT E M E N T\n\nThe data that support the findings of this study are available from the corresponding author upon reasonable request.\n\n\nO RCI D\n\n\nShanshan Dong\n\nhttps://orcid.org/0000-0002-1605-7066\n\nZhengmao Hu https://orcid.org/0000-0002-3921-8205\n\n\nR E FE R E N C E S\n\n\nHEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.\n\n\nprepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-\n\n\nTotal RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in\n\n\nSLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in\n\n\nCells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.\n\n\nSLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.\n\n\n3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).\n\nFigure 4 ,\n4SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.\n\n\nin the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.\n\n\nThis work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].\n\n\nShanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). 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J Ophthalmol. 2019;2019:1-7.\n\nAdditional supporting information may be found online in the Supporting Information section. How to cite this article. S U Pp O Rti N G I N Fo R M Ati O N ; Dong, S Tian, Q Zhu, T , S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Dong S, Tian Q, Zhu T, et al.\n\nSLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. 10.1111/jcmm.16803J Cell Mol Med. 25SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. J Cell Mol Med. 2021;25:8432- 8441. https://doi.org/10.1111/jcmm.16803\n"},"annotations_abstract":{"kind":"list like","value":["High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGFβ pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGFβ signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGFβ signalling and ECM synthesis in the KO cells. The fact that TGFβ signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGFβ signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGFβ signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGFβ signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.K E Y W O R D SECM, high myopia, SLC39A5, Smad, TGFβ signalling, zinc"],"string":"[\n \"High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGFβ pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGFβ signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGFβ signalling and ECM synthesis in the KO cells. The fact that TGFβ signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGFβ signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGFβ signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGFβ signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.K E Y W O R D SECM, high myopia, SLC39A5, Smad, TGFβ signalling, zinc\"\n]"},"annotations_author":{"kind":"list like","value":["Shanshan Dong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Qi Tian \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Tengfei Zhu \nDepartment of Critical Care Medicine\nShenzhen Third People's Hospital\nShenzhenGuangdongChina\n","| Kangli Wang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Ganting Lei \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Yanling Liu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Haofeng Xiong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","| Lu Shen \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Meng Wang ","Rongjuan Zhao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Huidan Wu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","| Bin Li \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nNational Clinical Research Center for Geriatric Disorders\nDepartment of Geriatrics\nXiangya Hospital\nCentral South University\nChangshaHunanChina\n","Qiumeng Zhang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","Yujun Yao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","| Hui Guo \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","| Kun Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nHunan Key Laboratory of Molecular Precisional Medicine\nCentral South University\nChangshaHunanChina\n","| Lu Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n","| Zhengmao Hu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nCenter for Medical Genetics\nHunan Key Laboratory of Animal Models for Human Diseases\nCentral South University\nCorrespondence Zhengmao Hu and Lu XiaChangshaHunanChina\n\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\n410078ChangshaHunanChina\n"],"string":"[\n \"Shanshan Dong \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Qi Tian \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Tengfei Zhu \\nDepartment of Critical Care Medicine\\nShenzhen Third People's Hospital\\nShenzhenGuangdongChina\\n\",\n \"| Kangli Wang \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Ganting Lei \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Yanling Liu \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Haofeng Xiong \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Lu Shen \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Meng Wang \",\n \"Rongjuan Zhao \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Huidan Wu \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Bin Li \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\\nNational Clinical Research Center for Geriatric Disorders\\nDepartment of Geriatrics\\nXiangya Hospital\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Qiumeng Zhang \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"Yujun Yao \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Hui Guo \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Kun Xia \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\\nHunan Key Laboratory of Molecular Precisional Medicine\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Lu Xia \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\",\n \"| Zhengmao Hu \\nCenter for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\\n\\nCenter for Medical Genetics\\nHunan Key Laboratory of Animal Models for Human Diseases\\nCentral South University\\nCorrespondence Zhengmao Hu and Lu XiaChangshaHunanChina\\n\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\n410078ChangshaHunanChina\\n\"\n]"},"annotations_authoraffiliation":{"kind":"list like","value":["Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of 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Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\",\n \"Center for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\",\n \"Center for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\",\n \"Center for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\",\n \"National Clinical Research Center for Geriatric Disorders\\nDepartment of Geriatrics\\nXiangya Hospital\\nCentral South University\\nChangshaHunanChina\",\n \"Center for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South University\\nChangshaHunanChina\",\n \"Center for Medical Genetics\\nSchool of Life Sciences\\nHunan Key Laboratory of Medical Genetics\\nCentral South 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Hu\"\n]"},"annotations_bibauthor":{"kind":"list like","value":["T L Young, ","S M Ronan, ","L A Drahozal, ","I G Morgan, ","K Ohno-Matsui, ","Saw Sm Myopia, ","B A Holden, ","T R Fricke, ","D A Wilson, ","E Dolgin, ","C J Hammond, ","H Snieder, ","C E Gilbert, ","T D Spector, ","A P Klein, ","B Suktitipat, ","P Duggal, ","X B Cai, ","S R Shen, ","D F Chen, ","Q Zhang, ","Z B Jin, ","J A Rada, ","S Shelton, ","T T Norton, ","A I Jobling, ","M Nguyen, ","A Gentle, ","N A Mcbrien, ","A I Jobling, ","R Wan, ","A Gentle, ","B V Bui, ","N A Mcbrien, ","B Y Chen, ","C Y Wang, ","W Y Chen, ","J X Ma, ","D S Lam, ","W S Lee, ","Y F Leung, ","Y Seko, ","H Shimokawa, ","T Tokoro, ","P C Wu, ","C L Tsai, ","G M Gordon, ","H Guo, ","Jin X Zhu, ","T , ","X B Cai, ","Y H Zheng, ","D F Chen, ","J Dufner-Beattie, ","Y M Kuo, ","J Gitschier, ","G K Andrews, ","F Wang, ","B E Kim, ","M J Petris, ","D J Eide, ","J Chai, ","J W Wu, ","N Yan, ","J Massague, ","N P Pavletich, ","Y Shi, ","C Y Feng, ","X Q Huang, ","X W Cheng, ","R H Wu, ","F Lu, ","Z B Jin, ","Z B Jin, ","J Wu, ","X F Huang, ","L Wan, ","B Deng, ","Z Wu, ","X Chen, ","D Jiang, ","J Li, ","X Xiao, ","R D Palmiter, ","L Huang, ","D J Eide, ","J Jeong, ","D J Eide, ","W Kou, ","H S Kolla, ","A Ortiz-Acevedo, ","D C Haines, ","M Junker, ","G R Dieckmann, ","L H Nguyen, ","T T Tran, ","Ltn Truong, ","H H Mai, ","T T Nguyen, ","D Pretzer, ","B Schulteis, ","Vander Velde, ","D G Smith, ","C D Mitchell, ","J W Manning, ","M C , ","T Mabrouk, ","G Lemay, ","C Iannuzzi, ","M Adrover, ","R Puglisi, ","R Yan, ","P A Temussi, ","A Pastore, ","K Kluska, ","J Adamczyk, ","A Krężel, ","S Z Potter, ","H Zhu, ","B F Shaw, ","T Knaus, ","M K Uhl, ","S Monschein, ","S Moratti, ","K Gruber, ","P Macheroux, ","J Massagué, ","J Seoane, ","D Wotton, ","K G Slepchenko, ","J M Holub, ","Y V Li, ","Y Shi, ","J Massagué, ","J Tan, ","Z H Deng, ","S Z Liu, ","J T Wang, ","C Huang, ","N Malachkova, ","D Yatsenko, ","G Ljudkevich, ","V Shkarupa, ","Y 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T L Young, S M Ronan, L A Drahozal, Am J Hum Genet. 63Young TL, Ronan SM, Drahozal LA, et al. Evidence that a locus for familial high myopia maps to chromosome 18p. Am J Hum Genet. 1998;63:109-119.",". I G Morgan, K Ohno-Matsui, Saw Sm Myopia, Lancet. 379Morgan IG, Ohno-Matsui K, Saw SM. Myopia. Lancet. 2012;379:1739-1748.","Global prevalence of myopia and high myopia and temporal trends from. B A Holden, T R Fricke, D A Wilson, Ophthalmology. 123Holden BA, Fricke TR, Wilson DA, et al. Global prevalence of myo- pia and high myopia and temporal trends from 2000 through 2050. Ophthalmology. 2016;123:1036-1042.","The myopia boom. E Dolgin, Nature. 519Dolgin E. The myopia boom. Nature. 2015;519:276-278.","Genes and environment in refractive error: the twin eye study. C J Hammond, H Snieder, C E Gilbert, T D Spector, Invest Ophthalmol Vis Sci. 42Hammond CJ, Snieder H, Gilbert CE, Spector TD. Genes and envi- ronment in refractive error: the twin eye study. Invest Ophthalmol Vis Sci. 2001;42:1232-1236.","Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study. A P Klein, B Suktitipat, P Duggal, Arch Ophthalmol. 127Klein AP, Suktitipat B, Duggal P, et al. Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study. Arch Ophthalmol. 2009;127:649-655.","An overview of myopia genetics. X B Cai, S R Shen, D F Chen, Q Zhang, Z B Jin, Exp Eye Res. 188107778Cai XB, Shen SR, Chen DF, Zhang Q, Jin ZB. An overview of myopia genetics. Exp Eye Res. 2019;188:107778.","The sclera and myopia. J A Rada, S Shelton, T T Norton, Exp Eye Res. 82Rada JA, Shelton S, Norton TT. The sclera and myopia. Exp Eye Res. 2006;82:185-200.","Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression. A I Jobling, M Nguyen, A Gentle, N A Mcbrien, J Biol Chem. 279Jobling AI, Nguyen M, Gentle A, McBrien NA. Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression. J Biol Chem. 2004;279:18121-18126.","Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. A I Jobling, R Wan, A Gentle, B V Bui, N A Mcbrien, Exp Eye Res. 88Jobling AI, Wan R, Gentle A, Bui BV, McBrien NA. Retinal and choroi- dal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. Exp Eye Res. 2009;88:458-466.","Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model. B Y Chen, C Y Wang, W Y Chen, J X Ma, Graefes Arch Clin Exp Ophthalmol. 251Chen BY, Wang CY, Chen WY, Ma JX. Altered TGF-β2 and bFGF expres- sion in scleral desmocytes from an experimentally-induced myopia guinea pig model. Graefes Arch Clin Exp Ophthalmol. 2013;251:1133-1144.","TGFbeta-induced factor: a candidate gene for high myopia. D S Lam, W S Lee, Y F Leung, Invest Ophthalmol Vis Sci. 44Lam DS, Lee WS, Leung YF, et al. TGFbeta-induced factor: a candidate gene for high myopia. Invest Ophthalmol Vis Sci. 2003;44:1012-1015.","Expression of bFGF and TGFbeta 2 in experimental myopia in chicks. Y Seko, H Shimokawa, T Tokoro, Invest Ophthalmol Vis Sci. 36Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF- beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36:1183-1187.","Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice. P C Wu, C L Tsai, G M Gordon, Mol Vis. 21Wu PC, Tsai CL, Gordon GM, et al. Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice. Mol Vis. 2015;21:138-147.","SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia. H Guo, Jin X Zhu, T , J Med Genet. 51Guo H, Jin X, Zhu T, et al. SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia. J Med Genet. 2014;51:518-525.","Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a crosssectional study in 731 Chinese patients. X B Cai, Y H Zheng, D F Chen, Invest Ophthalmol Vis Sci. 60Cai XB, Zheng YH, Chen DF, et al. Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a cross- sectional study in 731 Chinese patients. Invest Ophthalmol Vis Sci. 2019;60:4052-4062.","The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5. J Dufner-Beattie, Y M Kuo, J Gitschier, G K Andrews, J Biol Chem. 279Dufner-Beattie J, Kuo YM, Gitschier J, Andrews GK. The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5. J Biol Chem. 2004;279:49082-49090.","The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells. F Wang, B E Kim, M J Petris, D J Eide, J Biol Chem. 279Wang F, Kim BE, Petris MJ, Eide DJ. The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polar- ized cells. J Biol Chem. 2004;279:51433-51441.","Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding. J Chai, J W Wu, N Yan, J Massague, N P Pavletich, Y Shi, J Biol Chem. 278Chai J, Wu JW, Yan N, Massague J, Pavletich NP, Shi Y. Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding. J Biol Chem. 2003;278:20327-20331.","Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients. C Y Feng, X Q Huang, X W Cheng, R H Wu, F Lu, Z B Jin, Sci Rep. 7Feng CY, Huang XQ, Cheng XW, Wu RH, Lu F, Jin ZB. Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients. Sci Rep. 2017;7:1-9.","Trio-based exome sequencing arrests de novo mutations in early-onset high myopia. Z B Jin, J Wu, X F Huang, Proc Natl Acad Sci. 114Jin ZB, Wu J, Huang XF, et al. Trio-based exome sequencing arrests de novo mutations in early-onset high myopia. Proc Natl Acad Sci USA. 2017;114:4219-4224.","Exome sequencing study of 20 patients with high myopia. L Wan, B Deng, Z Wu, X Chen, PeerJ. 65552Wan L, Deng B, Wu Z, Chen X. Exome sequencing study of 20 pa- tients with high myopia. PeerJ. 2018;6:e5552.","Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with earlyonset high myopia by exome sequencing. D Jiang, J Li, X Xiao, Invest Ophthalmol Vis Sci. 56Jiang D, Li J, Xiao X, et al. Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with early- onset high myopia by exome sequencing. Invest Ophthalmol Vis Sci. 2014;56:339-345.","Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. R D Palmiter, L Huang, Pflugers Arch. 447Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. 2004;447:744-751.","Zinc transporters and the cellular trafficking of zinc. D J Eide, Biochem Biophys Acta. 1763Eide DJ. Zinc transporters and the cellular trafficking of zinc. Biochem Biophys Acta. 2006;1763:711-722.","The SLC39 family of zinc transporters. J Jeong, D J Eide, Mol Aspects Med. 34Jeong J, Eide DJ. The SLC39 family of zinc transporters. Mol Aspects Med. 2013;34:612-619.","Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36. W Kou, H S Kolla, A Ortiz-Acevedo, D C Haines, M Junker, G R Dieckmann, J Biol Inorg Chem. 10Kou W, Kolla HS, Ortiz-Acevedo A, Haines DC, Junker M, Dieckmann GR. Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36. J Biol Inorg Chem. 2005;10:167-180.","Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability. L H Nguyen, T T Tran, Ltn Truong, H H Mai, T T Nguyen, Biochemistry. 59Nguyen LH, Tran TT, Truong LTN, Mai HH, Nguyen TT. Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability. Biochemistry. 2020;59:1378-1390.","Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom. D Pretzer, B Schulteis, Vander Velde, D G Smith, C D Mitchell, J W Manning, M C , Pharm Res. 9Pretzer D, Schulteis B, Vander Velde DG, Smith CD, Mitchell JW, Manning MC. Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom. Pharm Res. 1992;9:870-877.","Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability. T Mabrouk, G Lemay, J Virol. 68Mabrouk T, Lemay G. Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability. J Virol. 1994;68:5287-5290.","The role of zinc in the stability of the marginally stable IscU scaffold protein. C Iannuzzi, M Adrover, R Puglisi, R Yan, P A Temussi, A Pastore, Protein Sci. 23Iannuzzi C, Adrover M, Puglisi R, Yan R, Temussi PA, Pastore A. The role of zinc in the stability of the marginally stable IscU scaffold protein. Protein Sci. 2014;23:1208-1219.","Metal binding properties, stability and reactivity of zinc fingers. K Kluska, J Adamczyk, A Krężel, Coord Chem Rev. 367Kluska K, Adamczyk J, Krężel A. Metal binding proper- ties, stability and reactivity of zinc fingers. Coord Chem Rev. 2018;367:18-64.","Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein. S Z Potter, H Zhu, B F Shaw, J Am Chem Soc. 129Potter SZ, Zhu H, Shaw BF, et al. Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substan- tially influences the structure and stability of the entire homodi- meric protein. J Am Chem Soc. 2007;129:4575-4583.","Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. T Knaus, M K Uhl, S Monschein, S Moratti, K Gruber, P Macheroux, Biochem Biophys Acta. 1844Knaus T, Uhl MK, Monschein S, Moratti S, Gruber K, Macheroux P. Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. Biochem Biophys Acta. 2014;1844:2298-2305.","Smad transcription factors. J Massagué, J Seoane, D Wotton, Genes Dev. 19Massagué J, Seoane J, Wotton D. Smad transcription factors. Genes Dev. 2005;19:2783-2810.","Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). K G Slepchenko, J M Holub, Y V Li, Cell Signal. 44Slepchenko KG, Holub JM, Li YV. Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). Cell Signal. 2018;44:148-157.","Mechanisms of TGF-β signaling from cell membrane to the nucleus. Y Shi, J Massagué, Cell. 113Shi Y, Massagué J. Mechanisms of TGF-β signaling from cell mem- brane to the nucleus. Cell. 2003;113:685-700.","TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. J Tan, Z H Deng, S Z Liu, J T Wang, C Huang, Curr Eye Res. 35Tan J, Deng ZH, Liu SZ, Wang JT, Huang C. TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. Curr Eye Res. 2010;35:37-44.","Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia. N Malachkova, D Yatsenko, G Ljudkevich, V Shkarupa, Oftalmologicheskii Zhurnal. 76Malachkova N, Yatsenko D, Ljudkevich G, Shkarupa V. Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myo- pia. Oftalmologicheskii Zhurnal. 2018;76:45-48.","Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. Y Mo, Y Wang, B Cao, J Zhang, G Ren, T Yang, J Tradit Chin Med Sci. 3Mo Y, Wang Y, Cao B, Zhang J, Ren G, Yang T. Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. J Tradit Chin Med Sci. 2016;3:124-132.","Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. H J Lin, C C Wei, C Y Chang, EBioMedicine. 10Lin HJ, Wei CC, Chang CY, et al. Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. EBioMedicine. 2016;10:269-281.","The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. T Fukada, N Civic, T Furuichi, PLoS One. 33642Fukada T, Civic N, Furuichi T, et al. The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. PLoS One. 2008;3:e3642.","Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia. M Fedor, B Urban, K Socha, J Ophthalmol. 2019Fedor M, Urban B, Socha K, et al. Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and ado- lescents with myopia. J Ophthalmol. 2019;2019:1-7.","Additional supporting information may be found online in the Supporting Information section. How to cite this article. S U Pp O Rti N G I N Fo R M Ati O N ; Dong, S Tian, Q Zhu, T , S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Dong S, Tian Q, Zhu T, et al.","SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. 10.1111/jcmm.16803J Cell Mol Med. 25SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. J Cell Mol Med. 2021;25:8432- 8441. https://doi.org/10.1111/jcmm.16803"],"string":"[\n \"Evidence that a locus for familial high myopia maps to chromosome 18p. T L Young, S M Ronan, L A Drahozal, Am J Hum Genet. 63Young TL, Ronan SM, Drahozal LA, et al. Evidence that a locus for familial high myopia maps to chromosome 18p. Am J Hum Genet. 1998;63:109-119.\",\n \". I G Morgan, K Ohno-Matsui, Saw Sm Myopia, Lancet. 379Morgan IG, Ohno-Matsui K, Saw SM. Myopia. Lancet. 2012;379:1739-1748.\",\n \"Global prevalence of myopia and high myopia and temporal trends from. B A Holden, T R Fricke, D A Wilson, Ophthalmology. 123Holden BA, Fricke TR, Wilson DA, et al. Global prevalence of myo- pia and high myopia and temporal trends from 2000 through 2050. Ophthalmology. 2016;123:1036-1042.\",\n \"The myopia boom. E Dolgin, Nature. 519Dolgin E. The myopia boom. Nature. 2015;519:276-278.\",\n \"Genes and environment in refractive error: the twin eye study. C J Hammond, H Snieder, C E Gilbert, T D Spector, Invest Ophthalmol Vis Sci. 42Hammond CJ, Snieder H, Gilbert CE, Spector TD. Genes and envi- ronment in refractive error: the twin eye study. Invest Ophthalmol Vis Sci. 2001;42:1232-1236.\",\n \"Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study. A P Klein, B Suktitipat, P Duggal, Arch Ophthalmol. 127Klein AP, Suktitipat B, Duggal P, et al. Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study. Arch Ophthalmol. 2009;127:649-655.\",\n \"An overview of myopia genetics. X B Cai, S R Shen, D F Chen, Q Zhang, Z B Jin, Exp Eye Res. 188107778Cai XB, Shen SR, Chen DF, Zhang Q, Jin ZB. An overview of myopia genetics. Exp Eye Res. 2019;188:107778.\",\n \"The sclera and myopia. J A Rada, S Shelton, T T Norton, Exp Eye Res. 82Rada JA, Shelton S, Norton TT. The sclera and myopia. Exp Eye Res. 2006;82:185-200.\",\n \"Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression. A I Jobling, M Nguyen, A Gentle, N A Mcbrien, J Biol Chem. 279Jobling AI, Nguyen M, Gentle A, McBrien NA. Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression. J Biol Chem. 2004;279:18121-18126.\",\n \"Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. A I Jobling, R Wan, A Gentle, B V Bui, N A Mcbrien, Exp Eye Res. 88Jobling AI, Wan R, Gentle A, Bui BV, McBrien NA. Retinal and choroi- dal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. Exp Eye Res. 2009;88:458-466.\",\n \"Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model. B Y Chen, C Y Wang, W Y Chen, J X Ma, Graefes Arch Clin Exp Ophthalmol. 251Chen BY, Wang CY, Chen WY, Ma JX. Altered TGF-β2 and bFGF expres- sion in scleral desmocytes from an experimentally-induced myopia guinea pig model. Graefes Arch Clin Exp Ophthalmol. 2013;251:1133-1144.\",\n \"TGFbeta-induced factor: a candidate gene for high myopia. D S Lam, W S Lee, Y F Leung, Invest Ophthalmol Vis Sci. 44Lam DS, Lee WS, Leung YF, et al. TGFbeta-induced factor: a candidate gene for high myopia. Invest Ophthalmol Vis Sci. 2003;44:1012-1015.\",\n \"Expression of bFGF and TGFbeta 2 in experimental myopia in chicks. Y Seko, H Shimokawa, T Tokoro, Invest Ophthalmol Vis Sci. 36Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF- beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36:1183-1187.\",\n \"Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice. P C Wu, C L Tsai, G M Gordon, Mol Vis. 21Wu PC, Tsai CL, Gordon GM, et al. Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice. Mol Vis. 2015;21:138-147.\",\n \"SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia. H Guo, Jin X Zhu, T , J Med Genet. 51Guo H, Jin X, Zhu T, et al. SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia. J Med Genet. 2014;51:518-525.\",\n \"Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a crosssectional study in 731 Chinese patients. X B Cai, Y H Zheng, D F Chen, Invest Ophthalmol Vis Sci. 60Cai XB, Zheng YH, Chen DF, et al. Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a cross- sectional study in 731 Chinese patients. Invest Ophthalmol Vis Sci. 2019;60:4052-4062.\",\n \"The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5. J Dufner-Beattie, Y M Kuo, J Gitschier, G K Andrews, J Biol Chem. 279Dufner-Beattie J, Kuo YM, Gitschier J, Andrews GK. The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5. J Biol Chem. 2004;279:49082-49090.\",\n \"The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells. F Wang, B E Kim, M J Petris, D J Eide, J Biol Chem. 279Wang F, Kim BE, Petris MJ, Eide DJ. The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polar- ized cells. J Biol Chem. 2004;279:51433-51441.\",\n \"Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding. J Chai, J W Wu, N Yan, J Massague, N P Pavletich, Y Shi, J Biol Chem. 278Chai J, Wu JW, Yan N, Massague J, Pavletich NP, Shi Y. Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding. J Biol Chem. 2003;278:20327-20331.\",\n \"Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients. C Y Feng, X Q Huang, X W Cheng, R H Wu, F Lu, Z B Jin, Sci Rep. 7Feng CY, Huang XQ, Cheng XW, Wu RH, Lu F, Jin ZB. Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients. Sci Rep. 2017;7:1-9.\",\n \"Trio-based exome sequencing arrests de novo mutations in early-onset high myopia. Z B Jin, J Wu, X F Huang, Proc Natl Acad Sci. 114Jin ZB, Wu J, Huang XF, et al. Trio-based exome sequencing arrests de novo mutations in early-onset high myopia. Proc Natl Acad Sci USA. 2017;114:4219-4224.\",\n \"Exome sequencing study of 20 patients with high myopia. L Wan, B Deng, Z Wu, X Chen, PeerJ. 65552Wan L, Deng B, Wu Z, Chen X. Exome sequencing study of 20 pa- tients with high myopia. PeerJ. 2018;6:e5552.\",\n \"Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with earlyonset high myopia by exome sequencing. D Jiang, J Li, X Xiao, Invest Ophthalmol Vis Sci. 56Jiang D, Li J, Xiao X, et al. Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with early- onset high myopia by exome sequencing. Invest Ophthalmol Vis Sci. 2014;56:339-345.\",\n \"Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. R D Palmiter, L Huang, Pflugers Arch. 447Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. 2004;447:744-751.\",\n \"Zinc transporters and the cellular trafficking of zinc. D J Eide, Biochem Biophys Acta. 1763Eide DJ. Zinc transporters and the cellular trafficking of zinc. Biochem Biophys Acta. 2006;1763:711-722.\",\n \"The SLC39 family of zinc transporters. J Jeong, D J Eide, Mol Aspects Med. 34Jeong J, Eide DJ. The SLC39 family of zinc transporters. Mol Aspects Med. 2013;34:612-619.\",\n \"Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36. W Kou, H S Kolla, A Ortiz-Acevedo, D C Haines, M Junker, G R Dieckmann, J Biol Inorg Chem. 10Kou W, Kolla HS, Ortiz-Acevedo A, Haines DC, Junker M, Dieckmann GR. Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36. J Biol Inorg Chem. 2005;10:167-180.\",\n \"Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability. L H Nguyen, T T Tran, Ltn Truong, H H Mai, T T Nguyen, Biochemistry. 59Nguyen LH, Tran TT, Truong LTN, Mai HH, Nguyen TT. Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability. Biochemistry. 2020;59:1378-1390.\",\n \"Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom. D Pretzer, B Schulteis, Vander Velde, D G Smith, C D Mitchell, J W Manning, M C , Pharm Res. 9Pretzer D, Schulteis B, Vander Velde DG, Smith CD, Mitchell JW, Manning MC. Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom. Pharm Res. 1992;9:870-877.\",\n \"Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability. T Mabrouk, G Lemay, J Virol. 68Mabrouk T, Lemay G. Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability. J Virol. 1994;68:5287-5290.\",\n \"The role of zinc in the stability of the marginally stable IscU scaffold protein. C Iannuzzi, M Adrover, R Puglisi, R Yan, P A Temussi, A Pastore, Protein Sci. 23Iannuzzi C, Adrover M, Puglisi R, Yan R, Temussi PA, Pastore A. The role of zinc in the stability of the marginally stable IscU scaffold protein. Protein Sci. 2014;23:1208-1219.\",\n \"Metal binding properties, stability and reactivity of zinc fingers. K Kluska, J Adamczyk, A Krężel, Coord Chem Rev. 367Kluska K, Adamczyk J, Krężel A. Metal binding proper- ties, stability and reactivity of zinc fingers. Coord Chem Rev. 2018;367:18-64.\",\n \"Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein. S Z Potter, H Zhu, B F Shaw, J Am Chem Soc. 129Potter SZ, Zhu H, Shaw BF, et al. Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substan- tially influences the structure and stability of the entire homodi- meric protein. J Am Chem Soc. 2007;129:4575-4583.\",\n \"Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. T Knaus, M K Uhl, S Monschein, S Moratti, K Gruber, P Macheroux, Biochem Biophys Acta. 1844Knaus T, Uhl MK, Monschein S, Moratti S, Gruber K, Macheroux P. Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. Biochem Biophys Acta. 2014;1844:2298-2305.\",\n \"Smad transcription factors. J Massagué, J Seoane, D Wotton, Genes Dev. 19Massagué J, Seoane J, Wotton D. Smad transcription factors. Genes Dev. 2005;19:2783-2810.\",\n \"Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). K G Slepchenko, J M Holub, Y V Li, Cell Signal. 44Slepchenko KG, Holub JM, Li YV. Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). Cell Signal. 2018;44:148-157.\",\n \"Mechanisms of TGF-β signaling from cell membrane to the nucleus. Y Shi, J Massagué, Cell. 113Shi Y, Massagué J. Mechanisms of TGF-β signaling from cell mem- brane to the nucleus. Cell. 2003;113:685-700.\",\n \"TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. J Tan, Z H Deng, S Z Liu, J T Wang, C Huang, Curr Eye Res. 35Tan J, Deng ZH, Liu SZ, Wang JT, Huang C. TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. Curr Eye Res. 2010;35:37-44.\",\n \"Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia. N Malachkova, D Yatsenko, G Ljudkevich, V Shkarupa, Oftalmologicheskii Zhurnal. 76Malachkova N, Yatsenko D, Ljudkevich G, Shkarupa V. Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myo- pia. Oftalmologicheskii Zhurnal. 2018;76:45-48.\",\n \"Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. Y Mo, Y Wang, B Cao, J Zhang, G Ren, T Yang, J Tradit Chin Med Sci. 3Mo Y, Wang Y, Cao B, Zhang J, Ren G, Yang T. Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. J Tradit Chin Med Sci. 2016;3:124-132.\",\n \"Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. H J Lin, C C Wei, C Y Chang, EBioMedicine. 10Lin HJ, Wei CC, Chang CY, et al. Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. EBioMedicine. 2016;10:269-281.\",\n \"The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. T Fukada, N Civic, T Furuichi, PLoS One. 33642Fukada T, Civic N, Furuichi T, et al. The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. PLoS One. 2008;3:e3642.\",\n \"Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia. M Fedor, B Urban, K Socha, J Ophthalmol. 2019Fedor M, Urban B, Socha K, et al. Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and ado- lescents with myopia. J Ophthalmol. 2019;2019:1-7.\",\n \"Additional supporting information may be found online in the Supporting Information section. How to cite this article. S U Pp O Rti N G I N Fo R M Ati O N ; Dong, S Tian, Q Zhu, T , S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Dong S, Tian Q, Zhu T, et al.\",\n \"SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. 10.1111/jcmm.16803J Cell Mol Med. 25SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. J Cell Mol Med. 2021;25:8432- 8441. https://doi.org/10.1111/jcmm.16803\"\n]"},"annotations_bibref":{"kind":"list like","value":["3,","4","5,","6","7","[8]","[9]","[10]","9","10","[10]","[11]","[12]","[13]","9,","10,","14","15","16","15","17,","18","(20)","15","16,","[20]","[21]","[22]","[23]","24","25","17,","18,","26","32","33","34","36","[38]","[39]","[40]","[41]","[8]","[9]","[10]","42","43"],"string":"[\n \"3,\",\n \"4\",\n \"5,\",\n \"6\",\n \"7\",\n \"[8]\",\n \"[9]\",\n \"[10]\",\n \"9\",\n \"10\",\n \"[10]\",\n \"[11]\",\n \"[12]\",\n \"[13]\",\n \"9,\",\n \"10,\",\n \"14\",\n \"15\",\n \"16\",\n \"15\",\n \"17,\",\n \"18\",\n \"(20)\",\n \"15\",\n \"16,\",\n \"[20]\",\n \"[21]\",\n \"[22]\",\n \"[23]\",\n \"24\",\n \"25\",\n \"17,\",\n \"18,\",\n \"26\",\n \"32\",\n \"33\",\n \"34\",\n \"36\",\n \"[38]\",\n \"[39]\",\n \"[40]\",\n \"[41]\",\n \"[8]\",\n \"[9]\",\n \"[10]\",\n \"42\",\n \"43\"\n]"},"annotations_bibtitle":{"kind":"list like","value":["Evidence that a locus for familial high myopia maps to chromosome 18p","Global prevalence of myopia and high myopia and temporal trends from","The myopia boom","Genes and environment in refractive error: the twin eye study","Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study","An overview of myopia genetics","The sclera and myopia","Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression","Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function","Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model","TGFbeta-induced factor: a candidate gene for high myopia","Expression of bFGF and TGFbeta 2 in experimental myopia in chicks","Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice","SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia","Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a crosssectional study in 731 Chinese patients","The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5","The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells","Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding","Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients","Trio-based exome sequencing arrests de novo mutations in early-onset high myopia","Exome sequencing study of 20 patients with high myopia","Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with earlyonset high myopia by exome sequencing","Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers","Zinc transporters and the cellular trafficking of zinc","The SLC39 family of zinc transporters","Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36","Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability","Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom","Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability","The role of zinc in the stability of the marginally stable IscU scaffold protein","Metal binding properties, stability and reactivity of zinc fingers","Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein","Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron","Smad transcription factors","Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ)","Mechanisms of TGF-β signaling from cell membrane to the nucleus","TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro","Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia","Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia","Role of chronic inflammation in myopia progression: clinical evidence and experimental validation","The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways","Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia","SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis"],"string":"[\n \"Evidence that a locus for familial high myopia maps to chromosome 18p\",\n \"Global prevalence of myopia and high myopia and temporal trends from\",\n \"The myopia boom\",\n \"Genes and environment in refractive error: the twin eye study\",\n \"Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study\",\n \"An overview of myopia genetics\",\n \"The sclera and myopia\",\n \"Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression\",\n \"Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function\",\n \"Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model\",\n \"TGFbeta-induced factor: a candidate gene for high myopia\",\n \"Expression of bFGF and TGFbeta 2 in experimental myopia in chicks\",\n \"Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice\",\n \"SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia\",\n \"Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a crosssectional study in 731 Chinese patients\",\n \"The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5\",\n \"The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells\",\n \"Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding\",\n \"Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients\",\n \"Trio-based exome sequencing arrests de novo mutations in early-onset high myopia\",\n \"Exome sequencing study of 20 patients with high myopia\",\n \"Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with earlyonset high myopia by exome sequencing\",\n \"Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers\",\n \"Zinc transporters and the cellular trafficking of zinc\",\n \"The SLC39 family of zinc transporters\",\n \"Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36\",\n \"Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability\",\n \"Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom\",\n \"Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability\",\n \"The role of zinc in the stability of the marginally stable IscU scaffold protein\",\n \"Metal binding properties, stability and reactivity of zinc fingers\",\n \"Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein\",\n \"Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron\",\n \"Smad transcription factors\",\n \"Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ)\",\n \"Mechanisms of TGF-β signaling from cell membrane to the nucleus\",\n \"TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro\",\n \"Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia\",\n \"Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia\",\n \"Role of chronic inflammation in myopia progression: clinical evidence and experimental validation\",\n \"The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways\",\n \"Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia\",\n \"SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["Am J Hum Genet","Lancet","Ophthalmology","Nature","Invest Ophthalmol Vis Sci","Arch Ophthalmol","Exp Eye Res","Exp Eye Res","J Biol Chem","Exp Eye Res","Graefes Arch Clin Exp Ophthalmol","Invest Ophthalmol Vis Sci","Invest Ophthalmol Vis Sci","Mol Vis","J Med Genet","Invest Ophthalmol Vis Sci","J Biol Chem","J Biol Chem","J Biol Chem","Sci Rep","Proc Natl Acad Sci","PeerJ","Invest Ophthalmol Vis Sci","Pflugers Arch","Biochem Biophys Acta","Mol Aspects Med","J Biol Inorg Chem","Biochemistry","Pharm Res","J Virol","Protein Sci","Coord Chem Rev","J Am Chem Soc","Biochem Biophys Acta","Genes Dev","Cell Signal","Cell","Curr Eye Res","Oftalmologicheskii Zhurnal","J Tradit Chin Med Sci","EBioMedicine","PLoS One","J Ophthalmol","Additional supporting information may be found online in the Supporting Information section. How to cite this article","J Cell Mol Med"],"string":"[\n \"Am J Hum Genet\",\n \"Lancet\",\n \"Ophthalmology\",\n \"Nature\",\n \"Invest Ophthalmol Vis Sci\",\n \"Arch Ophthalmol\",\n \"Exp Eye Res\",\n \"Exp Eye Res\",\n \"J Biol Chem\",\n \"Exp Eye Res\",\n \"Graefes Arch Clin Exp Ophthalmol\",\n \"Invest Ophthalmol Vis Sci\",\n \"Invest Ophthalmol Vis Sci\",\n \"Mol Vis\",\n \"J Med Genet\",\n \"Invest Ophthalmol Vis Sci\",\n \"J Biol Chem\",\n \"J Biol Chem\",\n \"J Biol Chem\",\n \"Sci Rep\",\n \"Proc Natl Acad Sci\",\n \"PeerJ\",\n \"Invest Ophthalmol Vis Sci\",\n \"Pflugers Arch\",\n \"Biochem Biophys Acta\",\n \"Mol Aspects Med\",\n \"J Biol Inorg Chem\",\n \"Biochemistry\",\n \"Pharm Res\",\n \"J Virol\",\n \"Protein Sci\",\n \"Coord Chem Rev\",\n \"J Am Chem Soc\",\n \"Biochem Biophys Acta\",\n \"Genes Dev\",\n \"Cell Signal\",\n \"Cell\",\n \"Curr Eye Res\",\n \"Oftalmologicheskii Zhurnal\",\n \"J Tradit Chin Med Sci\",\n \"EBioMedicine\",\n \"PLoS One\",\n \"J Ophthalmol\",\n \"Additional supporting information may be found online in the Supporting Information section. How to cite this article\",\n \"J Cell Mol Med\"\n]"},"annotations_figure":{"kind":"list like","value":["\n\nHEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.","\n\nprepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-","\n\nTotal RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in","\n\nSLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in","\n\nCells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.","\n\nSLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.","\n\n3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).","\nFigure 4 ,\n4SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.","\n\nin the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.","\n\nThis work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].","\n\nShanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead)."],"string":"[\n \"\\n\\nHEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.\",\n \"\\n\\nprepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-\",\n \"\\n\\nTotal RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in\",\n \"\\n\\nSLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in\",\n \"\\n\\nCells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.\",\n \"\\n\\nSLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.\",\n \"\\n\\n3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).\",\n \"\\nFigure 4 ,\\n4SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.\",\n \"\\n\\nin the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.\",\n \"\\n\\nThis work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].\",\n \"\\n\\nShanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead).\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["HEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.","prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-","Total RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in","SLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in","Cells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.","SLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.","3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).","SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.","in the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.","This work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].","Shanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead)."],"string":"[\n \"HEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.\",\n \"prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-\",\n \"Total RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in\",\n \"SLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in\",\n \"Cells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.\",\n \"SLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.\",\n \"3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).\",\n \"SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.\",\n \"in the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.\",\n \"This work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].\",\n \"Shanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead).\"\n]"},"annotations_figureref":{"kind":"list like","value":["Figure 1A","Figure 1B)","Figure S1","Figure 1C","Figures S3 and S4","Figure S5","(Figure S5)","Figures S6 and S7)","Figure S8","Figure 3","Figure 3A","Figure","Figure S9)","Figure S10)","Figure S11"],"string":"[\n \"Figure 1A\",\n \"Figure 1B)\",\n \"Figure S1\",\n \"Figure 1C\",\n \"Figures S3 and S4\",\n \"Figure S5\",\n \"(Figure S5)\",\n \"Figures S6 and S7)\",\n \"Figure S8\",\n \"Figure 3\",\n \"Figure 3A\",\n \"Figure\",\n \"Figure S9)\",\n \"Figure S10)\",\n \"Figure S11\"\n]"},"annotations_formula":{"kind":"list like","value":[],"string":"[]"},"annotations_paragraph":{"kind":"list like","value":["Myopia is a condition of refractive error causing light to focus in front of the retina, with axial elongation as its most common pathology. High myopia, clinically defined as a refractive error of at least −6.00 diopters (D) or an axial length >26 mm, 1 is one of the leading causes of visual impairment worldwide 2,3 with a significantly increased prevalence. 3,4 Thus, the prevention of high myopia has become a global public health problem. Pedigree analyses and twin studies identified high myopia highly heritable. 5,6 Even though numerous loci (1q41, 4q25, 8p23, etc.) and causative genes (ZNF644, CCDC111, SCO2, etc.) have been identified, 7 the pathogenic mechanism of high myopia remains unclear.","Recently, scleral extracellular matrix (ECM) synthesis or remodelling has been proposed as an underlying cause of high myopia. [8][9][10] According to this hypothesis, scleral collagen synthesis is regulated during myopia progression, 9 while scleral ECM is remodelled in myopic animal. 10 ECM synthesis and remodelling are targets of the TGFβ signalling pathway, one of the most reproducibly dysregulated pathways in myopia development. [10][11][12][13] Total or isoform-specific scleral TGFβ is frequently altered during myopia progression. 9,10,14 In the previous study, we first identified SLC39A5 as a high myopia-associated gene, and mutations of SLC39A5 dysregulated the BMP/TGFβ pathway. 15 Further research confirmed SLC39A5 as one of the top three genes contributing to nonsyndromic high myopia. 16 This study aimed to investigate the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia. We found that SLC39A5 played a key role in maintaining intracellular zinc homeostasis. SLC39A5 depletion-induced zinc deficiency directly destabilized Smad proteins, and Smads instability further impaired TGFβ signalling-mediated ECM synthesis, thus contributing to the pathogenesis of high myopia. ","According to the protocols from Zhang laboratory (http://www. genom e-engin eering.org/gecko/), target-specific sgRNA was cloned into the lentiCRISPRv2 vector (sgRNAs are listed in Table S1). Then, lentiviruses were packaged in HEK293T cells by co-transfection of CRISPR-sgRNA/pVSVG/pspAX2. The supernatants containing viral particles were collected 48 h after transfection and filtered (0.22 μm pore size). Then, HEK293 cells were transduced with viral supernatant supplemented and selected with 1 μg/ml puromycin.","After transfection, cells were detached and seeded in 10 ml Petri dishes at a density of 200 cells per plate. Puromycin (2 µg/ml) was added to allow the selection of positive clones during the following two weeks. Positive clones were isolated and transferred to sixwell plates. The SLC39A5 knockout clones were validated by direct Sanger sequencing (primers are listed in Table S2). The negative control cell line was generated from HEK293 transformed with the identical lentiviral vector lacking specific sgRNAs.","Zinc quantification assay (Abcam Cat# ab102507) was performed for accurate measurement of zinc levels according to the manufacturer's instructions. In brief, cells were harvested with NP-40 lysis buffer (EDTA-free), and proteins were deproteinized by an equal volume of 7% TCA Solution Buffer. The supernatants were transferred to new clean tubes after centrifugation. 200 μl Zinc Reaction Buffer was applied to 50 μl tested samples in a 96-well plate. After a 10-min incubation at room temperature, the OD 560 was measured in a microplate reader. The zinc standard curve was plotted after subtracting the background value, and the tested sample concentration was calculated from the standard curve.","Total RNA for RNA-seq library was extracted using TRIzol (Invitrogen Cat# AM9738), validated on a NanoDrop 1000 Spectrophotometer (Thermo) and quantitated using Qubit (Thermo). RNA-seq libraries were ing data were filtered with SOAPnuke (v1.5.2), then mapped to the reference genome using HISAT2 (v2.0.4) and aligned to the reference coding gene set using Bowtie2 (v2.2.5). Differential gene expression analysis between the target and reference sets of treatments was determined using DESeq2 (https://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq.html). Enrichment analysis of annotated different expressed genes was performed with the Phyper (https://en.wikip edia.org/wiki/ Hyper geome tric_distr ibution) based on the hypergeometric test. Table S3). All samples were run in triplicate. qRT-PCR data were analysed by LightCycler 96 software. Relative candidate gene mRNA levels were normalized to those of β-actin or GAPDH. A value of p < 0.05 was considered to be statistically significant.","The Smad1 zinc-binding site substitutions were generated to assess the correlation between zinc-binding capacity and protein stability. Smad1 WT plasmid was brought from OriGene (OriGene Cat# RC200299), and the four zinc-binding site substitutions (C64A, C109A, C121A and H126A) were separately generated using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in Table S4). Table S5).","Finally, the rescue sequence was cloned into the pLVX-IRES-puro vector to construct the KO rescue plasmid.","SLC30A1 shRNA interference plasmids were also constructed to rescue the poor intracellular zinc level of SLC39A5 KO cells by suppressing the zinc efflux baseline. The three targeting sequences were obtained from the siRNA sequence database (Thermo) and then generated into the pFUGW-lentiviral vector between the XbaI and BamHI restriction sites, respectively (primers are listed in Table S6). ","Cells were seeded on specific coverslips in 12-well plates.","The cell slides were fixed in 4% paraformaldehyde for 10 min.","Permeabilization was performed with 0.1% PBST (phosphatebuffered saline and 0.1% Triton) for 15 min. After blocking with 5% BSA for 1 h, the cell slides were incubated with primary antibody at recommended dilutions overnight in 4°C and stained with Alex Fluor 488-conjugated second antibody (Jackson Cat# 111-545-144) for 1 h protected from light. DAPI was applied for 1 min as a nuclear marker. Images were acquired with an Eclipse TCS-SP5 inverted confocal microscope (Leica).","HEK293 cells were seeded into the 2-well culture insert (Ibidi Cat# 81176) pre-coated by Matrigel. After cell attachment for approximately 24 h to form an optically confluent monolayer, the culture insert was removed to create the wound gap and the cells were then cultured with serum-free medium. The wound gap closure was monitored by taking pictures with an Eclipse inverted microscope (Leica) at different time points.","GraphPad Prism was used to calculate and plot the mean ± SEM of measured quantities. Significances were assessed by two-way analysis of variance (ANOVA)/Mann-Whitney U test (nonparametric)/Student's t test (parametric), p-value < 0.05 was considered to indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.","Two SLC39A5 knockout (KO) cell lines were developed using the CRISPR-Cas9 system in the human embryonic kidney cell line (HEK293) ( Figure 1A). Cell morphology anomalies were markedly observed in the SLC39A5-depleted cells. After regular growth for 2 days, the KO cells revealed a tendency to form small cell aggregates instead of a typical optically confluent monolayer ( Figure 1B).","This phenotype suggested a potential impairment or abnormality of ECM generation. Considering the influence of ECM dynamics on cell migration, invasion and morphogenesis, we immediately investigated the cellular motility and migratory capacity of the SLC39A5depleted HEK293 cells. Scratch assay was performed in both groups.","A 2-well culture insert generated a uniform gap in the confluent monolayer, and wound healing was imaged at different time points.","The results showed that the gap in the control group was completely healed after 24 h, while the wound gap in the KO group was not able to close ( Figure S1; Figure 1C).","To investigate the overall alteration caused by SLC39A5 depletion, RNA-sequencing (RNA-seq) was performed using a Immunofluorescence also verified the decrease in these ECM proteins ( Figures S3 and S4).","The transcription of collagens, FN and LN is closely related to TGFβ signalling, and we have already determined that mutations of SLC39A5 dysregulated TGFβ signalling previously. 15 Considering the transcriptional downregulation of collagens, FN and LN in SLC39A5 KO cells, we hypothesized that reduced SLC39A5 expression would inhibit the TGFβ signalling pathway. Hence, we examined the expression of Smad proteins, which lie on the centre of the TGFβ signalling pathway. The results showed an evident upregulation of total Receptor-Smads (R-Smads) (Smad1, Smad2/3) expression along with a significant decrease in phosphorylated Smad expression (pSmad1/5/9, pSmad2/3) ( Figure S5). Additionally, Co-Smad (Smad4) expression was also increased, while inhibitor-Smad (Smad7) was not affected (Figure S5), suggesting a suppressed activation of the TGFβ signalling, which was confirmed through immunofluorescence ( Figures S6 and S7). The qRT-PCR performed to assess the transcriptional levels of Smad1, Smad2 and Smad4 in KO cells showed increased mRNA expression of the tested proteins ( Figure S8). To validate the phenotype of inhibited TGFβ signalling activation and insufficient ECM synthesis resulting from SLC39A5 depletion, rescue experiments were carefully carried out. As shown in Figure 3, re-expression of SLC39A5 restored intracellular zinc level ( Figure 3A). TGFβ signalling activation and ECM synthesis were also rescued ( Figure ","The TGFβ pathway is a zinc-regulated pathway ( Figure S9), while SLC39A5 is a zinc transporter. 17,18 These facts prompted us to confirm the involvement of zinc homeostasis in TGFβ signalling alteration in SLC39A5 KO cells. Thus, zinc quantification was performed to detect intracellular zinc levels. As shown in Thus, we selected the most widely expressed zinc efflux protein SLC30A1 (20), as a target. Surprisingly, silencing SLC30A1 counterbalanced the influence of SLC39A5 depletion, which significantly reverted intracellular zinc level close to normal ( Figure S10) and normalized the TGFβ signalling pathway and ECM synthesis ( Figure S11).","SLC39A5 was first identified as a high myopia pathogenic gene in our previous study 15 and was later confirmed as one of the top three genes contributing to nonsyndromic high myopia. 16,[20][21][22][23] However, the mechanisms by which SLC39A5 contributes to high myopia are not well understood. Here, we demonstrated that SLC39A5 may play a role in the pathogenesis of high myopia by modulating TGFβ signalling-mediated ECM synthesis through the regulation of intracellular zinc homeostasis. SLC39A5 depletion significantly lowered the intracellular zinc level, which destabilized Smad proteins, and further inhibited TGFβ signalling-mediated ECM synthesis, which ultimately contributed to the pathogenesis of high myopia.","Zinc homeostasis is crucial for all organisms as it serves as a catalytic or structural cofactor for multiple types of proteins. Zinc homeostasis depends on the transport of zinc in both directions across the plasma membrane and both in and out of various vesicular compartments. 24 The SLC39 family facilitates zinc transport into the cytoplasm in the opposite direction to SLC30 family members. 25 Within this network, SLC39A5 is a specific zinc transporter. 17,18,26 Our work confirmed the zinc uptake characteristic of SLC39A5, in which SLC39A5 depletion caused an intracellular decline in zinc levels in HEK293 or lymphocyte cells. Once zinc homeostasis was disrupted, the catalytic or structural stability of proteins was impaired, suggesting that SLC39A5 depletion, indirectly, caused this impairment.","Zinc has a profound effect on stabilization of zinc-binding proteins, 27-31 since the proteins responsible for cellular zinc buffering will bind zinc transiently; therefore, their function will strongly depend on the cellular zinc status. 32 It has been proposed that zinc chelation affects protein stability through various mechanisms. The human SOD1 protein requires zinc binding to facilitate the restructuring of apoproteins, 33 while the putative protease of Bacteroides thetaiotaomicron (ppBat) requires zinc chelation to link the β-strands to α-helices in protein structures. 34 In our study, the R-Smads and Co-Smad, which are pivotal to the BMP/TGFβ signalling pathway, 35 F I G U R E 2 SLC39A5 depletion impairs extracellular matrix (ECM) synthesis. (A) shows the pathway enrichment analysis for the differentially expressed genes between the control and KO subpopulations of HEK293 cells. Pathway analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics Resources, with an enrichment p-value cut-off of 0.01. (B) shows the qRT-PCR validation of the differentially expressed genes of the ECM members indicated by RNA-seq. (C-E) shows the expression of several ECM components (COL1, COL2, COL4, FN and LN) in the supernatant, whole-cell lysate or intracellular lysate (trypsin digested) of wild-type (WT) and SLC39A5 knockout (KO) HEK293 cells. All tested ECM components were decreased in both the whole lysate and intracellular lysate. (B) and (C) show statistical analyses of the ECM components in the whole lysate and intracellular lysate, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are zinc-binding proteins. We found that Smad proteins were rapidly degraded during SLC39A5 deletion-induced zinc deficiency.","Further results suggested that loose zinc coupling, facilitated by a mutant ion-binding site, directly destabilized the exogenous Smad1 proteins, which clearly confirms the necessity of zinc chelation in maintaining the protein stability of Smads, while disturbances in zinc homeostasis induce protein instability.","Zinc binding has also been implicated in protein phosphorylation and subcellular translocation. 36 Non-zinc-bound protein kinase C delta (PKCδ) showed a significantly different structure near its phosphorylation site, compared with when it was zinc-bound. Subcellular translocation of PKCδ was also influenced by intracellular zinc. These findings expand our understanding of the phosphorylation decrease in R-Smads during SLC39A5-depleted conditions.","Since R-Smads are directly phosphorylated by upstream receptor kinases, which then form heteromeric complexes with Smad4 to translocate into the nucleus, 37 the zinc deficiency induced by SLC39A5 depletion may barely maintain the Smad protein in a favourable structure for phosphorylation, and may lead to nucleus translocation retention, further impairing the TGFβ signalling.","It is well recognized that TGFβ signalling directly affects ECM dynamics or triggers it when mediating other biological processes. [38][39][40][41] Herein, suppressed TGFβ signalling in the zinc-deficient conditions restrained ECM synthesis in HEK293. Recently, ECM synthesis and remodelling have attracted significant attention in high myopia aetiology studies. [8][9][10]42 We found that the transcription of all ECM proteins tested (COL1, COL2, COL4, FN and LN) was decreased under zinc-deficient conditions. ECM-induced cell morphogenesis According to this hypothesis, the SLC39A5-induced zinc deficiency suppresses TGFβ signalling, leading to insufficient ECM synthesis, which may ultimately contribute to high myopia development. We further validated this hypothesis by performing rescue experiments.","As expected, of SLC39A5 re-expression, which recovers zinc influx, and SLC30A1 silencing, which blocks zinc efflux, could identically rescue the alteration of R-Smad phosphorylation and ECM synthesis in SLC39A5-depleted HEK293 cells, by re-establishing zinc homeostasis.","Upon reviewing the relationship between zinc and high myopia, we found plenty of indications of zinc involvement in high myopia development. 43 First, zinc is present abundantly in the ocular system, including the retina and choroid; thus, zinc deficiency produces a variety of ocular manifestations. Furthermore, a number of high myopia gene products are zinc-related proteins.","Among them, ZNF644, ZFHX1B, ZC3H11B and SCO2 are zinc finger proteins, and HGF and IGF1 are positively regulated by intracellular zinc concentration. Additionally, certain pathogenic genes (LAMA1 and TGFB1) may even be regulated by intracellular zinc, according to the zinc-TGFβ signalling-ECM synthesis flow.","Finally, multiple small molecules that serve as chaperones for disease-related genes or pathways are zinc-related proteins. Smad proteins, together with their anchors (SARA and HGS), are either zinc-binding or zinc finger proteins. These findings strongly highlight the correlation between zinc and high myopia.","In summary, our data suggest that depletion of SLC39A5 induces zinc deficiency, which restrains TGFβ signalling-mediated ECM synthesis, thus possibly contributing to high myopia pathogenesis. Zinc homeostasis appears to be a dominant aetiological factor of high myopia development. ","The authors confirm that there are no conflicts of interest. ","The data that support the findings of this study are available from the corresponding author upon reasonable request.","https://orcid.org/0000-0002-1605-7066","Zhengmao Hu https://orcid.org/0000-0002-3921-8205"],"string":"[\n \"Myopia is a condition of refractive error causing light to focus in front of the retina, with axial elongation as its most common pathology. High myopia, clinically defined as a refractive error of at least −6.00 diopters (D) or an axial length >26 mm, 1 is one of the leading causes of visual impairment worldwide 2,3 with a significantly increased prevalence. 3,4 Thus, the prevention of high myopia has become a global public health problem. Pedigree analyses and twin studies identified high myopia highly heritable. 5,6 Even though numerous loci (1q41, 4q25, 8p23, etc.) and causative genes (ZNF644, CCDC111, SCO2, etc.) have been identified, 7 the pathogenic mechanism of high myopia remains unclear.\",\n \"Recently, scleral extracellular matrix (ECM) synthesis or remodelling has been proposed as an underlying cause of high myopia. [8][9][10] According to this hypothesis, scleral collagen synthesis is regulated during myopia progression, 9 while scleral ECM is remodelled in myopic animal. 10 ECM synthesis and remodelling are targets of the TGFβ signalling pathway, one of the most reproducibly dysregulated pathways in myopia development. [10][11][12][13] Total or isoform-specific scleral TGFβ is frequently altered during myopia progression. 9,10,14 In the previous study, we first identified SLC39A5 as a high myopia-associated gene, and mutations of SLC39A5 dysregulated the BMP/TGFβ pathway. 15 Further research confirmed SLC39A5 as one of the top three genes contributing to nonsyndromic high myopia. 16 This study aimed to investigate the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia. We found that SLC39A5 played a key role in maintaining intracellular zinc homeostasis. SLC39A5 depletion-induced zinc deficiency directly destabilized Smad proteins, and Smads instability further impaired TGFβ signalling-mediated ECM synthesis, thus contributing to the pathogenesis of high myopia. \",\n \"According to the protocols from Zhang laboratory (http://www. genom e-engin eering.org/gecko/), target-specific sgRNA was cloned into the lentiCRISPRv2 vector (sgRNAs are listed in Table S1). Then, lentiviruses were packaged in HEK293T cells by co-transfection of CRISPR-sgRNA/pVSVG/pspAX2. The supernatants containing viral particles were collected 48 h after transfection and filtered (0.22 μm pore size). Then, HEK293 cells were transduced with viral supernatant supplemented and selected with 1 μg/ml puromycin.\",\n \"After transfection, cells were detached and seeded in 10 ml Petri dishes at a density of 200 cells per plate. Puromycin (2 µg/ml) was added to allow the selection of positive clones during the following two weeks. Positive clones were isolated and transferred to sixwell plates. The SLC39A5 knockout clones were validated by direct Sanger sequencing (primers are listed in Table S2). The negative control cell line was generated from HEK293 transformed with the identical lentiviral vector lacking specific sgRNAs.\",\n \"Zinc quantification assay (Abcam Cat# ab102507) was performed for accurate measurement of zinc levels according to the manufacturer's instructions. In brief, cells were harvested with NP-40 lysis buffer (EDTA-free), and proteins were deproteinized by an equal volume of 7% TCA Solution Buffer. The supernatants were transferred to new clean tubes after centrifugation. 200 μl Zinc Reaction Buffer was applied to 50 μl tested samples in a 96-well plate. After a 10-min incubation at room temperature, the OD 560 was measured in a microplate reader. The zinc standard curve was plotted after subtracting the background value, and the tested sample concentration was calculated from the standard curve.\",\n \"Total RNA for RNA-seq library was extracted using TRIzol (Invitrogen Cat# AM9738), validated on a NanoDrop 1000 Spectrophotometer (Thermo) and quantitated using Qubit (Thermo). RNA-seq libraries were ing data were filtered with SOAPnuke (v1.5.2), then mapped to the reference genome using HISAT2 (v2.0.4) and aligned to the reference coding gene set using Bowtie2 (v2.2.5). Differential gene expression analysis between the target and reference sets of treatments was determined using DESeq2 (https://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq.html). Enrichment analysis of annotated different expressed genes was performed with the Phyper (https://en.wikip edia.org/wiki/ Hyper geome tric_distr ibution) based on the hypergeometric test. Table S3). All samples were run in triplicate. qRT-PCR data were analysed by LightCycler 96 software. Relative candidate gene mRNA levels were normalized to those of β-actin or GAPDH. A value of p < 0.05 was considered to be statistically significant.\",\n \"The Smad1 zinc-binding site substitutions were generated to assess the correlation between zinc-binding capacity and protein stability. Smad1 WT plasmid was brought from OriGene (OriGene Cat# RC200299), and the four zinc-binding site substitutions (C64A, C109A, C121A and H126A) were separately generated using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in Table S4). Table S5).\",\n \"Finally, the rescue sequence was cloned into the pLVX-IRES-puro vector to construct the KO rescue plasmid.\",\n \"SLC30A1 shRNA interference plasmids were also constructed to rescue the poor intracellular zinc level of SLC39A5 KO cells by suppressing the zinc efflux baseline. The three targeting sequences were obtained from the siRNA sequence database (Thermo) and then generated into the pFUGW-lentiviral vector between the XbaI and BamHI restriction sites, respectively (primers are listed in Table S6). \",\n \"Cells were seeded on specific coverslips in 12-well plates.\",\n \"The cell slides were fixed in 4% paraformaldehyde for 10 min.\",\n \"Permeabilization was performed with 0.1% PBST (phosphatebuffered saline and 0.1% Triton) for 15 min. After blocking with 5% BSA for 1 h, the cell slides were incubated with primary antibody at recommended dilutions overnight in 4°C and stained with Alex Fluor 488-conjugated second antibody (Jackson Cat# 111-545-144) for 1 h protected from light. DAPI was applied for 1 min as a nuclear marker. Images were acquired with an Eclipse TCS-SP5 inverted confocal microscope (Leica).\",\n \"HEK293 cells were seeded into the 2-well culture insert (Ibidi Cat# 81176) pre-coated by Matrigel. After cell attachment for approximately 24 h to form an optically confluent monolayer, the culture insert was removed to create the wound gap and the cells were then cultured with serum-free medium. The wound gap closure was monitored by taking pictures with an Eclipse inverted microscope (Leica) at different time points.\",\n \"GraphPad Prism was used to calculate and plot the mean ± SEM of measured quantities. Significances were assessed by two-way analysis of variance (ANOVA)/Mann-Whitney U test (nonparametric)/Student's t test (parametric), p-value < 0.05 was considered to indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.\",\n \"Two SLC39A5 knockout (KO) cell lines were developed using the CRISPR-Cas9 system in the human embryonic kidney cell line (HEK293) ( Figure 1A). Cell morphology anomalies were markedly observed in the SLC39A5-depleted cells. After regular growth for 2 days, the KO cells revealed a tendency to form small cell aggregates instead of a typical optically confluent monolayer ( Figure 1B).\",\n \"This phenotype suggested a potential impairment or abnormality of ECM generation. Considering the influence of ECM dynamics on cell migration, invasion and morphogenesis, we immediately investigated the cellular motility and migratory capacity of the SLC39A5depleted HEK293 cells. Scratch assay was performed in both groups.\",\n \"A 2-well culture insert generated a uniform gap in the confluent monolayer, and wound healing was imaged at different time points.\",\n \"The results showed that the gap in the control group was completely healed after 24 h, while the wound gap in the KO group was not able to close ( Figure S1; Figure 1C).\",\n \"To investigate the overall alteration caused by SLC39A5 depletion, RNA-sequencing (RNA-seq) was performed using a Immunofluorescence also verified the decrease in these ECM proteins ( Figures S3 and S4).\",\n \"The transcription of collagens, FN and LN is closely related to TGFβ signalling, and we have already determined that mutations of SLC39A5 dysregulated TGFβ signalling previously. 15 Considering the transcriptional downregulation of collagens, FN and LN in SLC39A5 KO cells, we hypothesized that reduced SLC39A5 expression would inhibit the TGFβ signalling pathway. Hence, we examined the expression of Smad proteins, which lie on the centre of the TGFβ signalling pathway. The results showed an evident upregulation of total Receptor-Smads (R-Smads) (Smad1, Smad2/3) expression along with a significant decrease in phosphorylated Smad expression (pSmad1/5/9, pSmad2/3) ( Figure S5). Additionally, Co-Smad (Smad4) expression was also increased, while inhibitor-Smad (Smad7) was not affected (Figure S5), suggesting a suppressed activation of the TGFβ signalling, which was confirmed through immunofluorescence ( Figures S6 and S7). The qRT-PCR performed to assess the transcriptional levels of Smad1, Smad2 and Smad4 in KO cells showed increased mRNA expression of the tested proteins ( Figure S8). To validate the phenotype of inhibited TGFβ signalling activation and insufficient ECM synthesis resulting from SLC39A5 depletion, rescue experiments were carefully carried out. As shown in Figure 3, re-expression of SLC39A5 restored intracellular zinc level ( Figure 3A). TGFβ signalling activation and ECM synthesis were also rescued ( Figure \",\n \"The TGFβ pathway is a zinc-regulated pathway ( Figure S9), while SLC39A5 is a zinc transporter. 17,18 These facts prompted us to confirm the involvement of zinc homeostasis in TGFβ signalling alteration in SLC39A5 KO cells. Thus, zinc quantification was performed to detect intracellular zinc levels. As shown in Thus, we selected the most widely expressed zinc efflux protein SLC30A1 (20), as a target. Surprisingly, silencing SLC30A1 counterbalanced the influence of SLC39A5 depletion, which significantly reverted intracellular zinc level close to normal ( Figure S10) and normalized the TGFβ signalling pathway and ECM synthesis ( Figure S11).\",\n \"SLC39A5 was first identified as a high myopia pathogenic gene in our previous study 15 and was later confirmed as one of the top three genes contributing to nonsyndromic high myopia. 16,[20][21][22][23] However, the mechanisms by which SLC39A5 contributes to high myopia are not well understood. Here, we demonstrated that SLC39A5 may play a role in the pathogenesis of high myopia by modulating TGFβ signalling-mediated ECM synthesis through the regulation of intracellular zinc homeostasis. SLC39A5 depletion significantly lowered the intracellular zinc level, which destabilized Smad proteins, and further inhibited TGFβ signalling-mediated ECM synthesis, which ultimately contributed to the pathogenesis of high myopia.\",\n \"Zinc homeostasis is crucial for all organisms as it serves as a catalytic or structural cofactor for multiple types of proteins. Zinc homeostasis depends on the transport of zinc in both directions across the plasma membrane and both in and out of various vesicular compartments. 24 The SLC39 family facilitates zinc transport into the cytoplasm in the opposite direction to SLC30 family members. 25 Within this network, SLC39A5 is a specific zinc transporter. 17,18,26 Our work confirmed the zinc uptake characteristic of SLC39A5, in which SLC39A5 depletion caused an intracellular decline in zinc levels in HEK293 or lymphocyte cells. Once zinc homeostasis was disrupted, the catalytic or structural stability of proteins was impaired, suggesting that SLC39A5 depletion, indirectly, caused this impairment.\",\n \"Zinc has a profound effect on stabilization of zinc-binding proteins, 27-31 since the proteins responsible for cellular zinc buffering will bind zinc transiently; therefore, their function will strongly depend on the cellular zinc status. 32 It has been proposed that zinc chelation affects protein stability through various mechanisms. The human SOD1 protein requires zinc binding to facilitate the restructuring of apoproteins, 33 while the putative protease of Bacteroides thetaiotaomicron (ppBat) requires zinc chelation to link the β-strands to α-helices in protein structures. 34 In our study, the R-Smads and Co-Smad, which are pivotal to the BMP/TGFβ signalling pathway, 35 F I G U R E 2 SLC39A5 depletion impairs extracellular matrix (ECM) synthesis. (A) shows the pathway enrichment analysis for the differentially expressed genes between the control and KO subpopulations of HEK293 cells. Pathway analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics Resources, with an enrichment p-value cut-off of 0.01. (B) shows the qRT-PCR validation of the differentially expressed genes of the ECM members indicated by RNA-seq. (C-E) shows the expression of several ECM components (COL1, COL2, COL4, FN and LN) in the supernatant, whole-cell lysate or intracellular lysate (trypsin digested) of wild-type (WT) and SLC39A5 knockout (KO) HEK293 cells. All tested ECM components were decreased in both the whole lysate and intracellular lysate. (B) and (C) show statistical analyses of the ECM components in the whole lysate and intracellular lysate, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are zinc-binding proteins. We found that Smad proteins were rapidly degraded during SLC39A5 deletion-induced zinc deficiency.\",\n \"Further results suggested that loose zinc coupling, facilitated by a mutant ion-binding site, directly destabilized the exogenous Smad1 proteins, which clearly confirms the necessity of zinc chelation in maintaining the protein stability of Smads, while disturbances in zinc homeostasis induce protein instability.\",\n \"Zinc binding has also been implicated in protein phosphorylation and subcellular translocation. 36 Non-zinc-bound protein kinase C delta (PKCδ) showed a significantly different structure near its phosphorylation site, compared with when it was zinc-bound. Subcellular translocation of PKCδ was also influenced by intracellular zinc. These findings expand our understanding of the phosphorylation decrease in R-Smads during SLC39A5-depleted conditions.\",\n \"Since R-Smads are directly phosphorylated by upstream receptor kinases, which then form heteromeric complexes with Smad4 to translocate into the nucleus, 37 the zinc deficiency induced by SLC39A5 depletion may barely maintain the Smad protein in a favourable structure for phosphorylation, and may lead to nucleus translocation retention, further impairing the TGFβ signalling.\",\n \"It is well recognized that TGFβ signalling directly affects ECM dynamics or triggers it when mediating other biological processes. [38][39][40][41] Herein, suppressed TGFβ signalling in the zinc-deficient conditions restrained ECM synthesis in HEK293. Recently, ECM synthesis and remodelling have attracted significant attention in high myopia aetiology studies. [8][9][10]42 We found that the transcription of all ECM proteins tested (COL1, COL2, COL4, FN and LN) was decreased under zinc-deficient conditions. ECM-induced cell morphogenesis According to this hypothesis, the SLC39A5-induced zinc deficiency suppresses TGFβ signalling, leading to insufficient ECM synthesis, which may ultimately contribute to high myopia development. We further validated this hypothesis by performing rescue experiments.\",\n \"As expected, of SLC39A5 re-expression, which recovers zinc influx, and SLC30A1 silencing, which blocks zinc efflux, could identically rescue the alteration of R-Smad phosphorylation and ECM synthesis in SLC39A5-depleted HEK293 cells, by re-establishing zinc homeostasis.\",\n \"Upon reviewing the relationship between zinc and high myopia, we found plenty of indications of zinc involvement in high myopia development. 43 First, zinc is present abundantly in the ocular system, including the retina and choroid; thus, zinc deficiency produces a variety of ocular manifestations. Furthermore, a number of high myopia gene products are zinc-related proteins.\",\n \"Among them, ZNF644, ZFHX1B, ZC3H11B and SCO2 are zinc finger proteins, and HGF and IGF1 are positively regulated by intracellular zinc concentration. Additionally, certain pathogenic genes (LAMA1 and TGFB1) may even be regulated by intracellular zinc, according to the zinc-TGFβ signalling-ECM synthesis flow.\",\n \"Finally, multiple small molecules that serve as chaperones for disease-related genes or pathways are zinc-related proteins. Smad proteins, together with their anchors (SARA and HGS), are either zinc-binding or zinc finger proteins. These findings strongly highlight the correlation between zinc and high myopia.\",\n \"In summary, our data suggest that depletion of SLC39A5 induces zinc deficiency, which restrains TGFβ signalling-mediated ECM synthesis, thus possibly contributing to high myopia pathogenesis. Zinc homeostasis appears to be a dominant aetiological factor of high myopia development. \",\n \"The authors confirm that there are no conflicts of interest. \",\n \"The data that support the findings of this study are available from the corresponding author upon reasonable request.\",\n \"https://orcid.org/0000-0002-1605-7066\",\n \"Zhengmao Hu https://orcid.org/0000-0002-3921-8205\"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["| INTRODUC TI ON","| MATERIAL S AND ME THODS","| Cell culture","| SLC39A5 knockout cell line construction","| Zinc quantification","| RNA-seq and analysis","| qRT-PCR validation","| Plasmid construction and transient transfection","| Immunoblotting","| Immunofluorescence","| Wound healing capacity test","| Statistical analysis","| RE SULTS","| SLC39A5 depletion impairs ECM synthesis","| SLC39A5 depletion suppresses TGFβ signalling","| SLC39A5 depletion induces zinc deficiency and destabilizes Smad protein","| DISCUSS ION","ACK N OWLED G EM ENTS","CO N FLI C T O F I NTE R E S T","AUTH O R CO NTR I B UTI O N","DATA AVA I L A B I L I T Y S TAT E M E N T","O RCI D","Shanshan Dong","R E FE R E N C E S","Figure 4 ,"],"string":"[\n \"| INTRODUC TI ON\",\n \"| MATERIAL S AND ME THODS\",\n \"| Cell culture\",\n \"| SLC39A5 knockout cell line construction\",\n \"| Zinc quantification\",\n \"| RNA-seq and analysis\",\n \"| qRT-PCR validation\",\n \"| Plasmid construction and transient transfection\",\n \"| Immunoblotting\",\n \"| Immunofluorescence\",\n \"| Wound healing capacity test\",\n \"| Statistical analysis\",\n \"| RE SULTS\",\n \"| SLC39A5 depletion impairs ECM synthesis\",\n \"| SLC39A5 depletion suppresses TGFβ signalling\",\n \"| SLC39A5 depletion induces zinc deficiency and destabilizes Smad protein\",\n \"| DISCUSS ION\",\n \"ACK N OWLED G EM ENTS\",\n \"CO N FLI C T O F I NTE R E S T\",\n \"AUTH O R CO NTR I B UTI O N\",\n \"DATA AVA I L A B I L I T Y S TAT E M E N T\",\n \"O RCI D\",\n \"Shanshan Dong\",\n \"R E FE R E N C E S\",\n \"Figure 4 ,\"\n]"},"annotations_table":{"kind":"list like","value":[],"string":"[]"},"annotations_tableref":{"kind":"list like","value":["Table S1","Table S2","Table S3","Table S4","Table S5","Table S6"],"string":"[\n \"Table S1\",\n \"Table S2\",\n \"Table S3\",\n \"Table S4\",\n \"Table S5\",\n \"Table S6\"\n]"},"annotations_title":{"kind":"list like","value":["O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis","O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis"],"string":"[\n \"O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis\",\n \"O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis\"\n]"},"annotations_venue":{"kind":"list like","value":["J Cell Mol Med"],"string":"[\n \"J Cell Mol Med\"\n]"}}},{"rowIdx":84744,"cells":{"corpusid":{"kind":"number","value":220888721,"string":"220,888,721"},"updated":{"kind":"string","value":"2022-01-23T02:19:35Z"},"content_source_oainfo_license":{"kind":"string","value":"CCBY"},"content_source_oainfo_openaccessurl":{"kind":"string","value":"https://nutritionandmetabolism.biomedcentral.com/track/pdf/10.1186/s12986-020-00480-w"},"content_source_oainfo_status":{"kind":"string","value":"GOLD"},"content_source_pdfsha":{"kind":"string","value":"01ce14faf78d6b8948a0ecf411868189efe5ecaf"},"content_source_pdfurls":{"kind":"null"},"externalids_acl":{"kind":"null"},"externalids_arxiv":{"kind":"null"},"externalids_dblp":{"kind":"null"},"externalids_doi":{"kind":"string","value":"10.1186/s12986-020-00480-w"},"externalids_mag":{"kind":"string","value":"3046886996"},"externalids_pubmed":{"kind":"string","value":"32774437"},"externalids_pubmedcentral":{"kind":"string","value":"7395973"},"content_text":{"kind":"string","value":"\nEffects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial\n\n\nXiaomin Sun \nXiao-Kai Ma \nLin Zhang \nZhen-Bo Cao \nEffects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial\n10.1186/s12986-020-00480-wR E S E A R C H Open Access\nBackground: Previous studies indicated that serum 25-hydroxyvitamin D [25(OH)D] concentrations are positively associated with physical activity levels independent of sun exposure. However, the effect of resistance training on serum 25(OH) D concentrations remains unclear. Thus, this study aimed to examine the effect of chronic resistance training on serum 25(OH) D concentrations and determine whether 25(OH) D concentration variations are influenced by body composition changes. Methods: Eighteen young men aged 19-39 years were randomly divided into a 12-week resistance training group (RT, n = 9) and non-exercise control group (CON, n = 9). The trial was undertaken in Shanghai University of Sport in Shanghai, China. Randomization and allocation to trial group were carried out by a central computer system. Serum 25(OH) D and intact parathyroid hormone concentrations were measured using commercially available enzymelinked immunosorbent assay kits. Body composition was measured by dual-energy X-ray absorptiometry.Results: The average serum 25(OH) D concentrations were 26.6 nmol/L at baseline. After the 12-week intervention program, serum 25(OH) D concentrations significantly increased in both groups. Serum 25(OH) D concentrations at midpoint (6-week) increased significantly only in the CON group (P < 0.01). From training midpoint to endpoint, a significantly greater increase in serum 25(OH) D concentrations was noted in the RT group (P-interaction = 0.043); 25(OH) D concentration changes (end-pre) were negatively related to fat-free mass (mid-pre) (r = − 0.565, P = 0.015) and muscle mass (mid-pre) (r = − 0.554, P = 0.017).Conclusions: There were no beneficial effects of the 12-week resistance training on serum 25(OH) D concentration in vitamin D deficient young men, and an indication that seasonal increase in serum 25(OH) D concentrations during the early phase of resistance training was transiently inhibited, which may partly be attributed to resistance training-induced muscle mass gain.\n\nIntroduction\n\nCirculating 25-hydroxyvitamin D [25(OH)D] is the major form of vitamin D, which is used in vitamin D status assessment. Accumulated studies indicated that vitamin D not only regulates calcium and phosphorus metabolism but also plays an important role in modifying cardiovascular diseases risk, and improving immune function and physical fitness [1][2][3][4].\n\nPrevious studies indicated that higher levels of physical activity or cardiorespiratory fitness were related to higher circulating 25(OH) D concentrations [5][6][7]. Although individuals who were physically active outdoors are more likely to have higher circulating 25(OH) D due to increased sun exposure [8,9], this relationship was still observed after adjustment for sunlight exposure [6,10]. Scragg et al. [5] found that higher levels of physical activity were associated with higher serum 25(OH) D concentrations not only during summer but also during winter, when the vitamin D synthesis from sun exposure is extremely limited [11]. Consistently, several intervention studies suggested that endurance exercise could increase circulating 25(OH) D or prevent its seasonal reduction [12][13][14]. These findings indicated that physical activity could directly increase 25(OH) D concentrations.\n\nResistance training (RT) promotes health benefits through increased skeletal muscle mass and qualitative adaptations [15]. In addition to adiposity tissue, muscle tissue was suggested to serve as an another important site to store 25(OH) D, and then it returns to the circulating when needed [16,17]. Nevertheless, Makanae and colleagues reported that intramuscular expression of CYP27B1, which catalyses the hydroxylation and activation of 25(OH) D, was significantly increased at 1 h and 3 h after resistance exercise, which may enhance local vitamin D metabolism in skeletal muscle [18]. It is well known that compared with endurance exercise, RT could not only reduce fat mass, but also it is more effective in increasing muscle mass and strength [19,20], which may largely affect vitamin D metabolism. However, whether serum 25(OH) D concentrations are affected by RT remains unclear.\n\nThis study aimed to evaluate the effect of chronic resistance training on serum 25(OH) D concentrations and to determine whether the effects are attributable to changes in body composition in healthy young men.\n\n\nMethods\n\n\nParticipants\n\nEighteen healthy men with no chronic diseases (aged 19 to 39 years) were included in this study, which was conducted from March to July 2016. Participants using vitamin D supplements or sunscreen regularly were excluded. Assessments were performed at 0 week (pre), 6 weeks (midpoint), and 12 weeks (endpoint). All subjects provided informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). The trial has been retrospectively registered at Chinese Clinical Trial Registry website (ChiCTR2000030876).\n\n\nExperimental protocol\n\nEighteen participants were randomly allocated to the following two groups by using a computer-generated random number sequence: resistance training group (RT) and no-exercise control group (CON). Participants in the RT group were instructed to exercise 2-3 times a week in a gymnasium. A supervised training session was performed in the afternoon between 1630 h and 2000 h during the 12 weeks of progressive resistance training. In the first 2 weeks, the participants were taught how to properly perform each movement. The resistance workload gradually changed from light to heavy, and the participants were instructed to complete 10 repetitions for each set of the following exercises: leg press, preacher curl, chest press, leg curl, and iso row, with 60-120-s rest period between two sets; additionally, they also performed three sets of 15-25 repetitions of abdominal crunch and back extension with their own body weight, with a 60-120-s rest period between two sets. At the first training session of the third week, the participants were instructed to perform strength testing, and 12 repetitions maximum (12RM) for each exercise were performed, except back extension and abdominal crunch. The workload for the next resistance training was calculated from the participants' previously determined 12RM. The participants were encouraged to perform back extension and abdominal crunch exercises 30 times from the first training session of the third week. When the strength training equipment at our gymnasium had been upgraded at the eighth week of training, the resistance training program was revised to include the following: leg extension, standing one-arm cable curl, standing cable chest press, leverage shoulder press, leverage high row, back extension, and abdominal crunch. These new components of the resistance training program were selected based on the principle of using similar muscle groups. The order of resistance exercise was randomized; however, training of the same muscle groups continuously was avoided. The participants were instructed not to undertake any formal exercise or change their levels of general physical activity and dietary habits.\n\n\nAnthropometric measurements\n\nHeight was measured with the participants wearing light clothing and being barefoot (HK6000-ST, HENGKANG JIAYE Technology Co., Ltd. CHN). Dual-energy X-ray absorptiometry (DXA Prodigy, GE Lunar Corp., Madison, WI, USA) was used for the measurement of whole and regional body composition, including total body mass, muscle mass, fat mass, fat percentage, and bone mineral density. Body mass index was calculated as body mass (kg) divided by height (m 2 ). Fat-free mass (FFM) was calculated as body mass minus fat mass.\n\n\nPhysical activity and sun exposure\n\nPhysical activity was measured using Actigraph GT3X+ accelerometers (ActiGraph, LLC, Pensacola, FL, USA). Participants were instructed to wear the accelerometers on their right hip at all times, except during water activities (swimming, showering) or sleeping, for 7 consecutive days at baseline. A wear time of ≥480 min/day was used as the criterion for a valid day, and ≥ 2 weekdays and 1 weekend day was used as the criteria for a valid 7-day period of accumulated data. Non-wear time was defined as consecutive periods of ≥60 min of zero accelerometer counts and excluded from the analyses. Data were recorded in 60-s epochs. A pragmatic cutoff of ≥2690 cpm was used to categorize moderatevigorous physical activity time [21,22]. The 7-day data collection for all subjects was fixed within 2 weeks. Outdoor time from 0900 h to 1600 h for 7 consecutive days was recorded and used as an alternative for sun exposure in the analysis.\n\n\nBlood analysis\n\nBlood samples were obtained after at least an 8-h overnight fasting period and subsequently centrifuged at 3000 rpm at 4°C for 10 min. Serum was collected and stored at − 80°C until analysis. Serum 25(OH) D and intact parathyroid hormone (iPTH) concentrations were measured in duplicate using commercially available enzyme-linked immunosorbent assay kits (25(OH) D, IDS, Bolton, UK; iPTH, DRG, Marburg, Germany) according to the manufacturer's instructions. The intraassay and inter-assay coefficients of variation were 9.1 and 7.7%, respectively, for serum 25(OH) D and 9.6 and 3.6%, respectively, for iPTH. Vitamin D deficiency is defined as 25(OH) D concentration < 50 nmol/L, and vitamin D insufficiency as 25(OH) D concentration < 75 nmol/L [23].\n\n\nStatistical analysis\n\nAll statistical analyses were performed using IBM SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were assessed for normality using a Kolmogorov-Smirnov test prior to all statistical analyses. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables were presented as median (interquartile range), unless otherwise indicated. The Student t test (for normally distributed variables) or the Mann-Whitney U test (for non-normally distributed variables) was used to evaluate the differences between groups. Two-factor repeat-measured analysis of variance (ANOVA) (group × time) was used to determine the effect of exercise on 25(OH) D, iPTH, and body composition indicators. A post hoc test with Bonferroni correction was performed to identify statistically significant differences among the mean values when a considerable interaction was identified. Pearson correlation and partial correlation coefficients were computed between 25(OH) D concentration changes and body composition indicators. The level of statistical significance was set at P < 0.05.\n\n\nResults\n\nThe participants' characteristics and blood parameters at baseline are presented in Table 1. The average serum 25(OH) D concentrations were 26.6 nmol/L, and all participants showed vitamin D deficiency. No significant difference in either body composition indicators or blood parameters was found between the groups (P > 0.05).\n\nAs shown in Table 2, significant interactions of group × time were found for FFM (P = 0.004) and muscle mass (P = 0.003). After resistance training, the FFM values (mid-pre, 2.7%, P = 0.003; end-pre, 3.0%, P = 0.004) and muscle mass (mid-pre, 3.0%, P = 0.002; end-pre, 3.4%, P = 0.004) significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in the CON group. In addition, we observed borderline significant interactions in body fat percentage, arm fat percentage, leg fat percentage, and trunk fat percentage ( Table 2).\n\nAs shown in Table 2 and Fig. 1, a significant group × time interaction was observed for serum 25(OH) D concentrations (P = 0.025). After the 12-week intervention, the mean serum 25(OH) D concentrations significantly increased to 36.7 ± 2.7 nmol/L in the RT group and 40.7 ± 2.7 nmol/L in the CON group. Moreover, in the CON group, higher serum 25(OH) D concentrations were observed at midpoint compared with the values at baseline (P < 0.01), whereas no notable differences were found in the RT group. From midpoint to endpoint, although serum 25(OH) D concentrations significantly increased in both groups, the increase was significantly greater in the RT group (25.8%) than in the CON group (9.4%) (P-interaction =0.043).\n\nCorrelation analyses were performed to identify the factors associated with the changes in 25(OH) D concentrations (Table 3). 25(OH) D concentration changes (end-pre) were negatively related to FFM (mid-pre) (P = 0.015) and muscle mass (mid-pre) (P = 0.017) changes (Fig. 2); a borderline association was observed between 25(OH) D concentration changes (mid-pre) and FFM (mid-pre) (P = 0.058) and muscle mass (mid-pre) (P = 0.057) changes. In addition, significant correlations of 25(OH) D concentration changes (end-pre) with FFM (mid-pre) (P = 0.039) and muscle mass (mid-pre) (P = 0.043) changes were observed after adjustment for group, age, and changes in sun exposure (end-pre), body weight (end-pre), and body fat percentage (end-pre).\n\n\nDiscussion\n\nIn this study, we found that the seasonal increase in serum 25(OH) D concentrations in young men was transiently inhibited during the early phase of RT; subsequently, serum 25(OH) D concentrations increased in both RT and CON groups, and the increase was observed to be greater in RT group than in CON group. The transient inhibition could be partly attributed to muscle mass and fat free mass gain induced by RT in the early phase.\n\nPrevious studies have reported significant positive associations of serum 25(OH) D concentrations with physical activity levels independent of sun exposure [6,10]. Bell et al. [24] found that 25(OH) D concentrations were higher in participants who had engaged in regular muscle-building exercise for at least 1 year compared to those in the control group. Consistently, our recent studies also observed that exercise training could increase serum 25(OH) D concentrations or prevent its seasonal reduction in young and old men [13,14]. However, the effect of RT on serum 25(OH) D concentrations remains unclear. Compared with endurance training, RT is more effective in increasing muscle mass and strength [24], which was also supported by our results. In the present study, we observed that after resistance training, body mass was reduced, while FFM and muscle mass were significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in CON group.\n\nIn addition to adiposity tissues, muscle tissues are also served as another important storage for 25(OH) D, which could enable optimal vitamin D status to maintain its physiological function during winter months [17]. Makanae and colleagues observed that intramuscular CYP27B1 was significantly increased after resistance exercise in skeletal muscle, which may enhance local vitamin D metabolism [18]. Srikuea et al. [25] also found that the CYP27B1 expression in the muscle increased significantly during skeletal muscle regeneration from injury. Thus, we speculated that the transient inhibition in serum 25(OH) D levels in RT group during the early phase of resistance training could be partly due to absorption of serum 25(OH) D in the increasing muscle mass and its use in muscle activity. 25(OH) D consumption in muscle may induce a compensatory increase in serum 25(OH) D concentrations; however, the concentrations were still lower than that in control group, which may due to the lack of vitamin D storage and short-term intervention period. Previous studies report that serum 25(OH) D concentrations gradually increase from around March to August [26]. This could partly explain the reason why serum 25(OH) D concentration was transiently inhibited in early Spring and followed by a marked increase in early Summer in our study. Future studies that aim at exploring the mechanism underlying the effect of resistance training on serum 25(OH) D concentrations are warranted. This study has several limitations. First, our study included only men with relatively lower serum 25(OH) D concentrations. Therefore, it remains to be established whether these findings are applicable to other subjects with higher 25(OH) D concentrations. Second, considering the seasonal variation of serum 25(OH) D concentration, the findings in our study have seasonal limitations. Third, at baseline resistance training group had relatively lower values of muscle mass and body fat percentage, although they are not statistically significant, which should be interpreted with caution. Fourth, we couldn't observe any relationship between muscle mass and 25(OH) D at three points, which may due to the small sample size. However, the muscle mass changes were significantly related with 25(OH) D concentration changes. Finally, although our sample size was relatively small, a post hoc power calculation would still give us a 90% chance, with an effect size of 0.58, to demonstrate the interaction effect of serum 25(OH) D concentrations, which was considered statistically significant at P < 0.05 at the endpoints.\n\n\nConclusions\n\nIn summary, serum 25(OH) D concentrations in vitamin D deficient young men was observed to be transiently inhibited during the early phase of resistance training and subsequently increased in the later phase. This finding could be partly attributed to the muscle mass and fat free mass gain induced by resistance training during the early phase. Our findings indicated that adequate administration of oral supplements or increased daily dietary vitamin D intake are encouraged to increase serum 25(OH) D concentrations, especially for those who exercise regularly. \n\nFig. 1\n1Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05\n\n\n25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum\n\nTable 1\n1Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activityVariable \nResistance training group (RT) (n = 9) \nNon-exercise control group (CON) (n = 9) \n\nAge (y) \n24.2 ± 3.1 \n26.7 ± 6.2 \n\nHeight (cm) \n1.74 ± 0.03 \n1.75 ± 0.06 \n\nWeight (kg) \n68.5 ± 9.7 \n68.7 ± 7.6 \n\nBMI (kg/m 2 ) \n22.5 ± 2.9 \n22.3 ± 1.9 \n\nFFM (kg) \n54.2 (49.2 -56.4 ) \n57.5 (51.1 -58.8 ) \n\nMuscle mass (kg) \n51.4 (46.6 -53.3 ) \n54.9 (48.4 -55.8 ) \n\nBody fat percentage (%) \n21.9 ± 6.9 \n18.8 ± 6.0 \n\nArms fat percentage (%) \n18.7 ± 7.1 \n14.4 ± 5.1 \n\nLegs fat percentage (%) \n20.5 ± 5.6 \n18.1 ± 5.2 \n\nTrunk fat percentage (%) \n25.1 ± 8.3 \n21.6 ± 7.3 \n\nBMD (g/cm 2 ) \n1.16 ± 0.07 \n1.18 ± 0.07 \n\n25(OH) D (nmol/L) \n26.9 ± 4.7 \n26.2 ± 4.1 \n\niPTH (pg/mL) \n54.9 ± 19.8 \n44.6 ± 23.8 \n\nMVPA (min/day) \n69.8 ± 32.2 \n65.8 ± 16.3 \n\nSun exposure (min/day) \n40.0 ± 27.9 \n50.0 ± 30.7 \n\n\n\nTable 2\n2Characteristics of the participants at baseline, midpoint and endpointVariable \nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \n\nBaseline \nMidpoint \nEndpoint \nBaseline \nMidpoint \nEndpoint \nTime Group Time × Group interaction \n\nWeight (kg) a \n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \n68.8 ± 3.1 \n68.7 ± 3.1 \n68.6 ± 3.0 \n0.156 0.958 0.762 \n\nBMI (kg/m 2 ) a \n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \n22.3 ± 0.9 \n22.2 ± 0.9 \n22.2 ± 0.8 \n0.273 0.768 0.455 \n\nFFM (kg) a \n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \n56.1 ± 1.8 \n55.8 ± 1.8 \n0.347 0.341 0.004 \n\nMuscle mass (kg) a \n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \n53.2 ± 1.7 \n53.0 ± 1.7 \n0.399 0.348 0.003 \n\nBody fat percentage a \n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \n18.3 ± 2.0 \n18.0 ± 1.9 \n18.3 ± 2.0 \n0.643 0.327 0.058 \n\nArms fat percentage a \n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \n13.7 ± 1.8 \n13.4 ± 1.8 \n13.6 ± 1.7 \n0.687 0.091 0.077 \n\nLegs fat percentage a \n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \n17.7 ± 1.7 \n17.2 ± 1.6 \n17.6 ± 1.7 \n0.846 0.378 0.064 \n\nTrunk fat percentage a \n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \n21.1 ± 2.5 \n20.8 ± 2.4 \n21.1 ± 2.4 \n0.484 0.397 0.075 \n\nBMD (g/cm 2 ) a \n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \n0.016 0.538 0.235 \n\n25(OH) D (nmol/L) b \n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \n\niPTH (pg/mL) b \n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \n37.6 ± 8.3 \n48.3 ± 6.1 \n51.5 ± 5.6 \n0.110 0.131 0.242 \n\nSun exposure (min/day) 40.0± 9.8 \n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \n73.5 ± 13.5 50.5 ± 15.2 \n0.015 0.889 0.493 \n\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \n\nc \n\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \nBoldface indicates significance (P < 0.05) \n\n\nTable 3\n3Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free massVariables \n25(OH)D mid-pre \n25(OH)D end-pre \n\nr \nP \nr \nP \n\nBody fat percentage \n\nmid-pre \n0.014 \n0.956 \n0.101 \n0.690 \n\nend-mid \n−0.217 \n0.387 \n−0.004 \n0.986 \n\nend-pre \n−0.090 \n0.722 \n0.064 \n0.800 \n\nFFM \n\nmid-pre \n−0.455 \n0.058 \n−0.565 \n0.015 \n\nend-mid \n0.121 \n0.632 \n0.037 \n0.883 \n\nend-pre \n−0.289 \n0.244 \n−0.424 \n0.080 \n\nMuscle mass \n\nmid-pre \n−0.456 \n0.057 \n−0.554 \n0.017 \n\nend-mid \n0.142 \n0.575 \n0.037 \n0.884 \n\nend-pre \n−0.280 \n0.261 \n−0.415 \n0.087 \n\n\nAcknowledgementsThe authors would like to thank all the participants who took part in the study.Authors' contributionsThe study was designed by ZBC and XKM; data were collected and analyzed by XKM and XS; data interpretation and manuscript preparation were undertaken by ZBC, XKM, XS, and LZ. All authors read and approved the final manuscript.FundingThis study was supported in part by a grant from the National Natural Science Foundation of China (Nos. 31571226 and 81703220).Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Ethics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). All subjects provided informed consent for inclusion before they participated in the study.Consent for publicationNot applicable.\nVitamin D -effects on skeletal and extraskeletal health and the need for supplementation. M Wacker, M F Holick, Nutrients. 51Wacker M, Holick MF. Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. Nutrients. 2013;5(1):111-48.\n\nVitamin D: beyond bone. S Christakos, M Hewison, D G Gardner, C L Wagner, I N Sergeev, E Rutten, Ann N Y Acad Sci. 1287Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al. Vitamin D: beyond bone. Ann N Y Acad Sci. 2013;1287:45-58.\n\nAssociation between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. X Sun, Z B Cao, Y Zhang, Y Ishimi, I Tabata, M Higuchi, Nutrients. 61Sun X, Cao ZB, Zhang Y, Ishimi Y, Tabata I, Higuchi M. Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. Nutrients. 2014;6(1):221-30.\n\nThe relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. X Sun, Z B Cao, K Tanisawa, T Ito, S Oshima, M Higuchi, Nutrients. 71Sun X, Cao ZB, Tanisawa K, Ito T, Oshima S, Higuchi M. The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. 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A Ardestani, B Parker, S Mathur, P Clarkson, L S Pescatello, H J Hoffman, Am J Cardiol. 1078Ardestani A, Parker B, Mathur S, Clarkson P, Pescatello LS, Hoffman HJ, et al. Relation of vitamin D level to maximal oxygen uptake in adults. Am J Cardiol. 2011;107(8):1246-9.\n\nAssociations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. A Burgaz, A Akesson, A Oster, K Michaelsson, A Wolk, Am J Clin Nutr. 865Burgaz A, Akesson A, Oster A, Michaelsson K, Wolk A. Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. Am J Clin Nutr. 2007;86(5):1399-404.\n\nThe contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. M G Kimlin, R M Lucas, S L Harrison, I Van Der Mei, B K Armstrong, D C Whiteman, Am J Epidemiol. 1797Kimlin MG, Lucas RM, Harrison SL, van der Mei I, Armstrong BK, Whiteman DC, et al. The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. Am J Epidemiol. 2014;179(7):864-74.\n\nDuration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women. M A Kluczynski, M J Lamonte, J A Mares, J Wactawski-Wende, A W Smith, C D Engelman, Ann Epidemiol. 216Kluczynski MA, Lamonte MJ, Mares JA, Wactawski-Wende J, Smith AW, Engelman CD, et al. Duration of physical activity and serum 25- hydroxyvitamin D status of postmenopausal women. Ann Epidemiol. 2011; 21(6):440-9.\n\nVitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. M F Holick, Clin Rev Bone Miner Metabol. 71Holick MF. Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. Clin Rev Bone Miner Metabol. 2009;7(1):2-19.\n\nEffect of antioxidants and exercise on bone metabolism. L Maïmoun, D Simar, C Caillaud, E Peruchon, C Sultan, M Rossi, J Sports Sci. 263Maïmoun L, Simar D, Caillaud C, Peruchon E, Sultan C, Rossi M, et al. Effect of antioxidants and exercise on bone metabolism. J Sports Sci. 2008;26(3): 251-8.\n\nEffect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. X Sun, Z B Cao, H Taniguchi, K Tanisawa, M Higuchi, J Clin Endocrinol Metab. 10211Sun X, Cao ZB, Taniguchi H, Tanisawa K, Higuchi M. Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. J Clin Endocrinol Metab. 2017;102(11):3937-44.\n\nEffects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. X Sun, Z B Cao, K Tanisawa, H Taniguchi, T Kubo, M Higuchi, Endocrine. 592Sun X, Cao ZB, Tanisawa K, Taniguchi H, Kubo T, Higuchi M. Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. Endocrine. 2018;59(2):330-7.\n\nResistance training is medicine: effects of strength training on health. W L Westcott, Curr Sports Med Rep. 114Westcott WL. Resistance training is medicine: effects of strength training on health. Curr Sports Med Rep. 2012;11(4):209-16.\n\nEvidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells. M Abboud, D A Puglisi, B N Davies, M Rybchyn, N P Whitehead, K E Brock, Endocrinology. 1549Abboud M, Puglisi DA, Davies BN, Rybchyn M, Whitehead NP, Brock KE, et al. Evidence for a specific uptake and retention mechanism for 25- hydroxyvitamin D (25OHD) in skeletal muscle cells. Endocrinology. 2013; 154(9):3022-30.\n\nThe role of skeletal muscle in maintaining vitamin D status in Winter. R S Mason, M S Rybchyn, M Abboud, T C Brennan-Speranza, D R Fraser, Curr Dev Nutr. 31087Mason RS, Rybchyn MS, Abboud M, Brennan-Speranza TC, Fraser DR. The role of skeletal muscle in maintaining vitamin D status in Winter. Curr Dev Nutr. 2019;3(10):nzz087.\n\nAcute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Y Makanae, R Ogasawara, K Sato, Y Takamura, K Matsutani, K Kido, Exp Physiol. 10010Makanae Y, Ogasawara R, Sato K, Takamura Y, Matsutani K, Kido K, et al. Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Exp Physiol. 2015;100(10):1168-76.\n\nEffects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. L H Willis, C A Slentz, L A Bateman, A T Shields, L W Piner, C W Bales, J Appl Physiol. 113121831Willis LH, Slentz CA, Bateman LA, Shields AT, Piner LW, Bales CW, et al. Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. J Appl Physiol. 2012;113(12):1831.\n\nResistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. B Strasser, U Siebert, W Schobersberger, Sports Med. 405Strasser B, Siebert U, Schobersberger W. Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. Sports Med. 2010;40(5):397-415.\n\nInter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. H Jarrett, L Fitzgerald, A C Routen, J Phys Act Health. 123Jarrett H, Fitzgerald L, Routen AC. Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. J Phys Act Health. 2014;12(3):382-7.\n\nValidation and comparison of ActiGraph activity monitors. J E Sasaki, D John, P S Freedson, J Sci Med Sport. 145Sasaki JE, John D, Freedson PS. Validation and comparison of ActiGraph activity monitors. J Sci Med Sport. 2011;14(5):411-6.\n\nEvaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. M F Holick, N C Binkley, H A Bischoff-Ferrari, C M Gordon, D A Hanley, R P Heaney, J Clin Endocrinol Metab. 967Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96(7):1911-30.\n\nThe effects of musclebuilding exercise on vitamin D and mineral metabolism. N H Bell, R N Godsen, D P Henry, J Shary, S Epstein, J Bone Miner Res. 34Bell NH, Godsen RN, Henry DP, Shary J, Epstein S. The effects of muscle- building exercise on vitamin D and mineral metabolism. J Bone Miner Res. 1988;3(4):369-73.\n\nVDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. R Srikuea, X Zhang, O K Park-Sarge, K A Esser, Am J Physiol Cell Physiol. 3034Srikuea R, Zhang X, Park-Sarge OK, Esser KA. VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. Am J Physiol Cell Physiol. 2012;303(4): C396-405.\n\nVariation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. T Kobayashi, T Okano, S Shida, K Okada, T Suginohara, H Nakao, J Nutr Sci Vitaminol (Tokyo). 293Kobayashi T, Okano T, Shida S, Okada K, Suginohara T, Nakao H, et al. Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. J Nutr Sci Vitaminol (Tokyo). 1983;29(3):271-81.\n\nPublisher's Note. Publisher's Note\n\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\n"},"annotations_abstract":{"kind":"list like","value":["Background: Previous studies indicated that serum 25-hydroxyvitamin D [25(OH)D] concentrations are positively associated with physical activity levels independent of sun exposure. However, the effect of resistance training on serum 25(OH) D concentrations remains unclear. Thus, this study aimed to examine the effect of chronic resistance training on serum 25(OH) D concentrations and determine whether 25(OH) D concentration variations are influenced by body composition changes. Methods: Eighteen young men aged 19-39 years were randomly divided into a 12-week resistance training group (RT, n = 9) and non-exercise control group (CON, n = 9). The trial was undertaken in Shanghai University of Sport in Shanghai, China. Randomization and allocation to trial group were carried out by a central computer system. Serum 25(OH) D and intact parathyroid hormone concentrations were measured using commercially available enzymelinked immunosorbent assay kits. Body composition was measured by dual-energy X-ray absorptiometry.Results: The average serum 25(OH) D concentrations were 26.6 nmol/L at baseline. After the 12-week intervention program, serum 25(OH) D concentrations significantly increased in both groups. Serum 25(OH) D concentrations at midpoint (6-week) increased significantly only in the CON group (P < 0.01). From training midpoint to endpoint, a significantly greater increase in serum 25(OH) D concentrations was noted in the RT group (P-interaction = 0.043); 25(OH) D concentration changes (end-pre) were negatively related to fat-free mass (mid-pre) (r = − 0.565, P = 0.015) and muscle mass (mid-pre) (r = − 0.554, P = 0.017).Conclusions: There were no beneficial effects of the 12-week resistance training on serum 25(OH) D concentration in vitamin D deficient young men, and an indication that seasonal increase in serum 25(OH) D concentrations during the early phase of resistance training was transiently inhibited, which may partly be attributed to resistance training-induced muscle mass gain."],"string":"[\n \"Background: Previous studies indicated that serum 25-hydroxyvitamin D [25(OH)D] concentrations are positively associated with physical activity levels independent of sun exposure. However, the effect of resistance training on serum 25(OH) D concentrations remains unclear. Thus, this study aimed to examine the effect of chronic resistance training on serum 25(OH) D concentrations and determine whether 25(OH) D concentration variations are influenced by body composition changes. Methods: Eighteen young men aged 19-39 years were randomly divided into a 12-week resistance training group (RT, n = 9) and non-exercise control group (CON, n = 9). The trial was undertaken in Shanghai University of Sport in Shanghai, China. Randomization and allocation to trial group were carried out by a central computer system. Serum 25(OH) D and intact parathyroid hormone concentrations were measured using commercially available enzymelinked immunosorbent assay kits. Body composition was measured by dual-energy X-ray absorptiometry.Results: The average serum 25(OH) D concentrations were 26.6 nmol/L at baseline. After the 12-week intervention program, serum 25(OH) D concentrations significantly increased in both groups. Serum 25(OH) D concentrations at midpoint (6-week) increased significantly only in the CON group (P < 0.01). From training midpoint to endpoint, a significantly greater increase in serum 25(OH) D concentrations was noted in the RT group (P-interaction = 0.043); 25(OH) D concentration changes (end-pre) were negatively related to fat-free mass (mid-pre) (r = − 0.565, P = 0.015) and muscle mass (mid-pre) (r = − 0.554, P = 0.017).Conclusions: There were no beneficial effects of the 12-week resistance training on serum 25(OH) D concentration in vitamin D deficient young men, and an indication that seasonal increase in serum 25(OH) D concentrations during the early phase of resistance training was transiently inhibited, which may partly be attributed to resistance training-induced muscle mass gain.\"\n]"},"annotations_author":{"kind":"list like","value":["Xiaomin Sun ","Xiao-Kai Ma ","Lin Zhang ","Zhen-Bo Cao "],"string":"[\n \"Xiaomin Sun \",\n \"Xiao-Kai Ma \",\n \"Lin Zhang \",\n \"Zhen-Bo Cao \"\n]"},"annotations_authoraffiliation":{"kind":"list like","value":[],"string":"[]"},"annotations_authorfirstname":{"kind":"list like","value":["Xiaomin","Xiao-Kai","Lin","Zhen-Bo"],"string":"[\n \"Xiaomin\",\n \"Xiao-Kai\",\n \"Lin\",\n \"Zhen-Bo\"\n]"},"annotations_authorlastname":{"kind":"list like","value":["Sun","Ma","Zhang","Cao"],"string":"[\n \"Sun\",\n \"Ma\",\n \"Zhang\",\n \"Cao\"\n]"},"annotations_bibauthor":{"kind":"list like","value":["M Wacker, ","M F Holick, ","S Christakos, ","M Hewison, ","D G Gardner, ","C L Wagner, ","I N Sergeev, ","E Rutten, ","X Sun, ","Z B Cao, ","Y Zhang, ","Y Ishimi, ","I Tabata, ","M Higuchi, ","X Sun, ","Z B Cao, ","K Tanisawa, ","T Ito, ","S Oshima, ","M Higuchi, ","R Scragg, ","I Holdaway, ","R Jackson, ","T Lim, ","D Scott, ","L Blizzard, ","J Fell, ","C Ding, ","T Winzenberg, ","G Jones, ","A Ardestani, ","B Parker, ","S Mathur, ","P Clarkson, ","L S Pescatello, ","H J Hoffman, ","A Burgaz, ","A Akesson, ","A Oster, ","K Michaelsson, ","A Wolk, ","M G Kimlin, ","R M Lucas, ","S L Harrison, ","I Van Der Mei, ","B K Armstrong, ","D C Whiteman, ","M A Kluczynski, ","M J Lamonte, ","J A Mares, ","J Wactawski-Wende, ","A W Smith, ","C D Engelman, ","M F Holick, ","L Maïmoun, ","D Simar, ","C Caillaud, ","E Peruchon, ","C Sultan, ","M Rossi, ","X Sun, ","Z B Cao, ","H Taniguchi, ","K Tanisawa, ","M Higuchi, ","X Sun, ","Z B Cao, ","K Tanisawa, ","H Taniguchi, ","T Kubo, ","M Higuchi, ","W L Westcott, ","M Abboud, ","D A Puglisi, ","B N Davies, ","M Rybchyn, ","N P Whitehead, ","K E Brock, ","R S Mason, ","M S Rybchyn, ","M Abboud, ","T C Brennan-Speranza, ","D R Fraser, ","Y Makanae, ","R Ogasawara, ","K Sato, ","Y Takamura, ","K Matsutani, ","K Kido, ","L H Willis, ","C A Slentz, ","L A Bateman, ","A T Shields, ","L W Piner, ","C W Bales, ","B Strasser, ","U Siebert, ","W Schobersberger, ","H Jarrett, ","L Fitzgerald, ","A C Routen, ","J E Sasaki, ","D John, ","P S Freedson, ","M F Holick, ","N C Binkley, ","H A Bischoff-Ferrari, ","C M Gordon, ","D A Hanley, ","R P Heaney, 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M Wacker, M F Holick, Nutrients. 51Wacker M, Holick MF. Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. Nutrients. 2013;5(1):111-48.","Vitamin D: beyond bone. S Christakos, M Hewison, D G Gardner, C L Wagner, I N Sergeev, E Rutten, Ann N Y Acad Sci. 1287Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al. Vitamin D: beyond bone. Ann N Y Acad Sci. 2013;1287:45-58.","Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. X Sun, Z B Cao, Y Zhang, Y Ishimi, I Tabata, M Higuchi, Nutrients. 61Sun X, Cao ZB, Zhang Y, Ishimi Y, Tabata I, Higuchi M. Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. Nutrients. 2014;6(1):221-30.","The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. X Sun, Z B Cao, K Tanisawa, T Ito, S Oshima, M Higuchi, Nutrients. 71Sun X, Cao ZB, Tanisawa K, Ito T, Oshima S, Higuchi M. The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. Nutrients. 2014;7(1):91-102.","Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. R Scragg, I Holdaway, R Jackson, T Lim, Ann Epidemiol. 25Scragg R, Holdaway I, Jackson R, Lim T. Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. Ann Epidemiol. 1992;2(5):697-703.","A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. D Scott, L Blizzard, J Fell, C Ding, T Winzenberg, G Jones, Clin Endocrinol. 735Scott D, Blizzard L, Fell J, Ding C, Winzenberg T, Jones G. A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. Clin Endocrinol. 2010;73(5): 581-7.","Relation of vitamin D level to maximal oxygen uptake in adults. A Ardestani, B Parker, S Mathur, P Clarkson, L S Pescatello, H J Hoffman, Am J Cardiol. 1078Ardestani A, Parker B, Mathur S, Clarkson P, Pescatello LS, Hoffman HJ, et al. Relation of vitamin D level to maximal oxygen uptake in adults. Am J Cardiol. 2011;107(8):1246-9.","Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. A Burgaz, A Akesson, A Oster, K Michaelsson, A Wolk, Am J Clin Nutr. 865Burgaz A, Akesson A, Oster A, Michaelsson K, Wolk A. Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. Am J Clin Nutr. 2007;86(5):1399-404.","The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. M G Kimlin, R M Lucas, S L Harrison, I Van Der Mei, B K Armstrong, D C Whiteman, Am J Epidemiol. 1797Kimlin MG, Lucas RM, Harrison SL, van der Mei I, Armstrong BK, Whiteman DC, et al. The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. Am J Epidemiol. 2014;179(7):864-74.","Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women. M A Kluczynski, M J Lamonte, J A Mares, J Wactawski-Wende, A W Smith, C D Engelman, Ann Epidemiol. 216Kluczynski MA, Lamonte MJ, Mares JA, Wactawski-Wende J, Smith AW, Engelman CD, et al. Duration of physical activity and serum 25- hydroxyvitamin D status of postmenopausal women. Ann Epidemiol. 2011; 21(6):440-9.","Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. M F Holick, Clin Rev Bone Miner Metabol. 71Holick MF. Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. Clin Rev Bone Miner Metabol. 2009;7(1):2-19.","Effect of antioxidants and exercise on bone metabolism. L Maïmoun, D Simar, C Caillaud, E Peruchon, C Sultan, M Rossi, J Sports Sci. 263Maïmoun L, Simar D, Caillaud C, Peruchon E, Sultan C, Rossi M, et al. Effect of antioxidants and exercise on bone metabolism. J Sports Sci. 2008;26(3): 251-8.","Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. X Sun, Z B Cao, H Taniguchi, K Tanisawa, M Higuchi, J Clin Endocrinol Metab. 10211Sun X, Cao ZB, Taniguchi H, Tanisawa K, Higuchi M. Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. J Clin Endocrinol Metab. 2017;102(11):3937-44.","Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. X Sun, Z B Cao, K Tanisawa, H Taniguchi, T Kubo, M Higuchi, Endocrine. 592Sun X, Cao ZB, Tanisawa K, Taniguchi H, Kubo T, Higuchi M. Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. Endocrine. 2018;59(2):330-7.","Resistance training is medicine: effects of strength training on health. W L Westcott, Curr Sports Med Rep. 114Westcott WL. Resistance training is medicine: effects of strength training on health. Curr Sports Med Rep. 2012;11(4):209-16.","Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells. M Abboud, D A Puglisi, B N Davies, M Rybchyn, N P Whitehead, K E Brock, Endocrinology. 1549Abboud M, Puglisi DA, Davies BN, Rybchyn M, Whitehead NP, Brock KE, et al. Evidence for a specific uptake and retention mechanism for 25- hydroxyvitamin D (25OHD) in skeletal muscle cells. Endocrinology. 2013; 154(9):3022-30.","The role of skeletal muscle in maintaining vitamin D status in Winter. R S Mason, M S Rybchyn, M Abboud, T C Brennan-Speranza, D R Fraser, Curr Dev Nutr. 31087Mason RS, Rybchyn MS, Abboud M, Brennan-Speranza TC, Fraser DR. The role of skeletal muscle in maintaining vitamin D status in Winter. Curr Dev Nutr. 2019;3(10):nzz087.","Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Y Makanae, R Ogasawara, K Sato, Y Takamura, K Matsutani, K Kido, Exp Physiol. 10010Makanae Y, Ogasawara R, Sato K, Takamura Y, Matsutani K, Kido K, et al. Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Exp Physiol. 2015;100(10):1168-76.","Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. L H Willis, C A Slentz, L A Bateman, A T Shields, L W Piner, C W Bales, J Appl Physiol. 113121831Willis LH, Slentz CA, Bateman LA, Shields AT, Piner LW, Bales CW, et al. Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. J Appl Physiol. 2012;113(12):1831.","Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. B Strasser, U Siebert, W Schobersberger, Sports Med. 405Strasser B, Siebert U, Schobersberger W. Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. Sports Med. 2010;40(5):397-415.","Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. H Jarrett, L Fitzgerald, A C Routen, J Phys Act Health. 123Jarrett H, Fitzgerald L, Routen AC. Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. J Phys Act Health. 2014;12(3):382-7.","Validation and comparison of ActiGraph activity monitors. J E Sasaki, D John, P S Freedson, J Sci Med Sport. 145Sasaki JE, John D, Freedson PS. Validation and comparison of ActiGraph activity monitors. J Sci Med Sport. 2011;14(5):411-6.","Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. M F Holick, N C Binkley, H A Bischoff-Ferrari, C M Gordon, D A Hanley, R P Heaney, J Clin Endocrinol Metab. 967Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96(7):1911-30.","The effects of musclebuilding exercise on vitamin D and mineral metabolism. N H Bell, R N Godsen, D P Henry, J Shary, S Epstein, J Bone Miner Res. 34Bell NH, Godsen RN, Henry DP, Shary J, Epstein S. The effects of muscle- building exercise on vitamin D and mineral metabolism. J Bone Miner Res. 1988;3(4):369-73.","VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. R Srikuea, X Zhang, O K Park-Sarge, K A Esser, Am J Physiol Cell Physiol. 3034Srikuea R, Zhang X, Park-Sarge OK, Esser KA. VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. Am J Physiol Cell Physiol. 2012;303(4): C396-405.","Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. T Kobayashi, T Okano, S Shida, K Okada, T Suginohara, H Nakao, J Nutr Sci Vitaminol (Tokyo). 293Kobayashi T, Okano T, Shida S, Okada K, Suginohara T, Nakao H, et al. Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. J Nutr Sci Vitaminol (Tokyo). 1983;29(3):271-81.","Publisher's Note. Publisher's Note","Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."],"string":"[\n \"Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. M Wacker, M F Holick, Nutrients. 51Wacker M, Holick MF. Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. Nutrients. 2013;5(1):111-48.\",\n \"Vitamin D: beyond bone. S Christakos, M Hewison, D G Gardner, C L Wagner, I N Sergeev, E Rutten, Ann N Y Acad Sci. 1287Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al. Vitamin D: beyond bone. Ann N Y Acad Sci. 2013;1287:45-58.\",\n \"Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. X Sun, Z B Cao, Y Zhang, Y Ishimi, I Tabata, M Higuchi, Nutrients. 61Sun X, Cao ZB, Zhang Y, Ishimi Y, Tabata I, Higuchi M. Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. Nutrients. 2014;6(1):221-30.\",\n \"The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. X Sun, Z B Cao, K Tanisawa, T Ito, S Oshima, M Higuchi, Nutrients. 71Sun X, Cao ZB, Tanisawa K, Ito T, Oshima S, Higuchi M. The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. Nutrients. 2014;7(1):91-102.\",\n \"Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. R Scragg, I Holdaway, R Jackson, T Lim, Ann Epidemiol. 25Scragg R, Holdaway I, Jackson R, Lim T. Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. Ann Epidemiol. 1992;2(5):697-703.\",\n \"A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. D Scott, L Blizzard, J Fell, C Ding, T Winzenberg, G Jones, Clin Endocrinol. 735Scott D, Blizzard L, Fell J, Ding C, Winzenberg T, Jones G. A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. Clin Endocrinol. 2010;73(5): 581-7.\",\n \"Relation of vitamin D level to maximal oxygen uptake in adults. A Ardestani, B Parker, S Mathur, P Clarkson, L S Pescatello, H J Hoffman, Am J Cardiol. 1078Ardestani A, Parker B, Mathur S, Clarkson P, Pescatello LS, Hoffman HJ, et al. Relation of vitamin D level to maximal oxygen uptake in adults. Am J Cardiol. 2011;107(8):1246-9.\",\n \"Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. A Burgaz, A Akesson, A Oster, K Michaelsson, A Wolk, Am J Clin Nutr. 865Burgaz A, Akesson A, Oster A, Michaelsson K, Wolk A. Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. Am J Clin Nutr. 2007;86(5):1399-404.\",\n \"The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. M G Kimlin, R M Lucas, S L Harrison, I Van Der Mei, B K Armstrong, D C Whiteman, Am J Epidemiol. 1797Kimlin MG, Lucas RM, Harrison SL, van der Mei I, Armstrong BK, Whiteman DC, et al. The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. Am J Epidemiol. 2014;179(7):864-74.\",\n \"Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women. M A Kluczynski, M J Lamonte, J A Mares, J Wactawski-Wende, A W Smith, C D Engelman, Ann Epidemiol. 216Kluczynski MA, Lamonte MJ, Mares JA, Wactawski-Wende J, Smith AW, Engelman CD, et al. Duration of physical activity and serum 25- hydroxyvitamin D status of postmenopausal women. Ann Epidemiol. 2011; 21(6):440-9.\",\n \"Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. M F Holick, Clin Rev Bone Miner Metabol. 71Holick MF. Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. Clin Rev Bone Miner Metabol. 2009;7(1):2-19.\",\n \"Effect of antioxidants and exercise on bone metabolism. L Maïmoun, D Simar, C Caillaud, E Peruchon, C Sultan, M Rossi, J Sports Sci. 263Maïmoun L, Simar D, Caillaud C, Peruchon E, Sultan C, Rossi M, et al. Effect of antioxidants and exercise on bone metabolism. J Sports Sci. 2008;26(3): 251-8.\",\n \"Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. X Sun, Z B Cao, H Taniguchi, K Tanisawa, M Higuchi, J Clin Endocrinol Metab. 10211Sun X, Cao ZB, Taniguchi H, Tanisawa K, Higuchi M. Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. J Clin Endocrinol Metab. 2017;102(11):3937-44.\",\n \"Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. X Sun, Z B Cao, K Tanisawa, H Taniguchi, T Kubo, M Higuchi, Endocrine. 592Sun X, Cao ZB, Tanisawa K, Taniguchi H, Kubo T, Higuchi M. Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. Endocrine. 2018;59(2):330-7.\",\n \"Resistance training is medicine: effects of strength training on health. W L Westcott, Curr Sports Med Rep. 114Westcott WL. Resistance training is medicine: effects of strength training on health. Curr Sports Med Rep. 2012;11(4):209-16.\",\n \"Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells. M Abboud, D A Puglisi, B N Davies, M Rybchyn, N P Whitehead, K E Brock, Endocrinology. 1549Abboud M, Puglisi DA, Davies BN, Rybchyn M, Whitehead NP, Brock KE, et al. Evidence for a specific uptake and retention mechanism for 25- hydroxyvitamin D (25OHD) in skeletal muscle cells. Endocrinology. 2013; 154(9):3022-30.\",\n \"The role of skeletal muscle in maintaining vitamin D status in Winter. R S Mason, M S Rybchyn, M Abboud, T C Brennan-Speranza, D R Fraser, Curr Dev Nutr. 31087Mason RS, Rybchyn MS, Abboud M, Brennan-Speranza TC, Fraser DR. The role of skeletal muscle in maintaining vitamin D status in Winter. Curr Dev Nutr. 2019;3(10):nzz087.\",\n \"Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Y Makanae, R Ogasawara, K Sato, Y Takamura, K Matsutani, K Kido, Exp Physiol. 10010Makanae Y, Ogasawara R, Sato K, Takamura Y, Matsutani K, Kido K, et al. Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Exp Physiol. 2015;100(10):1168-76.\",\n \"Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. L H Willis, C A Slentz, L A Bateman, A T Shields, L W Piner, C W Bales, J Appl Physiol. 113121831Willis LH, Slentz CA, Bateman LA, Shields AT, Piner LW, Bales CW, et al. Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. J Appl Physiol. 2012;113(12):1831.\",\n \"Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. B Strasser, U Siebert, W Schobersberger, Sports Med. 405Strasser B, Siebert U, Schobersberger W. Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. Sports Med. 2010;40(5):397-415.\",\n \"Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. H Jarrett, L Fitzgerald, A C Routen, J Phys Act Health. 123Jarrett H, Fitzgerald L, Routen AC. Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. J Phys Act Health. 2014;12(3):382-7.\",\n \"Validation and comparison of ActiGraph activity monitors. J E Sasaki, D John, P S Freedson, J Sci Med Sport. 145Sasaki JE, John D, Freedson PS. Validation and comparison of ActiGraph activity monitors. J Sci Med Sport. 2011;14(5):411-6.\",\n \"Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. M F Holick, N C Binkley, H A Bischoff-Ferrari, C M Gordon, D A Hanley, R P Heaney, J Clin Endocrinol Metab. 967Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96(7):1911-30.\",\n \"The effects of musclebuilding exercise on vitamin D and mineral metabolism. N H Bell, R N Godsen, D P Henry, J Shary, S Epstein, J Bone Miner Res. 34Bell NH, Godsen RN, Henry DP, Shary J, Epstein S. The effects of muscle- building exercise on vitamin D and mineral metabolism. J Bone Miner Res. 1988;3(4):369-73.\",\n \"VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. R Srikuea, X Zhang, O K Park-Sarge, K A Esser, Am J Physiol Cell Physiol. 3034Srikuea R, Zhang X, Park-Sarge OK, Esser KA. VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. Am J Physiol Cell Physiol. 2012;303(4): C396-405.\",\n \"Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. T Kobayashi, T Okano, S Shida, K Okada, T Suginohara, H Nakao, J Nutr Sci Vitaminol (Tokyo). 293Kobayashi T, Okano T, Shida S, Okada K, Suginohara T, Nakao H, et al. Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. J Nutr Sci Vitaminol (Tokyo). 1983;29(3):271-81.\",\n \"Publisher's Note. Publisher's Note\",\n \"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\"\n]"},"annotations_bibref":{"kind":"list like","value":["[1]","[2]","[3]","[4]","[5]","[6]","[7]","[8,","9]","[6,","10]","[5]","[11]","[12]","[13]","[14]","[15]","[16,","17]","[18]","[19,","20]","[21,","22]","[23]","[6,","10]","[24]","[13,","14]","[24]","[17]","[18]","[25]","[26]"],"string":"[\n \"[1]\",\n \"[2]\",\n \"[3]\",\n \"[4]\",\n \"[5]\",\n \"[6]\",\n \"[7]\",\n \"[8,\",\n \"9]\",\n \"[6,\",\n \"10]\",\n \"[5]\",\n \"[11]\",\n \"[12]\",\n \"[13]\",\n \"[14]\",\n \"[15]\",\n \"[16,\",\n \"17]\",\n \"[18]\",\n \"[19,\",\n \"20]\",\n \"[21,\",\n \"22]\",\n \"[23]\",\n \"[6,\",\n \"10]\",\n \"[24]\",\n \"[13,\",\n \"14]\",\n \"[24]\",\n \"[17]\",\n \"[18]\",\n \"[25]\",\n \"[26]\"\n]"},"annotations_bibtitle":{"kind":"list like","value":["Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation","Vitamin D: beyond bone","Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults","The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men","Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population","A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults","Relation of vitamin D level to maximal oxygen uptake in adults","Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter","The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study","Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women","Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D","Effect of antioxidants and exercise on bone metabolism","Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults","Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men","Resistance training is medicine: effects of strength training on health","Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells","The role of skeletal muscle in maintaining vitamin D status in Winter","Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle","Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults","Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism","Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults","Validation and comparison of ActiGraph activity monitors","Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline","The effects of musclebuilding exercise on vitamin D and mineral metabolism","VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation","Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects"],"string":"[\n \"Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation\",\n \"Vitamin D: beyond bone\",\n \"Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults\",\n \"The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men\",\n \"Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population\",\n \"A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults\",\n \"Relation of vitamin D level to maximal oxygen uptake in adults\",\n \"Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter\",\n \"The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study\",\n \"Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women\",\n \"Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D\",\n \"Effect of antioxidants and exercise on bone metabolism\",\n \"Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults\",\n \"Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men\",\n \"Resistance training is medicine: effects of strength training on health\",\n \"Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells\",\n \"The role of skeletal muscle in maintaining vitamin D status in Winter\",\n \"Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle\",\n \"Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults\",\n \"Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism\",\n \"Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults\",\n \"Validation and comparison of ActiGraph activity monitors\",\n \"Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline\",\n \"The effects of musclebuilding exercise on vitamin D and mineral metabolism\",\n \"VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation\",\n \"Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects\"\n]"},"annotations_bibvenue":{"kind":"list like","value":["Nutrients","Ann N Y Acad Sci","Nutrients","Nutrients","Ann Epidemiol","Clin Endocrinol","Am J Cardiol","Am J Clin Nutr","Am J Epidemiol","Ann Epidemiol","Clin Rev Bone Miner Metabol","J Sports Sci","J Clin Endocrinol Metab","Endocrine","Curr Sports Med Rep","Endocrinology","Curr Dev Nutr","Exp Physiol","J Appl Physiol","Sports Med","J Phys Act Health","J Sci Med Sport","J Clin Endocrinol Metab","J Bone Miner Res","Am J Physiol Cell Physiol","J Nutr Sci Vitaminol (Tokyo)","Publisher's Note","Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations"],"string":"[\n \"Nutrients\",\n \"Ann N Y Acad Sci\",\n \"Nutrients\",\n \"Nutrients\",\n \"Ann Epidemiol\",\n \"Clin Endocrinol\",\n \"Am J Cardiol\",\n \"Am J Clin Nutr\",\n \"Am J Epidemiol\",\n \"Ann Epidemiol\",\n \"Clin Rev Bone Miner Metabol\",\n \"J Sports Sci\",\n \"J Clin Endocrinol Metab\",\n \"Endocrine\",\n \"Curr Sports Med Rep\",\n \"Endocrinology\",\n \"Curr Dev Nutr\",\n \"Exp Physiol\",\n \"J Appl Physiol\",\n \"Sports Med\",\n \"J Phys Act Health\",\n \"J Sci Med Sport\",\n \"J Clin Endocrinol Metab\",\n \"J Bone Miner Res\",\n \"Am J Physiol Cell Physiol\",\n \"J Nutr Sci Vitaminol (Tokyo)\",\n \"Publisher's Note\",\n \"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations\"\n]"},"annotations_figure":{"kind":"list like","value":["\nFig. 1\n1Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05","\n\n25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum","\nTable 1\n1Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activityVariable \nResistance training group (RT) (n = 9) \nNon-exercise control group (CON) (n = 9) \n\nAge (y) \n24.2 ± 3.1 \n26.7 ± 6.2 \n\nHeight (cm) \n1.74 ± 0.03 \n1.75 ± 0.06 \n\nWeight (kg) \n68.5 ± 9.7 \n68.7 ± 7.6 \n\nBMI (kg/m 2 ) \n22.5 ± 2.9 \n22.3 ± 1.9 \n\nFFM (kg) \n54.2 (49.2 -56.4 ) \n57.5 (51.1 -58.8 ) \n\nMuscle mass (kg) \n51.4 (46.6 -53.3 ) \n54.9 (48.4 -55.8 ) \n\nBody fat percentage (%) \n21.9 ± 6.9 \n18.8 ± 6.0 \n\nArms fat percentage (%) \n18.7 ± 7.1 \n14.4 ± 5.1 \n\nLegs fat percentage (%) \n20.5 ± 5.6 \n18.1 ± 5.2 \n\nTrunk fat percentage (%) \n25.1 ± 8.3 \n21.6 ± 7.3 \n\nBMD (g/cm 2 ) \n1.16 ± 0.07 \n1.18 ± 0.07 \n\n25(OH) D (nmol/L) \n26.9 ± 4.7 \n26.2 ± 4.1 \n\niPTH (pg/mL) \n54.9 ± 19.8 \n44.6 ± 23.8 \n\nMVPA (min/day) \n69.8 ± 32.2 \n65.8 ± 16.3 \n\nSun exposure (min/day) \n40.0 ± 27.9 \n50.0 ± 30.7 \n\n","\nTable 2\n2Characteristics of the participants at baseline, midpoint and endpointVariable \nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \n\nBaseline \nMidpoint \nEndpoint \nBaseline \nMidpoint \nEndpoint \nTime Group Time × Group interaction \n\nWeight (kg) a \n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \n68.8 ± 3.1 \n68.7 ± 3.1 \n68.6 ± 3.0 \n0.156 0.958 0.762 \n\nBMI (kg/m 2 ) a \n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \n22.3 ± 0.9 \n22.2 ± 0.9 \n22.2 ± 0.8 \n0.273 0.768 0.455 \n\nFFM (kg) a \n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \n56.1 ± 1.8 \n55.8 ± 1.8 \n0.347 0.341 0.004 \n\nMuscle mass (kg) a \n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \n53.2 ± 1.7 \n53.0 ± 1.7 \n0.399 0.348 0.003 \n\nBody fat percentage a \n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \n18.3 ± 2.0 \n18.0 ± 1.9 \n18.3 ± 2.0 \n0.643 0.327 0.058 \n\nArms fat percentage a \n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \n13.7 ± 1.8 \n13.4 ± 1.8 \n13.6 ± 1.7 \n0.687 0.091 0.077 \n\nLegs fat percentage a \n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \n17.7 ± 1.7 \n17.2 ± 1.6 \n17.6 ± 1.7 \n0.846 0.378 0.064 \n\nTrunk fat percentage a \n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \n21.1 ± 2.5 \n20.8 ± 2.4 \n21.1 ± 2.4 \n0.484 0.397 0.075 \n\nBMD (g/cm 2 ) a \n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \n0.016 0.538 0.235 \n\n25(OH) D (nmol/L) b \n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \n\niPTH (pg/mL) b \n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \n37.6 ± 8.3 \n48.3 ± 6.1 \n51.5 ± 5.6 \n0.110 0.131 0.242 \n\nSun exposure (min/day) 40.0± 9.8 \n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \n73.5 ± 13.5 50.5 ± 15.2 \n0.015 0.889 0.493 \n\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \n\nc \n\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \nBoldface indicates significance (P < 0.05) \n","\nTable 3\n3Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free massVariables \n25(OH)D mid-pre \n25(OH)D end-pre \n\nr \nP \nr \nP \n\nBody fat percentage \n\nmid-pre \n0.014 \n0.956 \n0.101 \n0.690 \n\nend-mid \n−0.217 \n0.387 \n−0.004 \n0.986 \n\nend-pre \n−0.090 \n0.722 \n0.064 \n0.800 \n\nFFM \n\nmid-pre \n−0.455 \n0.058 \n−0.565 \n0.015 \n\nend-mid \n0.121 \n0.632 \n0.037 \n0.883 \n\nend-pre \n−0.289 \n0.244 \n−0.424 \n0.080 \n\nMuscle mass \n\nmid-pre \n−0.456 \n0.057 \n−0.554 \n0.017 \n\nend-mid \n0.142 \n0.575 \n0.037 \n0.884 \n\nend-pre \n−0.280 \n0.261 \n−0.415 \n0.087 \n\n"],"string":"[\n \"\\nFig. 1\\n1Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05\",\n \"\\n\\n25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum\",\n \"\\nTable 1\\n1Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activityVariable \\nResistance training group (RT) (n = 9) \\nNon-exercise control group (CON) (n = 9) \\n\\nAge (y) \\n24.2 ± 3.1 \\n26.7 ± 6.2 \\n\\nHeight (cm) \\n1.74 ± 0.03 \\n1.75 ± 0.06 \\n\\nWeight (kg) \\n68.5 ± 9.7 \\n68.7 ± 7.6 \\n\\nBMI (kg/m 2 ) \\n22.5 ± 2.9 \\n22.3 ± 1.9 \\n\\nFFM (kg) \\n54.2 (49.2 -56.4 ) \\n57.5 (51.1 -58.8 ) \\n\\nMuscle mass (kg) \\n51.4 (46.6 -53.3 ) \\n54.9 (48.4 -55.8 ) \\n\\nBody fat percentage (%) \\n21.9 ± 6.9 \\n18.8 ± 6.0 \\n\\nArms fat percentage (%) \\n18.7 ± 7.1 \\n14.4 ± 5.1 \\n\\nLegs fat percentage (%) \\n20.5 ± 5.6 \\n18.1 ± 5.2 \\n\\nTrunk fat percentage (%) \\n25.1 ± 8.3 \\n21.6 ± 7.3 \\n\\nBMD (g/cm 2 ) \\n1.16 ± 0.07 \\n1.18 ± 0.07 \\n\\n25(OH) D (nmol/L) \\n26.9 ± 4.7 \\n26.2 ± 4.1 \\n\\niPTH (pg/mL) \\n54.9 ± 19.8 \\n44.6 ± 23.8 \\n\\nMVPA (min/day) \\n69.8 ± 32.2 \\n65.8 ± 16.3 \\n\\nSun exposure (min/day) \\n40.0 ± 27.9 \\n50.0 ± 30.7 \\n\\n\",\n \"\\nTable 2\\n2Characteristics of the participants at baseline, midpoint and endpointVariable \\nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \\n\\nBaseline \\nMidpoint \\nEndpoint \\nBaseline \\nMidpoint \\nEndpoint \\nTime Group Time × Group interaction \\n\\nWeight (kg) a \\n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \\n68.8 ± 3.1 \\n68.7 ± 3.1 \\n68.6 ± 3.0 \\n0.156 0.958 0.762 \\n\\nBMI (kg/m 2 ) a \\n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \\n22.3 ± 0.9 \\n22.2 ± 0.9 \\n22.2 ± 0.8 \\n0.273 0.768 0.455 \\n\\nFFM (kg) a \\n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \\n56.1 ± 1.8 \\n55.8 ± 1.8 \\n0.347 0.341 0.004 \\n\\nMuscle mass (kg) a \\n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \\n53.2 ± 1.7 \\n53.0 ± 1.7 \\n0.399 0.348 0.003 \\n\\nBody fat percentage a \\n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \\n18.3 ± 2.0 \\n18.0 ± 1.9 \\n18.3 ± 2.0 \\n0.643 0.327 0.058 \\n\\nArms fat percentage a \\n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \\n13.7 ± 1.8 \\n13.4 ± 1.8 \\n13.6 ± 1.7 \\n0.687 0.091 0.077 \\n\\nLegs fat percentage a \\n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \\n17.7 ± 1.7 \\n17.2 ± 1.6 \\n17.6 ± 1.7 \\n0.846 0.378 0.064 \\n\\nTrunk fat percentage a \\n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \\n21.1 ± 2.5 \\n20.8 ± 2.4 \\n21.1 ± 2.4 \\n0.484 0.397 0.075 \\n\\nBMD (g/cm 2 ) a \\n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \\n0.016 0.538 0.235 \\n\\n25(OH) D (nmol/L) b \\n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \\n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \\n\\niPTH (pg/mL) b \\n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \\n37.6 ± 8.3 \\n48.3 ± 6.1 \\n51.5 ± 5.6 \\n0.110 0.131 0.242 \\n\\nSun exposure (min/day) 40.0± 9.8 \\n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \\n73.5 ± 13.5 50.5 ± 15.2 \\n0.015 0.889 0.493 \\n\\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \\nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \\n\\nc \\n\\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \\nBoldface indicates significance (P < 0.05) \\n\",\n \"\\nTable 3\\n3Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free massVariables \\n25(OH)D mid-pre \\n25(OH)D end-pre \\n\\nr \\nP \\nr \\nP \\n\\nBody fat percentage \\n\\nmid-pre \\n0.014 \\n0.956 \\n0.101 \\n0.690 \\n\\nend-mid \\n−0.217 \\n0.387 \\n−0.004 \\n0.986 \\n\\nend-pre \\n−0.090 \\n0.722 \\n0.064 \\n0.800 \\n\\nFFM \\n\\nmid-pre \\n−0.455 \\n0.058 \\n−0.565 \\n0.015 \\n\\nend-mid \\n0.121 \\n0.632 \\n0.037 \\n0.883 \\n\\nend-pre \\n−0.289 \\n0.244 \\n−0.424 \\n0.080 \\n\\nMuscle mass \\n\\nmid-pre \\n−0.456 \\n0.057 \\n−0.554 \\n0.017 \\n\\nend-mid \\n0.142 \\n0.575 \\n0.037 \\n0.884 \\n\\nend-pre \\n−0.280 \\n0.261 \\n−0.415 \\n0.087 \\n\\n\"\n]"},"annotations_figurecaption":{"kind":"list like","value":["Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05","25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum","Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activity","Characteristics of the participants at baseline, midpoint and endpoint","Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free mass"],"string":"[\n \"Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05\",\n \"25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum\",\n \"Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activity\",\n \"Characteristics of the participants at baseline, midpoint and endpoint\",\n \"Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free mass\"\n]"},"annotations_figureref":{"kind":"list like","value":["Fig. 1","(Fig. 2)"],"string":"[\n \"Fig. 1\",\n \"(Fig. 2)\"\n]"},"annotations_formula":{"kind":"list like","value":[],"string":"[]"},"annotations_paragraph":{"kind":"list like","value":["Circulating 25-hydroxyvitamin D [25(OH)D] is the major form of vitamin D, which is used in vitamin D status assessment. Accumulated studies indicated that vitamin D not only regulates calcium and phosphorus metabolism but also plays an important role in modifying cardiovascular diseases risk, and improving immune function and physical fitness [1][2][3][4].","Previous studies indicated that higher levels of physical activity or cardiorespiratory fitness were related to higher circulating 25(OH) D concentrations [5][6][7]. Although individuals who were physically active outdoors are more likely to have higher circulating 25(OH) D due to increased sun exposure [8,9], this relationship was still observed after adjustment for sunlight exposure [6,10]. Scragg et al. [5] found that higher levels of physical activity were associated with higher serum 25(OH) D concentrations not only during summer but also during winter, when the vitamin D synthesis from sun exposure is extremely limited [11]. Consistently, several intervention studies suggested that endurance exercise could increase circulating 25(OH) D or prevent its seasonal reduction [12][13][14]. These findings indicated that physical activity could directly increase 25(OH) D concentrations.","Resistance training (RT) promotes health benefits through increased skeletal muscle mass and qualitative adaptations [15]. In addition to adiposity tissue, muscle tissue was suggested to serve as an another important site to store 25(OH) D, and then it returns to the circulating when needed [16,17]. Nevertheless, Makanae and colleagues reported that intramuscular expression of CYP27B1, which catalyses the hydroxylation and activation of 25(OH) D, was significantly increased at 1 h and 3 h after resistance exercise, which may enhance local vitamin D metabolism in skeletal muscle [18]. It is well known that compared with endurance exercise, RT could not only reduce fat mass, but also it is more effective in increasing muscle mass and strength [19,20], which may largely affect vitamin D metabolism. However, whether serum 25(OH) D concentrations are affected by RT remains unclear.","This study aimed to evaluate the effect of chronic resistance training on serum 25(OH) D concentrations and to determine whether the effects are attributable to changes in body composition in healthy young men.","Eighteen healthy men with no chronic diseases (aged 19 to 39 years) were included in this study, which was conducted from March to July 2016. Participants using vitamin D supplements or sunscreen regularly were excluded. Assessments were performed at 0 week (pre), 6 weeks (midpoint), and 12 weeks (endpoint). All subjects provided informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). The trial has been retrospectively registered at Chinese Clinical Trial Registry website (ChiCTR2000030876).","Eighteen participants were randomly allocated to the following two groups by using a computer-generated random number sequence: resistance training group (RT) and no-exercise control group (CON). Participants in the RT group were instructed to exercise 2-3 times a week in a gymnasium. A supervised training session was performed in the afternoon between 1630 h and 2000 h during the 12 weeks of progressive resistance training. In the first 2 weeks, the participants were taught how to properly perform each movement. The resistance workload gradually changed from light to heavy, and the participants were instructed to complete 10 repetitions for each set of the following exercises: leg press, preacher curl, chest press, leg curl, and iso row, with 60-120-s rest period between two sets; additionally, they also performed three sets of 15-25 repetitions of abdominal crunch and back extension with their own body weight, with a 60-120-s rest period between two sets. At the first training session of the third week, the participants were instructed to perform strength testing, and 12 repetitions maximum (12RM) for each exercise were performed, except back extension and abdominal crunch. The workload for the next resistance training was calculated from the participants' previously determined 12RM. The participants were encouraged to perform back extension and abdominal crunch exercises 30 times from the first training session of the third week. When the strength training equipment at our gymnasium had been upgraded at the eighth week of training, the resistance training program was revised to include the following: leg extension, standing one-arm cable curl, standing cable chest press, leverage shoulder press, leverage high row, back extension, and abdominal crunch. These new components of the resistance training program were selected based on the principle of using similar muscle groups. The order of resistance exercise was randomized; however, training of the same muscle groups continuously was avoided. The participants were instructed not to undertake any formal exercise or change their levels of general physical activity and dietary habits.","Height was measured with the participants wearing light clothing and being barefoot (HK6000-ST, HENGKANG JIAYE Technology Co., Ltd. CHN). Dual-energy X-ray absorptiometry (DXA Prodigy, GE Lunar Corp., Madison, WI, USA) was used for the measurement of whole and regional body composition, including total body mass, muscle mass, fat mass, fat percentage, and bone mineral density. Body mass index was calculated as body mass (kg) divided by height (m 2 ). Fat-free mass (FFM) was calculated as body mass minus fat mass.","Physical activity was measured using Actigraph GT3X+ accelerometers (ActiGraph, LLC, Pensacola, FL, USA). Participants were instructed to wear the accelerometers on their right hip at all times, except during water activities (swimming, showering) or sleeping, for 7 consecutive days at baseline. A wear time of ≥480 min/day was used as the criterion for a valid day, and ≥ 2 weekdays and 1 weekend day was used as the criteria for a valid 7-day period of accumulated data. Non-wear time was defined as consecutive periods of ≥60 min of zero accelerometer counts and excluded from the analyses. Data were recorded in 60-s epochs. A pragmatic cutoff of ≥2690 cpm was used to categorize moderatevigorous physical activity time [21,22]. The 7-day data collection for all subjects was fixed within 2 weeks. Outdoor time from 0900 h to 1600 h for 7 consecutive days was recorded and used as an alternative for sun exposure in the analysis.","Blood samples were obtained after at least an 8-h overnight fasting period and subsequently centrifuged at 3000 rpm at 4°C for 10 min. Serum was collected and stored at − 80°C until analysis. Serum 25(OH) D and intact parathyroid hormone (iPTH) concentrations were measured in duplicate using commercially available enzyme-linked immunosorbent assay kits (25(OH) D, IDS, Bolton, UK; iPTH, DRG, Marburg, Germany) according to the manufacturer's instructions. The intraassay and inter-assay coefficients of variation were 9.1 and 7.7%, respectively, for serum 25(OH) D and 9.6 and 3.6%, respectively, for iPTH. Vitamin D deficiency is defined as 25(OH) D concentration < 50 nmol/L, and vitamin D insufficiency as 25(OH) D concentration < 75 nmol/L [23].","All statistical analyses were performed using IBM SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were assessed for normality using a Kolmogorov-Smirnov test prior to all statistical analyses. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables were presented as median (interquartile range), unless otherwise indicated. The Student t test (for normally distributed variables) or the Mann-Whitney U test (for non-normally distributed variables) was used to evaluate the differences between groups. Two-factor repeat-measured analysis of variance (ANOVA) (group × time) was used to determine the effect of exercise on 25(OH) D, iPTH, and body composition indicators. A post hoc test with Bonferroni correction was performed to identify statistically significant differences among the mean values when a considerable interaction was identified. Pearson correlation and partial correlation coefficients were computed between 25(OH) D concentration changes and body composition indicators. The level of statistical significance was set at P < 0.05.","The participants' characteristics and blood parameters at baseline are presented in Table 1. The average serum 25(OH) D concentrations were 26.6 nmol/L, and all participants showed vitamin D deficiency. No significant difference in either body composition indicators or blood parameters was found between the groups (P > 0.05).","As shown in Table 2, significant interactions of group × time were found for FFM (P = 0.004) and muscle mass (P = 0.003). After resistance training, the FFM values (mid-pre, 2.7%, P = 0.003; end-pre, 3.0%, P = 0.004) and muscle mass (mid-pre, 3.0%, P = 0.002; end-pre, 3.4%, P = 0.004) significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in the CON group. In addition, we observed borderline significant interactions in body fat percentage, arm fat percentage, leg fat percentage, and trunk fat percentage ( Table 2).","As shown in Table 2 and Fig. 1, a significant group × time interaction was observed for serum 25(OH) D concentrations (P = 0.025). After the 12-week intervention, the mean serum 25(OH) D concentrations significantly increased to 36.7 ± 2.7 nmol/L in the RT group and 40.7 ± 2.7 nmol/L in the CON group. Moreover, in the CON group, higher serum 25(OH) D concentrations were observed at midpoint compared with the values at baseline (P < 0.01), whereas no notable differences were found in the RT group. From midpoint to endpoint, although serum 25(OH) D concentrations significantly increased in both groups, the increase was significantly greater in the RT group (25.8%) than in the CON group (9.4%) (P-interaction =0.043).","Correlation analyses were performed to identify the factors associated with the changes in 25(OH) D concentrations (Table 3). 25(OH) D concentration changes (end-pre) were negatively related to FFM (mid-pre) (P = 0.015) and muscle mass (mid-pre) (P = 0.017) changes (Fig. 2); a borderline association was observed between 25(OH) D concentration changes (mid-pre) and FFM (mid-pre) (P = 0.058) and muscle mass (mid-pre) (P = 0.057) changes. In addition, significant correlations of 25(OH) D concentration changes (end-pre) with FFM (mid-pre) (P = 0.039) and muscle mass (mid-pre) (P = 0.043) changes were observed after adjustment for group, age, and changes in sun exposure (end-pre), body weight (end-pre), and body fat percentage (end-pre).","In this study, we found that the seasonal increase in serum 25(OH) D concentrations in young men was transiently inhibited during the early phase of RT; subsequently, serum 25(OH) D concentrations increased in both RT and CON groups, and the increase was observed to be greater in RT group than in CON group. The transient inhibition could be partly attributed to muscle mass and fat free mass gain induced by RT in the early phase.","Previous studies have reported significant positive associations of serum 25(OH) D concentrations with physical activity levels independent of sun exposure [6,10]. Bell et al. [24] found that 25(OH) D concentrations were higher in participants who had engaged in regular muscle-building exercise for at least 1 year compared to those in the control group. Consistently, our recent studies also observed that exercise training could increase serum 25(OH) D concentrations or prevent its seasonal reduction in young and old men [13,14]. However, the effect of RT on serum 25(OH) D concentrations remains unclear. Compared with endurance training, RT is more effective in increasing muscle mass and strength [24], which was also supported by our results. In the present study, we observed that after resistance training, body mass was reduced, while FFM and muscle mass were significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in CON group.","In addition to adiposity tissues, muscle tissues are also served as another important storage for 25(OH) D, which could enable optimal vitamin D status to maintain its physiological function during winter months [17]. Makanae and colleagues observed that intramuscular CYP27B1 was significantly increased after resistance exercise in skeletal muscle, which may enhance local vitamin D metabolism [18]. Srikuea et al. [25] also found that the CYP27B1 expression in the muscle increased significantly during skeletal muscle regeneration from injury. Thus, we speculated that the transient inhibition in serum 25(OH) D levels in RT group during the early phase of resistance training could be partly due to absorption of serum 25(OH) D in the increasing muscle mass and its use in muscle activity. 25(OH) D consumption in muscle may induce a compensatory increase in serum 25(OH) D concentrations; however, the concentrations were still lower than that in control group, which may due to the lack of vitamin D storage and short-term intervention period. Previous studies report that serum 25(OH) D concentrations gradually increase from around March to August [26]. This could partly explain the reason why serum 25(OH) D concentration was transiently inhibited in early Spring and followed by a marked increase in early Summer in our study. Future studies that aim at exploring the mechanism underlying the effect of resistance training on serum 25(OH) D concentrations are warranted. This study has several limitations. First, our study included only men with relatively lower serum 25(OH) D concentrations. Therefore, it remains to be established whether these findings are applicable to other subjects with higher 25(OH) D concentrations. Second, considering the seasonal variation of serum 25(OH) D concentration, the findings in our study have seasonal limitations. Third, at baseline resistance training group had relatively lower values of muscle mass and body fat percentage, although they are not statistically significant, which should be interpreted with caution. Fourth, we couldn't observe any relationship between muscle mass and 25(OH) D at three points, which may due to the small sample size. However, the muscle mass changes were significantly related with 25(OH) D concentration changes. Finally, although our sample size was relatively small, a post hoc power calculation would still give us a 90% chance, with an effect size of 0.58, to demonstrate the interaction effect of serum 25(OH) D concentrations, which was considered statistically significant at P < 0.05 at the endpoints.","In summary, serum 25(OH) D concentrations in vitamin D deficient young men was observed to be transiently inhibited during the early phase of resistance training and subsequently increased in the later phase. This finding could be partly attributed to the muscle mass and fat free mass gain induced by resistance training during the early phase. Our findings indicated that adequate administration of oral supplements or increased daily dietary vitamin D intake are encouraged to increase serum 25(OH) D concentrations, especially for those who exercise regularly. "],"string":"[\n \"Circulating 25-hydroxyvitamin D [25(OH)D] is the major form of vitamin D, which is used in vitamin D status assessment. Accumulated studies indicated that vitamin D not only regulates calcium and phosphorus metabolism but also plays an important role in modifying cardiovascular diseases risk, and improving immune function and physical fitness [1][2][3][4].\",\n \"Previous studies indicated that higher levels of physical activity or cardiorespiratory fitness were related to higher circulating 25(OH) D concentrations [5][6][7]. Although individuals who were physically active outdoors are more likely to have higher circulating 25(OH) D due to increased sun exposure [8,9], this relationship was still observed after adjustment for sunlight exposure [6,10]. Scragg et al. [5] found that higher levels of physical activity were associated with higher serum 25(OH) D concentrations not only during summer but also during winter, when the vitamin D synthesis from sun exposure is extremely limited [11]. Consistently, several intervention studies suggested that endurance exercise could increase circulating 25(OH) D or prevent its seasonal reduction [12][13][14]. These findings indicated that physical activity could directly increase 25(OH) D concentrations.\",\n \"Resistance training (RT) promotes health benefits through increased skeletal muscle mass and qualitative adaptations [15]. In addition to adiposity tissue, muscle tissue was suggested to serve as an another important site to store 25(OH) D, and then it returns to the circulating when needed [16,17]. Nevertheless, Makanae and colleagues reported that intramuscular expression of CYP27B1, which catalyses the hydroxylation and activation of 25(OH) D, was significantly increased at 1 h and 3 h after resistance exercise, which may enhance local vitamin D metabolism in skeletal muscle [18]. It is well known that compared with endurance exercise, RT could not only reduce fat mass, but also it is more effective in increasing muscle mass and strength [19,20], which may largely affect vitamin D metabolism. However, whether serum 25(OH) D concentrations are affected by RT remains unclear.\",\n \"This study aimed to evaluate the effect of chronic resistance training on serum 25(OH) D concentrations and to determine whether the effects are attributable to changes in body composition in healthy young men.\",\n \"Eighteen healthy men with no chronic diseases (aged 19 to 39 years) were included in this study, which was conducted from March to July 2016. Participants using vitamin D supplements or sunscreen regularly were excluded. Assessments were performed at 0 week (pre), 6 weeks (midpoint), and 12 weeks (endpoint). All subjects provided informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). The trial has been retrospectively registered at Chinese Clinical Trial Registry website (ChiCTR2000030876).\",\n \"Eighteen participants were randomly allocated to the following two groups by using a computer-generated random number sequence: resistance training group (RT) and no-exercise control group (CON). Participants in the RT group were instructed to exercise 2-3 times a week in a gymnasium. A supervised training session was performed in the afternoon between 1630 h and 2000 h during the 12 weeks of progressive resistance training. In the first 2 weeks, the participants were taught how to properly perform each movement. The resistance workload gradually changed from light to heavy, and the participants were instructed to complete 10 repetitions for each set of the following exercises: leg press, preacher curl, chest press, leg curl, and iso row, with 60-120-s rest period between two sets; additionally, they also performed three sets of 15-25 repetitions of abdominal crunch and back extension with their own body weight, with a 60-120-s rest period between two sets. At the first training session of the third week, the participants were instructed to perform strength testing, and 12 repetitions maximum (12RM) for each exercise were performed, except back extension and abdominal crunch. The workload for the next resistance training was calculated from the participants' previously determined 12RM. The participants were encouraged to perform back extension and abdominal crunch exercises 30 times from the first training session of the third week. When the strength training equipment at our gymnasium had been upgraded at the eighth week of training, the resistance training program was revised to include the following: leg extension, standing one-arm cable curl, standing cable chest press, leverage shoulder press, leverage high row, back extension, and abdominal crunch. These new components of the resistance training program were selected based on the principle of using similar muscle groups. The order of resistance exercise was randomized; however, training of the same muscle groups continuously was avoided. The participants were instructed not to undertake any formal exercise or change their levels of general physical activity and dietary habits.\",\n \"Height was measured with the participants wearing light clothing and being barefoot (HK6000-ST, HENGKANG JIAYE Technology Co., Ltd. CHN). Dual-energy X-ray absorptiometry (DXA Prodigy, GE Lunar Corp., Madison, WI, USA) was used for the measurement of whole and regional body composition, including total body mass, muscle mass, fat mass, fat percentage, and bone mineral density. Body mass index was calculated as body mass (kg) divided by height (m 2 ). Fat-free mass (FFM) was calculated as body mass minus fat mass.\",\n \"Physical activity was measured using Actigraph GT3X+ accelerometers (ActiGraph, LLC, Pensacola, FL, USA). Participants were instructed to wear the accelerometers on their right hip at all times, except during water activities (swimming, showering) or sleeping, for 7 consecutive days at baseline. A wear time of ≥480 min/day was used as the criterion for a valid day, and ≥ 2 weekdays and 1 weekend day was used as the criteria for a valid 7-day period of accumulated data. Non-wear time was defined as consecutive periods of ≥60 min of zero accelerometer counts and excluded from the analyses. Data were recorded in 60-s epochs. A pragmatic cutoff of ≥2690 cpm was used to categorize moderatevigorous physical activity time [21,22]. The 7-day data collection for all subjects was fixed within 2 weeks. Outdoor time from 0900 h to 1600 h for 7 consecutive days was recorded and used as an alternative for sun exposure in the analysis.\",\n \"Blood samples were obtained after at least an 8-h overnight fasting period and subsequently centrifuged at 3000 rpm at 4°C for 10 min. Serum was collected and stored at − 80°C until analysis. Serum 25(OH) D and intact parathyroid hormone (iPTH) concentrations were measured in duplicate using commercially available enzyme-linked immunosorbent assay kits (25(OH) D, IDS, Bolton, UK; iPTH, DRG, Marburg, Germany) according to the manufacturer's instructions. The intraassay and inter-assay coefficients of variation were 9.1 and 7.7%, respectively, for serum 25(OH) D and 9.6 and 3.6%, respectively, for iPTH. Vitamin D deficiency is defined as 25(OH) D concentration < 50 nmol/L, and vitamin D insufficiency as 25(OH) D concentration < 75 nmol/L [23].\",\n \"All statistical analyses were performed using IBM SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were assessed for normality using a Kolmogorov-Smirnov test prior to all statistical analyses. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables were presented as median (interquartile range), unless otherwise indicated. The Student t test (for normally distributed variables) or the Mann-Whitney U test (for non-normally distributed variables) was used to evaluate the differences between groups. Two-factor repeat-measured analysis of variance (ANOVA) (group × time) was used to determine the effect of exercise on 25(OH) D, iPTH, and body composition indicators. A post hoc test with Bonferroni correction was performed to identify statistically significant differences among the mean values when a considerable interaction was identified. Pearson correlation and partial correlation coefficients were computed between 25(OH) D concentration changes and body composition indicators. The level of statistical significance was set at P < 0.05.\",\n \"The participants' characteristics and blood parameters at baseline are presented in Table 1. The average serum 25(OH) D concentrations were 26.6 nmol/L, and all participants showed vitamin D deficiency. No significant difference in either body composition indicators or blood parameters was found between the groups (P > 0.05).\",\n \"As shown in Table 2, significant interactions of group × time were found for FFM (P = 0.004) and muscle mass (P = 0.003). After resistance training, the FFM values (mid-pre, 2.7%, P = 0.003; end-pre, 3.0%, P = 0.004) and muscle mass (mid-pre, 3.0%, P = 0.002; end-pre, 3.4%, P = 0.004) significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in the CON group. In addition, we observed borderline significant interactions in body fat percentage, arm fat percentage, leg fat percentage, and trunk fat percentage ( Table 2).\",\n \"As shown in Table 2 and Fig. 1, a significant group × time interaction was observed for serum 25(OH) D concentrations (P = 0.025). After the 12-week intervention, the mean serum 25(OH) D concentrations significantly increased to 36.7 ± 2.7 nmol/L in the RT group and 40.7 ± 2.7 nmol/L in the CON group. Moreover, in the CON group, higher serum 25(OH) D concentrations were observed at midpoint compared with the values at baseline (P < 0.01), whereas no notable differences were found in the RT group. From midpoint to endpoint, although serum 25(OH) D concentrations significantly increased in both groups, the increase was significantly greater in the RT group (25.8%) than in the CON group (9.4%) (P-interaction =0.043).\",\n \"Correlation analyses were performed to identify the factors associated with the changes in 25(OH) D concentrations (Table 3). 25(OH) D concentration changes (end-pre) were negatively related to FFM (mid-pre) (P = 0.015) and muscle mass (mid-pre) (P = 0.017) changes (Fig. 2); a borderline association was observed between 25(OH) D concentration changes (mid-pre) and FFM (mid-pre) (P = 0.058) and muscle mass (mid-pre) (P = 0.057) changes. In addition, significant correlations of 25(OH) D concentration changes (end-pre) with FFM (mid-pre) (P = 0.039) and muscle mass (mid-pre) (P = 0.043) changes were observed after adjustment for group, age, and changes in sun exposure (end-pre), body weight (end-pre), and body fat percentage (end-pre).\",\n \"In this study, we found that the seasonal increase in serum 25(OH) D concentrations in young men was transiently inhibited during the early phase of RT; subsequently, serum 25(OH) D concentrations increased in both RT and CON groups, and the increase was observed to be greater in RT group than in CON group. The transient inhibition could be partly attributed to muscle mass and fat free mass gain induced by RT in the early phase.\",\n \"Previous studies have reported significant positive associations of serum 25(OH) D concentrations with physical activity levels independent of sun exposure [6,10]. Bell et al. [24] found that 25(OH) D concentrations were higher in participants who had engaged in regular muscle-building exercise for at least 1 year compared to those in the control group. Consistently, our recent studies also observed that exercise training could increase serum 25(OH) D concentrations or prevent its seasonal reduction in young and old men [13,14]. However, the effect of RT on serum 25(OH) D concentrations remains unclear. Compared with endurance training, RT is more effective in increasing muscle mass and strength [24], which was also supported by our results. In the present study, we observed that after resistance training, body mass was reduced, while FFM and muscle mass were significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in CON group.\",\n \"In addition to adiposity tissues, muscle tissues are also served as another important storage for 25(OH) D, which could enable optimal vitamin D status to maintain its physiological function during winter months [17]. Makanae and colleagues observed that intramuscular CYP27B1 was significantly increased after resistance exercise in skeletal muscle, which may enhance local vitamin D metabolism [18]. Srikuea et al. [25] also found that the CYP27B1 expression in the muscle increased significantly during skeletal muscle regeneration from injury. Thus, we speculated that the transient inhibition in serum 25(OH) D levels in RT group during the early phase of resistance training could be partly due to absorption of serum 25(OH) D in the increasing muscle mass and its use in muscle activity. 25(OH) D consumption in muscle may induce a compensatory increase in serum 25(OH) D concentrations; however, the concentrations were still lower than that in control group, which may due to the lack of vitamin D storage and short-term intervention period. Previous studies report that serum 25(OH) D concentrations gradually increase from around March to August [26]. This could partly explain the reason why serum 25(OH) D concentration was transiently inhibited in early Spring and followed by a marked increase in early Summer in our study. Future studies that aim at exploring the mechanism underlying the effect of resistance training on serum 25(OH) D concentrations are warranted. This study has several limitations. First, our study included only men with relatively lower serum 25(OH) D concentrations. Therefore, it remains to be established whether these findings are applicable to other subjects with higher 25(OH) D concentrations. Second, considering the seasonal variation of serum 25(OH) D concentration, the findings in our study have seasonal limitations. Third, at baseline resistance training group had relatively lower values of muscle mass and body fat percentage, although they are not statistically significant, which should be interpreted with caution. Fourth, we couldn't observe any relationship between muscle mass and 25(OH) D at three points, which may due to the small sample size. However, the muscle mass changes were significantly related with 25(OH) D concentration changes. Finally, although our sample size was relatively small, a post hoc power calculation would still give us a 90% chance, with an effect size of 0.58, to demonstrate the interaction effect of serum 25(OH) D concentrations, which was considered statistically significant at P < 0.05 at the endpoints.\",\n \"In summary, serum 25(OH) D concentrations in vitamin D deficient young men was observed to be transiently inhibited during the early phase of resistance training and subsequently increased in the later phase. This finding could be partly attributed to the muscle mass and fat free mass gain induced by resistance training during the early phase. Our findings indicated that adequate administration of oral supplements or increased daily dietary vitamin D intake are encouraged to increase serum 25(OH) D concentrations, especially for those who exercise regularly. \"\n]"},"annotations_publisher":{"kind":"list like","value":[],"string":"[]"},"annotations_sectionheader":{"kind":"list like","value":["Introduction","Methods","Participants","Experimental protocol","Anthropometric measurements","Physical activity and sun exposure","Blood analysis","Statistical analysis","Results","Discussion","Conclusions","Fig. 1","Table 1","Table 2","Table 3"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Participants\",\n \"Experimental protocol\",\n \"Anthropometric measurements\",\n \"Physical activity and sun exposure\",\n \"Blood analysis\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Fig. 1\",\n \"Table 1\",\n \"Table 2\",\n \"Table 3\"\n]"},"annotations_table":{"kind":"list like","value":["Variable \nResistance training group (RT) (n = 9) \nNon-exercise control group (CON) (n = 9) \n\nAge (y) \n24.2 ± 3.1 \n26.7 ± 6.2 \n\nHeight (cm) \n1.74 ± 0.03 \n1.75 ± 0.06 \n\nWeight (kg) \n68.5 ± 9.7 \n68.7 ± 7.6 \n\nBMI (kg/m 2 ) \n22.5 ± 2.9 \n22.3 ± 1.9 \n\nFFM (kg) \n54.2 (49.2 -56.4 ) \n57.5 (51.1 -58.8 ) \n\nMuscle mass (kg) \n51.4 (46.6 -53.3 ) \n54.9 (48.4 -55.8 ) \n\nBody fat percentage (%) \n21.9 ± 6.9 \n18.8 ± 6.0 \n\nArms fat percentage (%) \n18.7 ± 7.1 \n14.4 ± 5.1 \n\nLegs fat percentage (%) \n20.5 ± 5.6 \n18.1 ± 5.2 \n\nTrunk fat percentage (%) \n25.1 ± 8.3 \n21.6 ± 7.3 \n\nBMD (g/cm 2 ) \n1.16 ± 0.07 \n1.18 ± 0.07 \n\n25(OH) D (nmol/L) \n26.9 ± 4.7 \n26.2 ± 4.1 \n\niPTH (pg/mL) \n54.9 ± 19.8 \n44.6 ± 23.8 \n\nMVPA (min/day) \n69.8 ± 32.2 \n65.8 ± 16.3 \n\nSun exposure (min/day) \n40.0 ± 27.9 \n50.0 ± 30.7 \n\n","Variable \nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \n\nBaseline \nMidpoint \nEndpoint \nBaseline \nMidpoint \nEndpoint \nTime Group Time × Group interaction \n\nWeight (kg) a \n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \n68.8 ± 3.1 \n68.7 ± 3.1 \n68.6 ± 3.0 \n0.156 0.958 0.762 \n\nBMI (kg/m 2 ) a \n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \n22.3 ± 0.9 \n22.2 ± 0.9 \n22.2 ± 0.8 \n0.273 0.768 0.455 \n\nFFM (kg) a \n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \n56.1 ± 1.8 \n55.8 ± 1.8 \n0.347 0.341 0.004 \n\nMuscle mass (kg) a \n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \n53.2 ± 1.7 \n53.0 ± 1.7 \n0.399 0.348 0.003 \n\nBody fat percentage a \n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \n18.3 ± 2.0 \n18.0 ± 1.9 \n18.3 ± 2.0 \n0.643 0.327 0.058 \n\nArms fat percentage a \n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \n13.7 ± 1.8 \n13.4 ± 1.8 \n13.6 ± 1.7 \n0.687 0.091 0.077 \n\nLegs fat percentage a \n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \n17.7 ± 1.7 \n17.2 ± 1.6 \n17.6 ± 1.7 \n0.846 0.378 0.064 \n\nTrunk fat percentage a \n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \n21.1 ± 2.5 \n20.8 ± 2.4 \n21.1 ± 2.4 \n0.484 0.397 0.075 \n\nBMD (g/cm 2 ) a \n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \n0.016 0.538 0.235 \n\n25(OH) D (nmol/L) b \n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \n\niPTH (pg/mL) b \n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \n37.6 ± 8.3 \n48.3 ± 6.1 \n51.5 ± 5.6 \n0.110 0.131 0.242 \n\nSun exposure (min/day) 40.0± 9.8 \n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \n73.5 ± 13.5 50.5 ± 15.2 \n0.015 0.889 0.493 \n\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \n\nc \n\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \nBoldface indicates significance (P < 0.05) \n","Variables \n25(OH)D mid-pre \n25(OH)D end-pre \n\nr \nP \nr \nP \n\nBody fat percentage \n\nmid-pre \n0.014 \n0.956 \n0.101 \n0.690 \n\nend-mid \n−0.217 \n0.387 \n−0.004 \n0.986 \n\nend-pre \n−0.090 \n0.722 \n0.064 \n0.800 \n\nFFM \n\nmid-pre \n−0.455 \n0.058 \n−0.565 \n0.015 \n\nend-mid \n0.121 \n0.632 \n0.037 \n0.883 \n\nend-pre \n−0.289 \n0.244 \n−0.424 \n0.080 \n\nMuscle mass \n\nmid-pre \n−0.456 \n0.057 \n−0.554 \n0.017 \n\nend-mid \n0.142 \n0.575 \n0.037 \n0.884 \n\nend-pre \n−0.280 \n0.261 \n−0.415 \n0.087 \n\n"],"string":"[\n \"Variable \\nResistance training group (RT) (n = 9) \\nNon-exercise control group (CON) (n = 9) \\n\\nAge (y) \\n24.2 ± 3.1 \\n26.7 ± 6.2 \\n\\nHeight (cm) \\n1.74 ± 0.03 \\n1.75 ± 0.06 \\n\\nWeight (kg) \\n68.5 ± 9.7 \\n68.7 ± 7.6 \\n\\nBMI (kg/m 2 ) \\n22.5 ± 2.9 \\n22.3 ± 1.9 \\n\\nFFM (kg) \\n54.2 (49.2 -56.4 ) \\n57.5 (51.1 -58.8 ) \\n\\nMuscle mass (kg) \\n51.4 (46.6 -53.3 ) \\n54.9 (48.4 -55.8 ) \\n\\nBody fat percentage (%) \\n21.9 ± 6.9 \\n18.8 ± 6.0 \\n\\nArms fat percentage (%) \\n18.7 ± 7.1 \\n14.4 ± 5.1 \\n\\nLegs fat percentage (%) \\n20.5 ± 5.6 \\n18.1 ± 5.2 \\n\\nTrunk fat percentage (%) \\n25.1 ± 8.3 \\n21.6 ± 7.3 \\n\\nBMD (g/cm 2 ) \\n1.16 ± 0.07 \\n1.18 ± 0.07 \\n\\n25(OH) D (nmol/L) \\n26.9 ± 4.7 \\n26.2 ± 4.1 \\n\\niPTH (pg/mL) \\n54.9 ± 19.8 \\n44.6 ± 23.8 \\n\\nMVPA (min/day) \\n69.8 ± 32.2 \\n65.8 ± 16.3 \\n\\nSun exposure (min/day) \\n40.0 ± 27.9 \\n50.0 ± 30.7 \\n\\n\",\n \"Variable \\nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \\n\\nBaseline \\nMidpoint \\nEndpoint \\nBaseline \\nMidpoint \\nEndpoint \\nTime Group Time × Group interaction \\n\\nWeight (kg) a \\n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \\n68.8 ± 3.1 \\n68.7 ± 3.1 \\n68.6 ± 3.0 \\n0.156 0.958 0.762 \\n\\nBMI (kg/m 2 ) a \\n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \\n22.3 ± 0.9 \\n22.2 ± 0.9 \\n22.2 ± 0.8 \\n0.273 0.768 0.455 \\n\\nFFM (kg) a \\n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \\n56.1 ± 1.8 \\n55.8 ± 1.8 \\n0.347 0.341 0.004 \\n\\nMuscle mass (kg) a \\n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \\n53.2 ± 1.7 \\n53.0 ± 1.7 \\n0.399 0.348 0.003 \\n\\nBody fat percentage a \\n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \\n18.3 ± 2.0 \\n18.0 ± 1.9 \\n18.3 ± 2.0 \\n0.643 0.327 0.058 \\n\\nArms fat percentage a \\n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \\n13.7 ± 1.8 \\n13.4 ± 1.8 \\n13.6 ± 1.7 \\n0.687 0.091 0.077 \\n\\nLegs fat percentage a \\n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \\n17.7 ± 1.7 \\n17.2 ± 1.6 \\n17.6 ± 1.7 \\n0.846 0.378 0.064 \\n\\nTrunk fat percentage a \\n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \\n21.1 ± 2.5 \\n20.8 ± 2.4 \\n21.1 ± 2.4 \\n0.484 0.397 0.075 \\n\\nBMD (g/cm 2 ) a \\n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \\n0.016 0.538 0.235 \\n\\n25(OH) D (nmol/L) b \\n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \\n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \\n\\niPTH (pg/mL) b \\n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \\n37.6 ± 8.3 \\n48.3 ± 6.1 \\n51.5 ± 5.6 \\n0.110 0.131 0.242 \\n\\nSun exposure (min/day) 40.0± 9.8 \\n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \\n73.5 ± 13.5 50.5 ± 15.2 \\n0.015 0.889 0.493 \\n\\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \\nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \\n\\nc \\n\\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \\nBoldface indicates significance (P < 0.05) \\n\",\n \"Variables \\n25(OH)D mid-pre \\n25(OH)D end-pre \\n\\nr \\nP \\nr \\nP \\n\\nBody fat percentage \\n\\nmid-pre \\n0.014 \\n0.956 \\n0.101 \\n0.690 \\n\\nend-mid \\n−0.217 \\n0.387 \\n−0.004 \\n0.986 \\n\\nend-pre \\n−0.090 \\n0.722 \\n0.064 \\n0.800 \\n\\nFFM \\n\\nmid-pre \\n−0.455 \\n0.058 \\n−0.565 \\n0.015 \\n\\nend-mid \\n0.121 \\n0.632 \\n0.037 \\n0.883 \\n\\nend-pre \\n−0.289 \\n0.244 \\n−0.424 \\n0.080 \\n\\nMuscle mass \\n\\nmid-pre \\n−0.456 \\n0.057 \\n−0.554 \\n0.017 \\n\\nend-mid \\n0.142 \\n0.575 \\n0.037 \\n0.884 \\n\\nend-pre \\n−0.280 \\n0.261 \\n−0.415 \\n0.087 \\n\\n\"\n]"},"annotations_tableref":{"kind":"list like","value":["Table 1","Table 2","Table 2)","Table 2","(Table 3"],"string":"[\n \"Table 1\",\n \"Table 2\",\n \"Table 2)\",\n \"Table 2\",\n \"(Table 3\"\n]"},"annotations_title":{"kind":"list like","value":["Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial","Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial"],"string":"[\n \"Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial\",\n \"Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial\"\n]"},"annotations_venue":{"kind":"list like","value":[],"string":"[]"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":847,"numItemsPerPage":100,"numTotalItems":84745,"offset":84700,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc0MzE1NzgwNSwic3ViIjoiL2RhdGFzZXRzL2NlbGxmYWJyaWsvY3VsdHVyZWRfbWVhdCIsImV4cCI6MTc0MzE2MTQwNSwiaXNzIjoiaHR0cHM6Ly9odWdnaW5nZmFjZS5jbyJ9.y7_jasBzBc7fWtuAvFavB-3nJaHOmH4nD-sXxYDqCe8CxOyEsUjDLyTwIancXRKNOAO6o30co1u7wLfRp2hIAw","displayUrls":true},"discussionsStats":{"closed":0,"open":1,"total":1},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false}">
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244,809,539 | 2021-12-31T06:13:10Z | CCBY | https://www.mdpi.com/2072-6643/13/12/4332/pdf | GOLD | 5435b7f6db393a09294bf016ce8dca48298e82df | null | null | null | null | 10.3390/nu13124332 | null | 34959884 | 8703777 |
In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract
Published: 30 November 2021
Ahreum Lee
Korea Institute of Brain Science
06022SeoulKorea
Yuki Sugiura
Department of Biochemistry and Integrative Medical Biology
School of Medicine
Keio University
160-8582TokyoJapan
Ik-Hyun Cho
Department of Convergence Korean Medical Science
College of Korean Medicine
Kyung Hee University
02447SeoulKorea
Noriko Setou
Department of Disaster Psychiatry
Fukushima Medical University
960-1295FukushimaJapan
Eugene Koh
Temasek Life Sciences Laboratories
117604Singapore, Singapore
Gyun Jee Song
Department of Medical Science
Catholic Kwandong University College of Medicine
25601GangneungKorea
Seungheun Lee
Korea Institute of Brain Science
06022SeoulKorea
Hyun-Jeong Yang
Korea Institute of Brain Science
06022SeoulKorea
Department of Integrative Health Care
University of Brain Education
31228CheonanKorea
In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract
Published: 30 November 202110.3390/nu13124332Received: 10 October 2021 Accepted: 27 November 2021nutrients Article
Citation: Lee, A.; Sugiura, Y.; Cho, I.-H.; Setou, N.; Koh, E.; Song, G.J.; Lee, S.; Yang, H.-J. In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract. Nutrients 2021, 13, 4332. https://doi.org/10.3390/ nu13124332 Academic Editors: Raffaella Canali and Fausta Natella
Introduction
Dendropanax is a genus of flowering plants in the family Araliaceae [1]. Although both Dendropanax morbiferus (DM) and Dendropanax trifidus (DT), which belong to this family, are native to eastern Asia, each specific habitat is distinct; the southern coast of the Korean Peninsula for DM [2][3][4], and Japan for DT [2,5]. However, according to previous PCR-Random Amplified Polymorphic DNA (RAPD) analysis of DM and DT, their genetic differences were not clear [2]. In a study of the fruit, sap color and leaf external morphologies of DM and DT, a clear distinction between the two was also not possible [6].
Materials and Methods
Mice
Eight-week-old mice of CrljOri:CD1 (ICR) lines were purchased from ORIENT BIO Inc. (Seongnam, Korea). After 1 week of rest, 9-week-old mice were used for the experiments. Mice were kept on a 12-h light-dark cycle with light period of 8:00-20:00 in a standard specific pathogen-free environment. Mice were provided with regular chow and water ad libitum. Each animal was anaesthetized by carbon dioxide inhalation before sampling. All experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by the University of Brain Education's Animal Care and Use Committee (Approval number: 2018-AE-01Y). Mouse number used in each experiment is indicated in Table S1.
Dendropanax trifidus Sap Preparation
Dendropanax trifidus (Thunb.) Makino ex H.Hara was collected from the forest in Simasi, Mieken, Japan and registered at Ibaraki Nature Museum: voucher number INM-2-212778. DT sap was dissolved in 100% EtOH, freeze dried and stored at −20 °C. The freezedried final product with viscosity was dissolved in EtOH to make a stock solution of 1.4 or 0.5 g/mL and stored at −20 °C. The extract is referred as DT sap in this paper.
In Vivo Toxicity Test
To determine the optimal concentration of DT sap for in vivo administration, shortterm effects of DT sap on survival as well as body and organ weights were examined when administered in a single dose of various concentrations (0, 1.2, 2.5, 4.8, 10.3, 21.3 mg/g, DT sap weight/body weight) to 9-week-old ICR mice by oral gavage. After the single injection, the mice were observed for several hours, and body weight was measured once per day for an initial 3 days. At day 20, body weight was measured again, and mice were sacrificed, thereafter organ (liver, spleen, kidney, lung, heart = and adrenal gland) weights were measured separately ( Figure S1). We further examined the effects of multiple injections with several lower concentrations (0, 0.5, 1 and 2.5 mg/g, DT sap weight/body weight) every day for 14 days to 9-week-old ICR mice by oral gavage. Mouse body weight was measured at day 0, 1, 3, 7 and 14, and the mice were sacrificed at day 14 to obtain organs for weight measurements, blood chemistry, Western blots and histochemistry. Before the sacrifice, all animals were fasted overnight ( Figure 1). Figure 1. Experimental design. Experimental procedures are briefly described in the scheme. Dendropanax Trifidus sap was extracted in 100% EtOH, freeze-dried and maintained at −20 °C. Its EtOH-dissolved solution was subjected to in vivo mouse studies. A single oral administration was conducted to limit the dose range of DT sap ( Figure S1). Based on this information, daily oral gavage of three concentrations was performed for 14 days. Survival rate and body weights were measured at day 0, 1, 3, 7 and 14 ( Figure 2). Organ weight measurement (Figure 3), blood chemistry ( Figure 4), histochemistry ( Figure 5) and Western blot analysis ( Figure 6) were performed on the samples of day 14. In order to analyze the chemical components of DT sap, it was subjected to non-targeted metabolome analysis ( Figure 7, Table 1). Figure 1. Experimental design. Experimental procedures are briefly described in the scheme. Dendropanax Trifidus sap was extracted in 100% EtOH, freeze-dried and maintained at −20 • C. Its EtOH-dissolved solution was subjected to in vivo mouse studies. A single oral administration was conducted to limit the dose range of DT sap ( Figure S1). Based on this information, daily oral gavage of three concentrations was performed for 14 days. Survival rate and body weights were measured at day 0, 1, 3, 7 and 14 ( Figure 2). Organ weight measurement (Figure 3), blood chemistry ( Figure 4), histochemistry ( Figure 5) and Western blot analysis ( Figure 6) were performed on the samples of day 14. In order to analyze the chemical components of DT sap, it was subjected to non-targeted metabolome analysis (Figure 7, Table 1).
3-cyclohexyl-N,N- dimethyl-3- phenylpropan-1- amine
C18H22O2 Trenbolone
Trienbolone; 17-beta-Hydroxyestra-4,9,11-trien-3one
C17H29NO 2,6-Di-tert- butyl-4-(di- methyla- minome- thyl)phenol N,N-dimethyl- 3,5-di-tert-bu- tyl-4-
Dendropanax trifidus Sap
Therefore, for the multiple administration regime, the highest concentration used among the tested concentrations was set lower than 4.8 mg/g (i.e., 2.5 mg/g). The indicated concentrations (0, 0.5, 1 and 2.5 mg/g) were applied every day by oral gavage for 14 days (Figure 2, N = 4 females and 5 males/concentration). At day 14 (i.e., end of the experimental period), the survival ratio was 100% for all the concentrations except the highest concentration (50% for female and 75% male at 2.5 mg/g, Figure 2A-C). There were no significant changes in mouse body weight by concentration and time ( Figure 2D-F). To examine the effects of the concentration on tissue weight in a multiple administration regime, mouse tissues were collected and measured at day 14 ( Figure 3). Using a oneway ANOVA against total organ weights, significant differences were found in the liver (p = 0.002, Figure 3A), spleen (p = 0.007, Figure 3B), kidney (p = 0.003, Figure 3C), heart (p = 0.001, Figure 3E), and with respect to concentration. By post hoc analysis (Holm-Sidak method), significant differences were found to be derived from one concentration (i.e., the highest concentration, 2.5 mg/g, p = 0.002 for kidney, 0.031 for heart, <0.001 for liver, 0.006 for spleen). There were no significant differences in the tissue weights at other concentrations. In addition, increasing DT sap concentration did not affect the lung and adrenal tissue weights. Analyzing by gender, the weights of female liver (one way ANOVA, p = 0.025, Figure 3AI), spleen (one way ANOVA, p = 0.043, Figure 3BI), kidney (one way ANOVA, p = 0.003, Figure 3CI), heart (Kruskal-Wallis one way ANOVA on Ranks, p = 0.016, Figure 3EI) were significantly changed and post hoc analysis revealed that these were due to the highest concentration of 2.5 mg/g (Holm-Sidak method, p = 0.039 for liver, p = 0.058 for spleen, p = 0.012 for kidney; Dunn's Method, p = 0.035 for heart). There were no significant weight differences in the other concentrations. In female lung and adrenal glands, no significant weight differences were found in all the concentrations. Figure S2 and Figure 3. Scale bar, 50 µm.
Blood Chemistry
DT sap extracts of the indicated concentrations were given to ICR mice for 14 days by daily oral gavage. Whole blood was withdrawn from the heart and maintained at room temperature (RT) for 30 min to clot, then centrifuged at 2000× g for 10 min in a refrigerated centrifuge, and the resulting supernatant (serum) was subjected to blood chemistry analysis. In the serum, the following parameters were measured by using a BS-200 Chemistry Analyzer (Mindray, China): GLU, glutamic pyruvate transaminase (GPT), glutamic oxalacetic transaminase (GOT), total protein (TP), blood urea nitrogen (BUN), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triglyceride (TG), cholesterol (CHOL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL).
Western Blot
Western blot was performed as previously described [21]. Briefly, tissues were lysed in RIPA lysis buffer (WSE-7420, ATTO, DAWINBIO Inc, Hanam, Korea), and centrifuged at 15,000 RPM for 15 min. The supernatant was quantified by Bradford assay, diluted with 5 × Sample buffer, boiled and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation. Proteins were transferred onto PVDF membranes and blocked with EZBlock Chemi (AE-1475, ATTO, Tokyo, Japan) for 1h at RT. Membranes were incubated with the primary antibody for overnight at 4 • C, washed and incubated with secondary antibody for 1h at RT, and washed. Proteins were visualized by Super Signal West Pico PLUS Chemiluminescent Substrate (34580, ThermoFisher Scientific, Waltham, MA, USA) and captured by Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). Images were analyzed by using Image J. For blotting, rabbit antibodies to the following antigens were purchased from Cell Signaling Technology (Danvers, MA, USA): phospho-IRS-1 (Ser1101) (2385), IRS-1 (2382), phospho-AMPKa (2531), AMPKa (2532), phosphor-Akt (4060) and Akt (9272). Rabbit antibodies to beta actin, Glut1 (ab652) and Glut4 (ab654) were purchased from Abcam (Cambridge, UK). For secondary antibody, horseradish peroxidase conjugate goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA) was used.
with respect to increasing DT sap concentration in liver tissues ( Figure 6A,C, p = 0.014, one way ANOVA). We noted that there was a non-significant slight increase in IRS/ꞵ-Actin levels with respect to increasing DT sap concentration ( Figure 6D). As Akt activation is known to act downstream of insulin signaling, we also measured Akt and pAkt levels. The levels of pAkt/Akt appear to alter at 0.5, 1 mg/g dose, without significant differences ( Figure 6E). Akt/ꞵ-Actin was not changed at 0.5 mg/g and decreased at 1 or 2.5 mg/g, without significant changes ( Figure 6F).
Histopathology
To assess the histopathological changes of the kidney and liver after DT sap extract administration, the ICR mice were anesthetized with CO 2 and then perfused intracardially with saline and cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The kidney and liver were removed, post-fixed, washed, dehydrated and embedded with melted paraffin wax [22]. Paraffin blocks were cut into 5-µm-thick sections using a Leica RM 2155 microtome (Leica Biosystems, Wetzlar, Germany). The paraffin sections were stained with hematoxylin-eosin (H&E) dye as previously described [22]. Images of stained sections were visualized and captured using a DP70 digital light microscope system (Olympus, Tokyo, Japan). The kidney and liver morphology were observed under 40x magnification. Table 1.
Discussion
The results of our toxicity study on survival ratio, body weight, organ weight, blood chemistry and histochemistry show that DT sap concentrations below 0.5 mg/mL were safe for long-term administration (Table S2). We also found that blood GLU levels were gradually reduced with increased dosage of DT sap in female mice, and that the AMPKmediated signaling was altered by DT sap administration in female mouse liver and spleen tissues. In addition, bioactive chemicals were identified from DT sap by LC-MS/MS. While a significant dose-dependent reduction in blood glucose was observed in female mice, it was not observed in male mice ( Figure 4AI,AII). However, 0.5 mg/g DT sap administration also reduced the mean value of blood glucose in males compared to the control, although it was not statistically significant, implying a possible gender difference in dose-response. Therefore, we cannot exclude the possibility of hypoglycemic effects of DT sap on male mice as well, warranting further studies. Reduction in blood GLU with corresponding increases of DT sap concentration ( Figure 4AI) may be related with the activation of the insulin signaling pathway [29] or AMPK signaling pathway [30]. Here, phosphorylation of IRS-1 and Akt for the insulin signaling pathway and phosphorylation of AMPK for the AMPK signaling pathway were investigated. Phosphorylation of IRS-1 (Ser1101) inhibits insulin signaling [24], therefore the reduction of pIRS-1(Ser1101)/IRS-1 by DT sap should result in a corresponding decrease in blood GLU, which is observed here ( Figure 6C). The level of pAkt/Akt, a downstream signaling factor of IRS-1, appears to increase according to DT sap concentration ( Figure 6I). These results suggest that DT sap appears to affect blood GLU regulation via activation of the insulin signaling pathway. Previous findings showed that the activation of AMPK suppresses gluconeogenesis [25]. In our study, pAMPK/AMPK levels were significantly increased at low DT sap concentrations (0.5 mg/g) in liver tissues ( Figure 6G), and exhibited a significant increase in conjunction with increasing DT sap concentration in spleen tissues ( Figure 6K). Therefore, reduced GLU production may also contribute to blood GLU reduction ( Figure 4AI).
We observed that pAMPK/AMPK, a key metabolic regulator, exhibited a common tendency of increase with respect to increasing DT sap concentrations in both liver and spleen tissues ( Figure 6G,K), In contrast, pAkt/Akt levels responded differently in liver and spleen tissues. While pAkt/Akt levels did not show a consistent change in liver tissue ( Figure 6E), the pAkt/Akt levels in the spleen tissue showed an increase, with respect to Table 1.
Non-Targeted Metabolome Analysis
Ethanol extracts of DT sap and Acer saccharum (AS) sap (Alleghanys Maple Farms Inc., Saint-Pacome, QC, Canada) were analyzed as previously described [23]. Briefly, for non-targeted analysis, metabolome data obtained by orbitrap-type MS (Q-Exactive Focus, Thermo Fisher Scientific, San Jose, CA, USA) connected to a HPLC (Ultimate3000 system, Thermo Fisher Scientific) with the discovery HS F5-3 column or an IC (ICS-5000+, Thermo Fisher Scientific) with the IonPac AS11-HC, 4-µm particle size column were analyzed. A Compound Discoverer 2.0 (Thermo Fisher Scientific) was used for the non-targeted metabolomics workflow. In brief, this software first aligned the total ion chromatograms of different samples along the retention time. Then, the detected features with an intensity of no less than 100,000 and an S/N larger than five in each set of data were extracted and merged into components. The resulting compounds were identified by both (i) formula prediction based on accurate m/z value and isotope peak patterns and (ii) MS/MS structural validation. Moreover, formula predicted signals were assigned into candidate compounds by database search (Chemspider database; http://www.chemspider.com/, accessed on 19 December 2019).
Statistics
Statistical analyses were performed using one way analysis of variance (ANOVA) with post hoc tests (Holm-Sidak method or Dunn's method), Kruskal-Wallis one way ANOVA on Ranks and Student's t-test.
Results
Effects on Survival Ratio and Body/Tissue Weight by a Single Administration of Dendropanax trifidus Sap
For assessing the in vivo toxicity of DT sap, a single administration regime was performed first in 9-week-old ICR mice by oral gavage, in order to limit the dose range. For the single administration ( Figure S1), DT sap of a wide range of concentrations (0, 1.2, 2.5, 4.8, 10.3 and 21.3 mg/g for DT sap weight/mouse body weight) were separately injected. Mice injected with the highest concentration of 21.3 mg/g died at day 2; however, mice administered with concentrations lower than 21.3 mg/g survived till the end of the experimental period (i.e., day 20 after the administration) ( Figure S1A). There were no significant changes in body weight for the initial three days after the injection ( Figure S1B). However, in the measurement at day 20 post-administration, the concentration of 10.3 mg/g increased total body weight and spleen weight compared to other concentrations ( Figure S1C,E). In the concentrations of ≤4.8 mg/g, total body weight and organ weights were not changed by the injection (Figure S1B-I).
Effects on Survival Ratio and Body/Tissue Weight by a Multiple Administration of Dendropanax trifidus Sap
Therefore, for the multiple administration regime, the highest concentration used among the tested concentrations was set lower than 4.8 mg/g (i.e., 2.5 mg/g). The indicated concentrations (0, 0.5, 1 and 2.5 mg/g) were applied every day by oral gavage for 14 days (Figure 2, N = 4 females and 5 males/concentration). At day 14 (i.e., end of the experimental period), the survival ratio was 100% for all the concentrations except the highest concentration (50% for female and 75% male at 2.5 mg/g, Figure 2A-C). There were no significant changes in mouse body weight by concentration and time ( Figure 2D-F).
To examine the effects of the concentration on tissue weight in a multiple administration regime, mouse tissues were collected and measured at day 14 ( Figure 3). Using a one-way ANOVA against total organ weights, significant differences were found in the liver (p = 0.002, Figure 3A), spleen (p = 0.007, Figure 3B), kidney (p = 0.003, Figure 3C), heart (p = 0.001, Figure 3E), and with respect to concentration. By post hoc analysis (Holm-Sidak method), significant differences were found to be derived from one concentration (i.e., the highest concentration, 2.5 mg/g, p = 0.002 for kidney, 0.031 for heart, <0.001 for liver, 0.006 for spleen). There were no significant differences in the tissue weights at other concentrations. In addition, increasing DT sap concentration did not affect the lung and adrenal tissue weights.
Analyzing by gender, the weights of female liver (one way ANOVA, p = 0.025, Figure 3AI), spleen (one way ANOVA, p = 0.043, Figure 3BI), kidney (one way ANOVA, p = 0.003, Figure 3CI), heart (Kruskal-Wallis one way ANOVA on Ranks, p = 0.016, Figure 3EI) were significantly changed and post hoc analysis revealed that these were due to the highest concentration of 2.5 mg/g (Holm-Sidak method, p = 0.039 for liver, p = 0.058 for spleen, p = 0.012 for kidney; Dunn's Method, p = 0.035 for heart). There were no significant weight differences in the other concentrations. In female lung and adrenal glands, no significant weight differences were found in all the concentrations.
In male mouse tissues, one-way ANOVA analysis showed significant differences in liver (p = 0.004, Figure 3AII), spleen (p = 0.049, Figure 3BII), kidney (p < 0.001, Figure 3CII), heart (p = 0.041, Figure 3EII) by DT sap concentrations, and subsequent post hoc analysis (Holm-Sidak method) showed that the difference was mainly derived from the 2.5 mg/g concentration, although differences were also found at 1 mg/g in kidney (liver, p = 0.003 (0 vs. 2.5 mg/g); kidney, p < 0.001(0 vs. 2.5 mg/g), p = 0.021 (0 vs. 1 mg/g); heart, p = 0.034 (0.5 vs. 2.5 mg/g)). There were no significant differences in these tissue weights in other concentrations. In male lung and adrenal glands, no differences were found in all the concentrations.
Reduction in Blood Glucose by Dendropanax trifidus Sap Injection
Next, we investigated the effect of DT sap concentration on blood chemistry after 14 days of daily oral administration (Figure 4). In total mouse blood chemistry analysis, there was a tendency of reduction in blood GLU with respect to increasing DT sap concentration (One way ANOVA, p = 0.060, Figure 4A). Other components in the blood chemistry did not show this tendency. In addition, when the data was individually analyzed by t-test in comparison with vehicle (0 mg/g)-injected mice, mice treated with 0.5 mg/g and 1 mg/g exhibited a tendency of reduction (p = 0.0628) and a significant reduction (p = 0.005), respectively. In female mice, there was a significant reduction in GLU corresponding with the increase of DT sap concentrations (One way ANOVA, p = 0.031, Figure 4AI). In addition, significant changes in GPT and ALP were observed (p = 0.045 and 0.004, respectively) and subsequent post hoc analysis showed a significant increase in ALP at 1 mg/g (p = 0.006, Figure 4FI), but not in GPT ( Figure 4BI). There were no significant changes in the other parameters (GOT, TP, BUN, LDH, TG, CHOL, LDL and HDL) with concentration ( Figure 4CI-KI). In addition, LDL showed a significant reduction in 1 mg/g compared to vehicle control in t-test (p = 0.01, Figure 4JI). Unlike female mice, no significant correlation was found in GLU in males (Kruskal-Wallis one way ANOVA on Ranks, p = 0.093), although GLU levels at 0.5 mg/g DT sap were reduced about half compared to the vehicle control ( Figure 4AII). No significant changes were found in the other parameters in males ( Figure 4BII~KII). In hematoxylin and eosin staining of mouse liver and kidney after 14 days of injection, no specific histopathological changes were observed by DT sap administration in both male and female mice ( Figures 5, S2 and S3, N = 5 per each concentration). To summarize, (1) DT sap did not affect mouse survival, body weight, tissue weight, blood chemistry and histochemistry at the concentration of ≤0.5 mg/g (Table S2), therefore the concentration of ≤0.5 mg/g is recommended for long term stable administration in mice. (2) A significant reduction was found in blood GLU with respect to increasing DT sap concentration in female mice ( Figure 4AI), suggesting the potential effects of DT sap on GLU metabolism.
Effects of Dendropanax trifidus Sap on AMPK-Mediated Signaling
To further investigate the effects of DT sap on GLU metabolism in female mice, Western blots were performed in liver tissues for several key signaling factors known to be involved in the regulation of blood GLU (Figures 6, S4 and S5). Previous studies showed that the phosphorylation of IRS-1 at Ser 1101 results in an inhibition of insulin signaling in the cell [24]. We observed that pIRS-1(Ser1101)/IRS-1 levels were significantly reduced with respect to increasing DT sap concentration in liver tissues ( Figure 6A,C, p = 0.014, one way ANOVA). We noted that there was a non-significant slight increase in IRS/β-Actin levels with respect to increasing DT sap concentration ( Figure 6D). As Akt activation is known to act downstream of insulin signaling, we also measured Akt and pAkt levels. The levels of pAkt/Akt appear to alter at 0.5, 1 mg/g dose, without significant differences ( Figure 6E). Akt/β-Actin was not changed at 0.5 mg/g and decreased at 1 or 2.5 mg/g, without significant changes ( Figure 6F).
Hepatic AMPK activation is known to induce a suppression in gluconeogenesis [25]. To see if the AMPK pathway is activated by DT sap, AMPK phosphorylation was investigated. Compared to the vehicle control, 0.5 mg/g DT sap injection over 14 days significantly increased pAMPK/AMPK in the liver tissues ( Figure 6A,G, Student's t-test, p = 0.032). Higher concentrations also increased pAMPK/AMPK level, but without significant changes. In addition, the AMPK/β-Actin level was significantly altered in liver tissue on application of DT sap ( Figure 6H, p = 0.001, one way ANOVA). Post hoc analysis showed significant reductions in AMPK/β-Actin at 0.5 and 1 mg/g (* p = 0.016 in 0 vs. 0.5 mg/g; * p = 0.001 in 0 vs. 1 mg/g, Holm-Sidak method).
The spleen is related with GLU homeostasis, and its absence induces diabetes in the long term [26,27]. Therefore, GLU signaling factors were investigated in the spleen as well. IRS-1 signaling could not be measured reliably in the spleen sample. However, pAkt/Akt levels were significantly increased along with an increase in DT sap concentrations ( Figure 6B,I, p = 0.034, one way ANOVA; * p = 0.039 in 0 vs. 1 mg/g, post hoc Holm-Sidak method). Akt/β-Actin was slightly reduced by DT sap administration without significant changes ( Figure 6J). The level of pAMPK/AMPK was significantly increased with increasing DT sap concentration ( Figure 6B,K, p = 0.049, one way ANOVA). The AMPK/β-Actin was not altered or slightly increased by the DT sap application in spleen tissues without significant differences ( Figure 6L). GLU transporters such as Glut4 and Glut1 also contribute to regulate GLU metabolism by facilitating the transport of GLU [28]. However, there was no significant changes in Glut4 and Glut1 levels by DT sap administration in both tissues ( Figure S4).
Component Analysis of Dendropanax trifidus Sap
To characterize the chemical composition of DT sap, two batches of DT sap extracts were subjected to LC-MS/MS. A total of 1080 chemicals were identified ( Figure 7A,B). Among them 1041 chemicals were fully or partially matched in more than one of seven tested annotation sources. The top three chemicals with the highest peak area (outside of the dotted red box in Figure 7A) were (1E,5E,9E)-1,5,9-Trime-thyl-1,5,9-cyclododecatriene, caryophyllene oxide and curcumene (Table 1). Seven more chemicals with the next highest peak area (dots with high values in Figure 7B) were 3,5-di-tert-butyl-4-hydroxybenzaldehyde, capsidiol, estradiol (or 19-norandroestenedione), bis [2-(2-butoxyethoxy)ethyl]adipate, trenbolone, o-tert-octylphenol and 5-ethyl-3,8-dimethyl-1,7-dihydroazulene (Table 1). Next, to identify DT sap-specific components, LC-MS/MS was performed and compared with the sap from another tree (i.e., Acer saccharum (AS)). DT sap-specific components were screened by fold change analysis with AS sap, and the consistency of the component analysis was confirmed by repeat experiments using different batches ( Figure 7C). In the graph of Figure 6C, dots of the shaded field (upper right quadrant) represent chemicals which are specifically abundant in DT sap compared to the AS sap, while dots of the other regions are chemicals which are specifically abundant in AS sap compared to DT sap, or unchanged. The intensity of 354 chemicals were higher in DT sap than in AS sap, while that of 726 chemicals were lower in DT sap. The top ten chemicals with the highest fold change was farnesol, gamfexine, (8z,11z,14z)-heptadecatrienoic acid, (1E,5E,9E)-1,5,9-Trime-thyl-1,5,9cyclododecatriene, o-tert-octylphenol, cumene, α-Eleostearic acid, oleanolic aldehyde, 2,6-Dimethyl-4-nonylphenol and 2,6-Di-tert-butyl-4-(dimethylaminome-thyl)phenol (Table 1). In addition to the above-mentioned chemicals, representative chemicals abundant in DT sap include 2-hydroxyestradiol, dienogest, isotretinoin, linoleic acid and costunolide (Table 1).
Discussion
The results of our toxicity study on survival ratio, body weight, organ weight, blood chemistry and histochemistry show that DT sap concentrations below 0.5 mg/mL were safe for long-term administration (Table S2). We also found that blood GLU levels were gradually reduced with increased dosage of DT sap in female mice, and that the AMPKmediated signaling was altered by DT sap administration in female mouse liver and spleen tissues. In addition, bioactive chemicals were identified from DT sap by LC-MS/MS. While a significant dose-dependent reduction in blood glucose was observed in female mice, it was not observed in male mice ( Figure 4AI,AII). However, 0.5 mg/g DT sap administration also reduced the mean value of blood glucose in males compared to the control, although it was not statistically significant, implying a possible gender difference in dose-response. Therefore, we cannot exclude the possibility of hypoglycemic effects of DT sap on male mice as well, warranting further studies. Reduction in blood GLU with corresponding increases of DT sap concentration ( Figure 4AI) may be related with the activation of the insulin signaling pathway [29] or AMPK signaling pathway [30]. Here, phosphorylation of IRS-1 and Akt for the insulin signaling pathway and phosphorylation of AMPK for the AMPK signaling pathway were investigated. Phosphorylation of IRS-1 (Ser1101) inhibits insulin signaling [24], therefore the reduction of pIRS-1(Ser1101)/IRS-1 by DT sap should result in a corresponding decrease in blood GLU, which is observed here ( Figure 6C). The level of pAkt/Akt, a downstream signaling factor of IRS-1, appears to increase according to DT sap concentration ( Figure 6I). These results suggest that DT sap appears to affect blood GLU regulation via activation of the insulin signaling pathway. Previous findings showed that the activation of AMPK suppresses gluconeogenesis [25]. In our study, pAMPK/AMPK levels were significantly increased at low DT sap concentrations (0.5 mg/g) in liver tissues ( Figure 6G), and exhibited a significant increase in conjunction with increasing DT sap concentration in spleen tissues ( Figure 6K). Therefore, reduced GLU production may also contribute to blood GLU reduction ( Figure 4AI).
We observed that pAMPK/AMPK, a key metabolic regulator, exhibited a common tendency of increase with respect to increasing DT sap concentrations in both liver and spleen tissues ( Figure 6G,K), In contrast, pAkt/Akt levels responded differently in liver and spleen tissues. While pAkt/Akt levels did not show a consistent change in liver tissue ( Figure 6E), the pAkt/Akt levels in the spleen tissue showed an increase, with respect to increasing DT sap concentration ( Figure 6I). At this stage, we cannot explain the divergence in pAkt/Akt between liver and spleen tissues, but this will be a matter of future investigation.
The β-actin-normalized AMPK level was significantly altered by DT sap and post hoc analysis showed that it was significantly reduced in 0.5 and 1 mg/g DT sap ( Figure 6H). The other total protein levels of IRS, Akt in liver as well as Akt, AMPK in spleen exhibited slight reductions at some dosage, but without significant differences ( Figure 6D,F,J,L). The reduction of total AMPK protein expression ( Figure 6H) might be a form of negative feedback to preserve cellular homeostasis by preventing the overactivation of AMPK signaling ( Figure 6G,K) induced by long-term DT sap administration. High dosage (2.5 mg/g) might disrupt cellular homeostasis, resulting in significant organ weight changes ( Figure 3) and lower survival rate (Figure 2), observed here.
Interestingly, chemicals enriched in DT sap as analyzed by LC-MS/MS included estradiol, its related metabolites or structurally similar substances (e.g., 2-hydroxyestradiol, trenbolone, dienogest) ( Table 1). It was previously shown that estradiol injections reduced fasting blood GLU levels in non-obese C57BL/6N mice with short-term ovariectomy, while AMPK was activated and gluconeogenic gene expression was reduced in liver tissues [31]. A 4-week subcutaneous injection of estradiol to ovariectomized female mice with STZinduced type 1 diabetes significantly suppressed blood GLU level and increased plasma insulin, suggesting protective effects of estradiol against STZ-induced diabetes [32]. Besides estradiol, various components were identified from DT sap, such as caryophyllene oxide, curcumene, 3, 5-di-tert-butyl-4-hydroxybenzaldehyde, trenbolone, farnesol, dienogest, 2-Hydroxyestradiol, isotretinoin, linoleic acid and costunolide (Table 1). Among them, 2-hydroxyestradiol is a metabolite of estradiol and directly activates AMPK in C2C12 myotubes [33], which attenuates the development of obesity and decreases the severity of diabetes [34]. A black pepper extract with a high content of caryophyllene improved GLU uptake in C2C12 myotubes [35]. Farnesol induced a significant reduction of postprandial hyperglycemia on alloxan-induced type 2 diabetic mice [36]. The function of linoleic acid relates with the regulation of body weight, and serum leptin in subjects with type 2 diabetes [37]. These components have potentials to affect GLU metabolism, which may be involved in the reduction of blood GLU by DT sap observed here. However, to identify the actual bioactive components, in vitro screening and in vivo tests using isolated compounds are required, which is expected as a follow-up study.
Conclusions
In this study, we examined the in vivo safety, hypoglycemic function and component analysis of DT sap. Toxicity tests in vivo confirmed safety in mice at less than 0.5 mg/g when administered for longer than 14 days. Our results showed that DT sap exhibited the effect of reducing blood GLU and that DT sap can be linked to mechanisms involved in blood GLU regulation at least partially by modulating IRS, Akt and AMPK signaling in liver and spleen tissues. From the LC-MS/MS analysis, DT sap included various bioactive chemicals, suggesting their potential contribution to the function of DT sap, warranting further studies. In future, studies on the effects of DT sap on diabetes and the identification of effective components are expected.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13124332/s1. Figure S1: Survival rates, body and organ weights by a single oral administration of Dendropanax trifidus (DT) sap, Figure S2: Morphology of liver tissue by Dendropanax trifidus (DT) sap administrations to mice, Figure S3: Morphology of kidney tissue by Dendropanax trifidus (DT) sap administrations to mice, Figure S4: Effects of Dendropanax trifidus (DT) sap administrations on the protein expression level of glucose transporters, Figure S5: Western blot Images of Figure 6, Table S1: Mouse number used in each experiment, Table S2: Summary of the concentration-dependent effects in vivo.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Figure 2 .of 18 Figure 2 .
2182Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days.(A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m. Nutrients 2021, 13, x FOR PEER REVIEW 6 Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days. (A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m.
Figure 3 .
3Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.
Figure 3 .
3Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.
Figure 4 .
4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.
Figure 5 .
5Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver;
Figure 4 .
4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.
Figure 4 .
4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice.
Figure 5 .Figure 5 .
55Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; (E-H) female liver; (I-L) male kidney; (M-P) female kidney. Representative pictures are shown. All pictures are provided in
Figure 6 .
6Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, ꞵ-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by ꞵ-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.
Figure 6 .
6Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, β-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by β-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by β-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by β-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by β-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by β-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.
Figure 7 .
7Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in
Figure 7 .
7Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in
Author Contributions: Conceptualization, H.-J.Y. and S.L.; methodology, A.L., Y.S., G.J.S., I.-H.C. and H.-J.Y.; software, Y.S.; validation, A.L., Y.S. and H.-J.Y.; formal analysis, A.L., Y.S.; investigation, A.L., Y.S. and N.S.; resources, S.L.; data curation, A.L. and Y.S.; writing-original draft preparation, H.-J.Y.; writing-review and editing, H.-J.Y. and E.K.; visualization, A.L. and H.-J.Y.; supervision, H.-J.Y.; project administration, H.-J.Y.; funding acquisition, H.-J.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3A04038150). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee of the University of Brain Education (Approval number: 2018-AE-01Y). Informed Consent Statement: N/A.
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular
Formula
Suggested
Compound
Synonyms
(Pubchem)
Suggested Structure
(Pubchem)
Annotation
MS/MS (Database
Search Score)
Molecular
Weight
Retention
Time (min)
Max. Area (Arbitrary
Unit, 1.0 × 10 8 )
Mean Group Area (Arbi-
trary Unit, 1.0 × 10 8 )
Mean [Log2 Fold
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryophyllene
oxide
(-)-Caryophyllene
oxide;
beta-Caryophyllene
oxide
Nutrients 2021, 13, x FOR PEER REVIEW
11 of 18
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular
Formula
Suggested
Compound
Synonyms
(Pubchem)
Suggested Structure
(Pubchem)
Annotation
MS/MS (Database
Search Score)
Molecular
Weight
Retention
Time (min)
Max. Area (Arbitrary
Unit, 1.0 × 10 8 )
Mean Group Area (Arbi-
trary Unit, 1.0 × 10 8 )
Mean [Log2 Fold
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22
Curcumene
Alpha-Curcumene;
2-Methyl-6-p-tolyl-2-
heptene
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular
Formula
Suggested
Compound
Synonyms
(Pubchem)
Suggested Structure
(Pubchem)
Annotation
MS/MS (Database
Search Score)
Molecular
Weight
Retention
Time (min)
Max. Area (Arbitrary
Unit, 1.0 × 10 8 )
Mean Group Area (Arbi-
trary Unit, 1.0 × 10 8 )
Mean [Log2 Fold
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-butyl-4-
hydroxybenzaldehyde
3,5-Di-t-butyl-4-
hydroxybenzaldehyde
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular
Formula
Suggested
Compound
Synonyms
(Pubchem)
Suggested Structure
(Pubchem)
Annotation
MS/MS (Database
Search Score)
Molecular
Weight
Retention
Time (min)
Max. Area (Arbitrary
Unit, 1.0 × 10 8 )
Mean Group Area (Arbi-
trary Unit, 1.0 × 10 8 )
Mean [Log2 Fold
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2
Capsidiol
(1R,3R,4S,4aR,6R)-6-
Isopropenyl-4,4a-
dimethyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphthalenediol
Table 1 .
1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular
Formula
Suggested
Compound
Synonyms
(Pubchem)
Suggested Structure
(Pubchem)
Annotation
MS/MS (Database
Search Score)
Molecular
Weight
Retention
Time (min)
Max. Area (Arbitrary
Unit, 1.0 × 10 8 )
Mean Group Area (Arbi-
trary Unit, 1.0 × 10 8 )
Mean [Log2 Fold
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-
Norandrostenedione
Estr-4-ene-3,17-dione;
19-Norandrost-4-ene-
3,17-dione;
Norandrostenedione
Formula Compound (Pubchem)
(Pubchem)
Search Score)
Weight Time (min)
Unit, 1.0 × 10 8 )
trary Unit, 1.0 × 10 8 )
Change (DT
Sap/AS Sap)]
Full
Partial
C15H24
(1E,5E,9E)-
1,5,9-Trime-
thyl-1,5,9-
cyclodode-
catriene
1,5,9-Trimethyl
cyclododeca-
triene
2
3
94.8 *
204.19
21.962
260.7
274.2
8.79
C15H24O
Caryo-
phyllene ox-
ide
(-)-Caryo-
phyllene oxide;
beta-Caryo-
phyllene oxide
3
3
93.6
220.18
22.201
136.9
171.4
6.51
C15H22 Curcumene
Alpha-Curcu-
mene;
2-Methyl-6-p-
tolyl-2-heptene
2
2
96.2 *
202.17
21.832
130.7
165.2
5.69
C15H22O2
3,5-di-tert-
butyl-4-hy-
droxyben-
zaldehyde
3,5-Di-t-butyl-
4-hydroxyben-
zaldehyde
2
3
85 *
234.16
20.281
63.91
50.75
3.59
C15H24O2 Capsidiol
(1R,3R,4S,4aR,6
R)-6-Isopro-
penyl-4,4a-di-
methyl-
1,2,3,4,4a,5,6,7-
octahydro-1,3-
naphtha-
lenediol
2
2
87.4 *
236.18
21.501
51.11
40.85
6.98
C18H24O2
19-Nor-
andros-
tenedione
Estr-4-ene-3,17-
dione;
19-Norandrost-
4-ene-3,17-di-
4
2
87.2
272.18
22.014
36.15
38.09
6.44
4
2
87.2
272.18
22.014
36.15
38.09
6.44
Estradiol
beta-Estradiol;
17beta-Estradiol
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one; Nor-
androstenedi-
one
Estradiol
beta-Estradiol;
17beta-Estra-
diol
C22H42O8
Bis [2-(2-
butoxyeth-
oxy)ethyl]
adipate
Dibutoxyethox-
yethyl adipate
2
1
68.8 *
434.29
21.792
36.42
36.30
1.49
C18H22O2 Trenbolone
17beta-
Trenbolone;
Trienbolone;
17-beta-Hy-
droxyestra-
4,9,11-trien-3-
one
3
3
84.1
270.16
21.313
39.92
28.62
6.43
C14H22O
o-tert-Oc-
tylphenol
t-octylphenol;
2-(2,4,4-trime-
thylpentan-2-
yl)phenol
2
1
80.6 *
206.17
21.866
31.28
27.76
7.79
C22H42O8
Bis [2-(2-
butoxyethoxy)ethyl]
adipate
Dibutoxyethoxyethyl
adipate
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one; Nor-
androstenedi-
one
Estradiol
beta-Estradiol;
17beta-Estra-
diol
C22H42O8
Bis [2-(2-
butoxyeth-
oxy)ethyl]
adipate
Dibutoxyethox-
yethyl adipate
2
1
68.8 *
434.29
21.792
36.42
36.30
1.49
C18H22O2 Trenbolone
17beta-
Trenbolone;
Trienbolone;
17-beta-Hy-
droxyestra-
4,9,11-trien-3-
one
3
3
84.1
270.16
21.313
39.92
28.62
6.43
C14H22O
o-tert-Oc-
tylphenol
t-octylphenol;
2-(2,4,4-trime-
thylpentan-2-
yl)phenol
2
1
80.6 *
206.17
21.866
31.28
27.76
7.79
5-Ethyl-3,8-5-Ethyl-3,8-di-
2
1
68.8 *
434.29
21.792
36.42
36.30
1.49
C18H22O2
Trenbolone
17beta-Trenbolone;
Trienbolone;
17-beta-
Hydroxyestra-4,9,11-
trien-3-one
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one; Nor-
androstenedi-
one
Estradiol
beta-Estradiol;
17beta-Estra-
diol
C22H42O8
Bis [2-(2-
butoxyeth-
oxy)ethyl]
adipate
Dibutoxyethox-
yethyl adipate
2
1
68.8 *
434.29
21.792
36.42
36.30
1.49
C18H22O2 Trenbolone
17beta-
Trenbolone;
Trienbolone;
17-beta-Hy-
droxyestra-
4,9,11-trien-3-
one
3
3
84.1
270.16
21.313
39.92
28.62
6.43
C14H22O
o-tert-Oc-
tylphenol
t-octylphenol;
2-(2,4,4-trime-
thylpentan-2-
yl)phenol
2
1
80.6 *
206.17
21.866
31.28
27.76
7.79
C14H18
5-Ethyl-3,8-
dimethyl-
1,7-dihydro-
azulene
5-Ethyl-3,8-di-
methyl-1,7-di-
hydroazulene
2
0
83.8 *
186.14
21.163
29.03
26.66
6.87
3
3
84.1
270.16
21.313
39.92
28.62
6.43
Table 1. Cont.Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum.hy-
droxybenzyla-
mine
2
0
78.7 *
263.22
21.865
4.091
3.238
7.20
C18H24O3
2-Hydroxy-
estradiol
2-OH-Estradiol;
2-hydroxy-es-
tradiol;
Estra-1,3,5(10)-
triene-
2,3,17beta-triol
2
2
81.6 *
288.17
21.268
22.30
17.78
4.08
C20H25NO
2
Dienogest
Dienogestrel;
Dinagest; En-
dometrion
3
0
63.1
311.19
21.276
22.67
16.19
6.85
2
2
81.6 *
288.17
21.268
22.30
17.78
4.08
C20H25NO2
Dienogest
Dienogestrel;
Dinagest;
Endometrion
C17H29NO methyla-
minome-
thyl)phenol
tyl-4-hy-
droxybenzyla-
mine
2
0
78.7 *
263.22
21.865
4.091
3.238
7.20
C18H24O3
2-Hydroxy-
estradiol
2-OH-Estradiol;
2-hydroxy-es-
tradiol;
Estra-1,3,5(10)-
triene-
2,3,17beta-triol
2
2
81.6 *
288.17
21.268
22.30
17.78
4.08
C20H25NO
2
Dienogest
Dienogestrel;
Dinagest; En-
dometrion
3
0
63.1
311.19
21.276
22.67
16.19
6.85
3
0
63.1
311.19
21.276
22.67
16.19
6.85
C20H28O2
Isotretinoin
13-cis-Retinoic acid;
3-cis-Vitamin A acid;
Accutane
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C20H28O2 Isotretinoin
13-cis-Retinoic
acid; 3-cis-Vita-
min A acid; Ac-
cutane
4
3
81.2
300.21
22.691
7.111
5.430
0.99
C18H32O2
Linoleic
acid
Linolic acid;
Telfairic acid;
cis,cis-Linoleic
acid
4
0
94.7 *
280.24
21.686
3.033
2.935
6.45
C15H20O2 Costunolide
(+)-costunolide;
Costunlide;
Costus lactone
2
2
94.8 *
232.16
21.149
5.355
2.636
4.68
4
3
81.2
300.21
22.691
7.111
5.430
0.99
C18H32O2
Linoleic acid
Linolic acid; Telfairic
acid; cis,cis-Linoleic
acid
Nutrients 2021, 13, x FOR PEER REVIEW
14 of 18
C20H28O2 Isotretinoin
13-cis-Retinoic
acid; 3-cis-Vita-
min A acid; Ac-
cutane
4
3
81.2
300.21
22.691
7.111
5.430
0.99
C18H32O2
Linoleic
acid
Linolic acid;
Telfairic acid;
cis,cis-Linoleic
acid
4
0
94.7 *
280.24
21.686
3.033
2.935
6.45
C15H20O2 Costunolide
(+)-costunolide;
Costunlide;
Costus lactone
2
2
94.8 *
232.16
21.149
5.355
2.636
4.68
4
0
94.7 *
280.24
21.686
3.033
2.935
6.45
C15H20O2
Costunolide
(+)-costunolide;
Costunlide; Costus
lactone
Nutrients 2021, 13, x FOR PEER REVIEW
14 of 18
C20H28O2 Isotretinoin
13-cis-Retinoic
acid; 3-cis-Vita-
min A acid; Ac-
cutane
4
3
81.2
300.21
22.691
7.111
5.430
0.99
C18H32O2
Linoleic
acid
Linolic acid;
Telfairic acid;
cis,cis-Linoleic
acid
4
0
94.7 *
280.24
21.686
3.033
2.935
6.45
C15H20O2 Costunolide
(+)-costunolide;
Costunlide;
Costus lactone
2
2
94.8 *
232.16
21.149
5.355
2.636
4.68
2
2
94.8 *
232.16
21.149
5.355
2.636
4.68
Nutrients 2021, 13, 4332
7 of 17
Acknowledgments:We thank Sung-Man Ahn for his assistance for registration of the plant to Ibaraki Nature Museum.Conflicts of Interest:The authors declare no conflict of interest.
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| [
"Citation: Lee, A.; Sugiura, Y.; Cho, I.-H.; Setou, N.; Koh, E.; Song, G.J.; Lee, S.; Yang, H.-J. In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract. Nutrients 2021, 13, 4332. https://doi.org/10.3390/ nu13124332 Academic Editors: Raffaella Canali and Fausta Natella"
] | [
"Ahreum Lee \nKorea Institute of Brain Science\n06022SeoulKorea\n",
"Yuki Sugiura \nDepartment of Biochemistry and Integrative Medical Biology\nSchool of Medicine\nKeio University\n160-8582TokyoJapan\n",
"Ik-Hyun Cho \nDepartment of Convergence Korean Medical Science\nCollege of Korean Medicine\nKyung Hee University\n02447SeoulKorea\n",
"Noriko Setou \nDepartment of Disaster Psychiatry\nFukushima Medical University\n960-1295FukushimaJapan\n",
"Eugene Koh \nTemasek Life Sciences Laboratories\n117604Singapore, Singapore\n",
"Gyun Jee Song \nDepartment of Medical Science\nCatholic Kwandong University College of Medicine\n25601GangneungKorea\n",
"Seungheun Lee \nKorea Institute of Brain Science\n06022SeoulKorea\n",
"Hyun-Jeong Yang \nKorea Institute of Brain Science\n06022SeoulKorea\n\nDepartment of Integrative Health Care\nUniversity of Brain Education\n31228CheonanKorea\n"
] | [
"Korea Institute of Brain Science\n06022SeoulKorea",
"Department of Biochemistry and Integrative Medical Biology\nSchool of Medicine\nKeio University\n160-8582TokyoJapan",
"Department of Convergence Korean Medical Science\nCollege of Korean Medicine\nKyung Hee University\n02447SeoulKorea",
"Department of Disaster Psychiatry\nFukushima Medical University\n960-1295FukushimaJapan",
"Temasek Life Sciences Laboratories\n117604Singapore, Singapore",
"Department of Medical Science\nCatholic Kwandong University College of Medicine\n25601GangneungKorea",
"Korea Institute of Brain Science\n06022SeoulKorea",
"Korea Institute of Brain Science\n06022SeoulKorea",
"Department of Integrative Health Care\nUniversity of Brain Education\n31228CheonanKorea"
] | [
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"T J Kim, ",
"D H Kim, ",
"K H Bae, ",
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"K Kim, ",
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"G J Song, ",
"H J Yang, ",
"E J Kim, ",
"M Jang, ",
"J H Choi, ",
"K S Park, ",
"I H Cho, ",
"M Miyajima, ",
"B Zhang, ",
"Y Sugiura, ",
"K Sonomura, ",
"M M Guerrini, ",
"Y Tsutsui, ",
"M Maruya, ",
"A Vogelzang, ",
"K Chamoto, ",
"K Honda, ",
"Y Li, ",
"T J Soos, ",
"X Li, ",
"J Wu, ",
"M Degennaro, ",
"X Sun, ",
"D R Kittman, ",
"M J Birnbaum, ",
"R D Polakiewicz, ",
"A Y Viana, ",
"H Sakoda, ",
"M Anai, ",
"M Fujishiro, ",
"H Ono, ",
"A Kushiyama, ",
"Y Fukushima, ",
"Y Sato, ",
"Y Oshida, ",
"Y Uchijima, ",
"E J Ley, ",
"M B Singer, ",
"M A Clond, ",
"T Johnson, ",
"M Bukur, ",
"R Chung, ",
"D R Margulies, ",
"A Salim, ",
"T A De Souza, ",
"D W De Souza, ",
"B S Siqueira, ",
"T Rentz, ",
"H R De Oliveria Emilio, ",
"S Grassiolli, ",
"A L Olson, ",
"J E Pessin, ",
"J Boucher, ",
"A Kleinridders, ",
"C R Kahn, ",
"J Li, ",
"L Zhong, ",
"F Wang, ",
"H Zhu, ",
"J Y Kim, ",
"K J Jo, ",
"O S Kim, ",
"B J Kim, ",
"D W Kang, ",
"K H Lee, ",
"H W Baik, ",
"M S Han, ",
"S K Lee, ",
"Y Li, ",
"J Huang, ",
"Y Yan, ",
"J Liang, ",
"Q Liang, ",
"Y Lu, ",
"L Zhao, ",
"H Li, ",
"T M D'eon, ",
"N H Rogers, ",
"Z S Stancheva, ",
"A S Greenberg, ",
"S P Tofovic, ",
"R K Dubey, ",
"E K Jackson, ",
"F Geddo, ",
"R Scandiffio, ",
"S Antoniotti, ",
"E Cottone, ",
"G Querio, ",
"M E Maffei, ",
"P Bovolin, ",
"M P Gallo, ",
"Pipenig, ",
"F Calzada, ",
"M Valdes, ",
"N Garcia-Hernandez, ",
"C Velazquez, ",
"E Barbosa, ",
"C Bustos-Brito, ",
"L Quijano, ",
"E Pina-Jimenez, ",
"J E Mendieta-Wejebe, ",
"M A Belury, ",
"A Mahon, ",
"S Banni, "
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] | [
"Phylogeny and Biogeography of Dendropanax (Araliaceae), an Amphi-Pacific Disjunct Genus between Tropical/Subtropical Asia and the Neotropics. R Li, J Wen, 10.1600/036364413X666606Syst. Bot. 38Li, R.; Wen, J. Phylogeny and Biogeography of Dendropanax (Araliaceae), an Amphi-Pacific Disjunct Genus between Tropi- cal/Subtropical Asia and the Neotropics. Syst. Bot. 2013, 38, 536-551. [CrossRef]",
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"Phylogeny and Biogeography of Dendropanax (Araliaceae), an Amphi-Pacific Disjunct Genus between Tropical/Subtropical Asia and the Neotropics",
"Phytogenetic relationships of the Dendropanax morbifera and D. trifidus based on PCR-RAPD",
"Plant geographical study for the plant of",
"Studies on the distribution of Dendropanax morbifera and component analysis of the golden lacquer",
"The major allergen of Dendropanax trifidus Makino",
"Taxonomic Appraisal of Dendropanax morbifera Leveille and D. trifidus (Thunb. ex Murray) Makino based on Morphological Characters",
"Foliar flavonoids of two sections of genus dendropanax in China",
"Phylogenetic Analysis of Dendropanax morbifera Using Nuclear Ribosomal DNA Internal Transcribed Spacer (ITS) Region Sequences",
"The allergens of Dendropanax trifidus Makino and Fatsia japonica Decne. et Planch. and evaluation of cross-reactions with other plants of the Araliaceae family",
"Antioxidant, cytotoxic, and antidiabetic activities of Dendropanax morbifera extract for production of health-oriented food materials",
"New Chemical Constituents from the Bark of Dendropanax morbifera Leveille and their Evaluation of Antioxidant Activities",
"Dendropanax morbifera Léveille extract ameliorates memory impairments and inflammatory responses in the hippocampus of streptozotocin-induced type 1 diabetic rats",
"Isolation and anticomplement activity of compounds from Dendropanax morbifera",
"Anti-Metastatic Effects of Plant Sap-Derived Extracellular Vesicles in a 3D Microfluidic Cancer Metastasis Model",
"Cytotoxic Effects of Plant Sap-Derived Extracellular Vesicles on Various Tumor Cell Types",
"Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic",
"Regulation of glucose transport by insulin: Traffic control of GLUT4",
"AMPK: Guardian of metabolism and mitochondrial homeostasis",
"Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPaseactivating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation",
"The effects of Korean red ginseng-derived components on oligodendrocyte lineage cells: Distinct facilitatory roles of the non-saponin and saponin fractions, and Rb1, in proliferation, differentiation and myelination",
"An Improved Dehydroepiandrosterone-Induced Rat Model of Polycystic Ovary Syndrome (PCOS): Post-pubertal Improve PCOS's Features",
"Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior",
"Protein Kinase C θ Inhibits Insulin Signaling by Phosphorylating IRS1 at Ser1101",
"Role of hepatic AMPK activation in glucose metabolism and dexamethasone-induced regulation of AMPK expression",
"Long-term effect of trauma splenectomy on blood glucose",
"Splenic participation in glycemic homeostasis in obese and non-obese male rats",
"Structure, function, and regulation of the mammalian facilitative glucose transporter gene family",
"Insulin receptor signaling in normal and insulin-resistant states",
"Dissecting the role of AMP-activated protein kinase in human diseases",
"Parenteral 17beta-estradiol decreases fasting blood glucose levels in non-obese mice with short-term ovariectomy",
"Preventative effects of resveratrol and estradiol on streptozotocin-induced diabetes in ovariectomized mice and the related mechanisms",
"Estradiol and the estradiol metabolite, 2-hydroxyestradiol, activate AMP-activated protein kinase in C2C12 myotubes",
"2-Hydroxyestradiol attenuates the development of obesity, the metabolic syndrome, and vascular and renal dysfunction in obese ZSF1 rats",
"R))-FL, a Fluid Extract of Black Pepper (Piper Nigrum L.) with a High Standardized Content of Trans-beta-Caryophyllene, Reduces Lipid Accumulation in 3T3-L1 Preadipocytes and Improves Glucose Uptake in C2C12 Myotubes",
"Antihyperglycemic activity of the leaves from annona diversifolia Safford. and farnesol on normal and alloxan-induced diabetic mice",
"The conjugated linoleic acid (CLA) isomer, t10c12-CLA, is inversely associated with changes in body weight and serum leptin in subjects with type 2 diabetes mellitus"
] | [
"Syst. Bot",
"Korean J. Genet",
"Cheju. Korean J. Plant Taxon",
"Korean J. Biotechnol. Bioeng",
"Contact Dermat",
"Korean J. Plant Taxon",
"Japan, and Korea. For. Sci. Technol",
"J. Life Sci",
"Contact Dermat",
"The Medicinal Plants of Korea",
"Afr. J. Biotechnol",
"Molecules",
"Mol. Cell. Toxicol",
"J. Ethnopharmacol",
"J. Funct. Biomater",
"J. Funct. Biomater",
"Am. J. Physiol. Endocrinol. Metab",
"Nat. Rev. Mol. Cell Biol",
"Nat. Rev. Mol. Cell Biol",
"J. Biol. Chem",
"J. Ginseng Res",
"Front. Endocrinol",
"Nat. Immunol",
"J. Biol. Chem",
"Diabetes Res. Clin. Pract",
"J. Surg. Res",
"Obes. Res. Clin. Pract",
"Annu. Rev. Nutr",
"Cold Spring Harb. Perspect. Biol",
"Acta Pharm. Sin. B",
"Life Sci",
"PLoS ONE",
"Obesity",
"J. Pharmacol. Exp. Ther",
"Nutrients",
"Phcog. Mag",
"J. Nutr"
] | [
"\nFigure 2 .of 18 Figure 2 .\n2182Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days.(A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m. Nutrients 2021, 13, x FOR PEER REVIEW 6 Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days. (A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m.",
"\nFigure 3 .\n3Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.",
"\nFigure 3 .\n3Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.",
"\nFigure 4 .\n4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.",
"\nFigure 5 .\n5Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver;",
"\nFigure 4 .\n4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.",
"\nFigure 4 .\n4Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice.",
"\nFigure 5 .Figure 5 .\n55Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; (E-H) female liver; (I-L) male kidney; (M-P) female kidney. Representative pictures are shown. All pictures are provided in",
"\nFigure 6 .\n6Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, ꞵ-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by ꞵ-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.",
"\nFigure 6 .\n6Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, β-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by β-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by β-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by β-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by β-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by β-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.",
"\nFigure 7 .\n7Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in",
"\nFigure 7 .\n7Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in",
"\n\nAuthor Contributions: Conceptualization, H.-J.Y. and S.L.; methodology, A.L., Y.S., G.J.S., I.-H.C. and H.-J.Y.; software, Y.S.; validation, A.L., Y.S. and H.-J.Y.; formal analysis, A.L., Y.S.; investigation, A.L., Y.S. and N.S.; resources, S.L.; data curation, A.L. and Y.S.; writing-original draft preparation, H.-J.Y.; writing-review and editing, H.-J.Y. and E.K.; visualization, A.L. and H.-J.Y.; supervision, H.-J.Y.; project administration, H.-J.Y.; funding acquisition, H.-J.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3A04038150). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee of the University of Brain Education (Approval number: 2018-AE-01Y). Informed Consent Statement: N/A.",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \nCaryophyllene \noxide \n\n(-)-Caryophyllene \noxide; \nbeta-Caryophyllene \noxide \n\nNutrients 2021, 13, x FOR PEER REVIEW \n11 of 18 \n\n",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 \nCurcumene \n\nAlpha-Curcumene; \n2-Methyl-6-p-tolyl-2-\nheptene \n\n",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n3,5-di-tert-butyl-4-\nhydroxybenzaldehyde \n\n3,5-Di-t-butyl-4-\nhydroxybenzaldehyde \n\n",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 \nCapsidiol \n\n(1R,3R,4S,4aR,6R)-6-\nIsopropenyl-4,4a-\ndimethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphthalenediol \n\n",
"\nTable 1 .\n1Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-\nNorandrostenedione \n\nEstr-4-ene-3,17-dione; \n19-Norandrost-4-ene-\n3,17-dione; \nNorandrostenedione \n\nFormula Compound (Pubchem) \n(Pubchem) \nSearch Score) \nWeight Time (min) \nUnit, 1.0 × 10 8 ) \ntrary Unit, 1.0 × 10 8 ) \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\nEstradiol \nbeta-Estradiol; \n17beta-Estradiol \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyethoxy)ethyl] \nadipate \n\nDibutoxyethoxyethyl \nadipate \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\n5-Ethyl-3,8-5-Ethyl-3,8-di-\n\n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 \nTrenbolone \n\n17beta-Trenbolone; \nTrienbolone; \n17-beta-\nHydroxyestra-4,9,11-\ntrien-3-one \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\nC14H18 \n\n5-Ethyl-3,8-\ndimethyl-\n1,7-dihydro-\nazulene \n\n5-Ethyl-3,8-di-\nmethyl-1,7-di-\nhydroazulene \n\n2 \n0 \n83.8 * \n186.14 \n21.163 \n29.03 \n26.66 \n6.87 \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n",
"\n\nTable 1. Cont.Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum.hy-\ndroxybenzyla-\nmine \n\n2 \n0 \n78.7 * \n263.22 \n21.865 \n4.091 \n3.238 \n7.20 \n\nC18H24O3 \n2-Hydroxy-\nestradiol \n\n2-OH-Estradiol; \n2-hydroxy-es-\ntradiol; \nEstra-1,3,5(10)-\ntriene-\n2,3,17beta-triol \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \n\nC20H25NO \n2 \nDienogest \n\nDienogestrel; \nDinagest; En-\ndometrion \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \nC20H25NO2 \nDienogest \n\nDienogestrel; \nDinagest; \nEndometrion \n\nC17H29NO methyla-\nminome-\nthyl)phenol \n\ntyl-4-hy-\ndroxybenzyla-\nmine \n\n2 \n0 \n78.7 * \n263.22 \n21.865 \n4.091 \n3.238 \n7.20 \n\nC18H24O3 \n2-Hydroxy-\nestradiol \n\n2-OH-Estradiol; \n2-hydroxy-es-\ntradiol; \nEstra-1,3,5(10)-\ntriene-\n2,3,17beta-triol \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \n\nC20H25NO \n2 \nDienogest \n\nDienogestrel; \nDinagest; En-\ndometrion \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\nC20H28O2 \nIsotretinoin \n\n13-cis-Retinoic acid; \n3-cis-Vitamin A acid; \nAccutane \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic acid \n\nLinolic acid; Telfairic \nacid; cis,cis-Linoleic \nacid \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 \nCostunolide \n\n(+)-costunolide; \nCostunlide; Costus \nlactone \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\nNutrients 2021, 13, 4332 \n\n7 of 17 \n\n"
] | [
"Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days.(A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m. Nutrients 2021, 13, x FOR PEER REVIEW 6 Effects on survival rate and body weight by Dendropanax trifidus (DT) sap daily administration in mice for two weeks. DT sap of the indicated concentrations was given to 9-week-old ICR mice by oral gavage every day for 14 days. (A-C) Survival rate at day 1, 3, 7 and 14 since the initial administration with respect to concentration of 0, 0.5, 1 and 2.5 mg/g (DT sap weight/body weight). (D-F) Body weight at day 0 (before administration), 1, 3, 7 and 14 with respect to concentration. (A,D) Total; (B,E) female; (C,F) male. N = 4-5 mice per group. Bars indicate mean ± s.e.m.",
"Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.",
"Effects on mouse organ weights by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Organ weights at day 14 depending on the concentration are shown: (A,AI,AII) liver; (B,BI,BII) spleen; (C,CI,CII) kidney; (D,DI,DII) lung; (E,EI,EII) heart; (F,FI,FII) adrenal gland. (A-F) Total; (AI-FI) female; (AII-FII) male. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analyses are indicated with asterisks. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bars indicate mean ± s.e.m.",
"Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.",
"Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver;",
"Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice. DT sap of the indicated concentrations was given to 9-week-old ICR mice (N = 4-5 per each group) by oral gavage every day for 14 days. Blood chemistry of following items was investigated: (A) GLU; (B) GPT; (C) GOT; (D) TP; (E) BUN; (F) ALP; (G) LDH; (H) TG; (I) CHOL; (J) LDL; (K) HDL. Data of total (A-K), female (AI-KI), male (AII-KII) mice are shown. Indicated p values are derived from a group difference in one way ANOVA. The significance of post hoc analysis is indicated with asterisks: **, p < 0.01. Bars indicate mean ± s.e.m.",
"Effects on blood chemistry by Dendropanax trifidus (DT) sap administrations in mice.",
"Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; Effects on histopathological morphology of liver and kidney by Dendropanax trifidus (DT) sap administration in mice. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice (N = 5 per each group) by oral gavage every day for 14 days. H&E staining was performed for the following tissues: (A-D) male liver; (E-H) female liver; (I-L) male kidney; (M-P) female kidney. Representative pictures are shown. All pictures are provided in",
"Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, ꞵ-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by ꞵ-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by ꞵ-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by ꞵ-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.",
"Effects on AMPK-mediated signaling after two weeks of daily oral administration of Dendropanax trifidus (DT) sap. DT sap of the indicated concentrations (0, 0.5, 1, 2.5 mg/g for DT sap weight/body weight) was given to 9-week-old ICR mice by oral gavage every day for 14 days.Western blot analysis on livers (A,C-H) or spleens (B,I-L) of the treated female mice. Blots of liver (A) or spleen (B) samples were incubated with antibodies to pIRS-1(ser1101), IRS-1, pAkt, Akt, pAMPK, AMPK, β-Actin, as indicated. (C) Relative value of pIRS-1 intensity normalized by general IRS-1 (p = 0.014, one way ANOVA; * p = 0.038 (1 mg/g), * p = 0.030 (2.5 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method). (D) Relative value of IRS-1 intensity normalized by β-Actin. p = 0.548, one way ANOVA. (E) Relative value of pAkt intensity normalized by general Akt. p = 0.157, one way ANOVA. (F) Relative value of Akt intensity normalized by β-Actin. p = 0.143, one way ANOVA. (G) Relative value of pAMPK intensity normalized by general AMPK. ## p = 0.007, compared with 0 mg/g, Student's t-test. (H) Relative value of AMPK intensity normalized by β-Actin. p = 0.001, one way ANOVA; * p = 0.016 (0.5 mg/g), * p = 0.001 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (I) Relative value of pAkt intensity normalized by general Akt. p = 0.034, one way ANOVA; * p = 0.039 (1 mg/g), compared with 0 mg/g, post hoc Holm-Sidak method. (J) Relative value of Akt intensity normalized by β-Actin. p = 0.262, one way ANOVA. (K) Relative value of pAMPK intensity normalized by general AMPK. p = 0.049, one way ANOVA. (L) Relative value of AMPK intensity normalized by β-Actin. p = 0.476, one way ANOVA. N = 3 (female) mice per group. 20 ug/lane. Bars indicate mean ± s.e.m. The significance of one-way ANOVA post-hoc tests is indicated with asterisks: *, p < 0.05.",
"Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in",
"Dendropanax trifidus (DT) sap-derived chemicals identified by LC-MS/MS. Dots indicate 1080 chemicals identified by LC-MS/MS. Among them, 1041 chemicals were matched in more than one of seven annotation sources. Two batches of DT sap (indicated as DT sap 1, DT sap 2) and Acer saccharum (AS) sap were used. (A,B) Group area, which indicates area under curve of the intensity peak, of DT sap 1 and 2 are plotted. The plot region within the red dotted box is enlarged in (B). (C) The intensity of each chemical was compared between DT sap and AS sap to provide a fold change of DT sap/AS sap. X and Y axis indicate Log2 [fold change (DT sap 2/AS sap)] and Log2 [fold change (DT sap 1/AS sap)]. Chemical intensity is higher in DT sap than in AS sap in the shaded field (354 chemicals) and vice versa in the bright field (726 chemicals) in the graph. Representative chemicals in the shaded field, with high group area, are listed in",
"Author Contributions: Conceptualization, H.-J.Y. and S.L.; methodology, A.L., Y.S., G.J.S., I.-H.C. and H.-J.Y.; software, Y.S.; validation, A.L., Y.S. and H.-J.Y.; formal analysis, A.L., Y.S.; investigation, A.L., Y.S. and N.S.; resources, S.L.; data curation, A.L. and Y.S.; writing-original draft preparation, H.-J.Y.; writing-review and editing, H.-J.Y. and E.K.; visualization, A.L. and H.-J.Y.; supervision, H.-J.Y.; project administration, H.-J.Y.; funding acquisition, H.-J.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3A04038150). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee of the University of Brain Education (Approval number: 2018-AE-01Y). Informed Consent Statement: N/A.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Representative chemical components of Dendropanax trifidus sap ethanol extracts identified by Liquid Chromatography-tandem Mass Spectrometry.",
"Table 1. Cont.Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum. Representative chemicals are listed. Annotation sources: Predicted Compositions; mzCloud Search; mzVault Search; Metabolika Search; ChemSpider Search; BioCyc Search; MassList Search. MS/MS (database search score): mzCloud Best Match; *, mzCloud Best Sim. Match. Abbreviations: DT, Dendropanax trifidus; AS, Acer saccharum."
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"3-cyclohexyl-N,N- dimethyl-3- phenylpropan-1- amine",
"C17H29NO 2,6-Di-tert- butyl-4-(di- methyla- minome- thyl)phenol N,N-dimethyl- 3,5-di-tert-bu- tyl-4-"
] | [
"Dendropanax is a genus of flowering plants in the family Araliaceae [1]. Although both Dendropanax morbiferus (DM) and Dendropanax trifidus (DT), which belong to this family, are native to eastern Asia, each specific habitat is distinct; the southern coast of the Korean Peninsula for DM [2][3][4], and Japan for DT [2,5]. However, according to previous PCR-Random Amplified Polymorphic DNA (RAPD) analysis of DM and DT, their genetic differences were not clear [2]. In a study of the fruit, sap color and leaf external morphologies of DM and DT, a clear distinction between the two was also not possible [6].",
"Eight-week-old mice of CrljOri:CD1 (ICR) lines were purchased from ORIENT BIO Inc. (Seongnam, Korea). After 1 week of rest, 9-week-old mice were used for the experiments. Mice were kept on a 12-h light-dark cycle with light period of 8:00-20:00 in a standard specific pathogen-free environment. Mice were provided with regular chow and water ad libitum. Each animal was anaesthetized by carbon dioxide inhalation before sampling. All experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by the University of Brain Education's Animal Care and Use Committee (Approval number: 2018-AE-01Y). Mouse number used in each experiment is indicated in Table S1.",
"Dendropanax trifidus (Thunb.) Makino ex H.Hara was collected from the forest in Simasi, Mieken, Japan and registered at Ibaraki Nature Museum: voucher number INM-2-212778. DT sap was dissolved in 100% EtOH, freeze dried and stored at −20 °C. The freezedried final product with viscosity was dissolved in EtOH to make a stock solution of 1.4 or 0.5 g/mL and stored at −20 °C. The extract is referred as DT sap in this paper.",
"To determine the optimal concentration of DT sap for in vivo administration, shortterm effects of DT sap on survival as well as body and organ weights were examined when administered in a single dose of various concentrations (0, 1.2, 2.5, 4.8, 10.3, 21.3 mg/g, DT sap weight/body weight) to 9-week-old ICR mice by oral gavage. After the single injection, the mice were observed for several hours, and body weight was measured once per day for an initial 3 days. At day 20, body weight was measured again, and mice were sacrificed, thereafter organ (liver, spleen, kidney, lung, heart = and adrenal gland) weights were measured separately ( Figure S1). We further examined the effects of multiple injections with several lower concentrations (0, 0.5, 1 and 2.5 mg/g, DT sap weight/body weight) every day for 14 days to 9-week-old ICR mice by oral gavage. Mouse body weight was measured at day 0, 1, 3, 7 and 14, and the mice were sacrificed at day 14 to obtain organs for weight measurements, blood chemistry, Western blots and histochemistry. Before the sacrifice, all animals were fasted overnight ( Figure 1). Figure 1. Experimental design. Experimental procedures are briefly described in the scheme. Dendropanax Trifidus sap was extracted in 100% EtOH, freeze-dried and maintained at −20 °C. Its EtOH-dissolved solution was subjected to in vivo mouse studies. A single oral administration was conducted to limit the dose range of DT sap ( Figure S1). Based on this information, daily oral gavage of three concentrations was performed for 14 days. Survival rate and body weights were measured at day 0, 1, 3, 7 and 14 ( Figure 2). Organ weight measurement (Figure 3), blood chemistry ( Figure 4), histochemistry ( Figure 5) and Western blot analysis ( Figure 6) were performed on the samples of day 14. In order to analyze the chemical components of DT sap, it was subjected to non-targeted metabolome analysis ( Figure 7, Table 1). Figure 1. Experimental design. Experimental procedures are briefly described in the scheme. Dendropanax Trifidus sap was extracted in 100% EtOH, freeze-dried and maintained at −20 • C. Its EtOH-dissolved solution was subjected to in vivo mouse studies. A single oral administration was conducted to limit the dose range of DT sap ( Figure S1). Based on this information, daily oral gavage of three concentrations was performed for 14 days. Survival rate and body weights were measured at day 0, 1, 3, 7 and 14 ( Figure 2). Organ weight measurement (Figure 3), blood chemistry ( Figure 4), histochemistry ( Figure 5) and Western blot analysis ( Figure 6) were performed on the samples of day 14. In order to analyze the chemical components of DT sap, it was subjected to non-targeted metabolome analysis (Figure 7, Table 1). ",
"Trienbolone; 17-beta-Hydroxyestra-4,9,11-trien-3one ",
"Therefore, for the multiple administration regime, the highest concentration used among the tested concentrations was set lower than 4.8 mg/g (i.e., 2.5 mg/g). The indicated concentrations (0, 0.5, 1 and 2.5 mg/g) were applied every day by oral gavage for 14 days (Figure 2, N = 4 females and 5 males/concentration). At day 14 (i.e., end of the experimental period), the survival ratio was 100% for all the concentrations except the highest concentration (50% for female and 75% male at 2.5 mg/g, Figure 2A-C). There were no significant changes in mouse body weight by concentration and time ( Figure 2D-F). To examine the effects of the concentration on tissue weight in a multiple administration regime, mouse tissues were collected and measured at day 14 ( Figure 3). Using a oneway ANOVA against total organ weights, significant differences were found in the liver (p = 0.002, Figure 3A), spleen (p = 0.007, Figure 3B), kidney (p = 0.003, Figure 3C), heart (p = 0.001, Figure 3E), and with respect to concentration. By post hoc analysis (Holm-Sidak method), significant differences were found to be derived from one concentration (i.e., the highest concentration, 2.5 mg/g, p = 0.002 for kidney, 0.031 for heart, <0.001 for liver, 0.006 for spleen). There were no significant differences in the tissue weights at other concentrations. In addition, increasing DT sap concentration did not affect the lung and adrenal tissue weights. Analyzing by gender, the weights of female liver (one way ANOVA, p = 0.025, Figure 3AI), spleen (one way ANOVA, p = 0.043, Figure 3BI), kidney (one way ANOVA, p = 0.003, Figure 3CI), heart (Kruskal-Wallis one way ANOVA on Ranks, p = 0.016, Figure 3EI) were significantly changed and post hoc analysis revealed that these were due to the highest concentration of 2.5 mg/g (Holm-Sidak method, p = 0.039 for liver, p = 0.058 for spleen, p = 0.012 for kidney; Dunn's Method, p = 0.035 for heart). There were no significant weight differences in the other concentrations. In female lung and adrenal glands, no significant weight differences were found in all the concentrations. Figure S2 and Figure 3. Scale bar, 50 µm.",
"DT sap extracts of the indicated concentrations were given to ICR mice for 14 days by daily oral gavage. Whole blood was withdrawn from the heart and maintained at room temperature (RT) for 30 min to clot, then centrifuged at 2000× g for 10 min in a refrigerated centrifuge, and the resulting supernatant (serum) was subjected to blood chemistry analysis. In the serum, the following parameters were measured by using a BS-200 Chemistry Analyzer (Mindray, China): GLU, glutamic pyruvate transaminase (GPT), glutamic oxalacetic transaminase (GOT), total protein (TP), blood urea nitrogen (BUN), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triglyceride (TG), cholesterol (CHOL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL).",
"Western blot was performed as previously described [21]. Briefly, tissues were lysed in RIPA lysis buffer (WSE-7420, ATTO, DAWINBIO Inc, Hanam, Korea), and centrifuged at 15,000 RPM for 15 min. The supernatant was quantified by Bradford assay, diluted with 5 × Sample buffer, boiled and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation. Proteins were transferred onto PVDF membranes and blocked with EZBlock Chemi (AE-1475, ATTO, Tokyo, Japan) for 1h at RT. Membranes were incubated with the primary antibody for overnight at 4 • C, washed and incubated with secondary antibody for 1h at RT, and washed. Proteins were visualized by Super Signal West Pico PLUS Chemiluminescent Substrate (34580, ThermoFisher Scientific, Waltham, MA, USA) and captured by Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). Images were analyzed by using Image J. For blotting, rabbit antibodies to the following antigens were purchased from Cell Signaling Technology (Danvers, MA, USA): phospho-IRS-1 (Ser1101) (2385), IRS-1 (2382), phospho-AMPKa (2531), AMPKa (2532), phosphor-Akt (4060) and Akt (9272). Rabbit antibodies to beta actin, Glut1 (ab652) and Glut4 (ab654) were purchased from Abcam (Cambridge, UK). For secondary antibody, horseradish peroxidase conjugate goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA) was used.",
"with respect to increasing DT sap concentration in liver tissues ( Figure 6A,C, p = 0.014, one way ANOVA). We noted that there was a non-significant slight increase in IRS/ꞵ-Actin levels with respect to increasing DT sap concentration ( Figure 6D). As Akt activation is known to act downstream of insulin signaling, we also measured Akt and pAkt levels. The levels of pAkt/Akt appear to alter at 0.5, 1 mg/g dose, without significant differences ( Figure 6E). Akt/ꞵ-Actin was not changed at 0.5 mg/g and decreased at 1 or 2.5 mg/g, without significant changes ( Figure 6F). ",
"To assess the histopathological changes of the kidney and liver after DT sap extract administration, the ICR mice were anesthetized with CO 2 and then perfused intracardially with saline and cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The kidney and liver were removed, post-fixed, washed, dehydrated and embedded with melted paraffin wax [22]. Paraffin blocks were cut into 5-µm-thick sections using a Leica RM 2155 microtome (Leica Biosystems, Wetzlar, Germany). The paraffin sections were stained with hematoxylin-eosin (H&E) dye as previously described [22]. Images of stained sections were visualized and captured using a DP70 digital light microscope system (Olympus, Tokyo, Japan). The kidney and liver morphology were observed under 40x magnification. Table 1.",
"The results of our toxicity study on survival ratio, body weight, organ weight, blood chemistry and histochemistry show that DT sap concentrations below 0.5 mg/mL were safe for long-term administration (Table S2). We also found that blood GLU levels were gradually reduced with increased dosage of DT sap in female mice, and that the AMPKmediated signaling was altered by DT sap administration in female mouse liver and spleen tissues. In addition, bioactive chemicals were identified from DT sap by LC-MS/MS. While a significant dose-dependent reduction in blood glucose was observed in female mice, it was not observed in male mice ( Figure 4AI,AII). However, 0.5 mg/g DT sap administration also reduced the mean value of blood glucose in males compared to the control, although it was not statistically significant, implying a possible gender difference in dose-response. Therefore, we cannot exclude the possibility of hypoglycemic effects of DT sap on male mice as well, warranting further studies. Reduction in blood GLU with corresponding increases of DT sap concentration ( Figure 4AI) may be related with the activation of the insulin signaling pathway [29] or AMPK signaling pathway [30]. Here, phosphorylation of IRS-1 and Akt for the insulin signaling pathway and phosphorylation of AMPK for the AMPK signaling pathway were investigated. Phosphorylation of IRS-1 (Ser1101) inhibits insulin signaling [24], therefore the reduction of pIRS-1(Ser1101)/IRS-1 by DT sap should result in a corresponding decrease in blood GLU, which is observed here ( Figure 6C). The level of pAkt/Akt, a downstream signaling factor of IRS-1, appears to increase according to DT sap concentration ( Figure 6I). These results suggest that DT sap appears to affect blood GLU regulation via activation of the insulin signaling pathway. Previous findings showed that the activation of AMPK suppresses gluconeogenesis [25]. In our study, pAMPK/AMPK levels were significantly increased at low DT sap concentrations (0.5 mg/g) in liver tissues ( Figure 6G), and exhibited a significant increase in conjunction with increasing DT sap concentration in spleen tissues ( Figure 6K). Therefore, reduced GLU production may also contribute to blood GLU reduction ( Figure 4AI).",
"We observed that pAMPK/AMPK, a key metabolic regulator, exhibited a common tendency of increase with respect to increasing DT sap concentrations in both liver and spleen tissues ( Figure 6G,K), In contrast, pAkt/Akt levels responded differently in liver and spleen tissues. While pAkt/Akt levels did not show a consistent change in liver tissue ( Figure 6E), the pAkt/Akt levels in the spleen tissue showed an increase, with respect to Table 1.",
"Ethanol extracts of DT sap and Acer saccharum (AS) sap (Alleghanys Maple Farms Inc., Saint-Pacome, QC, Canada) were analyzed as previously described [23]. Briefly, for non-targeted analysis, metabolome data obtained by orbitrap-type MS (Q-Exactive Focus, Thermo Fisher Scientific, San Jose, CA, USA) connected to a HPLC (Ultimate3000 system, Thermo Fisher Scientific) with the discovery HS F5-3 column or an IC (ICS-5000+, Thermo Fisher Scientific) with the IonPac AS11-HC, 4-µm particle size column were analyzed. A Compound Discoverer 2.0 (Thermo Fisher Scientific) was used for the non-targeted metabolomics workflow. In brief, this software first aligned the total ion chromatograms of different samples along the retention time. Then, the detected features with an intensity of no less than 100,000 and an S/N larger than five in each set of data were extracted and merged into components. The resulting compounds were identified by both (i) formula prediction based on accurate m/z value and isotope peak patterns and (ii) MS/MS structural validation. Moreover, formula predicted signals were assigned into candidate compounds by database search (Chemspider database; http://www.chemspider.com/, accessed on 19 December 2019).",
"Statistical analyses were performed using one way analysis of variance (ANOVA) with post hoc tests (Holm-Sidak method or Dunn's method), Kruskal-Wallis one way ANOVA on Ranks and Student's t-test.",
"For assessing the in vivo toxicity of DT sap, a single administration regime was performed first in 9-week-old ICR mice by oral gavage, in order to limit the dose range. For the single administration ( Figure S1), DT sap of a wide range of concentrations (0, 1.2, 2.5, 4.8, 10.3 and 21.3 mg/g for DT sap weight/mouse body weight) were separately injected. Mice injected with the highest concentration of 21.3 mg/g died at day 2; however, mice administered with concentrations lower than 21.3 mg/g survived till the end of the experimental period (i.e., day 20 after the administration) ( Figure S1A). There were no significant changes in body weight for the initial three days after the injection ( Figure S1B). However, in the measurement at day 20 post-administration, the concentration of 10.3 mg/g increased total body weight and spleen weight compared to other concentrations ( Figure S1C,E). In the concentrations of ≤4.8 mg/g, total body weight and organ weights were not changed by the injection (Figure S1B-I).",
"Therefore, for the multiple administration regime, the highest concentration used among the tested concentrations was set lower than 4.8 mg/g (i.e., 2.5 mg/g). The indicated concentrations (0, 0.5, 1 and 2.5 mg/g) were applied every day by oral gavage for 14 days (Figure 2, N = 4 females and 5 males/concentration). At day 14 (i.e., end of the experimental period), the survival ratio was 100% for all the concentrations except the highest concentration (50% for female and 75% male at 2.5 mg/g, Figure 2A-C). There were no significant changes in mouse body weight by concentration and time ( Figure 2D-F).",
"To examine the effects of the concentration on tissue weight in a multiple administration regime, mouse tissues were collected and measured at day 14 ( Figure 3). Using a one-way ANOVA against total organ weights, significant differences were found in the liver (p = 0.002, Figure 3A), spleen (p = 0.007, Figure 3B), kidney (p = 0.003, Figure 3C), heart (p = 0.001, Figure 3E), and with respect to concentration. By post hoc analysis (Holm-Sidak method), significant differences were found to be derived from one concentration (i.e., the highest concentration, 2.5 mg/g, p = 0.002 for kidney, 0.031 for heart, <0.001 for liver, 0.006 for spleen). There were no significant differences in the tissue weights at other concentrations. In addition, increasing DT sap concentration did not affect the lung and adrenal tissue weights.",
"Analyzing by gender, the weights of female liver (one way ANOVA, p = 0.025, Figure 3AI), spleen (one way ANOVA, p = 0.043, Figure 3BI), kidney (one way ANOVA, p = 0.003, Figure 3CI), heart (Kruskal-Wallis one way ANOVA on Ranks, p = 0.016, Figure 3EI) were significantly changed and post hoc analysis revealed that these were due to the highest concentration of 2.5 mg/g (Holm-Sidak method, p = 0.039 for liver, p = 0.058 for spleen, p = 0.012 for kidney; Dunn's Method, p = 0.035 for heart). There were no significant weight differences in the other concentrations. In female lung and adrenal glands, no significant weight differences were found in all the concentrations.",
"In male mouse tissues, one-way ANOVA analysis showed significant differences in liver (p = 0.004, Figure 3AII), spleen (p = 0.049, Figure 3BII), kidney (p < 0.001, Figure 3CII), heart (p = 0.041, Figure 3EII) by DT sap concentrations, and subsequent post hoc analysis (Holm-Sidak method) showed that the difference was mainly derived from the 2.5 mg/g concentration, although differences were also found at 1 mg/g in kidney (liver, p = 0.003 (0 vs. 2.5 mg/g); kidney, p < 0.001(0 vs. 2.5 mg/g), p = 0.021 (0 vs. 1 mg/g); heart, p = 0.034 (0.5 vs. 2.5 mg/g)). There were no significant differences in these tissue weights in other concentrations. In male lung and adrenal glands, no differences were found in all the concentrations.",
"Next, we investigated the effect of DT sap concentration on blood chemistry after 14 days of daily oral administration (Figure 4). In total mouse blood chemistry analysis, there was a tendency of reduction in blood GLU with respect to increasing DT sap concentration (One way ANOVA, p = 0.060, Figure 4A). Other components in the blood chemistry did not show this tendency. In addition, when the data was individually analyzed by t-test in comparison with vehicle (0 mg/g)-injected mice, mice treated with 0.5 mg/g and 1 mg/g exhibited a tendency of reduction (p = 0.0628) and a significant reduction (p = 0.005), respectively. In female mice, there was a significant reduction in GLU corresponding with the increase of DT sap concentrations (One way ANOVA, p = 0.031, Figure 4AI). In addition, significant changes in GPT and ALP were observed (p = 0.045 and 0.004, respectively) and subsequent post hoc analysis showed a significant increase in ALP at 1 mg/g (p = 0.006, Figure 4FI), but not in GPT ( Figure 4BI). There were no significant changes in the other parameters (GOT, TP, BUN, LDH, TG, CHOL, LDL and HDL) with concentration ( Figure 4CI-KI). In addition, LDL showed a significant reduction in 1 mg/g compared to vehicle control in t-test (p = 0.01, Figure 4JI). Unlike female mice, no significant correlation was found in GLU in males (Kruskal-Wallis one way ANOVA on Ranks, p = 0.093), although GLU levels at 0.5 mg/g DT sap were reduced about half compared to the vehicle control ( Figure 4AII). No significant changes were found in the other parameters in males ( Figure 4BII~KII). In hematoxylin and eosin staining of mouse liver and kidney after 14 days of injection, no specific histopathological changes were observed by DT sap administration in both male and female mice ( Figures 5, S2 and S3, N = 5 per each concentration). To summarize, (1) DT sap did not affect mouse survival, body weight, tissue weight, blood chemistry and histochemistry at the concentration of ≤0.5 mg/g (Table S2), therefore the concentration of ≤0.5 mg/g is recommended for long term stable administration in mice. (2) A significant reduction was found in blood GLU with respect to increasing DT sap concentration in female mice ( Figure 4AI), suggesting the potential effects of DT sap on GLU metabolism.",
"To further investigate the effects of DT sap on GLU metabolism in female mice, Western blots were performed in liver tissues for several key signaling factors known to be involved in the regulation of blood GLU (Figures 6, S4 and S5). Previous studies showed that the phosphorylation of IRS-1 at Ser 1101 results in an inhibition of insulin signaling in the cell [24]. We observed that pIRS-1(Ser1101)/IRS-1 levels were significantly reduced with respect to increasing DT sap concentration in liver tissues ( Figure 6A,C, p = 0.014, one way ANOVA). We noted that there was a non-significant slight increase in IRS/β-Actin levels with respect to increasing DT sap concentration ( Figure 6D). As Akt activation is known to act downstream of insulin signaling, we also measured Akt and pAkt levels. The levels of pAkt/Akt appear to alter at 0.5, 1 mg/g dose, without significant differences ( Figure 6E). Akt/β-Actin was not changed at 0.5 mg/g and decreased at 1 or 2.5 mg/g, without significant changes ( Figure 6F).",
"Hepatic AMPK activation is known to induce a suppression in gluconeogenesis [25]. To see if the AMPK pathway is activated by DT sap, AMPK phosphorylation was investigated. Compared to the vehicle control, 0.5 mg/g DT sap injection over 14 days significantly increased pAMPK/AMPK in the liver tissues ( Figure 6A,G, Student's t-test, p = 0.032). Higher concentrations also increased pAMPK/AMPK level, but without significant changes. In addition, the AMPK/β-Actin level was significantly altered in liver tissue on application of DT sap ( Figure 6H, p = 0.001, one way ANOVA). Post hoc analysis showed significant reductions in AMPK/β-Actin at 0.5 and 1 mg/g (* p = 0.016 in 0 vs. 0.5 mg/g; * p = 0.001 in 0 vs. 1 mg/g, Holm-Sidak method).",
"The spleen is related with GLU homeostasis, and its absence induces diabetes in the long term [26,27]. Therefore, GLU signaling factors were investigated in the spleen as well. IRS-1 signaling could not be measured reliably in the spleen sample. However, pAkt/Akt levels were significantly increased along with an increase in DT sap concentrations ( Figure 6B,I, p = 0.034, one way ANOVA; * p = 0.039 in 0 vs. 1 mg/g, post hoc Holm-Sidak method). Akt/β-Actin was slightly reduced by DT sap administration without significant changes ( Figure 6J). The level of pAMPK/AMPK was significantly increased with increasing DT sap concentration ( Figure 6B,K, p = 0.049, one way ANOVA). The AMPK/β-Actin was not altered or slightly increased by the DT sap application in spleen tissues without significant differences ( Figure 6L). GLU transporters such as Glut4 and Glut1 also contribute to regulate GLU metabolism by facilitating the transport of GLU [28]. However, there was no significant changes in Glut4 and Glut1 levels by DT sap administration in both tissues ( Figure S4).",
"To characterize the chemical composition of DT sap, two batches of DT sap extracts were subjected to LC-MS/MS. A total of 1080 chemicals were identified ( Figure 7A,B). Among them 1041 chemicals were fully or partially matched in more than one of seven tested annotation sources. The top three chemicals with the highest peak area (outside of the dotted red box in Figure 7A) were (1E,5E,9E)-1,5,9-Trime-thyl-1,5,9-cyclododecatriene, caryophyllene oxide and curcumene (Table 1). Seven more chemicals with the next highest peak area (dots with high values in Figure 7B) were 3,5-di-tert-butyl-4-hydroxybenzaldehyde, capsidiol, estradiol (or 19-norandroestenedione), bis [2-(2-butoxyethoxy)ethyl]adipate, trenbolone, o-tert-octylphenol and 5-ethyl-3,8-dimethyl-1,7-dihydroazulene (Table 1). Next, to identify DT sap-specific components, LC-MS/MS was performed and compared with the sap from another tree (i.e., Acer saccharum (AS)). DT sap-specific components were screened by fold change analysis with AS sap, and the consistency of the component analysis was confirmed by repeat experiments using different batches ( Figure 7C). In the graph of Figure 6C, dots of the shaded field (upper right quadrant) represent chemicals which are specifically abundant in DT sap compared to the AS sap, while dots of the other regions are chemicals which are specifically abundant in AS sap compared to DT sap, or unchanged. The intensity of 354 chemicals were higher in DT sap than in AS sap, while that of 726 chemicals were lower in DT sap. The top ten chemicals with the highest fold change was farnesol, gamfexine, (8z,11z,14z)-heptadecatrienoic acid, (1E,5E,9E)-1,5,9-Trime-thyl-1,5,9cyclododecatriene, o-tert-octylphenol, cumene, α-Eleostearic acid, oleanolic aldehyde, 2,6-Dimethyl-4-nonylphenol and 2,6-Di-tert-butyl-4-(dimethylaminome-thyl)phenol (Table 1). In addition to the above-mentioned chemicals, representative chemicals abundant in DT sap include 2-hydroxyestradiol, dienogest, isotretinoin, linoleic acid and costunolide (Table 1).",
"The results of our toxicity study on survival ratio, body weight, organ weight, blood chemistry and histochemistry show that DT sap concentrations below 0.5 mg/mL were safe for long-term administration (Table S2). We also found that blood GLU levels were gradually reduced with increased dosage of DT sap in female mice, and that the AMPKmediated signaling was altered by DT sap administration in female mouse liver and spleen tissues. In addition, bioactive chemicals were identified from DT sap by LC-MS/MS. While a significant dose-dependent reduction in blood glucose was observed in female mice, it was not observed in male mice ( Figure 4AI,AII). However, 0.5 mg/g DT sap administration also reduced the mean value of blood glucose in males compared to the control, although it was not statistically significant, implying a possible gender difference in dose-response. Therefore, we cannot exclude the possibility of hypoglycemic effects of DT sap on male mice as well, warranting further studies. Reduction in blood GLU with corresponding increases of DT sap concentration ( Figure 4AI) may be related with the activation of the insulin signaling pathway [29] or AMPK signaling pathway [30]. Here, phosphorylation of IRS-1 and Akt for the insulin signaling pathway and phosphorylation of AMPK for the AMPK signaling pathway were investigated. Phosphorylation of IRS-1 (Ser1101) inhibits insulin signaling [24], therefore the reduction of pIRS-1(Ser1101)/IRS-1 by DT sap should result in a corresponding decrease in blood GLU, which is observed here ( Figure 6C). The level of pAkt/Akt, a downstream signaling factor of IRS-1, appears to increase according to DT sap concentration ( Figure 6I). These results suggest that DT sap appears to affect blood GLU regulation via activation of the insulin signaling pathway. Previous findings showed that the activation of AMPK suppresses gluconeogenesis [25]. In our study, pAMPK/AMPK levels were significantly increased at low DT sap concentrations (0.5 mg/g) in liver tissues ( Figure 6G), and exhibited a significant increase in conjunction with increasing DT sap concentration in spleen tissues ( Figure 6K). Therefore, reduced GLU production may also contribute to blood GLU reduction ( Figure 4AI).",
"We observed that pAMPK/AMPK, a key metabolic regulator, exhibited a common tendency of increase with respect to increasing DT sap concentrations in both liver and spleen tissues ( Figure 6G,K), In contrast, pAkt/Akt levels responded differently in liver and spleen tissues. While pAkt/Akt levels did not show a consistent change in liver tissue ( Figure 6E), the pAkt/Akt levels in the spleen tissue showed an increase, with respect to increasing DT sap concentration ( Figure 6I). At this stage, we cannot explain the divergence in pAkt/Akt between liver and spleen tissues, but this will be a matter of future investigation.",
"The β-actin-normalized AMPK level was significantly altered by DT sap and post hoc analysis showed that it was significantly reduced in 0.5 and 1 mg/g DT sap ( Figure 6H). The other total protein levels of IRS, Akt in liver as well as Akt, AMPK in spleen exhibited slight reductions at some dosage, but without significant differences ( Figure 6D,F,J,L). The reduction of total AMPK protein expression ( Figure 6H) might be a form of negative feedback to preserve cellular homeostasis by preventing the overactivation of AMPK signaling ( Figure 6G,K) induced by long-term DT sap administration. High dosage (2.5 mg/g) might disrupt cellular homeostasis, resulting in significant organ weight changes ( Figure 3) and lower survival rate (Figure 2), observed here.",
"Interestingly, chemicals enriched in DT sap as analyzed by LC-MS/MS included estradiol, its related metabolites or structurally similar substances (e.g., 2-hydroxyestradiol, trenbolone, dienogest) ( Table 1). It was previously shown that estradiol injections reduced fasting blood GLU levels in non-obese C57BL/6N mice with short-term ovariectomy, while AMPK was activated and gluconeogenic gene expression was reduced in liver tissues [31]. A 4-week subcutaneous injection of estradiol to ovariectomized female mice with STZinduced type 1 diabetes significantly suppressed blood GLU level and increased plasma insulin, suggesting protective effects of estradiol against STZ-induced diabetes [32]. Besides estradiol, various components were identified from DT sap, such as caryophyllene oxide, curcumene, 3, 5-di-tert-butyl-4-hydroxybenzaldehyde, trenbolone, farnesol, dienogest, 2-Hydroxyestradiol, isotretinoin, linoleic acid and costunolide (Table 1). Among them, 2-hydroxyestradiol is a metabolite of estradiol and directly activates AMPK in C2C12 myotubes [33], which attenuates the development of obesity and decreases the severity of diabetes [34]. A black pepper extract with a high content of caryophyllene improved GLU uptake in C2C12 myotubes [35]. Farnesol induced a significant reduction of postprandial hyperglycemia on alloxan-induced type 2 diabetic mice [36]. The function of linoleic acid relates with the regulation of body weight, and serum leptin in subjects with type 2 diabetes [37]. These components have potentials to affect GLU metabolism, which may be involved in the reduction of blood GLU by DT sap observed here. However, to identify the actual bioactive components, in vitro screening and in vivo tests using isolated compounds are required, which is expected as a follow-up study.",
"In this study, we examined the in vivo safety, hypoglycemic function and component analysis of DT sap. Toxicity tests in vivo confirmed safety in mice at less than 0.5 mg/g when administered for longer than 14 days. Our results showed that DT sap exhibited the effect of reducing blood GLU and that DT sap can be linked to mechanisms involved in blood GLU regulation at least partially by modulating IRS, Akt and AMPK signaling in liver and spleen tissues. From the LC-MS/MS analysis, DT sap included various bioactive chemicals, suggesting their potential contribution to the function of DT sap, warranting further studies. In future, studies on the effects of DT sap on diabetes and the identification of effective components are expected.",
"Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13124332/s1. Figure S1: Survival rates, body and organ weights by a single oral administration of Dendropanax trifidus (DT) sap, Figure S2: Morphology of liver tissue by Dendropanax trifidus (DT) sap administrations to mice, Figure S3: Morphology of kidney tissue by Dendropanax trifidus (DT) sap administrations to mice, Figure S4: Effects of Dendropanax trifidus (DT) sap administrations on the protein expression level of glucose transporters, Figure S5: Western blot Images of Figure 6, Table S1: Mouse number used in each experiment, Table S2: Summary of the concentration-dependent effects in vivo. ",
"The data presented in this study are available on request from the corresponding author."
] | [] | [
"Introduction",
"Materials and Methods",
"Mice",
"Dendropanax trifidus Sap Preparation",
"In Vivo Toxicity Test",
"C18H22O2 Trenbolone",
"Dendropanax trifidus Sap",
"Blood Chemistry",
"Western Blot",
"Histopathology",
"Discussion",
"Non-Targeted Metabolome Analysis",
"Statistics",
"Results",
"Effects on Survival Ratio and Body/Tissue Weight by a Single Administration of Dendropanax trifidus Sap",
"Effects on Survival Ratio and Body/Tissue Weight by a Multiple Administration of Dendropanax trifidus Sap",
"Reduction in Blood Glucose by Dendropanax trifidus Sap Injection",
"Effects of Dendropanax trifidus Sap on AMPK-Mediated Signaling",
"Component Analysis of Dendropanax trifidus Sap",
"Discussion",
"Conclusions",
"Data Availability Statement:",
"Figure 2 .of 18 Figure 2 .",
"Figure 3 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 .",
"Figure 4 .",
"Figure 4 .",
"Figure 5 .Figure 5 .",
"Figure 6 .",
"Figure 6 .",
"Figure 7 .",
"Figure 7 .",
"Table 1 .",
"Table 1 .",
"Table 1 .",
"Table 1 .",
"Table 1 .",
"Table 1 ."
] | [
"Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \nCaryophyllene \noxide \n\n(-)-Caryophyllene \noxide; \nbeta-Caryophyllene \noxide \n\nNutrients 2021, 13, x FOR PEER REVIEW \n11 of 18 \n\n",
"Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 \nCurcumene \n\nAlpha-Curcumene; \n2-Methyl-6-p-tolyl-2-\nheptene \n\n",
"Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n3,5-di-tert-butyl-4-\nhydroxybenzaldehyde \n\n3,5-Di-t-butyl-4-\nhydroxybenzaldehyde \n\n",
"Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 \nCapsidiol \n\n(1R,3R,4S,4aR,6R)-6-\nIsopropenyl-4,4a-\ndimethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphthalenediol \n\n",
"Molecular \nFormula \n\nSuggested \nCompound \n\nSynonyms \n(Pubchem) \n\nSuggested Structure \n(Pubchem) \n\nAnnotation \nMS/MS (Database \nSearch Score) \n\nMolecular \nWeight \n\nRetention \nTime (min) \n\nMax. Area (Arbitrary \nUnit, 1.0 × 10 8 ) \n\nMean Group Area (Arbi-\ntrary Unit, 1.0 × 10 8 ) \n\nMean [Log2 Fold \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-\nNorandrostenedione \n\nEstr-4-ene-3,17-dione; \n19-Norandrost-4-ene-\n3,17-dione; \nNorandrostenedione \n\nFormula Compound (Pubchem) \n(Pubchem) \nSearch Score) \nWeight Time (min) \nUnit, 1.0 × 10 8 ) \ntrary Unit, 1.0 × 10 8 ) \nChange (DT \nSap/AS Sap)] \nFull \nPartial \n\nC15H24 \n\n(1E,5E,9E)-\n1,5,9-Trime-\nthyl-1,5,9-\ncyclodode-\ncatriene \n\n1,5,9-Trimethyl \ncyclododeca-\ntriene \n\n2 \n3 \n94.8 * \n204.19 \n21.962 \n260.7 \n274.2 \n8.79 \n\nC15H24O \n\nCaryo-\nphyllene ox-\nide \n\n(-)-Caryo-\nphyllene oxide; \nbeta-Caryo-\nphyllene oxide \n\n3 \n3 \n93.6 \n220.18 \n22.201 \n136.9 \n171.4 \n6.51 \n\nC15H22 Curcumene \n\nAlpha-Curcu-\nmene; \n2-Methyl-6-p-\ntolyl-2-heptene \n\n2 \n2 \n96.2 * \n202.17 \n21.832 \n130.7 \n165.2 \n5.69 \n\nC15H22O2 \n\n3,5-di-tert-\nbutyl-4-hy-\ndroxyben-\nzaldehyde \n\n3,5-Di-t-butyl-\n4-hydroxyben-\nzaldehyde \n\n2 \n3 \n85 * \n234.16 \n20.281 \n63.91 \n50.75 \n3.59 \n\nC15H24O2 Capsidiol \n\n(1R,3R,4S,4aR,6 \nR)-6-Isopro-\npenyl-4,4a-di-\nmethyl-\n1,2,3,4,4a,5,6,7-\noctahydro-1,3-\nnaphtha-\nlenediol \n\n2 \n2 \n87.4 * \n236.18 \n21.501 \n51.11 \n40.85 \n6.98 \n\nC18H24O2 \n\n19-Nor-\nandros-\ntenedione \n\nEstr-4-ene-3,17-\ndione; \n19-Norandrost-\n4-ene-3,17-di-\n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\n4 \n2 \n87.2 \n272.18 \n22.014 \n36.15 \n38.09 \n6.44 \n\nEstradiol \nbeta-Estradiol; \n17beta-Estradiol \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyethoxy)ethyl] \nadipate \n\nDibutoxyethoxyethyl \nadipate \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\n5-Ethyl-3,8-5-Ethyl-3,8-di-\n\n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 \nTrenbolone \n\n17beta-Trenbolone; \nTrienbolone; \n17-beta-\nHydroxyestra-4,9,11-\ntrien-3-one \n\nNutrients 2021, 13, x FOR PEER REVIEW \n12 of 18 \n\none; Nor-\nandrostenedi-\none \n\nEstradiol \n\nbeta-Estradiol; \n17beta-Estra-\ndiol \n\nC22H42O8 \n\nBis [2-(2-\nbutoxyeth-\noxy)ethyl] \nadipate \n\nDibutoxyethox-\nyethyl adipate \n2 \n1 \n68.8 * \n434.29 \n21.792 \n36.42 \n36.30 \n1.49 \n\nC18H22O2 Trenbolone \n\n17beta-\nTrenbolone; \nTrienbolone; \n17-beta-Hy-\ndroxyestra-\n4,9,11-trien-3-\none \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n\nC14H22O \no-tert-Oc-\ntylphenol \n\nt-octylphenol; \n2-(2,4,4-trime-\nthylpentan-2-\nyl)phenol \n\n2 \n1 \n80.6 * \n206.17 \n21.866 \n31.28 \n27.76 \n7.79 \n\nC14H18 \n\n5-Ethyl-3,8-\ndimethyl-\n1,7-dihydro-\nazulene \n\n5-Ethyl-3,8-di-\nmethyl-1,7-di-\nhydroazulene \n\n2 \n0 \n83.8 * \n186.14 \n21.163 \n29.03 \n26.66 \n6.87 \n\n3 \n3 \n84.1 \n270.16 \n21.313 \n39.92 \n28.62 \n6.43 \n",
"hy-\ndroxybenzyla-\nmine \n\n2 \n0 \n78.7 * \n263.22 \n21.865 \n4.091 \n3.238 \n7.20 \n\nC18H24O3 \n2-Hydroxy-\nestradiol \n\n2-OH-Estradiol; \n2-hydroxy-es-\ntradiol; \nEstra-1,3,5(10)-\ntriene-\n2,3,17beta-triol \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \n\nC20H25NO \n2 \nDienogest \n\nDienogestrel; \nDinagest; En-\ndometrion \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \nC20H25NO2 \nDienogest \n\nDienogestrel; \nDinagest; \nEndometrion \n\nC17H29NO methyla-\nminome-\nthyl)phenol \n\ntyl-4-hy-\ndroxybenzyla-\nmine \n\n2 \n0 \n78.7 * \n263.22 \n21.865 \n4.091 \n3.238 \n7.20 \n\nC18H24O3 \n2-Hydroxy-\nestradiol \n\n2-OH-Estradiol; \n2-hydroxy-es-\ntradiol; \nEstra-1,3,5(10)-\ntriene-\n2,3,17beta-triol \n\n2 \n2 \n81.6 * \n288.17 \n21.268 \n22.30 \n17.78 \n4.08 \n\nC20H25NO \n2 \nDienogest \n\nDienogestrel; \nDinagest; En-\ndometrion \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\n3 \n0 \n63.1 \n311.19 \n21.276 \n22.67 \n16.19 \n6.85 \n\nC20H28O2 \nIsotretinoin \n\n13-cis-Retinoic acid; \n3-cis-Vitamin A acid; \nAccutane \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic acid \n\nLinolic acid; Telfairic \nacid; cis,cis-Linoleic \nacid \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 \nCostunolide \n\n(+)-costunolide; \nCostunlide; Costus \nlactone \n\nNutrients 2021, 13, x FOR PEER REVIEW \n14 of 18 \n\nC20H28O2 Isotretinoin \n\n13-cis-Retinoic \nacid; 3-cis-Vita-\nmin A acid; Ac-\ncutane \n\n4 \n3 \n81.2 \n300.21 \n22.691 \n7.111 \n5.430 \n0.99 \n\nC18H32O2 \nLinoleic \nacid \n\nLinolic acid; \nTelfairic acid; \ncis,cis-Linoleic \nacid \n\n4 \n0 \n94.7 * \n280.24 \n21.686 \n3.033 \n2.935 \n6.45 \n\nC15H20O2 Costunolide \n\n(+)-costunolide; \nCostunlide; \nCostus lactone \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\n2 \n2 \n94.8 * \n232.16 \n21.149 \n5.355 \n2.636 \n4.68 \n\nNutrients 2021, 13, 4332 \n\n7 of 17 \n\n"
] | [
"Table S1",
"Table 1",
"Table 1",
"Table 1",
"(Table S2",
"Table 1",
"(Table S2",
"(Table 1)",
"(Table 1)",
"(Table 1",
"(Table 1)",
"(Table S2",
"Table 1)",
"(Table 1)",
"Table S1",
"Table S2"
] | [
"In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract",
"In Vivo Hypoglycemic Effects, Potential Mechanisms and LC-MS/MS Analysis of Dendropanax Trifidus Sap Extract"
] | [] |
212,022 | 2022-07-24T11:17:25Z | CCBY | http://www.jbc.org/content/288/47/34041.full.pdf | HYBRID | 8aba2c28a26107a70e97cd436f53d02aa3d7158f | null | null | null | null | 10.1074/jbc.m113.518019 | 1976474458 | 24106267 | null |
Phosphorylation of Angiomotin by Lats1/2 Kinases Inhibits F-actin Binding, Cell Migration, and Angiogenesis *
Published, JBC Papers in Press, October 8, 2013,
Xiaoming Dai
Peilu She
Department of Genetics
School of Life Sciences
State Key Laboratory of Genetic Engineering
Fudan University
200433ShanghaiChina
Fangtao Chi
Ying Feng
Huan Liu
Daqing Jin
Department of Genetics
School of Life Sciences
State Key Laboratory of Genetic Engineering
Fudan University
200433ShanghaiChina
Yiqiang Zhao
Xiaocan Guo
Dandan Jiang
Kun-Liang Guan
Department of Pharmacology
Moores Cancer Center
University of California at San Diego
La Jolla92093-0815California
Tao P Zhong
Department of Genetics
School of Life Sciences
State Key Laboratory of Genetic Engineering
Fudan University
200433ShanghaiChina
Bin Zhao
From the ‡ Life Sciences Institute and Innovation Center for Cell Biology
Zhejiang University
310058HangzhouZhejiangChina
Phosphorylation of Angiomotin by Lats1/2 Kinases Inhibits F-actin Binding, Cell Migration, and Angiogenesis *
Published, JBC Papers in Press, October 8, 2013,10.1074/jbc.M113.518019Received for publication, September 11, 2013, and in revised form, October 8, 2013
Background: Substrates of the Hippo pathway kinases Lats1/2 are largely unknown besides YAP/TAZ. Results: Phosphorylation of angiomotin by Lats1/2 inhibits interaction with F-actin thus impairs cell migration and angiogenesis. Conclusion: AMOTp130 is a physiological and functional substrate of Lats1/2 and the Hippo pathway. Significance: Demonstrating how identification of novel substrates would facilitate understanding the physiology of the Hippo pathway.
The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/ TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.
Organ size homeostasis is a remarkable feature of multicellular organisms. In the last decade, the Hippo pathway has been found to play a key role in control of organ size (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). At the center stage of this pathway is the Mst1/2-Lats1/2 kinase cascade. Upstream signals such as cell adhesion, cytoskeleton remodeling, lysophosphatidic acid (LPA) 2 and its respective G-protein-coupled receptors (GPCRs) were found to regulate the Hippo pathway (15)(16)(17)(18)(19)(20)(21)(22)(23). YAP transcription co-activator and its paralog TAZ are the best known Hippo pathway targets mediating gene expression regulation and organ size control (22, 24 -27). Nevertheless, the various upstream input signals suggest rich functions of the pathway. Indeed, Lats1/2 were reported to regulate cellular processes such as cell differentiation, cytokinesis, senescence, autophagy, centrosome duplica-tion, and neuron dendritic tiling (28 -33). It is unlikely that YAP/TAZ inactivation mediates all these functions. However, YAP/TAZ-independent functions of the Hippo pathway were poorly studied.
Here we report that the angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and zebrafish embryonic angiogenesis in vivo. These studies identified AMOT as a critical effector of the Hippo pathway downstream of GPCR signaling in regulation of cell migration and angiogenesis.
EXPERIMENTAL PROCEDURES
Antibodies, Plasmids, and Other Materials-We obtained anti-AMOT antibodies from Bethyl Lab; anti-Lats1 and anti-Lats2 from Cell Signaling Technologies; anti-GST from Genscript; anti-␣-tubulin, anti-Flag, and anti-vinculin from Sigma; anti-thiophosphate ester from Epitomics; anti-HSP90 from BD Biosciences; anti-HA and anti-Myc from Covance; Alexa Fluor 488-or 594-conjugated secondary antibodies and Alexa Fluor 488-phalloidin from Invitrogen; Horseradish peroxidase-conjugated secondary antibodies from GE Healthcare. Anti-phos-phoAMOTp130 (S175) antibody was generated by immunizing rabbits with phospho-peptide KQGHVRSLS(p)ERL. Human AMOTp130 were subcloned from other vectors into pCMV-Flag, pcDNA3-HA, pGEX-KG, and pQCXIH vectors. Human AMOTp80, mouse AMOTL1 and AMOTL2 were subcloned into pcDNA3-HA and pCMV-Flag vectors. Human AMOTp130 mutants were generated by site-directed mutagenesis. Other plasmids were described before (16,22,34). Phos-tag conjugated acrylamide was purchased from Wako Chemicals. All other chemicals were from Sigma.
Cell Culture, Transfection, and Viral Infection-HEK293, HEK293T, COS7, HEK293P, and HUVEC cells were cultured in DMEM (Invitrogen) containing 10% FBS (Invitrogen) and 50 g/ml penicillin/streptomycin (P/S). Transfection with Lipofectamine (Invitrogen) was performed according to the manufacturer's instructions. For viral infection, HEK293P cells were transfected with viral constructs and packaging plasmids. 48 h later, viral supernatant was supplemented with 5 g/ml polybrene, filtered through a 0.45 m filter, and used to infect target cells.
Immunoprecipitation and Kinase Assay-For Lats2 kinase assays, HEK293 cells were transfected with indicated plasmids. 48 h post-transfection, cells were lysed with lysis buffer (50 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na 3 VO 4 , protease inhibitor mixture (Roche), 1 mM DTT, 1 mM PMSF) and immunoprecipitated with anti-HA antibody. The immunoprecipitates were washed three times with lysis buffer, once with wash buffer (40 mM HEPES, 200 mM NaCl), and once with kinase assay buffer (30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl 2 ). The immunoprecipitated Lats2 was subjected to kinase assay in the presence of 500 M ATP or ATP-␥S and 1 g of recombinant GST-AMOTp130 purified from Escherichia coli as substrates. The reaction mixtures were incubated for 30 min at 30°C. For detection by antithiophosphate-ester antibody, the reaction mixtures were further supplemented with 2.5 mM PNBM. Alkylating reactions were allowed to proceed for 1 h at room temperature. The reactions were terminated with SDS sample buffer and boiled before analysis by SDS-PAGE.
Actin Spin-down Assay-Actin Binding Protein Biochem Kit was obtained from Cytoskeleton. The assay was performed following the manufacturer's instructions. Briefly, F-actin was polymerized and mixed with GST-AMOTp130 proteins purified from E. coli. The mixture was then centrifuged at 150,000 ϫ g for 1.5 h at 24°C. Supernatants and pellets were then collected and processed for electrophoresis. Proteins were visualized by Coomassie Blue staining or Western blots.
Cell Migration Assay-Cell migration assay was performed using BD Falcon Cell culture inserts for 24-well plates with 8.0 m pore size. Bottom sides of filters were pre-coated with 20 mg/ml fibronectin. HUVEC cells were serum-starved for 12 h and then seeded into the upper chambers of the inserts at 4 ϫ 10 4 cells/well in serum-free medium, and lower chambers were filled with serum-free or 10% FBS-containing medium. After 12 h, cells were stained with 0.5% crystal violet. Cells in upper chambers were carefully removed, and bottom sides of the chambers were pictured.
Immunofluorescence Staining-Cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100. Cells may be treated with 100 g/ml digitonin for 5 min as indicated. After blocking in 2% BSA for 30 min, slides were incubated with first antibody diluted in 1% BSA overnight at 4°C. After washing with PBS, slides were incubated with Alexa Fluor 488-or 594-conjugated secondary antibodies for 1.5 h. For staining of F-actin, cells were incubated with Alexa Fluor 488-phalloidin for 1.5 h. The slides were then washed and mounted.
RNA Interference-Short interfering RNA (siRNA) oligonucleotides toward human Lats1, Lats2, AMOT, and control siRNA toward firefly luciferase were transfected into indicated cells using Lipofectamine RNAiMax Reagent (Invitrogen) following the manufacturer's instructions. Cells were analyzed 72 h post-transfection.
Zebrafish Maintenance and Angiogenesis Assay-Zebrafish embryos were produced by pairwise matings, raised at 28.5°C and staged as described (35). A transgenic Tg(flk:GFP) line was used in this study (36). Sense-capped mRNAs were synthesized using mMESSAGE mMACHINE system (Ambion) according to the manufacturer's instructions. Plasmids encoding human AMOTp130, AMOTp130-SA, AMOTp130-S.D., mouse Lats2, or Lats2-KR were digested with XhoI and transcribed with T7 polymerase. Poly (A) tails (Takara Bio Cat. No. 2181) were added to the synthetic mRNAs. Synthesized mRNAs were purified using the MEGAclear Kit (Ambion). Antisense morpholino oligonucleotide AMOT-MO (5Ј-CCACTGACACAACTAC-CACCAAGTG-3Ј) (37) was synthesized by Gene Tools, LLC. Synthetic mRNAs and morpholinos were microinjected into zebrafish embryos at the one-two-cell stages as described (38). Injection doses were as following: 8.2 ng AMOT-MO; 8.2 ng AMOT-MOϩ400pg hAMOTp130-WT/SA/S.D.; 5.6 ng AMOT-MOϩ373pg hAMOTp130-WT/SA ϩ80pg mLats2-WT/KR. Vascular phenotypes were imaged using a Zeiss Observer Z1 microscope.
RESULTS
The Hippo Pathway Phosphorylates AMOT Family Proteins-
The AMOT family proteins localize to specific cellular compartments and regulate cell migration and proliferation (37, 39 -41). AMOT is a component of the Hippo pathway capable of inhibiting YAP through direct binding and Lats1/2 activation (34,(42)(43)(44). Surprisingly, we found that Lats2 induced a dramatic electrophoretic mobility shift of AMOTp130 comparable to that of YAP on Phos-tag-containing gels which specifically retards phosphorylated proteins (Fig. 1A). Furthermore, Lats2 also induced dramatic up-shift of other AMOT family proteins AMOTL1 and AMOTL2 (Fig. 1A). The Lats2-induced mobility shift of AMOTp130 was eliminated by treatment of the protein with lambda protein phosphatase (Fig. 1B). In addition, co-transfection of AMOTp130 with other Hippo pathway components Mst2, Sav, Mob, and Lats2 induced AMOTp130 mobility shift in a synergistic manner (Fig. 1C). These experiments indicate that Lats1/2 in the Hippo pathway could induce phosphorylation of AMOT family proteins.
We then examined whether Lats2 could phosphorylate AMOT in vitro. In this assay, the kinase utilizes ATP-␥S as phosphate donor to generate thiophosphorylated substrate, which in turn reacts with p-nitrobenzyl mesylate (PNBM) to form a thiophosphate-ester (45). As detected by a thiophosphate-ester-specific antibody, recombinant AMOTp130 could be phosphorylated by Lats2 in vitro (Fig. 1D). The phosphorylation is more efficient when Mob and Sav were co-transfected. In contrast, the kinase inactive Lats2-KR could not phosphorylate AMOTp130. Thus, Lats2 phosphorylates AMOTp130 both in vitro and in vivo.
Lats2 Phosphorylates AMOT on Serine 175-Previous studies have revealed the optimal Lats1/2 phosphorylation target consensus motif as HXRXXS (22,25). Interestingly, we found one HXRXXS motif, and one HXKXXS motif, also permissive for phosphorylation by Lats1/2, on AMOTp130 ( Fig. 2A). Both motifs are conserved in AMOT family members and across species ( Fig. 2A). As shown in Fig. 2B, mutation of Ser-175 in the N-terminal motif to alanine largely repressed Lats2-induced up-shift of AMOTp130, suggesting that this reside is phosphorylated by Lats2. Further mutation of Ser-859 eliminates the mild shift of S175A, suggesting that Ser-859 is also phosphorylated by Lats2. However, individual mutation of Ser-859 did not repress the up-shift possibly due to limited sensitivity of the Phos-tag method. The histidine in the HXRXXS motif is crucial for substrate recognition. Indeed, similar to S175A, mutation of H170 largely abolished AMOTp130 upshift in response to Lats2 (Fig. 2B). These experiments demonstrate that phosphorylation of Ser-175 is largely responsible for Lats2-induced up-shift of AMOTp130.
To further confirm that AMOTp130 Ser-175 is indeed phosphorylated by Lats2, we generated an anti-pAMOT (Ser-175) antibody. As expected, this antibody detected phosphorylation of ectopically expressed AMOTp130, which was markedly enhanced by Lats2 co-expression and eliminated by phosphatase treatment (Fig. 2C). Furthermore, AMOTp130 is phosphorylated on Ser-175 by Lats2 but not Lats2-KR in vitro (Fig. 2D). Thus, AMOTp130 is phosphorylated on Ser-175 by Lats2 in vitro and in vivo.
GPCR Signaling Inhibits AMOTp130 Phosphorylation Mediated by the Hippo Pathway-Following identification of the phosphorylation site we further examined the role of the Hippo pathway in physiological regulation of AMOT. Consistent with the inhibition of the Hippo pathway by serum and LPA, these stimulations inhibit endogenous AMOTp130 phosphorylation on Ser-175 (Fig. 2, E and F). LPA in serum represses the Hippo pathway via activation of GPCRs such as LPAR2, and the coupled G-proteins such as G q and G 12 (16). Consistently, expression of LPAR2 or active QL mutants of G q or G 12 potently inhibits endogenous AMOTp130 phosphorylation in serumdeprived cells (Fig. 2G). Furthermore, siRNA knockdown of Lats1 and Lats2 largely eliminates AMOTp130 phosphorylation on Ser-175 in serum-deprived cells (Fig. 2H). Integrity of the actin cytoskeleton is required for GPCR-induced repression of the Hippo pathway (16). Indeed, disruption of F-actin induced AMOTp130 phosphorylation in cells cultured in serum-rich medium (Fig. 2I). Taken together, serum and GPCR signaling regulate AMOTp130 phosphorylation on Ser-175 through Lats1/2 kinases.
Phosphorylation of AMOTp130 on Ser-175 Inhibits Its Interaction with F-actin-AMOTp130 is known to colocalize with F-actin in cells dependent on its N-terminal domain containing Ser-175 (46). By immunofluorescence staining, we indeed observed colocalization of ectopically expressed AMOTp130 with F-actin filaments (Fig. 3A). At lower expression level of AMOT, the AMOT-F-actin filaments closely resemble actin stress fibers (Fig. 3A). However, at higher expression level, the filaments are thicker possibly represent AMOT-F-actin filament bundles (Fig. 3A, middle panel). In support of a real stress fiber identity of AMOT-F-actin filaments, they are resistant to digitonin treatment before fixation and are anchored to focal NOVEMBER 22, 2013 • VOLUME 288 • NUMBER 47 adhesions (Fig. 3, B and C). Interestingly, expression of Lats2 disrupted the filamentous AMOTp130 and results in diffused and irregular dots-like localization of AMOT (Fig. 3, A and F). Thus phosphorylation of AMOTp130 on Ser-175 by Lats2 may disrupt AMOT-F-actin interaction. Consistently, the S175A mutant colocalizes with F-actin in a manner insensitive to Lats2 (Fig. 3, D and F). More strikingly, irrespective to Lats2 expression, the phospho-mimetic S175D mutant exhibited dots-like cytoplasmic localization (Fig. 3, E and F). Importantly, using two independent antibodies, we demonstrated that endogenous AMOT colocalizes with F-actin in 293T cells (Fig. 4, A, B, D). The filamentous local- Experiments were similar to that in Fig. 1D except that regular ATP was used. E, serum inhibits phosphorylation of endogenous AMOTp130 on Ser-175. Serum-starved 293T cells were stimulated with 10% FBS for the indicated time before harvest. F, LPA inhibits phosphorylation of endogenous AMOTp130 on Ser-175. Serum-starved 293T cells were stimulated with serum or LPA for 1 h before harvest. G, GPCR signaling inhibits Ser-175 phosphorylation of endogenous AMOTp130. 293T cells were transfected with indicated plasmids and serum-starved overnight before harvest. H, knockdown of Lats1 and Lats2 represses endogenous AMOTp130 phosphorylation on Ser-175. HEK293T cells were transfected with scramble or Lats1-and Lats2-specific siRNAs. Cells were serum-starved overnight before harvest. I, disruption of F-actin induces phosphorylation of AMOTp130. HEK293T cells cultured in serum-rich medium were treated with 1 g/ml Latrunculin B (LatB) for 1 h as indicated before being harvested for Western blot analysis.
The Hippo Pathway Inhibits AMOT in Cell Migration
FIGURE 3. Phosphorylation of AMOTp130 on Ser-175 inhibits interaction with F-actin.
A, Lats2 inhibits colocalization of AMOTp130 with F-actin. Transfected COS7 cells were stained with anti-Flag antibody for localization of AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, colocalization of AMOTp130 with F-actin in digitonin-treated cells. Flag-AMOTp130 transfected Cos7 cells were treated with digitonin before fixation and stained as that in A. C, AMOT-F-actin filaments are anchored to focal adhesions. COS7 cells expressing wild-type or mutant AMOTs were co-stained with anti-Flag for AMOT and anti-vinculin to visualize focal adhesions. D, phospho-deficient mutant of AMOTp130 constitutively colocalizes with F-actin. Experiments were similar to these in A except the use of S175A mutant. E, phospho-mimetic mutant of AMOTp130 loses colocalization with F-actin. Experiments were similar to these in A except the use of S175D mutant. F, quantification of AMOT-F-actin colocalization. 120 cells were quantified for each transfection. Thick filament is similar to Fig. 3A, middle panel; thin filament is similar to that in Fig. 3A, top panel; non filamentous is similar to that in Fig. 3E, top panel.
. Phosphorylation inhibits endogenous AMOTp130-F-actin interaction and direct AMOTp130-F-actin interaction in vitro.
A, endogenous AMOT colocalizes with F-actin. Scramble or AMOT-specific siRNA transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, endogenous AMOT colocalizes with F-actin. Experiments were similar to these in A except that endogenous AMOT was stained with anti-AMOT antibody #2. C, Lats2 inhibits AMOT colocalization with F-actin in a kinase-dependent manner. HA-Lats2 wild-type or KR transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130 and anti-HA for Lats2 expression. D, quantification of filamentous localization of endogenous AMOT. Cells with or without filamentous AMOT in A and C were quantified. 120 cells were quantified for each sample. E, phospho-mimetic mutation inhibits AMOTp130 association with F-actin in vitro. Recombinant GST-AMOT wild-type or N-terminal fragment (N) were subjected to in vitro actin spin-down assay. (S), supernatant; (P), pellet. Actin was examined by Coomassie Blue staining and GST-AMOT was determined by Western blots. Asterisks denote nonspecific proteins. F, AMOTp130 does not co-immunoprecipitate with G-actin. HEK293 cells were transfected as indicated. Transfection amount was adjusted to make soluble AMOT levels comparable. Lysates were immunoprecipitated with anti-Flag antibody. The presence of endogenous actin and Flag-AMOTp130 in immunoprecipitates and lysates were examined by Western blots. G, phosphorylation of wild-type AMOTp130 by Lats2 does not affect interaction between AMOTp130 and YAP. HEK293 cells were transfected as indicated. AMOTp130 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of YAP wild-type or 5SA mutant was examined by anti-Flag Western blots. AMOTp130 phosphorylation was shown by electrophoretic mobility shift on Phos-tag containing gel.
ization of endogenous AMOT is also disrupted by Lats2 in a kinase-dependent manner (Fig. 4, C and D). These data demonstrate that phosphorylation of AMOTp130 by Lats2 on Ser-175 inhibits AMOTp130-F-actin interaction in vivo.
To test the possibility of a direct interaction between AMOTp130 and F-actin we performed in vitro F-actin spindown assay. In this assay, F-actin binding proteins would cosediment with pre-polymerized F-actin. We found that addition of F-actin brought all recombinant AMOTp130 or the N-terminal fragment into the pellet fraction indicating direct binding of AMOTp130 to F-actin (Fig. 4E). However, the phospho-mimetic S175D mutant largely remains in the supernatant (Fig. 4E). These results suggest that phosphorylation of AMOTp130 on Ser-175 would be inhibitory on the direct interaction of AMOTp130 with F-actin in vitro. In contrast, we did not observe any interaction of AMOTp130 with monomeric G-actin by co-immunoprecipitation (Fig. 4F), suggesting that AMOTp130 specifically interacts with F-actin. These results indicate that AMOTp130 directly interacts with F-actin under negative regulation by phosphorylation of Ser-175.
Phosphorylation of AMOTp130 on Ser-175 Interferes with Stress Fiber and Focal Adhesion Formation and Inhibits Endothelial Cell
Migration-AMOTp130 is known to directly bind and inhibit YAP (34,43,44). However, although AMOT-SA mutant show some decreased interaction with YAP, the interaction between YAP and wild-type AMOT is not significantly affected by AMOT phosphorylation (Fig. 4G). AMOT is also known for its roles in endothelial cell morphology and migration (47). Therefore we examined the function of AMOTp130 phosphorylation by Lats1/2 in endothelial cells. In agreement FIGURE 5. Phosphorylation of AMOTp130 on Ser-175 inhibits endothelial cell migration. A, knockdown efficiency of AMOT siRNAs. AMOT, CTGF, and Cyr61 levels in siRNA transfected HUVEC cells were determined by realtime RT-PCR. B, phospho-mimetic mutant of AMOT is unable to compensate endogenous AMOT in actin stress fiber and focal adhesion formation. Control or AMOT-expressing stable cells transfected with siRNAs were serum-starved overnight and then seeded on fibronectin-coated coverslips for 2 h in serum-containing medium. Focal adhesions and F-actin were stained with anti-vinculin and Alex-aFluor488-Phalloidin, respectively. C, phospho-mimetic AMOT mutant could not rescue cell migration defects caused by loss of AMOT. Cells the same as these in B were examined in transwell migration assay. D, size of 60 FAs from the staining of each stable cell as these in B was quantified by ImageJ and drawn into scatter plot. The median number was indicated. E, number of migrated cells in C was quantified. Experiments performed were in duplicates.
with our previous observations, knockdown of AMOT induces expression of YAP target genes CTGF and Cyr61 (Fig. 5A). Interestingly, consistent with a direct association of AMOT with F-actin, knockdown of AMOT leads to reduced stress fibers in cells, which also correlates with reduced focal adhesions (Fig. 5, B and D). The defects are partially rescued by re-expression of AMOTp80 and better rescued by re-expression of AMOTp130 (Fig. 5, B and D). Interestingly, co-expression of AMOTp130 wild-type or SA mutant but not the SD mutant together with AMOTp80 rescues stress fiber and focal adhesion formation to a level similar to control cells (Fig. 5, B and D). Thus phosphorylation of AMOTp130 plays an inhibitory role on stress fiber and focal adhesion formation.
Remodeling of actin cytoskeleton and cell adhesion is crucial to cell migration. Consistent with alterations of the cytoskeleton and focal adhesions, knockdown of AMOT largely inhibits endothelial cell migration (Fig. 5, C and E). Again, we observed that co-expression of AMOTp130 wild-type or SA mutant but not the SD mutant together with AMOTp80 rescues the cell migration defects (Fig. 5, C and E). This suggests that phosphorylation of AMOT on Ser-175 inhibits cell migration. We then further investigated the role of AMOT in cell migration downstream of the Hippo signaling. Serum starvation is well known to strongly inhibit cell migration in vitro. However, we found that cell migration in starvation condition is partially but significantly rescued by knockdown of Lats1 and Lats2 (Fig. 6, A and C). This result suggests that activation of Lats1/2 by serum starvation due to blunted GPCR signaling is an important reason for repression of cell migration. More importantly, simultaneous knockdown of AMOT blocks cell migration induced by loss of Lats1/2 expression (Fig. 6, A and C). Thus inhibition of the Hippo pathway leads to cell migration in an AMOT-dependent manner. In correlation with cell migration, knockdown of Lats1/2 also induces actin stress fiber and focal adhesion formation in serum-starved cells in an AMOT-dependent manner (Fig. 6, B and D). Our data suggest that AMOTp130 is important for endothelial cell migration under negative regulation by Lats1/2-induced phosphorylation.
Phosphorylation of AMOTp130 on Ser-175 Inhibits Angiogenesis in Zebrafish-AMOT is known to control endothelial cell migration and angiogenesis in zebrafish (37). Thus we further investigated the effects of AMOTp130 phosphorylation on angiogenesis in zebrafish Tg(flk:GFP) transgenic embryos, in which GFP expression is driven by endothelial specific promoter flk. Consistent with the previous study (37), amot knockdown via injection of AMOT antisense morpholino-oligonu- cleotide (AMOT-MO) caused angiogenesis defects in AMOT morphants, in which intersegmental vessels (ISV) were disrupted and failed to reach the dorsolateral region at 30 h postfertilization (hpf) (Fig. 7-2). At 36 hpf, some ISV had still not arrived the most dorsolateral position so that the dorsal longitudinal anastomotic vessel (DLAV) formation was disrupted ( Fig. 7-2). Nevertheless, the angiogenesis defects are rescued by co-injection of mRNAs encoding human AMOTp130 ( Fig. 7-3) or the phospho-deficient SA mutant (Fig. 7-4) but not the phospho-mimetic SD mutant (Fig. 7-5). Furthermore, the angiogenic activity of AMOTp130 is inhibited by co-injection of mRNAs encoding Lats2 (Fig. 7-6) but not Lats2-KR ( Fig. 7-7). In addition, the angiogenic activity of AMOTp130-SA is insensitive to Lats2 (Fig. 7-8). The overall development and morphologies of injected embryos were comparable to control embryos at bud stage and 30 hpf (data not shown). These results indicate that Lats2 could inhibit the angiogenic function of AMOTp130 in vivo through phosphorylation of Ser-175.
DISCUSSION
Biological functions of kinases are largely determined by substrate selectivity. However, known substrates of the Lats1/2 kinases are very limited. Besides YAP/TAZ, several other proteins have been suggested as Lats1/2 substrates including Dyrk1a, MYPT1, and Snail1 (48 -50). In this study, we identified AMOT family proteins to be new substrates of Lats1/2 with canonical target consensus motifs being phosphorylated by Lats1/2 to comparable levels as YAP/TAZ. Importantly, this phosphorylation is responsive to GPCR signaling and plays a regulatory role on AMOT interaction with F-actin thus regu-lates cell migration. While this report was being prepared, an independent study reported the phosphorylation of AMOT by Lats1/2 in promoting AMOT-Lats1/2 interaction, and its role in cell fate specification in preimplantation mouse embryos (51). Together, these studies suggest AMOT as an important effector of the Hippo pathway. We anticipate more substrates of Lats1/2 to be discovered to explain the ever expanding functions of the Hippo pathway.
AMOTp130 also plays a regulatory role in the Hippo pathway through both physical binding with YAP/TAZ and promotion of Lats1/2 activity (34,(42)(43)(44). In addition, AMOT also interacts with Merlin, an upstream component of the Hippo pathway (40). We demonstrated that the association between wild-type AMOTp130 and YAP is not regulated by AMOTp130 phosphorylation. However, it was shown that the phospho-mimetic AMOTp130 has elevated interaction with Lats1/2 (51). Therefore there is possibly a feedback loop consisting of the two roles of AMOTp130 as both a regulator and an effector of the Hippo pathway, although the physiological context and the exact mechanism would need further investigation. What we have found in this study also indicate that the phosphorylated and dephosphorylated AMOTp130 are both functional with differential roles. While the phosphorylated AMOT may promote Hippo signaling, the dephosphorylated AMOTp130 function as an F-actin interacting protein to promote stress fiber formation and cell migration.
YAP/TAZ are downstream effectors of the Hippo pathway and function through regulation of gene transcription (52). YAP/TAZ also promotes cell migration likely through regulation of gene expression and initiation of an EMT program (53). However, our study identified AMOTp130 as a novel mediator of the Hippo pathway in inhibiting cell migration. Phosphorylation of AMOTp130 by Lats1/2 directly inhibits F-actin binding and correlates with reduced actin stress fiber and focal adhesion formation. Considering the direct role of F-actin and focal adhesion in cell migration, the role of AMOTp130 in cell migration is likely structural. Migration promoting functions of AMOT and YAP/TAZ could be reconciled in at least two ways. First, the expression pattern of AMOT and YAP/TAZ may dictate their functions in different tissues and cell types. Second, in cells expressing both of these proteins, AMOTp130 and YAP/ TAZ may mediate acute and prolonged effects of the Hippo pathway on cell migration in a cooperative manner (Fig. 8). Whether YAP/TAZ plays a role in endothelial cell migration is unknown. However, we demonstrated in this report that Lats1/2 play a role in endothelial cell migration in a manner dependent on AMOT phosphorylation. More importantly we demonstrated in vivo for the first time that the Hippo pathway may regulate developmental angiogenesis through AMOT phosphorylation. It would be interesting to investigate whether AMOT phosphorylation by the Hippo pathway also functions in pathological angiogenesis such as that in cancer.
FIGURE 1 .
1The Hippo pathway phosphorylates angiomotin family proteins. A, Lats2 induces mobility shift of AMOT family proteins. HEK293 cells were transfected with plasmids encoding Flag-YAP or AMOT family proteins with or without Lats2 co-transfection. Cell lysates were resolved on Phos-tag-containing gels. Vps34 is a negative control. B, phosphatase eliminates AMOT mobility shift induced by Lats2. Flag-AMOTp130 was immunoprecipitated from transfected cells and treated with lambda protein phosphatase as indicated. Samples were analyzed as that in A. C, hippo pathway proteins synergistically induces AMOTp130 mobility shift. Lysates of transfected cells were resolved on Phos-tag-containing gels for mobility shift of Flag-AMOTp130. D, Lats2 phosphorylates AMOTp130 in vitro. In vitro phosphorylation of GST-AMOTp130 by immunoprecipitated Lats2 was performed. (S), short exposure; (L), long exposure.
FIGURE 2 .
2AMOTp130 is phosphorylated on Ser-175 by Lats1/2 in response to GPCR signaling. A, domain organization of AMOTp130 and alignment of AMOT family proteins from different species. B, phosphorylation of Ser-175 leads to Lats2-induced AMOTp130 up-shift. Lysates of transfected HEK293 cells were analyzed by Western blots. C, Ser-175 of AMOTp130 is phosphorylated by Lats2. AMOTp130 phosphorylation was detected by pAMOT (S175)-specific antibody. D, Lats2 phosphorylates AMOTp130 on Ser-175 in vitro.
FIGURE 4
4FIGURE 4. Phosphorylation inhibits endogenous AMOTp130-F-actin interaction and direct AMOTp130-F-actin interaction in vitro. A, endogenous AMOT colocalizes with F-actin. Scramble or AMOT-specific siRNA transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, endogenous AMOT colocalizes with F-actin. Experiments were similar to these in A except that endogenous AMOT was stained with anti-AMOT antibody #2. C, Lats2 inhibits AMOT colocalization with F-actin in a kinase-dependent manner. HA-Lats2 wild-type or KR transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130 and anti-HA for Lats2 expression. D, quantification of filamentous localization of endogenous AMOT. Cells with or without filamentous AMOT in A and C were quantified. 120 cells were quantified for each sample. E, phospho-mimetic mutation inhibits AMOTp130 association with F-actin in vitro. Recombinant GST-AMOT wild-type or N-terminal fragment (N) were subjected to in vitro actin spin-down assay. (S), supernatant; (P), pellet. Actin was examined by Coomassie Blue staining and GST-AMOT was determined by Western blots. Asterisks denote nonspecific proteins. F, AMOTp130 does not co-immunoprecipitate with G-actin. HEK293 cells were transfected as indicated. Transfection amount was adjusted to make soluble AMOT levels comparable. Lysates were immunoprecipitated with anti-Flag antibody. The presence of endogenous actin and Flag-AMOTp130 in immunoprecipitates and lysates were examined by Western blots. G, phosphorylation of wild-type AMOTp130 by Lats2 does not affect interaction between AMOTp130 and YAP. HEK293 cells were transfected as indicated. AMOTp130 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of YAP wild-type or 5SA mutant was examined by anti-Flag Western blots. AMOTp130 phosphorylation was shown by electrophoretic mobility shift on Phos-tag containing gel.
FIGURE 6 .
6Knockdown of Lats1/2 induces cell migration, actin stress fiber and focal adhesion in an AMOT-dependent manner.A, Lats1/2 plays a role in serum deprivation-induced migration retardation by repressing AMOT. siRNA-transfected HUVEC cells were serum starved and then seeded in transwell inserts in serum-free medium. The lower chambers were filled with serum-containing or serum-free medium as indicated. Cells on bottom sides of the transwells were stained after 12 h. B, Lats1/2 plays a role in serum deprivation-induced stress fiber and focal adhesion deformation by repressing AMOT. Cells were the same as these in A. Cells were serum starved overnight and then seeded on fibronectin-coated coverslips for 2 h either in serum-containing medium or serum-free medium as indicated. Focal adhesions and F-actin were stained with anti-vinculin and AlexaFluor488-Phalloidin respectively. C, number of migrated cells in A was quantified. Experiments were in duplicates. D, size of 25 FAs from the staining of each stable cell as these in B was quantified by ImageJ and drawn into scatter plot. The median number was indicated.
FIGURE 7 .
7Phosphorylation of AMOTp130 by Lats1/2 regulates angiogenesis in zebrafish. The fluorescent microscopy analyses revealed the development of ISV and DLAV at 30 hpf and 36 hpf in the trunk region of control Tg(flk:GFP) embryos and embryos injected with AMOT-MO or a combination of AMOT-MO and mRNAs encoding human AMOTp130, Lats2 or their mutants. Red asterisk: defective ISV; red arrow: defective DLAV. ISV: intersegmental vessels. DLAV: dorsal longitudinal anastomotic vessels. Embryos were orientated with anterior to the left. Histogram depicted the quantification of normal and angiogenesis defective embryos at 30 hpf. n, embryo number.
FIGURE 8 .
8A model of Lats1/2 in regulation of cell migration through phosphorylation of AMOT and YAP. Phosphorylation of AMOT by Lats1/2 inhibits F-actin association and then actin stress fiber and focal adhesion formation. These effects of AMOT on cell structure and cell adhesion may work independently or in cooperation with YAP-dependent gene transcription alteration downstream of Lats1/2 to regulate cell migration.
Acknowledgment-We thank Dr. Bin Zhou for providing HUVEC cells.
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| [
"Background: Substrates of the Hippo pathway kinases Lats1/2 are largely unknown besides YAP/TAZ. Results: Phosphorylation of angiomotin by Lats1/2 inhibits interaction with F-actin thus impairs cell migration and angiogenesis. Conclusion: AMOTp130 is a physiological and functional substrate of Lats1/2 and the Hippo pathway. Significance: Demonstrating how identification of novel substrates would facilitate understanding the physiology of the Hippo pathway."
] | [
"Xiaoming Dai ",
"Peilu She \nDepartment of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina\n",
"Fangtao Chi ",
"Ying Feng ",
"Huan Liu ",
"Daqing Jin \nDepartment of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina\n",
"Yiqiang Zhao ",
"Xiaocan Guo ",
"Dandan Jiang ",
"Kun-Liang Guan \nDepartment of Pharmacology\nMoores Cancer Center\nUniversity of California at San Diego\nLa Jolla92093-0815California\n",
"Tao P Zhong \nDepartment of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina\n",
"Bin Zhao ",
"\nFrom the ‡ Life Sciences Institute and Innovation Center for Cell Biology\nZhejiang University\n310058HangzhouZhejiangChina\n"
] | [
"Department of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina",
"Department of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina",
"Department of Pharmacology\nMoores Cancer Center\nUniversity of California at San Diego\nLa Jolla92093-0815California",
"Department of Genetics\nSchool of Life Sciences\nState Key Laboratory of Genetic Engineering\nFudan University\n200433ShanghaiChina",
"From the ‡ Life Sciences Institute and Innovation Center for Cell Biology\nZhejiang University\n310058HangzhouZhejiangChina"
] | [
"Xiaoming",
"Peilu",
"Fangtao",
"Ying",
"Huan",
"Daqing",
"Yiqiang",
"Xiaocan",
"Dandan",
"Kun-Liang",
"Tao",
"P",
"Bin"
] | [
"Dai",
"She",
"Chi",
"Feng",
"Liu",
"Jin",
"Zhao",
"Guo",
"Jiang",
"Guan",
"Zhong",
"Zhao"
] | [
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"J R Yates, ",
"M J Fernandes, ",
"D Mccollum, ",
"T Oka, ",
"A P Schmitt, ",
"M Sudol, ",
"W Wang, ",
"J Huang, ",
"Chen , ",
"J , ",
"K Shah, ",
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"M Ernkvist, ",
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"N , ",
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"M Ito, ",
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"H Saya, ",
"S Kuninaka, ",
"K Zhang, ",
"E Rodriguez-Aznar, ",
"N Yabuta, ",
"R J Owen, ",
"J M Mingot, ",
"H Nojima, ",
"M A Nieto, ",
"G D Longmore, ",
"Y Hirate, ",
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"K Inoue, ",
"A Suzuki, ",
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"M Adachi, ",
"K Chida, ",
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"Y Marikawa, ",
"K Nakao, ",
"A Shimono, ",
"H Sasaki, ",
"B Zhao, ",
"X Ye, ",
"J Yu, ",
"L Li, ",
"W Li, ",
"S Li, ",
"J D Lin, ",
"C Y Wang, ",
"A M Chinnaiyan, ",
"Z C Lai, ",
"K L Guan, ",
"M Overholtzer, ",
"J Zhang, ",
"G A Smolen, ",
"B Muir, ",
"W Li, ",
"D C Sgroi, ",
"C X Deng, ",
"J S Brugge, ",
"D A Haber, "
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] | [
"The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. B Zhao, L Li, Q Lei, K L Guan, Genes Dev. 24Zhao, B., Li, L., Lei, Q., and Guan, K. L. (2010) The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. Genes Dev. 24, 862-874",
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"Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. D Zhou, C Conrad, F Xia, J S Park, B Payer, Y Yin, G Y Lauwers, W Thasler, J T Lee, J Avruch, N Bardeesy, Cancer Cell. 16Zhou, D., Conrad, C., Xia, F., Park, J. S., Payer, B., Yin, Y., Lauwers, G. Y., Thasler, W., Lee, J. T., Avruch, J., and Bardeesy, N. (2009) Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 16, 425-438",
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"hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. S Wu, J Huang, J Dong, D Pan, Cell. 114Wu, S., Huang, J., Dong, J., and Pan, D. (2003) hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apo- ptosis in conjunction with salvador and warts. Cell 114, 445-456",
"The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. J Jia, W Zhang, B Wang, R Trinko, J Jiang, Genes Dev. 17Jia, J., Zhang, W., Wang, B., Trinko, R., and Jiang, J. (2003) The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev. 17, 2514 -2519",
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"Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. B Zhao, L Li, L Wang, C Y Wang, J Yu, K L Guan, Genes Dev. 26Zhao, B., Li, L., Wang, L., Wang, C. Y., Yu, J., and Guan, K. L. (2012) Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 26, 54 -68",
"Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. F X Yu, B Zhao, N Panupinthu, J L Jewell, I Lian, L H Wang, J Zhao, H Yuan, K Tumaneng, H Li, X D Fu, G B Mills, K L Guan, Cell. 150Yu, F. X., Zhao, B., Panupinthu, N., Jewell, J. L., Lian, I., Wang, L. H., Zhao, J., Yuan, H., Tumaneng, K., Li, H., Fu, X. D., Mills, G. B., and Guan, K. L. (2012) Regulation of the Hippo-YAP pathway by G-protein-coupled re- ceptor signaling. Cell 150, 780 -791",
"Regulation of the Hippo-YAP pathway by protease-activated receptors. J.-S Mo, F.-X Yu, R Gong, J H Brown, K.-L Guan, PARsMo, J.-S., Yu, F.-X., Gong, R., Brown, J. H., and Guan, K.-L. (2012) Regu- lation of the Hippo-YAP pathway by protease-activated receptors (PARs).",
". Genes Dev. 26Genes Dev. 26, 2138 -2143",
"Hippo pathway regulation by cell morphology and stress fibers. K Wada, K Itoga, T Okano, S Yonemura, H Sasaki, Development. 138Wada, K., Itoga, K., Okano, T., Yonemura, S., and Sasaki, H. (2011) Hippo pathway regulation by cell morphology and stress fibers. Development 138, 3907-3914",
"Modulating F-actin organization induces organ growth by affecting the Hippo pathway. L Sansores-Garcia, W Bossuyt, K Wada, S Yonemura, C Tao, H Sasaki, G Halder, EMBO J. 30Sansores-Garcia, L., Bossuyt, W., Wada, K., Yonemura, S., Tao, C., Sasaki, H., and Halder, G. (2011) Modulating F-actin organization induces organ growth by affecting the Hippo pathway. EMBO J. 30, 2325-2335",
"Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila. B G Fernández, P Gaspar, C Brás-Pereira, B Jezowska, S R Rebelo, Janody , F , Development. 138Fernández, B. G., Gaspar, P., Brás-Pereira, C., Jezowska, B., Rebelo, S. R., and Janody, F. (2011) Actin-Capping Protein and the Hippo pathway reg- ulate F-actin and tissue growth in Drosophila. Development 138, 2337-2346",
"Role of YAP/TAZ in mechanotransduction. S Dupont, L Morsut, M Aragona, E Enzo, S Giulitti, M Cordenonsi, F Zanconato, J Le Digabel, M Forcato, S Bicciato, N Elvassore, S Piccolo, Nature. 474Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S., Elvassore, N., and Piccolo, S. (2011) Role of YAP/TAZ in mechanotransduction. Nature 474, 179 -183",
". B Zhao, X Wei, W Li, R S Udan, Q Yang, J Kim, J Xie, T Ikenoue, J Yu, L Li, P Zheng, K Ye, A Chinnaiyan, G Halder, Z C Lai, Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z. C., and",
"Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. K L Guan, Genes Dev. 21Guan, K. L. (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747-2761",
"Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP. E Miller, J Yang, M Deran, C Wu, A I Su, G M Bonamy, J Liu, E C Peters, X Wu, Chem. Biol. 19Miller, E., Yang, J., DeRan, M., Wu, C., Su, A. I., Bonamy, G. M., Liu, J., Peters, E. C., and Wu, X. (2012) Identification of serum-derived sphingo- sine-1-phosphate as a small molecule regulator of YAP. Chem. Biol. 19, 955-962",
"A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(-TRCP). B Zhao, L Li, K Tumaneng, C Y Wang, K L Guan, Genes Dev. 24Zhao, B., Li, L., Tumaneng, K., Wang, C. Y., and Guan, K. L. (2010) A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(-TRCP). Genes Dev. 24, 72-85",
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"TAZ promotes cell proliferation and epithelialmesenchymal transition and is inhibited by the hippo pathway. Q Y Lei, H Zhang, B Zhao, Z Y Zha, F Bai, X H Pei, S Zhao, Y Xiong, K L Guan, Mol. Cell. Biol. 28Lei, Q. Y., Zhang, H., Zhao, B., Zha, Z. Y., Bai, F., Pei, X. H., Zhao, S., Xiong, Y., and Guan, K. L. (2008) TAZ promotes cell proliferation and epithelial- mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426 -2436",
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"LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1. X Yang, K Yu, Y Hao, D M Li, R Stewart, K L Insogna, T Xu, Nature Cell Biol. 6Yang, X., Yu, K., Hao, Y., Li, D. M., Stewart, R., Insogna, K. L., and Xu, T. (2004) LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1. Nature Cell Biol. 6, 609 -617",
"The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors. T Mikeladze-Dvali, M F Wernet, D Pistillo, E O Mazzoni, A A Teleman, Y W Chen, S Cohen, C Desplan, Cell. 122Mikeladze-Dvali, T., Wernet, M. F., Pistillo, D., Mazzoni, E. O., Teleman, A. A., Chen, Y. W., Cohen, S., and Desplan, C. (2005) The growth regula- tors warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors. Cell 122, 775-787",
"Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. A Takahashi, N Ohtani, K Yamakoshi, S Iida, H Tahara, K Nakayama, K I Nakayama, T Ide, H Saya, E Hara, Nature Cell Biol. 8Takahashi, A., Ohtani, N., Yamakoshi, K., Iida, S., Tahara, H., Nakayama, K., Nakayama, K. I., Ide, T., Saya, H., and Hara, E. (2006) Mitogenic sig- nalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nature Cell Biol. 8, 1291-1297",
"Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. S Dutta, E H Baehrecke, Curr. Biol. 18Dutta, S., and Baehrecke, E. H. (2008) Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. Curr. Biol. 18, 1466 -1475",
"The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance. K Emoto, J Z Parrish, L Y Jan, Jan , Y N , Nature. 443Emoto, K., Parrish, J. Z., Jan, L. Y., and Jan, Y. N. (2006) The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and main- tenance. Nature 443, 210 -213",
"Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity. J P Mcpherson, L Tamblyn, A Elia, E Migon, A Shehabeldin, E Matysiak-Zablocki, B Lemmers, L Salmena, A Hakem, J Fish, F Kassam, J Squire, B G Bruneau, M P Hande, R Hakem, EMBO J. 23McPherson, J. P., Tamblyn, L., Elia, A., Migon, E., Shehabeldin, A., Maty- siak-Zablocki, E., Lemmers, B., Salmena, L., Hakem, A., Fish, J., Kassam, F., Squire, J., Bruneau, B. G., Hande, M. P., and Hakem, R. (2004) Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity. EMBO J. 23, 3677-3688",
"Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. B Zhao, L Li, Q Lu, L H Wang, C Y Liu, Q Lei, K L Guan, Genes Dev. 25Zhao, B., Li, L., Lu, Q., Wang, L. H., Liu, C. Y., Lei, Q., and Guan, K. L. (2011) Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 25, 51-63",
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"Angiomotin regulates endothelial cell migration during embryonic angiogenesis. K Aase, M Ernkvist, L Ebarasi, L Jakobsson, A Majumdar, C Yi, O Birot, Y Ming, A Kvanta, D Edholm, P Aspenström, J Kissil, L Claesson-Welsh, A Shimono, L Holmgren, Genes Dev. 21Aase, K., Ernkvist, M., Ebarasi, L., Jakobsson, L., Majumdar, A., Yi, C., Birot, O., Ming, Y., Kvanta, A., Edholm, D., Aspenström, P., Kissil, J., Claesson-Welsh, L., Shimono, A., and Holmgren, L. (2007) Angiomotin regulates endothelial cell migration during embryonic angiogenesis. Genes Dev. 21, 2055-2068",
"Gridlock signalling pathway fashions the first embryonic artery. T P Zhong, S Childs, J P Leu, M C Fishman, Nature. 414Zhong, T. P., Childs, S., Leu, J. P., and Fishman, M. C. (2001) Gridlock signalling pathway fashions the first embryonic artery. Nature 414, 216 -220",
"A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. C D Wells, J P Fawcett, A Traweger, Y Yamanaka, M Goudreault, K Elder, S Kulkarni, G Gish, C Virag, C Lim, K Colwill, A Starostine, P Metalnikov, T Pawson, Cell. 125Wells, C. D., Fawcett, J. P., Traweger, A., Yamanaka, Y., Goudreault, M., Elder, K., Kulkarni, S., Gish, G., Virag, C., Lim, C., Colwill, K., Starostine, A., Metalnikov, P., and Pawson, T. (2006) A Rich1/Amot complex regu- lates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. Cell 125, 535-548",
"A tight junction-associated Merlin-angiomotin complex mediates Merlin's regulation of mitogenic signaling and tumor suppressive functions. C Yi, S Troutman, D Fera, A Stemmer-Rachamimov, J L Avila, N Christian, N L Persson, A Shimono, D W Speicher, R Marmorstein, L Holmgren, J L Kissil, Cancer Cell. 19Yi, C., Troutman, S., Fera, D., Stemmer-Rachamimov, A., Avila, J. L., Christian, N., Persson, N. L., Shimono, A., Speicher, D. W., Marmorstein, R., Holmgren, L., and Kissil, J. L. (2011) A tight junction-associated Mer- lin-angiomotin complex mediates Merlin's regulation of mitogenic signal- ing and tumor suppressive functions. Cancer Cell 19, 527-540",
"The adaptor protein AMOT promotes the proliferation of mammary epithelial cells via the prolonged activation of the extracellular signal-regulated kinases. W P Ranahan, Z Han, W Smith-Kinnaman, S C Nabinger, B Heller, B S Herbert, R Chan, C D Wells, Cancer Res. 71Ranahan, W. P., Han, Z., Smith-Kinnaman, W., Nabinger, S. C., Heller, B., Herbert, B. S., Chan, R., and Wells, C. D. (2011) The adaptor protein AMOT promotes the proliferation of mammary epithelial cells via the prolonged activation of the extracellular signal-regulated kinases. Cancer Res. 71, 2203-2211",
"Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor. M Paramasivam, A Sarkeshik, J R Yates, M J Fernandes, D Mccollum, Mol. Biol. Cell. 22Paramasivam, M., Sarkeshik, A., Yates, J. R., 3rd, Fernandes, M. J., and McCollum, D. (2011) Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor. Mol. Biol. Cell 22, 3725-3733",
"Opposing roles of angiomotin-like-1 and zona occludens-2 on pro-apoptotic function of YAP. T Oka, A P Schmitt, M Sudol, Oncogene. 31Oka, T., Schmitt, A. P., and Sudol, M. (2012) Opposing roles of angiomo- tin-like-1 and zona occludens-2 on pro-apoptotic function of YAP. Onco- gene 31, 128 -134",
"Angiomotin-like proteins associate with and negatively regulate YAP1. W Wang, J Huang, Chen , J , J. Biol. Chem. 286Wang, W., Huang, J., and Chen, J. (2011) Angiomotin-like proteins asso- ciate with and negatively regulate YAP1. J. Biol. Chem. 286, 4364 -4370",
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"-angiomotin associates to actin and controls endothelial cell shape. M Ernkvist, K Aase, C Ukomadu, J Wohlschlegel, R Blackman, N Veitonmäki, A Bratt, A Dutta, L Holmgren, FEBS J. 273130Ernkvist, M., Aase, K., Ukomadu, C., Wohlschlegel, J., Blackman, R., Veitonmäki, N., Bratt, A., Dutta, A., and Holmgren, L. (2006) p130-angi- omotin associates to actin and controls endothelial cell shape. FEBS J 273, 2000 -2011",
"Differential roles of p80-and p130-angiomotin in the switch between migration and stabilization of endothelial cells. M Ernkvist, O Birot, I Sinha, N Veitonmaki, S Nyströom, K Aase, L Holmgren, Biochim. Biophys. Acta. 1783Ernkvist, M., Birot, O., Sinha, I., Veitonmaki, N., Nyströom, S., Aase, K., and Holmgren, L. (2008) Differential roles of p80-and p130-angiomotin in the switch between migration and stabilization of endothelial cells. Biochim. Biophys. Acta 1783, 429 -437",
"A kinase shRNA screen links LATS2 and the pRB tumor suppressor. K Tschöp, A R Conery, L Litovchick, J A Decaprio, J Settleman, E Harlow, Dyson , N , Genes Dev. 25Tschöp, K., Conery, A. R., Litovchick, L., Decaprio, J. A., Settleman, J., Harlow, E., and Dyson, N. (2011) A kinase shRNA screen links LATS2 and the pRB tumor suppressor. Genes Dev. 25, 814 -830",
"LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression. T Chiyoda, N Sugiyama, T Shimizu, H Naoe, Y Kobayashi, J Ishizawa, Y Arima, H Tsuda, M Ito, K Kaibuchi, D Aoki, Y Ishihama, H Saya, S Kuninaka, J. Cell Biol. 197Chiyoda, T., Sugiyama, N., Shimizu, T., Naoe, H., Kobayashi, Y., Ishizawa, J., Arima, Y., Tsuda, H., Ito, M., Kaibuchi, K., Aoki, D., Ishihama, Y., Saya, H., and Kuninaka, S. (2012) LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression. J. Cell Biol. 197, 625-641",
"Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation. K Zhang, E Rodriguez-Aznar, N Yabuta, R J Owen, J M Mingot, H Nojima, M A Nieto, G D Longmore, EMBO J. 31Zhang, K., Rodriguez-Aznar, E., Yabuta, N., Owen, R. J., Mingot, J. M., Nojima, H., Nieto, M. A., and Longmore, G. D. (2012) Lats2 kinase poten- tiates Snail1 activity by promoting nuclear retention upon phosphoryla- tion. EMBO J. 31, 29 -43",
"Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Y Hirate, S Hirahara, K Inoue, A Suzuki, V B Alarcon, K Akimoto, T Hirai, T Hara, M Adachi, K Chida, S Ohno, Y Marikawa, K Nakao, A Shimono, H Sasaki, Curr. Biol. 23Hirate, Y., Hirahara, S., Inoue, K., Suzuki, A., Alarcon, V. B., Akimoto, K., Hirai, T., Hara, T., Adachi, M., Chida, K., Ohno, S., Marikawa, Y., Nakao, K., Shimono, A., and Sasaki, H. (2013) Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Curr. Biol. 23, 1181-1194",
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"The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version",
"Elucidation of a universal size-control mechanism in Drosophila and mammals",
"Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene",
"YAP1 increases organ size and expands undifferentiated progenitor cells",
"hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts",
"The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis",
"The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis",
"Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway",
"The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila",
") salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines",
"The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation",
"Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase",
"Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila",
"Control of cell proliferation and apoptosis by mob as tumor suppressor, mats",
"Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis",
"Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling",
"Hippo pathway regulation by cell morphology and stress fibers",
"Modulating F-actin organization induces organ growth by affecting the Hippo pathway",
"Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila",
"Role of YAP/TAZ in mechanotransduction",
"Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control",
"Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP",
"A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(-TRCP)",
"Tumor suppressor LATS1 is a negative regulator of oncogene YAP",
"TAZ promotes cell proliferation and epithelialmesenchymal transition and is inhibited by the hippo pathway",
"A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells",
"LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1",
"The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors",
"Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence",
"Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila",
"The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance",
"Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity",
"Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein",
"Stages of embryonic development of the zebrafish",
"Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay",
"Angiomotin regulates endothelial cell migration during embryonic angiogenesis",
"Gridlock signalling pathway fashions the first embryonic artery",
"A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells",
"A tight junction-associated Merlin-angiomotin complex mediates Merlin's regulation of mitogenic signaling and tumor suppressive functions",
"The adaptor protein AMOT promotes the proliferation of mammary epithelial cells via the prolonged activation of the extracellular signal-regulated kinases",
"Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor",
"Opposing roles of angiomotin-like-1 and zona occludens-2 on pro-apoptotic function of YAP",
"Angiomotin-like proteins associate with and negatively regulate YAP1",
"A chemical genetic approach for the identification of direct substrates of protein kinases",
"-angiomotin associates to actin and controls endothelial cell shape",
"Differential roles of p80-and p130-angiomotin in the switch between migration and stabilization of endothelial cells",
"A kinase shRNA screen links LATS2 and the pRB tumor suppressor",
"LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression",
"Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation",
"Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos",
"TEAD mediates YAP-dependent gene induction and growth control",
"Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon"
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"\nFIGURE 1 .\n1The Hippo pathway phosphorylates angiomotin family proteins. A, Lats2 induces mobility shift of AMOT family proteins. HEK293 cells were transfected with plasmids encoding Flag-YAP or AMOT family proteins with or without Lats2 co-transfection. Cell lysates were resolved on Phos-tag-containing gels. Vps34 is a negative control. B, phosphatase eliminates AMOT mobility shift induced by Lats2. Flag-AMOTp130 was immunoprecipitated from transfected cells and treated with lambda protein phosphatase as indicated. Samples were analyzed as that in A. C, hippo pathway proteins synergistically induces AMOTp130 mobility shift. Lysates of transfected cells were resolved on Phos-tag-containing gels for mobility shift of Flag-AMOTp130. D, Lats2 phosphorylates AMOTp130 in vitro. In vitro phosphorylation of GST-AMOTp130 by immunoprecipitated Lats2 was performed. (S), short exposure; (L), long exposure.",
"\nFIGURE 2 .\n2AMOTp130 is phosphorylated on Ser-175 by Lats1/2 in response to GPCR signaling. A, domain organization of AMOTp130 and alignment of AMOT family proteins from different species. B, phosphorylation of Ser-175 leads to Lats2-induced AMOTp130 up-shift. Lysates of transfected HEK293 cells were analyzed by Western blots. C, Ser-175 of AMOTp130 is phosphorylated by Lats2. AMOTp130 phosphorylation was detected by pAMOT (S175)-specific antibody. D, Lats2 phosphorylates AMOTp130 on Ser-175 in vitro.",
"\nFIGURE 4\n4FIGURE 4. Phosphorylation inhibits endogenous AMOTp130-F-actin interaction and direct AMOTp130-F-actin interaction in vitro. A, endogenous AMOT colocalizes with F-actin. Scramble or AMOT-specific siRNA transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, endogenous AMOT colocalizes with F-actin. Experiments were similar to these in A except that endogenous AMOT was stained with anti-AMOT antibody #2. C, Lats2 inhibits AMOT colocalization with F-actin in a kinase-dependent manner. HA-Lats2 wild-type or KR transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130 and anti-HA for Lats2 expression. D, quantification of filamentous localization of endogenous AMOT. Cells with or without filamentous AMOT in A and C were quantified. 120 cells were quantified for each sample. E, phospho-mimetic mutation inhibits AMOTp130 association with F-actin in vitro. Recombinant GST-AMOT wild-type or N-terminal fragment (N) were subjected to in vitro actin spin-down assay. (S), supernatant; (P), pellet. Actin was examined by Coomassie Blue staining and GST-AMOT was determined by Western blots. Asterisks denote nonspecific proteins. F, AMOTp130 does not co-immunoprecipitate with G-actin. HEK293 cells were transfected as indicated. Transfection amount was adjusted to make soluble AMOT levels comparable. Lysates were immunoprecipitated with anti-Flag antibody. The presence of endogenous actin and Flag-AMOTp130 in immunoprecipitates and lysates were examined by Western blots. G, phosphorylation of wild-type AMOTp130 by Lats2 does not affect interaction between AMOTp130 and YAP. HEK293 cells were transfected as indicated. AMOTp130 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of YAP wild-type or 5SA mutant was examined by anti-Flag Western blots. AMOTp130 phosphorylation was shown by electrophoretic mobility shift on Phos-tag containing gel.",
"\nFIGURE 6 .\n6Knockdown of Lats1/2 induces cell migration, actin stress fiber and focal adhesion in an AMOT-dependent manner.A, Lats1/2 plays a role in serum deprivation-induced migration retardation by repressing AMOT. siRNA-transfected HUVEC cells were serum starved and then seeded in transwell inserts in serum-free medium. The lower chambers were filled with serum-containing or serum-free medium as indicated. Cells on bottom sides of the transwells were stained after 12 h. B, Lats1/2 plays a role in serum deprivation-induced stress fiber and focal adhesion deformation by repressing AMOT. Cells were the same as these in A. Cells were serum starved overnight and then seeded on fibronectin-coated coverslips for 2 h either in serum-containing medium or serum-free medium as indicated. Focal adhesions and F-actin were stained with anti-vinculin and AlexaFluor488-Phalloidin respectively. C, number of migrated cells in A was quantified. Experiments were in duplicates. D, size of 25 FAs from the staining of each stable cell as these in B was quantified by ImageJ and drawn into scatter plot. The median number was indicated.",
"\nFIGURE 7 .\n7Phosphorylation of AMOTp130 by Lats1/2 regulates angiogenesis in zebrafish. The fluorescent microscopy analyses revealed the development of ISV and DLAV at 30 hpf and 36 hpf in the trunk region of control Tg(flk:GFP) embryos and embryos injected with AMOT-MO or a combination of AMOT-MO and mRNAs encoding human AMOTp130, Lats2 or their mutants. Red asterisk: defective ISV; red arrow: defective DLAV. ISV: intersegmental vessels. DLAV: dorsal longitudinal anastomotic vessels. Embryos were orientated with anterior to the left. Histogram depicted the quantification of normal and angiogenesis defective embryos at 30 hpf. n, embryo number.",
"\nFIGURE 8 .\n8A model of Lats1/2 in regulation of cell migration through phosphorylation of AMOT and YAP. Phosphorylation of AMOT by Lats1/2 inhibits F-actin association and then actin stress fiber and focal adhesion formation. These effects of AMOT on cell structure and cell adhesion may work independently or in cooperation with YAP-dependent gene transcription alteration downstream of Lats1/2 to regulate cell migration."
] | [
"The Hippo pathway phosphorylates angiomotin family proteins. A, Lats2 induces mobility shift of AMOT family proteins. HEK293 cells were transfected with plasmids encoding Flag-YAP or AMOT family proteins with or without Lats2 co-transfection. Cell lysates were resolved on Phos-tag-containing gels. Vps34 is a negative control. B, phosphatase eliminates AMOT mobility shift induced by Lats2. Flag-AMOTp130 was immunoprecipitated from transfected cells and treated with lambda protein phosphatase as indicated. Samples were analyzed as that in A. C, hippo pathway proteins synergistically induces AMOTp130 mobility shift. Lysates of transfected cells were resolved on Phos-tag-containing gels for mobility shift of Flag-AMOTp130. D, Lats2 phosphorylates AMOTp130 in vitro. In vitro phosphorylation of GST-AMOTp130 by immunoprecipitated Lats2 was performed. (S), short exposure; (L), long exposure.",
"AMOTp130 is phosphorylated on Ser-175 by Lats1/2 in response to GPCR signaling. A, domain organization of AMOTp130 and alignment of AMOT family proteins from different species. B, phosphorylation of Ser-175 leads to Lats2-induced AMOTp130 up-shift. Lysates of transfected HEK293 cells were analyzed by Western blots. C, Ser-175 of AMOTp130 is phosphorylated by Lats2. AMOTp130 phosphorylation was detected by pAMOT (S175)-specific antibody. D, Lats2 phosphorylates AMOTp130 on Ser-175 in vitro.",
"FIGURE 4. Phosphorylation inhibits endogenous AMOTp130-F-actin interaction and direct AMOTp130-F-actin interaction in vitro. A, endogenous AMOT colocalizes with F-actin. Scramble or AMOT-specific siRNA transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, endogenous AMOT colocalizes with F-actin. Experiments were similar to these in A except that endogenous AMOT was stained with anti-AMOT antibody #2. C, Lats2 inhibits AMOT colocalization with F-actin in a kinase-dependent manner. HA-Lats2 wild-type or KR transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130 and anti-HA for Lats2 expression. D, quantification of filamentous localization of endogenous AMOT. Cells with or without filamentous AMOT in A and C were quantified. 120 cells were quantified for each sample. E, phospho-mimetic mutation inhibits AMOTp130 association with F-actin in vitro. Recombinant GST-AMOT wild-type or N-terminal fragment (N) were subjected to in vitro actin spin-down assay. (S), supernatant; (P), pellet. Actin was examined by Coomassie Blue staining and GST-AMOT was determined by Western blots. Asterisks denote nonspecific proteins. F, AMOTp130 does not co-immunoprecipitate with G-actin. HEK293 cells were transfected as indicated. Transfection amount was adjusted to make soluble AMOT levels comparable. Lysates were immunoprecipitated with anti-Flag antibody. The presence of endogenous actin and Flag-AMOTp130 in immunoprecipitates and lysates were examined by Western blots. G, phosphorylation of wild-type AMOTp130 by Lats2 does not affect interaction between AMOTp130 and YAP. HEK293 cells were transfected as indicated. AMOTp130 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of YAP wild-type or 5SA mutant was examined by anti-Flag Western blots. AMOTp130 phosphorylation was shown by electrophoretic mobility shift on Phos-tag containing gel.",
"Knockdown of Lats1/2 induces cell migration, actin stress fiber and focal adhesion in an AMOT-dependent manner.A, Lats1/2 plays a role in serum deprivation-induced migration retardation by repressing AMOT. siRNA-transfected HUVEC cells were serum starved and then seeded in transwell inserts in serum-free medium. The lower chambers were filled with serum-containing or serum-free medium as indicated. Cells on bottom sides of the transwells were stained after 12 h. B, Lats1/2 plays a role in serum deprivation-induced stress fiber and focal adhesion deformation by repressing AMOT. Cells were the same as these in A. Cells were serum starved overnight and then seeded on fibronectin-coated coverslips for 2 h either in serum-containing medium or serum-free medium as indicated. Focal adhesions and F-actin were stained with anti-vinculin and AlexaFluor488-Phalloidin respectively. C, number of migrated cells in A was quantified. Experiments were in duplicates. D, size of 25 FAs from the staining of each stable cell as these in B was quantified by ImageJ and drawn into scatter plot. The median number was indicated.",
"Phosphorylation of AMOTp130 by Lats1/2 regulates angiogenesis in zebrafish. The fluorescent microscopy analyses revealed the development of ISV and DLAV at 30 hpf and 36 hpf in the trunk region of control Tg(flk:GFP) embryos and embryos injected with AMOT-MO or a combination of AMOT-MO and mRNAs encoding human AMOTp130, Lats2 or their mutants. Red asterisk: defective ISV; red arrow: defective DLAV. ISV: intersegmental vessels. DLAV: dorsal longitudinal anastomotic vessels. Embryos were orientated with anterior to the left. Histogram depicted the quantification of normal and angiogenesis defective embryos at 30 hpf. n, embryo number.",
"A model of Lats1/2 in regulation of cell migration through phosphorylation of AMOT and YAP. Phosphorylation of AMOT by Lats1/2 inhibits F-actin association and then actin stress fiber and focal adhesion formation. These effects of AMOT on cell structure and cell adhesion may work independently or in cooperation with YAP-dependent gene transcription alteration downstream of Lats1/2 to regulate cell migration."
] | [
"(Fig. 1A)",
"(Fig. 1A)",
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"Fig. 2A)",
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"(Fig. 2B)",
"(Fig. 2C)",
"(Fig. 2D)",
"(Fig. 2, E and F)",
"(Fig. 2G)",
"(Fig. 2H)",
"(Fig. 2I)",
"(Fig. 3A)",
"(Fig. 3A)",
"(Fig. 3A, middle panel)",
"(Fig. 3, B and C)",
"(Fig. 3, A and F)",
"(Fig. 3, D and F)",
"(Fig. 3, E and F)",
"(Fig. 4, A, B, D)",
"Fig. 1D",
"Fig. 3A",
"Fig. 3A, top panel;",
"Fig. 3E",
"(Fig. 4, C and D)",
"(Fig. 4E)",
"(Fig. 4E)",
"(Fig. 4F)",
"(Fig. 4G)",
"(Fig. 5A)",
"(Fig. 5, B and D)",
"(Fig. 5, B and D)",
"(Fig. 5, B and D)",
"(Fig. 5, C and E)",
"(Fig. 5, C and E)",
"(Fig. 6",
"(Fig. 6, A and C)",
"(Fig. 6, B and D)",
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"Fig. 7-2)",
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"(Fig. 8)"
] | [] | [
"The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/ TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.",
"Organ size homeostasis is a remarkable feature of multicellular organisms. In the last decade, the Hippo pathway has been found to play a key role in control of organ size (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). At the center stage of this pathway is the Mst1/2-Lats1/2 kinase cascade. Upstream signals such as cell adhesion, cytoskeleton remodeling, lysophosphatidic acid (LPA) 2 and its respective G-protein-coupled receptors (GPCRs) were found to regulate the Hippo pathway (15)(16)(17)(18)(19)(20)(21)(22)(23). YAP transcription co-activator and its paralog TAZ are the best known Hippo pathway targets mediating gene expression regulation and organ size control (22, 24 -27). Nevertheless, the various upstream input signals suggest rich functions of the pathway. Indeed, Lats1/2 were reported to regulate cellular processes such as cell differentiation, cytokinesis, senescence, autophagy, centrosome duplica-tion, and neuron dendritic tiling (28 -33). It is unlikely that YAP/TAZ inactivation mediates all these functions. However, YAP/TAZ-independent functions of the Hippo pathway were poorly studied.",
"Here we report that the angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and zebrafish embryonic angiogenesis in vivo. These studies identified AMOT as a critical effector of the Hippo pathway downstream of GPCR signaling in regulation of cell migration and angiogenesis.",
"Antibodies, Plasmids, and Other Materials-We obtained anti-AMOT antibodies from Bethyl Lab; anti-Lats1 and anti-Lats2 from Cell Signaling Technologies; anti-GST from Genscript; anti-␣-tubulin, anti-Flag, and anti-vinculin from Sigma; anti-thiophosphate ester from Epitomics; anti-HSP90 from BD Biosciences; anti-HA and anti-Myc from Covance; Alexa Fluor 488-or 594-conjugated secondary antibodies and Alexa Fluor 488-phalloidin from Invitrogen; Horseradish peroxidase-conjugated secondary antibodies from GE Healthcare. Anti-phos-phoAMOTp130 (S175) antibody was generated by immunizing rabbits with phospho-peptide KQGHVRSLS(p)ERL. Human AMOTp130 were subcloned from other vectors into pCMV-Flag, pcDNA3-HA, pGEX-KG, and pQCXIH vectors. Human AMOTp80, mouse AMOTL1 and AMOTL2 were subcloned into pcDNA3-HA and pCMV-Flag vectors. Human AMOTp130 mutants were generated by site-directed mutagenesis. Other plasmids were described before (16,22,34). Phos-tag conjugated acrylamide was purchased from Wako Chemicals. All other chemicals were from Sigma.",
"Cell Culture, Transfection, and Viral Infection-HEK293, HEK293T, COS7, HEK293P, and HUVEC cells were cultured in DMEM (Invitrogen) containing 10% FBS (Invitrogen) and 50 g/ml penicillin/streptomycin (P/S). Transfection with Lipofectamine (Invitrogen) was performed according to the manufacturer's instructions. For viral infection, HEK293P cells were transfected with viral constructs and packaging plasmids. 48 h later, viral supernatant was supplemented with 5 g/ml polybrene, filtered through a 0.45 m filter, and used to infect target cells.",
"Immunoprecipitation and Kinase Assay-For Lats2 kinase assays, HEK293 cells were transfected with indicated plasmids. 48 h post-transfection, cells were lysed with lysis buffer (50 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na 3 VO 4 , protease inhibitor mixture (Roche), 1 mM DTT, 1 mM PMSF) and immunoprecipitated with anti-HA antibody. The immunoprecipitates were washed three times with lysis buffer, once with wash buffer (40 mM HEPES, 200 mM NaCl), and once with kinase assay buffer (30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl 2 ). The immunoprecipitated Lats2 was subjected to kinase assay in the presence of 500 M ATP or ATP-␥S and 1 g of recombinant GST-AMOTp130 purified from Escherichia coli as substrates. The reaction mixtures were incubated for 30 min at 30°C. For detection by antithiophosphate-ester antibody, the reaction mixtures were further supplemented with 2.5 mM PNBM. Alkylating reactions were allowed to proceed for 1 h at room temperature. The reactions were terminated with SDS sample buffer and boiled before analysis by SDS-PAGE.",
"Actin Spin-down Assay-Actin Binding Protein Biochem Kit was obtained from Cytoskeleton. The assay was performed following the manufacturer's instructions. Briefly, F-actin was polymerized and mixed with GST-AMOTp130 proteins purified from E. coli. The mixture was then centrifuged at 150,000 ϫ g for 1.5 h at 24°C. Supernatants and pellets were then collected and processed for electrophoresis. Proteins were visualized by Coomassie Blue staining or Western blots.",
"Cell Migration Assay-Cell migration assay was performed using BD Falcon Cell culture inserts for 24-well plates with 8.0 m pore size. Bottom sides of filters were pre-coated with 20 mg/ml fibronectin. HUVEC cells were serum-starved for 12 h and then seeded into the upper chambers of the inserts at 4 ϫ 10 4 cells/well in serum-free medium, and lower chambers were filled with serum-free or 10% FBS-containing medium. After 12 h, cells were stained with 0.5% crystal violet. Cells in upper chambers were carefully removed, and bottom sides of the chambers were pictured.",
"Immunofluorescence Staining-Cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100. Cells may be treated with 100 g/ml digitonin for 5 min as indicated. After blocking in 2% BSA for 30 min, slides were incubated with first antibody diluted in 1% BSA overnight at 4°C. After washing with PBS, slides were incubated with Alexa Fluor 488-or 594-conjugated secondary antibodies for 1.5 h. For staining of F-actin, cells were incubated with Alexa Fluor 488-phalloidin for 1.5 h. The slides were then washed and mounted.",
"RNA Interference-Short interfering RNA (siRNA) oligonucleotides toward human Lats1, Lats2, AMOT, and control siRNA toward firefly luciferase were transfected into indicated cells using Lipofectamine RNAiMax Reagent (Invitrogen) following the manufacturer's instructions. Cells were analyzed 72 h post-transfection.",
"Zebrafish Maintenance and Angiogenesis Assay-Zebrafish embryos were produced by pairwise matings, raised at 28.5°C and staged as described (35). A transgenic Tg(flk:GFP) line was used in this study (36). Sense-capped mRNAs were synthesized using mMESSAGE mMACHINE system (Ambion) according to the manufacturer's instructions. Plasmids encoding human AMOTp130, AMOTp130-SA, AMOTp130-S.D., mouse Lats2, or Lats2-KR were digested with XhoI and transcribed with T7 polymerase. Poly (A) tails (Takara Bio Cat. No. 2181) were added to the synthetic mRNAs. Synthesized mRNAs were purified using the MEGAclear Kit (Ambion). Antisense morpholino oligonucleotide AMOT-MO (5Ј-CCACTGACACAACTAC-CACCAAGTG-3Ј) (37) was synthesized by Gene Tools, LLC. Synthetic mRNAs and morpholinos were microinjected into zebrafish embryos at the one-two-cell stages as described (38). Injection doses were as following: 8.2 ng AMOT-MO; 8.2 ng AMOT-MOϩ400pg hAMOTp130-WT/SA/S.D.; 5.6 ng AMOT-MOϩ373pg hAMOTp130-WT/SA ϩ80pg mLats2-WT/KR. Vascular phenotypes were imaged using a Zeiss Observer Z1 microscope.",
"The AMOT family proteins localize to specific cellular compartments and regulate cell migration and proliferation (37, 39 -41). AMOT is a component of the Hippo pathway capable of inhibiting YAP through direct binding and Lats1/2 activation (34,(42)(43)(44). Surprisingly, we found that Lats2 induced a dramatic electrophoretic mobility shift of AMOTp130 comparable to that of YAP on Phos-tag-containing gels which specifically retards phosphorylated proteins (Fig. 1A). Furthermore, Lats2 also induced dramatic up-shift of other AMOT family proteins AMOTL1 and AMOTL2 (Fig. 1A). The Lats2-induced mobility shift of AMOTp130 was eliminated by treatment of the protein with lambda protein phosphatase (Fig. 1B). In addition, co-transfection of AMOTp130 with other Hippo pathway components Mst2, Sav, Mob, and Lats2 induced AMOTp130 mobility shift in a synergistic manner (Fig. 1C). These experiments indicate that Lats1/2 in the Hippo pathway could induce phosphorylation of AMOT family proteins.",
"We then examined whether Lats2 could phosphorylate AMOT in vitro. In this assay, the kinase utilizes ATP-␥S as phosphate donor to generate thiophosphorylated substrate, which in turn reacts with p-nitrobenzyl mesylate (PNBM) to form a thiophosphate-ester (45). As detected by a thiophosphate-ester-specific antibody, recombinant AMOTp130 could be phosphorylated by Lats2 in vitro (Fig. 1D). The phosphorylation is more efficient when Mob and Sav were co-transfected. In contrast, the kinase inactive Lats2-KR could not phosphorylate AMOTp130. Thus, Lats2 phosphorylates AMOTp130 both in vitro and in vivo.",
"Lats2 Phosphorylates AMOT on Serine 175-Previous studies have revealed the optimal Lats1/2 phosphorylation target consensus motif as HXRXXS (22,25). Interestingly, we found one HXRXXS motif, and one HXKXXS motif, also permissive for phosphorylation by Lats1/2, on AMOTp130 ( Fig. 2A). Both motifs are conserved in AMOT family members and across species ( Fig. 2A). As shown in Fig. 2B, mutation of Ser-175 in the N-terminal motif to alanine largely repressed Lats2-induced up-shift of AMOTp130, suggesting that this reside is phosphorylated by Lats2. Further mutation of Ser-859 eliminates the mild shift of S175A, suggesting that Ser-859 is also phosphorylated by Lats2. However, individual mutation of Ser-859 did not repress the up-shift possibly due to limited sensitivity of the Phos-tag method. The histidine in the HXRXXS motif is crucial for substrate recognition. Indeed, similar to S175A, mutation of H170 largely abolished AMOTp130 upshift in response to Lats2 (Fig. 2B). These experiments demonstrate that phosphorylation of Ser-175 is largely responsible for Lats2-induced up-shift of AMOTp130.",
"To further confirm that AMOTp130 Ser-175 is indeed phosphorylated by Lats2, we generated an anti-pAMOT (Ser-175) antibody. As expected, this antibody detected phosphorylation of ectopically expressed AMOTp130, which was markedly enhanced by Lats2 co-expression and eliminated by phosphatase treatment (Fig. 2C). Furthermore, AMOTp130 is phosphorylated on Ser-175 by Lats2 but not Lats2-KR in vitro (Fig. 2D). Thus, AMOTp130 is phosphorylated on Ser-175 by Lats2 in vitro and in vivo.",
"GPCR Signaling Inhibits AMOTp130 Phosphorylation Mediated by the Hippo Pathway-Following identification of the phosphorylation site we further examined the role of the Hippo pathway in physiological regulation of AMOT. Consistent with the inhibition of the Hippo pathway by serum and LPA, these stimulations inhibit endogenous AMOTp130 phosphorylation on Ser-175 (Fig. 2, E and F). LPA in serum represses the Hippo pathway via activation of GPCRs such as LPAR2, and the coupled G-proteins such as G q and G 12 (16). Consistently, expression of LPAR2 or active QL mutants of G q or G 12 potently inhibits endogenous AMOTp130 phosphorylation in serumdeprived cells (Fig. 2G). Furthermore, siRNA knockdown of Lats1 and Lats2 largely eliminates AMOTp130 phosphorylation on Ser-175 in serum-deprived cells (Fig. 2H). Integrity of the actin cytoskeleton is required for GPCR-induced repression of the Hippo pathway (16). Indeed, disruption of F-actin induced AMOTp130 phosphorylation in cells cultured in serum-rich medium (Fig. 2I). Taken together, serum and GPCR signaling regulate AMOTp130 phosphorylation on Ser-175 through Lats1/2 kinases.",
"Phosphorylation of AMOTp130 on Ser-175 Inhibits Its Interaction with F-actin-AMOTp130 is known to colocalize with F-actin in cells dependent on its N-terminal domain containing Ser-175 (46). By immunofluorescence staining, we indeed observed colocalization of ectopically expressed AMOTp130 with F-actin filaments (Fig. 3A). At lower expression level of AMOT, the AMOT-F-actin filaments closely resemble actin stress fibers (Fig. 3A). However, at higher expression level, the filaments are thicker possibly represent AMOT-F-actin filament bundles (Fig. 3A, middle panel). In support of a real stress fiber identity of AMOT-F-actin filaments, they are resistant to digitonin treatment before fixation and are anchored to focal NOVEMBER 22, 2013 • VOLUME 288 • NUMBER 47 adhesions (Fig. 3, B and C). Interestingly, expression of Lats2 disrupted the filamentous AMOTp130 and results in diffused and irregular dots-like localization of AMOT (Fig. 3, A and F). Thus phosphorylation of AMOTp130 on Ser-175 by Lats2 may disrupt AMOT-F-actin interaction. Consistently, the S175A mutant colocalizes with F-actin in a manner insensitive to Lats2 (Fig. 3, D and F). More strikingly, irrespective to Lats2 expression, the phospho-mimetic S175D mutant exhibited dots-like cytoplasmic localization (Fig. 3, E and F). Importantly, using two independent antibodies, we demonstrated that endogenous AMOT colocalizes with F-actin in 293T cells (Fig. 4, A, B, D). The filamentous local- Experiments were similar to that in Fig. 1D except that regular ATP was used. E, serum inhibits phosphorylation of endogenous AMOTp130 on Ser-175. Serum-starved 293T cells were stimulated with 10% FBS for the indicated time before harvest. F, LPA inhibits phosphorylation of endogenous AMOTp130 on Ser-175. Serum-starved 293T cells were stimulated with serum or LPA for 1 h before harvest. G, GPCR signaling inhibits Ser-175 phosphorylation of endogenous AMOTp130. 293T cells were transfected with indicated plasmids and serum-starved overnight before harvest. H, knockdown of Lats1 and Lats2 represses endogenous AMOTp130 phosphorylation on Ser-175. HEK293T cells were transfected with scramble or Lats1-and Lats2-specific siRNAs. Cells were serum-starved overnight before harvest. I, disruption of F-actin induces phosphorylation of AMOTp130. HEK293T cells cultured in serum-rich medium were treated with 1 g/ml Latrunculin B (LatB) for 1 h as indicated before being harvested for Western blot analysis.",
"A, Lats2 inhibits colocalization of AMOTp130 with F-actin. Transfected COS7 cells were stained with anti-Flag antibody for localization of AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, colocalization of AMOTp130 with F-actin in digitonin-treated cells. Flag-AMOTp130 transfected Cos7 cells were treated with digitonin before fixation and stained as that in A. C, AMOT-F-actin filaments are anchored to focal adhesions. COS7 cells expressing wild-type or mutant AMOTs were co-stained with anti-Flag for AMOT and anti-vinculin to visualize focal adhesions. D, phospho-deficient mutant of AMOTp130 constitutively colocalizes with F-actin. Experiments were similar to these in A except the use of S175A mutant. E, phospho-mimetic mutant of AMOTp130 loses colocalization with F-actin. Experiments were similar to these in A except the use of S175D mutant. F, quantification of AMOT-F-actin colocalization. 120 cells were quantified for each transfection. Thick filament is similar to Fig. 3A, middle panel; thin filament is similar to that in Fig. 3A, top panel; non filamentous is similar to that in Fig. 3E, top panel. ",
"A, endogenous AMOT colocalizes with F-actin. Scramble or AMOT-specific siRNA transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130, AlexaFluor488-Phalloidin for F-actin, and DAPI for cell nuclei. B, endogenous AMOT colocalizes with F-actin. Experiments were similar to these in A except that endogenous AMOT was stained with anti-AMOT antibody #2. C, Lats2 inhibits AMOT colocalization with F-actin in a kinase-dependent manner. HA-Lats2 wild-type or KR transfected HEK293T cells were stained with anti-AMOT antibody #1 for localization of endogenous AMOTp130 and anti-HA for Lats2 expression. D, quantification of filamentous localization of endogenous AMOT. Cells with or without filamentous AMOT in A and C were quantified. 120 cells were quantified for each sample. E, phospho-mimetic mutation inhibits AMOTp130 association with F-actin in vitro. Recombinant GST-AMOT wild-type or N-terminal fragment (N) were subjected to in vitro actin spin-down assay. (S), supernatant; (P), pellet. Actin was examined by Coomassie Blue staining and GST-AMOT was determined by Western blots. Asterisks denote nonspecific proteins. F, AMOTp130 does not co-immunoprecipitate with G-actin. HEK293 cells were transfected as indicated. Transfection amount was adjusted to make soluble AMOT levels comparable. Lysates were immunoprecipitated with anti-Flag antibody. The presence of endogenous actin and Flag-AMOTp130 in immunoprecipitates and lysates were examined by Western blots. G, phosphorylation of wild-type AMOTp130 by Lats2 does not affect interaction between AMOTp130 and YAP. HEK293 cells were transfected as indicated. AMOTp130 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of YAP wild-type or 5SA mutant was examined by anti-Flag Western blots. AMOTp130 phosphorylation was shown by electrophoretic mobility shift on Phos-tag containing gel.",
"ization of endogenous AMOT is also disrupted by Lats2 in a kinase-dependent manner (Fig. 4, C and D). These data demonstrate that phosphorylation of AMOTp130 by Lats2 on Ser-175 inhibits AMOTp130-F-actin interaction in vivo.",
"To test the possibility of a direct interaction between AMOTp130 and F-actin we performed in vitro F-actin spindown assay. In this assay, F-actin binding proteins would cosediment with pre-polymerized F-actin. We found that addition of F-actin brought all recombinant AMOTp130 or the N-terminal fragment into the pellet fraction indicating direct binding of AMOTp130 to F-actin (Fig. 4E). However, the phospho-mimetic S175D mutant largely remains in the supernatant (Fig. 4E). These results suggest that phosphorylation of AMOTp130 on Ser-175 would be inhibitory on the direct interaction of AMOTp130 with F-actin in vitro. In contrast, we did not observe any interaction of AMOTp130 with monomeric G-actin by co-immunoprecipitation (Fig. 4F), suggesting that AMOTp130 specifically interacts with F-actin. These results indicate that AMOTp130 directly interacts with F-actin under negative regulation by phosphorylation of Ser-175.",
"Migration-AMOTp130 is known to directly bind and inhibit YAP (34,43,44). However, although AMOT-SA mutant show some decreased interaction with YAP, the interaction between YAP and wild-type AMOT is not significantly affected by AMOT phosphorylation (Fig. 4G). AMOT is also known for its roles in endothelial cell morphology and migration (47). Therefore we examined the function of AMOTp130 phosphorylation by Lats1/2 in endothelial cells. In agreement FIGURE 5. Phosphorylation of AMOTp130 on Ser-175 inhibits endothelial cell migration. A, knockdown efficiency of AMOT siRNAs. AMOT, CTGF, and Cyr61 levels in siRNA transfected HUVEC cells were determined by realtime RT-PCR. B, phospho-mimetic mutant of AMOT is unable to compensate endogenous AMOT in actin stress fiber and focal adhesion formation. Control or AMOT-expressing stable cells transfected with siRNAs were serum-starved overnight and then seeded on fibronectin-coated coverslips for 2 h in serum-containing medium. Focal adhesions and F-actin were stained with anti-vinculin and Alex-aFluor488-Phalloidin, respectively. C, phospho-mimetic AMOT mutant could not rescue cell migration defects caused by loss of AMOT. Cells the same as these in B were examined in transwell migration assay. D, size of 60 FAs from the staining of each stable cell as these in B was quantified by ImageJ and drawn into scatter plot. The median number was indicated. E, number of migrated cells in C was quantified. Experiments performed were in duplicates.",
"with our previous observations, knockdown of AMOT induces expression of YAP target genes CTGF and Cyr61 (Fig. 5A). Interestingly, consistent with a direct association of AMOT with F-actin, knockdown of AMOT leads to reduced stress fibers in cells, which also correlates with reduced focal adhesions (Fig. 5, B and D). The defects are partially rescued by re-expression of AMOTp80 and better rescued by re-expression of AMOTp130 (Fig. 5, B and D). Interestingly, co-expression of AMOTp130 wild-type or SA mutant but not the SD mutant together with AMOTp80 rescues stress fiber and focal adhesion formation to a level similar to control cells (Fig. 5, B and D). Thus phosphorylation of AMOTp130 plays an inhibitory role on stress fiber and focal adhesion formation.",
"Remodeling of actin cytoskeleton and cell adhesion is crucial to cell migration. Consistent with alterations of the cytoskeleton and focal adhesions, knockdown of AMOT largely inhibits endothelial cell migration (Fig. 5, C and E). Again, we observed that co-expression of AMOTp130 wild-type or SA mutant but not the SD mutant together with AMOTp80 rescues the cell migration defects (Fig. 5, C and E). This suggests that phosphorylation of AMOT on Ser-175 inhibits cell migration. We then further investigated the role of AMOT in cell migration downstream of the Hippo signaling. Serum starvation is well known to strongly inhibit cell migration in vitro. However, we found that cell migration in starvation condition is partially but significantly rescued by knockdown of Lats1 and Lats2 (Fig. 6, A and C). This result suggests that activation of Lats1/2 by serum starvation due to blunted GPCR signaling is an important reason for repression of cell migration. More importantly, simultaneous knockdown of AMOT blocks cell migration induced by loss of Lats1/2 expression (Fig. 6, A and C). Thus inhibition of the Hippo pathway leads to cell migration in an AMOT-dependent manner. In correlation with cell migration, knockdown of Lats1/2 also induces actin stress fiber and focal adhesion formation in serum-starved cells in an AMOT-dependent manner (Fig. 6, B and D). Our data suggest that AMOTp130 is important for endothelial cell migration under negative regulation by Lats1/2-induced phosphorylation.",
"Phosphorylation of AMOTp130 on Ser-175 Inhibits Angiogenesis in Zebrafish-AMOT is known to control endothelial cell migration and angiogenesis in zebrafish (37). Thus we further investigated the effects of AMOTp130 phosphorylation on angiogenesis in zebrafish Tg(flk:GFP) transgenic embryos, in which GFP expression is driven by endothelial specific promoter flk. Consistent with the previous study (37), amot knockdown via injection of AMOT antisense morpholino-oligonu- cleotide (AMOT-MO) caused angiogenesis defects in AMOT morphants, in which intersegmental vessels (ISV) were disrupted and failed to reach the dorsolateral region at 30 h postfertilization (hpf) (Fig. 7-2). At 36 hpf, some ISV had still not arrived the most dorsolateral position so that the dorsal longitudinal anastomotic vessel (DLAV) formation was disrupted ( Fig. 7-2). Nevertheless, the angiogenesis defects are rescued by co-injection of mRNAs encoding human AMOTp130 ( Fig. 7-3) or the phospho-deficient SA mutant (Fig. 7-4) but not the phospho-mimetic SD mutant (Fig. 7-5). Furthermore, the angiogenic activity of AMOTp130 is inhibited by co-injection of mRNAs encoding Lats2 (Fig. 7-6) but not Lats2-KR ( Fig. 7-7). In addition, the angiogenic activity of AMOTp130-SA is insensitive to Lats2 (Fig. 7-8). The overall development and morphologies of injected embryos were comparable to control embryos at bud stage and 30 hpf (data not shown). These results indicate that Lats2 could inhibit the angiogenic function of AMOTp130 in vivo through phosphorylation of Ser-175.",
"Biological functions of kinases are largely determined by substrate selectivity. However, known substrates of the Lats1/2 kinases are very limited. Besides YAP/TAZ, several other proteins have been suggested as Lats1/2 substrates including Dyrk1a, MYPT1, and Snail1 (48 -50). In this study, we identified AMOT family proteins to be new substrates of Lats1/2 with canonical target consensus motifs being phosphorylated by Lats1/2 to comparable levels as YAP/TAZ. Importantly, this phosphorylation is responsive to GPCR signaling and plays a regulatory role on AMOT interaction with F-actin thus regu-lates cell migration. While this report was being prepared, an independent study reported the phosphorylation of AMOT by Lats1/2 in promoting AMOT-Lats1/2 interaction, and its role in cell fate specification in preimplantation mouse embryos (51). Together, these studies suggest AMOT as an important effector of the Hippo pathway. We anticipate more substrates of Lats1/2 to be discovered to explain the ever expanding functions of the Hippo pathway.",
"AMOTp130 also plays a regulatory role in the Hippo pathway through both physical binding with YAP/TAZ and promotion of Lats1/2 activity (34,(42)(43)(44). In addition, AMOT also interacts with Merlin, an upstream component of the Hippo pathway (40). We demonstrated that the association between wild-type AMOTp130 and YAP is not regulated by AMOTp130 phosphorylation. However, it was shown that the phospho-mimetic AMOTp130 has elevated interaction with Lats1/2 (51). Therefore there is possibly a feedback loop consisting of the two roles of AMOTp130 as both a regulator and an effector of the Hippo pathway, although the physiological context and the exact mechanism would need further investigation. What we have found in this study also indicate that the phosphorylated and dephosphorylated AMOTp130 are both functional with differential roles. While the phosphorylated AMOT may promote Hippo signaling, the dephosphorylated AMOTp130 function as an F-actin interacting protein to promote stress fiber formation and cell migration.",
"YAP/TAZ are downstream effectors of the Hippo pathway and function through regulation of gene transcription (52). YAP/TAZ also promotes cell migration likely through regulation of gene expression and initiation of an EMT program (53). However, our study identified AMOTp130 as a novel mediator of the Hippo pathway in inhibiting cell migration. Phosphorylation of AMOTp130 by Lats1/2 directly inhibits F-actin binding and correlates with reduced actin stress fiber and focal adhesion formation. Considering the direct role of F-actin and focal adhesion in cell migration, the role of AMOTp130 in cell migration is likely structural. Migration promoting functions of AMOT and YAP/TAZ could be reconciled in at least two ways. First, the expression pattern of AMOT and YAP/TAZ may dictate their functions in different tissues and cell types. Second, in cells expressing both of these proteins, AMOTp130 and YAP/ TAZ may mediate acute and prolonged effects of the Hippo pathway on cell migration in a cooperative manner (Fig. 8). Whether YAP/TAZ plays a role in endothelial cell migration is unknown. However, we demonstrated in this report that Lats1/2 play a role in endothelial cell migration in a manner dependent on AMOT phosphorylation. More importantly we demonstrated in vivo for the first time that the Hippo pathway may regulate developmental angiogenesis through AMOT phosphorylation. It would be interesting to investigate whether AMOT phosphorylation by the Hippo pathway also functions in pathological angiogenesis such as that in cancer."
] | [] | [
"EXPERIMENTAL PROCEDURES",
"RESULTS",
"The Hippo Pathway Phosphorylates AMOT Family Proteins-",
"The Hippo Pathway Inhibits AMOT in Cell Migration",
"FIGURE 3. Phosphorylation of AMOTp130 on Ser-175 inhibits interaction with F-actin.",
". Phosphorylation inhibits endogenous AMOTp130-F-actin interaction and direct AMOTp130-F-actin interaction in vitro.",
"Phosphorylation of AMOTp130 on Ser-175 Interferes with Stress Fiber and Focal Adhesion Formation and Inhibits Endothelial Cell",
"DISCUSSION",
"FIGURE 1 .",
"FIGURE 2 .",
"FIGURE 4",
"FIGURE 6 .",
"FIGURE 7 .",
"FIGURE 8 ."
] | [] | [] | [
"Phosphorylation of Angiomotin by Lats1/2 Kinases Inhibits F-actin Binding, Cell Migration, and Angiogenesis *",
"Phosphorylation of Angiomotin by Lats1/2 Kinases Inhibits F-actin Binding, Cell Migration, and Angiogenesis *"
] | [] |
13,607,679 | 2022-03-19T02:11:53Z | CCBY | https://www.nature.com/articles/oncsis201744.pdf | GOLD | d41524968a4c76cafd86fb96a5a4994424a8d3ee | null | null | null | null | 10.1038/oncsis.2017.44 | 2621792783 | 28604765 | 5519193 |
MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells
2017
C Rattanasinchai
B J Llewellyn
S E Conrad
K A Gallo
MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells
6345201710.1038/oncsis.2017.44OPEN ORIGINAL ARTICLE
Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood.Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), which is accompanied by elevation of matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced ER+ BC cell invasion is abrogated by FRA-1 silencing, demonstrating that MLK3 drives invasion through FRA-1. Conversely, in metastatic TNBC models, high FRA-1 levels are significantly reduced upon depletion of MLK3 by either gene silencing or by the CRISPR/Cas9n editing approach. Furthermore, ablation of MLK3 or MLK inhibitor treatment decreases expression of both MMP-1 and MMP-9. Consistent with the role of tumor cell-derived MMP-1 in endothelial permeability and transendothelial migration, both of these are reduced in MLK3-depleted TNBC cells. In addition, MLK inhibitor treatment or MLK3 depletion, which downregulates MMP-9 expression, renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells derived from TNBC-bearing mice display increased levels of FRA-1 and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in vascular intravasation. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMPs axis suggest that MLK3 is a promising therapeutic target for treatment of TNBC. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al
INTRODUCTION
Metastatic breast cancer is responsible for nearly half a million deaths worldwide 1 and 40 000 deaths in the United States, 2 annually. A major contributor is a lack of efficacy of the current standard treatments in preventing and treating metastatic disease. Breast cancer metastasis is a multistep process initiated by cancer cells within a primary tumor that gain invasive capacity. These cancer cells must breach the basement membrane, invade through extracellular matrix and intravasate into blood vessels. The cells that intravasate into the bloodstream, circulating tumor cells (CTCs), must survive in the circulation, extravasate to a distant site and colonize to form metastatic lesions. 3 Of the major clinical breast cancer subtypes, triple-negative breast cancer (TNBC) is considered the most aggressive and has the highest rate of metastasis and early recurrence. 4 Given the relative dearth of targeted therapies for treating TNBC, standard treatment relies on surgical removal, adjuvant radiotherapy and toxic chemotherapy.
Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that transduces signals from multiple cell surface receptors to activate MAPK cascades in a context-dependent manner. 5,6 Activated MAPKs directly phosphorylate cytosolic substrates or undergo nuclear translocation to regulate transcription factors, including activating protein-1 (AP-1). 5,6 MLK3 is critical for TNBC metastasis. 7,8 We have shown in TNBC models that MLK3 mediates JNK-dependent paxillin phosphorylation to facilitate focal adhesion turnover and cell migration. 8 In addition, MLK3 signaling leads to JNK-mediated c-JUN phosphorylation, 9 which activates AP-1-mediated gene expression.
AP-1 transcription factors comprise, usually heterodimeric, combinations of JUN and FOS family members including c-JUN, JUN-B, JUN-D, c-FOS, FOS-B, FRA-1 and FRA-2. 10 Aberrant AP-1 activity regulates genes that promote cancer progression. 11,12 Among AP-1 members, high levels of FRA-1 are associated with poor prognosis in TNBC. 11,13 FRA-1 is elevated in TNBC cell lines compared with estrogen receptor-positive breast cancer (ER+ BC) cell lines; 14,15 and is required for proliferation, 15 epithelial-tomesenchymal transition, 13,16 invasion 17,18 and metastasis. 19 Invasion genes controlled by FRA-1 include matrix metalloproteinases (MMPs), the zinc-dependent endopeptidases involved in matrix degradation and extracellular matrix remodeling. 20 Elevated levels of several MMPs are found in many types of solid tumors; these MMPs have crucial roles in multiple steps of tumor progression including tumor growth, angiogenesis, invasion and metastasis. 21 In this study, we demonstrate that MLK3 is a key regulator of FRA-1 expression in both ER+ BC and TNBC models. Furthermore, we show that the MLK3-FRA-1 axis controls levels of MMP-1 and MMP-9. Consistent with the roles of these MMPs, loss of MLK3 blocks Matrigel invasion as well as transendothelial migration of highly aggressive 4T1 cells. Importantly, an MLK inhibitor diminishes FRA-1 and its target genes, MMP-1 and MMP-9, in TNBC cells suggesting that targeting MLK3 may interfere with metastatic progression.
RESULTS
MLK3 is required for FRA-1 expression in breast cancer cells High FRA-1 levels are found in aggressive TNBC, whereas ER+ BC cell lines typically have low FRA-1 levels and are poorly invasive. 14,15 To examine whether MLK3 promotes FRA-1 expression in ER+ BC cells, we utilized MCF7 cells engineered to overexpress MLK3 upon treatment with the transcriptional inducer AP21967 (MCF7iMLK3). 9,22,23 As shown in Figure 1a, induced MLK3 expression increases FRA-1 protein expression, and drives cell migration in both transwell 9 and wound-healing assays (Supplementary Figure 1). As FRA-1 is an AP-1-regulated gene, 24 and MLK3 is known to activate AP-1, 9 quantitative reverse transcribed PCR (qRT-PCR) analysis was performed to determine FRA-1 transcript levels. As shown in Figure 1b, MLK3 robustly increases the FRA-1 transcript level. Consistent with our previous findings, 22 ectopically expressed MLK3 is active as judged by phospho-MLK3 immunoblotting (Figure 1a). To assess whether MLK3 catalytic activity is required for FRA-1 expression, vectors encoding wild-type MLK3 or a kinase inactive mutant MLK3 K144M 23 were transiently introduced into MCF7 cells. Wild-type MLK3 drives FRA-1 expression, whereas ectopic expression of equivalent protein levels of MLK3 K144M fails to upregulate FRA-1 ( Figure 1c). As shown in Figure 1d, overexpression of wild-type, active MLK3 in ER+ ZR-75-1 cells also drives FRA-1 expression. Thus, high levels of active MLK3 can upregulate FRA-1 expression in multiple ER+ BC cells.
In complementary experiments, we investigated the requirement for MLK3 in metastatic TNBC cell lines, which possess high endogenous levels of FRA-1. 15 MLK3 silencing in highly invasive TNBC SUM-159 cells reduces both FRA-1 protein ( Figure 2a) and mRNA (Figure 2b) levels, compared with SUM-159 cells transfected with control siRNA. To evaluate the function of MLK3 in the highly metastatic murine TNBC 4T1 model, we first generated MLK3 gene knockout 4T1-luc2 cells using the CRISPR/Cas9n (nickase) 25 system (Supplementary Figure 2A). Three MLK3-knockout (MLK3 KO) clones, 4T1KO-1, 4T1KO-2 and 4T1KO-3, as well as a wild-type (WT) clone that maintained MLK3 expression, were confirmed by sequencing (Supplementary Figure 2B). All three 4T1KO clones lack MLK3 expression and show decreased FRA-1 protein expression in contrast to parental 4T1 cells and the WT clone ( Figure 2c). Based on qRT-PCR analysis, FRA-1 mRNA transcripts are also reduced in MLK3 KO 4T1 cells compared with parental cells (Figure 2d). MCF7iMLK3 cells treated with vehicle or 50 nM AP21967 to induce MLK3 expression for 24 h, (c) MCF7 cells were transiently transfected with a wild-type MLK3 (pRK-MLK3) or a kinase dead MLK3 variant (pRK-MLK3-K144M) for 24 h, and (d) ZR-75-1 cells transiently transfected with pRK-MLK3 expression vector for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. The mRNAs were subjected to qRT-PCR with primers for the indicated genes. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; NS, not statistically significant; **P o0.01.
Nuclear FRA-1 expression, observed in parental 4T1 cells, is nearly absent in the 4T1KO-1 cells (Figure 2c). To validate the specificity of CRISPR MLK3 KO, a rescue experiment was performed by transiently transfecting a bi-cistronic pCMS-EGFP-MLK3 vector. EGFP-positive cells, which coexpress MLK3, regain FRA-1 expression (Figure 4e, Supplementary Figure 2C). Because 4T1 cells are poorly transfectable, multiple slides were used to score 100 EGFP-positive cells for FRA-1 expression. As shown in Figure 2f, 98% of the EGFP-positive 4T1KO-1 cells, which also express ectopic MLK3, regain nuclear FRA-1 staining.
To assess whether blockade of MLK activity reduces FRA-1 levels in highly invasive breast cancer cells, SUM-159 and 4T1 cells were treated with either CEP-1347 or URMC-099, MLK inhibitors with unrelated chemical structures, for 24 h and subjected to immunoblot analysis. As shown in Figure 3a and MLK3-activated JNK and ERK contribute to FRA-1 regulation Our data show that active MLK3 induces expression of FRA-1. Ectopic expression of wild-type MLK3 in MCF7 cells increases both JNK and ERK activation, as judged by the levels of phospho-JNK and phospho-ERK. It is well established that MLK3 utilizes its catalytic activity to regulate JNK activity. 8,9 However, MLK3 can activate ERK either through either kinase-dependent signaling 26 or kinase-independent scaffolding, 27 depending upon context. As shown in Figure 3a, wild-type MLK3 increases both JNK and ERK activities and drives FRA-1 expression, but equivalent levels of the kinase dead mutant MLK3-K144M fail to upregulate FRA-1 and do not significantly increase JNK or ERK activity.
To determine which MAPK signaling pathways are required for maintaining basal levels of endogenous FRA-1 in TNBC cells, cells were treated with small molecule inhibitors that block specific Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. The mRNAs were subjected to qRT-PCR with primers for the indicated genes. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate. (e and f) Parental 4T1 cells or 4T1KO-1 cells were transfected with bi-cistronic vector expressing EGFP and MLK3 (pCMS-EGFP-MLK3) for 24-48 h and were subjected to immunofluorescence staining using a FRA-1 antibody. FRA-1 staining is shown in red and GFP, which indicates co-expression of MLK3, is shown in green. Nuclei were counterstained with DAPI (blue); Scale bar, 25 μm; NS, not statistically significant; **P o0.01.
breast cancer cell lines, perhaps reflecting the established antagonism between the JNK and p38 pathways. 28 In a time course, treatment with either the JNK or MEK inhibitor resulted in a reduction of basal FRA-1 protein levels over time (Supplementary Figure 5), consistent with what has been observed on MEK inhibitor treatment of colon cancer cells. 29 In this experiment, SP600125 effectively blocked JNK activity as judged by phospho-c-JUN levels, but had no impact on phospho-ERK. The MEK inhibitor, U0126, efficiently blocked ERK activation over the entire time course, although a slight diminution of phospho-c-JUN at later time points was observed, suggesting that inhibition of the ERK pathway may indirectly downregulate phospho-c-JUN, as has been shown in Ras-overexpressing thyroid cells. 30 Treatment with either CEP-1347, SP600125 or U0126, for 24 h reduced FRA-1 transcript levels, as determined by qRT-PCR ( Figure 4d), suggesting that both the JNK and ERK pathways contribute to FRA-1 transcriptional regulation.
MLK3 increases MMP-1 and MMP-9 through FRA-1 FRA-1 is an oncogenic member of the AP-1 transcription factor family, 31 which regulates expression of genes involved in cancer progression, including MMPs. As MLK3 regulates both FRA-1 and cancer cell invasion, we hypothesized that it may control the expression of FRA-1-dependent MMPs, such as MMP-1, 15,17,32 MMP-2 33,34 and MMP-9. 17 As shown in Figure 5a, induced expression of MLK3 upregulates MMP-1 and MMP-9 mRNA but reduces MMP-2 mRNA levels. Both cytosolic and secreted MMP-1 protein can be detected on induction of MLK3 expression in MCF7iMLK3 cells, reflecting the strong upregulation of MMP-1 mRNA. Importantly, the increase in MMP-1 protein is abrogated by FRA-1 gene silencing ( Figure 5b).
The mRNA levels of the same MMPs were evaluated in the 4T1 line and 4T1KO clones. By qRT-PCR analysis, MMP-1a, the functional ortholog of human MMP-1, 35
MLK3 regulates cancer cell invasion and transendothelial migration
To investigate the requirement of FRA-1 in MLK3-driven cancer cell invasion, the impact of FRA-1 silencing on Matrigel transwell invasion of MCF7iMLK3 cells was determined. As shown in Figure 6a-c, induced expression of MLK3 in MCF7iMLK3 cells upregulates FRA-1 protein levels and enhances invasion. Silencing of FRA-1 reduces MLK3-induced invasion, indicating that FRA-1 functions downstream of MLK3 in this context. Of note, we previously found a role for MLK3 in JNK-mediated phosphorylation of paxillin in MCF10A and TNBC cells. 8 In MCF7 cells, we observe a modest increase in phospho-paxillin on MLK3 expression, which is unaffected by FRA-1-silencing (Supplementary Figure 6) suggesting that MLK3 regulation of paxillin is independent of FRA-1, and consistent with the idea that MLK3 controls multiple pathways in cancer cell migration and invasion.
In TNBC models, tumor cell-derived MMP-9 is required for Matrigel invasion and for formation of pulmonary metastases. 37-39 MMP-1 is crucial for transendothelial migration and vascular intravasation. 40 As MLK3 deletion decreases both MMP-1 and MMP-9 levels, we tested whether deletion of MLK3 impacts Matrigel invasion and transendothelial migration in TNBC cells. Indeed three independent 4T1 MLK3 KO clones show impaired invasion through Matrigel compared with parental 4T1 cells. Treatment with CEP-1347 similarly inhibits Matrigel invasion (Figure 6d) of 4T1 cells. We then performed gelatin zymography to evaluate levels of secreted MMP-9. Notably, MMP-9 can be distinguished from the other major gelatinase MMP-2, based on molecular weights of 92 kDa and 72 kDa, respectively. Gelatin zymography of conditioned medium from 4T1 cells shows a single predominant band corresponding to MMP-9; and secreted MMP-9 levels are reduced in conditioned media from 4T1 cells treated with CEP-1347 or from multiple 4T1 MLK3 KO clones (Figure 6e). (Figure 6g). These data indicate that MLK3 contributes to cancer cell-induced endothelial permeability and is required for transendothelial migration.
FRA-1 and MMP-1a are upregulated in circulating tumor cells derived from TNBC tumors The established role of MMP-1 in cancer intravasation, coupled with our findings that MLK3 controls MMP-1 levels, prompted us to evaluate the components of the MLK3-FRA-1-MMP-1 signaling axis in CTCs. A clonogenic assay was used to isolate CTCs from the blood of mice bearing 4T1-luc2 mammary tumors and associated metastases. By phase contrast imaging, isolated CTC lines (4T1-CTC) show distinct morphology compared with the morphology of the parental 4T1 cells. Many of these cells are able to detach and re-attach to tissue culture plates (Supplementary Figure 8). Bioluminescence imaging demonstrated that the isolated CTC lines retain luciferase activity, confirming their origin from the 4T1-luc2 tumors (Supplementary Figure 8). Both 4T1-CTC lines show increased FRA-1 and MMP-1a mRNA expression, compared with parental 4T1 cells. Furthermore in the 4T1-CTC lines, FRA-1 and MMP-1a levels are dependent upon MLK activity, as their levels are decreased by CEP-1347 treatment (Figure 7). Co-expression and mutual exclusivity analysis (Supplementary Figure 9) revealed a statistically significant tendency towards co-occurrence of MLK3 and FRA-1 gene expression, as well as MMP-1 and FRA-1.
DISCUSSION
Metastasis is overwhelmingly the cause of breast cancer-related death, yet the complexity of the metastatic process makes it therapeutically challenging to treat. 41 Previous studies in our lab and others have demonstrated that MLK3 is crucial for TNBC metastasis. 7,8 Herein, we utilized the CRISPR/Cas9 approach to deplete MLK3 in the highly aggressive 4T1 mammary cancer model to elucidate the role(s) of MLK3 in discrete steps of metastasis and to identify the key signaling pathways through which MLK3 regulates these events. Mechanistically, we have deciphered a novel function for MLK3 in controlling FRA-1 in breast cancer cells. In this context, both JNK and ERK signal downstream of MLK3 (Figure 4, Supplementary Figure 3) to enhance FRA-1 expression. FRA-1 regulation is complex. JNK is well known to phosphorylate c-JUN, 42 the AP-1 member, which is required for transcription of FRA-1. 43 ERK, in turn, phosphorylates FRA-1, which enhances the stability of the FRA-1/c-JUN heterodimer. 14,29 Thus MLK3 signaling is well poised to regulate FRA-1/c-JUN-mediated transcription.
FRA-1 has emerged as a key driver of metastatic progression in multiple cancer types including breast cancer, 11,14,15,17 lung cancer, 18 colorectal cancer 16,44,45 and glioblastoma. 46 FRA-1 not only controls expression of genes involved in cell motility, epithelialto-mesenchymal transition 13,45,47,48 and cell invasion, 15,17,32,49 but it also controls proliferation, 15,50,51 metastatic outgrowth 44 and the stem cell phenotype of cancer cells. 52 Several FRA-1-regulated genes 15,17,32,49 have been demonstrated to facilitate cancer cell invasion including MMP-1, MMP-2 and MMP-9. 17,18 Zhan et al. 53 have previously reported that, in ovarian cancer cells, MLK3 is important for expression of MMP-1, -2, -9 and -12 and is required for ovarian cancer invasion. Our studies show that in TNBC models, MLK3 deletion decreases both MMP-1 and MMP-9 expression ( Figure 5) but does not significantly affect MMP-2 levels. Functionally, MMP-9 facilitates extracellular matrix remodeling and basement membrane degradation and, like MLK3, is critical for TNBC metastasis. 37 Likewise, MMP-9 silencing in multiple TNBC lines has been shown to block Matrigel invasion, 37 analogous to our findings that MLK3 deletion or CEP-1347 treatment inhibits Matrigel invasion ( Figure 6). In TNBC, hematogenous metastasis, which requires transendothelial migration 54 is more common than lymphatic spread. 55 During vascular intravasation and extravasation, cancer cells must disrupt endothelial barrier integrity and transmigrate through the endothelial layer. MMP-1 is an interstitial collagenase-I required for fibrillar collagen remodeling. [56][57][58] However non-collagenolytic mechanisms of MMP-1 are implicated in transendothelial migration of tumor cells. 40 In epidermoid cancer, for instance, tumor cell-derived MMP-1 increases endothelial barrier permeability by proteolytically activating the endothelial thrombin receptor PAR-1 and facilitating transendothelial migration. 40 In our study, TNBC cells induce endothelial barrier permeability and transmigrate through an endothelial barrier, whereas, MLK3-deleted tumor cells fail to induce permeability and their transendothelial migration is impaired ( Figure 6). These findings suggest that one mechanism through which MLK3 facilitates TNBC metastasis could be through vascular intravasation and, possibly, extravasation.
CTCs have risen to prominence as potential prognostic and predictive biomarkers for metastatic burden, metastatic recurrence and therapeutic response. [59][60][61][62][63][64][65][66][67][68][69][70] Our finding that 4T1-CTCs have elevated mRNA levels of FRA-1 and MMP-1a compared with parental 4T1 cells (Figure 7) is important in light of several lines of evidence supporting a key role for MMP-1 in CTCs, epithelial-to-mesenchymal transition and metastatic progression. Furthermore, MMP-1 was identified as a key gene upregulated in infiltratative self-seeding CTCs. 71 Elevated MMP-1 was also observed in TNBC MDA-MB-231 subclones selected for their ability to metastasize to lung 72 and brain. 73 Recently, single cell gene expression analysis studies utilizing TNBC patient-derived xenograft models showed that MMP-1, as well as components of a proliferative gene signature, was significantly increased in late stage, high burden metastatic cells compared with early stage, low burden metastatic cells. 74 These data, along with the role of MMP-1 in vascular intravasation, suggest that MMP-1 is not only required for early stages of metastatic process, but may also contribute to colonization.
TNBC is considered the most aggressive subtype of breast cancer; however, therapeutic options are limited. A major challenge is to identify important targetable signaling pathways in TNBC. MMP-1 expression is significantly elevated in aggressive breast tumors and correlates with both tumor size and grade 75 pointing to MMP-1 as a promising therapeutic target. Indeed, MMPs, including MMP-1, have a long history as targets for cancer therapeutics yet early clinical trials using MMP inhibitors were unsuccessful due, in part, to inadequate preclinical and clinical design, lack of drug specificity and high toxicity. 76,77 Recently The mRNAs were subjected to qRT-PCR with primers to the indicated genes. Relative mRNA expression is displayed as the mean ± s.d. from at least three independent experiments performed in triplicate. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. Con, Control; CL, cellular lysate; CM, concentrated conditioned medium; NS, not statistically significant; *Po 0.05; **P o0.01.
Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al FRA-1, an upstream regulator of MMP-1, has also emerged as a key driver of cancer progression; however, transcription factors are not readily druggable. In one study, an existing inhibitor of the FRA-1 regulated gene, adenosine receptor A 2B , ADORA2B, was shown to block formation of lung metastases in a TNBC experimental metastasis xenograft model. 19 However, it is unclear whether targeting a single FRA-1-regulated gene will always be sufficient to halt breast cancer progression, as FRA-1 controls a suite of genes involved in cancer invasion and metastasis. 45 Based on our findings, we propose that an alternative strategy would be to target MLK3, an upstream regulator of FRA-1.
Multiple MLK inhibitors exist, including CEP-1347 78 and URMC-099. 79 Our data show that both of these MLK inhibitors, built upon different chemical scaffolds, reduce FRA-1 levels in 4T1 cells. In addition, either CEP-1347 treatment or MLK3 deletion reduces FRA-1, MMP-1, and MMP-9 expression to similar levels in multiple TNBC cells, indicating that MLK3, specifically, controls FRA-1, MMP-1 and MMP-9 expression. CEP-1347 progressed through Phase II/III clinical trials for Parkinson's disease, and although it failed to delay progression, no significant toxicity was observed, 80 suggesting that it could potentially be repurposed for breast cancer treatment.
In summary, we provide evidence that MLK3 signaling is a crucial regulator of FRA-1 and its target genes, MMP-1 and MMP-9 in models of TNBC. As a consequence, depletion or inhibition of MLK3 in TNBC cells impairs both Matrigel invasion and transendothelial migration. Consistent with these findings, FRA-1 and MMP-1 are upregulated in isolated CTC lines from a TNBC
RNA interference and plasmid transfection
For siRNAs, Mission siRNA Universal Negative control #1, siRNA duplexes targeting human MLK3 (5′-CUGACUGCCACUCAUGGUG-3′ and its antisense) 9,81 and human FRA-1 (5′-GGGCAGUGACGUCUGGAG-3′ and its antisense) 15 were from Sigma-Aldrich. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent.
Plasmids, pRK-MLK3 or pRK-MLK3 K144M, were previously described. 23 Lipofectamine 2000 and Lipofectamine 3000 were used to transfect MCF7 and ZR-75-1 cells, respectively. In a recovery assay of MLK3-knockout 4T1 cells, pCMV-EGFP-MLK3 82 expression vector was reverse-transfected into 4T1KO-1 cells using Lipofectamine 3000.
CRISPR-Cas9n constructs
The MLK3 CRISPR construct was generated based on a previously described protocol. 83 Briefly, two pairs of guide RNAs for a CRISPR-Cas9n construct were designed (crispr.mit.edu) to target exon 1 of murine MLK3 (Supplementary Figure 2) and were cloned into pSpCas9n(BB)-2A-GFP (PX461) 83 (a gift from Dr Feng Zhang; Addgene plasmid #48140). After reverse transfection using Lipofectamine 3000, GFP-positive clones were screened for MLK3 deletion by immunoblotting. Genomic DNAs from selected clones were collected and amplified using forward primer 5′-ATGGAGCCCTTGAAGAACCT-3′ and reverse primer 5′-ACGGTAGA CCTTGCCGAAG-3′. Purified PCR products were subjected to TOPO TA cloning (Invitrogen). At least five clones were subjected to nucleotide sequencing to identify the genomic alterations.
Immunoblot analysis
Cellular lysates were prepared in lysis buffer (1% NP-40, 150 mM sodium chloride and 50 mM Tris, pH 8.0) and immunoblotting was performed as described. 8,9 Immunofluorescence analysis Immunofluorescence staining was performed as previously described. 8 Images were acquired and, if indicated, quantified from N4100 cells per group, using an Olympus fluorescence microscope and MetaMorph software.
Gelatin zymography 4T1 cells or their derivatives (2.5 × 10 5 ) were seeded to 35 mm culture dishes. The following day, the cells were incubated in serum-free medium for 24 h, in the presence of CEP-1347, as indicated. Conditioned media corresponding to equal cellular equivalents were loaded onto and run through 10% polyacrylamide gels containing 1 mg/ml gelatin. The gels were incubated for 1 h in 2.5% Triton X-100, developed for 24 h at 37°C in 50 mM Tris-HCl buffer, pH 7.6, containing 5 mM CaCl 2 and 200 mM NaCl, and finally stained for 16 h with Simple Blue SafeStain (Invitrogen). After 30 min destaining with water, gels were scanned and the images were processed using Image J software.
Quantitative real-time PCR Total RNAs were extracted using the RNeasy kit (Qiagen, Valencia, CA, USA) and cDNA synthesis was performed using a cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time qPCR was performed using either PerfeCTa SyBR green superMix (Quanta, Gaithersburg, MD, USA) or SYBR Green Master mix (Applied Biosystems). Specific primer sequences were designed using PrimerBank 84 (see Supplementary Table 1 for full list of primers).
In vitro Matrigel invasion assay
In vitro Matrigel invasion assay was performed as previously described. 8,9 Briefly, MCF7iMLK3 cells were transfected with 50 nM Control siRNA or MLK3 siRNA as described above for 16 h, and then serum-deprived for an additional 8 h in the presence of vehicle or 25 nM AP21957. The cells (1 × 10 5 ) were seeded into the upper chamber of 5 μm Matrigel-coated transwell chamber and allowed to invade in the presence of vehicle or 25 nM AP21967 for 24 h. For 4T1 cells and their derivatives, cells were serum-deprived overnight and 2 × 10 4 cells introduced into the upper chamber and allowed to invade for 24 h toward 10% FBS in the presence of indicated inhibitors. Mitomycin C (2 μg/ml) was included to eliminate possible effects of cell proliferation. Experiments performed in duplicate were repeated at least three times.
Transendothelial migration and endothelial permeability
Transendothelial migration was assessed essentially as described. 40,85 Briefly, 1 × 10 5 EA.hy926 cells were grown as a confluent monolayer on 5 μm pore transwell inserts for 1-2 days and 1 × 10 5 4T1 cells, or their derivatives, that had been deprived of serum for 18 h were introduced to the upper chamber. 4T1 cells were allowed to migrate toward 10% FBS for 24 h. To measure permeability of the endothelial layer, 5(6)-Carboxyfluorescein (6-FAM)-conjugated albumin (10 μM final concentration) was added to the upper chamber and culture medium was collected from the bottom chamber after 30 min at 37 o C. Permeability was determined by measuring the fluorescence due to leakage of 6-FAM-conjugated albumin (excitation = 495 nm, emission = 520 nm) into the bottom chamber. To measure transendothelial migration, the cells inside the transwell inserts were wiped out with the cotton swabs, and the extent of migration through the transwell membranes was then determined by relative bioluminescence activity.
CTC isolation from the 4T1 tumor bearing mice
This experiment was carried out in accordance with standard protocols approved by All University Committee on Animal Use and Care at Michigan State University. Briefly, puromycin-resistant 4T1-luc2 cells (7.5 × 10 5 cells) were injected into the fourth mammary gland of 8-week-old female athymic nu/nu mice (N = 2). After 24 days, the two mice were killed and CTCs were isolated as previously described. 86 Briefly, 200 μl blood collected by cardiac puncture was cultured in RPMI-1640 supplemented with 20% FBS, 2 μg/ml puromycin, and penicillin/streptomycin for 10 days. Approximately 40 colonies were obtained from each blood sample, pooled and propagated as populations, named 4T1-CTC#1 and 4T1-CTC#2.
Statistical analysis
Results are expressed as mean ± standard deviation (s.d.
). An unpaired, two-tailed Student's t-test was used to calculate the P-value, and P o0.05 is considered statistically significant. At least three independent experiments were performed unless otherwise noted.
Figure 1 .
1Ectopic expression of MLK3 drives FRA-1 expression in ER+ BC cells. Cellular lysates and/or mRNAs were collected from (a and b)
Figure 3b ,
3btreatment with either MLK inhibitor downregulates FRA-1 protein in both SUM-159 and 4T1 cells. Similarly, immunofluorescence staining of SUM-159 and 4T1 cells treated with CEP-1347 revealed loss of FRA-1 staining (Figure 3c and d), similar to the phenotype of the 4T1KO-1 cells. These findings further support a requirement for active MLK3 in FRA-1 expression in highly invasive breast cancer cell lines. Of note, MLK inhibitor treatment is sufficient to reduce both JNK and ERK signaling in 4T1 cells (SupplementaryFigure 3). Further, overexpression, silencing or inhibition of MLK3 in breast cancer cells had no effect on levels of the related transcription factor c-FOS, reflecting specificity in MLK3 control of FRA-1 (SupplementaryFigure 4).
MAPK pathways, including SP600125 (JNK), U0126 (MEK/ERK) or SB203580 (P38), and the impact on FRA-1 protein levels was assessed. Treatment with the MLK, JNK or ERK inhibitor significantly decreased basal, endogenous FRA-1 levels in SUM-159 (Figure 4b) and 4T1 cells (Figure 4c). The P38 MAPK inhibitor SB203580 increased FRA-1 expression in both invasive
Figure 2 .
2MLK3 is required for FRA-1 expression in TNBC cells. Cellular lysates and/or mRNA samples were collected from (a and b) SUM-159 cells treated with 50 nM control siRNA or MLK3 siRNA for 24 h, (c) parental 4T1, WT clone and three 4T1 CRISPR MLK3-knockout clones (KO-1, KO-2 and KO-3) and (d) parental 4T1 or 4T1KO-1 cells. Cellular lysates were subjected to immunoblotting with indicated antibodies.
is ablated in 4T1KO-1 and 4T1KO-2, compared with control parental 4T1 cells (Figure 5c). Loss of MLK3 decreases MMP-9 mRNA levels by 60 and 50% in 4T1KO-1 and 4T1KO-2 cells, respectively, compared with parental 4T1 cells. Similar effects on mRNA levels of MMPs were observed in 4T1 cells treated with CEP-1347 (Figure 5d). SUM-159 TNBC cells, which express MMP-1 but not MMP-9, 36 show decreased MMP-1 mRNA levels upon MLK3 gene silencing or MLK inhibitor treatment (Figure 5e and f).
Figure 3 .
3Pharmacological inhibition of MLKs reduces FRA-1 protein expression in TNBC lines. (a) SUM-159 cells and (b) 4T1 cells were treated with vehicle, 400 nM CEP-1347 or 400 nM URMC-099 for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (c) SUM-159 cells and (d) 4T1 cells were seeded on coverslips, treated ± 400 nM CEP-1347 for 24 h, and subjected to immunofluorescence staining against FRA-1 antibody (green). Nuclei were counterstained with DAPI (blue); Scale bar, 25 μm in c and 50 μm in d. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al Transendothelial migration assays were performed to assess the ability of 4T1 or 4T1KO-1 cells to disrupt and invade through a confluent endothelial cell monolayer. Migration of luciferaseexpressing cancer cells through the endothelial cell monolayer was quantified by bioluminescence imaging. The 4T1KO-1 clone was chosen for these experiments because its bioluminescence activity is comparable to that of parental 4T1 cells (Supplementary Figure 7). Parental 4T1 cells increase permeability of the endothelial cell layer as measured by leakage of fluorescently labeled albumin from the upper into the lower transwell chamber, whereas 4T1KO-1 cells show markedly reduced endothelial permeability (Figure 6f). Compared with parental 4T1 cells, 4T1KO-1 cells show a fourfold reduction in transendothelial migration
Figure 4 .
4Both JNK and ERK pathways contribute to FRA-1 expression through MLK3 signaling. (a) MCF7 cells were transiently transfected with a wild-type MLK3 (pRK-MLK3) or a kinase dead MLK3 variant (pRK-MLK3-K144M) for 24 h, (b) SUM-159 and (c) 4T1 cells were treated with vehicle, 400 nM CEP-1347 (MLK inhibitor), 15 μM SP600125 (JNK inhibitor), 10 μM U0126 (MEK/ERK inhibitor) or 10 μM SB203580 (P38 inhibitor) for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (d) The mRNAs from 4T1 cells treated with vehicle, 400 nM CEP-1347, 15 μM SP600125 and 10 μM U0126 for 24 h were subjected to qRT-PCR analysis with FRA-1 primers.Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; NS, not statistically significant; **P o0.01.
Figure 5 .
5MLK3 induces MMP-1 and MMP-9 expression. Cellular lysates or mRNAs, as indicated, were isolated from (a) MCF7iMLK3 cells treated with vehicle or 50 nM AP21967 for 24 h, (b) MCF7iMLK3 cells treated with vehicle or with 50 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, for 24 h, (c) parental 4T1 cells and two 4T1 MLK3-knockout clones (4T1KO-1 and 4T1KO-2), (d) 4T1 cells treated with vehicle or 400 nM CEP-1347 for 24 h, and SUM-159 cells treated with (e) 50 nM control or MLK3 siRNA for 24 h, or (f) vehicle or 400 nM CEP-1347 for 24 h.
Figure 6 .
6MLK3 regulates cancer cell invasion and transendothelial migration. (a-c) MCF7iMLK3 cells treated with vehicle or with 25 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, and (d) parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3) were subjected to an in vitro Matrigel transwell invasion assay for 24 h. Relative cell invasion, with control set at 100%, is expressed as mean ± s.d. from three independent experiments. (e) A representative image from three independent experiments of gelatin zymography of conditioned medium from parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3). (f and g) Parental 4T1 and 4T1KO-1 were subjected to transendothelial migration toward 10% FBS. Endothelial permeability was assessed using 5(6)-Carboxyfluorescein (6-FAM)-conjugated albumin, and transendothelial migration was assessed using bioluminescence imaging as described in the 'Materials and Methods' section. Relative 6-FAM conjugated albumin concentration and bioluminescence activity are expressed mean ± s.d. from three independent experiments performed in triplicate. **P o0.01. *Po0.1. NS, not significant. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al model. Importantly the MLK inhibitor, CEP-1347, which blocks invasion, also reduces FRA-1 and MMP-1 in CTC lines. Taken together, our data reveal important roles of MLK3 during basement membrane degradation and transendothelial migration and suggest that MLK3 inhibitors may be a useful addition to the limited armament for combating TNBC. MATERIALS AND METHODS Chemicals and antibodies Chemicals. 5(6)-Carboxyfluorescein (6-FAM), bovine serum albumin, gelatin and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St Louis, MO, USA). SP600125, U0126 and SB203580 were from Calbiochem (San Diego, CA, USA). CEP-1347 and CEP-11004 were generously provided by Cephalon, Inc., a wholly owned subsidiary of Teva Pharmaceuticals, Ltd (North Wales, PA, USA). AP21967 was provided by Ariad Pharmaceuticals (Cambridge, MA, USA). Calcein AM and Simple Blue SafeStain were from Invitrogen (Carlsbad, CA, USA). Antibodies. Anti-MLK3 (A-20) (for detection of murine MLK3), anti-FRA-1 (R-20), anti-JNK1/3 (C-17), anti-ERK1 (K-23), anti-P38 (C-20), anti-actin (C-2), anti-p-c-JUN (S63)(KM-1) and anti-c-JUN (H-79) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-MLK3 (C-terminal) (for detecting human MLK3) was from Epitomics (Cambridge, MA, USA).Anti-p-MLK3, anti-p-ERK-1/2 (T202/Y204)(E-10), anti-p-JNK1/2 (T183/Y185) (81E11) and anti-p-P38 (T180/Y182) (#9216) were obtained from Cell Signaling (Danvers, MA, USA). Anti-MMP-1 (#36665 R) was purchased from R&D systems (Minneapolis, MN, USA), anti-p-paxillin S178 (#A300-100 A) was purchased from Bethyl Laboratory (Montgomery, TX, USA). IRDye 800CW goat anti-mouse IgG, IRDye 680 goat anti-rabbit IgG and IRDye 800CW donkey anti-goat IgG were from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and 546 was from Invitrogen and used for immunofluorescence staining.Cell lines MCF7, obtained from ATCC (Manassas, VA, USA), have been recently authenticated and were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). MCF7iMLK3 cells engineered to inducibly express MLK3 were previously described. 9,22 ZR-75-1 (from ATCC) and 4T1-luc2 (Perkin Elmer, Waltham, MA, USA) cells were maintained in RPMI-1640 (Gibco, Life Technology, Grand Island, NY, USA) with 10% FBS. SUM-159-GFP cells (a gift from Dr Chengfeng Yang (University of Kentucky)) were maintained in Ham's F-12 (Gibco) supplemented with 5% FBS, 5 μg/ml insulin, 1 μg/ml hydrocortisone and containing penicillin/streptomycin. The cell lines were routinely tested for mycoplasma contamination.
Figure 7 .
7FRA-1 and MMP-1a are upregulated in 4T1-derived circulating tumor cells. The mRNAs isolated from parental 4T1 cells and from circulating tumor cell lines, 4T1-CTC#1 and 4T1-CTC#2, treated with vehicle or 400 nM CEP-1347 for 24 h were subjected to qRT-PCR analysis using FRA-1-and MMP-1a-specific primers. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; **P o0.01.
© The Author(s) 2017Supplementary Information accompanies this paper on the Oncogenesis website (http://www.nature.com/oncsis)
Oncogenesis (2017), 1 -11
ACKNOWLEDGEMENTSCR is the recipient of Thai Royal Government fellowship and Aitch Foundation Cancer Fellowship. BJL is an MSU Professorial Assistantship Undergraduate Research Program fellow.CONFLICT OF INTERESTThe authors declare no conflict of interest.Oncogenesis is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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| [
"Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood.Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), which is accompanied by elevation of matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced ER+ BC cell invasion is abrogated by FRA-1 silencing, demonstrating that MLK3 drives invasion through FRA-1. Conversely, in metastatic TNBC models, high FRA-1 levels are significantly reduced upon depletion of MLK3 by either gene silencing or by the CRISPR/Cas9n editing approach. Furthermore, ablation of MLK3 or MLK inhibitor treatment decreases expression of both MMP-1 and MMP-9. Consistent with the role of tumor cell-derived MMP-1 in endothelial permeability and transendothelial migration, both of these are reduced in MLK3-depleted TNBC cells. In addition, MLK inhibitor treatment or MLK3 depletion, which downregulates MMP-9 expression, renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells derived from TNBC-bearing mice display increased levels of FRA-1 and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in vascular intravasation. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMPs axis suggest that MLK3 is a promising therapeutic target for treatment of TNBC. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al"
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"F Galtier, ",
"D Chalbos, ",
"H Liu, ",
"G Ren, ",
"T Wang, ",
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"N Mori, ",
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"B Krishnamachary, ",
"L Jiang, ",
"K Glunde, ",
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"A Hockla, ",
"E Miller, ",
"S Ran, ",
"D C Radisky, ",
"E S Radisky, ",
"Z S Zeng, ",
"A M Cohen, ",
"J G Guillem, ",
"H S Lee, ",
"A W Ha, ",
"W K Kim, ",
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"E I Deryugina, ",
"I Rimann, ",
"E Zajac, ",
"T A Kupriyanova, ",
"L H Engelholm, ",
"N Sethi, ",
"Y Kang, ",
"G L Johnson, ",
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"S Peddakama, ",
"Q Zhang, ",
"D V Kalvakolanu, ",
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"A M Liu, ",
"J Chadee, ",
"D N , ",
"N Reymond, ",
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"R A Mohammed, ",
"I O Ellis, ",
"A M Mahmmod, ",
"E C Hawkes, ",
"A R Green, ",
"E A Rakha, ",
"T Klein, ",
"R Bischoff, ",
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"F C Bidard, ",
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"V Dieras, ",
"C Mathiot, ",
"L Mignot, ",
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"J Benca, ",
"M Yu, ",
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"B S Wittner, ",
"S L Stott, ",
"M E Smas, ",
"D T Ting, ",
"M Tewes, ",
"B Aktas, ",
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"S Mueller, ",
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"T Rau, ",
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"Y L Ottaviano, ",
"D F Hayes, ",
"M Cristofanilli, ",
"G T Budd, ",
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"A Stopeck, ",
"M C Miller, ",
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"S Jackson, ",
"K R Hess, ",
"De Giorgi, ",
"U Mego, ",
"M , ",
"M Cristofanilli, ",
"D F Hayes, ",
"G T Budd, ",
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"A Stopeck, ",
"J M Reuben, ",
"M Cristofanilli, ",
"G T Budd, ",
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"A Stopeck, ",
"J Matera, ",
"M C Miller, ",
"M Cristofanilli, ",
"K R Broglio, ",
"V Guarneri, ",
"S Jackson, ",
"H A Fritsche, ",
"R Islam, ",
"G T Budd, ",
"M Cristofanilli, ",
"M J Ellis, ",
"A Stopeck, ",
"E Borden, ",
"M C Miller, ",
"M-Y Kim, ",
"T Oskarsson, ",
"S Acharyya, ",
"D X Nguyen, ",
"Xhf Zhang, ",
"L Norton, ",
"A J Minn, ",
"G P Gupta, ",
"P M Siegel, ",
"P D Bos, ",
"W Shu, ",
"D D Giri, ",
"P D Bos, ",
"Xh-F Zhang, ",
"C Nadal, ",
"W Shu, ",
"R R Gomis, ",
"D X Nguyen, ",
"D A Lawson, ",
"N R Bhakta, ",
"K Kessenbrock, ",
"K D Prummel, ",
"Y Yu, ",
"K Takai, ",
"P M Mcgowan, ",
"M J Duffy, ",
"J Cathcart, ",
"A Pulkoski-Gross, ",
"J Cao, ",
"R E Vandenbroucke, ",
"C Libert, ",
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"J P Finn, ",
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"J T Durkin, ",
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"G , ",
"D F Marker, ",
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"C J , ",
"D N Chadee, ",
"J M Kyriakis, ",
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"N V Kukekov, ",
"L A Greene, ",
"F A Ran, ",
"P D Hsu, ",
"J Wright, ",
"V Agarwala, ",
"D A Scott, ",
"F Zhang, ",
"X Wang, ",
"A Spandidos, ",
"H Wang, ",
"B Seed, ",
"J Kim, ",
"S H Thorne, ",
"L Sun, ",
"B Huang, ",
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"Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop. Z Xu, N V Kukekov, L A Greene, Mol Cell Biol. 25Xu Z, Kukekov NV, Greene LA. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop. Mol Cell Biol 2005; 25: 9949-9959.",
"Genome engineering using the CRISPR-Cas9 system. F A Ran, P D Hsu, J Wright, V Agarwala, D A Scott, F Zhang, Nat Protoc. 8Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8: 2281-2308.",
"PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. X Wang, A Spandidos, H Wang, B Seed, Nucleic Acids Res. 40Wang X, Spandidos A, Wang H, Seed B. PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res 2012; 40: D1144-D1149.",
"Sustained inhibition of PKC[alpha] reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model. J Kim, S H Thorne, L Sun, B Huang, D Mochly-Rosen, Oncogene. 30Kim J, Thorne SH, Sun L, Huang B, Mochly-Rosen D. Sustained inhibition of PKC[alpha] reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model. Oncogene 2011; 30: 323-333.",
"Disruption of the SRC-1 gene in mice suppresses breast cancer metastasis without affecting primary tumor formation. S Wang, Y Yuan, L Liao, S Q Kuang, J C Tien, O'malley Bw, Proc Natl Acad Sci. 106Wang S, Yuan Y, Liao L, Kuang SQ, Tien JC, O'Malley BW et al. Disruption of the SRC-1 gene in mice suppresses breast cancer metastasis without affecting primary tumor formation. Proc Natl Acad Sci USA 2009; 106: 151-156."
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"Antagonistic control of cell fates by JNK and p38-MAPK signaling",
"Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer",
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"Fra-1 a target for cancer prevention or intervention",
"Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells",
"A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers",
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"Mixed lineage kinase inhibitor CEP-1347 fails to delay disability in early Parkinson disease",
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] | [
"\nFigure 1 .\n1Ectopic expression of MLK3 drives FRA-1 expression in ER+ BC cells. Cellular lysates and/or mRNAs were collected from (a and b)",
"\nFigure 3b ,\n3btreatment with either MLK inhibitor downregulates FRA-1 protein in both SUM-159 and 4T1 cells. Similarly, immunofluorescence staining of SUM-159 and 4T1 cells treated with CEP-1347 revealed loss of FRA-1 staining (Figure 3c and d), similar to the phenotype of the 4T1KO-1 cells. These findings further support a requirement for active MLK3 in FRA-1 expression in highly invasive breast cancer cell lines. Of note, MLK inhibitor treatment is sufficient to reduce both JNK and ERK signaling in 4T1 cells (SupplementaryFigure 3). Further, overexpression, silencing or inhibition of MLK3 in breast cancer cells had no effect on levels of the related transcription factor c-FOS, reflecting specificity in MLK3 control of FRA-1 (SupplementaryFigure 4).",
"\n\nMAPK pathways, including SP600125 (JNK), U0126 (MEK/ERK) or SB203580 (P38), and the impact on FRA-1 protein levels was assessed. Treatment with the MLK, JNK or ERK inhibitor significantly decreased basal, endogenous FRA-1 levels in SUM-159 (Figure 4b) and 4T1 cells (Figure 4c). The P38 MAPK inhibitor SB203580 increased FRA-1 expression in both invasive",
"\nFigure 2 .\n2MLK3 is required for FRA-1 expression in TNBC cells. Cellular lysates and/or mRNA samples were collected from (a and b) SUM-159 cells treated with 50 nM control siRNA or MLK3 siRNA for 24 h, (c) parental 4T1, WT clone and three 4T1 CRISPR MLK3-knockout clones (KO-1, KO-2 and KO-3) and (d) parental 4T1 or 4T1KO-1 cells. Cellular lysates were subjected to immunoblotting with indicated antibodies.",
"\n\nis ablated in 4T1KO-1 and 4T1KO-2, compared with control parental 4T1 cells (Figure 5c). Loss of MLK3 decreases MMP-9 mRNA levels by 60 and 50% in 4T1KO-1 and 4T1KO-2 cells, respectively, compared with parental 4T1 cells. Similar effects on mRNA levels of MMPs were observed in 4T1 cells treated with CEP-1347 (Figure 5d). SUM-159 TNBC cells, which express MMP-1 but not MMP-9, 36 show decreased MMP-1 mRNA levels upon MLK3 gene silencing or MLK inhibitor treatment (Figure 5e and f).",
"\nFigure 3 .\n3Pharmacological inhibition of MLKs reduces FRA-1 protein expression in TNBC lines. (a) SUM-159 cells and (b) 4T1 cells were treated with vehicle, 400 nM CEP-1347 or 400 nM URMC-099 for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (c) SUM-159 cells and (d) 4T1 cells were seeded on coverslips, treated ± 400 nM CEP-1347 for 24 h, and subjected to immunofluorescence staining against FRA-1 antibody (green). Nuclei were counterstained with DAPI (blue); Scale bar, 25 μm in c and 50 μm in d. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al Transendothelial migration assays were performed to assess the ability of 4T1 or 4T1KO-1 cells to disrupt and invade through a confluent endothelial cell monolayer. Migration of luciferaseexpressing cancer cells through the endothelial cell monolayer was quantified by bioluminescence imaging. The 4T1KO-1 clone was chosen for these experiments because its bioluminescence activity is comparable to that of parental 4T1 cells (Supplementary Figure 7). Parental 4T1 cells increase permeability of the endothelial cell layer as measured by leakage of fluorescently labeled albumin from the upper into the lower transwell chamber, whereas 4T1KO-1 cells show markedly reduced endothelial permeability (Figure 6f). Compared with parental 4T1 cells, 4T1KO-1 cells show a fourfold reduction in transendothelial migration",
"\nFigure 4 .\n4Both JNK and ERK pathways contribute to FRA-1 expression through MLK3 signaling. (a) MCF7 cells were transiently transfected with a wild-type MLK3 (pRK-MLK3) or a kinase dead MLK3 variant (pRK-MLK3-K144M) for 24 h, (b) SUM-159 and (c) 4T1 cells were treated with vehicle, 400 nM CEP-1347 (MLK inhibitor), 15 μM SP600125 (JNK inhibitor), 10 μM U0126 (MEK/ERK inhibitor) or 10 μM SB203580 (P38 inhibitor) for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (d) The mRNAs from 4T1 cells treated with vehicle, 400 nM CEP-1347, 15 μM SP600125 and 10 μM U0126 for 24 h were subjected to qRT-PCR analysis with FRA-1 primers.Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; NS, not statistically significant; **P o0.01.",
"\nFigure 5 .\n5MLK3 induces MMP-1 and MMP-9 expression. Cellular lysates or mRNAs, as indicated, were isolated from (a) MCF7iMLK3 cells treated with vehicle or 50 nM AP21967 for 24 h, (b) MCF7iMLK3 cells treated with vehicle or with 50 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, for 24 h, (c) parental 4T1 cells and two 4T1 MLK3-knockout clones (4T1KO-1 and 4T1KO-2), (d) 4T1 cells treated with vehicle or 400 nM CEP-1347 for 24 h, and SUM-159 cells treated with (e) 50 nM control or MLK3 siRNA for 24 h, or (f) vehicle or 400 nM CEP-1347 for 24 h.",
"\nFigure 6 .\n6MLK3 regulates cancer cell invasion and transendothelial migration. (a-c) MCF7iMLK3 cells treated with vehicle or with 25 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, and (d) parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3) were subjected to an in vitro Matrigel transwell invasion assay for 24 h. Relative cell invasion, with control set at 100%, is expressed as mean ± s.d. from three independent experiments. (e) A representative image from three independent experiments of gelatin zymography of conditioned medium from parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3). (f and g) Parental 4T1 and 4T1KO-1 were subjected to transendothelial migration toward 10% FBS. Endothelial permeability was assessed using 5(6)-Carboxyfluorescein (6-FAM)-conjugated albumin, and transendothelial migration was assessed using bioluminescence imaging as described in the 'Materials and Methods' section. Relative 6-FAM conjugated albumin concentration and bioluminescence activity are expressed mean ± s.d. from three independent experiments performed in triplicate. **P o0.01. *Po0.1. NS, not significant. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al model. Importantly the MLK inhibitor, CEP-1347, which blocks invasion, also reduces FRA-1 and MMP-1 in CTC lines. Taken together, our data reveal important roles of MLK3 during basement membrane degradation and transendothelial migration and suggest that MLK3 inhibitors may be a useful addition to the limited armament for combating TNBC. MATERIALS AND METHODS Chemicals and antibodies Chemicals. 5(6)-Carboxyfluorescein (6-FAM), bovine serum albumin, gelatin and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St Louis, MO, USA). SP600125, U0126 and SB203580 were from Calbiochem (San Diego, CA, USA). CEP-1347 and CEP-11004 were generously provided by Cephalon, Inc., a wholly owned subsidiary of Teva Pharmaceuticals, Ltd (North Wales, PA, USA). AP21967 was provided by Ariad Pharmaceuticals (Cambridge, MA, USA). Calcein AM and Simple Blue SafeStain were from Invitrogen (Carlsbad, CA, USA). Antibodies. Anti-MLK3 (A-20) (for detection of murine MLK3), anti-FRA-1 (R-20), anti-JNK1/3 (C-17), anti-ERK1 (K-23), anti-P38 (C-20), anti-actin (C-2), anti-p-c-JUN (S63)(KM-1) and anti-c-JUN (H-79) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-MLK3 (C-terminal) (for detecting human MLK3) was from Epitomics (Cambridge, MA, USA).Anti-p-MLK3, anti-p-ERK-1/2 (T202/Y204)(E-10), anti-p-JNK1/2 (T183/Y185) (81E11) and anti-p-P38 (T180/Y182) (#9216) were obtained from Cell Signaling (Danvers, MA, USA). Anti-MMP-1 (#36665 R) was purchased from R&D systems (Minneapolis, MN, USA), anti-p-paxillin S178 (#A300-100 A) was purchased from Bethyl Laboratory (Montgomery, TX, USA). IRDye 800CW goat anti-mouse IgG, IRDye 680 goat anti-rabbit IgG and IRDye 800CW donkey anti-goat IgG were from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and 546 was from Invitrogen and used for immunofluorescence staining.Cell lines MCF7, obtained from ATCC (Manassas, VA, USA), have been recently authenticated and were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). MCF7iMLK3 cells engineered to inducibly express MLK3 were previously described. 9,22 ZR-75-1 (from ATCC) and 4T1-luc2 (Perkin Elmer, Waltham, MA, USA) cells were maintained in RPMI-1640 (Gibco, Life Technology, Grand Island, NY, USA) with 10% FBS. SUM-159-GFP cells (a gift from Dr Chengfeng Yang (University of Kentucky)) were maintained in Ham's F-12 (Gibco) supplemented with 5% FBS, 5 μg/ml insulin, 1 μg/ml hydrocortisone and containing penicillin/streptomycin. The cell lines were routinely tested for mycoplasma contamination.",
"\nFigure 7 .\n7FRA-1 and MMP-1a are upregulated in 4T1-derived circulating tumor cells. The mRNAs isolated from parental 4T1 cells and from circulating tumor cell lines, 4T1-CTC#1 and 4T1-CTC#2, treated with vehicle or 400 nM CEP-1347 for 24 h were subjected to qRT-PCR analysis using FRA-1-and MMP-1a-specific primers. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; **P o0.01."
] | [
"Ectopic expression of MLK3 drives FRA-1 expression in ER+ BC cells. Cellular lysates and/or mRNAs were collected from (a and b)",
"treatment with either MLK inhibitor downregulates FRA-1 protein in both SUM-159 and 4T1 cells. Similarly, immunofluorescence staining of SUM-159 and 4T1 cells treated with CEP-1347 revealed loss of FRA-1 staining (Figure 3c and d), similar to the phenotype of the 4T1KO-1 cells. These findings further support a requirement for active MLK3 in FRA-1 expression in highly invasive breast cancer cell lines. Of note, MLK inhibitor treatment is sufficient to reduce both JNK and ERK signaling in 4T1 cells (SupplementaryFigure 3). Further, overexpression, silencing or inhibition of MLK3 in breast cancer cells had no effect on levels of the related transcription factor c-FOS, reflecting specificity in MLK3 control of FRA-1 (SupplementaryFigure 4).",
"MAPK pathways, including SP600125 (JNK), U0126 (MEK/ERK) or SB203580 (P38), and the impact on FRA-1 protein levels was assessed. Treatment with the MLK, JNK or ERK inhibitor significantly decreased basal, endogenous FRA-1 levels in SUM-159 (Figure 4b) and 4T1 cells (Figure 4c). The P38 MAPK inhibitor SB203580 increased FRA-1 expression in both invasive",
"MLK3 is required for FRA-1 expression in TNBC cells. Cellular lysates and/or mRNA samples were collected from (a and b) SUM-159 cells treated with 50 nM control siRNA or MLK3 siRNA for 24 h, (c) parental 4T1, WT clone and three 4T1 CRISPR MLK3-knockout clones (KO-1, KO-2 and KO-3) and (d) parental 4T1 or 4T1KO-1 cells. Cellular lysates were subjected to immunoblotting with indicated antibodies.",
"is ablated in 4T1KO-1 and 4T1KO-2, compared with control parental 4T1 cells (Figure 5c). Loss of MLK3 decreases MMP-9 mRNA levels by 60 and 50% in 4T1KO-1 and 4T1KO-2 cells, respectively, compared with parental 4T1 cells. Similar effects on mRNA levels of MMPs were observed in 4T1 cells treated with CEP-1347 (Figure 5d). SUM-159 TNBC cells, which express MMP-1 but not MMP-9, 36 show decreased MMP-1 mRNA levels upon MLK3 gene silencing or MLK inhibitor treatment (Figure 5e and f).",
"Pharmacological inhibition of MLKs reduces FRA-1 protein expression in TNBC lines. (a) SUM-159 cells and (b) 4T1 cells were treated with vehicle, 400 nM CEP-1347 or 400 nM URMC-099 for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (c) SUM-159 cells and (d) 4T1 cells were seeded on coverslips, treated ± 400 nM CEP-1347 for 24 h, and subjected to immunofluorescence staining against FRA-1 antibody (green). Nuclei were counterstained with DAPI (blue); Scale bar, 25 μm in c and 50 μm in d. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al Transendothelial migration assays were performed to assess the ability of 4T1 or 4T1KO-1 cells to disrupt and invade through a confluent endothelial cell monolayer. Migration of luciferaseexpressing cancer cells through the endothelial cell monolayer was quantified by bioluminescence imaging. The 4T1KO-1 clone was chosen for these experiments because its bioluminescence activity is comparable to that of parental 4T1 cells (Supplementary Figure 7). Parental 4T1 cells increase permeability of the endothelial cell layer as measured by leakage of fluorescently labeled albumin from the upper into the lower transwell chamber, whereas 4T1KO-1 cells show markedly reduced endothelial permeability (Figure 6f). Compared with parental 4T1 cells, 4T1KO-1 cells show a fourfold reduction in transendothelial migration",
"Both JNK and ERK pathways contribute to FRA-1 expression through MLK3 signaling. (a) MCF7 cells were transiently transfected with a wild-type MLK3 (pRK-MLK3) or a kinase dead MLK3 variant (pRK-MLK3-K144M) for 24 h, (b) SUM-159 and (c) 4T1 cells were treated with vehicle, 400 nM CEP-1347 (MLK inhibitor), 15 μM SP600125 (JNK inhibitor), 10 μM U0126 (MEK/ERK inhibitor) or 10 μM SB203580 (P38 inhibitor) for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. (d) The mRNAs from 4T1 cells treated with vehicle, 400 nM CEP-1347, 15 μM SP600125 and 10 μM U0126 for 24 h were subjected to qRT-PCR analysis with FRA-1 primers.Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; NS, not statistically significant; **P o0.01.",
"MLK3 induces MMP-1 and MMP-9 expression. Cellular lysates or mRNAs, as indicated, were isolated from (a) MCF7iMLK3 cells treated with vehicle or 50 nM AP21967 for 24 h, (b) MCF7iMLK3 cells treated with vehicle or with 50 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, for 24 h, (c) parental 4T1 cells and two 4T1 MLK3-knockout clones (4T1KO-1 and 4T1KO-2), (d) 4T1 cells treated with vehicle or 400 nM CEP-1347 for 24 h, and SUM-159 cells treated with (e) 50 nM control or MLK3 siRNA for 24 h, or (f) vehicle or 400 nM CEP-1347 for 24 h.",
"MLK3 regulates cancer cell invasion and transendothelial migration. (a-c) MCF7iMLK3 cells treated with vehicle or with 25 nM AP21967 plus either 50 nM control or FRA-1 siRNA, as indicated, and (d) parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3) were subjected to an in vitro Matrigel transwell invasion assay for 24 h. Relative cell invasion, with control set at 100%, is expressed as mean ± s.d. from three independent experiments. (e) A representative image from three independent experiments of gelatin zymography of conditioned medium from parental 4T1 cells treated with vehicle or 400 nM CEP-1347 and MLK3-KO 4T1 clones (KO-1, KO-2 and KO-3). (f and g) Parental 4T1 and 4T1KO-1 were subjected to transendothelial migration toward 10% FBS. Endothelial permeability was assessed using 5(6)-Carboxyfluorescein (6-FAM)-conjugated albumin, and transendothelial migration was assessed using bioluminescence imaging as described in the 'Materials and Methods' section. Relative 6-FAM conjugated albumin concentration and bioluminescence activity are expressed mean ± s.d. from three independent experiments performed in triplicate. **P o0.01. *Po0.1. NS, not significant. Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al model. Importantly the MLK inhibitor, CEP-1347, which blocks invasion, also reduces FRA-1 and MMP-1 in CTC lines. Taken together, our data reveal important roles of MLK3 during basement membrane degradation and transendothelial migration and suggest that MLK3 inhibitors may be a useful addition to the limited armament for combating TNBC. MATERIALS AND METHODS Chemicals and antibodies Chemicals. 5(6)-Carboxyfluorescein (6-FAM), bovine serum albumin, gelatin and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St Louis, MO, USA). SP600125, U0126 and SB203580 were from Calbiochem (San Diego, CA, USA). CEP-1347 and CEP-11004 were generously provided by Cephalon, Inc., a wholly owned subsidiary of Teva Pharmaceuticals, Ltd (North Wales, PA, USA). AP21967 was provided by Ariad Pharmaceuticals (Cambridge, MA, USA). Calcein AM and Simple Blue SafeStain were from Invitrogen (Carlsbad, CA, USA). Antibodies. Anti-MLK3 (A-20) (for detection of murine MLK3), anti-FRA-1 (R-20), anti-JNK1/3 (C-17), anti-ERK1 (K-23), anti-P38 (C-20), anti-actin (C-2), anti-p-c-JUN (S63)(KM-1) and anti-c-JUN (H-79) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-MLK3 (C-terminal) (for detecting human MLK3) was from Epitomics (Cambridge, MA, USA).Anti-p-MLK3, anti-p-ERK-1/2 (T202/Y204)(E-10), anti-p-JNK1/2 (T183/Y185) (81E11) and anti-p-P38 (T180/Y182) (#9216) were obtained from Cell Signaling (Danvers, MA, USA). Anti-MMP-1 (#36665 R) was purchased from R&D systems (Minneapolis, MN, USA), anti-p-paxillin S178 (#A300-100 A) was purchased from Bethyl Laboratory (Montgomery, TX, USA). IRDye 800CW goat anti-mouse IgG, IRDye 680 goat anti-rabbit IgG and IRDye 800CW donkey anti-goat IgG were from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and 546 was from Invitrogen and used for immunofluorescence staining.Cell lines MCF7, obtained from ATCC (Manassas, VA, USA), have been recently authenticated and were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). MCF7iMLK3 cells engineered to inducibly express MLK3 were previously described. 9,22 ZR-75-1 (from ATCC) and 4T1-luc2 (Perkin Elmer, Waltham, MA, USA) cells were maintained in RPMI-1640 (Gibco, Life Technology, Grand Island, NY, USA) with 10% FBS. SUM-159-GFP cells (a gift from Dr Chengfeng Yang (University of Kentucky)) were maintained in Ham's F-12 (Gibco) supplemented with 5% FBS, 5 μg/ml insulin, 1 μg/ml hydrocortisone and containing penicillin/streptomycin. The cell lines were routinely tested for mycoplasma contamination.",
"FRA-1 and MMP-1a are upregulated in 4T1-derived circulating tumor cells. The mRNAs isolated from parental 4T1 cells and from circulating tumor cell lines, 4T1-CTC#1 and 4T1-CTC#2, treated with vehicle or 400 nM CEP-1347 for 24 h were subjected to qRT-PCR analysis using FRA-1-and MMP-1a-specific primers. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; **P o0.01."
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"Metastatic breast cancer is responsible for nearly half a million deaths worldwide 1 and 40 000 deaths in the United States, 2 annually. A major contributor is a lack of efficacy of the current standard treatments in preventing and treating metastatic disease. Breast cancer metastasis is a multistep process initiated by cancer cells within a primary tumor that gain invasive capacity. These cancer cells must breach the basement membrane, invade through extracellular matrix and intravasate into blood vessels. The cells that intravasate into the bloodstream, circulating tumor cells (CTCs), must survive in the circulation, extravasate to a distant site and colonize to form metastatic lesions. 3 Of the major clinical breast cancer subtypes, triple-negative breast cancer (TNBC) is considered the most aggressive and has the highest rate of metastasis and early recurrence. 4 Given the relative dearth of targeted therapies for treating TNBC, standard treatment relies on surgical removal, adjuvant radiotherapy and toxic chemotherapy.",
"Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that transduces signals from multiple cell surface receptors to activate MAPK cascades in a context-dependent manner. 5,6 Activated MAPKs directly phosphorylate cytosolic substrates or undergo nuclear translocation to regulate transcription factors, including activating protein-1 (AP-1). 5,6 MLK3 is critical for TNBC metastasis. 7,8 We have shown in TNBC models that MLK3 mediates JNK-dependent paxillin phosphorylation to facilitate focal adhesion turnover and cell migration. 8 In addition, MLK3 signaling leads to JNK-mediated c-JUN phosphorylation, 9 which activates AP-1-mediated gene expression.",
"AP-1 transcription factors comprise, usually heterodimeric, combinations of JUN and FOS family members including c-JUN, JUN-B, JUN-D, c-FOS, FOS-B, FRA-1 and FRA-2. 10 Aberrant AP-1 activity regulates genes that promote cancer progression. 11,12 Among AP-1 members, high levels of FRA-1 are associated with poor prognosis in TNBC. 11,13 FRA-1 is elevated in TNBC cell lines compared with estrogen receptor-positive breast cancer (ER+ BC) cell lines; 14,15 and is required for proliferation, 15 epithelial-tomesenchymal transition, 13,16 invasion 17,18 and metastasis. 19 Invasion genes controlled by FRA-1 include matrix metalloproteinases (MMPs), the zinc-dependent endopeptidases involved in matrix degradation and extracellular matrix remodeling. 20 Elevated levels of several MMPs are found in many types of solid tumors; these MMPs have crucial roles in multiple steps of tumor progression including tumor growth, angiogenesis, invasion and metastasis. 21 In this study, we demonstrate that MLK3 is a key regulator of FRA-1 expression in both ER+ BC and TNBC models. Furthermore, we show that the MLK3-FRA-1 axis controls levels of MMP-1 and MMP-9. Consistent with the roles of these MMPs, loss of MLK3 blocks Matrigel invasion as well as transendothelial migration of highly aggressive 4T1 cells. Importantly, an MLK inhibitor diminishes FRA-1 and its target genes, MMP-1 and MMP-9, in TNBC cells suggesting that targeting MLK3 may interfere with metastatic progression.",
"MLK3 is required for FRA-1 expression in breast cancer cells High FRA-1 levels are found in aggressive TNBC, whereas ER+ BC cell lines typically have low FRA-1 levels and are poorly invasive. 14,15 To examine whether MLK3 promotes FRA-1 expression in ER+ BC cells, we utilized MCF7 cells engineered to overexpress MLK3 upon treatment with the transcriptional inducer AP21967 (MCF7iMLK3). 9,22,23 As shown in Figure 1a, induced MLK3 expression increases FRA-1 protein expression, and drives cell migration in both transwell 9 and wound-healing assays (Supplementary Figure 1). As FRA-1 is an AP-1-regulated gene, 24 and MLK3 is known to activate AP-1, 9 quantitative reverse transcribed PCR (qRT-PCR) analysis was performed to determine FRA-1 transcript levels. As shown in Figure 1b, MLK3 robustly increases the FRA-1 transcript level. Consistent with our previous findings, 22 ectopically expressed MLK3 is active as judged by phospho-MLK3 immunoblotting (Figure 1a). To assess whether MLK3 catalytic activity is required for FRA-1 expression, vectors encoding wild-type MLK3 or a kinase inactive mutant MLK3 K144M 23 were transiently introduced into MCF7 cells. Wild-type MLK3 drives FRA-1 expression, whereas ectopic expression of equivalent protein levels of MLK3 K144M fails to upregulate FRA-1 ( Figure 1c). As shown in Figure 1d, overexpression of wild-type, active MLK3 in ER+ ZR-75-1 cells also drives FRA-1 expression. Thus, high levels of active MLK3 can upregulate FRA-1 expression in multiple ER+ BC cells.",
"In complementary experiments, we investigated the requirement for MLK3 in metastatic TNBC cell lines, which possess high endogenous levels of FRA-1. 15 MLK3 silencing in highly invasive TNBC SUM-159 cells reduces both FRA-1 protein ( Figure 2a) and mRNA (Figure 2b) levels, compared with SUM-159 cells transfected with control siRNA. To evaluate the function of MLK3 in the highly metastatic murine TNBC 4T1 model, we first generated MLK3 gene knockout 4T1-luc2 cells using the CRISPR/Cas9n (nickase) 25 system (Supplementary Figure 2A). Three MLK3-knockout (MLK3 KO) clones, 4T1KO-1, 4T1KO-2 and 4T1KO-3, as well as a wild-type (WT) clone that maintained MLK3 expression, were confirmed by sequencing (Supplementary Figure 2B). All three 4T1KO clones lack MLK3 expression and show decreased FRA-1 protein expression in contrast to parental 4T1 cells and the WT clone ( Figure 2c). Based on qRT-PCR analysis, FRA-1 mRNA transcripts are also reduced in MLK3 KO 4T1 cells compared with parental cells (Figure 2d). MCF7iMLK3 cells treated with vehicle or 50 nM AP21967 to induce MLK3 expression for 24 h, (c) MCF7 cells were transiently transfected with a wild-type MLK3 (pRK-MLK3) or a kinase dead MLK3 variant (pRK-MLK3-K144M) for 24 h, and (d) ZR-75-1 cells transiently transfected with pRK-MLK3 expression vector for 24 h. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. The mRNAs were subjected to qRT-PCR with primers for the indicated genes. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate; NS, not statistically significant; **P o0.01.",
"Nuclear FRA-1 expression, observed in parental 4T1 cells, is nearly absent in the 4T1KO-1 cells (Figure 2c). To validate the specificity of CRISPR MLK3 KO, a rescue experiment was performed by transiently transfecting a bi-cistronic pCMS-EGFP-MLK3 vector. EGFP-positive cells, which coexpress MLK3, regain FRA-1 expression (Figure 4e, Supplementary Figure 2C). Because 4T1 cells are poorly transfectable, multiple slides were used to score 100 EGFP-positive cells for FRA-1 expression. As shown in Figure 2f, 98% of the EGFP-positive 4T1KO-1 cells, which also express ectopic MLK3, regain nuclear FRA-1 staining.",
"To assess whether blockade of MLK activity reduces FRA-1 levels in highly invasive breast cancer cells, SUM-159 and 4T1 cells were treated with either CEP-1347 or URMC-099, MLK inhibitors with unrelated chemical structures, for 24 h and subjected to immunoblot analysis. As shown in Figure 3a and MLK3-activated JNK and ERK contribute to FRA-1 regulation Our data show that active MLK3 induces expression of FRA-1. Ectopic expression of wild-type MLK3 in MCF7 cells increases both JNK and ERK activation, as judged by the levels of phospho-JNK and phospho-ERK. It is well established that MLK3 utilizes its catalytic activity to regulate JNK activity. 8,9 However, MLK3 can activate ERK either through either kinase-dependent signaling 26 or kinase-independent scaffolding, 27 depending upon context. As shown in Figure 3a, wild-type MLK3 increases both JNK and ERK activities and drives FRA-1 expression, but equivalent levels of the kinase dead mutant MLK3-K144M fail to upregulate FRA-1 and do not significantly increase JNK or ERK activity.",
"To determine which MAPK signaling pathways are required for maintaining basal levels of endogenous FRA-1 in TNBC cells, cells were treated with small molecule inhibitors that block specific Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. The mRNAs were subjected to qRT-PCR with primers for the indicated genes. Relative mRNA expression is displayed as mean ± s.d. from at least three independent experiments performed in triplicate. (e and f) Parental 4T1 cells or 4T1KO-1 cells were transfected with bi-cistronic vector expressing EGFP and MLK3 (pCMS-EGFP-MLK3) for 24-48 h and were subjected to immunofluorescence staining using a FRA-1 antibody. FRA-1 staining is shown in red and GFP, which indicates co-expression of MLK3, is shown in green. Nuclei were counterstained with DAPI (blue); Scale bar, 25 μm; NS, not statistically significant; **P o0.01.",
"breast cancer cell lines, perhaps reflecting the established antagonism between the JNK and p38 pathways. 28 In a time course, treatment with either the JNK or MEK inhibitor resulted in a reduction of basal FRA-1 protein levels over time (Supplementary Figure 5), consistent with what has been observed on MEK inhibitor treatment of colon cancer cells. 29 In this experiment, SP600125 effectively blocked JNK activity as judged by phospho-c-JUN levels, but had no impact on phospho-ERK. The MEK inhibitor, U0126, efficiently blocked ERK activation over the entire time course, although a slight diminution of phospho-c-JUN at later time points was observed, suggesting that inhibition of the ERK pathway may indirectly downregulate phospho-c-JUN, as has been shown in Ras-overexpressing thyroid cells. 30 Treatment with either CEP-1347, SP600125 or U0126, for 24 h reduced FRA-1 transcript levels, as determined by qRT-PCR ( Figure 4d), suggesting that both the JNK and ERK pathways contribute to FRA-1 transcriptional regulation.",
"MLK3 increases MMP-1 and MMP-9 through FRA-1 FRA-1 is an oncogenic member of the AP-1 transcription factor family, 31 which regulates expression of genes involved in cancer progression, including MMPs. As MLK3 regulates both FRA-1 and cancer cell invasion, we hypothesized that it may control the expression of FRA-1-dependent MMPs, such as MMP-1, 15,17,32 MMP-2 33,34 and MMP-9. 17 As shown in Figure 5a, induced expression of MLK3 upregulates MMP-1 and MMP-9 mRNA but reduces MMP-2 mRNA levels. Both cytosolic and secreted MMP-1 protein can be detected on induction of MLK3 expression in MCF7iMLK3 cells, reflecting the strong upregulation of MMP-1 mRNA. Importantly, the increase in MMP-1 protein is abrogated by FRA-1 gene silencing ( Figure 5b).",
"The mRNA levels of the same MMPs were evaluated in the 4T1 line and 4T1KO clones. By qRT-PCR analysis, MMP-1a, the functional ortholog of human MMP-1, 35 ",
"To investigate the requirement of FRA-1 in MLK3-driven cancer cell invasion, the impact of FRA-1 silencing on Matrigel transwell invasion of MCF7iMLK3 cells was determined. As shown in Figure 6a-c, induced expression of MLK3 in MCF7iMLK3 cells upregulates FRA-1 protein levels and enhances invasion. Silencing of FRA-1 reduces MLK3-induced invasion, indicating that FRA-1 functions downstream of MLK3 in this context. Of note, we previously found a role for MLK3 in JNK-mediated phosphorylation of paxillin in MCF10A and TNBC cells. 8 In MCF7 cells, we observe a modest increase in phospho-paxillin on MLK3 expression, which is unaffected by FRA-1-silencing (Supplementary Figure 6) suggesting that MLK3 regulation of paxillin is independent of FRA-1, and consistent with the idea that MLK3 controls multiple pathways in cancer cell migration and invasion.",
"In TNBC models, tumor cell-derived MMP-9 is required for Matrigel invasion and for formation of pulmonary metastases. 37-39 MMP-1 is crucial for transendothelial migration and vascular intravasation. 40 As MLK3 deletion decreases both MMP-1 and MMP-9 levels, we tested whether deletion of MLK3 impacts Matrigel invasion and transendothelial migration in TNBC cells. Indeed three independent 4T1 MLK3 KO clones show impaired invasion through Matrigel compared with parental 4T1 cells. Treatment with CEP-1347 similarly inhibits Matrigel invasion (Figure 6d) of 4T1 cells. We then performed gelatin zymography to evaluate levels of secreted MMP-9. Notably, MMP-9 can be distinguished from the other major gelatinase MMP-2, based on molecular weights of 92 kDa and 72 kDa, respectively. Gelatin zymography of conditioned medium from 4T1 cells shows a single predominant band corresponding to MMP-9; and secreted MMP-9 levels are reduced in conditioned media from 4T1 cells treated with CEP-1347 or from multiple 4T1 MLK3 KO clones (Figure 6e). (Figure 6g). These data indicate that MLK3 contributes to cancer cell-induced endothelial permeability and is required for transendothelial migration.",
"FRA-1 and MMP-1a are upregulated in circulating tumor cells derived from TNBC tumors The established role of MMP-1 in cancer intravasation, coupled with our findings that MLK3 controls MMP-1 levels, prompted us to evaluate the components of the MLK3-FRA-1-MMP-1 signaling axis in CTCs. A clonogenic assay was used to isolate CTCs from the blood of mice bearing 4T1-luc2 mammary tumors and associated metastases. By phase contrast imaging, isolated CTC lines (4T1-CTC) show distinct morphology compared with the morphology of the parental 4T1 cells. Many of these cells are able to detach and re-attach to tissue culture plates (Supplementary Figure 8). Bioluminescence imaging demonstrated that the isolated CTC lines retain luciferase activity, confirming their origin from the 4T1-luc2 tumors (Supplementary Figure 8). Both 4T1-CTC lines show increased FRA-1 and MMP-1a mRNA expression, compared with parental 4T1 cells. Furthermore in the 4T1-CTC lines, FRA-1 and MMP-1a levels are dependent upon MLK activity, as their levels are decreased by CEP-1347 treatment (Figure 7). Co-expression and mutual exclusivity analysis (Supplementary Figure 9) revealed a statistically significant tendency towards co-occurrence of MLK3 and FRA-1 gene expression, as well as MMP-1 and FRA-1.",
"Metastasis is overwhelmingly the cause of breast cancer-related death, yet the complexity of the metastatic process makes it therapeutically challenging to treat. 41 Previous studies in our lab and others have demonstrated that MLK3 is crucial for TNBC metastasis. 7,8 Herein, we utilized the CRISPR/Cas9 approach to deplete MLK3 in the highly aggressive 4T1 mammary cancer model to elucidate the role(s) of MLK3 in discrete steps of metastasis and to identify the key signaling pathways through which MLK3 regulates these events. Mechanistically, we have deciphered a novel function for MLK3 in controlling FRA-1 in breast cancer cells. In this context, both JNK and ERK signal downstream of MLK3 (Figure 4, Supplementary Figure 3) to enhance FRA-1 expression. FRA-1 regulation is complex. JNK is well known to phosphorylate c-JUN, 42 the AP-1 member, which is required for transcription of FRA-1. 43 ERK, in turn, phosphorylates FRA-1, which enhances the stability of the FRA-1/c-JUN heterodimer. 14,29 Thus MLK3 signaling is well poised to regulate FRA-1/c-JUN-mediated transcription.",
"FRA-1 has emerged as a key driver of metastatic progression in multiple cancer types including breast cancer, 11,14,15,17 lung cancer, 18 colorectal cancer 16,44,45 and glioblastoma. 46 FRA-1 not only controls expression of genes involved in cell motility, epithelialto-mesenchymal transition 13,45,47,48 and cell invasion, 15,17,32,49 but it also controls proliferation, 15,50,51 metastatic outgrowth 44 and the stem cell phenotype of cancer cells. 52 Several FRA-1-regulated genes 15,17,32,49 have been demonstrated to facilitate cancer cell invasion including MMP-1, MMP-2 and MMP-9. 17,18 Zhan et al. 53 have previously reported that, in ovarian cancer cells, MLK3 is important for expression of MMP-1, -2, -9 and -12 and is required for ovarian cancer invasion. Our studies show that in TNBC models, MLK3 deletion decreases both MMP-1 and MMP-9 expression ( Figure 5) but does not significantly affect MMP-2 levels. Functionally, MMP-9 facilitates extracellular matrix remodeling and basement membrane degradation and, like MLK3, is critical for TNBC metastasis. 37 Likewise, MMP-9 silencing in multiple TNBC lines has been shown to block Matrigel invasion, 37 analogous to our findings that MLK3 deletion or CEP-1347 treatment inhibits Matrigel invasion ( Figure 6). In TNBC, hematogenous metastasis, which requires transendothelial migration 54 is more common than lymphatic spread. 55 During vascular intravasation and extravasation, cancer cells must disrupt endothelial barrier integrity and transmigrate through the endothelial layer. MMP-1 is an interstitial collagenase-I required for fibrillar collagen remodeling. [56][57][58] However non-collagenolytic mechanisms of MMP-1 are implicated in transendothelial migration of tumor cells. 40 In epidermoid cancer, for instance, tumor cell-derived MMP-1 increases endothelial barrier permeability by proteolytically activating the endothelial thrombin receptor PAR-1 and facilitating transendothelial migration. 40 In our study, TNBC cells induce endothelial barrier permeability and transmigrate through an endothelial barrier, whereas, MLK3-deleted tumor cells fail to induce permeability and their transendothelial migration is impaired ( Figure 6). These findings suggest that one mechanism through which MLK3 facilitates TNBC metastasis could be through vascular intravasation and, possibly, extravasation.",
"CTCs have risen to prominence as potential prognostic and predictive biomarkers for metastatic burden, metastatic recurrence and therapeutic response. [59][60][61][62][63][64][65][66][67][68][69][70] Our finding that 4T1-CTCs have elevated mRNA levels of FRA-1 and MMP-1a compared with parental 4T1 cells (Figure 7) is important in light of several lines of evidence supporting a key role for MMP-1 in CTCs, epithelial-to-mesenchymal transition and metastatic progression. Furthermore, MMP-1 was identified as a key gene upregulated in infiltratative self-seeding CTCs. 71 Elevated MMP-1 was also observed in TNBC MDA-MB-231 subclones selected for their ability to metastasize to lung 72 and brain. 73 Recently, single cell gene expression analysis studies utilizing TNBC patient-derived xenograft models showed that MMP-1, as well as components of a proliferative gene signature, was significantly increased in late stage, high burden metastatic cells compared with early stage, low burden metastatic cells. 74 These data, along with the role of MMP-1 in vascular intravasation, suggest that MMP-1 is not only required for early stages of metastatic process, but may also contribute to colonization.",
"TNBC is considered the most aggressive subtype of breast cancer; however, therapeutic options are limited. A major challenge is to identify important targetable signaling pathways in TNBC. MMP-1 expression is significantly elevated in aggressive breast tumors and correlates with both tumor size and grade 75 pointing to MMP-1 as a promising therapeutic target. Indeed, MMPs, including MMP-1, have a long history as targets for cancer therapeutics yet early clinical trials using MMP inhibitors were unsuccessful due, in part, to inadequate preclinical and clinical design, lack of drug specificity and high toxicity. 76,77 Recently The mRNAs were subjected to qRT-PCR with primers to the indicated genes. Relative mRNA expression is displayed as the mean ± s.d. from at least three independent experiments performed in triplicate. Cellular lysates were subjected to immunoblotting with indicated antibodies. Western blot quantification of the indicated protein normalized to actin is expressed as mean ± s.d. from at least three independent experiments. Con, Control; CL, cellular lysate; CM, concentrated conditioned medium; NS, not statistically significant; *Po 0.05; **P o0.01.",
"Critical role of MLK3 in metastasis of TNBC C Rattanasinchai et al FRA-1, an upstream regulator of MMP-1, has also emerged as a key driver of cancer progression; however, transcription factors are not readily druggable. In one study, an existing inhibitor of the FRA-1 regulated gene, adenosine receptor A 2B , ADORA2B, was shown to block formation of lung metastases in a TNBC experimental metastasis xenograft model. 19 However, it is unclear whether targeting a single FRA-1-regulated gene will always be sufficient to halt breast cancer progression, as FRA-1 controls a suite of genes involved in cancer invasion and metastasis. 45 Based on our findings, we propose that an alternative strategy would be to target MLK3, an upstream regulator of FRA-1.",
"Multiple MLK inhibitors exist, including CEP-1347 78 and URMC-099. 79 Our data show that both of these MLK inhibitors, built upon different chemical scaffolds, reduce FRA-1 levels in 4T1 cells. In addition, either CEP-1347 treatment or MLK3 deletion reduces FRA-1, MMP-1, and MMP-9 expression to similar levels in multiple TNBC cells, indicating that MLK3, specifically, controls FRA-1, MMP-1 and MMP-9 expression. CEP-1347 progressed through Phase II/III clinical trials for Parkinson's disease, and although it failed to delay progression, no significant toxicity was observed, 80 suggesting that it could potentially be repurposed for breast cancer treatment.",
"In summary, we provide evidence that MLK3 signaling is a crucial regulator of FRA-1 and its target genes, MMP-1 and MMP-9 in models of TNBC. As a consequence, depletion or inhibition of MLK3 in TNBC cells impairs both Matrigel invasion and transendothelial migration. Consistent with these findings, FRA-1 and MMP-1 are upregulated in isolated CTC lines from a TNBC ",
"For siRNAs, Mission siRNA Universal Negative control #1, siRNA duplexes targeting human MLK3 (5′-CUGACUGCCACUCAUGGUG-3′ and its antisense) 9,81 and human FRA-1 (5′-GGGCAGUGACGUCUGGAG-3′ and its antisense) 15 were from Sigma-Aldrich. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent.",
"Plasmids, pRK-MLK3 or pRK-MLK3 K144M, were previously described. 23 Lipofectamine 2000 and Lipofectamine 3000 were used to transfect MCF7 and ZR-75-1 cells, respectively. In a recovery assay of MLK3-knockout 4T1 cells, pCMV-EGFP-MLK3 82 expression vector was reverse-transfected into 4T1KO-1 cells using Lipofectamine 3000.",
"The MLK3 CRISPR construct was generated based on a previously described protocol. 83 Briefly, two pairs of guide RNAs for a CRISPR-Cas9n construct were designed (crispr.mit.edu) to target exon 1 of murine MLK3 (Supplementary Figure 2) and were cloned into pSpCas9n(BB)-2A-GFP (PX461) 83 (a gift from Dr Feng Zhang; Addgene plasmid #48140). After reverse transfection using Lipofectamine 3000, GFP-positive clones were screened for MLK3 deletion by immunoblotting. Genomic DNAs from selected clones were collected and amplified using forward primer 5′-ATGGAGCCCTTGAAGAACCT-3′ and reverse primer 5′-ACGGTAGA CCTTGCCGAAG-3′. Purified PCR products were subjected to TOPO TA cloning (Invitrogen). At least five clones were subjected to nucleotide sequencing to identify the genomic alterations.",
"Cellular lysates were prepared in lysis buffer (1% NP-40, 150 mM sodium chloride and 50 mM Tris, pH 8.0) and immunoblotting was performed as described. 8,9 Immunofluorescence analysis Immunofluorescence staining was performed as previously described. 8 Images were acquired and, if indicated, quantified from N4100 cells per group, using an Olympus fluorescence microscope and MetaMorph software.",
"Gelatin zymography 4T1 cells or their derivatives (2.5 × 10 5 ) were seeded to 35 mm culture dishes. The following day, the cells were incubated in serum-free medium for 24 h, in the presence of CEP-1347, as indicated. Conditioned media corresponding to equal cellular equivalents were loaded onto and run through 10% polyacrylamide gels containing 1 mg/ml gelatin. The gels were incubated for 1 h in 2.5% Triton X-100, developed for 24 h at 37°C in 50 mM Tris-HCl buffer, pH 7.6, containing 5 mM CaCl 2 and 200 mM NaCl, and finally stained for 16 h with Simple Blue SafeStain (Invitrogen). After 30 min destaining with water, gels were scanned and the images were processed using Image J software.",
"Quantitative real-time PCR Total RNAs were extracted using the RNeasy kit (Qiagen, Valencia, CA, USA) and cDNA synthesis was performed using a cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time qPCR was performed using either PerfeCTa SyBR green superMix (Quanta, Gaithersburg, MD, USA) or SYBR Green Master mix (Applied Biosystems). Specific primer sequences were designed using PrimerBank 84 (see Supplementary Table 1 for full list of primers). ",
"In vitro Matrigel invasion assay was performed as previously described. 8,9 Briefly, MCF7iMLK3 cells were transfected with 50 nM Control siRNA or MLK3 siRNA as described above for 16 h, and then serum-deprived for an additional 8 h in the presence of vehicle or 25 nM AP21957. The cells (1 × 10 5 ) were seeded into the upper chamber of 5 μm Matrigel-coated transwell chamber and allowed to invade in the presence of vehicle or 25 nM AP21967 for 24 h. For 4T1 cells and their derivatives, cells were serum-deprived overnight and 2 × 10 4 cells introduced into the upper chamber and allowed to invade for 24 h toward 10% FBS in the presence of indicated inhibitors. Mitomycin C (2 μg/ml) was included to eliminate possible effects of cell proliferation. Experiments performed in duplicate were repeated at least three times.",
"Transendothelial migration was assessed essentially as described. 40,85 Briefly, 1 × 10 5 EA.hy926 cells were grown as a confluent monolayer on 5 μm pore transwell inserts for 1-2 days and 1 × 10 5 4T1 cells, or their derivatives, that had been deprived of serum for 18 h were introduced to the upper chamber. 4T1 cells were allowed to migrate toward 10% FBS for 24 h. To measure permeability of the endothelial layer, 5(6)-Carboxyfluorescein (6-FAM)-conjugated albumin (10 μM final concentration) was added to the upper chamber and culture medium was collected from the bottom chamber after 30 min at 37 o C. Permeability was determined by measuring the fluorescence due to leakage of 6-FAM-conjugated albumin (excitation = 495 nm, emission = 520 nm) into the bottom chamber. To measure transendothelial migration, the cells inside the transwell inserts were wiped out with the cotton swabs, and the extent of migration through the transwell membranes was then determined by relative bioluminescence activity.",
"This experiment was carried out in accordance with standard protocols approved by All University Committee on Animal Use and Care at Michigan State University. Briefly, puromycin-resistant 4T1-luc2 cells (7.5 × 10 5 cells) were injected into the fourth mammary gland of 8-week-old female athymic nu/nu mice (N = 2). After 24 days, the two mice were killed and CTCs were isolated as previously described. 86 Briefly, 200 μl blood collected by cardiac puncture was cultured in RPMI-1640 supplemented with 20% FBS, 2 μg/ml puromycin, and penicillin/streptomycin for 10 days. Approximately 40 colonies were obtained from each blood sample, pooled and propagated as populations, named 4T1-CTC#1 and 4T1-CTC#2.",
"Results are expressed as mean ± standard deviation (s.d.",
"). An unpaired, two-tailed Student's t-test was used to calculate the P-value, and P o0.05 is considered statistically significant. At least three independent experiments were performed unless otherwise noted."
] | [] | [
"INTRODUCTION",
"RESULTS",
"MLK3 regulates cancer cell invasion and transendothelial migration",
"DISCUSSION",
"RNA interference and plasmid transfection",
"CRISPR-Cas9n constructs",
"Immunoblot analysis",
"In vitro Matrigel invasion assay",
"Transendothelial migration and endothelial permeability",
"CTC isolation from the 4T1 tumor bearing mice",
"Statistical analysis",
"Figure 1 .",
"Figure 3b ,",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 .",
"Figure 6 .",
"Figure 7 ."
] | [] | [
"Supplementary Table 1"
] | [
"MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells",
"MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells"
] | [] |
13,946,681 | 2022-03-18T21:25:44Z | CCBY | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0066070&type=printable | GOLD | 42835499ae44ea4824cb8b5c0ddf3547fda855c7 | null | null | null | null | 10.1371/journal.pone.0066070 | 2008596564 | 23741524 | 3669147 |
The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs
Published May 31, 2013
Maarja-Liisa Nairismä
Department of Molecular Biology and Genetics
Aarhus University
AarhusDenmark
Annette Fü
Department of Molecular Biology and Genetics
Aarhus University
AarhusDenmark
JesperRodrigo Labouriau
Department of Molecular Biology and Genetics
Aarhus University
AarhusDenmark
Bertram Bramsen
Department of Molecular Biology and Genetics
Aarhus University
AarhusDenmark
Ernst-Martin Fü
Department of Molecular Biology and Genetics
Aarhus University
AarhusDenmark
The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs
Published May 31, 2013Received May 11, 2012; Accepted May 6, 2013;Editor: Thomas Preiss, The John Curtin School of Medical Research, Australia Funding: This work was supported by The Danish Cancer Society grant DP08036 (www.cancer.dk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Upregulation of the proto-oncogene Twist1 is highly correlated with acquired drug resistance and poor prognosis in human cancers. Altered expression of this multifunctional transcription factor is also associated with inherited skeletal malformations. The mammalian Twist1 39UTRs are highly conserved and contain a number of potential regulatory elements including miRNA target sites. We analyzed the translational regulation of TWIST1 using luciferase reporter assays in a variety of cell lines. Among several miRNAs tested, miR-145a-5p, miR-151-5p and a combination of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p were able to significantly repress Twist1 translation. This phenomena was confirmed with both exogenous and endogenous miRNAs and was dependent on the presence of the predicted target sites in the 39UTR. Furthermore, the repression was sensitive to LNA-modified miRNA antagonists and resulted in decreased migratory potential of murine embryonic fibroblast cells. Understanding the in vivo mechanisms of this oncogene's regulation might open up a possibility for therapeutic interference by gene specific cancer therapies. Citation: Nairismägi M-L, Fü chtbauer A, Labouriau R, Bramsen JB, Fü chtbauer E-M (2013) The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs. PLoS ONE 8(5): e66070.
Introduction
The evolutionary conserved basic helix-loop-helix (bHLH) transcription factor TWIST1 is a multifunctional proto-oncogene with a strong correlation to poor prognosis. TWIST1 is able to inhibit c-MYC induced apoptosis [1] and directly regulates the expression of several other oncogenes such as GLI1 [2], miR-10b [3] and AKT2 [4]. Overexpression of TWIST1 has been observed in various types of cancer such as breast, prostate, gastric, pancreatic and bladder cancer, hepatocarcinoma, rhabdomyosarcoma, and glioma and is often associated with more aggressive phenotypes, and acquired drug resistance (reviewed in [5]).
Twist1 expression is regulated by a complex network of signals and has been described as an integrator of SHH, FGF and BMP signaling [6]. In mice, a genomic fragment containing 6 kb upstream and 1.5 kb downstream of the Twist1 gene is not sufficient to recapitulate Twist1 expression during embryogenesis (unpublished data) which is consistent with the observation that a translocation breakpoint 3 kb downstream of the Twist1 gene creates a comparable haploid insufficiency as the null allele [7]. However, a number of transcription factor binding sites have been identified in the Twist1 upstream region and direct binding of transcriptional activators like NF-kB [8] and repressors like Prospero-related homeobox 1 (PROX1) [9] has been shown.
TWIST1 is directly upregulated by hypoxia-inducible factor-1a (HIF-1a) and HIF-2a [10,11]. Intratumoral hypoxia is correlated with radiation therapy resistance and enhanced metastatic potential [12]. TWIST1 promotes tumor metastasis [10] by epithelial-mesenchymal transition (EMT) [13] and formation of invadopodia, the specialized membrane protrusions for extracellular matrix degradation [14]. TWIST1 is linked to the transcription factor SNAIL, which also induces EMT, in a positive feedback loop [15]. In addition, co-expression of TWIST1, HIF-1a and SNAIL has been correlated with metastasis and poor prognosis in primary tumors of head and neck squamous cell carcinoma (HNSCC) patients [10].
The wide spectrum of TWIST1 functions might be explained by the fact that TWIST1 is able to act both as a transcriptional activator [16] and inhibitor [17,18].
Originally, the Twist gene was discovered as a mutation disturbing cellular motility and EMT during gastrulation in Drosophila melanogaster [19,20]. A comparable phenotype is observed in murine homozygous Twist1 null mutant embryos in which the cephalic neural crest cells are unable to form a functional mesenchyme [21]. Haploid insufficiency causes Saethre-Chotzen syndrome (polysyndactyly and craniosynostosis) in humans and comparable symptoms in mice [7,22].
During mammalian embryogenesis, Twist1 mRNA precedes TWIST1 protein expression, indicating a translational control of Twist1 [23,24]. The same phenomena was recently observed in MCF-10ANeoT cells that had undergone EMT [25]. It is therefore interesting that many of the biological processes in which TWIST1 is involved, are associated with regulation by miRNAs, a hallmark of translational control. MiRNAs are endogenous small non-coding RNAs that regulate gene expression post-transcriptionally by modulating mRNA translation or stability. They influence cellular processes such as EMT, apoptosis, and differentiation, which all are essential for development and cancer [26,27]. MiRNA-mediated regulation of gene expression is complex as one mRNA is usually targeted by several miRNAs and one miRNA can target several mRNAs. Furthermore, miRNAs can act synergistically leading to higher levels of repression [28].
Here we show that the 39UTRs of mammalian Twist1 genes contain conserved miRNA target sites, which make them sensitive to regulation by several miRNAs, individually and in cooperative combination. Understanding the exact mechanism of Twist1 regulation is important as it may allow to utilize this physiological process to be utilized therapeutically.
Results
Analysis of Twist1 39UTR
Translational regulation of mRNAs is typically mediated through evolutionary conserved regulatory regions within the UTRs. To test if this is the case for Twist1, we compared the conservation of coding sequence (CDS) and 39UTR of Twist1 mRNA among selected amniotes. 59UTRs were not included since the full-length sequences are not available for all species. Limiting the investigation to amniotes gave us the possibility to also compare the conservation of Twist1 with that of Twist2 (Table 1), a highly related gene that is differently expressed but functionally largely equivalent to Twist1 [29][30][31].
The two genes are highly similar in their coding regions, genomic structure and length of the 39UTR. However, the 39UTR of Twist2 is not related to that of Twist1 (percent of identity 30%) and is, between species, remarkably less conserved compared to the unusually highly conserved 39UTR of Twist1 indicating a functional selection of this sequence that is almost as stringent as for the CDS. Furthermore, the 39UTR of Twist1 contains considerably more potential regulatory sequences, namely four nuclear polyadenylation signals (pA1-4 where pA3 and pA4 overlap), two cytoplasmic polyadenylation elements (CPEs), one AU-rich sequence and a number of putative miRNA target sites predicted by several algorithms (TargetScan, miRBase and PicTar). All but one of the identified potential regulatory sequences (miR-15b-3p) are 100% conserved between mouse and human Twist1 39UTR sequences (Fig. 1) and a large number are conserved in a wide range of mammalian species (Table 2). In addition, out of 18 miRNAs predicted to target Twist1, only one (miR-145a-5p) putatively targets Twist2 as well (Table 2).
MicroRNAs are targeting the Twist1 39UTR
The presence of multiple potential miRNA target sites in the 39UTR of Twist1 led us to investigate whether Twist1 expression could be regulated by miRNAs. The following miRNAs were tested for their potential to repress Twist1 translation in the human lung carcinoma cell line H1299: miR-33, miR-145a, miR-151, miR-326, miR-337, miR-361, miR-378a, miR-381, miR-409 and miR-543 (Fig. 1).
Murine pre-miRNA sequences were cloned into pdCMV2-EGFP expression vector and their correct processing was confirmed by Northern Blot (not shown). To determine whether any of the selected miRNAs are able to repress the expression of TWIST1, the miRNAs were tested individually or in pairwise combinations in a luciferase reporter assay using a construct in which the Firefly luciferase CDS was followed by the murine Twist1 39UTR sequence. MiR-485 and miR-609 have no target site in the 39UTR of Twist1 and were used as negative (mock) controls to calculate the unrepressed expression level of the reporter in these cells (Fig. 2). Expression of miR-145a resulted in a significant downregulation of the reporter by 44% (p,0.006). In addition, the co-expression of two pairs of miRNAs also led to a significantly increased repression: miR-151 + miR-337 resulted in a synergistic inhibition of 78% and miR-145a + miR-151 repressed TWIST1 expression by 61% (p,0.006). Notably, miR-337 alone had no effect while miR-151 only had a weak effect which in this assay was not statistically significant (p,0.15; see discussion).
In order to confirm the location and functionality of the predicted miRNA target sites within the 39UTR of Twist1, we mutated some of them to restriction enzyme recognition sites. Transfection of the wild type (wt) or mutant reporter with the corresponding miRNA confirmed that the repressive effects depended on the presence of the miRNA target sites. As shown in Fig. 3, mutating the target site of miR-145a-5p or miR-151-5p resulted in a loss of repression whereas lack of a binding site for miR-337-3p had no effect. When both miR-145a and miR-151 were present, mutating either of the target sites caused a comparable loss of repression. Interestingly, miR-337 was only able to repress the 39UTR reporter when miR-151 and its target site both were present. This confirms that miRNAs and their target sites both are necessary to repress the expression of TWIST1 protein.
MicroRNAs can act by translational inhibition
MicroRNAs primarily act by destabilizing target mRNA through decapping and/or deadenylation. However, a smaller subset of targets is rather translationally repressed leaving the mRNA levels unaltered (reviewed in [32]). As the uneven ratio between Twist1 mRNA and TWIST1 protein in the mouse embryo [23,24] indicated translational inhibition, we compared the mRNA and protein ratios in cells treated with a combination of synthetic miR-151-5p and miR-337-3p precursors. Using high concentrations of pre-miR-151-5p and pre-miR-337-3p (above 10 nM) we consistently found comparable decrease in both the mRNA and protein level of the Twist1 39UTR reporter (Fig. 4A). To more closely mimic the physiological conditions, we established stable pools of cells harboring the Twist1 39UTR reporter and transfected them using low concentrations (5 nM) of synthetic miR-151-5p and miR-337-3p precursors. Under such conditions, we observed a 50% reduction of luciferase reporter activity and hardly any reduction in the corresponding mRNA level upon miRNA co-transfection (Fig. 4B). This indicates that the synergistic effect of miR-151-5p and miR-337-3p is mainly due to translational inhibition and not RNA degradation. A corresponding analysis using the endogenous TWIST1 protein was technically not possible as we found all available TWIST1 specific antibodies to also react with unidentified proteins in murine cells not expressing TWIST1, a specificity issue not seen in human cells [25].
Twist1 reporter is downregulated by endogenous microRNAs
To test whether endogenous miRNAs are able to target the murine Twist1 39UTR reporter, we analyzed the expression pattern of miRNAs of interest in some commonly used mouse cell lines: NIH-3T3, C3H/10T1/2 and C 2 C 12 (Fig. 5). Since miR-145a-5p, miR-151-5p and miR-337-3p all were expressed in NIH-3T3 and C3H/10T1/2 but not in C 2 C 12 cells, we selected the first two lines for further analysis.
We then analyzed the effect of endogenous miRNAs on the wt and mutant Twist1 39UTR reporters. As shown in Fig. 6A and 6B, endogenous miR-145a-5p, miR-151-5p and miR-337-3p are targeting Twist1 39UTR in both cell lines, as mutating their target sites led to a statistically significant increase of reporter activities. This was confirmed by using locked nucleic acid (LNA)-modified miRNA antagonists to block each of these endogenous miRNAs. Inhibition of miR-145a-5p, miR-151-5p and miR-337-3p resulted in a decreased repression, i.e. an elevated expression of the reporter protein in both cell lines ( Fig. 6C and 6D), confirming that the endogenous murine miRNAs are able to target the Twist1 39UTR.
MiR-151-5p and miR-337-3p reduce the mobility of murine embryonic fibroblast cells There is no known cellular function which depends exclusively on TWIST1 and thus would give an all-or-none phenotype upon removal of TWIST1. We therefore decided to test whether the combination of miR-151-5p and miR-337-3p also effects the biological function of endogenous TWIST1, by investigating the ability of murine embryonic fibroblasts to migrate through an 8 mm pore filter. This type of assay correlates well with the EMT promotion by TWIST1 and has been used in similar studies [25]. Compared to cells treated with a scrambled control miRNA, cells treated with a combination of miR-151-5p and miR-337-3p migrated significantly less (Fig.7). Despite the small absolute difference, which probably indicates the limited contribution of TWIST1 to the mobility of these cells, the effect was highly significant. The probabilities of cell migration were estimated as 0.898 (95% bootstrap CI 0.896-0.913) for the miR-151-5p + miR-337-3p treated cells and 0.918 (95% bootstrap CI 0.914-0.922) for the control treated cells, and a bootstrap permutation test (p-value 0.000135) showed that the difference between the probabilities is statistically significant.
+ + + + 2 + 2 miR-137-3p + + + + 2 + 2 miR-145a-5p + + + + 2 2 + miR-151-5p + + + + 2 + 2 miR-214-5p + + + + 2 2 2 miR-326-3p + + + + 2 2 2 miR-337-3p + + + + 2 + 2 miR-361-5p + + + + 2 2 2 miR-378a-5p + + + + 2 2 2 miR-381-3p + + + + 2 + 2 miR-409-3p + + + + 2 2 2 miR-450b-5p + + + + 2 + 2 miR-508-3p + + + + 2 2 2 miR-543-3p + + + + 2 2 2 miR-576-5p + + + + 2 2 2 miR-580 + + + + 2 2 2 miR-591 + + + + 2 2 2
MicroRNAs underlined were tested in this study. doi:10.1371/journal.pone.0066070.t002
Discussion
In this study, we report a new mechanism for the regulation of TWIST1 expression. TWIST1 is a key player in tumorigenesis and metastasis and its overexpression has mostly been correlated with progressed stages of cancer and drug resistance [33]. Furthermore, suppression of TWIST1 expression in tumor cells can lead to inhibition of metastatic potential [13].
We analyzed the presence of conserved miRNA target sites in the 39UTR of Twist1 in selected amniotes (Table 1 and 2) and identified miR-145a-5p, miR-151-5p and the combinations of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p as the strongest regulators of murine Twist1 (Fig. 2 and 3). For the initial screen, we used human H1299 cells, which are easy to transfect. It should be noted, however, that a strong expression of endogenous miRNAs in these cells might cover the effect of exogenous miRNAs tested and thus give false negative results. In fact, we assume that the relatively high expression of endogenous miR-151-5p in H1299 cells (publically available miRNA microarray dataset; GEO accession number GSE30075) explains the significant difference between the miR-151-5p-mediated repressive effects shown in figures 2 and 3.
The distance between miR-145a-5p and miR-151-5p target sites is 394 nt, too long to expect a synergistic interaction [34]. Indeed, the effect of their combination was only slightly stronger than the additive effect of their independent action. In contrast, the combinatorial effect of miR-151-5p and miR-337-3p clearly is synergistic, as miR-337-3p alone was not able to downregulate the Twist1 39UTR reporter but only did so when miR-151-5p was present (Fig. 3). Importantly, this synergistic effect was not observed if the miR-151-5p target site was mutated. The 6 nt distance between these two miRNA target sites is within the optimal range for two cooperating miRNAs (between 6 to 40 nt) [34]. In addition, miR-337-3p has no complementary sequence to Twist1 59 of its seed region leaving this sequence free for miR-151-5p to bind. Interestingly, the target sites for miR-151-5p and miR-337-3p will still be present, even if the previously reported shortening of 39UTRs in cancer cells [35] also occurs with TWIST1 and either the first or the second pA signal should be used.
By mutating miRNA target sites and inhibiting miRNA binding using specific LNA-modified oligonucleotides, we confirmed the effect of the above described miRNAs on the Twist1 39UTR (Fig. 6) and demonstrated that the physiological expression levels of endogenous miRNAs are sufficient for a significant repression of Twist1 which most likely is due to translational inhibition rather than degradation of Twist1 mRNA (Fig. 4B). MiR-337 always required miR-151 to be present in order to have an effect. While this work was under revision, miR-214 [36], miR-300, miR-539 and miR-543 [37] have also been reported to target the TWIST1 39UTR. While two of them, miR-539 and miR-300 have no target site in the murine Twist1 39UTR, a third, miR-543 did not show a significant effect in our screen. This might be due to quantitative differences between the transfection of a miRNA expression vector and application of miRNA-mimics. It would be interesting to see whether these miRNAs also target TWIST1 by inhibiting translation or by RNA destabilization. During development, miR-145a-5p functions as a critical switch in promoting smooth muscle differentiation and its downregulation is associated with proliferation [38]. MiR-145a-5p is a known tumor suppressor with a strong inhibitory effect on cancer cell proliferation that is downregulated in a variety of tumors including bladder, breast, colon, colorectal, gastric, lung, oral, ovarian, and prostate cancers, hepatocellular and nasopharyngeal carcinoma and pituitary tumors [39], all of which are associated with increased levels of TWIST1. In cervical cancer cells, miR-145a-5p was shown to inhibit growth, invasion and therapy resistance [40], problems often observed in tumors in which TWIST1 is upregulated, and ectopic delivery into mouse pancreatic xenograft models resulted in the inhibition of tumor growth [41,42].
MiR-151 targets the thrombopoietin receptor MPL [43], a gene known to be downregulated in myeloproliferative neoplasms [44]. In hepatocellular carcinoma (HCC), miR-151-5p has been correlated with cell migration and intrahepatic metastasis [45]. However, miR-151 has been shown to be both upregulated (HCC, nasopharyngeal carcinoma) and downregulated (pappillary carcinoma, acute myeloid leukemia) in different types of cancer [46]. This dual function probably reflects the differences in gene regulation that leads to the various types of cancer. It would thus be interesting to investigate whether the expression of miR-151 in tumors correlates with the expression level of TWIST1.
MiR-337 is upregulated during bone formation [47] whereas TWIST1 is downregulated [48] and overexpression of Twist1 is known to inhibit osteoblast differentiation [49]. This suggests miR-337 as a potential inhibitory factor regulating Twist1 during bone formation. In cancer, miR-337 has been shown to be overexpressed in older melanoma patients compared to younger ones [50] and is negatively associated with survival in advanced ovarian cancer [51]. So far, nothing is known about the co-expression of miR-151 and miR-337. In light of our results, it would be interesting to see whether miR-337 is able to acquire a tumor suppressor function when co-expressed with miR-151 in TWIST1 positive tumors.
TWIST1 itself is also known to regulate the expression of several miRNAs, many of which are cancer-related. E.g. the oncogenic miR-10b is directly activated by TWIST1 and promotes the invasion and metastasis through repressing HOXD10 expression [3]. Also, the miR-200 family and miR-205 were shown to be repressed by TWIST1 [52]. These miRNAs are frequently silenced in advanced cancer [53,54]. They target ZEB1 and ZEB2, transcriptional repressors of E-cadherin, and thereby inhibit EMT and tumor invasion [55]. Recently, miR-223, overexpressed in metastatic gastric cancer cells, was also found to be induced by TWIST1 [56]. Considering its repression by miRNAs as demonstrated in this study, TWIST1 appears as a central player in the regulating network of miRNAs and transcription factors necessary for cellular homeostasis.
Recently, RNAi-mediated silencing of TWIST1 was shown to suppress the proliferation of human cervical cancer cells and to improve the chemosensitivity to cisplatin treatment, indicating a novel therapeutic strategy to overcome drug resistance [57]. Likewise, Burns et al. demonstrated a strong therapeutic effect in oncogene driven non-small cell lung cancer after silencing TWIST1 expression [58]. Our results about the miRNA regulation of TWIST1 provide an alternative approach to suppress this potent oncogene utilizing an endogenous mechanism.
The reduced mobility of miR-151-5p + miR-337-3p treated embryonic fibroblasts further shows the therapeutic potential in the combination of these two miRNAs (Fig. 7). The relatively small absolute difference in the migration probability is most likely explained by the fact that cell motility is a property influenced by a number of different factors. However, the high statistical significance of the difference indicates that the treatment with miR-151-5p and miR-337-3p has a reliable effect.
In summary, we have shown that TWIST1 is regulated by miR-145a-5p, miR-151-5p and miR-337-3p. The additive and synergistic effects of these miRNAs could reduce unwanted 'off target' effects and might open up new possibilities to specifically interfere with TWIST1 translation in therapeutic approaches.
Materials and Methods
Sequence comparison
Sequence comparison was done with CLC Main Workbench Version 5.7 (CLC bio). Parameters for alignments were: gap open cost = 10 and gap extension cost = 1. The following sequences were pairwise compared: Twist1: human (GeneBank: NM_00047); cow (GeneBank: XM_002686684); mouse (GeneBank: NM_011658); chicken (GeneBank: NM_204739; 39UTR without nt 845-873 and 899-971 which correspond to 2 stretches of sequence not found in any other species). Twist2: human (GeneBank: NM_057179); cow (GeneBank: NM_001083748); mouse (GeneBank: NM_007855); chicken (GeneBank: NM_204679). In addition, Twist1 sequences used for miRNA target site analysis were: dog (GeneBank: XM_857736); frog (GeneBank: NM_001085883).
Cell lines and constructs
All cell lines were obtained from American Type Culture Collection and cultured at 37uC in 5% CO 2 . H1299 cells were maintained in RPMI 1640 (Gibco) and NIH-3T3, C3H/10T1/2 and C 2 C 12 cells in DMEM (Gibco), all supplemented with 10% FCS, 100 mg/ml streptomycin and 100 U/ml penicillin (Gibco).
Murine pre-miRNA sequences with about 200 nt 59 and 39 flanking regions were amplified from C57Bl/6J mouse genomic Mouse Twist1 39UTR sequence (GeneBank: NC_000078; genomic DNA position 34,643,544 to 34,645,282) was cloned into the XbaI/NotI sites of pGL3-control vector (Promega). This sequence includes the unique intron of the Twist1 gene and thus represents a construct which will report all aspects of Twist1 mRNA processing including splicing. PRluc-N2 (PerkinElmer) encoding Renilla luciferase was used for normalization.
Due to high fluctuations in the pGL3-control transfection efficiencies, mouse Twist1 39UTR sequence (GeneBank: NM_011658; nucleotide position 926-1634) was cloned into XhoI/NotI sites in psiCheck2 vector (Promega) which expresses both Firefly and Renilla luciferases. MiRNA target sites were subsequently mutated to restriction enzyme recognition sites: miR-145a-5p to SacI; miR-151-5p to AgeI; miR-337-3p to SalI.
Stable pools of cells expressing psiCheck2-Twist1-39UTR were established as described in [59].
Transfections and luciferase assays
All transfections were carried out in triplicates on 48-well plates in three independent experiments and assayed after 48 h using the Dual-Luciferase Reporter Assay System Kit (Promega). 48 h was chosen to allow the cells to recover from the transfection and grow up to ,80-100% confluence at the time of measurement.
In the miRNA screen, 10 4 /cm 2 H1299 cells were transfected with 250 ng pdCMV2-EGFP-miR-X (or 125 ng each if two miRNAs were combined), 50 ng pRluc-N2 and 50 ng pGL3-Twist1-39UTR or pGL3-control using TransIT-LT1 transfection reagent (Mirus) according to manufacturer's guidelines. For normalization, the Firefly luciferase activity was divided by the Renilla luciferase activity. Triplicates were averaged and normalized to the pGL3-control.
In the target site mutation assay, 10 4 /cm 2 H1299 cells were transfected with 250 ng (or 125 ng each) miRNA construct and 50 ng wt or mutant psiCheck2-Twist1-39UTR using TransIT-LT1 transfection reagent (Mirus) according to manufacturer's instructions. For normalization, Renilla luciferase activity was divided by Firefly luciferase activity and the mutant-transfected cells were subsequently normalized to the respective wt 39UTR reporter treated with miRNA.
The transfection efficiency of miRNA-encoding vectors was monitored by the expression of EGFP with the Leica DM fluorescence microscope. The efficiency was always above 60%. 10 4 /cm 2 NIH-3T3 and C3H/10T1/2 cells were transfected with 50 ng psiCheck2-Twist1-39UTR using Fugene HD transfection reagent (Roche) according to manufacturer's guidelines. 50 nM miRCURY LNA microRNA inhibitors (Exiqon) were added in the inhibition assay: mmu-miR-145a-5p (139465-00), hsa-miR-151-5p (414654-00), mmu-miR-337-3p (139530-00) and a Scrambled negative control (199004-00). For normalization, Renilla luciferase activity was divided by Firefly luciferase activity.
10,000 stable psiCheck2-Twist1-39UTR-expressing cells per 96well plate well were transfected while seeding in 100 ml serum-free RPMI 1640 (Gibco) using 0.2 ml Lipofectamine 2000 (Invitrogen) and 20 or 5 nM Pre-miR miRNA Precursor (Applied Biosystems) per well: pre-mmu-miR-151 (PM11537), pre-mmu-miR-337 (PM12817) and Scrambled Negative Control #1 (AM17110). Simultaneously, 30,000 cells/well were transfected on a 24-well plate for RNA extraction and analysis using more reagents, accordingly. After 4 h of transfection, 10% FCS (Gibco) of total volume was added to each well. Renilla luciferase mRNA and protein levels were measured after 48 h.
Quantitative PCR
Total RNA was purified using TRIzol (Invitrogen) according to the manufacturer's instructions. 500 ng of total RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen) using 200 ng random hexamer primer (Roche).
Renilla luciferase expression was quantified using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and 25 ng cDNA as template. The primers used were: R-luc_fwd (59 gttccctaacaccgagttcgtg 39), R-luc_rev (59 ctccacgaagctcttgatgtac 39), F-luc_fwd (59 ctgatcgacaaggacggctgg 39), F-luc_rev (59 ggtagcccttgtacttgatcag 39). Renilla luciferase mRNA levels were normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control. Relative quantification of mRNA levels was performed using the DDCt-method.
For miRNA analysis, 25 ng of total RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). QPCR was carried out in triplicates using 1.33 ml cDNA and TaqMan Universal PCR Master Mix (Applied Biosystems). The TaqMan MicroRNA Assays used were: hsa-miR-145a-5p (002278), hsa-miR-151-5p (002642), mmu-miR-337-3p (002532) and U6 snRNA (001973). Relative quantities of miRNA were calculated by the DCt method.
Cell migration assay 3T3 immortalized murine embryonic fibroblasts [60] were plated on a 12-well plate at a density of 10 4 /cm 2 in 1 ml complete growth medium. While seeding, cells were transiently transfected with a total of 20 nM pre-mmu-miR-151-5p (PM11537) and premmu-miR-337-3p (PM12817) or with Scrambled Negative Control #1 (AM17110; all Applied Biosystems) using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. After 24 h, cells were trypsinized, resuspended in 400 ml medium, distributed equally on 2 cell culture inserts 200 ml each (24-well, 8.0 mm pore size, Falcon, 35 3097) and then placed in a 24-well plate (Falcon, 35 3504) which contained 600 ml medium per well. The 2 inserts were designated as 'A,over' and 'B,under' to study the analysis of non-migrated and migrated cells, respectively. Cells were allowed to migrate for 21 h, washed with PBS and fixed in 4% freshly dissolved paraformaldehyde for 30 min at room temperature. Following 26 washing with PBS, the cells on the lower surface of insert A and the cells on the upper surface of insert B were wiped off with a cotton swap. The insert membranes were cut off and embedded in ProLong Gold Antifade Reagent with DAPI (Invitrogen). Migrated (under) and non-migrated (over) cells were counted in 10 (primary magnification 106) and 5 (primary magnification 56) random areas, respectively, on micrographs taken with a Leica DM fluorescence microscope.
Statistical analysis
Statistical significance for the transfection experiments was calculated using Student's t-test and 2-sided p-values.
The effect of the treatment on the probability of cell migration was studied in the following way: Denote the counts of the migrated cells and the non-migrated cells for the m th observed microscopic field for the r th replication subject to the t th treatment by M mrt and S mrt , respectively. The expected number of migrated and non-migrated cells are given by E(M mrt ) = p t U rt and E(S mrt ) = (1-p t ) U rt /40, respectively. Here p t is the probability of migration common to all the observations subject to the t th treatment and U rt is the expected number of cells transferred from the suspension associated to the rt th replicate. The factor 1/40 arises from the fact that different macroscopic field areas were observed for the independent determinations of the migrated and non-migrated cells. Using a first order Taylor expansion yields that the expectation of P t = Mmrt /(M mrt + S mrt ) is the probability p t , therefore P t is an unbiased estimate of p t . The effect of the treatment on the probability of cell migration was then formally tested using the Monte Carlo permutation test [61] with 1,000,000 bootstrap permutations. Moreover, 95% bootstrap confidence intervals for the probabilities of cell migration were constructed with non-parametric bootstrap [61], with 10,000 parametric bootstrap each.
Figure 1 .
1Alignment of human and murine Twist1 39UTR sequences. Yellow represents the conserved nucleotides in the 39UTR of mouse (upper), human (lower), cow, pig and dog Twist1. Polyadenylation signals are indicated in red, CPEs in green, AU-rich sequence in brown and potential miRNA target sites in blue. In overlapping sequences, potential miRNA target sites are individualized by underlining. AU: AU-rich sequence, CPE: cytoplasmic polyadenylation element, H.: Homo sapiens, M.: Mus musculus, pA: nuclear polyadenylation signal. doi:10.1371/journal.pone.0066070.g001 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org
Figure 2 .
2MicroRNAs repress the Twist1 39UTR reporter. H1299 cells were transfected with the indicated miRNA, pRluc-N2 and pGL3-Twist1-39UTR Firefly luciferase reporter or the empty pGL3-control vector and analyzed after 48 h. Firefly luciferase activities were normalized to the Renilla luciferase activities which served as internal standards, averages of triplicates were calculated and results were normalized to empty pGL3-control vector. The dashed line indicates the unrepressed expression level of the reporter (0,176; calculated from the average of two negative controls (*), miR-485 and miR-609). Statistical significance of miRNA effects was calculated by comparison with this average using Student's t-test. doi:10.1371/journal.pone.0066070.g002 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org
Figure 3 .
3MicroRNA target sites are necessary for the repression of Twist1 39UTR reporter. MiRNAs of interest were co-transfected with either wt or the corresponding mutant of psiCheck2-Twist1-39UTR reporter into H1299 cells. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the respective wt+miRNA. Statistical significance was calculated by using Student's ttest and by comparing the mutant 39UTR values with the wt 39UTR reporter. doi:10.1371/journal.pone.0066070.g003
Figure 4 .
4MicroRNAs lead to translational inhibition of Twist1 39UTR reporter. (A) H1299 cells were co-transfected with wt psiCheck2-Twist1-39UTR reporter and 20 nM synthetic precursor miRNAs. (B) H1299 cells stably expressing the psiCheck2-Twist1-39UTR reporter were transfected with 5 nM synthetic precursor miRNAs. Renilla luciferase activity was measured after 48 h and normalized to the Firefly luciferase activity. Renilla luciferase mRNA levels were measured by qPCR, normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control. doi:10.1371/journal.pone.0066070.g004 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org
Figure 5 .
5MicroRNA expression levels in different mouse cell lines. Endogenous miRNA expression levels in NIH-3T3, C3H/10T1/2 and C 2 C 12 cells were measured by qPCR using TaqMan probes, and normalized to U6 snRNA. Due to the high expression of miR-145a-5p, the relative values are presented on a logarithmic scale. doi:10.1371/journal.pone.0066070.g005
Figure 6 .
6Endogenous microRNAs reduce the activity of Twist1 39UTR reporter. NIH-3T3 (A) and C3H/10T1/2 (B) cells were transfected with wt or mutant psiCheck2-Twist1-39UTR reporter only. Endogenous miRNAs in NIH-3T3 (C) and C3H/10T1/2 (D) cells were inhibited by co-transfection of 50 nM miRCURY LNA microRNA inhibitors and wt psiCheck2-Twist1-39UTR reporter. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the wt 39UTR (A-B) or wt+anti-Scrambled (C-D). Statistical significance was calculated by using Student's t-test and by comparing the mutant Twist1 39UTR reporters or miRNA specific antagonists with the wt 39UTR reporter or a scrambled control (*), respectively. doi:10.1371/journal.pone.0066070.g006 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org All of the miRNAs shown to repress TWIST1 expression have been demonstrated to be involved both in embryonic development and cancer.
Figure 7 .
7MicroRNAs 151-5p and 337-3p reduce the mobility of murine embryonic fibroblast cells. Dot-plot superposed with the box-plot of the predicted probabilities of cell migration for observations from miR-151-5p + miR-337-3p (red) and scrambled control (blue) treated cells (p-value 0.000135). Note the presence of miR-151-5p + miR-337-3p treated cells with extreme low estimates of probability of cell migration. doi:10.1371/journal.pone.two miRNAs used as negative controls are: hsa-miR-485 (MI0002469) and hsa-miR-609 (MI0003622). Primers used for cloning were: mmu-miR-33-upper (59ctgtcagcggccggaagcctactcgggcaatgtgcc 39), mmu-miR-33-lower (59 gctgcgatcgtcgacggaagcagctagttctaacctcc 39), mmu-miR-145a-upper (59 agctatgcggccggactctacgcacatgaaatgcttcttcc 39), mmu-miR-145a-lower (59 gctgcgatcgtcgacggattggatgtaatataaatgaagcaaacc 39), mmu-miR-151-upper (59 ctgtcagcggccgctttagggcctgagaatatcttga 39), mmu-miR-151-lower (59 gggaccgcgtcgacgaagcaaacctcaataacagaactc 39), mmu-miR-326-upper (59 cctgtagcggccgctactccgcatagccactgaga 39), mmu-miR-326-lower (59 gcggatccgtcgacctagcccagggccatatacatg 39), mmu-miR-337-upper (59 ctgtcagcggccgccatgagagtttataagaagtgagg 39), mmu-miR-337-lower (59 gggaccgcgtcgacgccaaggaatgatctcaggtatgg 39), mmu-miR-361-upper (59 agctatgcggccgcttaa-catgccttggtttgcaga39), mmu-miR-361-lower (59 acctagcggtcgacctttgctgctttctttgcttcc 39), mmu-miR-378a-upper (59 cctgtagcggccggtaattgataccaggagctttgcagcc 39), mmu-miR-378alower (59 acctagcggtcgacccagacagctcacatgcaaacacagg 39), mmu-miR-381-upper (59 catgtagcggccgccaatctgttgtaacatctgccagt 39), mmu-miR-381-lower (59 gctgcgatcgtcgacagaacatgcacacttgggtac 39), mmu-miR-409-upper (59 cctgtagcggccggggaaggctttagatattcttggaagg 39), mmu-miR-409-lower (59 acctagcggtcgaccacggtcgatctcccttcaagtaccagc 39), mmu-miR-543-upper (59 agctatgcggccgggagactccaaagacctccccaaagg 39) and mmu-miR-543lower (59 gggaccgcgtcgacgtggaggagggaggagggagcaggagcc 39).
Table 1 .
1Pairwise comparison of Twist1 and Twist2 mRNA sequence domains of three selected amniotes with the corresponding human sequence. Numbers represent% sequence identity.Twist1 CDS 1 Twist1 39UTR 2 Twist2 CDS 1 Twist2 39UTR 2
Human
100
100
100
100
Cow
95
92
98
68
Mouse
93
87
97
65
Chicken
84
54
80
32
1
CDS: coding sequence; 2 UTR: untranslated region.
doi:10.1371/journal.pone.0066070.t001
Table 2 .
2Conservation of microRNA target sites in selected amniotes and the presence in Twist2 39UTR.MiRNA target site/Species Human
Mouse
Cow
Dog
Chicken
Frog
Targeting Twist2
miR-15b-3p
+
2
+
+
2
2
2
miR-33-5p
PLOS ONE | www.plosone.org
May 2013 | Volume 8 | Issue 5 | e66070
AcknowledgmentsWe thank Agata Magdziarz and Urzula Kania for help in cloning some of the miRNAs and Twist1 39UTR reporters, Claus Bus and Tine Birch for technical support and Karen Colbjørn Søgaard for the critical reading of the manuscript.Author Contributions
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Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro. L Dyrskjot, M S Ostenfeld, J B Bramsen, A N Silahtaroglu, P Lamy, Cancer Res. 69Dyrskjot L, Ostenfeld MS, Bramsen JB, Silahtaroglu AN, Lamy P, et al. (2009) Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro. Cancer Res 69: 4851-4860.
The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. P A Gregory, A G Bert, E L Paterson, S C Barry, A Tsykin, Nat Cell Biol. 10Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, et al. (2008) The miR- 200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10: 593-601.
) miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3. X Li, Y Zhang, H Zhang, X Liu, T Gong, Mol Cancer Res. 9Li X, Zhang Y, Zhang H, Liu X, Gong T, et al. (2011) miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3. Mol Cancer Res 9: 824-833.
Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells. K Zhu, L Chen, X Han, J Wang, Oncol Rep. 27Zhu K, Chen L, Han X, Wang J (2012) Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells. Oncol Rep 27: 1027-1034.
Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer. T F Burns, I Dobromilskaya, S C Murphy, R P Gajula, S Thiyagarajan, Mol Cancer Res. In pressBurns TF, Dobromilskaya I, Murphy SC, Gajula RP, Thiyagarajan S, et al. (2013) Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer.Mol Cancer Res: In press .
A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. J B Bramsen, M M Pakula, T B Hansen, C Bus, N Langkjaer, Nucleic Acids Res. 38Bramsen JB, Pakula MM, Hansen TB, Bus C, Langkjaer N, et al. (2010) A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. Nucleic Acids Res 38: 5761-5773.
Septin9 is involved in septin filament formation and cellular stability. A Füchtbauer, L B Lassen, A B Jensen, J Howard, Quiroga Ade, S , Biol Chem. 392Füchtbauer A, Lassen LB, Jensen AB, Howard J, Quiroga Ade S, et al. (2011) Septin9 is involved in septin filament formation and cellular stability. Biol Chem 392: 769-777.
A C Davison, D V Hinkley, Bootstrap Methods and Their Application. NY, USACambridge University Press193Davison AC, Hinkley DV (1997) Bootstrap Methods and Their Application. NY, USA : Cambridge University Press.161-175, 193 p.
| [
"Upregulation of the proto-oncogene Twist1 is highly correlated with acquired drug resistance and poor prognosis in human cancers. Altered expression of this multifunctional transcription factor is also associated with inherited skeletal malformations. The mammalian Twist1 39UTRs are highly conserved and contain a number of potential regulatory elements including miRNA target sites. We analyzed the translational regulation of TWIST1 using luciferase reporter assays in a variety of cell lines. Among several miRNAs tested, miR-145a-5p, miR-151-5p and a combination of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p were able to significantly repress Twist1 translation. This phenomena was confirmed with both exogenous and endogenous miRNAs and was dependent on the presence of the predicted target sites in the 39UTR. Furthermore, the repression was sensitive to LNA-modified miRNA antagonists and resulted in decreased migratory potential of murine embryonic fibroblast cells. Understanding the in vivo mechanisms of this oncogene's regulation might open up a possibility for therapeutic interference by gene specific cancer therapies. Citation: Nairismägi M-L, Fü chtbauer A, Labouriau R, Bramsen JB, Fü chtbauer E-M (2013) The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs. PLoS ONE 8(5): e66070."
] | [
"Maarja-Liisa Nairismä \nDepartment of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark\n",
"Annette Fü \nDepartment of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark\n",
"JesperRodrigo Labouriau \nDepartment of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark\n",
"Bertram Bramsen \nDepartment of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark\n",
"Ernst-Martin Fü \nDepartment of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark\n"
] | [
"Department of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark",
"Department of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark",
"Department of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark",
"Department of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark",
"Department of Molecular Biology and Genetics\nAarhus University\nAarhusDenmark"
] | [
"Maarja-Liisa",
"Annette",
"Rodrigo",
"Bertram",
"Ernst-Martin"
] | [
"Nairismä",
"Fü",
"Labouriau",
"Bramsen",
"Fü"
] | [
"R Maestro, ",
"Dei Tos, ",
"A P Hamamori, ",
"Y Krasnokutsky, ",
"S Sartorelli, ",
"V , ",
"E H Villavicencio, ",
"J W Yoon, ",
"D J Frank, ",
"E M Füchtbauer, ",
"D O Walterhouse, ",
"L Ma, ",
"J Teruya-Feldstein, ",
"R A Weinberg, ",
"G Z Cheng, ",
"W Zhang, ",
"L H Wang, ",
"Q Qin, ",
"Y Xu, ",
"T He, ",
"C Qin, ",
"J Xu, ",
"C Hornik, ",
"B Brand-Saberi, ",
"S Rudloff, ",
"B Christ, ",
"E M Füchtbauer, ",
"I Krebs, ",
"I Weis, ",
"M Hudler, ",
"J M Rommens, ",
"H Roth, ",
"C W Li, ",
"W Xia, ",
"L Huo, ",
"S O Lim, ",
"Y Wu, ",
"T M Chang, ",
"W C Hung, ",
"M H Yang, ",
"M Z Wu, ",
"S H Chiou, ",
"P M Chen, ",
"S Y Chang, ",
"E H Gort, ",
"G Van Haaften, ",
"I Verlaan, ",
"A J Groot, ",
"R H Plasterk, ",
"A L Harris, ",
"J Yang, ",
"S A Mani, ",
"J L Donaher, ",
"S Ramaswamy, ",
"R A Itzykson, ",
"M A Eckert, ",
"T M Lwin, ",
"A T Chang, ",
"J Kim, ",
"E Danis, ",
"N Dave, ",
"S Guaita-Esteruelas, ",
"S Gutarra, ",
"A Frias, ",
"M Beltran, ",
"K B Laursen, ",
"E Mielke, ",
"P Iannaccone, ",
"E M Füchtbauer, ",
"M Hebrok, ",
"A Füchtbauer, ",
"E M Füchtbauer, ",
"D B Spicer, ",
"J Rhee, ",
"W L Cheung, ",
"A B Lassar, ",
"B Thisse, ",
"C Stoetzel, ",
"C Gorostiza-Thisse, ",
"F Perrin-Schmitt, ",
"P Simpson, ",
"Z F Chen, ",
"R R Behringer, ",
"V El Ghouzzi, ",
"Le Merrer, ",
"M Perrin-Schmitt, ",
"F Lajeunie, ",
"E Benit, ",
"P , ",
"E M Fü Chtbauer, ",
"I Gitelman, ",
"M L Nairismä Gi, ",
"A Vislovukh, ",
"Q Meng, ",
"G Kratassiouk, ",
"C Beldiman, ",
"A E Erson, ",
"E M Petty, ",
"A Cano, ",
"M A Nieto, ",
"J G Doench, ",
"P A Sharp, ",
"M Scaal, ",
"E M Füchtbauer, ",
"B Brand-Saberi, ",
"L Li, ",
"P Cserjesi, ",
"E N Olson, ",
"H L Franco, ",
"J Casasnovas, ",
"J R Rodriguez-Medina, ",
"C L Cadilla, ",
"K Kapinas, ",
"A M Delany, ",
"H Peinado, ",
"D Olmeda, ",
"A Cano, ",
"A Grimson, ",
"K K Farh, ",
"W K Johnston, ",
"P Garrett-Engele, ",
"L P Lim, ",
"C Mayr, ",
"D P Bartel, ",
"B Li, ",
"Q Han, ",
"Y Zhu, ",
"Y Yu, ",
"J Wang, ",
"C L Haga, ",
"D G Phinney, ",
"K R Cordes, ",
"N T Sheehy, ",
"M P White, ",
"E C Berry, ",
"S U Morton, ",
"J Zhang, ",
"H Guo, ",
"G Qian, ",
"S Ge, ",
"H Ji, ",
"M Shi, ",
"L Du, ",
"D Liu, ",
"L Qian, ",
"M Hu, ",
"D Pramanik, ",
"N R Campbell, ",
"C Karikari, ",
"R Chivukula, ",
"O A Kent, ",
"O A Kent, ",
"R R Chivukula, ",
"M Mullendore, ",
"E A Wentzel, ",
"G Feldmann, ",
"M Girardot, ",
"C Pecquet, ",
"S Boukour, ",
"L Knoops, ",
"A Ferrant, ",
"A R Moliterno, ",
"W D Hankins, ",
"J L Spivak, ",
"J Ding, ",
"S Huang, ",
"S Wu, ",
"Y Zhao, ",
"L Liang, ",
"Y Wang, ",
"C G Lee, ",
"P Annalisa, ",
"P Furio, ",
"Z Ilaria, ",
"Anna A Luca, ",
"S , ",
"S S Murray, ",
"C A Glackin, ",
"K A Winters, ",
"D Gazit, ",
"A J Kahn, ",
"P Bialek, ",
"B Kern, ",
"X Yang, ",
"M Schrock, ",
"D Sosic, ",
"D M Jukic, ",
"U N Rao, ",
"L Kelly, ",
"J S Skaf, ",
"L M Drogowski, ",
"K K Shih, ",
"L X Qin, ",
"E J Tanner, ",
"Q Zhou, ",
"M Bisogna, ",
"E D Wiklund, ",
"J B Bramsen, ",
"T Hulf, ",
"L Dyrskjot, ",
"R Ramanathan, ",
"F Gottardo, ",
"C G Liu, ",
"M Ferracin, ",
"G A Calin, ",
"M Fassan, ",
"L Dyrskjot, ",
"M S Ostenfeld, ",
"J B Bramsen, ",
"A N Silahtaroglu, ",
"P Lamy, ",
"P A Gregory, ",
"A G Bert, ",
"E L Paterson, ",
"S C Barry, ",
"A Tsykin, ",
"X Li, ",
"Y Zhang, ",
"H Zhang, ",
"X Liu, ",
"T Gong, ",
"K Zhu, ",
"L Chen, ",
"X Han, ",
"J Wang, ",
"T F Burns, ",
"I Dobromilskaya, ",
"S C Murphy, ",
"R P Gajula, ",
"S Thiyagarajan, ",
"J B Bramsen, ",
"M M Pakula, ",
"T B Hansen, ",
"C Bus, ",
"N Langkjaer, ",
"A Füchtbauer, ",
"L B Lassen, ",
"A B Jensen, ",
"J Howard, ",
"Quiroga Ade, ",
"S , ",
"A C Davison, ",
"D V Hinkley, "
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"Yoon",
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"Füchtbauer",
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"Huo",
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"Chang",
"Hung",
"Yang",
"Wu",
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] | [
"Twist is a potential oncogene that inhibits apoptosis. R Maestro, Dei Tos, A P Hamamori, Y Krasnokutsky, S Sartorelli, V , Genes Dev. 13Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, et al. (1999) Twist is a potential oncogene that inhibits apoptosis. Genes Dev 13: 2207-2217.",
"Cooperative E-box regulation of human GLI1 by TWIST and USF. E H Villavicencio, J W Yoon, D J Frank, E M Füchtbauer, D O Walterhouse, Genesis. 32Villavicencio EH, Yoon JW, Frank DJ, Füchtbauer EM, Walterhouse DO, et al. (2002) Cooperative E-box regulation of human GLI1 by TWIST and USF. Genesis 32: 247-258.",
"Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. L Ma, J Teruya-Feldstein, R A Weinberg, Nature. 449Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449: 682-688.",
"Regulation of cancer cell survival, migration, and invasion by Twist: AKT2 comes to interplay. G Z Cheng, W Zhang, L H Wang, Cancer Res. 68Cheng GZ, Zhang W, Wang LH (2008) Regulation of cancer cell survival, migration, and invasion by Twist: AKT2 comes to interplay. Cancer Res 68: 957-960.",
"Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms. Q Qin, Y Xu, T He, C Qin, J Xu, Cell Res. 22Qin Q, Xu Y, He T, Qin C, Xu J (2012) Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms. Cell Res 22: 90-106.",
"Twist is an integrator of SHH, FGF, and BMP signaling. C Hornik, B Brand-Saberi, S Rudloff, B Christ, E M Füchtbauer, Anat Embryol (Berl). 209Hornik C, Brand-Saberi B, Rudloff S, Christ B, Füchtbauer EM (2004) Twist is an integrator of SHH, FGF, and BMP signaling. Anat Embryol (Berl) 209: 31- 39.",
"Translocation breakpoint maps 5 kb 39 from TWIST in a patient affected with Saethre-Chotzen syndrome. I Krebs, I Weis, M Hudler, J M Rommens, H Roth, Hum Mol Genet. 6Krebs I, Weis I, Hudler M, Rommens JM, Roth H, et al. (1997) Translocation breakpoint maps 5 kb 39 from TWIST in a patient affected with Saethre- Chotzen syndrome. Hum Mol Genet 6: 1079-1086.",
"Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1. C W Li, W Xia, L Huo, S O Lim, Y Wu, Cancer Res. 72Li CW, Xia W, Huo L, Lim SO, Wu Y, et al. (2012) Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1. Cancer Res 72: 1290-1300.",
"Transcriptional repression of TWIST1 gene by Prospero-related homeobox 1 inhibits invasiveness of hepatocellular carcinoma cells. T M Chang, W C Hung, FEBS Lett. 586Chang TM, Hung WC (2012) Transcriptional repression of TWIST1 gene by Prospero-related homeobox 1 inhibits invasiveness of hepatocellular carcinoma cells. FEBS Lett 586: 3746-3752.",
"Direct regulation of TWIST by HIF-1alpha promotes metastasis. M H Yang, M Z Wu, S H Chiou, P M Chen, S Y Chang, Nat Cell Biol. 10Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, et al. (2008) Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol 10: 295-305.",
"The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. E H Gort, G Van Haaften, I Verlaan, A J Groot, R H Plasterk, Oncogene. 27Gort EH, van Haaften G, Verlaan I, Groot AJ, Plasterk RH, et al. (2008) The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. Oncogene 27: 1501-1510.",
"Hypoxia--a key regulatory factor in tumour growth. A L Harris, Nat Rev Cancer. 2Harris AL (2002) Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2: 38-47.",
"Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. J Yang, S A Mani, J L Donaher, S Ramaswamy, R A Itzykson, Cell. 117Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, et al. (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117: 927-939.",
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"Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition. N Dave, S Guaita-Esteruelas, S Gutarra, A Frias, M Beltran, J Biol Chem. 286Dave N, Guaita-Esteruelas S, Gutarra S, Frias A, Beltran M, et al. (2011) Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition. J Biol Chem 286: 12024-12032.",
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"Down-regulation of miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist. B Li, Q Han, Y Zhu, Y Yu, J Wang, FEBS J. 279Li B, Han Q, Zhu Y, Yu Y, Wang J, et al. (2012) Down-regulation of miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist. FEBS J 279: 2393-2398.",
"MicroRNAs in the imprinted DLK1-DIO3 region repress the epithelial-to-mesenchymal transition by targeting the TWIST1 protein signaling network. C L Haga, D G Phinney, J Biol Chem. 287Haga CL, Phinney DG (2012) MicroRNAs in the imprinted DLK1-DIO3 region repress the epithelial-to-mesenchymal transition by targeting the TWIST1 protein signaling network. J Biol Chem 287: 42695-42707.",
") miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. K R Cordes, N T Sheehy, M P White, E C Berry, S U Morton, Nature. 460Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, et al. (2009) miR- 145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460: 705-710.",
"MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network. J Zhang, H Guo, G Qian, S Ge, H Ji, Mol Cancer. 9211Zhang J, Guo H, Qian G, Ge S, Ji H, et al. (2010) MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network. Mol Cancer 9: 211.",
"Glucocorticoid regulation of a novel HPV-E6-p53-miR-145 pathway modulates invasion and therapy resistance of cervical cancer cells. M Shi, L Du, D Liu, L Qian, M Hu, J Pathol. 228Shi M, Du L, Liu D, Qian L, Hu M, et al. (2012) Glucocorticoid regulation of a novel HPV-E6-p53-miR-145 pathway modulates invasion and therapy resis- tance of cervical cancer cells. J Pathol 228: 148-157.",
"Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice. D Pramanik, N R Campbell, C Karikari, R Chivukula, O A Kent, Mol Cancer Ther. 10Pramanik D, Campbell NR, Karikari C, Chivukula R, Kent OA, et al. (2011) Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice. Mol Cancer Ther 10: 1470-1480.",
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"Microrna profiling analysis of differences between the melanoma of young adults and older adults. D M Jukic, U N Rao, L Kelly, J S Skaf, L M Drogowski, J Transl Med. 827Jukic DM, Rao UN, Kelly L, Skaf JS, Drogowski LM, et al. (2010) Microrna profiling analysis of differences between the melanoma of young adults and older adults. J Transl Med 8: 27.",
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"The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. P A Gregory, A G Bert, E L Paterson, S C Barry, A Tsykin, Nat Cell Biol. 10Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, et al. (2008) The miR- 200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10: 593-601.",
") miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3. X Li, Y Zhang, H Zhang, X Liu, T Gong, Mol Cancer Res. 9Li X, Zhang Y, Zhang H, Liu X, Gong T, et al. (2011) miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3. Mol Cancer Res 9: 824-833.",
"Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells. K Zhu, L Chen, X Han, J Wang, Oncol Rep. 27Zhu K, Chen L, Han X, Wang J (2012) Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells. Oncol Rep 27: 1027-1034.",
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"Twist is a potential oncogene that inhibits apoptosis",
"Cooperative E-box regulation of human GLI1 by TWIST and USF",
"Tumour invasion and metastasis initiated by microRNA-10b in breast cancer",
"Regulation of cancer cell survival, migration, and invasion by Twist: AKT2 comes to interplay",
"Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms",
"Twist is an integrator of SHH, FGF, and BMP signaling",
"Translocation breakpoint maps 5 kb 39 from TWIST in a patient affected with Saethre-Chotzen syndrome",
"Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1",
"Transcriptional repression of TWIST1 gene by Prospero-related homeobox 1 inhibits invasiveness of hepatocellular carcinoma cells",
"Direct regulation of TWIST by HIF-1alpha promotes metastasis",
"The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha",
"Hypoxia--a key regulatory factor in tumour growth",
"Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis",
"Twist1-induced invadopodia formation promotes tumor metastasis",
"Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition",
"Mechanism of transcriptional activation by the proto-oncogene Twist1",
"Repression of muscle-specific gene activation by the murine Twist protein",
"Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist",
"Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos",
"Maternal-Zygotic Gene Interactions during Formation of the Dorsoventral Pattern in Drosophila Embryos",
"twist is required in head mesenchyme for cranial neural tube morphogenesis",
"Mutations of the TWIST gene in the Saethre-Chotzen syndrome",
"Expression of M-twist during postimplantation development of the mouse",
"Twist protein in mouse embryogenesis",
"Translational control of TWIST1 expression in MCF-10A cell lines recapitulating breast cancer progression",
"MicroRNAs in development and disease",
"Non-coding RNAs take centre stage in epithelial-tomesenchymal transition",
"Specificity of microRNA target selection in translational repression",
") cDermo-1 expression indicates a role in avian skin development",
"Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis",
"Redundant or separate entities?--roles of Twist1 and Twist2 as molecular switches during gene transcription",
"MicroRNA biogenesis and regulation of bone remodeling",
"Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?",
"MicroRNA targeting specificity in mammals: determinants beyond seed pairing",
"Widespread shortening of 39UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells",
"Down-regulation of miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist",
"MicroRNAs in the imprinted DLK1-DIO3 region repress the epithelial-to-mesenchymal transition by targeting the TWIST1 protein signaling network",
") miR-145 and miR-143 regulate smooth muscle cell fate and plasticity",
"MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network",
"Glucocorticoid regulation of a novel HPV-E6-p53-miR-145 pathway modulates invasion and therapy resistance of cervical cancer cells",
"Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice",
"Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway",
"miR-28 is a thrombopoietin receptor targeting microRNA detected in a fraction of myeloproliferative neoplasm patient platelets",
"Impaired expression of the thrombopoietin receptor by platelets from patients with polycythemia vera",
"Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA",
"MicroRNA and cancer--focus on apoptosis",
"Anorganic bovine bone and a silicate-based synthetic bone activate different microRNAs",
"Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells",
"A twist code determines the onset of osteoblast differentiation",
"Microrna profiling analysis of differences between the melanoma of young adults and older adults",
"A microRNA survival signature (MiSS) for advanced ovarian cancer",
"Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer",
"Micro-RNA profiling in kidney and bladder cancers",
"Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro",
"The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1",
") miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3",
"Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells",
"Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer",
"A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects",
"Septin9 is involved in septin filament formation and cellular stability"
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"\nFigure 1 .\n1Alignment of human and murine Twist1 39UTR sequences. Yellow represents the conserved nucleotides in the 39UTR of mouse (upper), human (lower), cow, pig and dog Twist1. Polyadenylation signals are indicated in red, CPEs in green, AU-rich sequence in brown and potential miRNA target sites in blue. In overlapping sequences, potential miRNA target sites are individualized by underlining. AU: AU-rich sequence, CPE: cytoplasmic polyadenylation element, H.: Homo sapiens, M.: Mus musculus, pA: nuclear polyadenylation signal. doi:10.1371/journal.pone.0066070.g001 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"\nFigure 2 .\n2MicroRNAs repress the Twist1 39UTR reporter. H1299 cells were transfected with the indicated miRNA, pRluc-N2 and pGL3-Twist1-39UTR Firefly luciferase reporter or the empty pGL3-control vector and analyzed after 48 h. Firefly luciferase activities were normalized to the Renilla luciferase activities which served as internal standards, averages of triplicates were calculated and results were normalized to empty pGL3-control vector. The dashed line indicates the unrepressed expression level of the reporter (0,176; calculated from the average of two negative controls (*), miR-485 and miR-609). Statistical significance of miRNA effects was calculated by comparison with this average using Student's t-test. doi:10.1371/journal.pone.0066070.g002 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"\nFigure 3 .\n3MicroRNA target sites are necessary for the repression of Twist1 39UTR reporter. MiRNAs of interest were co-transfected with either wt or the corresponding mutant of psiCheck2-Twist1-39UTR reporter into H1299 cells. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the respective wt+miRNA. Statistical significance was calculated by using Student's ttest and by comparing the mutant 39UTR values with the wt 39UTR reporter. doi:10.1371/journal.pone.0066070.g003",
"\nFigure 4 .\n4MicroRNAs lead to translational inhibition of Twist1 39UTR reporter. (A) H1299 cells were co-transfected with wt psiCheck2-Twist1-39UTR reporter and 20 nM synthetic precursor miRNAs. (B) H1299 cells stably expressing the psiCheck2-Twist1-39UTR reporter were transfected with 5 nM synthetic precursor miRNAs. Renilla luciferase activity was measured after 48 h and normalized to the Firefly luciferase activity. Renilla luciferase mRNA levels were measured by qPCR, normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control. doi:10.1371/journal.pone.0066070.g004 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"\nFigure 5 .\n5MicroRNA expression levels in different mouse cell lines. Endogenous miRNA expression levels in NIH-3T3, C3H/10T1/2 and C 2 C 12 cells were measured by qPCR using TaqMan probes, and normalized to U6 snRNA. Due to the high expression of miR-145a-5p, the relative values are presented on a logarithmic scale. doi:10.1371/journal.pone.0066070.g005",
"\nFigure 6 .\n6Endogenous microRNAs reduce the activity of Twist1 39UTR reporter. NIH-3T3 (A) and C3H/10T1/2 (B) cells were transfected with wt or mutant psiCheck2-Twist1-39UTR reporter only. Endogenous miRNAs in NIH-3T3 (C) and C3H/10T1/2 (D) cells were inhibited by co-transfection of 50 nM miRCURY LNA microRNA inhibitors and wt psiCheck2-Twist1-39UTR reporter. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the wt 39UTR (A-B) or wt+anti-Scrambled (C-D). Statistical significance was calculated by using Student's t-test and by comparing the mutant Twist1 39UTR reporters or miRNA specific antagonists with the wt 39UTR reporter or a scrambled control (*), respectively. doi:10.1371/journal.pone.0066070.g006 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org All of the miRNAs shown to repress TWIST1 expression have been demonstrated to be involved both in embryonic development and cancer.",
"\nFigure 7 .\n7MicroRNAs 151-5p and 337-3p reduce the mobility of murine embryonic fibroblast cells. Dot-plot superposed with the box-plot of the predicted probabilities of cell migration for observations from miR-151-5p + miR-337-3p (red) and scrambled control (blue) treated cells (p-value 0.000135). Note the presence of miR-151-5p + miR-337-3p treated cells with extreme low estimates of probability of cell migration. doi:10.1371/journal.pone.two miRNAs used as negative controls are: hsa-miR-485 (MI0002469) and hsa-miR-609 (MI0003622). Primers used for cloning were: mmu-miR-33-upper (59ctgtcagcggccggaagcctactcgggcaatgtgcc 39), mmu-miR-33-lower (59 gctgcgatcgtcgacggaagcagctagttctaacctcc 39), mmu-miR-145a-upper (59 agctatgcggccggactctacgcacatgaaatgcttcttcc 39), mmu-miR-145a-lower (59 gctgcgatcgtcgacggattggatgtaatataaatgaagcaaacc 39), mmu-miR-151-upper (59 ctgtcagcggccgctttagggcctgagaatatcttga 39), mmu-miR-151-lower (59 gggaccgcgtcgacgaagcaaacctcaataacagaactc 39), mmu-miR-326-upper (59 cctgtagcggccgctactccgcatagccactgaga 39), mmu-miR-326-lower (59 gcggatccgtcgacctagcccagggccatatacatg 39), mmu-miR-337-upper (59 ctgtcagcggccgccatgagagtttataagaagtgagg 39), mmu-miR-337-lower (59 gggaccgcgtcgacgccaaggaatgatctcaggtatgg 39), mmu-miR-361-upper (59 agctatgcggccgcttaa-catgccttggtttgcaga39), mmu-miR-361-lower (59 acctagcggtcgacctttgctgctttctttgcttcc 39), mmu-miR-378a-upper (59 cctgtagcggccggtaattgataccaggagctttgcagcc 39), mmu-miR-378alower (59 acctagcggtcgacccagacagctcacatgcaaacacagg 39), mmu-miR-381-upper (59 catgtagcggccgccaatctgttgtaacatctgccagt 39), mmu-miR-381-lower (59 gctgcgatcgtcgacagaacatgcacacttgggtac 39), mmu-miR-409-upper (59 cctgtagcggccggggaaggctttagatattcttggaagg 39), mmu-miR-409-lower (59 acctagcggtcgaccacggtcgatctcccttcaagtaccagc 39), mmu-miR-543-upper (59 agctatgcggccgggagactccaaagacctccccaaagg 39) and mmu-miR-543lower (59 gggaccgcgtcgacgtggaggagggaggagggagcaggagcc 39).",
"\nTable 1 .\n1Pairwise comparison of Twist1 and Twist2 mRNA sequence domains of three selected amniotes with the corresponding human sequence. Numbers represent% sequence identity.Twist1 CDS 1 Twist1 39UTR 2 Twist2 CDS 1 Twist2 39UTR 2 \n\nHuman \n100 \n100 \n100 \n100 \n\nCow \n95 \n92 \n98 \n68 \n\nMouse \n93 \n87 \n97 \n65 \n\nChicken \n84 \n54 \n80 \n32 \n\n1 \n\nCDS: coding sequence; 2 UTR: untranslated region. \ndoi:10.1371/journal.pone.0066070.t001 \n",
"\nTable 2 .\n2Conservation of microRNA target sites in selected amniotes and the presence in Twist2 39UTR.MiRNA target site/Species Human \nMouse \nCow \nDog \nChicken \nFrog \nTargeting Twist2 \n\nmiR-15b-3p \n+ \n2 \n+ \n+ \n2 \n2 \n2 \n\nmiR-33-5p \n"
] | [
"Alignment of human and murine Twist1 39UTR sequences. Yellow represents the conserved nucleotides in the 39UTR of mouse (upper), human (lower), cow, pig and dog Twist1. Polyadenylation signals are indicated in red, CPEs in green, AU-rich sequence in brown and potential miRNA target sites in blue. In overlapping sequences, potential miRNA target sites are individualized by underlining. AU: AU-rich sequence, CPE: cytoplasmic polyadenylation element, H.: Homo sapiens, M.: Mus musculus, pA: nuclear polyadenylation signal. doi:10.1371/journal.pone.0066070.g001 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"MicroRNAs repress the Twist1 39UTR reporter. H1299 cells were transfected with the indicated miRNA, pRluc-N2 and pGL3-Twist1-39UTR Firefly luciferase reporter or the empty pGL3-control vector and analyzed after 48 h. Firefly luciferase activities were normalized to the Renilla luciferase activities which served as internal standards, averages of triplicates were calculated and results were normalized to empty pGL3-control vector. The dashed line indicates the unrepressed expression level of the reporter (0,176; calculated from the average of two negative controls (*), miR-485 and miR-609). Statistical significance of miRNA effects was calculated by comparison with this average using Student's t-test. doi:10.1371/journal.pone.0066070.g002 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"MicroRNA target sites are necessary for the repression of Twist1 39UTR reporter. MiRNAs of interest were co-transfected with either wt or the corresponding mutant of psiCheck2-Twist1-39UTR reporter into H1299 cells. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the respective wt+miRNA. Statistical significance was calculated by using Student's ttest and by comparing the mutant 39UTR values with the wt 39UTR reporter. doi:10.1371/journal.pone.0066070.g003",
"MicroRNAs lead to translational inhibition of Twist1 39UTR reporter. (A) H1299 cells were co-transfected with wt psiCheck2-Twist1-39UTR reporter and 20 nM synthetic precursor miRNAs. (B) H1299 cells stably expressing the psiCheck2-Twist1-39UTR reporter were transfected with 5 nM synthetic precursor miRNAs. Renilla luciferase activity was measured after 48 h and normalized to the Firefly luciferase activity. Renilla luciferase mRNA levels were measured by qPCR, normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control. doi:10.1371/journal.pone.0066070.g004 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org",
"MicroRNA expression levels in different mouse cell lines. Endogenous miRNA expression levels in NIH-3T3, C3H/10T1/2 and C 2 C 12 cells were measured by qPCR using TaqMan probes, and normalized to U6 snRNA. Due to the high expression of miR-145a-5p, the relative values are presented on a logarithmic scale. doi:10.1371/journal.pone.0066070.g005",
"Endogenous microRNAs reduce the activity of Twist1 39UTR reporter. NIH-3T3 (A) and C3H/10T1/2 (B) cells were transfected with wt or mutant psiCheck2-Twist1-39UTR reporter only. Endogenous miRNAs in NIH-3T3 (C) and C3H/10T1/2 (D) cells were inhibited by co-transfection of 50 nM miRCURY LNA microRNA inhibitors and wt psiCheck2-Twist1-39UTR reporter. Firefly luciferase activities were measured after 48 h, normalized to Renilla luciferase activities and subsequently to the wt 39UTR (A-B) or wt+anti-Scrambled (C-D). Statistical significance was calculated by using Student's t-test and by comparing the mutant Twist1 39UTR reporters or miRNA specific antagonists with the wt 39UTR reporter or a scrambled control (*), respectively. doi:10.1371/journal.pone.0066070.g006 TWIST1 Regulation by Micro-RNA PLOS ONE | www.plosone.org All of the miRNAs shown to repress TWIST1 expression have been demonstrated to be involved both in embryonic development and cancer.",
"MicroRNAs 151-5p and 337-3p reduce the mobility of murine embryonic fibroblast cells. Dot-plot superposed with the box-plot of the predicted probabilities of cell migration for observations from miR-151-5p + miR-337-3p (red) and scrambled control (blue) treated cells (p-value 0.000135). Note the presence of miR-151-5p + miR-337-3p treated cells with extreme low estimates of probability of cell migration. doi:10.1371/journal.pone.two miRNAs used as negative controls are: hsa-miR-485 (MI0002469) and hsa-miR-609 (MI0003622). Primers used for cloning were: mmu-miR-33-upper (59ctgtcagcggccggaagcctactcgggcaatgtgcc 39), mmu-miR-33-lower (59 gctgcgatcgtcgacggaagcagctagttctaacctcc 39), mmu-miR-145a-upper (59 agctatgcggccggactctacgcacatgaaatgcttcttcc 39), mmu-miR-145a-lower (59 gctgcgatcgtcgacggattggatgtaatataaatgaagcaaacc 39), mmu-miR-151-upper (59 ctgtcagcggccgctttagggcctgagaatatcttga 39), mmu-miR-151-lower (59 gggaccgcgtcgacgaagcaaacctcaataacagaactc 39), mmu-miR-326-upper (59 cctgtagcggccgctactccgcatagccactgaga 39), mmu-miR-326-lower (59 gcggatccgtcgacctagcccagggccatatacatg 39), mmu-miR-337-upper (59 ctgtcagcggccgccatgagagtttataagaagtgagg 39), mmu-miR-337-lower (59 gggaccgcgtcgacgccaaggaatgatctcaggtatgg 39), mmu-miR-361-upper (59 agctatgcggccgcttaa-catgccttggtttgcaga39), mmu-miR-361-lower (59 acctagcggtcgacctttgctgctttctttgcttcc 39), mmu-miR-378a-upper (59 cctgtagcggccggtaattgataccaggagctttgcagcc 39), mmu-miR-378alower (59 acctagcggtcgacccagacagctcacatgcaaacacagg 39), mmu-miR-381-upper (59 catgtagcggccgccaatctgttgtaacatctgccagt 39), mmu-miR-381-lower (59 gctgcgatcgtcgacagaacatgcacacttgggtac 39), mmu-miR-409-upper (59 cctgtagcggccggggaaggctttagatattcttggaagg 39), mmu-miR-409-lower (59 acctagcggtcgaccacggtcgatctcccttcaagtaccagc 39), mmu-miR-543-upper (59 agctatgcggccgggagactccaaagacctccccaaagg 39) and mmu-miR-543lower (59 gggaccgcgtcgacgtggaggagggaggagggagcaggagcc 39).",
"Pairwise comparison of Twist1 and Twist2 mRNA sequence domains of three selected amniotes with the corresponding human sequence. Numbers represent% sequence identity.",
"Conservation of microRNA target sites in selected amniotes and the presence in Twist2 39UTR."
] | [
"(Fig. 1)",
"(Fig. 1)",
"(Fig. 2)",
"Fig. 3",
"(Fig. 4A)",
"(Fig. 4B",
"(Fig. 5",
"Fig. 6A",
"Fig. 6C and 6D)",
"(Fig.7)",
"(Fig. 2 and 3)",
"(Fig. 3)",
"(Fig. 6)",
"(Fig. 4B)",
"(Fig. 7)"
] | [
"+ + + + 2 + 2 miR-137-3p + + + + 2 + 2 miR-145a-5p + + + + 2 2 + miR-151-5p + + + + 2 + 2 miR-214-5p + + + + 2 2 2 miR-326-3p + + + + 2 2 2 miR-337-3p + + + + 2 + 2 miR-361-5p + + + + 2 2 2 miR-378a-5p + + + + 2 2 2 miR-381-3p + + + + 2 + 2 miR-409-3p + + + + 2 2 2 miR-450b-5p + + + + 2 + 2 miR-508-3p + + + + 2 2 2 miR-543-3p + + + + 2 2 2 miR-576-5p + + + + 2 2 2 miR-580 + + + + 2 2 2 miR-591 + + + + 2 2 2"
] | [
"The evolutionary conserved basic helix-loop-helix (bHLH) transcription factor TWIST1 is a multifunctional proto-oncogene with a strong correlation to poor prognosis. TWIST1 is able to inhibit c-MYC induced apoptosis [1] and directly regulates the expression of several other oncogenes such as GLI1 [2], miR-10b [3] and AKT2 [4]. Overexpression of TWIST1 has been observed in various types of cancer such as breast, prostate, gastric, pancreatic and bladder cancer, hepatocarcinoma, rhabdomyosarcoma, and glioma and is often associated with more aggressive phenotypes, and acquired drug resistance (reviewed in [5]).",
"Twist1 expression is regulated by a complex network of signals and has been described as an integrator of SHH, FGF and BMP signaling [6]. In mice, a genomic fragment containing 6 kb upstream and 1.5 kb downstream of the Twist1 gene is not sufficient to recapitulate Twist1 expression during embryogenesis (unpublished data) which is consistent with the observation that a translocation breakpoint 3 kb downstream of the Twist1 gene creates a comparable haploid insufficiency as the null allele [7]. However, a number of transcription factor binding sites have been identified in the Twist1 upstream region and direct binding of transcriptional activators like NF-kB [8] and repressors like Prospero-related homeobox 1 (PROX1) [9] has been shown.",
"TWIST1 is directly upregulated by hypoxia-inducible factor-1a (HIF-1a) and HIF-2a [10,11]. Intratumoral hypoxia is correlated with radiation therapy resistance and enhanced metastatic potential [12]. TWIST1 promotes tumor metastasis [10] by epithelial-mesenchymal transition (EMT) [13] and formation of invadopodia, the specialized membrane protrusions for extracellular matrix degradation [14]. TWIST1 is linked to the transcription factor SNAIL, which also induces EMT, in a positive feedback loop [15]. In addition, co-expression of TWIST1, HIF-1a and SNAIL has been correlated with metastasis and poor prognosis in primary tumors of head and neck squamous cell carcinoma (HNSCC) patients [10].",
"The wide spectrum of TWIST1 functions might be explained by the fact that TWIST1 is able to act both as a transcriptional activator [16] and inhibitor [17,18].",
"Originally, the Twist gene was discovered as a mutation disturbing cellular motility and EMT during gastrulation in Drosophila melanogaster [19,20]. A comparable phenotype is observed in murine homozygous Twist1 null mutant embryos in which the cephalic neural crest cells are unable to form a functional mesenchyme [21]. Haploid insufficiency causes Saethre-Chotzen syndrome (polysyndactyly and craniosynostosis) in humans and comparable symptoms in mice [7,22].",
"During mammalian embryogenesis, Twist1 mRNA precedes TWIST1 protein expression, indicating a translational control of Twist1 [23,24]. The same phenomena was recently observed in MCF-10ANeoT cells that had undergone EMT [25]. It is therefore interesting that many of the biological processes in which TWIST1 is involved, are associated with regulation by miRNAs, a hallmark of translational control. MiRNAs are endogenous small non-coding RNAs that regulate gene expression post-transcriptionally by modulating mRNA translation or stability. They influence cellular processes such as EMT, apoptosis, and differentiation, which all are essential for development and cancer [26,27]. MiRNA-mediated regulation of gene expression is complex as one mRNA is usually targeted by several miRNAs and one miRNA can target several mRNAs. Furthermore, miRNAs can act synergistically leading to higher levels of repression [28].",
"Here we show that the 39UTRs of mammalian Twist1 genes contain conserved miRNA target sites, which make them sensitive to regulation by several miRNAs, individually and in cooperative combination. Understanding the exact mechanism of Twist1 regulation is important as it may allow to utilize this physiological process to be utilized therapeutically.",
"Translational regulation of mRNAs is typically mediated through evolutionary conserved regulatory regions within the UTRs. To test if this is the case for Twist1, we compared the conservation of coding sequence (CDS) and 39UTR of Twist1 mRNA among selected amniotes. 59UTRs were not included since the full-length sequences are not available for all species. Limiting the investigation to amniotes gave us the possibility to also compare the conservation of Twist1 with that of Twist2 (Table 1), a highly related gene that is differently expressed but functionally largely equivalent to Twist1 [29][30][31].",
"The two genes are highly similar in their coding regions, genomic structure and length of the 39UTR. However, the 39UTR of Twist2 is not related to that of Twist1 (percent of identity 30%) and is, between species, remarkably less conserved compared to the unusually highly conserved 39UTR of Twist1 indicating a functional selection of this sequence that is almost as stringent as for the CDS. Furthermore, the 39UTR of Twist1 contains considerably more potential regulatory sequences, namely four nuclear polyadenylation signals (pA1-4 where pA3 and pA4 overlap), two cytoplasmic polyadenylation elements (CPEs), one AU-rich sequence and a number of putative miRNA target sites predicted by several algorithms (TargetScan, miRBase and PicTar). All but one of the identified potential regulatory sequences (miR-15b-3p) are 100% conserved between mouse and human Twist1 39UTR sequences (Fig. 1) and a large number are conserved in a wide range of mammalian species (Table 2). In addition, out of 18 miRNAs predicted to target Twist1, only one (miR-145a-5p) putatively targets Twist2 as well (Table 2).",
"The presence of multiple potential miRNA target sites in the 39UTR of Twist1 led us to investigate whether Twist1 expression could be regulated by miRNAs. The following miRNAs were tested for their potential to repress Twist1 translation in the human lung carcinoma cell line H1299: miR-33, miR-145a, miR-151, miR-326, miR-337, miR-361, miR-378a, miR-381, miR-409 and miR-543 (Fig. 1).",
"Murine pre-miRNA sequences were cloned into pdCMV2-EGFP expression vector and their correct processing was confirmed by Northern Blot (not shown). To determine whether any of the selected miRNAs are able to repress the expression of TWIST1, the miRNAs were tested individually or in pairwise combinations in a luciferase reporter assay using a construct in which the Firefly luciferase CDS was followed by the murine Twist1 39UTR sequence. MiR-485 and miR-609 have no target site in the 39UTR of Twist1 and were used as negative (mock) controls to calculate the unrepressed expression level of the reporter in these cells (Fig. 2). Expression of miR-145a resulted in a significant downregulation of the reporter by 44% (p,0.006). In addition, the co-expression of two pairs of miRNAs also led to a significantly increased repression: miR-151 + miR-337 resulted in a synergistic inhibition of 78% and miR-145a + miR-151 repressed TWIST1 expression by 61% (p,0.006). Notably, miR-337 alone had no effect while miR-151 only had a weak effect which in this assay was not statistically significant (p,0.15; see discussion).",
"In order to confirm the location and functionality of the predicted miRNA target sites within the 39UTR of Twist1, we mutated some of them to restriction enzyme recognition sites. Transfection of the wild type (wt) or mutant reporter with the corresponding miRNA confirmed that the repressive effects depended on the presence of the miRNA target sites. As shown in Fig. 3, mutating the target site of miR-145a-5p or miR-151-5p resulted in a loss of repression whereas lack of a binding site for miR-337-3p had no effect. When both miR-145a and miR-151 were present, mutating either of the target sites caused a comparable loss of repression. Interestingly, miR-337 was only able to repress the 39UTR reporter when miR-151 and its target site both were present. This confirms that miRNAs and their target sites both are necessary to repress the expression of TWIST1 protein.",
"MicroRNAs primarily act by destabilizing target mRNA through decapping and/or deadenylation. However, a smaller subset of targets is rather translationally repressed leaving the mRNA levels unaltered (reviewed in [32]). As the uneven ratio between Twist1 mRNA and TWIST1 protein in the mouse embryo [23,24] indicated translational inhibition, we compared the mRNA and protein ratios in cells treated with a combination of synthetic miR-151-5p and miR-337-3p precursors. Using high concentrations of pre-miR-151-5p and pre-miR-337-3p (above 10 nM) we consistently found comparable decrease in both the mRNA and protein level of the Twist1 39UTR reporter (Fig. 4A). To more closely mimic the physiological conditions, we established stable pools of cells harboring the Twist1 39UTR reporter and transfected them using low concentrations (5 nM) of synthetic miR-151-5p and miR-337-3p precursors. Under such conditions, we observed a 50% reduction of luciferase reporter activity and hardly any reduction in the corresponding mRNA level upon miRNA co-transfection (Fig. 4B). This indicates that the synergistic effect of miR-151-5p and miR-337-3p is mainly due to translational inhibition and not RNA degradation. A corresponding analysis using the endogenous TWIST1 protein was technically not possible as we found all available TWIST1 specific antibodies to also react with unidentified proteins in murine cells not expressing TWIST1, a specificity issue not seen in human cells [25]. ",
"To test whether endogenous miRNAs are able to target the murine Twist1 39UTR reporter, we analyzed the expression pattern of miRNAs of interest in some commonly used mouse cell lines: NIH-3T3, C3H/10T1/2 and C 2 C 12 (Fig. 5). Since miR-145a-5p, miR-151-5p and miR-337-3p all were expressed in NIH-3T3 and C3H/10T1/2 but not in C 2 C 12 cells, we selected the first two lines for further analysis.",
"We then analyzed the effect of endogenous miRNAs on the wt and mutant Twist1 39UTR reporters. As shown in Fig. 6A and 6B, endogenous miR-145a-5p, miR-151-5p and miR-337-3p are targeting Twist1 39UTR in both cell lines, as mutating their target sites led to a statistically significant increase of reporter activities. This was confirmed by using locked nucleic acid (LNA)-modified miRNA antagonists to block each of these endogenous miRNAs. Inhibition of miR-145a-5p, miR-151-5p and miR-337-3p resulted in a decreased repression, i.e. an elevated expression of the reporter protein in both cell lines ( Fig. 6C and 6D), confirming that the endogenous murine miRNAs are able to target the Twist1 39UTR.",
"MiR-151-5p and miR-337-3p reduce the mobility of murine embryonic fibroblast cells There is no known cellular function which depends exclusively on TWIST1 and thus would give an all-or-none phenotype upon removal of TWIST1. We therefore decided to test whether the combination of miR-151-5p and miR-337-3p also effects the biological function of endogenous TWIST1, by investigating the ability of murine embryonic fibroblasts to migrate through an 8 mm pore filter. This type of assay correlates well with the EMT promotion by TWIST1 and has been used in similar studies [25]. Compared to cells treated with a scrambled control miRNA, cells treated with a combination of miR-151-5p and miR-337-3p migrated significantly less (Fig.7). Despite the small absolute difference, which probably indicates the limited contribution of TWIST1 to the mobility of these cells, the effect was highly significant. The probabilities of cell migration were estimated as 0.898 (95% bootstrap CI 0.896-0.913) for the miR-151-5p + miR-337-3p treated cells and 0.918 (95% bootstrap CI 0.914-0.922) for the control treated cells, and a bootstrap permutation test (p-value 0.000135) showed that the difference between the probabilities is statistically significant. ",
"MicroRNAs underlined were tested in this study. doi:10.1371/journal.pone.0066070.t002 ",
"In this study, we report a new mechanism for the regulation of TWIST1 expression. TWIST1 is a key player in tumorigenesis and metastasis and its overexpression has mostly been correlated with progressed stages of cancer and drug resistance [33]. Furthermore, suppression of TWIST1 expression in tumor cells can lead to inhibition of metastatic potential [13].",
"We analyzed the presence of conserved miRNA target sites in the 39UTR of Twist1 in selected amniotes (Table 1 and 2) and identified miR-145a-5p, miR-151-5p and the combinations of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p as the strongest regulators of murine Twist1 (Fig. 2 and 3). For the initial screen, we used human H1299 cells, which are easy to transfect. It should be noted, however, that a strong expression of endogenous miRNAs in these cells might cover the effect of exogenous miRNAs tested and thus give false negative results. In fact, we assume that the relatively high expression of endogenous miR-151-5p in H1299 cells (publically available miRNA microarray dataset; GEO accession number GSE30075) explains the significant difference between the miR-151-5p-mediated repressive effects shown in figures 2 and 3.",
"The distance between miR-145a-5p and miR-151-5p target sites is 394 nt, too long to expect a synergistic interaction [34]. Indeed, the effect of their combination was only slightly stronger than the additive effect of their independent action. In contrast, the combinatorial effect of miR-151-5p and miR-337-3p clearly is synergistic, as miR-337-3p alone was not able to downregulate the Twist1 39UTR reporter but only did so when miR-151-5p was present (Fig. 3). Importantly, this synergistic effect was not observed if the miR-151-5p target site was mutated. The 6 nt distance between these two miRNA target sites is within the optimal range for two cooperating miRNAs (between 6 to 40 nt) [34]. In addition, miR-337-3p has no complementary sequence to Twist1 59 of its seed region leaving this sequence free for miR-151-5p to bind. Interestingly, the target sites for miR-151-5p and miR-337-3p will still be present, even if the previously reported shortening of 39UTRs in cancer cells [35] also occurs with TWIST1 and either the first or the second pA signal should be used.",
"By mutating miRNA target sites and inhibiting miRNA binding using specific LNA-modified oligonucleotides, we confirmed the effect of the above described miRNAs on the Twist1 39UTR (Fig. 6) and demonstrated that the physiological expression levels of endogenous miRNAs are sufficient for a significant repression of Twist1 which most likely is due to translational inhibition rather than degradation of Twist1 mRNA (Fig. 4B). MiR-337 always required miR-151 to be present in order to have an effect. While this work was under revision, miR-214 [36], miR-300, miR-539 and miR-543 [37] have also been reported to target the TWIST1 39UTR. While two of them, miR-539 and miR-300 have no target site in the murine Twist1 39UTR, a third, miR-543 did not show a significant effect in our screen. This might be due to quantitative differences between the transfection of a miRNA expression vector and application of miRNA-mimics. It would be interesting to see whether these miRNAs also target TWIST1 by inhibiting translation or by RNA destabilization. During development, miR-145a-5p functions as a critical switch in promoting smooth muscle differentiation and its downregulation is associated with proliferation [38]. MiR-145a-5p is a known tumor suppressor with a strong inhibitory effect on cancer cell proliferation that is downregulated in a variety of tumors including bladder, breast, colon, colorectal, gastric, lung, oral, ovarian, and prostate cancers, hepatocellular and nasopharyngeal carcinoma and pituitary tumors [39], all of which are associated with increased levels of TWIST1. In cervical cancer cells, miR-145a-5p was shown to inhibit growth, invasion and therapy resistance [40], problems often observed in tumors in which TWIST1 is upregulated, and ectopic delivery into mouse pancreatic xenograft models resulted in the inhibition of tumor growth [41,42].",
"MiR-151 targets the thrombopoietin receptor MPL [43], a gene known to be downregulated in myeloproliferative neoplasms [44]. In hepatocellular carcinoma (HCC), miR-151-5p has been correlated with cell migration and intrahepatic metastasis [45]. However, miR-151 has been shown to be both upregulated (HCC, nasopharyngeal carcinoma) and downregulated (pappillary carcinoma, acute myeloid leukemia) in different types of cancer [46]. This dual function probably reflects the differences in gene regulation that leads to the various types of cancer. It would thus be interesting to investigate whether the expression of miR-151 in tumors correlates with the expression level of TWIST1.",
"MiR-337 is upregulated during bone formation [47] whereas TWIST1 is downregulated [48] and overexpression of Twist1 is known to inhibit osteoblast differentiation [49]. This suggests miR-337 as a potential inhibitory factor regulating Twist1 during bone formation. In cancer, miR-337 has been shown to be overexpressed in older melanoma patients compared to younger ones [50] and is negatively associated with survival in advanced ovarian cancer [51]. So far, nothing is known about the co-expression of miR-151 and miR-337. In light of our results, it would be interesting to see whether miR-337 is able to acquire a tumor suppressor function when co-expressed with miR-151 in TWIST1 positive tumors.",
"TWIST1 itself is also known to regulate the expression of several miRNAs, many of which are cancer-related. E.g. the oncogenic miR-10b is directly activated by TWIST1 and promotes the invasion and metastasis through repressing HOXD10 expression [3]. Also, the miR-200 family and miR-205 were shown to be repressed by TWIST1 [52]. These miRNAs are frequently silenced in advanced cancer [53,54]. They target ZEB1 and ZEB2, transcriptional repressors of E-cadherin, and thereby inhibit EMT and tumor invasion [55]. Recently, miR-223, overexpressed in metastatic gastric cancer cells, was also found to be induced by TWIST1 [56]. Considering its repression by miRNAs as demonstrated in this study, TWIST1 appears as a central player in the regulating network of miRNAs and transcription factors necessary for cellular homeostasis.",
"Recently, RNAi-mediated silencing of TWIST1 was shown to suppress the proliferation of human cervical cancer cells and to improve the chemosensitivity to cisplatin treatment, indicating a novel therapeutic strategy to overcome drug resistance [57]. Likewise, Burns et al. demonstrated a strong therapeutic effect in oncogene driven non-small cell lung cancer after silencing TWIST1 expression [58]. Our results about the miRNA regulation of TWIST1 provide an alternative approach to suppress this potent oncogene utilizing an endogenous mechanism.",
"The reduced mobility of miR-151-5p + miR-337-3p treated embryonic fibroblasts further shows the therapeutic potential in the combination of these two miRNAs (Fig. 7). The relatively small absolute difference in the migration probability is most likely explained by the fact that cell motility is a property influenced by a number of different factors. However, the high statistical significance of the difference indicates that the treatment with miR-151-5p and miR-337-3p has a reliable effect.",
"In summary, we have shown that TWIST1 is regulated by miR-145a-5p, miR-151-5p and miR-337-3p. The additive and synergistic effects of these miRNAs could reduce unwanted 'off target' effects and might open up new possibilities to specifically interfere with TWIST1 translation in therapeutic approaches.",
"Sequence comparison was done with CLC Main Workbench Version 5.7 (CLC bio). Parameters for alignments were: gap open cost = 10 and gap extension cost = 1. The following sequences were pairwise compared: Twist1: human (GeneBank: NM_00047); cow (GeneBank: XM_002686684); mouse (GeneBank: NM_011658); chicken (GeneBank: NM_204739; 39UTR without nt 845-873 and 899-971 which correspond to 2 stretches of sequence not found in any other species). Twist2: human (GeneBank: NM_057179); cow (GeneBank: NM_001083748); mouse (GeneBank: NM_007855); chicken (GeneBank: NM_204679). In addition, Twist1 sequences used for miRNA target site analysis were: dog (GeneBank: XM_857736); frog (GeneBank: NM_001085883).",
"All cell lines were obtained from American Type Culture Collection and cultured at 37uC in 5% CO 2 . H1299 cells were maintained in RPMI 1640 (Gibco) and NIH-3T3, C3H/10T1/2 and C 2 C 12 cells in DMEM (Gibco), all supplemented with 10% FCS, 100 mg/ml streptomycin and 100 U/ml penicillin (Gibco).",
"Murine pre-miRNA sequences with about 200 nt 59 and 39 flanking regions were amplified from C57Bl/6J mouse genomic Mouse Twist1 39UTR sequence (GeneBank: NC_000078; genomic DNA position 34,643,544 to 34,645,282) was cloned into the XbaI/NotI sites of pGL3-control vector (Promega). This sequence includes the unique intron of the Twist1 gene and thus represents a construct which will report all aspects of Twist1 mRNA processing including splicing. PRluc-N2 (PerkinElmer) encoding Renilla luciferase was used for normalization.",
"Due to high fluctuations in the pGL3-control transfection efficiencies, mouse Twist1 39UTR sequence (GeneBank: NM_011658; nucleotide position 926-1634) was cloned into XhoI/NotI sites in psiCheck2 vector (Promega) which expresses both Firefly and Renilla luciferases. MiRNA target sites were subsequently mutated to restriction enzyme recognition sites: miR-145a-5p to SacI; miR-151-5p to AgeI; miR-337-3p to SalI.",
"Stable pools of cells expressing psiCheck2-Twist1-39UTR were established as described in [59].",
"All transfections were carried out in triplicates on 48-well plates in three independent experiments and assayed after 48 h using the Dual-Luciferase Reporter Assay System Kit (Promega). 48 h was chosen to allow the cells to recover from the transfection and grow up to ,80-100% confluence at the time of measurement.",
"In the miRNA screen, 10 4 /cm 2 H1299 cells were transfected with 250 ng pdCMV2-EGFP-miR-X (or 125 ng each if two miRNAs were combined), 50 ng pRluc-N2 and 50 ng pGL3-Twist1-39UTR or pGL3-control using TransIT-LT1 transfection reagent (Mirus) according to manufacturer's guidelines. For normalization, the Firefly luciferase activity was divided by the Renilla luciferase activity. Triplicates were averaged and normalized to the pGL3-control.",
"In the target site mutation assay, 10 4 /cm 2 H1299 cells were transfected with 250 ng (or 125 ng each) miRNA construct and 50 ng wt or mutant psiCheck2-Twist1-39UTR using TransIT-LT1 transfection reagent (Mirus) according to manufacturer's instructions. For normalization, Renilla luciferase activity was divided by Firefly luciferase activity and the mutant-transfected cells were subsequently normalized to the respective wt 39UTR reporter treated with miRNA.",
"The transfection efficiency of miRNA-encoding vectors was monitored by the expression of EGFP with the Leica DM fluorescence microscope. The efficiency was always above 60%. 10 4 /cm 2 NIH-3T3 and C3H/10T1/2 cells were transfected with 50 ng psiCheck2-Twist1-39UTR using Fugene HD transfection reagent (Roche) according to manufacturer's guidelines. 50 nM miRCURY LNA microRNA inhibitors (Exiqon) were added in the inhibition assay: mmu-miR-145a-5p (139465-00), hsa-miR-151-5p (414654-00), mmu-miR-337-3p (139530-00) and a Scrambled negative control (199004-00). For normalization, Renilla luciferase activity was divided by Firefly luciferase activity.",
"10,000 stable psiCheck2-Twist1-39UTR-expressing cells per 96well plate well were transfected while seeding in 100 ml serum-free RPMI 1640 (Gibco) using 0.2 ml Lipofectamine 2000 (Invitrogen) and 20 or 5 nM Pre-miR miRNA Precursor (Applied Biosystems) per well: pre-mmu-miR-151 (PM11537), pre-mmu-miR-337 (PM12817) and Scrambled Negative Control #1 (AM17110). Simultaneously, 30,000 cells/well were transfected on a 24-well plate for RNA extraction and analysis using more reagents, accordingly. After 4 h of transfection, 10% FCS (Gibco) of total volume was added to each well. Renilla luciferase mRNA and protein levels were measured after 48 h.",
"Total RNA was purified using TRIzol (Invitrogen) according to the manufacturer's instructions. 500 ng of total RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen) using 200 ng random hexamer primer (Roche).",
"Renilla luciferase expression was quantified using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and 25 ng cDNA as template. The primers used were: R-luc_fwd (59 gttccctaacaccgagttcgtg 39), R-luc_rev (59 ctccacgaagctcttgatgtac 39), F-luc_fwd (59 ctgatcgacaaggacggctgg 39), F-luc_rev (59 ggtagcccttgtacttgatcag 39). Renilla luciferase mRNA levels were normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control. Relative quantification of mRNA levels was performed using the DDCt-method.",
"For miRNA analysis, 25 ng of total RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). QPCR was carried out in triplicates using 1.33 ml cDNA and TaqMan Universal PCR Master Mix (Applied Biosystems). The TaqMan MicroRNA Assays used were: hsa-miR-145a-5p (002278), hsa-miR-151-5p (002642), mmu-miR-337-3p (002532) and U6 snRNA (001973). Relative quantities of miRNA were calculated by the DCt method.",
"Cell migration assay 3T3 immortalized murine embryonic fibroblasts [60] were plated on a 12-well plate at a density of 10 4 /cm 2 in 1 ml complete growth medium. While seeding, cells were transiently transfected with a total of 20 nM pre-mmu-miR-151-5p (PM11537) and premmu-miR-337-3p (PM12817) or with Scrambled Negative Control #1 (AM17110; all Applied Biosystems) using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. After 24 h, cells were trypsinized, resuspended in 400 ml medium, distributed equally on 2 cell culture inserts 200 ml each (24-well, 8.0 mm pore size, Falcon, 35 3097) and then placed in a 24-well plate (Falcon, 35 3504) which contained 600 ml medium per well. The 2 inserts were designated as 'A,over' and 'B,under' to study the analysis of non-migrated and migrated cells, respectively. Cells were allowed to migrate for 21 h, washed with PBS and fixed in 4% freshly dissolved paraformaldehyde for 30 min at room temperature. Following 26 washing with PBS, the cells on the lower surface of insert A and the cells on the upper surface of insert B were wiped off with a cotton swap. The insert membranes were cut off and embedded in ProLong Gold Antifade Reagent with DAPI (Invitrogen). Migrated (under) and non-migrated (over) cells were counted in 10 (primary magnification 106) and 5 (primary magnification 56) random areas, respectively, on micrographs taken with a Leica DM fluorescence microscope.",
"Statistical significance for the transfection experiments was calculated using Student's t-test and 2-sided p-values.",
"The effect of the treatment on the probability of cell migration was studied in the following way: Denote the counts of the migrated cells and the non-migrated cells for the m th observed microscopic field for the r th replication subject to the t th treatment by M mrt and S mrt , respectively. The expected number of migrated and non-migrated cells are given by E(M mrt ) = p t U rt and E(S mrt ) = (1-p t ) U rt /40, respectively. Here p t is the probability of migration common to all the observations subject to the t th treatment and U rt is the expected number of cells transferred from the suspension associated to the rt th replicate. The factor 1/40 arises from the fact that different macroscopic field areas were observed for the independent determinations of the migrated and non-migrated cells. Using a first order Taylor expansion yields that the expectation of P t = Mmrt /(M mrt + S mrt ) is the probability p t , therefore P t is an unbiased estimate of p t . The effect of the treatment on the probability of cell migration was then formally tested using the Monte Carlo permutation test [61] with 1,000,000 bootstrap permutations. Moreover, 95% bootstrap confidence intervals for the probabilities of cell migration were constructed with non-parametric bootstrap [61], with 10,000 parametric bootstrap each."
] | [] | [
"Introduction",
"Results",
"Analysis of Twist1 39UTR",
"MicroRNAs are targeting the Twist1 39UTR",
"MicroRNAs can act by translational inhibition",
"Twist1 reporter is downregulated by endogenous microRNAs",
"Discussion",
"Materials and Methods",
"Sequence comparison",
"Cell lines and constructs",
"Transfections and luciferase assays",
"Quantitative PCR",
"Statistical analysis",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 .",
"Figure 6 .",
"Figure 7 .",
"Table 1 .",
"Table 2 ."
] | [
"Twist1 CDS 1 Twist1 39UTR 2 Twist2 CDS 1 Twist2 39UTR 2 \n\nHuman \n100 \n100 \n100 \n100 \n\nCow \n95 \n92 \n98 \n68 \n\nMouse \n93 \n87 \n97 \n65 \n\nChicken \n84 \n54 \n80 \n32 \n\n1 \n\nCDS: coding sequence; 2 UTR: untranslated region. \ndoi:10.1371/journal.pone.0066070.t001 \n",
"MiRNA target site/Species Human \nMouse \nCow \nDog \nChicken \nFrog \nTargeting Twist2 \n\nmiR-15b-3p \n+ \n2 \n+ \n+ \n2 \n2 \n2 \n\nmiR-33-5p \n"
] | [
"(Table 1)",
"(Table 2",
"(Table 2)",
"(Table 1"
] | [
"The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs",
"The Proto-Oncogene TWIST1 Is Regulated by MicroRNAs"
] | [] |
91,185,514 | 2022-02-23T07:13:15Z | CCBY | https://www.mdpi.com/2073-4360/9/8/329/pdf | GOLD | 17c2b45b4e408770abdbf311b4617beb39bb72ad | null | null | null | null | 10.3390/polym9080329 | 2739866199 | 30971004 | 6418683 |
The Influence of Calcium Glycerophosphate (GPCa) Modifier on Physicochemical, Mechanical, and Biological Performance of Polyurethanes Applicable as Biomaterials for Bone Tissue Scaffolds Fabrication
2017
Justyna Kucińska-Lipka
Department of Polymer Technology
Faculty of Chemistry
Gdansk University of Technology
Narutowicza St. 11/1280-233GdanskPoland
Iga Gubanska [email protected].
Department of Polymer Technology
Faculty of Chemistry
Gdansk University of Technology
Narutowicza St. 11/1280-233GdanskPoland
Olexandr Korchynskyi
Institute of Cell Biology
National Academy Science of Ukraine
14/16 Drahomanov Str79005LvivUkraine
Centre for Innovative Research in Medical and Natural Sciences, Rzeszow University and Medical Faculty
35-959RzeszowPoland
Khrystyna Malysheva [email protected].
Institute of Cell Biology
National Academy Science of Ukraine
14/16 Drahomanov Str79005LvivUkraine
Marcin Kostrzewa [email protected]
Department of Organic Materials Technology
Technical University of Radom
26-600RadomPoland
Damian Włodarczyk
Institute of Physics, Polish Academy of Science
Division of Physics and Technology of Wide-Band-Gap Semiconductor Nanostructures
Al. Lotnikow 32/4602-668WarsawPoland
Jakub Karczewski
Faculty of Applied Physics and Mathematics
Gdansk University of Technology
Narutowicza St. 11/1280-233GdanskPoland
Helena Janik [email protected].
Department of Polymer Technology
Faculty of Chemistry
Gdansk University of Technology
Narutowicza St. 11/1280-233GdanskPoland
The Influence of Calcium Glycerophosphate (GPCa) Modifier on Physicochemical, Mechanical, and Biological Performance of Polyurethanes Applicable as Biomaterials for Bone Tissue Scaffolds Fabrication
Polymers
9329201710.3390/polym9080329Received: 23 June 2017; Accepted: 28 July 2017;polymers Article 2 of 21polyurethanebone tissue engineeringcalcium glycerolphosphate saltmechanical propertiescontact angleSEMEDXcalcificationsolvent casting/particulate leachingTIPS
In this paper we describe the synthesis of poly(ester ether urethane)s (PEEURs) by using selected raw materials to reach a biocompatible polyurethane (PU) for biomedical applications. PEEURs were synthesized by using aliphatic 1,6-hexamethylene diisocyanate (HDI), poly(ethylene glycol) (PEG), α,ω-dihydroxy(ethylene-butylene adipate) (Polios), 1,4-butanediol (BDO) as a chain extender and calcium glycerolphosphate salt (GPCa) as a modifier used to stimulate bone tissue regeneration. The obtained unmodified (PURs) and modified with GPCa (PURs-M) PEEURs were studied by various techniques. It was confirmed that urethane prepolymer reacts with GPCa modifier. Further analysis of the obtained PURs and PURs-M by Fourier transform infrared (FTIR) and Raman spectroscopy revealed the chemical composition typical for PUs by the confirmed presence of urethane bonds. Moreover, the FTIR and Raman spectra indicated that GPCa was incorporated into the main PU chain at least at one-side. The scanning electron microscopy (SEM) analysis of the PURs-M surface was in good agreement with the FTIR and Raman analysis due to the fact that inclusions were observed only at 20% of its surface, which were related to the non-reacted GPCa enclosed in the PUR matrix as filler. Further studies of hydrophilicity, mechanical properties, biocompatibility, short term-interactions, and calcification study lead to the final conclusion that the obtained PURs-M may by suitable candidate material for further scaffold fabrication. Scaffolds were prepared by the solvent casting/particulate leaching technique (SC/PL) combined with thermally-induced phase separation (TIPS). Such porous scaffolds had satisfactory pore sizes (36-100 µm) and porosity (77-82%) so as to be considered as suitable templates for bone tissue regeneration.
Introduction
Tissue scaffolds, designed for tissues regeneration, are three-dimensional porous structures, which serve as biological tissue substitutes that enable the functional performance of the regenerated tissue to be restored, maintained or improved [1]. Tissue engineering (TE) applies both natural and synthetic polymers [2], metals [3], ceramics [4] and bioactive glasses [5]. Biomaterials used for the purpose of tissue engineering (TE) have to meet strict requirements such as biocompatibility. Moreover, these materials may reveal some bioactive behavior, which could stimulate proper tissue regeneration [6]. The 3D scaffold has to provide an adequate support for the regenerated tissue, thus the mechanical characteristics of the used biomaterials are an important feature. In addition the gradual degradation of the scaffold is necessary for proper tissue restoration [7,8]. However, in order to meet the requirements of an ideal tissue scaffold material and to combine the best mechanical properties with bioactivity and ability to degrade in the human body environment, new composite materials, which are the combination of polymers with different types of fillers, are being developed [1,8]. Polyurethanes (PU) according to their superior characteristics of proper physicochemical, mechanical, and biological properties seem to meet all of these requirements for use in TE as materials for scaffold fabrication [1,6].
PUs have been widely developed in the field of biomedical devices, thus their modification is well known [1,6,9]. The most common PU modifications for bone tissue engineering take place mainly by the introduction of the filler into the PU matrix. The most often reported fillers are calcium phosphates like hydroxyapatite [4,10,11], nanohydroxyapatite [12,13], and β-tricalcium phosphate [14,15]. Recently anew solution for PU modification was proposed in the form of calcium glycerophosphate [16], bioactive glass [5] or carbon nanotubes [17].
Calcium glycerophosphate is the calcium salt of glycerophosphoric acid. This compound has been approved for use by the Ministry of Health [18] as a nutrient, a component of dietary supplements or mineral food products, and has been considered as a safe ingredient/food additive by the US Food and Drug Administration [19]. In addition to the previously listed applications, calcium glycerophosphate is also used as an ingredient in toothpaste [20], dental varnishes [21], as well as electrolytes used for mineralization of hydrogels for bone regeneration [22] or surface modification of titanium bone implants [3].
There are several examples in the literature, describing the synthesis of PU composites with the use of fillers mentioned above leading to the desired changes in mechanical properties of the material, as well as contributing to its bioactivity improvement [4,[10][11][12][13][14][15][16][17].The effect of calcium glycerolphosphate salt (GPCa) as a filler on tissue scaffolds properties which received biodegradable polyurethane foams for bone graft substitutes was recognized by Gorna et al. [16]. The PU system studied by Gorna et al. was derived from 1,6-hexamethylene diisocyanate, poly(ethylene oxide)diol, poly(ε-caprolactone)diol, amine-based polyol or sucrose-based polyol, water as a chain extender and foaming agent, catalysts, citric acid as a calcium complexing agent, lecithin or solutions of vitamin D 3 as surfactants, and various inorganic fillers. One of the fillers used was GPCa, others were calcium carbonate and hydroxyapatite [16]. Recently, Kavanaugh et al. [23] proved as well that segmented polyurethanes prepared with β-glycerol phosphate as a biologically active chain extender, supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation. The PU system studied by Kavanaugh et al. was synthesized by using poly(ε-caprolactone)diol, 4,4 -methylene bis(cyclohexyl isocyanate), and biologically active compounds such as ascorbic acid, L-glutamine, β-glycerol phosphate, and dexamethasone as chain extenders [23]. In brief, the use of β-glycerol phosphate as a chain extender has improved the biological activity of polyurethane applicable as a material for bone regeneration. Furthermore, glycerophosphates have high potential for mineralization, proper adhesion and proliferation and therefore the attempted GPCa chain PU contrasts with the approach by Gornal et al. [16] where it was used as a filler.
In this paper we describe the synthesis and characterization of the PEEURs designed for bone tissue regeneration. PEEURs were synthesized by using 1,6-hexamethylene diisocyanate (HDI), poly(ethylene glycol) (PEG), α,ω-dihydroxy(ethylene-butylene adipate) (Polios), 1,4-butanediol Polymers 2017, 9,329 3 of 21 (BDO) as a chain extender and GPCa as a modifier used to stimulate bone tissue regeneration. The obtained unmodified (PUR) and GPCa modified (PURs-M) PEEURs were studied with various techniques in order to confirm the reactivity of GPCa with the urethane prepolymer, to study its chemical composition, surface morphology, hydrophilic character, mechanical properties, in vitro biocompatibility and short-term interactions with selected acidic, basic and oxidative environment. Moreover the calcification study was performed to establish if the GPCa modifier improved the bioactive character of the obtained PUR-M materials. Furthermore, with the use of selected samples, porous scaffolds were obtained by using the SC/PL technique combined with TIPS. According to performed studies the obtained PURs-M may be a suitable candidate for bone tissue engineering and further studies are being developed by our team in this field.
Experimental
Poly(ester ether urethane)s Synthesis
PEEURs were synthesized by the standard two step polymerization procedure with urethane prepolymer intermediate [1,6]. The urethane prepolymer was obtained in the reaction of polyester α,ω-dihydroxy(ethylene-butylene adipate) (PEBA, trade name Polios 55/20; Purinova, Bydgoszcz, Poland) (63 wt %),poly(ethylene glycol) (PEG) (14 wt %) and aliphatic 1,6-hexamethylene diisocyanate (HDI) (Sigma Aldrich, Poznań, Poland) (23 wt % ). In the second step the chain extender-1,4-butanediol (BDO) (POCH, Gliwice, Poland)-Was added to the urethane prepolymer to obtain PEEUs with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of the chain extender BDO equal to NCO/OH = 0.9:1.
The synthesis of modified PEEURs (PUR-M) was as follows: In the first step the urethane prepolymer was obtained in the reaction between PEBA, PEG, and HDI. In the second step the 10 wt % of GPCa (Sigma Aldrich, Poznań, Poland) calculated per mass of the prepolymer was added at 80 • C and stirred for 4 h (the mass ratio of urethane prepolymer to GPCa was equal to 1:0.25). In the next step the chain extender BDO was added to obtain PUR-M with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of chain extender BDO equal to NCO/OH = 0.9:1. Reaction of unmodified and modified polyurethanes is presented in Figure 1.
Polymers 2017, 9,329 3 of 21 obtained unmodified (PUR) and GPCa modified (PURs-M) PEEURs were studied with various techniques in order to confirm the reactivity of GPCa with the urethane prepolymer, to study its chemical composition, surface morphology, hydrophilic character, mechanical properties, in vitro biocompatibility and short-term interactions with selected acidic, basic and oxidative environment. Moreover the calcification study was performed to establish if the GPCa modifier improved the bioactive character of the obtained PUR-M materials. Furthermore, with the use of selected samples, porous scaffolds were obtained by using the SC/PL technique combined with TIPS. According to performed studies the obtained PURs-M may be a suitable candidate for bone tissue engineering and further studies are being developed by our team in this field.
Experimental
Poly(ester ether urethane)s Synthesis
PEEURs were synthesized by the standard two step polymerization procedure with urethane prepolymer intermediate [1,6]. The urethane prepolymer was obtained in the reaction of polyester α,ωdihydroxy(ethylene-butylene adipate) (PEBA, trade name Polios 55/20; Purinova, Bydgoszcz, Poland) (63 wt %),poly(ethylene glycol) (PEG) (14 wt %) and aliphatic 1,6-hexamethylene diisocyanate (HDI) (Sigma Aldrich, Poznań, Poland) (23 wt % ). In the second step the chain extender-1,4-butanediol (BDO) (POCH, Gliwice, Poland)-Was added to the urethane prepolymer to obtain PEEUs with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of the chain extender BDO equal to NCO/OH= 0.9:1.
The synthesis of modified PEEURs (PUR-M) was as follows: In the first step the urethane prepolymer was obtained in the reaction between PEBA, PEG, and HDI. In the second step the 10 wt % of GPCa (Sigma Aldrich, Poznań, Poland) calculated per mass of the prepolymer was added at 80 °C and stirred for 4 h (the mass ratio of urethane prepolymer to GPCa was equal to 1:0.25). In the next step the chain extender BDO was added to obtain PUR-M with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of chain extender BDO equal to NCO/OH = 0.9:1. Reaction of unmodified and modified polyurethanes is presented in Figure 1.
Characterization Methods
Indications of Free Isocyanate Groups (FNCO) by the Acidimetric Method
Indication of free isocyanate groups is a standard procedure in the case of PURs obtained by a two-step polymerization method. Its aim is to establish the time, after which the requiredamount of unreacted diisocyanate groups (NCO) in the prepolymerization reaction takes place. The determination of free isocyanate groups (FNCO, %) was performed according to the PN-EN 1242:2006 standard.
Fourier Transform Infrared Spectroscopy (FTIR)
The FTIR of the solid PUR and PUR-M was performed by an FTIR Nicolet 8700 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with Specac's Golden Gate module and single reflection diamond ATR unit to determine the influence of the processing technique on the composition of the obtained materials. The studied spectral range was from 4000 to 500 cm −1 averaging 254 scans per sample with a resolution of 4 cm −1 .
Raman Spectroscopy
Raman spectra were performed by using Monovista CRS + spectrometer provided by S&I Ltd., (Warstein, Germany) which was operated by VistaControl 4.1 software (S&I Ltd., (Warstein, Germany). Data about the polymers' structure was obtained by using a 532 nm green laser with the scanning power reduced to 10 mV. The best suitable grating was set to 1800 grooves per mm and was chosen to provide resolution of about 0.5 cm −1 . The slit was set to standard 100 micrometers and the objective used to scan in Point by Point, 2D mode was a long focal-length Olympus with 100 magnification rate. All spectra were obtained by gaining two accumulations within 10 s time frame. Each scanning point from which the Raman spectra were taken had approximately1 μm 2 surface.
Characterization Methods
Indications of Free Isocyanate Groups (F NCO ) by the Acidimetric Method
Indication of free isocyanate groups is a standard procedure in the case of PURs obtained by a two-step polymerization method. Its aim is to establish the time, after which the requiredamount of unreacted diisocyanate groups (NCO) in the prepolymerization reaction takes place. The determination of free isocyanate groups (F NCO , %) was performed according to the PN-EN 1242:2006 standard.
Fourier Transform Infrared Spectroscopy (FTIR)
The FTIR of the solid PUR and PUR-M was performed by an FTIR Nicolet 8700 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with Specac's Golden Gate module and single reflection diamond ATR unit to determine the influence of the processing technique on the composition of the obtained materials. The studied spectral range was from 4000 to 500 cm −1 averaging 254 scans per sample with a resolution of 4 cm −1 .
Raman Spectroscopy
Raman spectra were performed by using Monovista CRS + spectrometer provided by S&I Ltd., (Warstein, Germany) which was operated by VistaControl 4.1 software (S&I Ltd., (Warstein, Germany). Data about the polymers' structure was obtained by using a 532 nm green laser with the scanning power reduced to 10 mV. The best suitable grating was set to 1800 grooves per mm and was chosen to provide resolution of about 0.5 cm −1 . The slit was set to standard 100 micrometers and the objective used to scan in Point by Point, 2D mode was a long focal-length Olympus with 100 magnification rate. All spectra were obtained by gaining two accumulations within 10 s time frame. Each scanning point from which the Raman spectra were taken had approximately 1 µm 2 surface.
Static Contact Angle Determination
The contact angle as well as the surface free energy of the materials' surfaces weredeterminedat room temperature by using a Kruss Goniometer G10 (KRÜSS GmbH, Hamburg, Germany) with drop shape analysis software. The water contact angle of the 10 samples was evaluated by static contact angle measurements using the sessile drop method. Surface free energy was calculated by using measurements performed according to the Acid-Base method (AB) [24] by static contact angle studied with the use of three liquids: Water, ethylene glycol, and formamide.
Mechanical Properties
Tensile strength (T SB ) and elongation at break (ε b ) were studied using the universal testing machine Zwick & Roell Z020 (Zwick Roell Polska Sp. z o.o. Sp.K., Wrocław, Poland) according to PN-EN ISO 527-2:2012 with a crosshead sped of 500 mm/min. Samples of sixwere used for this study.
Hardness was measured by using the Shore method according to PN-EN ISO 868:2004. Obtained data were presented with Shore degree ( • Sh D and • Sh A). Samples of 10 were used for this study.
Short-Term Interactions Study Performed in Selected Environments
PURs were cut into round samples of 0.5 cm 2 area. Prepared samples were dried and weighed in a thermobalance (Radwag, Radom, Poland) (RADWAG MAX50/SX) set at 60 • C. Then, 6 samples of each studied PUR materials were placed in a 24-well cell culture plate filled with selected media: Oxidative solution of 0.1 M CoCl 2 /20% H 2 O 2 ; acidic solution of 2 N HCl or basic solution of 5 M NaOH. Samples were incubated in the selected media at 37 • C. The mass change of the samples was examined after 15 days for oxidative, acidic, and basic media. Samples mass change measurement was as follows: Samples were taken out from the container and put into a paper sheet to reduce the medium excess. Then, samples were placed in the thermobalance (set at 60 • C) where they were weighed to constant mass. The results are the arithmetic mean of six measurements.
The changes at the PUR and PUR-M surface were monitored by optical microscopy (OM) performed with the use of a Bresser microscope(Bresser GmbH, Rhede, Germany) at a magnification of 20×.
In Vitro Cytocompatibility
The cytotoxicity assay was performed by using selected PUR and PUR-M samples. To examine the cytotoxicity of the obtained materials, the extract was prepared and tested with the use of a C2C12 cell line according to ISO 10993-5:2009 standard. In order to obtain extract the sterile PUR or PUR-M samples were incubated at the ratio 1:100 (w/v) in cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), L-glutamine (1% solution in medium) (Gibco), 1% antibiotic-antimycotic mixture for 24 h at 37 • C under continuous steering.
Cell Viability Assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for assessing cell metabolic activity. C2C12 cells were split into 24-well plates at 30,000 cells per well and grown for 24 h in 500 µL of culture medium. Cells were incubated for 72 h with the matrices' extracts. Then, the MTT assay of viable cells was used in accordance with the manufacturer's recommendations (Sigma Aldrich, St. Louis, MO, USA). The reaction product was quantitatively determined by an Absorbance Reader BioTek EL*800 (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 570 nm. The viability of the untreated cells was counted as 100.
Calcification Study
Golomb and Wagner's Compound was used to perform the calcification study. The calcification metastable solution consisted of 3.87 millimole (mM) CaCl 2 , 2.32 mM K 2 HPO 4 , yielding a ratio of calcium to phosphate (Ca/PO 4 ) = 1.67, and 0.05 M Tris Buffer (in this study C 4 H 11 NO 3 ) dissolved in one mL of reverse osmosis (RO) water [25]. PUR and PUR-M samples were cut into round samples of 0.5 cm 2 area. Prepared samples were dried and weighed in a thermobalance (RADWAG MAX50/SX) set at 60 • C. Then, 6 samples of each studied PUR materials were placed in a 24-well cell culture plate filled with Golomb and Wagner's Compound. The progress of the calcification was studied by SEM and EDX after 21 days.
Scaffold Fabrication
PUR or PUR-M was dissolved in 1,4-dioxane (POCH, Gliwice, Poland) at a concentration of 20% w/v. Then, sodium chloride, of crystal size in the range of 0.6-0.4 µm, was added to the polyurethane solution until complete saturation of the solution occurred. Formulated PUR (or PUR-M)-salt saturated solution was transferred into the stainless steel mold of the size 2.5 cm × 2.5 cm × 2.5 cm and placed at −20 • C for 24 h to direct the solvent crystallization and to fabricate scaffolds of local anisotropy where the porosity of the scaffolds was of controlled pore size and porosity [26][27][28][29]. Then scaffolds were removed from the mold and immersed in warm (40-50 • C) bidistilled water, where for 7 days the sodium chloride crystals were washed out. The water was changed twice a day. Finally, the samples were dried at 60 • C for 24 h.
Results and Discussion
The impact of the prepolymer modification time on the decrease of the free isocyanate groups in it is outlined below. Table 1 presents the influence of the prepolymer modification time on the decrease of the free isocyanate groups present in it. Table 1 shows that the prepolymerization reaction takes place in a similar way for both PUR and PUR-M materials until the 4th hour of the synthesis, when the GPCa modifier was added to the system. Incorporation of the GPCa modifier into the system caused a slightly higher F NCO decrease for PUR-M in comparison to PUR after 1 h of modification (8.54 ± 0.02% and 8.43 ± 0.03% respectively). After 5 h of prepolymer modification with GPCa the F NCO reached the previously established 8 wt % and the reaction was recognized as finished. It can be concluded that the NCO groups of diisocyanate react with OH groups present in the GPCa modifier at the adjusted modification conditions. It suggests that GPCa was incorporated into the PUR structure through covalent bonds. Analysis of FTIR spectra ( Figure 2) revealed the presence of functional groups characteristic for PURs composition; i.e., urethane linkages (see Table 2). Thus, conditions designed to carry out PURs synthesis were suitable and provided PUR product. The expanded base of the NH stretching band (3392 and 3326 cm −1 ) for PURs suggested the presence of "free" and hydrogen bonded HS in the obtained material respectively [30,31]. In the case of PUR-M the expanded base of the NH stretching band was observed as well, but its intensity relating to the "free" NH (3389 cm −1 ) in the PUR-M composition was decreased. Conversely to the PURs the PUR-M revealed the well-shaped NH stretching band related to the moderate and strong hydrogen bonds present in the HS of PUR chains [32,33]. The observed C=O stretching band confirmed the presence of ester and urethane linkages, which were "free" (1728 cm −1 ) and hydrogen bonded (1680 cm −1 ) for both PUR and PUR-M [32,33]. It can be pointed out here that the PUR-M appears to have a large number of hydrogen bonded urethane linkages in comparison to PURs. Performed FTIR analysis confirmed the presence of urethane linkages. Furthermore, the FTIR spectra of PUR-M showed that GPCa modification represents a higher level of hydrogen bonds between HS in PUR chains. Some differences in band intensities (1462-945 cm −1 ) between PURs and PURs-M were observed which might be related as well to the presence of GPCa molecules in the PUR system [34]. Analysis of FTIR spectra ( Figure 2) revealed the presence of functional groups characteristic for PURs composition; i.e., urethane linkages (see Table 2). Thus, conditions designed to carry out PURs synthesis were suitable and provided PUR product. The expanded base of the NH stretching band (3392 and 3326 cm −1 ) for PURs suggested the presence of "free" and hydrogen bonded HS in the obtained material respectively [30,31]. In the case of PUR-M the expanded base of the NH stretching band was observed as well, but its intensity relating to the "free" NH (3389 cm −1 ) in the PUR-M composition was decreased. Conversely to the PURs the PUR-M revealed the well-shaped NH stretching band related to the moderate and strong hydrogen bonds present in the HS of PUR chains [32,33]. The observed C=O stretching band confirmed the presence of ester and urethane linkages, which were "free" (1728 cm −1 ) and hydrogen bonded (1680 cm −1 ) for both PUR and PUR-M [32,33]. It can be pointed out here that the PUR-M appears to have a large number of hydrogen bonded urethane linkages in comparison to PURs. Performed FTIR analysis confirmed the presence of urethane linkages. Furthermore, the FTIR spectra of PUR-M showed that GPCa modification represents a higher level of hydrogen bonds between HS in PUR chains. Some differences in band intensities (1462-945 cm −1 ) between PURs and PURs-M were observed which might be related as well to the presence of GPCa molecules in the PUR system [34]. Table 2. Spectral data and band assignments of FTIR analysis presented in Figure 2.
Fourier Transform Infrared Spectroscopy (FTIR)
Raman Spectroscopy
In Figure 3 the Raman spectra of obtained PUR and PUR-M are presented, of which the band assignments are given in Tables 3 and 4. δCH2, δNH δOH out of the plane deformation of CH2 and CH3 groups as well as NH and OH groups
Raman Spectroscopy
In Figure 3 the Raman spectra of obtained PUR and PUR-M are presented, of which the band assignments are given in Tables 3 and 4. The performed Raman spectroscopy results were complimentary to the FTIR study (Tables 4 and 5). Thus, it confirmed the presence of strongly hydrogen bonded urethane groups in the PUR-M structure as well as introduction of the GPCa modifier into the main chain of PUR at least at one-side. Figure 4 shows the SEM images of the GPCa modifier and the obtained PUR and PUR-M surface. Figure 4a shows an image of GPCa used as a modifier. The particles resemble a spherical shape and their sizes are in the range of 6.754-7.952 µm. Figure 4b shows the image of the PURs surface, which is rather homogeneous and without visible inclusions. In Figure 4c it can be seen that the surface of PUR-M modified with GPCa is homogenous over 80% with little inclusions visible at the surface top. These inclusions can be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix. It suggests that a large amount of used GPCa modifier was incorporated into the PUR structure by covalent bonding, which is consistent with FTIR and Raman spectroscopy. and their sizes are in the range of 6.754-7.952 μm. Figure 4b shows the image of the PURs surface, which is rather homogeneous and without visible inclusions. In Figure 4c it can be seen that the surface of PUR-M modified with GPCa is homogenous over 80% with little inclusions visible at the surface top. These inclusions can be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix. It suggests that a large amount of used GPCa modifier was incorporated into the PUR structure by covalent bonding, which is consistent with FTIR and Raman spectroscopy.
Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy(SEM/EDX)
Contact Angle
In Table 5 are gathered the values of the contact angles and total surface free energy studied for the obtained PUR and PUR-M.
The obtained PURs and PURs-M had water contact angles in the range from 72° (PUR) to 57° (PU-M) ( Table 5). The contact angles studied in formamide and ethylene glycol were slightly lower in comparison to the values of the water contact angle. Addition of GPCa caused a decrease of the contact angle independently of the solvent used in the study. The total surface free energy is higher for PURs-M (59 mN/m) than for PURs (32 mN/m). This trend was observed independently of the method used for its examination (Table 5).
Mechanical Properties
The TSb, εb and hardness of the obtained PUR and PUR-M are shown in the Figure 5.
The TSb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of εb the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 °ShD for PURs and 35 ± 2 °ShD for PURs-M).
Contact Angle
In Table 5 are gathered the values of the contact angles and total surface free energy studied for the obtained PUR and PUR-M.
The obtained PURs and PURs-M had water contact angles in the range from 72 • (PUR) to 57 • (PU-M) ( Table 5). The contact angles studied in formamide and ethylene glycol were slightly lower in comparison to the values of the water contact angle. Addition of GPCa caused a decrease of the contact angle independently of the solvent used in the study. The total surface free energy is higher for PURs-M (59 mN/m) than for PURs (32 mN/m). This trend was observed independently of the method used for its examination (Table 5).
Mechanical Properties
The T Sb , ε b and hardness of the obtained PUR and PUR-M are shown in the Figure 5.
The T Sb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of ε b the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 • ShD for PURs and 35 ± 2 • ShD for PURs-M).
Mechanical Properties
The TSb, εb and hardness of the obtained PUR and PUR-M are shown in the Figure 5. The TSb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of εb the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 °ShD for PURs and 35 ± 2 °ShD for PURs-M). Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.
In Vitro Cytocompatibility
Short-Term Interactions Study Performed in Selected Environments
Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) caused the increase of degradation rate of about 4% in comparison to PURs. In the case of acidic environment the mass loss was over 30% for both PURs and PURs-M, and this mass loss was higher Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.
In Vitro Cytocompatibility
In Vitro Cytocompatibility
Short-Term Interactions Study Performed in Selected Environments
Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.
Short-Term Interactions Study Performed in Selected Environments
Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) caused the increase of degradation rate of about 4% in comparison to PURs. In the case of acidic environment the mass loss was over 30% for both PURs and PURs-M, and this mass loss was higher by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of short-term interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.
Polymers 2017, 9,329 12 of 21 by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of shortterm interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.
Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment. by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of shortterm interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.
Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment.
Calcification Study
SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress. Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment.
Calcification Study
SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9.
Calcification Study
SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress.
Fabrication of PUR and PURs-M Scaffolds
Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds.
Polymers 2017, 9,329 16 of 21
Fabrication of PUR and PURs-M Scaffolds
Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds. Obtained PUR had pore sizes in the range of 36-100 μm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 μm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay. Obtained PUR had pore sizes in the range of 36-100 µm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 µm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay.
Fabrication of PUR and PURs-M Scaffolds
Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds. Obtained PUR had pore sizes in the range of 36-100 μm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 μm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay. The main conclusion coming from the analysis of Figure 13 is the fact that the obtained PURs-M possesses better biocompatibility than PURs. Thus, it confirms the beneficial effects of the employed GPCa modifier. The proliferation of cells of the PUR-M extracts was observed at concentrations between 25-75%. PURs had lower cells viability in comparison to the PURs-M. In the case of undiluted extracts (100%) the cells viability was comparable for both PUR and PUR-M scaffolds.
Discussion
Bone tissue engineering is a demanding field of strictly described requirements of biomaterials, which may be used for bone tissue scaffold fabrication. Accordingly of the many biomaterials used in this field PU seems to be the most suitable candidate. This is due to its ease of modification to attain a bioactive material as well as its suitable mechanical property design related to the raw materials selection for its synthesis [14,[44][45][46][47].
In this paper we described the synthesis of PEEURs carried out with the use of selected raw materials such as aliphatic HDI, polyester (Polios,) and polyether (PEG) polyols, with BDO chain extender to reach the requirements of biocompatible biomaterials for medical applications. The GPCa modifier was selected according to the literature, which describes it as a compound that can improve the bioactivity of the material as well as stimulating bone tissue regeneration. The successful synthesis of PURs was confirmed by FTIR and Raman spectroscopy, which revealed the formation of urethane bonds. Application of GPCa modifier improved hydrogen-bond formation in the PURs-M structure compared to the PURs (see FTIR analysis). Spectroscopic studies and F NCO determination confirmed the fact that GPCa is partially covalently bonded with the PUR chain. This is possible due to the hydroxyl groups present in the GPCa chemical structure. The SEM image of the PUR-M surface was in good agreement with the FTIR analysis due to the fact that it revealed the presence of a homogenous surface (about 80%) of this material, with only little inclusions visible at the top. The presence of these inclusions could be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix in the form of the filler. This filling effect of GPCa occurring was beneficial in the case of the biocompatibility and calcification study and may be useful as bioactive stimulant to improve bone tissue regeneration. The contact angle was decreased for the PURs-M (57 • ) in comparison to the PURs (72 • ). Thus, the addition of GPCa improved in a superior way the hydrophilic characteristic of the obtained materials, which may be beneficial for cell growth. According to Guelcher et al. the contact angle of the polymer surface in the range of 45-76 • supports the attachment of mammalian cells, which can have a beneficial influence on the absorption of albumin and a preservative coupler of live tissues [48]. The total surface free energy (59 mN/m for PURs-M and 32 mN/m for PURs respectively) was in good agreement with the contact angle study and is suitable for cell growth only for PURs-M according to the references [11,49,50]. In further studies the total surface free energy can be increased by increased GPCa addition into the materials. Most researchers have come to the conclusion that rather a surface charge (either positive, or negative) is the most important, not the surface free energy [49]. According to this, a modification of a hydrophilic but not-charged polyurethane surface with bipolar calcium β-glycerophosphate is a good option for the optimization of cellular adherence at the PU surface. The mechanical properties of the obtained PURs (T Sb = 12 MPa, ε b = 390 ± 13%) and PURs-M (T Sb = 18 MPa, ε b = 280 ± 15%) were more suitable for bone tissue engineering in the case of PUR-M. The materials used in bone regeneration must have a T Sb in the range of 1.5-38 MPa for human cancellous bone regeneration or 35-283 MPa for human cortical bone regeneration [51]. Thus, the obtained PURs and PURs-M may find an application as biomaterials for scaffold fabrication of human cancellous bone. The hardness was comparable for both PURs and PURs-M (31 ± 3 • ShD for PURs and 35 ± 2 • ShD for PURs-M). The short term interactions study showed that both PURs and PURs-M are sensitive to basic and acidic environment, while in an oxidative one they remain stable. Thus, this is consistent with references [52]. The observed superior mass decrease, in the case of basic (over 50%) and acidic (over 30%) environment, after 15 days of the short-term interactions study may be a sign of the first step of material degradation called defragmentation [53]. This was confirmed by the optical microscopy studies, which showed defragmentation of the obtained PURs and more favorably PURs-M. The SEM and EDX verification of the calcification study showed significant improvement in the progress of calcification, the process needed in the case of bone regeneration [11,16,35,54], for PURs-M. Thus, the GPCa modification is a superior factor, which improves calcification, which was indicated by the peaks of Ca present in the EDX spectra. After the calcification study the calcium amount at the materials' surfaces increased over 50% for PURs and 110% on PURs-M. According to the satisfactory physicochemical, mechanical, and biological characteristics of the obtained PURs and PURs-M the fabrication of the scaffolds was performed by using the SC/PL technique combined with TIPS. Obtained PUR scaffolds had pore sizes in the range of 36-100 µm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had a pore size in the range of 50-100 µm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references, for using such scaffolds in bone tissue engineering applications. Forthermore, in the case of PURs-M scaffolds particles of GCPa modifier were visible, which were not washed out during the scaffold fabrication process. Thus, according to the results of the MTT assay, it can be concluded that PURs-M may be a suitable candidate for bone tisue engineering applications [43].
Conclusions
In this paper we reported the synthesis and characteristic of PEEURs, obtained by using raw materials (HDI, Polios, PEG, and BDO), selected to reach high biocompatibility of the materials. Moreover, we successfully modified the PEEUR chains by incorporating into them GPCa modifier, confirmed by various techniques. The stronger hydrogen bonding, lower contact angle, and higher total surface free energy as well as the more suitable mechanical properties and good biocompatibility of PURs-M let to the conclusion that these materials possess satisfactory characteristics of materials dedicated to bone tissue engineering. Further studies of calcification of these materials indicated the superior effect of GPCa on these processes. Moreover, the short term interactions study revealed that the obtained PUR-M materials undergo gradual degradation in selected basic and acidic environment by chain defragmentation and such degradable materials are being widely developed for tissue scaffolds. The obtained porous scaffolds, by using the SC/PL technique combined with TIPS, represented suitable pore sizes (36-100 µm) and porosity (77-82%) to serve as templates for bone tissue regeneration. Moreover, the biocompatibility of PURs-M scaffolds was superior in comparison to PURs. Thus, further studies in this direction will be developed in our team.
Figure 1 .Figure 1 .
11ContSynthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).
2. 2 . 4 .
24Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40(Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.2.2.5. Static Contact Angle Determination
Figure 1 .
1Synthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).
2. 2 . 4 .
24Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40 (Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.
Figure 2 Figure 2 .
22presents the FTIR spectra of PUR and PUR-M. FTIR spectra of PUR (black) and PUR-M (red).
1238s 1216s νC-(C=O)-O stretching vibrations of -C-(C=O)-O-of ester group, not hydrogen bonded
Figure 2 .
2FTIR spectra of PUR (black) and PUR-M (red).
stretching of NH groups, hydrogen bonded withthe C=O of the ester group present in macrodiol and in aliphatic asymmetric and symmetric CH 2 groups present in the PUR chain and in GCPa modifier 1728m 1728s νC=O stretching of C=O in the ester and urethane groups, which were not bonded
Figure 3 .
3Raman spectra of PUR and PUR-M.
Figure 3 .
3Raman spectra of PUR and PUR-M.
of CH 2 groups in different positions and deforming bending vibrations outside of the plane of N-H groups 610 deforming vibrations outside of the plane of ester groups and their fluctuations * [35-38].
vibrations of N-H in urethane groups (as for II amides), P-O-Ca stretch 2925, 2883 the strongest polarized stretching vibrations of asymmetric and symmetric CH 2 groups present in PUR chains. Analogic CH 2 in H 2 C-O-P-O stretch phonons included 1733 stretching vibrations of carbonyl groups present in macrodiol Polios 55/20 1684 stretching vibrations of carbonyl groups C=O in urethane (HDI-BDO) 1483, 1452, 1442, 1421 deforming and scissoring vibrations of CH 2 groups in both polymer and GPCa 1302, 1261 swinging and bending vibrations of CH 2 out of the plane groups in polymer and filler with additional C-N stretch at the end 1126, 1131 stretching asymmetric vibrations of C-O-C in ester groups 1040,1080 complex CCCC stretch in branched alkanes with symmetric PO 4 and C-O-P stretch 959, 939, 882, 835 swinging vibrations of CH 2 groups in different positions and NH, CH bending deformation vibrations outside of the plane of urethane groups 580,614 deforming vibrations outside of the plane of ester groups with eventual phosphoester and PO 4 fluctuations * [39-41].
Figure 4 .
4SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).
Figure 4 .
4SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).
Figure 5 .
5The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.
Figure 6
6shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.
Figure 6 .
6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Figure 5 .
5The T Sb (a), ε b (b), and hardness (c) of obtained PUR and PUR-M.
Figure 6 Figure 5 .
65shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility. The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.
Figure 6
6shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations.Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.
Figure 6 .
6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Figure 6 .
6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Figure 7 .Figure 7 .
77Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).
Figure 7 .Figure 8 .Figure 8 .
788Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). ContOptical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).
Figure 9 .
9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.
Figure 8 .
8Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).
Figure 8 .
8Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).
Figure 9 .
9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.
Figure 9 .
9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.
Figure 10 .
10The EDX of PURs (a) and PURs-M (b) before calcification study.
Figure 10 .
10The EDX of PURs (a) and PURs-M (b) before calcification study.
Figure 11 .
11The EDX of PURs (a) and PURs-M (b) after calcification study.
Figure 11 .
11The EDX of PURs (a) and PURs-M (b) after calcification study.
Figure 12 .
12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.
Figure 13 .
13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Figure 12 .
12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.
Figure 12 .
12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.
Figure 13 .
13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Figure 13 .
13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).
Table 1 .
1The impact of the prepolymer modification time on the decrease of free isocyanate groups present in it.Time of
prepolymeryzation (h)
Content of the free isocyanate groups
in the unmodified prepolymer (NCO )
Content of the free isocyanate groups
in the modified prepolymer (NCO )
PEEUR
PEEUR-M
0
10.14 ± 0.03
10.14 ± 0.03
1
9.04 ± 0.01
9.04 ± 0.01
2
8.83 ± 0.03
8.83 ± 0.03
3
8.54 ± 0.03
8.54 ± 0.03
4
8.54 ± 0.02
8.54 ± 0.02
5
8.54 ± 0.02
8.43 ± 0.01 *
6
8.53 ± 0.03
8.33 ± 0.03
7
8.53 ± 0.01
8.14 ± 0.03
8
8.53 ± 0.02
8.12 ± 0.02
9
8.53 ± 0.01
8.12 ± 0.03
* addition of the GPCa modifier.
Table 2 .
2Spectral data and band assignments of FTIR analysis presented inFigure 2.PUR
PUR-M
Band
Description
Wavelength (cm −1 )
3392
3326w
3380
3318w
νNH
stretching of NH groups, hydrogen bonded withthe C=O of the
ester group present in macrodiol and in GCPa modifier
2942m
2868m
2942m
2868m
νCH2
stretching of aliphatic asymmetric and symmetric CH2 groups
present in the PUR chain and in GCPa modifier
1728m
1728s
νC=O
stretching of C=O in the ester and urethane groups, which were
not bonded
1680m
1680s
νC=O
stretching of C=O groups which formed hydrogen bonds
1535m
1535s
νC-N
stretching between CN in urethane group
1462w
1416w
1350w
1459m
1415m
1347m
δCH2
deformation vibrations of aliphatic CH2 groups present in the
PUR and GPCa modifier: bending, wagging, scissoring
in plane
1260w
1214m
Table 2 .
2Cont.1680m
1680s
νC=O
stretching of C=O groups which formed hydrogen bonds
1535m
1535s
νC-N
stretching between CN in urethane group
1462w
1416w
1350w
1459m
1415m
1347m
δCH 2
deformation vibrations of aliphatic CH 2 groups present in the
PUR and GPCa modifier: bending, wagging, scissoring
in plane
1260w
1214m
1238s
1216s
νC-(C=O)-O
stretching vibrations of -C-(C=O)-O-of ester group, not
hydrogen bonded
1170m
1170s
νNH-(C=O)-O
stretching vibrations of -NH-(C=O)-O-of urethane group
1135m
1080m
1058m
947w
1134s
1061s
993s
949s
νC-(C=O)-O
νC-O
stretching vibration of hydrogen bonded -C-(C=O)-O-,
868w
813w
775w
731w
636w
867s
777s
730s
638s
δCH 2 ,
δNH
δOH
out of the plane deformation of CH 2 and CH 3 groups as well
as NH and OH groups
Table 3 .
3Raman spectra band assignments for obtained PURs.Wavelength (cm −1 )
Assignments *
3328
stretching vibrations of N-H in urethane groups (as for II-ary amides)
2925, 2890
the strongest polarized stretching vibrations of asymmetric and symmetric CH2
groups present in PUR chains.
1735, 1685
stretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and
urethane groups (as for II-ary amides) respectively
1480, 1450,
1443, 1424
strong planar deforming vibrations (scissoring) of CH2 groups
Table 3 .
3Raman spectra band assignments for obtained PURs.Wavelength (cm −1 )
Assignments *
3328
stretching vibrations of N-H in urethane groups (as for II-ary amides)
2925, 2890
the strongest polarized stretching vibrations of asymmetric and symmetric CH 2
groups present in PUR chains.
1735, 1685
stretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and
urethane groups (as for II-ary amides) respectively
1480, 1450,
1443, 1424
strong planar deforming vibrations (scissoring) of CH 2 groups
Table 4 .
4Raman spectra band assignments for GPCa modified PURs.
Table 5 .
5Contact angle and total surface free energy of obtained PURs and PURs-M.Symbol
Contact angle (°)
Surface energy (mN/m)
Formamide
Ethylene glycol
Water
Acid-part
Base-part
Total surface
free energy
PUR
63.6 ± 2
68.2 ± 1
72.1 ± 2
0.04
20.83
31.82
PUR-M
35.8 ± 4
47.5 ± 3
57 ± 3
14.51
21.72
59.09
Table 5 .
5Contact angle and total surface free energy of obtained PURs and PURs-M.Symbol
Contact angle ( • )
Surface energy (mN/m)
Formamide
Ethylene glycol
Water
Acid-part
Base-part
Total surface
free energy
PUR
63.6 ± 2
68.2 ± 1
72.1 ± 2
0.04
20.83
31.82
PUR-M
35.8 ± 4
47.5 ± 3
57 ± 3
14.51
21.72
59.09
Table 6 .
6The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment.Sample
Extracted mass (%)
5 M NaOH
2 N HCl
0.1 M CoCl 2 /20% H 2 O 2
PUR
50.9 ± 0.2
38.62 ± 0.14
4.25 ± 0.08
PUR -M
54.7 ± 0.1
35.81 ± 0.11
2.03 ± 0.12
Table 6 .
6Themass loss of the PURs and PURs-M after 15 days of short-term interactions study
performed with selected media of the acidic, basic, and oxidative environment.
Sample
Extracted mass (%)
5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2
PUR
50.9 ± 0.2 38.62 ± 0.14
4.25 ± 0.08
PUR -M
54.7 ± 0.1 35.81 ± 0.11
2.03 ± 0.12
Table 6 .
6The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment.Sample
Extracted mass (%)
5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2
PUR
50.9 ± 0.2 38.62 ± 0.14
4.25 ± 0.08
PUR -M
54.7 ± 0.1 35.81 ± 0.11
2.03 ± 0.12
Polymers 2017,9, 329
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Conflicts of Interest:The authors declare no conflict of interest.
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| [
"In this paper we describe the synthesis of poly(ester ether urethane)s (PEEURs) by using selected raw materials to reach a biocompatible polyurethane (PU) for biomedical applications. PEEURs were synthesized by using aliphatic 1,6-hexamethylene diisocyanate (HDI), poly(ethylene glycol) (PEG), α,ω-dihydroxy(ethylene-butylene adipate) (Polios), 1,4-butanediol (BDO) as a chain extender and calcium glycerolphosphate salt (GPCa) as a modifier used to stimulate bone tissue regeneration. The obtained unmodified (PURs) and modified with GPCa (PURs-M) PEEURs were studied by various techniques. It was confirmed that urethane prepolymer reacts with GPCa modifier. Further analysis of the obtained PURs and PURs-M by Fourier transform infrared (FTIR) and Raman spectroscopy revealed the chemical composition typical for PUs by the confirmed presence of urethane bonds. Moreover, the FTIR and Raman spectra indicated that GPCa was incorporated into the main PU chain at least at one-side. The scanning electron microscopy (SEM) analysis of the PURs-M surface was in good agreement with the FTIR and Raman analysis due to the fact that inclusions were observed only at 20% of its surface, which were related to the non-reacted GPCa enclosed in the PUR matrix as filler. Further studies of hydrophilicity, mechanical properties, biocompatibility, short term-interactions, and calcification study lead to the final conclusion that the obtained PURs-M may by suitable candidate material for further scaffold fabrication. Scaffolds were prepared by the solvent casting/particulate leaching technique (SC/PL) combined with thermally-induced phase separation (TIPS). Such porous scaffolds had satisfactory pore sizes (36-100 µm) and porosity (77-82%) so as to be considered as suitable templates for bone tissue regeneration."
] | [
"Justyna Kucińska-Lipka \nDepartment of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland\n",
"Iga Gubanska [email protected]. \nDepartment of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland\n",
"Olexandr Korchynskyi \nInstitute of Cell Biology\nNational Academy Science of Ukraine\n14/16 Drahomanov Str79005LvivUkraine\n\nCentre for Innovative Research in Medical and Natural Sciences, Rzeszow University and Medical Faculty\n35-959RzeszowPoland\n",
"Khrystyna Malysheva [email protected]. \nInstitute of Cell Biology\nNational Academy Science of Ukraine\n14/16 Drahomanov Str79005LvivUkraine\n",
"Marcin Kostrzewa [email protected] \nDepartment of Organic Materials Technology\nTechnical University of Radom\n26-600RadomPoland\n",
"Damian Włodarczyk \nInstitute of Physics, Polish Academy of Science\nDivision of Physics and Technology of Wide-Band-Gap Semiconductor Nanostructures\nAl. Lotnikow 32/4602-668WarsawPoland\n",
"Jakub Karczewski \nFaculty of Applied Physics and Mathematics\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland\n",
"Helena Janik [email protected]. \nDepartment of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland\n"
] | [
"Department of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland",
"Department of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland",
"Institute of Cell Biology\nNational Academy Science of Ukraine\n14/16 Drahomanov Str79005LvivUkraine",
"Centre for Innovative Research in Medical and Natural Sciences, Rzeszow University and Medical Faculty\n35-959RzeszowPoland",
"Institute of Cell Biology\nNational Academy Science of Ukraine\n14/16 Drahomanov Str79005LvivUkraine",
"Department of Organic Materials Technology\nTechnical University of Radom\n26-600RadomPoland",
"Institute of Physics, Polish Academy of Science\nDivision of Physics and Technology of Wide-Band-Gap Semiconductor Nanostructures\nAl. Lotnikow 32/4602-668WarsawPoland",
"Faculty of Applied Physics and Mathematics\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland",
"Department of Polymer Technology\nFaculty of Chemistry\nGdansk University of Technology\nNarutowicza St. 11/1280-233GdanskPoland"
] | [
"Justyna",
"Iga",
"Olexandr",
"Khrystyna",
"Marcin",
"Damian",
"Jakub",
"Helena"
] | [
"Kucińska-Lipka",
"Gubanska",
"Korchynskyi",
"Malysheva",
"Kostrzewa",
"Włodarczyk",
"Karczewski",
"Janik"
] | [
"J Kucinska-Lipka, ",
"I Gubanska, ",
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"F C Sampaio, ",
"A Lussi, ",
"T E L Douglas, ",
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"I Senderek, ",
"A Skwarczyńska, ",
"V M J I Cuijpers, ",
"Z Modrzejewska, ",
"M Lewandowska-Szumieł, ",
"P Dubruel, ",
"T E Kavanaugh, ",
"A Y Clark, ",
"L H Chan-Chan, ",
"M Ramírez-Saldaña, ",
"R F Vargas-Coronado, ",
"J M Cervantes-Uc, ",
"P Alves, ",
"J F J Coelho, ",
"J Haack, ",
"A Rota, ",
"A Bruinink, ",
"M H Gil, ",
"P Boloori Zadeh, ",
"S C Corbett, ",
"H Nayeb-Hashemi, ",
"A Silvestri, ",
"M Boffito, ",
"S Sartori, ",
"G Ciardelli, ",
"T Courtney, ",
"M S Sacks, ",
"J Stankus, ",
"J Guan, ",
"W R Wagner, ",
"A Karchin, ",
"F I Simonovsky, ",
"B D Ratner, ",
"J E Sanders, ",
"T Matsuda, ",
"M Ihara, ",
"H Inoguchi, ",
"I K Kwon, ",
"K Takamizawa, ",
"S Kidoaki, ",
"M M Coleman, ",
"K H Lee, ",
"D J Skrovanek, ",
"P C Painter, ",
"M M Coleman, ",
"D J Skrovanek, ",
"J Hu, ",
"P C Painter, ",
"M Spirkova, ",
"R Poręba, ",
"J Pavlicevic, ",
"L Kobera, ",
"J Baldrian, ",
"M Pakarek, ",
"I Yilgor, ",
"E Yilgor, ",
"I G Guler, ",
"T C Ward, ",
"G L Wilkes, ",
"Yohannan Panicker, ",
"C Tresa Varghese, ",
"H Philip, ",
"D Ft-Ir, ",
"Sers Ft-Raman, ",
"C Spectra Of Vitamin, ",
"A Asefnejad, ",
"M T Khorasani, ",
"A Behnamghader, ",
"B Farsadzadeh, ",
"S Bonakdar, ",
"J Xu, ",
"T Wu, ",
"C Peng, ",
"S Adegbite, ",
"S L Cooke, ",
"A R Whittington, ",
"M Strankowski, ",
"D Włodarczyk, ",
"Ł Piszczyk, ",
"J Strankowska, ",
"J E Gough, ",
"I Notingher, ",
"L L Hench, ",
"I Notingher, ",
"J E Gough, ",
"L L Hench, ",
"C Silve, ",
"E Lopez, ",
"B Vidal, ",
"D C Smith, ",
"S Camprasse, ",
"G Camprasse, ",
"G Couly, ",
"B R Barrioni, ",
"S M De Carvalho, ",
"R L Oréfice, ",
"A A R De Oliveira, ",
"M D M Pereira, ",
"S Gogolewski, ",
"K Gorna, ",
"A S Turner, ",
"A R Amini, ",
"C T Laurencin, ",
"S P Nukavarapu, ",
"J Henkel, ",
"M A Woodruff, ",
"D R Epari, ",
"R Steck, ",
"V Glatt, ",
"I C Dickinson, ",
"P F Choong, ",
"M A Schuetz, ",
"D W Hutmacher, ",
"X Yu, ",
"X Tang, ",
"S V Gohil, ",
"C T Laurencin, ",
"M Bil, ",
"J Ryszkowska, ",
"P Woźniak, ",
"K J Kurzydłowski, ",
"M Lewandowska-Szumieł, ",
"S A Guelcher, ",
"A Srinivasan, ",
"J E Dumas, ",
"J E Didier, ",
"S Mcbride, ",
"J O Hollinger, ",
"M K Chaudhury, ",
"P Król, ",
"B Król, ",
"Z Sheikh, ",
"S Najeeb, ",
"Z Khurshid, ",
"V Verma, ",
"H Rashid, ",
"M Glogauer, ",
"S H Teoh, ",
"Z G Tang, ",
"G W Hastings, ",
"W Murphy, ",
"J Black, ",
"G Hastings, ",
"F Montini-Ballarin, ",
"P C Caracciolo, ",
"G Rivero, ",
"G A Abraham, ",
"S Gogolewski, "
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"Synthesis and characterization of cycloaliphatic hydrophilic polyurethanes, modified with L-ascorbic acid, as materials for soft tissue regeneration",
"Fabrication of polyurethane and polyurethane based composite fibres by the electrospinning technique for soft tissue engineering of cardiovascular system",
"Surface characteristics of titanium anodized in the four different types of electrolyte",
"Cytocompatibility and osteogenesis evaluation of HA/GCPU composite as scaffolds for bone tissue engineering",
"Biodegradable polyurethane composite scaffolds containing Bioglass?? for bone tissue engineering",
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"A review: Fabrication of porous polyurethane scaffolds",
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"Electrospun polyurethane/hydroxyapatite bioactive Scaffolds for bone tissue engineering: The role of solvent and hydroxyapatite particles",
"Degradation and biocompatibility of porous nano-hydroxyapatite/polyurethane composite scaffold for bone tissue engineering",
"In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering",
"Synthesis of two-component injectable polyurethanes for bone tissue engineering",
"Biodegradable injectable polyurethanes: Synthesis and evaluation for orthopaedic applications",
"Preparation, degradation, and calcification of biodegradable polyurethane foams for bone graft substitutes",
"Polyurethane foams electrophoretically coated with carbon nanotubes for tissue engineering scaffolds",
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"Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy",
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"Mechano-active scaffold design of small-diameter artificial graft made of electrospun segmented polyurethane fabrics",
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"Manufacturing of biodegradable polyurethane scaffolds based on polycaprolactone using a phase separation method: Physical properties and in vitro assay",
"Influence of acid and alkali pre-treatments on thermal degradation behaviour of polyisocyanurate foam and its carbon morphology",
"Influence of therapeutic radiation on polycaprolactone and polyurethane biomaterials",
"Polyurethane Nanocomposites Containing Reduced Graphene Oxide, FTIR, Raman, and XRD Studies",
"Osteoblast attachment and mineralized nodule formation on rough and smooth 45S5 bioactive glass monoliths",
"Study of osteoblasts mineralisation in-vitro by Raman micro-spectroscopy",
"Nacre initiates biomineralization by human osteoblasts maintained In Vitro",
"Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications",
"Regeneration of bicortical defects in the iliac crest of estrogen-deficient sheep, using new biodegradable polyurethane bone graft substitutes",
"Bone Tissue Engineering: Recent Advances and Challenges",
"Bone Regeneration Based on Tissue Engineering Conceptions-A 21st Century Perspective",
"Biomaterials for Bone Regenerative Engineering",
"Optimization of the structure of polyurethanes for bone tissue engineering applications",
"Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates",
"Interfacial interaction between low energy surfaces",
"Surface free energy of polyurethane coatings with improved hydrophobicity",
"Biodegradable materials for bone repair and tissue engineering applications",
"Chapter 3 Thermoplastic Polymers In Biomedical Applications: Structures, Properties and Processing",
"In vitro degradation of electrospun poly(l-lactic acid)/segmented poly(ester urethane) blends",
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"Acta Biomater",
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"Biomed. Mater",
"Polish Online System of Legal Acts",
"Available online: www.fda.gov (accessed on",
"J. Dent",
"Clin. Oral Investig",
"Mater. Lett",
"J. Mater. Sci. Mater. Med",
"Eur. Polym. J",
"Mater. Sci. Eng. C",
"Macromol. Biosci",
"Biomaterials",
"Acta Biomater",
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"\nFigure 1 .Figure 1 .\n11ContSynthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).",
"\n2. 2 . 4 .\n24Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40(Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.2.2.5. Static Contact Angle Determination",
"\nFigure 1 .\n1Synthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).",
"\n2. 2 . 4 .\n24Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40 (Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.",
"\nFigure 2 Figure 2 .\n22presents the FTIR spectra of PUR and PUR-M. FTIR spectra of PUR (black) and PUR-M (red).",
"\n\n1238s 1216s νC-(C=O)-O stretching vibrations of -C-(C=O)-O-of ester group, not hydrogen bonded",
"\nFigure 2 .\n2FTIR spectra of PUR (black) and PUR-M (red).",
"\n\nstretching of NH groups, hydrogen bonded withthe C=O of the ester group present in macrodiol and in aliphatic asymmetric and symmetric CH 2 groups present in the PUR chain and in GCPa modifier 1728m 1728s νC=O stretching of C=O in the ester and urethane groups, which were not bonded",
"\nFigure 3 .\n3Raman spectra of PUR and PUR-M.",
"\nFigure 3 .\n3Raman spectra of PUR and PUR-M.",
"\n\nof CH 2 groups in different positions and deforming bending vibrations outside of the plane of N-H groups 610 deforming vibrations outside of the plane of ester groups and their fluctuations * [35-38].",
"\n\nvibrations of N-H in urethane groups (as for II amides), P-O-Ca stretch 2925, 2883 the strongest polarized stretching vibrations of asymmetric and symmetric CH 2 groups present in PUR chains. Analogic CH 2 in H 2 C-O-P-O stretch phonons included 1733 stretching vibrations of carbonyl groups present in macrodiol Polios 55/20 1684 stretching vibrations of carbonyl groups C=O in urethane (HDI-BDO) 1483, 1452, 1442, 1421 deforming and scissoring vibrations of CH 2 groups in both polymer and GPCa 1302, 1261 swinging and bending vibrations of CH 2 out of the plane groups in polymer and filler with additional C-N stretch at the end 1126, 1131 stretching asymmetric vibrations of C-O-C in ester groups 1040,1080 complex CCCC stretch in branched alkanes with symmetric PO 4 and C-O-P stretch 959, 939, 882, 835 swinging vibrations of CH 2 groups in different positions and NH, CH bending deformation vibrations outside of the plane of urethane groups 580,614 deforming vibrations outside of the plane of ester groups with eventual phosphoester and PO 4 fluctuations * [39-41].",
"\nFigure 4 .\n4SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).",
"\nFigure 4 .\n4SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).",
"\nFigure 5 .\n5The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.",
"\nFigure 6\n6shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.",
"\nFigure 6 .\n6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nFigure 5 .\n5The T Sb (a), ε b (b), and hardness (c) of obtained PUR and PUR-M.",
"\nFigure 6 Figure 5 .\n65shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility. The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.",
"\nFigure 6\n6shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations.Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.",
"\nFigure 6 .\n6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nFigure 6 .\n6The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nFigure 7 .Figure 7 .\n77Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"\nFigure 7 .Figure 8 .Figure 8 .\n788Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). ContOptical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"\nFigure 9 .\n9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"\nFigure 8 .\n8Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"\nFigure 8 .\n8Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"\nFigure 9 .\n9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"\nFigure 9 .\n9SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"\nFigure 10 .\n10The EDX of PURs (a) and PURs-M (b) before calcification study.",
"\nFigure 10 .\n10The EDX of PURs (a) and PURs-M (b) before calcification study.",
"\nFigure 11 .\n11The EDX of PURs (a) and PURs-M (b) after calcification study.",
"\nFigure 11 .\n11The EDX of PURs (a) and PURs-M (b) after calcification study.",
"\nFigure 12 .\n12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"\nFigure 13 .\n13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nFigure 12 .\n12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"\nFigure 12 .\n12The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"\nFigure 13 .\n13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nFigure 13 .\n13The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"\nTable 1 .\n1The impact of the prepolymer modification time on the decrease of free isocyanate groups present in it.Time of \nprepolymeryzation (h) \n\nContent of the free isocyanate groups \nin the unmodified prepolymer (NCO ) \n\nContent of the free isocyanate groups \nin the modified prepolymer (NCO ) \n\nPEEUR \nPEEUR-M \n\n0 \n10.14 ± 0.03 \n10.14 ± 0.03 \n1 \n9.04 ± 0.01 \n9.04 ± 0.01 \n2 \n8.83 ± 0.03 \n8.83 ± 0.03 \n3 \n8.54 ± 0.03 \n8.54 ± 0.03 \n4 \n8.54 ± 0.02 \n8.54 ± 0.02 \n5 \n8.54 ± 0.02 \n8.43 ± 0.01 * \n6 \n8.53 ± 0.03 \n8.33 ± 0.03 \n7 \n8.53 ± 0.01 \n8.14 ± 0.03 \n8 \n8.53 ± 0.02 \n8.12 ± 0.02 \n9 \n8.53 ± 0.01 \n8.12 ± 0.03 \n\n* addition of the GPCa modifier. \n\n",
"\nTable 2 .\n2Spectral data and band assignments of FTIR analysis presented inFigure 2.PUR \nPUR-M \nBand \nDescription \nWavelength (cm −1 ) \n3392 \n3326w \n\n3380 \n3318w \nνNH \nstretching of NH groups, hydrogen bonded withthe C=O of the \nester group present in macrodiol and in GCPa modifier \n2942m \n2868m \n\n2942m \n2868m \nνCH2 \nstretching of aliphatic asymmetric and symmetric CH2 groups \npresent in the PUR chain and in GCPa modifier \n\n1728m \n1728s \nνC=O \nstretching of C=O in the ester and urethane groups, which were \nnot bonded \n1680m \n1680s \nνC=O \nstretching of C=O groups which formed hydrogen bonds \n1535m \n1535s \nνC-N \nstretching between CN in urethane group \n1462w \n1416w \n1350w \n\n1459m \n1415m \n1347m \n\nδCH2 \n\ndeformation vibrations of aliphatic CH2 groups present in the \nPUR and GPCa modifier: bending, wagging, scissoring \nin plane \n1260w \n1214m \n\n",
"\nTable 2 .\n2Cont.1680m \n1680s \nνC=O \nstretching of C=O groups which formed hydrogen bonds \n\n1535m \n1535s \nνC-N \nstretching between CN in urethane group \n\n1462w \n1416w \n1350w \n\n1459m \n1415m \n1347m \n\nδCH 2 \n\ndeformation vibrations of aliphatic CH 2 groups present in the \nPUR and GPCa modifier: bending, wagging, scissoring \nin plane \n\n1260w \n1214m \n\n1238s \n1216s \nνC-(C=O)-O \nstretching vibrations of -C-(C=O)-O-of ester group, not \nhydrogen bonded \n\n1170m \n1170s \nνNH-(C=O)-O \nstretching vibrations of -NH-(C=O)-O-of urethane group \n\n1135m \n1080m \n1058m \n947w \n\n1134s \n1061s \n993s \n949s \n\nνC-(C=O)-O \nνC-O \nstretching vibration of hydrogen bonded -C-(C=O)-O-, \n\n868w \n813w \n775w \n731w \n636w \n\n867s \n777s \n730s \n638s \n\nδCH 2 , \nδNH \nδOH \n\nout of the plane deformation of CH 2 and CH 3 groups as well \nas NH and OH groups \n\n",
"\nTable 3 .\n3Raman spectra band assignments for obtained PURs.Wavelength (cm −1 ) \nAssignments * \n\n3328 \nstretching vibrations of N-H in urethane groups (as for II-ary amides) \n\n2925, 2890 \nthe strongest polarized stretching vibrations of asymmetric and symmetric CH2 \n\ngroups present in PUR chains. \n\n1735, 1685 \nstretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and \n\nurethane groups (as for II-ary amides) respectively \n\n1480, 1450, \n\n1443, 1424 \nstrong planar deforming vibrations (scissoring) of CH2 groups \n\n",
"\nTable 3 .\n3Raman spectra band assignments for obtained PURs.Wavelength (cm −1 ) \nAssignments * \n\n3328 \nstretching vibrations of N-H in urethane groups (as for II-ary amides) \n\n2925, 2890 \nthe strongest polarized stretching vibrations of asymmetric and symmetric CH 2 \ngroups present in PUR chains. \n\n1735, 1685 \nstretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and \nurethane groups (as for II-ary amides) respectively \n1480, 1450, \n1443, 1424 \nstrong planar deforming vibrations (scissoring) of CH 2 groups \n\n",
"\nTable 4 .\n4Raman spectra band assignments for GPCa modified PURs.",
"\nTable 5 .\n5Contact angle and total surface free energy of obtained PURs and PURs-M.Symbol \n\nContact angle (°) \nSurface energy (mN/m) \n\nFormamide \nEthylene glycol \nWater \nAcid-part \nBase-part \nTotal surface \nfree energy \nPUR \n63.6 ± 2 \n68.2 ± 1 \n72.1 ± 2 \n0.04 \n20.83 \n31.82 \nPUR-M \n35.8 ± 4 \n47.5 ± 3 \n57 ± 3 \n14.51 \n21.72 \n59.09 \n\n",
"\nTable 5 .\n5Contact angle and total surface free energy of obtained PURs and PURs-M.Symbol \n\nContact angle ( • ) \nSurface energy (mN/m) \n\nFormamide \nEthylene glycol \nWater \nAcid-part \nBase-part \nTotal surface \nfree energy \n\nPUR \n63.6 ± 2 \n68.2 ± 1 \n72.1 ± 2 \n0.04 \n20.83 \n31.82 \nPUR-M \n35.8 ± 4 \n47.5 ± 3 \n57 ± 3 \n14.51 \n21.72 \n59.09 \n\n",
"\nTable 6 .\n6The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment.Sample \nExtracted mass (%) \n\n5 M NaOH \n2 N HCl \n0.1 M CoCl 2 /20% H 2 O 2 \n\nPUR \n50.9 ± 0.2 \n38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 \n35.81 ± 0.11 \n2.03 ± 0.12 \n\n",
"\nTable 6 .\n6Themass loss of the PURs and PURs-M after 15 days of short-term interactions study \nperformed with selected media of the acidic, basic, and oxidative environment. \n\nSample \nExtracted mass (%) \n\n5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2 \nPUR \n50.9 ± 0.2 38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 35.81 ± 0.11 \n2.03 ± 0.12 \n\n",
"\nTable 6 .\n6The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment.Sample \nExtracted mass (%) \n5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2 \nPUR \n50.9 ± 0.2 38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 35.81 ± 0.11 \n2.03 ± 0.12 \n\n"
] | [
"ContSynthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).",
"Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40(Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.2.2.5. Static Contact Angle Determination",
"Synthesis of unmodified (a) and calcium glycerolphosphate (GPCa)-modified (b) poly(ester ether urethane)s (PEEURs).",
"Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX) SEM was performed with the use of a Zeiss Scanning Electron Microscope EVO-40 (Jena, Germany) (different magnifications were used: 3000×, 1000×, and 250×). The SEM instrument was integrated with an energy dispersive X-ray (EDX) microanalyzer (Jena, Germany) for elemental analysis. Prior to the study PURs were covered with a conductive layer of gold by sputter coater Quorum 150T E. To study the morphology of the obtained PURs, PURs-M and the scaffolds ImageJ ® software (U.S. National Institutes of Health, Bethesda, MA, USA) was used.",
"presents the FTIR spectra of PUR and PUR-M. FTIR spectra of PUR (black) and PUR-M (red).",
"1238s 1216s νC-(C=O)-O stretching vibrations of -C-(C=O)-O-of ester group, not hydrogen bonded",
"FTIR spectra of PUR (black) and PUR-M (red).",
"stretching of NH groups, hydrogen bonded withthe C=O of the ester group present in macrodiol and in aliphatic asymmetric and symmetric CH 2 groups present in the PUR chain and in GCPa modifier 1728m 1728s νC=O stretching of C=O in the ester and urethane groups, which were not bonded",
"Raman spectra of PUR and PUR-M.",
"Raman spectra of PUR and PUR-M.",
"of CH 2 groups in different positions and deforming bending vibrations outside of the plane of N-H groups 610 deforming vibrations outside of the plane of ester groups and their fluctuations * [35-38].",
"vibrations of N-H in urethane groups (as for II amides), P-O-Ca stretch 2925, 2883 the strongest polarized stretching vibrations of asymmetric and symmetric CH 2 groups present in PUR chains. Analogic CH 2 in H 2 C-O-P-O stretch phonons included 1733 stretching vibrations of carbonyl groups present in macrodiol Polios 55/20 1684 stretching vibrations of carbonyl groups C=O in urethane (HDI-BDO) 1483, 1452, 1442, 1421 deforming and scissoring vibrations of CH 2 groups in both polymer and GPCa 1302, 1261 swinging and bending vibrations of CH 2 out of the plane groups in polymer and filler with additional C-N stretch at the end 1126, 1131 stretching asymmetric vibrations of C-O-C in ester groups 1040,1080 complex CCCC stretch in branched alkanes with symmetric PO 4 and C-O-P stretch 959, 939, 882, 835 swinging vibrations of CH 2 groups in different positions and NH, CH bending deformation vibrations outside of the plane of urethane groups 580,614 deforming vibrations outside of the plane of ester groups with eventual phosphoester and PO 4 fluctuations * [39-41].",
"SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).",
"SEM images of the GPCa modifier (a) and the surface of PUR (b) and PUR-M (c).",
"The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.",
"shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.",
"The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"The T Sb (a), ε b (b), and hardness (c) of obtained PUR and PUR-M.",
"shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations. Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility. The TSb (a), εb (b), and hardness (c) of obtained PUR and PUR-M.",
"shows the MTT assay results performed by using PURs and PURs-M extracts at different concentrations.Performed in vitro cell studies revealed good biocompatibility of the obtained PUR and PUR-M materials independent of the extract concentration. In the case of extract concentrations in the range of 25-75% as light improvement of cell growth was noted for PURs-M in comparison to the controls. Only in the case of undiluted extracts (100%) was the cell viability of PURs and PURs-M slightly lower in comparison to the controls, but still in the range of good biocompatibility.",
"The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"The effect of PUR and PUR-M extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"Optical microscope images of PURs before (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d). ContOptical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"Optical microscope images of PURs-M after (a) and after 15 days of short-term interactions study performed in the selected environments: basic (b); acidic (c); and oxidative (d).",
"SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"SEM images of PUR (a) and PUR-M (b) before calcification study and images of PUR (c) and PUR-M (d) after calcification study.",
"The EDX of PURs (a) and PURs-M (b) before calcification study.",
"The EDX of PURs (a) and PURs-M (b) before calcification study.",
"The EDX of PURs (a) and PURs-M (b) after calcification study.",
"The EDX of PURs (a) and PURs-M (b) after calcification study.",
"The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"The PUR (a) and PUR-M (b) scaffold fabricated by SC/PL in combination with TIPS.",
"The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"The effect of PUR and PUR-M scaffolds' extracts on the in vitro growth of C2C12 cells studied by MTT assay after 72 h (* p < 0.05).",
"The impact of the prepolymer modification time on the decrease of free isocyanate groups present in it.",
"Spectral data and band assignments of FTIR analysis presented inFigure 2.",
"Cont.",
"Raman spectra band assignments for obtained PURs.",
"Raman spectra band assignments for obtained PURs.",
"Raman spectra band assignments for GPCa modified PURs.",
"Contact angle and total surface free energy of obtained PURs and PURs-M.",
"Contact angle and total surface free energy of obtained PURs and PURs-M.",
"The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment.",
"The",
"The mass loss of the PURs and PURs-M after 15 days of short-term interactions study performed with selected media of the acidic, basic, and oxidative environment."
] | [
"Figure 1",
"Figure 1",
"Figure 2",
"Figure 2",
"Figure 2",
"Figure 3",
"Figure 3",
"Figure 4",
"Figure 4a",
"Figure 4b",
"Figure 4c",
"Figure 4b",
"Figure 4c",
"Figure 5",
"Figure 5",
"Figure 5",
"Figures 7 and 8",
"Figures 7 and 8",
"(Figure 8",
"Figures 7 and 8",
"(Figure 8",
"Figure 9",
"Figure 9",
"Figures 10 and 11",
"Figure 10",
"Figure 11",
"(Figure 8",
"Figure 9",
"Figure 9",
"Figure 9",
"Figures 10 and 11",
"Figure 10",
"Figure 11",
"Figure 9",
"Figures 10 and 11",
"Figure 10",
"Figure 11",
"Figure 12",
"Figure 12",
"Figure 13",
"Figure 13",
"Figure 12",
"Figure 13",
"Figure 13"
] | [] | [
"Tissue scaffolds, designed for tissues regeneration, are three-dimensional porous structures, which serve as biological tissue substitutes that enable the functional performance of the regenerated tissue to be restored, maintained or improved [1]. Tissue engineering (TE) applies both natural and synthetic polymers [2], metals [3], ceramics [4] and bioactive glasses [5]. Biomaterials used for the purpose of tissue engineering (TE) have to meet strict requirements such as biocompatibility. Moreover, these materials may reveal some bioactive behavior, which could stimulate proper tissue regeneration [6]. The 3D scaffold has to provide an adequate support for the regenerated tissue, thus the mechanical characteristics of the used biomaterials are an important feature. In addition the gradual degradation of the scaffold is necessary for proper tissue restoration [7,8]. However, in order to meet the requirements of an ideal tissue scaffold material and to combine the best mechanical properties with bioactivity and ability to degrade in the human body environment, new composite materials, which are the combination of polymers with different types of fillers, are being developed [1,8]. Polyurethanes (PU) according to their superior characteristics of proper physicochemical, mechanical, and biological properties seem to meet all of these requirements for use in TE as materials for scaffold fabrication [1,6].",
"PUs have been widely developed in the field of biomedical devices, thus their modification is well known [1,6,9]. The most common PU modifications for bone tissue engineering take place mainly by the introduction of the filler into the PU matrix. The most often reported fillers are calcium phosphates like hydroxyapatite [4,10,11], nanohydroxyapatite [12,13], and β-tricalcium phosphate [14,15]. Recently anew solution for PU modification was proposed in the form of calcium glycerophosphate [16], bioactive glass [5] or carbon nanotubes [17].",
"Calcium glycerophosphate is the calcium salt of glycerophosphoric acid. This compound has been approved for use by the Ministry of Health [18] as a nutrient, a component of dietary supplements or mineral food products, and has been considered as a safe ingredient/food additive by the US Food and Drug Administration [19]. In addition to the previously listed applications, calcium glycerophosphate is also used as an ingredient in toothpaste [20], dental varnishes [21], as well as electrolytes used for mineralization of hydrogels for bone regeneration [22] or surface modification of titanium bone implants [3].",
"There are several examples in the literature, describing the synthesis of PU composites with the use of fillers mentioned above leading to the desired changes in mechanical properties of the material, as well as contributing to its bioactivity improvement [4,[10][11][12][13][14][15][16][17].The effect of calcium glycerolphosphate salt (GPCa) as a filler on tissue scaffolds properties which received biodegradable polyurethane foams for bone graft substitutes was recognized by Gorna et al. [16]. The PU system studied by Gorna et al. was derived from 1,6-hexamethylene diisocyanate, poly(ethylene oxide)diol, poly(ε-caprolactone)diol, amine-based polyol or sucrose-based polyol, water as a chain extender and foaming agent, catalysts, citric acid as a calcium complexing agent, lecithin or solutions of vitamin D 3 as surfactants, and various inorganic fillers. One of the fillers used was GPCa, others were calcium carbonate and hydroxyapatite [16]. Recently, Kavanaugh et al. [23] proved as well that segmented polyurethanes prepared with β-glycerol phosphate as a biologically active chain extender, supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation. The PU system studied by Kavanaugh et al. was synthesized by using poly(ε-caprolactone)diol, 4,4 -methylene bis(cyclohexyl isocyanate), and biologically active compounds such as ascorbic acid, L-glutamine, β-glycerol phosphate, and dexamethasone as chain extenders [23]. In brief, the use of β-glycerol phosphate as a chain extender has improved the biological activity of polyurethane applicable as a material for bone regeneration. Furthermore, glycerophosphates have high potential for mineralization, proper adhesion and proliferation and therefore the attempted GPCa chain PU contrasts with the approach by Gornal et al. [16] where it was used as a filler.",
"In this paper we describe the synthesis and characterization of the PEEURs designed for bone tissue regeneration. PEEURs were synthesized by using 1,6-hexamethylene diisocyanate (HDI), poly(ethylene glycol) (PEG), α,ω-dihydroxy(ethylene-butylene adipate) (Polios), 1,4-butanediol Polymers 2017, 9,329 3 of 21 (BDO) as a chain extender and GPCa as a modifier used to stimulate bone tissue regeneration. The obtained unmodified (PUR) and GPCa modified (PURs-M) PEEURs were studied with various techniques in order to confirm the reactivity of GPCa with the urethane prepolymer, to study its chemical composition, surface morphology, hydrophilic character, mechanical properties, in vitro biocompatibility and short-term interactions with selected acidic, basic and oxidative environment. Moreover the calcification study was performed to establish if the GPCa modifier improved the bioactive character of the obtained PUR-M materials. Furthermore, with the use of selected samples, porous scaffolds were obtained by using the SC/PL technique combined with TIPS. According to performed studies the obtained PURs-M may be a suitable candidate for bone tissue engineering and further studies are being developed by our team in this field.",
"PEEURs were synthesized by the standard two step polymerization procedure with urethane prepolymer intermediate [1,6]. The urethane prepolymer was obtained in the reaction of polyester α,ω-dihydroxy(ethylene-butylene adipate) (PEBA, trade name Polios 55/20; Purinova, Bydgoszcz, Poland) (63 wt %),poly(ethylene glycol) (PEG) (14 wt %) and aliphatic 1,6-hexamethylene diisocyanate (HDI) (Sigma Aldrich, Poznań, Poland) (23 wt % ). In the second step the chain extender-1,4-butanediol (BDO) (POCH, Gliwice, Poland)-Was added to the urethane prepolymer to obtain PEEUs with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of the chain extender BDO equal to NCO/OH = 0.9:1.",
"The synthesis of modified PEEURs (PUR-M) was as follows: In the first step the urethane prepolymer was obtained in the reaction between PEBA, PEG, and HDI. In the second step the 10 wt % of GPCa (Sigma Aldrich, Poznań, Poland) calculated per mass of the prepolymer was added at 80 • C and stirred for 4 h (the mass ratio of urethane prepolymer to GPCa was equal to 1:0.25). In the next step the chain extender BDO was added to obtain PUR-M with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of chain extender BDO equal to NCO/OH = 0.9:1. Reaction of unmodified and modified polyurethanes is presented in Figure 1.",
"Polymers 2017, 9,329 3 of 21 obtained unmodified (PUR) and GPCa modified (PURs-M) PEEURs were studied with various techniques in order to confirm the reactivity of GPCa with the urethane prepolymer, to study its chemical composition, surface morphology, hydrophilic character, mechanical properties, in vitro biocompatibility and short-term interactions with selected acidic, basic and oxidative environment. Moreover the calcification study was performed to establish if the GPCa modifier improved the bioactive character of the obtained PUR-M materials. Furthermore, with the use of selected samples, porous scaffolds were obtained by using the SC/PL technique combined with TIPS. According to performed studies the obtained PURs-M may be a suitable candidate for bone tissue engineering and further studies are being developed by our team in this field.",
"PEEURs were synthesized by the standard two step polymerization procedure with urethane prepolymer intermediate [1,6]. The urethane prepolymer was obtained in the reaction of polyester α,ωdihydroxy(ethylene-butylene adipate) (PEBA, trade name Polios 55/20; Purinova, Bydgoszcz, Poland) (63 wt %),poly(ethylene glycol) (PEG) (14 wt %) and aliphatic 1,6-hexamethylene diisocyanate (HDI) (Sigma Aldrich, Poznań, Poland) (23 wt % ). In the second step the chain extender-1,4-butanediol (BDO) (POCH, Gliwice, Poland)-Was added to the urethane prepolymer to obtain PEEUs with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of the chain extender BDO equal to NCO/OH= 0.9:1.",
"The synthesis of modified PEEURs (PUR-M) was as follows: In the first step the urethane prepolymer was obtained in the reaction between PEBA, PEG, and HDI. In the second step the 10 wt % of GPCa (Sigma Aldrich, Poznań, Poland) calculated per mass of the prepolymer was added at 80 °C and stirred for 4 h (the mass ratio of urethane prepolymer to GPCa was equal to 1:0.25). In the next step the chain extender BDO was added to obtain PUR-M with a molar ratio of free isocyanate groups (NCO) (in the urethane prepolymer) to hydroxyl groups (OH) of chain extender BDO equal to NCO/OH = 0.9:1. Reaction of unmodified and modified polyurethanes is presented in Figure 1. ",
"Indication of free isocyanate groups is a standard procedure in the case of PURs obtained by a two-step polymerization method. Its aim is to establish the time, after which the requiredamount of unreacted diisocyanate groups (NCO) in the prepolymerization reaction takes place. The determination of free isocyanate groups (FNCO, %) was performed according to the PN-EN 1242:2006 standard.",
"The FTIR of the solid PUR and PUR-M was performed by an FTIR Nicolet 8700 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with Specac's Golden Gate module and single reflection diamond ATR unit to determine the influence of the processing technique on the composition of the obtained materials. The studied spectral range was from 4000 to 500 cm −1 averaging 254 scans per sample with a resolution of 4 cm −1 .",
"Raman spectra were performed by using Monovista CRS + spectrometer provided by S&I Ltd., (Warstein, Germany) which was operated by VistaControl 4.1 software (S&I Ltd., (Warstein, Germany). Data about the polymers' structure was obtained by using a 532 nm green laser with the scanning power reduced to 10 mV. The best suitable grating was set to 1800 grooves per mm and was chosen to provide resolution of about 0.5 cm −1 . The slit was set to standard 100 micrometers and the objective used to scan in Point by Point, 2D mode was a long focal-length Olympus with 100 magnification rate. All spectra were obtained by gaining two accumulations within 10 s time frame. Each scanning point from which the Raman spectra were taken had approximately1 μm 2 surface. ",
"Indication of free isocyanate groups is a standard procedure in the case of PURs obtained by a two-step polymerization method. Its aim is to establish the time, after which the requiredamount of unreacted diisocyanate groups (NCO) in the prepolymerization reaction takes place. The determination of free isocyanate groups (F NCO , %) was performed according to the PN-EN 1242:2006 standard.",
"The FTIR of the solid PUR and PUR-M was performed by an FTIR Nicolet 8700 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with Specac's Golden Gate module and single reflection diamond ATR unit to determine the influence of the processing technique on the composition of the obtained materials. The studied spectral range was from 4000 to 500 cm −1 averaging 254 scans per sample with a resolution of 4 cm −1 .",
"Raman spectra were performed by using Monovista CRS + spectrometer provided by S&I Ltd., (Warstein, Germany) which was operated by VistaControl 4.1 software (S&I Ltd., (Warstein, Germany). Data about the polymers' structure was obtained by using a 532 nm green laser with the scanning power reduced to 10 mV. The best suitable grating was set to 1800 grooves per mm and was chosen to provide resolution of about 0.5 cm −1 . The slit was set to standard 100 micrometers and the objective used to scan in Point by Point, 2D mode was a long focal-length Olympus with 100 magnification rate. All spectra were obtained by gaining two accumulations within 10 s time frame. Each scanning point from which the Raman spectra were taken had approximately 1 µm 2 surface. ",
"The contact angle as well as the surface free energy of the materials' surfaces weredeterminedat room temperature by using a Kruss Goniometer G10 (KRÜSS GmbH, Hamburg, Germany) with drop shape analysis software. The water contact angle of the 10 samples was evaluated by static contact angle measurements using the sessile drop method. Surface free energy was calculated by using measurements performed according to the Acid-Base method (AB) [24] by static contact angle studied with the use of three liquids: Water, ethylene glycol, and formamide.",
"Tensile strength (T SB ) and elongation at break (ε b ) were studied using the universal testing machine Zwick & Roell Z020 (Zwick Roell Polska Sp. z o.o. Sp.K., Wrocław, Poland) according to PN-EN ISO 527-2:2012 with a crosshead sped of 500 mm/min. Samples of sixwere used for this study.",
"Hardness was measured by using the Shore method according to PN-EN ISO 868:2004. Obtained data were presented with Shore degree ( • Sh D and • Sh A). Samples of 10 were used for this study.",
"PURs were cut into round samples of 0.5 cm 2 area. Prepared samples were dried and weighed in a thermobalance (Radwag, Radom, Poland) (RADWAG MAX50/SX) set at 60 • C. Then, 6 samples of each studied PUR materials were placed in a 24-well cell culture plate filled with selected media: Oxidative solution of 0.1 M CoCl 2 /20% H 2 O 2 ; acidic solution of 2 N HCl or basic solution of 5 M NaOH. Samples were incubated in the selected media at 37 • C. The mass change of the samples was examined after 15 days for oxidative, acidic, and basic media. Samples mass change measurement was as follows: Samples were taken out from the container and put into a paper sheet to reduce the medium excess. Then, samples were placed in the thermobalance (set at 60 • C) where they were weighed to constant mass. The results are the arithmetic mean of six measurements.",
"The changes at the PUR and PUR-M surface were monitored by optical microscopy (OM) performed with the use of a Bresser microscope(Bresser GmbH, Rhede, Germany) at a magnification of 20×.",
"The cytotoxicity assay was performed by using selected PUR and PUR-M samples. To examine the cytotoxicity of the obtained materials, the extract was prepared and tested with the use of a C2C12 cell line according to ISO 10993-5:2009 standard. In order to obtain extract the sterile PUR or PUR-M samples were incubated at the ratio 1:100 (w/v) in cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), L-glutamine (1% solution in medium) (Gibco), 1% antibiotic-antimycotic mixture for 24 h at 37 • C under continuous steering.",
"MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for assessing cell metabolic activity. C2C12 cells were split into 24-well plates at 30,000 cells per well and grown for 24 h in 500 µL of culture medium. Cells were incubated for 72 h with the matrices' extracts. Then, the MTT assay of viable cells was used in accordance with the manufacturer's recommendations (Sigma Aldrich, St. Louis, MO, USA). The reaction product was quantitatively determined by an Absorbance Reader BioTek EL*800 (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 570 nm. The viability of the untreated cells was counted as 100.",
"Golomb and Wagner's Compound was used to perform the calcification study. The calcification metastable solution consisted of 3.87 millimole (mM) CaCl 2 , 2.32 mM K 2 HPO 4 , yielding a ratio of calcium to phosphate (Ca/PO 4 ) = 1.67, and 0.05 M Tris Buffer (in this study C 4 H 11 NO 3 ) dissolved in one mL of reverse osmosis (RO) water [25]. PUR and PUR-M samples were cut into round samples of 0.5 cm 2 area. Prepared samples were dried and weighed in a thermobalance (RADWAG MAX50/SX) set at 60 • C. Then, 6 samples of each studied PUR materials were placed in a 24-well cell culture plate filled with Golomb and Wagner's Compound. The progress of the calcification was studied by SEM and EDX after 21 days.",
"PUR or PUR-M was dissolved in 1,4-dioxane (POCH, Gliwice, Poland) at a concentration of 20% w/v. Then, sodium chloride, of crystal size in the range of 0.6-0.4 µm, was added to the polyurethane solution until complete saturation of the solution occurred. Formulated PUR (or PUR-M)-salt saturated solution was transferred into the stainless steel mold of the size 2.5 cm × 2.5 cm × 2.5 cm and placed at −20 • C for 24 h to direct the solvent crystallization and to fabricate scaffolds of local anisotropy where the porosity of the scaffolds was of controlled pore size and porosity [26][27][28][29]. Then scaffolds were removed from the mold and immersed in warm (40-50 • C) bidistilled water, where for 7 days the sodium chloride crystals were washed out. The water was changed twice a day. Finally, the samples were dried at 60 • C for 24 h.",
"The impact of the prepolymer modification time on the decrease of the free isocyanate groups in it is outlined below. Table 1 presents the influence of the prepolymer modification time on the decrease of the free isocyanate groups present in it. Table 1 shows that the prepolymerization reaction takes place in a similar way for both PUR and PUR-M materials until the 4th hour of the synthesis, when the GPCa modifier was added to the system. Incorporation of the GPCa modifier into the system caused a slightly higher F NCO decrease for PUR-M in comparison to PUR after 1 h of modification (8.54 ± 0.02% and 8.43 ± 0.03% respectively). After 5 h of prepolymer modification with GPCa the F NCO reached the previously established 8 wt % and the reaction was recognized as finished. It can be concluded that the NCO groups of diisocyanate react with OH groups present in the GPCa modifier at the adjusted modification conditions. It suggests that GPCa was incorporated into the PUR structure through covalent bonds. Analysis of FTIR spectra ( Figure 2) revealed the presence of functional groups characteristic for PURs composition; i.e., urethane linkages (see Table 2). Thus, conditions designed to carry out PURs synthesis were suitable and provided PUR product. The expanded base of the NH stretching band (3392 and 3326 cm −1 ) for PURs suggested the presence of \"free\" and hydrogen bonded HS in the obtained material respectively [30,31]. In the case of PUR-M the expanded base of the NH stretching band was observed as well, but its intensity relating to the \"free\" NH (3389 cm −1 ) in the PUR-M composition was decreased. Conversely to the PURs the PUR-M revealed the well-shaped NH stretching band related to the moderate and strong hydrogen bonds present in the HS of PUR chains [32,33]. The observed C=O stretching band confirmed the presence of ester and urethane linkages, which were \"free\" (1728 cm −1 ) and hydrogen bonded (1680 cm −1 ) for both PUR and PUR-M [32,33]. It can be pointed out here that the PUR-M appears to have a large number of hydrogen bonded urethane linkages in comparison to PURs. Performed FTIR analysis confirmed the presence of urethane linkages. Furthermore, the FTIR spectra of PUR-M showed that GPCa modification represents a higher level of hydrogen bonds between HS in PUR chains. Some differences in band intensities (1462-945 cm −1 ) between PURs and PURs-M were observed which might be related as well to the presence of GPCa molecules in the PUR system [34]. Analysis of FTIR spectra ( Figure 2) revealed the presence of functional groups characteristic for PURs composition; i.e., urethane linkages (see Table 2). Thus, conditions designed to carry out PURs synthesis were suitable and provided PUR product. The expanded base of the NH stretching band (3392 and 3326 cm −1 ) for PURs suggested the presence of \"free\" and hydrogen bonded HS in the obtained material respectively [30,31]. In the case of PUR-M the expanded base of the NH stretching band was observed as well, but its intensity relating to the \"free\" NH (3389 cm −1 ) in the PUR-M composition was decreased. Conversely to the PURs the PUR-M revealed the well-shaped NH stretching band related to the moderate and strong hydrogen bonds present in the HS of PUR chains [32,33]. The observed C=O stretching band confirmed the presence of ester and urethane linkages, which were \"free\" (1728 cm −1 ) and hydrogen bonded (1680 cm −1 ) for both PUR and PUR-M [32,33]. It can be pointed out here that the PUR-M appears to have a large number of hydrogen bonded urethane linkages in comparison to PURs. Performed FTIR analysis confirmed the presence of urethane linkages. Furthermore, the FTIR spectra of PUR-M showed that GPCa modification represents a higher level of hydrogen bonds between HS in PUR chains. Some differences in band intensities (1462-945 cm −1 ) between PURs and PURs-M were observed which might be related as well to the presence of GPCa molecules in the PUR system [34]. Table 2. Spectral data and band assignments of FTIR analysis presented in Figure 2. ",
"In Figure 3 the Raman spectra of obtained PUR and PUR-M are presented, of which the band assignments are given in Tables 3 and 4. δCH2, δNH δOH out of the plane deformation of CH2 and CH3 groups as well as NH and OH groups",
"In Figure 3 the Raman spectra of obtained PUR and PUR-M are presented, of which the band assignments are given in Tables 3 and 4. The performed Raman spectroscopy results were complimentary to the FTIR study (Tables 4 and 5). Thus, it confirmed the presence of strongly hydrogen bonded urethane groups in the PUR-M structure as well as introduction of the GPCa modifier into the main chain of PUR at least at one-side. Figure 4 shows the SEM images of the GPCa modifier and the obtained PUR and PUR-M surface. Figure 4a shows an image of GPCa used as a modifier. The particles resemble a spherical shape and their sizes are in the range of 6.754-7.952 µm. Figure 4b shows the image of the PURs surface, which is rather homogeneous and without visible inclusions. In Figure 4c it can be seen that the surface of PUR-M modified with GPCa is homogenous over 80% with little inclusions visible at the surface top. These inclusions can be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix. It suggests that a large amount of used GPCa modifier was incorporated into the PUR structure by covalent bonding, which is consistent with FTIR and Raman spectroscopy. and their sizes are in the range of 6.754-7.952 μm. Figure 4b shows the image of the PURs surface, which is rather homogeneous and without visible inclusions. In Figure 4c it can be seen that the surface of PUR-M modified with GPCa is homogenous over 80% with little inclusions visible at the surface top. These inclusions can be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix. It suggests that a large amount of used GPCa modifier was incorporated into the PUR structure by covalent bonding, which is consistent with FTIR and Raman spectroscopy. ",
"In Table 5 are gathered the values of the contact angles and total surface free energy studied for the obtained PUR and PUR-M.",
"The obtained PURs and PURs-M had water contact angles in the range from 72° (PUR) to 57° (PU-M) ( Table 5). The contact angles studied in formamide and ethylene glycol were slightly lower in comparison to the values of the water contact angle. Addition of GPCa caused a decrease of the contact angle independently of the solvent used in the study. The total surface free energy is higher for PURs-M (59 mN/m) than for PURs (32 mN/m). This trend was observed independently of the method used for its examination (Table 5). ",
"The TSb, εb and hardness of the obtained PUR and PUR-M are shown in the Figure 5.",
"The TSb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of εb the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 °ShD for PURs and 35 ± 2 °ShD for PURs-M). ",
"In Table 5 are gathered the values of the contact angles and total surface free energy studied for the obtained PUR and PUR-M.",
"The obtained PURs and PURs-M had water contact angles in the range from 72 • (PUR) to 57 • (PU-M) ( Table 5). The contact angles studied in formamide and ethylene glycol were slightly lower in comparison to the values of the water contact angle. Addition of GPCa caused a decrease of the contact angle independently of the solvent used in the study. The total surface free energy is higher for PURs-M (59 mN/m) than for PURs (32 mN/m). This trend was observed independently of the method used for its examination (Table 5). ",
"The T Sb , ε b and hardness of the obtained PUR and PUR-M are shown in the Figure 5.",
"The T Sb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of ε b the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 • ShD for PURs and 35 ± 2 • ShD for PURs-M).",
"The TSb, εb and hardness of the obtained PUR and PUR-M are shown in the Figure 5. The TSb of PURs-M (18 MPa) was 6 MPa higher than for PURs (12 MPa). In the case of εb the introduction of GPCa modifier caused a decrease of this property from 390 ± 13% (PURs) to 280 ± 15% (PURs-M). The hardness was comparable for both PURs and PURs-M (31 ± 3 °ShD for PURs and 35 ± 2 °ShD for PURs-M). Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.",
"Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) caused the increase of degradation rate of about 4% in comparison to PURs. In the case of acidic environment the mass loss was over 30% for both PURs and PURs-M, and this mass loss was higher Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.",
"Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) Table 6 shows the mass loss of PURs and PURs-M noted after the short-term interactions study (15 days) performed with the selected media of acidic, basic, and oxidative environment.",
"Indicated mass loss was noted for both PUR and PUR-M materials (Table 6). Thus, both types of obtained materials may be considered as possibly degradable. In a strongly basic environment both materials had similar values of mass loss, which was over 50%. The GPCa modification (PURs-M) caused the increase of degradation rate of about 4% in comparison to PURs. In the case of acidic environment the mass loss was over 30% for both PURs and PURs-M, and this mass loss was higher by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of short-term interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.",
"Polymers 2017, 9,329 12 of 21 by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of shortterm interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.",
"Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment. by 3% for PURs than for PURs-M. In the oxidative environment PURs-M were slightly more stable than PURs, but the mass loss did not exceed 5%. The surface changes of PURs and PURs-M were monitored before and after the 15 days of shortterm interactions study. Figures 7 and 8 show the surface changes at the time of the performed study.",
"Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment. ",
"SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress. Optical microscopy showed the visible changes, which took place on the surface top of the obtained PURs and PURs-M after 15 days of the short-term interactions study performed for different environments (Figure 8). It can be observed that PUR-M materials are more sensitive in the basic and acidic environment due to the fact that defragmentation of these materials occurs. The indicated materials' defragmentation, according to the references, may concern an initial step of polymer degradation [32,42]. The optical microscopy images confirmed that both PURs and PURs-M are resistant to the oxidative environment.",
"SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9. ",
"SEM images of PURs and PURs-M before and after the calcification study are presented in Figure 9. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress. As can be noted in Figure 9 the PURs do not have an excessive ability to calcification. This is in contrast to the PURs-M, on whose surface a significant deposition of calcium was indicated. The confirmation of the calcification progress on the obtained materials surface was achieved due to the EDX study, which is presented in Figures 10 and 11. Figure 10 showes that PURs do not contain calcium at their surface prior to the calcification study. In the case of PURs-M the calcium content is approximately 50% which is related to the presence of GPCa modifier. After the calcification study ( Figure 11) the calcium amount at the materials' surfaces increase by over 50% for PURs and 110% on PURs-M. Thus, the GPCa modification has a significant impact on the calcification progress. ",
"Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds.",
"Polymers 2017, 9,329 16 of 21",
"Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds. Obtained PUR had pore sizes in the range of 36-100 μm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 μm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay. Obtained PUR had pore sizes in the range of 36-100 µm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 µm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay. ",
"Obtained PURs and PURs-M met the requirements of biomaterials used for bone tissue engineering. Their mechanical, physicochemical biological characteristic was suitable for bone tissue engineering. Thus, in the next step we made an attempt to fabricate, with the use of PURs and PURs-M, the porous scaffold by using SC/PL combined with TIPS. Figure 12 shows the SEM micrographs of the obtained porous scaffolds. Obtained PUR had pore sizes in the range of 36-100 μm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had pore size in the range of 50-100 μm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references [8], for using such scaffolds in bone tissue engineering applications. Furthermore, in the case of PURs-M scaffolds, particles of the GCPa modifier were visible. Thus, they were not washed out of the scaffold during the scaffold fabrication process and according to the biocompatibility and calcification study, this may be a useful compound stimulating the regeneration of native bone tissue [43] According to the proper morphology of the obtained porous scaffolds their biocompatibility was studied as well. Figure 13 shows the cells viability after 72 h of MTT assay. The main conclusion coming from the analysis of Figure 13 is the fact that the obtained PURs-M possesses better biocompatibility than PURs. Thus, it confirms the beneficial effects of the employed GPCa modifier. The proliferation of cells of the PUR-M extracts was observed at concentrations between 25-75%. PURs had lower cells viability in comparison to the PURs-M. In the case of undiluted extracts (100%) the cells viability was comparable for both PUR and PUR-M scaffolds.",
"Bone tissue engineering is a demanding field of strictly described requirements of biomaterials, which may be used for bone tissue scaffold fabrication. Accordingly of the many biomaterials used in this field PU seems to be the most suitable candidate. This is due to its ease of modification to attain a bioactive material as well as its suitable mechanical property design related to the raw materials selection for its synthesis [14,[44][45][46][47].",
"In this paper we described the synthesis of PEEURs carried out with the use of selected raw materials such as aliphatic HDI, polyester (Polios,) and polyether (PEG) polyols, with BDO chain extender to reach the requirements of biocompatible biomaterials for medical applications. The GPCa modifier was selected according to the literature, which describes it as a compound that can improve the bioactivity of the material as well as stimulating bone tissue regeneration. The successful synthesis of PURs was confirmed by FTIR and Raman spectroscopy, which revealed the formation of urethane bonds. Application of GPCa modifier improved hydrogen-bond formation in the PURs-M structure compared to the PURs (see FTIR analysis). Spectroscopic studies and F NCO determination confirmed the fact that GPCa is partially covalently bonded with the PUR chain. This is possible due to the hydroxyl groups present in the GPCa chemical structure. The SEM image of the PUR-M surface was in good agreement with the FTIR analysis due to the fact that it revealed the presence of a homogenous surface (about 80%) of this material, with only little inclusions visible at the top. The presence of these inclusions could be related to the GPCa, which did not react with prepolymer and was partially enclosed in the polyurethane matrix in the form of the filler. This filling effect of GPCa occurring was beneficial in the case of the biocompatibility and calcification study and may be useful as bioactive stimulant to improve bone tissue regeneration. The contact angle was decreased for the PURs-M (57 • ) in comparison to the PURs (72 • ). Thus, the addition of GPCa improved in a superior way the hydrophilic characteristic of the obtained materials, which may be beneficial for cell growth. According to Guelcher et al. the contact angle of the polymer surface in the range of 45-76 • supports the attachment of mammalian cells, which can have a beneficial influence on the absorption of albumin and a preservative coupler of live tissues [48]. The total surface free energy (59 mN/m for PURs-M and 32 mN/m for PURs respectively) was in good agreement with the contact angle study and is suitable for cell growth only for PURs-M according to the references [11,49,50]. In further studies the total surface free energy can be increased by increased GPCa addition into the materials. Most researchers have come to the conclusion that rather a surface charge (either positive, or negative) is the most important, not the surface free energy [49]. According to this, a modification of a hydrophilic but not-charged polyurethane surface with bipolar calcium β-glycerophosphate is a good option for the optimization of cellular adherence at the PU surface. The mechanical properties of the obtained PURs (T Sb = 12 MPa, ε b = 390 ± 13%) and PURs-M (T Sb = 18 MPa, ε b = 280 ± 15%) were more suitable for bone tissue engineering in the case of PUR-M. The materials used in bone regeneration must have a T Sb in the range of 1.5-38 MPa for human cancellous bone regeneration or 35-283 MPa for human cortical bone regeneration [51]. Thus, the obtained PURs and PURs-M may find an application as biomaterials for scaffold fabrication of human cancellous bone. The hardness was comparable for both PURs and PURs-M (31 ± 3 • ShD for PURs and 35 ± 2 • ShD for PURs-M). The short term interactions study showed that both PURs and PURs-M are sensitive to basic and acidic environment, while in an oxidative one they remain stable. Thus, this is consistent with references [52]. The observed superior mass decrease, in the case of basic (over 50%) and acidic (over 30%) environment, after 15 days of the short-term interactions study may be a sign of the first step of material degradation called defragmentation [53]. This was confirmed by the optical microscopy studies, which showed defragmentation of the obtained PURs and more favorably PURs-M. The SEM and EDX verification of the calcification study showed significant improvement in the progress of calcification, the process needed in the case of bone regeneration [11,16,35,54], for PURs-M. Thus, the GPCa modification is a superior factor, which improves calcification, which was indicated by the peaks of Ca present in the EDX spectra. After the calcification study the calcium amount at the materials' surfaces increased over 50% for PURs and 110% on PURs-M. According to the satisfactory physicochemical, mechanical, and biological characteristics of the obtained PURs and PURs-M the fabrication of the scaffolds was performed by using the SC/PL technique combined with TIPS. Obtained PUR scaffolds had pore sizes in the range of 36-100 µm and a porosity of approximately 77%. On the other hand, PUR-M scaffolds had a pore size in the range of 50-100 µm and a porosity of approximately 82%. This morphological characteristic is suitable, according to the references, for using such scaffolds in bone tissue engineering applications. Forthermore, in the case of PURs-M scaffolds particles of GCPa modifier were visible, which were not washed out during the scaffold fabrication process. Thus, according to the results of the MTT assay, it can be concluded that PURs-M may be a suitable candidate for bone tisue engineering applications [43].",
"In this paper we reported the synthesis and characteristic of PEEURs, obtained by using raw materials (HDI, Polios, PEG, and BDO), selected to reach high biocompatibility of the materials. Moreover, we successfully modified the PEEUR chains by incorporating into them GPCa modifier, confirmed by various techniques. The stronger hydrogen bonding, lower contact angle, and higher total surface free energy as well as the more suitable mechanical properties and good biocompatibility of PURs-M let to the conclusion that these materials possess satisfactory characteristics of materials dedicated to bone tissue engineering. Further studies of calcification of these materials indicated the superior effect of GPCa on these processes. Moreover, the short term interactions study revealed that the obtained PUR-M materials undergo gradual degradation in selected basic and acidic environment by chain defragmentation and such degradable materials are being widely developed for tissue scaffolds. The obtained porous scaffolds, by using the SC/PL technique combined with TIPS, represented suitable pore sizes (36-100 µm) and porosity (77-82%) to serve as templates for bone tissue regeneration. Moreover, the biocompatibility of PURs-M scaffolds was superior in comparison to PURs. Thus, further studies in this direction will be developed in our team."
] | [] | [
"Introduction",
"Experimental",
"Poly(ester ether urethane)s Synthesis",
"Experimental",
"Poly(ester ether urethane)s Synthesis",
"Characterization Methods",
"Indications of Free Isocyanate Groups (FNCO) by the Acidimetric Method",
"Fourier Transform Infrared Spectroscopy (FTIR)",
"Raman Spectroscopy",
"Characterization Methods",
"Indications of Free Isocyanate Groups (F NCO ) by the Acidimetric Method",
"Fourier Transform Infrared Spectroscopy (FTIR)",
"Raman Spectroscopy",
"Static Contact Angle Determination",
"Mechanical Properties",
"Short-Term Interactions Study Performed in Selected Environments",
"In Vitro Cytocompatibility",
"Cell Viability Assay",
"Calcification Study",
"Scaffold Fabrication",
"Results and Discussion",
"Fourier Transform Infrared Spectroscopy (FTIR)",
"Raman Spectroscopy",
"Raman Spectroscopy",
"Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy(SEM/EDX)",
"Contact Angle",
"Mechanical Properties",
"Contact Angle",
"Mechanical Properties",
"Mechanical Properties",
"In Vitro Cytocompatibility",
"Short-Term Interactions Study Performed in Selected Environments",
"In Vitro Cytocompatibility",
"In Vitro Cytocompatibility",
"Short-Term Interactions Study Performed in Selected Environments",
"Short-Term Interactions Study Performed in Selected Environments",
"Calcification Study",
"Calcification Study",
"Calcification Study",
"Fabrication of PUR and PURs-M Scaffolds",
"Fabrication of PUR and PURs-M Scaffolds",
"Fabrication of PUR and PURs-M Scaffolds",
"Discussion",
"Conclusions",
"Figure 1 .Figure 1 .",
"2. 2 . 4 .",
"Figure 1 .",
"2. 2 . 4 .",
"Figure 2 Figure 2 .",
"Figure 2 .",
"Figure 3 .",
"Figure 3 .",
"Figure 4 .",
"Figure 4 .",
"Figure 5 .",
"Figure 6",
"Figure 6 .",
"Figure 5 .",
"Figure 6 Figure 5 .",
"Figure 6",
"Figure 6 .",
"Figure 6 .",
"Figure 7 .Figure 7 .",
"Figure 7 .Figure 8 .Figure 8 .",
"Figure 9 .",
"Figure 8 .",
"Figure 8 .",
"Figure 9 .",
"Figure 9 .",
"Figure 10 .",
"Figure 10 .",
"Figure 11 .",
"Figure 11 .",
"Figure 12 .",
"Figure 13 .",
"Figure 12 .",
"Figure 12 .",
"Figure 13 .",
"Figure 13 .",
"Table 1 .",
"Table 2 .",
"Table 2 .",
"Table 3 .",
"Table 3 .",
"Table 4 .",
"Table 5 .",
"Table 5 .",
"Table 6 .",
"Table 6 .",
"Table 6 ."
] | [
"Time of \nprepolymeryzation (h) \n\nContent of the free isocyanate groups \nin the unmodified prepolymer (NCO ) \n\nContent of the free isocyanate groups \nin the modified prepolymer (NCO ) \n\nPEEUR \nPEEUR-M \n\n0 \n10.14 ± 0.03 \n10.14 ± 0.03 \n1 \n9.04 ± 0.01 \n9.04 ± 0.01 \n2 \n8.83 ± 0.03 \n8.83 ± 0.03 \n3 \n8.54 ± 0.03 \n8.54 ± 0.03 \n4 \n8.54 ± 0.02 \n8.54 ± 0.02 \n5 \n8.54 ± 0.02 \n8.43 ± 0.01 * \n6 \n8.53 ± 0.03 \n8.33 ± 0.03 \n7 \n8.53 ± 0.01 \n8.14 ± 0.03 \n8 \n8.53 ± 0.02 \n8.12 ± 0.02 \n9 \n8.53 ± 0.01 \n8.12 ± 0.03 \n\n* addition of the GPCa modifier. \n\n",
"PUR \nPUR-M \nBand \nDescription \nWavelength (cm −1 ) \n3392 \n3326w \n\n3380 \n3318w \nνNH \nstretching of NH groups, hydrogen bonded withthe C=O of the \nester group present in macrodiol and in GCPa modifier \n2942m \n2868m \n\n2942m \n2868m \nνCH2 \nstretching of aliphatic asymmetric and symmetric CH2 groups \npresent in the PUR chain and in GCPa modifier \n\n1728m \n1728s \nνC=O \nstretching of C=O in the ester and urethane groups, which were \nnot bonded \n1680m \n1680s \nνC=O \nstretching of C=O groups which formed hydrogen bonds \n1535m \n1535s \nνC-N \nstretching between CN in urethane group \n1462w \n1416w \n1350w \n\n1459m \n1415m \n1347m \n\nδCH2 \n\ndeformation vibrations of aliphatic CH2 groups present in the \nPUR and GPCa modifier: bending, wagging, scissoring \nin plane \n1260w \n1214m \n\n",
"1680m \n1680s \nνC=O \nstretching of C=O groups which formed hydrogen bonds \n\n1535m \n1535s \nνC-N \nstretching between CN in urethane group \n\n1462w \n1416w \n1350w \n\n1459m \n1415m \n1347m \n\nδCH 2 \n\ndeformation vibrations of aliphatic CH 2 groups present in the \nPUR and GPCa modifier: bending, wagging, scissoring \nin plane \n\n1260w \n1214m \n\n1238s \n1216s \nνC-(C=O)-O \nstretching vibrations of -C-(C=O)-O-of ester group, not \nhydrogen bonded \n\n1170m \n1170s \nνNH-(C=O)-O \nstretching vibrations of -NH-(C=O)-O-of urethane group \n\n1135m \n1080m \n1058m \n947w \n\n1134s \n1061s \n993s \n949s \n\nνC-(C=O)-O \nνC-O \nstretching vibration of hydrogen bonded -C-(C=O)-O-, \n\n868w \n813w \n775w \n731w \n636w \n\n867s \n777s \n730s \n638s \n\nδCH 2 , \nδNH \nδOH \n\nout of the plane deformation of CH 2 and CH 3 groups as well \nas NH and OH groups \n\n",
"Wavelength (cm −1 ) \nAssignments * \n\n3328 \nstretching vibrations of N-H in urethane groups (as for II-ary amides) \n\n2925, 2890 \nthe strongest polarized stretching vibrations of asymmetric and symmetric CH2 \n\ngroups present in PUR chains. \n\n1735, 1685 \nstretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and \n\nurethane groups (as for II-ary amides) respectively \n\n1480, 1450, \n\n1443, 1424 \nstrong planar deforming vibrations (scissoring) of CH2 groups \n\n",
"Wavelength (cm −1 ) \nAssignments * \n\n3328 \nstretching vibrations of N-H in urethane groups (as for II-ary amides) \n\n2925, 2890 \nthe strongest polarized stretching vibrations of asymmetric and symmetric CH 2 \ngroups present in PUR chains. \n\n1735, 1685 \nstretching vibrations of carbonyl groups present in macrodiol Polios 55/20 and \nurethane groups (as for II-ary amides) respectively \n1480, 1450, \n1443, 1424 \nstrong planar deforming vibrations (scissoring) of CH 2 groups \n\n",
"Symbol \n\nContact angle (°) \nSurface energy (mN/m) \n\nFormamide \nEthylene glycol \nWater \nAcid-part \nBase-part \nTotal surface \nfree energy \nPUR \n63.6 ± 2 \n68.2 ± 1 \n72.1 ± 2 \n0.04 \n20.83 \n31.82 \nPUR-M \n35.8 ± 4 \n47.5 ± 3 \n57 ± 3 \n14.51 \n21.72 \n59.09 \n\n",
"Symbol \n\nContact angle ( • ) \nSurface energy (mN/m) \n\nFormamide \nEthylene glycol \nWater \nAcid-part \nBase-part \nTotal surface \nfree energy \n\nPUR \n63.6 ± 2 \n68.2 ± 1 \n72.1 ± 2 \n0.04 \n20.83 \n31.82 \nPUR-M \n35.8 ± 4 \n47.5 ± 3 \n57 ± 3 \n14.51 \n21.72 \n59.09 \n\n",
"Sample \nExtracted mass (%) \n\n5 M NaOH \n2 N HCl \n0.1 M CoCl 2 /20% H 2 O 2 \n\nPUR \n50.9 ± 0.2 \n38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 \n35.81 ± 0.11 \n2.03 ± 0.12 \n\n",
"mass loss of the PURs and PURs-M after 15 days of short-term interactions study \nperformed with selected media of the acidic, basic, and oxidative environment. \n\nSample \nExtracted mass (%) \n\n5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2 \nPUR \n50.9 ± 0.2 38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 35.81 ± 0.11 \n2.03 ± 0.12 \n\n",
"Sample \nExtracted mass (%) \n5 M NaOH 2 N HCl 0.1 M CoCl2/20% H2O2 \nPUR \n50.9 ± 0.2 38.62 ± 0.14 \n4.25 ± 0.08 \nPUR -M \n54.7 ± 0.1 35.81 ± 0.11 \n2.03 ± 0.12 \n\n"
] | [
"Table 1",
"Table 1",
"Table 2",
"Table 2",
"Table 2",
"Tables 3 and 4",
"Tables 3 and 4",
"(Tables 4 and 5",
"Table 5",
"Table 5",
"(Table 5",
"Table 5",
"Table 5",
"(Table 5",
"Table 6",
"(Table 6",
"Table 6",
"(Table 6",
"Table 6",
"(Table 6)"
] | [
"The Influence of Calcium Glycerophosphate (GPCa) Modifier on Physicochemical, Mechanical, and Biological Performance of Polyurethanes Applicable as Biomaterials for Bone Tissue Scaffolds Fabrication",
"The Influence of Calcium Glycerophosphate (GPCa) Modifier on Physicochemical, Mechanical, and Biological Performance of Polyurethanes Applicable as Biomaterials for Bone Tissue Scaffolds Fabrication"
] | [
"Polymers"
] |
17,981,576 | 2022-03-15T16:18:38Z | CCBY | http://downloads.hindawi.com/journals/jl/2011/702853.pdf | GOLD | d9bdb2f1aef2d8e9fc5211a42ef515041851aaf7 | null | null | null | null | 10.1155/2011/702853 | 1977516421 | 21837278 | 3151505 |
Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?
Hindawi Publishing CorporationCopyright Hindawi Publishing Corporation
Oksana Tehlivets [email protected]
Institute of Molecular Biosciences
University of Graz
Humboldtstrasse 50/II8010GrazAustria
Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?
Journal of Lipids
Hindawi Publishing Corporation20111110.1155/2011/702853Received 1 March 2011; Accepted 4 June 2011Review Article Correspondence should be addressed to Oksana Tehlivets, oksana. Academic Editor: George Leondaritis
Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed.
Introduction
The first indication that sulfur amino acid metabolism is linked to atherosclerosis came from observations in 1953 demonstrating that pathogenic cholesterol concentrations and experimental atherogenesis in monkeys can be inhibited by dietary methionine [1]. Since the early 60s elevated Hcy levels in blood (hyperhomocysteinemia) caused by different deficiencies of sulfur amino acid metabolism were reported to be associated with vascular disease and, in particular, with atherosclerotic plaque formation [2,3]. Today, Hcy is recognized by many studies as a strong, independent and causal risk factor for atherosclerosis [4][5][6][7][8], although there is still controversy on the underlying metabolic connections [9]. In addition to its association with vascular diseases, Hcy is also linked to neurological disorders [10], aging [11], and all-cause mortality [12]. Understanding the pathological mechanisms triggered by Hcy is, therefore, essential for understanding its role in several disease states.
Numerous mechanisms have been proposed that explain pathological changes associated with elevated Hcy levels (reviewed in [3]). Several of them, for example, protein homocysteinylation and oxidative stress, are directly triggered by Hcy. However, not Hcy, but rather AdoHcy, an immediate precursor of Hcy (Figure 1), emerged as a more sensitive indicator of cardiovascular disease during the last decade [13,14]. Supporting the potentially pathogenic role of AdoHcy, studies in yeast showed that indeed AdoHcy is more toxic than Hcy to cells that are deficient in Hcy catabolism [15].
AdoHcy is synthesized as a universal byproduct of AdoMet-dependent methyltransferase reactions ( Figure 1). It is a strong competitive inhibitor of many AdoMetdependent methyltransferases [16] and, therefore, has to be removed to sustain these reactions. The only eukaryotic enzyme capable of AdoHcy catabolism, S-adenosyl-Lhomocysteine hydrolase (Sah1 in yeast, AHCY in mammals), catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. The equilibrium of S-adenosyl-L-homocysteine hydrolase-catalyzed reaction lies far in the direction of synthesis, and both Hcy and adenosine have to be quickly metabolized in order to drive the net hydrolysis of AdoHcy [17]. Therefore, accumulation of hydrolytic products of the S-adenosyl-L-homocysteine hydrolase-catalyzed reaction, in particular Hcy, results in AdoHcy synthesis and accumulation showing that AdoHcy is not only the precursor, but also the product of Hcy metabolism in vivo [18][19][20].
Changes at the epigenetic level are the most extensively studied consequences of methylation deficiency [21][22][23][24]. However, phospholipid methylation that requires three sequential AdoMet-dependent methylation steps to synthesize one molecule of phosphatidylcholine (PC) from phosphatidylethanolamine (PE), the predominant way for PC synthesis in yeast, in particular, in the absence of choline and ethanolamine in the culture medium, is the major consumer of AdoMet. Phospholipid methylation is also the major consumer of AdoMet in mice, since the loss of phosphatidylethanolamine N-methyltransferase (PEMT) in PEMT −/− knockout mice leads to a 50% decrease in plasma Hcy levels [25]. Reexamination of methylation metabolism in humans also revealed that phospholipid methylation, but not creatine synthesis, as was assumed previously, accounts for the major part of AdoMet being utilized in the human body [26].
While PE methylation is the predominant way to synthesize phospholipids in yeast, phospholipid synthesis by the de novo methylation pathway is primarily present in the liver in mammals, where it constitutes 30% of PC production and account for estimated 10 μmol and 1,65 mmol PEMTderived PC secreted into bile per day in mice and humans, respectively [26,27]. However, other mammalian tissues and cells are also capable of phospholipid methylation including brain, skeletal muscle, adipose tissues, fibroblasts, arterial smooth muscle cells, endothelial cells, macrophages, and erythrocytes [28][29][30][31][32][33][34][35][36][37]. The evolutionary conservation of phospholipid methylation suggests its essential role in some specific functions in different cell types. For instance, phospholipid methylation is enhanced in hypertrophied myocardium, correlates with the level of β-adrenergic receptors [38,39] and is stimulated by isoproterenol, a potent cardiac stimulant [40]. In contrast, phospholipid methylation is inhibited by quinidine, an antiarrhythmic drug that causes repression of myocardial contractility [41]. Phospholipid methylation was also observed in microsome preparations from aorta [42,43] and was suggested to affect membrane fluidity and function of membrane calcium channels in aorta [42,43] as well as in heart [40]. Moreover, phospholipid methylation appears to be coupled to Ca 2+ influx and von Willebrand factor release in endothelial cells [35]. In accordance, it was shown that increased methylation of phospholipids is required for an influx of Ca 2+ and subsequent release of histamine in mast cells [44]. Furthermore, Ca 2+ influx was correlated with the release of arachidonic acid in rabbit neutrophils and human fibroblasts, which also appears to require phospholipid methylation [32,45]. Requirement of phospholipid methylation for polyunsaturated fatty acid metabolism was also observed in the brain [46]. It was reported that developing, remyelinating, and diabetic brain exhibits increased synthesis of PC by the de novo methylation pathway in comparison with normal adult brain [47,48]. Phospholipid methylation was shown to be linked to diabetes [49][50][51] and neurological disorders [52,53] also in other studies.
PEMT mRNA and protein levels increase substantially in differentiating adipocytes [30]. It was shown very recently that phospholipid methylation is required for lipid droplet formation and stability in 3T3-L1 adipocytes, and high-fat challenge induces PEMT expression in adipose tissue [54]. Moreover, PEMT and the CDP-choline pathway for PC synthesis are both required for the secretion of very-low-density lipoproteins [55][56][57]. While cells lacking the rate-limiting enzyme of the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase, do not survive [57], deficiency of phospholipid methylation in PEMT −/− mice under choline deprivation results in development of hepatic steatosis followed by steatohepatitis and hyperacute liver failure [58] and is lethal within 5 days [59]. Moreover, deficiency in phospholipid methylation, but not in the synthesis of PC by the CDP-choline pathway, protects from diet-induced obesity in mice due to increased energy utilization suggesting that PEMT plays a role in whole energy metabolism and is linked to insulin signaling [60]. Finally, an isoform of phosphatidylethanolamine N-methyltransferase, PEMT2, appears to be involved in the control of hepatocyte cell division, since its inactivation is associated with several types of liver cell proliferation including tumorigenesis [61]. Vice versa, rat hepatoma cell growth is suppressed by PEMT2 expression [62].
Sensitivity of phospholipid methylation to AdoHcy accumulation [16,18,63] as well as numerous correlations reported for phospholipid methylation pathway suggests that interference with this reaction in Hcy-associated pathology may lead to widespread defects, what indeed seems to be the case. In particular, elevated Hcy levels were found to trigger deregulation of lipid metabolism in yeast and mammalian cells [18,64]. A mechanism of deregulation of lipid metabolism and lipid-associated cellular functions in hyperhomocysteinemia mediated by AdoHcy accumulation and subsequent inhibition of phospholipid methylation is proposed in this paper.
Role of Homocysteine in the Methylation Cycle
Homocysteine is a sulfur-containing amino acid, which does not occur in proteins, but is found at the intersection of methylation and transsulfuration metabolism ( Figure 1, reviewed in [65]). Hcy is formed during methionine metabolism by S-adenosyl-L-homocysteine hydrolase that catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. To be kept in the methylation cycle, Hcy has to be remethylated to methionine, which can be further activated to AdoMet and used by over 50 AdoMet-dependent methyltransferases that release AdoHcy as a by-product after the methyl transfer reaction. The ratio of AdoMet to AdoHcy, that is, the ratio of the substrate versus the specific inhibitor of AdoMet-dependent methyltransferases, is indicative of the cellular methylation potential [66].
In addition to its remethylation to methionine, Hcy can be subjected to transsulfuration leading to the synthesis of cysteine, which is also a precursor of glutathione, an essential cellular defense molecule in oxidative stress response [65]. This pathway irreversibly withdraws Hcy from the methylation cycle. An alternative way for Hcy metabolism is the reversal of the reaction catalyzed by S-adenosyl-Lhomocysteine hydrolase. This occurs upon accumulation of the hydrolytic products of the reaction, in particular Hcy, and leads to AdoHcy synthesis and accumulation [19,[67][68][69]. Thus, elevated Hcy levels via accumulation of AdoHcy lead to the disruption of the methylation cycle and, potentially, to methylation deficiency.
Deficiency in cystathionine β-synthase (CBS), the first and rate-limiting enzyme of the transsulfuration pathway ( Figure 1), is the major cause of severe hyperhomocysteinemia followed by genetic defects of folate and cobalamin metabolism that is involved in Hcy remethylation [65]. These pathological conditions lead to the plasma Hcy levels of more than 100 μmol/L [65] and are rare in comparison with mild hyperhomocysteinemia that is caused by dietary deficiencies of the vitamin cofactors required for Hcy catabolism -folic acid, vitamins B 6 and B 12 , and characterized by the plasma Hcy levels of 15-25 μmol/L [70]. Vitamin B 6 is required for the activity of CBS. Folic acid and vitamin B 6 are required for the activity of methionine synthase catalyzing 5-methyltetrahydrofolate-dependent remethylation of Hcy to methionine (Figure 1). While vitamin supplementation appeared to be a straightforward strategy to reduce/prevent cardiovascular events, this possibility was studied in several large trials. However, it was observed that vitamins, while capable of lowering elevated plasma Hcy levels, do not reduce the rates of vascular events [71]. Several potential mechanisms that might explain this result by offsetting the positive effect of Hcy-lowering therapy were subsequently proposed. These include promotion of cell proliferation by folic acid through its role in the synthesis of thymidine, increase of the methylation potential leading to changes in gene expression, and increase in the levels of asymmetric dimethylarginine that inhibit the activity of nitric oxide synthase [71]. An additional possibility is that, not Hcy, but rather a related metabolite could be a trigger of some pathological changes associated with elevated Hcy levels. Possibly explaining the failure of Hcy-lowering vitamins to reduce vascular events, it was recently reported that supplementation with B-vitamins including folate does not efficiently lower plasma AdoHcy levels [72], presumably due to elevation of AdoMet-dependent methylation.
AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast
In yeast, the synthesis of PC from PE by the de novo phospholipid methylation pathway is particularly sensitive to AdoHcy accumulation [18,63]. Both inhibition of S-adenosyl-Lhomocysteine hydrolase and Hcy supplementation results in AdoHcy accumulation and inhibition of phospholipid methylation in yeast [18]. However, not only phospholipid methylation, but also a methylation-independent branch of lipid metabolism, namely, TAG synthesis, is affected by Ado-Hcy accumulation in yeast: yeast cells deficient in AdoHcy catabolism or supplemented with Hcy massively accumulate TAG [18]. Supporting the causal role of impaired phospholipid methylation in the deregulation of TAG metabolism in response to AdoHcy accumulation, it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of PE to PC, cho2 and opi3, also accumulate TAG [18]. TAG is known to play an important role in buffering excess fatty acids [73]. Therefore, TAG accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to TAG synthesis in methylation deficiency in yeast. Another observation as well supports accumulation of fatty acids under these conditions. In addition to TAG metabolism, transcriptional regulation of phospholipid biosynthesis is also affected in yeast mutants deficient in AdoHcy catabolism. Impaired phospholipid methylation in Sah1-depleted cells unable to hydrolyze AdoHcy or in cho2 and opi3 mutants leads to upregulation of genes, which have an inositol-sensitive upstream regulatory sequence (UAS INO ) in their promoter regions, indicating accumulation of the phospholipid precursor, phosphatidic acid, in the ER [18]. ACC1 encoding acetyl-CoA carboxylase, the first and ratelimiting enzyme of fatty acid biosynthesis, is also a subject to UAS INO -mediated regulation, suggesting upregulation of the de novo fatty acid biosynthesis in response to AdoHcy accumulation. Moreover, Sah1 depletion also affects sterol synthesis in yeast, leading to 4-fold elevated squalene levels and suggesting accumulation of early precursors of ergosterol biosynthesis under these conditions (Tehlivets, Kohlwein, unpublished). Taken together, inhibition of phospholipid methylation induced by AdoHcy accumulation appears to lead to upregulation of fatty acid, TAG, and sterol biosynthetic pathways in yeast.
Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals
AdoHcy inhibits phosphatidylethanolamine N-methyltransferase in vitro and in vivo also in mammals [28,37,74]. Similarly as in yeast, deficiency of phospholipid methylation in PEMT −/− knockout mice leads to a rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in the liver, in the absence of choline supplementation [75]. However, TAG accumulation in the livers of these animals appears to be at least in part due to decreased TAG secretion from hepatocytes [55]. Elevated levels of Hcy are as well linked to deregulation of lipid metabolism in mammals. CBS −/− knockout mice exhibit severe hyperhomocysteinemia and accumulate Ado-Hcy in all tissues tested [68,69]. These mutant animals show elevated TAG and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [76,77]. Another genetic disorder that results in moderately elevated Hcy levels, methylenetetrahydrofolate reductase (MTHFR) deficiency, leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [78,79]. Dietary-induced hyperhomocysteinemia in mice also causes fatty liver, further supporting the role of Hcy in deregulation of lipid metabolism in mammals [64]. In these mice as well as in the CBS −/− knockout mice lipid accumulates in liver rather than in serum [64,76].
Preferable accumulation of lipids in the liver and, possibly, other tissues in hyperhomocysteinemia suggests that other mechanisms than those associated with elevation of circulating lipids are responsible for the development of cardiovascular disease under these conditions. Indeed, conventional risk factors including hypercholesterolemia accounts only for approximately 50% of all cases of cardiovascular disease, while 40% of patiens diagnosed with premature coronary artery disease, peripheral vascular disease or venous thrombosis exhibit hyperhomocysteinemia [80]. In accordance, unlike typical lipid-rich atherosclerotic plagues, vascular lesions associated with hyperhomocysteinemia are lipid-poor, fibrous plaques [81,82], greatly outnumbering fatty atherosclerotic lesions [80].
In animal models of hyperhomocysteinemia atherosclerotic lesions are rare. They are found only in the MTHFR −/− knockout mice that exhibit aortic lipid accumulation reminiscent of early atherosclerotic lesions [2,78,83], however, not in, for example, CBS −/− knockout mice. This discrepancy might be due to disruption of two different Hcy utilizing pathways in these animals. While 5-methyltetrahydrofolatedependent Hcy remethylation occurs in all mammalian cells, transsulfuration of Hcy occurs primarily in the liver and kidney [65]. Thus, impairment of 5-methyltetrahydrofolatedependent Hcy remethylation in MTHFR −/− knockout mice may differently affect Hcy metabolism in comparison to the deficiency in the first step of Hcy transsulfuration in CBS −/− knockout mice. The observation that dietary (methionine or Hcy supplementation) or genetically (CBS gene deletion) induced hyperhomocysteinemia in apoE-deficient (apoE −/− ) mice leads to development of larger and more advanced atherosclerotic lesions clearly demonstrates a causal relationship between elevated Hcy levels and atherosclerosis [83]. In contrast, lack of PEMT was shown to reduce significantly plasma VLDL and to attenuate atherosclerosis in both PEMT −/− /Ldlr −/− mice deficient in PEMT and LDL receptors as well as in PEMT −/− /ApoE −/− mice [84,85].
Role of the Unfolded Protein Response in Hyperhomocysteinemia and Atherosclerosis
Elevated Hcy levels induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in a variety of mammalian cells. These include cultured human hepatocytes, vascular endothelial and aortic smooth muscle cells as well as liver cells of the CBS −/− knockout mice [64,[86][87][88]. Furthermore, elevated Hcy levels lead to activation of the sterol regulatory element-binding proteins (SREBPs), which function to activate genes encoding enzymes in cholesterol, fatty acid, and TAG metabolism and uptake, both in cultured mammalian cell lines as well as in the livers of the CBS −/− knockout mice [64,86]. ER stress appears to play a direct role in the activation of TAG and cholesterol biosynthesis, since overexpression of the ER chaperone GRP78/BiP was reported to inhibit Hcy-induced SREBP-1 gene expression in mammalian cell cultures [64] as well as in mice [89] and lead to reduction of the hepatic steatosis in leptin-deficient (ob/ob) mice [89]. SREBP-1 overcomes translation inhibition induced by UPR through an internal ribosome entry site (IRS), similarly to GRP78 [90].
Confirming the causal role of Hcy in UPR induction and deregulation of lipid metabolism, a decrease of elevated plasma Hcy levels is accompanied by a decrease in hepatic lipids and ER stress response [91]. A strong correlation between lipid metabolism, ER stress response and elevated Hcy levels is also evident form a literature mining approach [92]. Further demonstration of close relationship between ER stress and Hcy metabolism came from the observation that MTHFR involved in Hcy remethylation is induced in response to ER stress [93]. Evolutionary conservation of the relationship between Hcy and UPR is shown by the induction of ER stress and activation of UPR in response to Hcy supplementation in yeast [94]. Finally, demonstrating its pathophysiological role, ER stress was shown to be strongly associated with accelerated atherosclerosis in hyperhomocysteinemic apoE-deficient mice [95], liver diseases [96] as well as hyperglycemia-induced atherosclerosis [97].
Deregulation of Fatty Acid Metabolism in Response to AdoHcy Accumulation
The UPR, as a conserved cellular stress response pathway, is aimed at restoring normal ER and secretory function as well as membrane trafficking upon impaired protein folding in the ER. The de novo methylation and the CDP-choline phospholipid biosynthetic pathways produce phospholipid species with distinct fatty acyl chain composition in yeast: the de novo phospholipid methylation pathway produces more unsaturated phospholipids [98,99]. Similarly, the PEMT-generated PC pool in mammals is also enriched in unsaturated fatty acids [100,101]. Supporting the role of PEMT in metabolism of unsaturated fatty acids PEM −/− knockout mice were reported to accumulate more saturated PC molecular species in the liver compared with the control littermates [60] and to exhibit dramatically reduced concentrations of polyunsaturated fatty acids in the plasma and in hepatic PC, independently of choline status [102]. Thus, beyond its role as a compensatory pathway for PC biosynthesis under conditions of choline deprivation, phospholipid methylation plays a crucial role in unsaturated fatty acid metabolism both in yeast and in mammals. The observation that deficiency of phospholipid methylation in cho2 and opi3 yeast mutants is synthetically lethal in the absence of a functional UPR [103] suggests an essential requirement of UPR in response to impaired phospholipid methylation. Thus, Hcy accumulation, which was shown to lead to AdoHcy-mediated inhibition of phospholipid methylation in yeast, may lead to accumulation of saturated fatty acids in membrane phospholipids-a potential pathological mechanism that might be shared by both yeast and mammals. Indeed, accumulation of saturated fatty acids in membrane phospholipids interferes with ER structure and integrity, induces ER stress and leads to cell death in mammalian cell cultures [104,105]. Accordingly, decreased membrane phospholipid desaturation due to stearoyl-CoA desaturase 1 knockdown induces UPR in HeLa cells [106]. Vice versa, overexpression of stearoyl-CoA desaturase attenuates palmitate-induced ER stress and protects from lipoapoptosis [107][108][109]. Treatment with the molecular chaperone 4phenyl butyrate, which is capable of stabilization of protein conformation, improvement of ER folding capacity and facilitation of protein trafficking, leads to abolishment of UPR induction in yeast subjected to lipid-induced ER stress [105]. This finding suggests that accumulation of saturated fatty acids in membrane phospholipids first leads to changes in the membrane environment followed by induction of ER stress and accumulation of misfolded protein(s) that, in turn, activate UPR [105]. Potential mechanisms involved in saturated fatty acid-induced UPR include depletion of ER Ca 2+ stores leading to decreased ER chaperone activity and protein misfolding, as well as interference with ER-to-Golgi trafficking [110]. Recently, proteomic studies showed that carboxypeptidase E, a key enzyme involved in processing [111] and sorting of insulin [112], is involved in induction of ER stress in β-cells in response to palmitate treatment [113]. Degradation of carboxypeptidase E in palmitateinduced ER stress is mediated by palmitate metabolism and Ca 2+ flux [113]. Alternatively, changes of the ER membrane environment may directly activate ER sensors IRE1, ATF6, and PERK or modulate the binding of the ER sensors to the ER chaperone GRP78 causing its dissociation and activation of UPR pathways.
Supporting the hypothesis that Hcy interferes with phospholipid acyl chain composition it was observed in humans that elevated plasma AdoHcy levels are negatively correlated with both PC content and the level of polyunsaturated fatty acids in PC, but not in PE, in red blood cells in Alzheimer's patients [114]. Elevated plasma Hcy levels were also shown to be associated with a decrease in polyunsaturated (docosahexaenoic) fatty acids in the plasma of healthy humans [115] and in the plasma and erythrocytes of cystic fibrosis patients; these individuals exhibit increased Hcy and AdoHcy levels as well as altered PE and PC metabolism [116].
Taken together, deficiency of phospholipid methylation caused by AdoHcy accumulation in Hcy-associated pathology appears to lead to an increase in saturated PC molecular species in ER membranes followed by ER stress, protein misfolding, induction of UPR, and activation of lipid metabolism ( Figure 2). Upregulation of lipid biosynthesis, which apparently should serve to compensate for suboptimal composition of membrane lipids, leads, however, to accumulation of fatty acids, TAG and sterols in the absence of functional phospholipid methylation. In addition to a role in formation of a specific pool of PC molecular species, phospholipid methylation is crucial for maintenance of a distinct PC/PE ratio important for cell integrity when dietary choline-supply is blunted. Decrease of the PC/PE ratio was reported to result in increased cell permeability of hepatocytes from PEMT −/− mice fed choline deficient diet leading to liver damage [75]. Similarly, decrease of PC or increase in PE was shown to lead to cell damage and/or death in several other mammalian cell types [117][118][119]. Finally, observation of different outcomes in hyperhomocysteinemic apoE −/− mice and both in PEMT −/− /Ldlr −/− and PEMT −/− /ApoE −/− mice suggests mechanisms besides inhibition of phospholipid methylation, for example, AdoHcy-dependent modulation of gene expression that may also contribute to the development of Hcy-dependent atherosclerosis.
Concluding Remarks
The metabolism of homocysteine, consequences of its accumulation as well as associated AdoHcy-triggered inhibition of AdoMet-dependent methylation are complex. In this paper a novel mechanism of Hcy-triggered deregulation Figure 2: A model of activation of UPR and upregulation of fatty acid, TAG, and sterol biosynthesis in response to inhibition of phospholipid methylation in hyperhomocysteinemia. Elevated Hcy levels via AdoHcy accumulation and inhibition of phospholipid methylation lead to accumulation of saturated PC molecular species in ER membranes followed by ER stress, UPR activation, and upregulation of fatty acid, TAG, and sterol biosynthesis. The enzymes involved in yeast and mammalian metabolism are shown in grey circles (see Figure 1). PE: phosphatidylethanolamine; PC: phosphatidylcholine; PEMT: phosphatidylethanolamine N-methyltransferase in mammals; Cho2 and Opi3: phosphatidylethanolamine N-methyltransferases in yeast. of lipid metabolism and UPR induction that is mediated through an AdoHcy-dependent inhibition of phospholipid methylation and based on experimental evidence derived from both yeast and mammalian systems is proposed. The observations made in yeast and mammals are summarized in Table 1.
S-adenosyl-L-homocysteine hydrolase is recognized since many years as a target for antiviral drug design [120]. Inhibitors that block AdoHcy hydrolysis are efficient against many types of viruses including Ebola and show other effects of pharmacological importance [120][121][122]. However, they are associated with high cytotoxicity due to interference with central metabolic pathways [121,122]. While using these inhibitors to study the effects of AdoHcy accumulation appears to be straightforward, ability of some of nucleoside inhibitors of S-adenosyl-L-homocysteine hydrolase to undergo metabolic phosphorylation to nucleotides may account for a part of their biological activities [121,122]. Inhibitors that would be able to block selectively homocysteine and adenosine conversion to AdoHcy are not available. However, based on current understanding of regulation of homocysteine and methionine metabolism (i) selective blockage of AdoHcy synthesis from homocysteine and adenosine that will relieve not only inhibition of phospholipid methylation but also many other AdoMetdependent methyltransferase reactions, combined with (ii) vitamin B 6 supplementation in order to accelerate homocystein catabolism by transsulfuration pathway, may serve as a way to reduce AdoHcy in hyperhomocysteinemia without elevation of AdoMet.
Many questions in the model presented in this paper are still unanswered. Is AdoHcy-mediated accumulation of saturated fatty acids in membrane lipids indeed the way by which elevated Hcy levels induce ER stress? What role do specific lipid precursors have in regulation of lipid metabolism in UPR? What is the impact of AdoHcy accumulation on other methylation reactions unrelated to lipid metabolism? What is the role of deficient phospholipid methylation in homocysteine-associated pathology beyond deregulation of fatty acid, TAG, sterol metabolism, and UPR induction? Elucidation of the molecular mechanisms triggered by elevated Hcy levels will undoubtfully improve our understanding of its pathological role in numerous diseases.
Abbreviations
AdoMet:
S-adenosyl-L-methionine AdoHcy:
S-adenosyl-L-homocysteine Hcy:
Homocysteine PC:
Phosphatidylcholine PE:
Phosphatidylethanolamine TAG:
Triacylglycerol UPR:
Unfolded protein response Sah1:
S-adenosyl-L-homocysteine hydrolase in yeast AHCY:
S-adenosyl-L-homocysteine hydrolase in mammals Cho2 and Opi3: Phosphatidylethanolamine N-methyltransferases in yeast PEMT:
Phosphatidylethanolamine N-methyltransferase in mammals CBS:
Cystathionine β-synthase MTHFR:
Methylenetetrahydrofolate reductase.
Figure 1 :
1Role of AdoHcy and Hcy in AdoMet-dependent methylation in yeast and mammals. The enzymes involved in yeast and mammalian metabolism are shown in grey circles. AdoMet: S-adenosyl-L-methionine; AdoHcy: S-adenosyl-L-homocysteine; Hcy: homocysteine; Met: methionine; CTT: cystathionine; in yeast: Sah1: S-adenosyl-L-homocysteine hydrolase; Sam1 and Sam2: Sadenosyl-L-methionine synthetases; Met6: methionine synthase; Str4: cystathionine β-synthase; Str1: cystathionine γ-lyase; Gsh1: γ-glutamylcysteine synthetase; in mammals: AHCY: S-adenosyl-L-homocysteine hydrolase; MAT: methionine adenosyltransferase; MFMT: 5-methyltetrahydrofolate homocysteine methyltransferase; BHMT: betaine homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; GSH: glutathione synthase.
6
Journal of LipidsUpregulation of fatty acid, TAG and sterol biosynthes sUPR
ER stress
PE
PC
Cho2
Opi3
PEMT
AdoMet
AdoHcy
Sam1/Sam2
MAT
MET
Hcy
Sah1/AHCY
Met6
MFMT/BHMT
CTT
Cysteine
Glutathione
Gsh1/GCS
Str1/CTH
Str4/CBS
Accumulation of SFA
in membrane phospholipids
i
Table 1 :
1Experimental evidence on deregulation of lipid metabolism and UPR induction under elevated homocysteine levels in yeast and mammals * .Hcy/AdoHcy levels are inversely correlated to the levels of unsaturated fatty acids + * The absence of a plus sign in some columns implies lack of data or nonapplicability.Experimental evidence
Yeast
Mammals
AdoHcy is formed in vivo in
response to elevated Hcy levels
+
+
AdoHcy is more toxic than Hcy
to cells deficient in Hcy
catabolism
+
AdoHcy represents a better
marker of cardiovascular risk
than Hcy
+
Phospholipid methylation is
quantitatively the major
consumer of AdoMet
+
+
Phospholipid methylation is
inhibited in response to Hcy
supplementation
+
Phospholipid methylation is
inhibited by AdoHcy
+
+
TAG is accumulating in response
to Hcy supplementation
+
+
TAG is accumulating in response
to deficiency in AdoHcy
hydrolysis
+
TAG is accumulating in response
to deficiency in phospholipid
methylation
+
+
UPR is inducted in response to
Hcy supplementation
+
+
The de novo phospholipid
methylation pathway produces
phospholipids enriched in
unsaturated fatty acids
+
+
ER stress is inducted by
accumulation of saturated fatty
acids in membrane
phospholipids
+
+
AcknowledgmentsThe author thanks Dr. Sepp D. Kohlwein, Institute of Molecular Biosciences, University of Graz for critically reading this paper and Dr. Ernst Steyrer, Institute of Molecular Biology and Biochemistry, Medical University Graz for the helpful comments. Work in the author's laboratory is supported by the Austrian Science Fund FWF, project P18094-B14.
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| [
"Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed.",
"Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed."
] | [
"Oksana Tehlivets [email protected] \nInstitute of Molecular Biosciences\nUniversity of Graz\nHumboldtstrasse 50/II8010GrazAustria\n",
"Oksana Tehlivets [email protected] \nInstitute of Molecular Biosciences\nUniversity of Graz\nHumboldtstrasse 50/II8010GrazAustria\n"
] | [
"Institute of Molecular Biosciences\nUniversity of Graz\nHumboldtstrasse 50/II8010GrazAustria",
"Institute of Molecular Biosciences\nUniversity of Graz\nHumboldtstrasse 50/II8010GrazAustria"
] | [
"Oksana",
"Oksana"
] | [
"Tehlivets",
"Tehlivets"
] | [
"G V Mann, ",
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"R Hosni, ",
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"H Refsum, ",
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"S A Christopher, ",
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"D Ingrosso, ",
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"H Wang, ",
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"I P Pogribny, ",
"M A Caudill, ",
"R Castro, ",
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"D E Vance, ",
"R L Jacobs, ",
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"D E Vance, ",
"R C Reitz, ",
"D J Mead, ",
"W H WelchJr, ",
"R W Kuncl, ",
"D B Drachman, ",
"Y Kishimoto, ",
"L K Cole, ",
"D E Vance, ",
"C Maziere, ",
"J C Maziere, ",
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"D L Bareis, ",
"V C Manganiello, ",
"F Hirata, ",
"M Vaughan, ",
"J Axelrod, ",
"M Waite, ",
"L Kucera, ",
"L King, ",
"S Crosland, ",
"A H Lichtenstein, ",
"J Walewski, ",
"P Brecher, ",
"C Franzblau, ",
"B Faris, ",
"A V Chobanian, ",
"P G De Groot, ",
"M D Gonsalves, ",
"C Loesberg, ",
"M F Van Buul-Wortelboer, ",
"W G Van Aken, ",
"J A Van Mourik, ",
"S Trajkovic-Bodennec, ",
"J Bodennec, ",
"A H Futerman, ",
"R C Reitz, ",
"D J Mead, ",
"R A Bjur, ",
"A H Greenhouse, ",
"W H WelchJr, ",
"C J Limas, ",
"V Panagia, ",
"K Okumura, ",
"K R Shah, ",
"N S Dhalla, ",
"Y Taira, ",
"V Panagia, ",
"K R Shah, ",
"R E Beamish, ",
"N S Dhalla, ",
"V Panagia, ",
"P K Ganguly, ",
"M P Gupta, ",
"Y Taira, ",
"N S Dhalla, ",
"R K Jaiswal, ",
"E J Landon, ",
"B V Sastry, ",
"E J Landon, ",
"L Owens, ",
"B V Sastry, ",
"T Ishizaka, ",
"F Hirata, ",
"K Ishizaka, ",
"J Axelrod, ",
"D L Bareis, ",
"F Hirata, ",
"E Schiffmann, ",
"J Axelrod, ",
"V Magret, ",
"L Elkhalil, ",
"F Nazih-Sanderson, ",
"S Tashiro, ",
"K Sudou, ",
"A Imoh, ",
"M Koide, ",
"Y Akazawa, ",
"J K Blusztajn, ",
"S H Zeisel, ",
"R J Wurtman, ",
"V Panagia, ",
"Y Taira, ",
"P K Ganguly, ",
"S Tung, ",
"N S Dhalla, ",
"Y Taira, ",
"P K Ganguly, ",
"V Panagia, ",
"N S Dhalla, ",
"C S Hartz, ",
"K M Nieman, ",
"R L Jacobs, ",
"D E Vance, ",
"K L Schalinske, ",
"Z Z Guan, ",
"Y N Wang, ",
"K Q Xiao, ",
"P S Hu, ",
"J L Liu, ",
"E S Lee, ",
"C G Charlton, ",
"G Hoerl, ",
"A Wagner, ",
"L K Cole, ",
"A A Noga, ",
"Y Zhao, ",
"D E Vance, ",
"A A Noga, ",
"D E Vance, ",
"D E Vance, ",
"Z Li, ",
"R L Jacobs, ",
"C J Walkey, ",
"L Yu, ",
"L B Agellon, ",
"D E Vance, ",
"Z Li, ",
"L B Agellon, ",
"D E Vance, ",
"R L Jacobs, ",
"Y Zhao, ",
"D P Koonen, ",
"L Tessitore, ",
"I Dianzani, ",
"Z Cui, ",
"D E Vance, ",
"Z Cui, ",
"M Houweling, ",
"D E Vance, ",
"P M Gaynor, ",
"G M Carman, ",
"G H Werstuck, ",
"S R Lentz, ",
"S Dayal, ",
"J D Finkelstein, ",
"J J Martin, ",
"D L Kramer, ",
"C W Porter, ",
"R T Borchardt, ",
"J R Sufrin, ",
"Y Isa, ",
"H Tsuge, ",
"T Hayakawa, ",
"S Dayal, ",
"T Bottiglieri, ",
"E Arning, ",
"S F Choumenkovitch, ",
"J Selhub, ",
"P J Bagley, ",
"D W Jacobsen, ",
"J Loscalzo, ",
"T J Green, ",
"C M Skeaff, ",
"J A Mcmahon, ",
"S D Kohlwein, ",
"J S Schanche, ",
"T Schanche, ",
"P M Ueland, ",
"Z Li, ",
"L B Agellon, ",
"T M Allen, ",
"K Namekata, ",
"Y Enokido, ",
"I Ishii, ",
"Y Nagai, ",
"T Harada, ",
"H Kimura, ",
"M Watanabe, ",
"J Osada, ",
"Y Aratani, ",
"Z Chen, ",
"A C Karaplis, ",
"S L Ackerman, ",
"B C Schwahn, ",
"Z Chen, ",
"M D Laryea, ",
"A B Lawrence De Koning, ",
"G H Werstuck, ",
"J Zhou, ",
"R C Austin, ",
"A P Burke, ",
"V Fonseca, ",
"F Kolodgie, ",
"A Zieske, ",
"L Fink, ",
"R Virmani, ",
"K S Mccully, ",
"J Zhou, ",
"R C Austin, ",
"Y Zhao, ",
"B Su, ",
"R L Jacobs, ",
"L K Cole, ",
"V W Dolinsky, ",
"J R Dyck, ",
"D E Vance, ",
"J Hamelet, ",
"K Demuth, ",
"J L Paul, ",
"J M Delabar, ",
"N Janel, ",
"P A Outinen, ",
"S K Sood, ",
"P C Liaw, ",
"K Kokame, ",
"H Kato, ",
"T Miyata, ",
"H L Kammoun, ",
"H Chabanon, ",
"I Hainault, ",
"S M Colgan, ",
"A A Hashimi, ",
"R C Austin, ",
"C Ji, ",
"N Kaplowitz, ",
"P Sharma, ",
"R D Senthilkumar, ",
"V Brahmachari, ",
"D Leclerc, ",
"R Rozen, ",
"A Kumar, ",
"L John, ",
"M M Alam, ",
"J Zhou, ",
"G H Werstuck, ",
"S Lhotak, ",
"C Ji, ",
"G H Werstuck, ",
"M I Khan, ",
"G Femia, ",
"H A Boumann, ",
"P T Chin, ",
"A J Heck, ",
"B De Kruijff, ",
"A I De Kroon, ",
"H A Boumann, ",
"M J Damen, ",
"C Versluis, ",
"A J Heck, ",
"B De Kruijff, ",
"A I De Kroon, ",
"M Tacconi, ",
"R J Wurtman, ",
"C J Delong, ",
"Y J Shen, ",
"M J Thomas, ",
"Z Cui, ",
"S M Watkins, ",
"X Zhu, ",
"S H Zeisel, ",
"M Costanzo, ",
"A Baryshnikova, ",
"J Bellay, ",
"N M Borradaile, ",
"X Han, ",
"J D Harp, ",
"S E Gale, ",
"D S Ory, ",
"J E Schaffer, ",
"L Pineau, ",
"J Colas, ",
"S Dupont, ",
"H Ariyama, ",
"N Kono, ",
"S Matsuda, ",
"T Inoue, ",
"H Arai, ",
"A Peter, ",
"C Weigert, ",
"H Staiger, ",
"C D Green, ",
"L K Olson, ",
"A K Busch, ",
"E Gurisik, ",
"D V Cordery, ",
"M Cnop, ",
"L Ladrière, ",
"M Igoillo-Esteve, ",
"R F Moura, ",
"D A Cunha, ",
"J K Naggert, ",
"L D Fricker, ",
"O Varlamov, ",
"S Dhanvantari, ",
"F S Shen, ",
"T Adams, ",
"K D Jeffrey, ",
"E U Alejandro, ",
"D S Luciani, ",
"M L Selley, ",
"D Li, ",
"N J Mann, ",
"A J Sinclair, ",
"S M Innis, ",
"A G Davidson, ",
"A Chen, ",
"R Dyer, ",
"S Melnyk, ",
"S J James, ",
"C L Yen, ",
"M H Mar, ",
"S H Zeisel, ",
"J A Post, ",
"J J Bijvelt, ",
"A J Verkleij, ",
"K A Da Costa, ",
"M Badea, ",
"L M Fischer, ",
"S H Zeisel, ",
"E De Clercq, ",
"M S Wolfe, ",
"R T Borchardt, ",
"P K Chiang, ",
"G V Mann, ",
"S B Andrus, ",
"N A Mc, ",
"F J Stare, ",
"J Zhou, ",
"G H Werstuck, ",
"S Lhotak, ",
"K S Mccully, ",
"K S Mccully, ",
"K S Mccully, ",
"A G Bostom, ",
"I H Rosenberg, ",
"H Silbershatz, ",
"H Refsum, ",
"P M Ueland, ",
"O Nygård, ",
"S E Vollset, ",
"D S Wald, ",
"M Law, ",
"J K Morris, ",
"P M Ueland, ",
"H Refsum, ",
"S A Beresford, ",
"S E Vollset, ",
"S Seshadri, ",
"A Beiser, ",
"J Selhub, ",
"M El-Sammak, ",
"M Kandil, ",
"S El-Hifni, ",
"R Hosni, ",
"M Ragab, ",
"S E Vollset, ",
"H Refsum, ",
"A , ",
"D M Kerins, ",
"M J Koury, ",
"A Capdevila, ",
"S Rana, ",
"C Wagner, ",
"C Liu, ",
"Q Wang, ",
"H Guo, ",
"S A Christopher, ",
"S Melnyk, ",
"S J James, ",
"W D Kruger, ",
"S Clarke, ",
"K Banfield, ",
"G De La Haba, ",
"G L Cantoni, ",
"N Malanovic, ",
"I Streith, ",
"H Wolinski, ",
"G Rechberger, ",
"S D Kohlwein, ",
"O Tehlivets, ",
"Y Isa, ",
"T Mishima, ",
"H Tsuge, ",
"T Hayakawa, ",
"D R Hoffman, ",
"D W Marion, ",
"W E Cornatzer, ",
"J A Duerre, ",
"D Ingrosso, ",
"A F Perna, ",
"M S Jamaluddin, ",
"X Yang, ",
"H Wang, ",
"S J James, ",
"S Melnyk, ",
"M Pogribna, ",
"I P Pogribny, ",
"M A Caudill, ",
"R Castro, ",
"I Rivera, ",
"H J Blom, ",
"C Jakobs, ",
"I T De Almeida, ",
"A A Noga, ",
"L M Stead, ",
"Y Zhao, ",
"M E Brosnan, ",
"J T Brosnan, ",
"D E Vance, ",
"L M Stead, ",
"J T Brosnan, ",
"M E Brosnan, ",
"D E Vance, ",
"R L Jacobs, ",
"J E Vance, ",
"D E Vance, ",
"R C Reitz, ",
"D J Mead, ",
"W H WelchJr, ",
"R W Kuncl, ",
"D B Drachman, ",
"Y Kishimoto, ",
"L K Cole, ",
"D E Vance, ",
"C Maziere, ",
"J C Maziere, ",
"L Mora, ",
"J Polonovski, ",
"D L Bareis, ",
"V C Manganiello, ",
"F Hirata, ",
"M Vaughan, ",
"J Axelrod, ",
"M Waite, ",
"L Kucera, ",
"L King, ",
"S Crosland, ",
"A H Lichtenstein, ",
"J Walewski, ",
"P Brecher, ",
"C Franzblau, ",
"B Faris, ",
"A V Chobanian, ",
"P G De Groot, ",
"M D Gonsalves, ",
"C Loesberg, ",
"M F Van Buul-Wortelboer, ",
"W G Van Aken, ",
"J A Van Mourik, ",
"S Trajkovic-Bodennec, ",
"J Bodennec, ",
"A H Futerman, ",
"R C Reitz, ",
"D J Mead, ",
"R A Bjur, ",
"A H Greenhouse, ",
"W H WelchJr, ",
"C J Limas, ",
"V Panagia, ",
"K Okumura, ",
"K R Shah, ",
"N S Dhalla, ",
"Y Taira, ",
"V Panagia, ",
"K R Shah, ",
"R E Beamish, ",
"N S Dhalla, ",
"V Panagia, ",
"P K Ganguly, ",
"M P Gupta, ",
"Y Taira, ",
"N S Dhalla, ",
"R K Jaiswal, ",
"E J Landon, ",
"B V Sastry, ",
"E J Landon, ",
"L Owens, ",
"B V Sastry, ",
"T Ishizaka, ",
"F Hirata, ",
"K Ishizaka, ",
"J Axelrod, ",
"D L Bareis, ",
"F Hirata, ",
"E Schiffmann, ",
"J Axelrod, ",
"V Magret, ",
"L Elkhalil, ",
"F Nazih-Sanderson, ",
"S Tashiro, ",
"K Sudou, ",
"A Imoh, ",
"M Koide, ",
"Y Akazawa, ",
"J K Blusztajn, ",
"S H Zeisel, ",
"R J Wurtman, ",
"V Panagia, ",
"Y Taira, ",
"P K Ganguly, ",
"S Tung, ",
"N S Dhalla, ",
"Y Taira, ",
"P K Ganguly, ",
"V Panagia, ",
"N S Dhalla, ",
"C S Hartz, ",
"K M Nieman, ",
"R L Jacobs, ",
"D E Vance, ",
"K L Schalinske, ",
"Z Z Guan, ",
"Y N Wang, ",
"K Q Xiao, ",
"P S Hu, ",
"J L Liu, ",
"E S Lee, ",
"C G Charlton, ",
"G Hoerl, ",
"A Wagner, ",
"L K Cole, ",
"A A Noga, ",
"Y Zhao, ",
"D E Vance, ",
"A A Noga, ",
"D E Vance, ",
"D E Vance, ",
"Z Li, ",
"R L Jacobs, ",
"C J Walkey, ",
"L Yu, ",
"L B Agellon, ",
"D E Vance, ",
"Z Li, ",
"L B Agellon, ",
"D E Vance, ",
"R L Jacobs, ",
"Y Zhao, ",
"D P Koonen, ",
"L Tessitore, ",
"I Dianzani, ",
"Z Cui, ",
"D E Vance, ",
"Z Cui, ",
"M Houweling, ",
"D E Vance, ",
"P M Gaynor, ",
"G M Carman, ",
"G H Werstuck, ",
"S R Lentz, ",
"S Dayal, ",
"J D Finkelstein, ",
"J J Martin, ",
"D L Kramer, ",
"C W Porter, ",
"R T Borchardt, ",
"J R Sufrin, ",
"Y Isa, ",
"H Tsuge, ",
"T Hayakawa, ",
"S Dayal, ",
"T Bottiglieri, ",
"E Arning, ",
"S F Choumenkovitch, ",
"J Selhub, ",
"P J Bagley, ",
"D W Jacobsen, ",
"J Loscalzo, ",
"T J Green, ",
"C M Skeaff, ",
"J A Mcmahon, ",
"S D Kohlwein, ",
"J S Schanche, ",
"T Schanche, ",
"P M Ueland, ",
"Z Li, ",
"L B Agellon, ",
"T M Allen, ",
"K Namekata, ",
"Y Enokido, ",
"I Ishii, ",
"Y Nagai, ",
"T Harada, ",
"H Kimura, ",
"M Watanabe, ",
"J Osada, ",
"Y Aratani, ",
"Z Chen, ",
"A C Karaplis, ",
"S L Ackerman, ",
"B C Schwahn, ",
"Z Chen, ",
"M D Laryea, ",
"A B Lawrence De Koning, ",
"G H Werstuck, ",
"J Zhou, ",
"R C Austin, ",
"A P Burke, ",
"V Fonseca, ",
"F Kolodgie, ",
"A Zieske, ",
"L Fink, ",
"R Virmani, ",
"K S Mccully, ",
"J Zhou, ",
"R C Austin, ",
"Y Zhao, ",
"B Su, ",
"R L Jacobs, ",
"L K Cole, ",
"V W Dolinsky, ",
"J R Dyck, ",
"D E Vance, ",
"J Hamelet, ",
"K Demuth, ",
"J L Paul, ",
"J M Delabar, ",
"N Janel, ",
"P A Outinen, ",
"S K Sood, ",
"P C Liaw, ",
"K Kokame, ",
"H Kato, ",
"T Miyata, ",
"H L Kammoun, ",
"H Chabanon, ",
"I Hainault, ",
"S M Colgan, ",
"A A Hashimi, ",
"R C Austin, ",
"C Ji, ",
"N Kaplowitz, ",
"P Sharma, ",
"R D Senthilkumar, ",
"V Brahmachari, ",
"D Leclerc, ",
"R Rozen, ",
"A Kumar, ",
"L John, ",
"M M Alam, ",
"J Zhou, ",
"G H Werstuck, ",
"S Lhotak, ",
"C Ji, ",
"G H Werstuck, ",
"M I Khan, ",
"G Femia, ",
"H A Boumann, ",
"P T Chin, ",
"A J Heck, ",
"B De Kruijff, ",
"A I De Kroon, ",
"H A Boumann, ",
"M J Damen, ",
"C Versluis, ",
"A J Heck, ",
"B De Kruijff, ",
"A I De Kroon, ",
"M Tacconi, ",
"R J Wurtman, ",
"C J Delong, ",
"Y J Shen, ",
"M J Thomas, ",
"Z Cui, ",
"S M Watkins, ",
"X Zhu, ",
"S H Zeisel, ",
"M Costanzo, ",
"A Baryshnikova, ",
"J Bellay, ",
"N M Borradaile, ",
"X Han, ",
"J D Harp, ",
"S E Gale, ",
"D S Ory, ",
"J E Schaffer, ",
"L Pineau, ",
"J Colas, ",
"S Dupont, ",
"H Ariyama, ",
"N Kono, ",
"S Matsuda, ",
"T Inoue, ",
"H Arai, ",
"A Peter, ",
"C Weigert, ",
"H Staiger, ",
"C D Green, ",
"L K Olson, ",
"A K Busch, ",
"E Gurisik, ",
"D V Cordery, ",
"M Cnop, ",
"L Ladrière, ",
"M Igoillo-Esteve, ",
"R F Moura, ",
"D A Cunha, ",
"J K Naggert, ",
"L D Fricker, ",
"O Varlamov, ",
"S Dhanvantari, ",
"F S Shen, ",
"T Adams, ",
"K D Jeffrey, ",
"E U Alejandro, ",
"D S Luciani, ",
"M L Selley, ",
"D Li, ",
"N J Mann, ",
"A J Sinclair, ",
"S M Innis, ",
"A G Davidson, ",
"A Chen, ",
"R Dyer, ",
"S Melnyk, ",
"S J James, ",
"C L Yen, ",
"M H Mar, ",
"S H Zeisel, ",
"J A Post, ",
"J J Bijvelt, ",
"A J Verkleij, ",
"K A Da Costa, ",
"M Badea, ",
"L M Fischer, ",
"S H Zeisel, ",
"E De Clercq, ",
"M S Wolfe, ",
"R T Borchardt, ",
"P K Chiang, "
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"Tashiro",
"Sudou",
"Imoh",
"Koide",
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"Guo",
"Christopher",
"Melnyk",
"James",
"Kruger",
"Clarke",
"Banfield",
"De La Haba",
"Cantoni",
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"Streith",
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"Rechberger",
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"Tehlivets",
"Isa",
"Mishima",
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"Hayakawa",
"Hoffman",
"Marion",
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"Duerre",
"Ingrosso",
"Perna",
"Jamaluddin",
"Yang",
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"James",
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"Pogribna",
"Pogribny",
"Caudill",
"Castro",
"Rivera",
"Blom",
"Jakobs",
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"Noga",
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"Cole",
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"Mora",
"Polonovski",
"Bareis",
"Manganiello",
"Hirata",
"Vaughan",
"Axelrod",
"Waite",
"Kucera",
"King",
"Crosland",
"Lichtenstein",
"Walewski",
"Brecher",
"Franzblau",
"Faris",
"Chobanian",
"De Groot",
"Gonsalves",
"Loesberg",
"Van Buul-Wortelboer",
"Van Aken",
"Van Mourik",
"Trajkovic-Bodennec",
"Bodennec",
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"Chen",
"Karaplis",
"Ackerman",
"Schwahn",
"Chen",
"Laryea",
"Lawrence De Koning",
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"Austin",
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"Ji",
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"A significant inverse relationship between concentrations of plasma homocysteine and phospholipid docosahexaenoic acid in healthy male subjects. D Li, N J Mann, A J Sinclair, Lipids. 411D. Li, N. J. Mann, and A. J. Sinclair, \"A significant inverse relationship between concentrations of plasma homocysteine and phospholipid docosahexaenoic acid in healthy male subjects,\" Lipids, vol. 41, no. 1, pp. 85-89, 2006.",
"Increased plasma homocysteine and S-adenosylhomocysteine and decreased methionine is associated with altered phosphatidylcholine and phosphatidylethanolamine in cystic fibrosis. S M Innis, A G Davidson, A Chen, R Dyer, S Melnyk, S J James, Journal of Pediatrics. 1433S. M. Innis, A. G. Davidson, A. Chen, R. Dyer, S. Mel- nyk, and S. J. James, \"Increased plasma homocysteine and S-adenosylhomocysteine and decreased methionine is associated with altered phosphatidylcholine and phos- phatidylethanolamine in cystic fibrosis,\" Journal of Pediatrics, vol. 143, no. 3, pp. 351-356, 2003.",
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] | [
"Experimental atherosclerosis in Cebus monkeys",
"Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice",
"Homocysteine, vitamins, and vascular disease prevention",
"Homocysteine and vascular disease",
"Homocysteine, folate, vitamin B6, and cardiovascular disease",
"Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: the Framingham study",
"Homocysteine and cardiovascular disease",
"Homocysteine and cardiovascular disease: evidence on causality from a metaanalysis",
"The controversy over homocysteine and cardiovascular risk",
"Plasma homocysteine as a risk factor for dementia and Alzheimer's disease",
"Elevated plasma homocysteine is positively associated with age independent of C677T mutation of the methylenetetrahydrofolate reductase gene in selected Egyptian subjects",
"Plasma total homocysteine and cardiovascular and noncardiovascular mortality: the Hordaland homocysteine study",
"Plasma S-adenosylhomocysteine is a more sensitive indicator of cardiovascular disease than plasma homocysteine",
"Plasma S-adenosylhomocysteine is a better biomarker of atherosclerosis than homocysteine in apolipoprotein E-deficient mice fed high dietary methionine",
"S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine β-synthase",
"S-adenosylmethionine-dependent methyltransferases",
"The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine",
"S-adenosyl-L-homocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast: implications for homocysteine as a risk factor of atherosclerosis",
"Increase in S-adenosylhomocysteine content and its effect on the S-adenosylhomocysteine hydrolase activity under transient high plasma homocysteine levels in rats",
"S-adenosylmethionine and S-adenosylhomocysteine metabolism in isolated rat liver. Effects of Lmethionine, L-homocysteine, and adenosine",
"Epigenetics in hyperhomocysteinemic states. A special focus on uremia",
"Hyperhomocysteinemia, DNA methylation and vascular disease",
"Elevation in S-Adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology",
"Homocysteine metabolism, hyperhomocysteinaemia and vascular disease: an overview",
"Plasma homocysteine is regulated by phospholipid methylation",
"Is it time to reevaluate methyl balance in humans?",
"Phospholipid biosynthesis in mammalian cells",
"Phospholipid methylation in brain membrane preparations: kinetic mechanism",
"Phospholipid methylation in skeletal muscle membranes",
"A role for Sp1 in transcriptional regulation of phosphatidylethanolamine Nmethyltransferase in liver and 3T3-L1 adipocytes",
"Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts",
"Bradykinin stimulates phospholipid methylation, calcium influx, prostaglandin formation, and cAMP accumulation in human fibroblasts",
"Lipid synthesis in cultured human embryonic fibroblasts",
"Phospholipid methylation in rabbit aorta",
"Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation",
"Phosphatidylcholine metabolism is altered in a monocytederived macrophage model of Gaucher disease but not in lymphocytes",
"Phosphatidylethanolamine Nmethyltransferase in human red blood cell membrane preparations. Kinetic mechanism",
"Effect of phospholipid methylation on βadrenergic receptors in the normal and hypertrophied rat myocardium",
"Modification of sarcolemmal phosphatidylethanolamine Nmethylation during heart hypertrophy",
"Stimulation of phospholipid N-methylation by isoproterenol in rat hearts",
"Alterations of phosphatidylethanolamine Nmethylation in rat heart by quinidine",
"Methylation of phospholipids in microsomes of the rat aorta",
"Effect of Lmethionine on contractile response, calcium influx and calcium channel blocking agents in the rat aorta",
"Stimulation of phospholipid methylation, Ca2+ influx, and histamine release by bridging of IgE receptors on rat mast cells",
"Phospholipid metabolism, calcium flux, and the receptormediated induction of chemotaxis in rabbit neutrophils",
"Entry of polyunsaturated fatty acids into the brain: evidence Journal of Lipids 9 that high-density lipoprotein-induced methylation of phosphatidylethanolamine and phospholipase A2 are involved",
"Phosphatidylethanolamine methyltransferase activity in developing, demyelinating, and diabetic mouse brain",
"Syn thesis of lecithin (phosphatidylcholine) from phosphatidylethanolamine in bovine brain",
"Alterations in phospholipid N-methylation of cardiac subcellular membranes due to experimentally induced diabetes in rats",
"Increased SR phospholipid N-methylation in skeletal muscle of diabetic rats",
"Hepatic phosphatidylethanolamine Nmethyltransferase expression is increased in diabetic rats",
"Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimer's disease",
"1-Methyl-4-phenyl-pyridinium increases S-adenosyl-L-methionine dependent phospholipid methylation",
"Sequential synthesis and methylation of phosphatidylethanolamine promotes lipid droplet biosynthesis and stability in tissue culture and in vivo",
"An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins",
"Insights into the requirement of phosphatidylcholine synthesis for liver function in mice",
"Hepatic phosphatidylethanolamine N-methyltransferase, unexpected roles in animal biochemistry and physiology",
"Biochemical and evolutionary significance of phospholipid methylation",
"Phosphatidylcholine homeostasis and liver failure",
"Impaired de novo choline synthesis explains why phosphatidylethanolamine Nmethyltransferase-deficient mice are protected from dietinduced obesity",
"Diminished expression of phosphatidylethanolamine N-methyltransferase 2 during hepatocarcinogenesis",
"Suppression of rat hepatoma cell growth by expression of phosphatidylethanolamine N-methyltransferase-2",
"Phospatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties",
"Homocysteineinduced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways",
"Homocysteine",
"Combined modulation of S-adenosylmethionine biosynthesis and S-adenosylhomocysteine metabolism enhances inhibition of nucleic acid methylation and L1210 cell growth",
"Effect of vitamin B6 deficiency on S-adenosylhomocysteine hydrolase activity as a target point for methionine metabolic regulation",
"Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine β-synthase-deficient mice",
"In the cystathionine β-synthase knockout mouse, elevations in total plasma homocysteine increase tissue S-adenosylhomocysteine, but responses of S-adenosylmethionine and DNA methylation are tissue specific",
"Homocysteine and vitamins in cardiovascular disease",
"Homocysteine trials-clear outcomes for complex reasons",
"Homocysteine-lowering vitamins do not lower plasma S-adenosylhomocysteine in older people with elevated homocysteine concentrations",
"Obese and anorexic yeasts: experimental models to understand the metabolic syndrome and lipotoxicity",
"Inhibition of phospholipid methylation in isolated rat hepatocytes by analogues of adenosine and S-adenosylhomocysteine",
"The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis",
"Abnormal lipid metabolism in cystathionine βsynthase-deficient mice, an animal model for hyperhomocysteinemia",
"Mice deficient in cystathionine β-synthase: animal models for mild and severe homocyst(e)inemia",
"Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition",
"Homocysteinebetaine interactions in a murine model of 5,10-methylenetetrahydrofolate reductase deficiency",
"Hyperhomocysteinemia and its role in the development of atherosclerosis",
"Increased serum homocysteine and sudden death resulting from coronary atherosclerosis with fibrous plaques",
"Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis",
"Contributions of hyperhomocysteinemia to atherosclerosis: causal relationship and potential mechanisms",
"Lack of phosphatidylethanolamine N-methyltransferase alters plasma VLDL phospholipids and attenuates atherosclerosis in mice",
"Impaired phosphatidylcholine biosynthesis reduces atherosclerosis and prevents lipotoxic cardiac dysfunction in ApoE-/-mice",
"Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice",
"Characterization of the stress-inducing effects of homocysteine",
"Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis: GRP78/BiP and novel genes",
"GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice",
"Endoplasmic reticulum stress and lipid dysregulation",
"Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice",
"Mining literature for a comprehensive pathway analysis: a case study for retrieval of homocysteine related genes for genetic and epigenetic studies",
"Endoplasmic reticulum stress increases the expression of methylenetetrahydrofolate reductase through the IRE1 transducer",
"Homocysteineand cysteine-mediated growth defect is not associated with induction of oxidative stress response genes in yeast",
"Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice",
"Dissection of endoplasmic reticulum stress signaling in alcoholic and non-alcoholic liver injury",
"Glucosamineinduced endoplasmic reticulum dysfunction is associated with accelerated atherosclerosis in a hyperglycemic mouse model",
"The yeast phospholipid N-methyltransferases catalyzing the synthesis of phosphatidylcholine preferentially convert Di-C16:1 substrates both in vivo and in vitro",
"The two biosynthetic routes leading to phosphatidylcholine in yeast produce different sets of molecular species. Evidence for lipid remodeling",
"Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids",
"Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway",
"Phosphatidylethanolamine-N-methyltransferase activity and dietary choline regulate liver-plasma lipid flux and essen-tial fatty acid metabolism in mice",
"The genetic landscape of a cell",
"Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death",
"Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids",
"Decrease in membrane phospholipid unsaturation induces unfolded protein response",
"Individual stearoyl-CoA desaturase 1 expression modulates endoplasmic reticulum stress and inflammation in human myotubes and is associated with skeletal muscle lipid storage and insulin sensitivity in vivo",
"Modulation of palmitateinduced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6",
"Increased fatty acid desaturation and enhanced expression of stearoyl coenzyme A desaturase protects pancreatic β-cells from lipoapoptosis",
"Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction",
"Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity",
"Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia",
"Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis",
"A metabolic link between S-adenosylhomocysteine and polyunsaturated fatty acid metabolism in Alzheimer's disease",
"A significant inverse relationship between concentrations of plasma homocysteine and phospholipid docosahexaenoic acid in healthy male subjects",
"Increased plasma homocysteine and S-adenosylhomocysteine and decreased methionine is associated with altered phosphatidylcholine and phosphatidylethanolamine in cystic fibrosis",
"Choline deficiencyinduced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase",
"Phosphatidylethanolamine and sarcolemmal damage during ischemia or metabolic inhibition of heart myocytes",
"Elevated serum creatine phosphokinase in choline-deficient humans: mechanistic studies in C2C12 mouse myoblasts",
"John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broadspectrum antiviral activity",
"S-Adenosyl-L-homocysteine hydrolase as a target for antiviral chemotherapy",
"Biological effects of inhibitors of S-adenosylhomocysteine hydrolase",
"Experimental atherosclerosis in Cebus monkeys",
"Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice",
"Homocysteine, vitamins, and vascular disease prevention",
"Homocysteine and vascular disease",
"Homocysteine, folate, vitamin B6, and cardiovascular disease",
"Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: the Framingham study",
"Homocysteine and cardiovascular disease",
"Homocysteine and cardiovascular disease: evidence on causality from a metaanalysis",
"The controversy over homocysteine and cardiovascular risk",
"Plasma homocysteine as a risk factor for dementia and Alzheimer's disease",
"Elevated plasma homocysteine is positively associated with age independent of C677T mutation of the methylenetetrahydrofolate reductase gene in selected Egyptian subjects",
"Plasma total homocysteine and cardiovascular and noncardiovascular mortality: the Hordaland homocysteine study",
"Plasma S-adenosylhomocysteine is a more sensitive indicator of cardiovascular disease than plasma homocysteine",
"Plasma S-adenosylhomocysteine is a better biomarker of atherosclerosis than homocysteine in apolipoprotein E-deficient mice fed high dietary methionine",
"S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine β-synthase",
"S-adenosylmethionine-dependent methyltransferases",
"The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine",
"S-adenosyl-L-homocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast: implications for homocysteine as a risk factor of atherosclerosis",
"Increase in S-adenosylhomocysteine content and its effect on the S-adenosylhomocysteine hydrolase activity under transient high plasma homocysteine levels in rats",
"S-adenosylmethionine and S-adenosylhomocysteine metabolism in isolated rat liver. Effects of Lmethionine, L-homocysteine, and adenosine",
"Epigenetics in hyperhomocysteinemic states. A special focus on uremia",
"Hyperhomocysteinemia, DNA methylation and vascular disease",
"Elevation in S-Adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology",
"Homocysteine metabolism, hyperhomocysteinaemia and vascular disease: an overview",
"Plasma homocysteine is regulated by phospholipid methylation",
"Is it time to reevaluate methyl balance in humans?",
"Phospholipid biosynthesis in mammalian cells",
"Phospholipid methylation in brain membrane preparations: kinetic mechanism",
"Phospholipid methylation in skeletal muscle membranes",
"A role for Sp1 in transcriptional regulation of phosphatidylethanolamine Nmethyltransferase in liver and 3T3-L1 adipocytes",
"Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts",
"Bradykinin stimulates phospholipid methylation, calcium influx, prostaglandin formation, and cAMP accumulation in human fibroblasts",
"Lipid synthesis in cultured human embryonic fibroblasts",
"Phospholipid methylation in rabbit aorta",
"Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation",
"Phosphatidylcholine metabolism is altered in a monocytederived macrophage model of Gaucher disease but not in lymphocytes",
"Phosphatidylethanolamine Nmethyltransferase in human red blood cell membrane preparations. Kinetic mechanism",
"Effect of phospholipid methylation on βadrenergic receptors in the normal and hypertrophied rat myocardium",
"Modification of sarcolemmal phosphatidylethanolamine Nmethylation during heart hypertrophy",
"Stimulation of phospholipid N-methylation by isoproterenol in rat hearts",
"Alterations of phosphatidylethanolamine Nmethylation in rat heart by quinidine",
"Methylation of phospholipids in microsomes of the rat aorta",
"Effect of Lmethionine on contractile response, calcium influx and calcium channel blocking agents in the rat aorta",
"Stimulation of phospholipid methylation, Ca2+ influx, and histamine release by bridging of IgE receptors on rat mast cells",
"Phospholipid metabolism, calcium flux, and the receptormediated induction of chemotaxis in rabbit neutrophils",
"Entry of polyunsaturated fatty acids into the brain: evidence Journal of Lipids 9 that high-density lipoprotein-induced methylation of phosphatidylethanolamine and phospholipase A2 are involved",
"Phosphatidylethanolamine methyltransferase activity in developing, demyelinating, and diabetic mouse brain",
"Syn thesis of lecithin (phosphatidylcholine) from phosphatidylethanolamine in bovine brain",
"Alterations in phospholipid N-methylation of cardiac subcellular membranes due to experimentally induced diabetes in rats",
"Increased SR phospholipid N-methylation in skeletal muscle of diabetic rats",
"Hepatic phosphatidylethanolamine Nmethyltransferase expression is increased in diabetic rats",
"Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimer's disease",
"1-Methyl-4-phenyl-pyridinium increases S-adenosyl-L-methionine dependent phospholipid methylation",
"Sequential synthesis and methylation of phosphatidylethanolamine promotes lipid droplet biosynthesis and stability in tissue culture and in vivo",
"An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins",
"Insights into the requirement of phosphatidylcholine synthesis for liver function in mice",
"Hepatic phosphatidylethanolamine N-methyltransferase, unexpected roles in animal biochemistry and physiology",
"Biochemical and evolutionary significance of phospholipid methylation",
"Phosphatidylcholine homeostasis and liver failure",
"Impaired de novo choline synthesis explains why phosphatidylethanolamine Nmethyltransferase-deficient mice are protected from dietinduced obesity",
"Diminished expression of phosphatidylethanolamine N-methyltransferase 2 during hepatocarcinogenesis",
"Suppression of rat hepatoma cell growth by expression of phosphatidylethanolamine N-methyltransferase-2",
"Phospatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties",
"Homocysteineinduced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways",
"Homocysteine",
"Combined modulation of S-adenosylmethionine biosynthesis and S-adenosylhomocysteine metabolism enhances inhibition of nucleic acid methylation and L1210 cell growth",
"Effect of vitamin B6 deficiency on S-adenosylhomocysteine hydrolase activity as a target point for methionine metabolic regulation",
"Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine β-synthase-deficient mice",
"In the cystathionine β-synthase knockout mouse, elevations in total plasma homocysteine increase tissue S-adenosylhomocysteine, but responses of S-adenosylmethionine and DNA methylation are tissue specific",
"Homocysteine and vitamins in cardiovascular disease",
"Homocysteine trials-clear outcomes for complex reasons",
"Homocysteine-lowering vitamins do not lower plasma S-adenosylhomocysteine in older people with elevated homocysteine concentrations",
"Obese and anorexic yeasts: experimental models to understand the metabolic syndrome and lipotoxicity",
"Inhibition of phospholipid methylation in isolated rat hepatocytes by analogues of adenosine and S-adenosylhomocysteine",
"The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis",
"Abnormal lipid metabolism in cystathionine βsynthase-deficient mice, an animal model for hyperhomocysteinemia",
"Mice deficient in cystathionine β-synthase: animal models for mild and severe homocyst(e)inemia",
"Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition",
"Homocysteinebetaine interactions in a murine model of 5,10-methylenetetrahydrofolate reductase deficiency",
"Hyperhomocysteinemia and its role in the development of atherosclerosis",
"Increased serum homocysteine and sudden death resulting from coronary atherosclerosis with fibrous plaques",
"Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis",
"Contributions of hyperhomocysteinemia to atherosclerosis: causal relationship and potential mechanisms",
"Lack of phosphatidylethanolamine N-methyltransferase alters plasma VLDL phospholipids and attenuates atherosclerosis in mice",
"Impaired phosphatidylcholine biosynthesis reduces atherosclerosis and prevents lipotoxic cardiac dysfunction in ApoE-/-mice",
"Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice",
"Characterization of the stress-inducing effects of homocysteine",
"Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis: GRP78/BiP and novel genes",
"GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice",
"Endoplasmic reticulum stress and lipid dysregulation",
"Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice",
"Mining literature for a comprehensive pathway analysis: a case study for retrieval of homocysteine related genes for genetic and epigenetic studies",
"Endoplasmic reticulum stress increases the expression of methylenetetrahydrofolate reductase through the IRE1 transducer",
"Homocysteineand cysteine-mediated growth defect is not associated with induction of oxidative stress response genes in yeast",
"Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice",
"Dissection of endoplasmic reticulum stress signaling in alcoholic and non-alcoholic liver injury",
"Glucosamineinduced endoplasmic reticulum dysfunction is associated with accelerated atherosclerosis in a hyperglycemic mouse model",
"The yeast phospholipid N-methyltransferases catalyzing the synthesis of phosphatidylcholine preferentially convert Di-C16:1 substrates both in vivo and in vitro",
"The two biosynthetic routes leading to phosphatidylcholine in yeast produce different sets of molecular species. Evidence for lipid remodeling",
"Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids",
"Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway",
"Phosphatidylethanolamine-N-methyltransferase activity and dietary choline regulate liver-plasma lipid flux and essen-tial fatty acid metabolism in mice",
"The genetic landscape of a cell",
"Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death",
"Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids",
"Decrease in membrane phospholipid unsaturation induces unfolded protein response",
"Individual stearoyl-CoA desaturase 1 expression modulates endoplasmic reticulum stress and inflammation in human myotubes and is associated with skeletal muscle lipid storage and insulin sensitivity in vivo",
"Modulation of palmitateinduced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6",
"Increased fatty acid desaturation and enhanced expression of stearoyl coenzyme A desaturase protects pancreatic β-cells from lipoapoptosis",
"Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction",
"Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity",
"Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia",
"Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis",
"A metabolic link between S-adenosylhomocysteine and polyunsaturated fatty acid metabolism in Alzheimer's disease",
"A significant inverse relationship between concentrations of plasma homocysteine and phospholipid docosahexaenoic acid in healthy male subjects",
"Increased plasma homocysteine and S-adenosylhomocysteine and decreased methionine is associated with altered phosphatidylcholine and phosphatidylethanolamine in cystic fibrosis",
"Choline deficiencyinduced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase",
"Phosphatidylethanolamine and sarcolemmal damage during ischemia or metabolic inhibition of heart myocytes",
"Elevated serum creatine phosphokinase in choline-deficient humans: mechanistic studies in C2C12 mouse myoblasts",
"John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broadspectrum antiviral activity",
"S-Adenosyl-L-homocysteine hydrolase as a target for antiviral chemotherapy",
"Biological effects of inhibitors of S-adenosylhomocysteine hydrolase"
] | [
"the National Academy of Sciences of the United States of America",
"the National Academy of Sciences of the United States of America",
"the National Academy of Sciences of the United States of America",
"the National Academy of Sciences of the United States of America",
"the National Academy of Sciences of the United States of America",
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"Journal of Biological Chemistry",
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"Journal of Biological Chemistry",
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"Pharmacology Biochemistry and Behavior",
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"Journal of Lipid Research",
"Journal of Biological Chemistry",
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"Journal of Biological Chemistry",
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"Cancer Research",
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"Journal of Biological Chemistry",
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"Human Molecular Genetics",
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"Arteriosclerosis, Thrombosis, and Vascular Biology",
"American Journal of Pathology",
"BioFactors",
"Arteriosclerosis, Thrombosis, and Vascular Biology",
"Circulation Research",
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"Diabetes, Obesity and Metabolism",
"Nature Genetics",
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"Neurobiology of Aging",
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"Journal of the Federation of American Societies for Experimental Biology",
"American Journal of Physiology",
"American Journal of Clinical Nutrition",
"Nucleosides, Nucleotides and Nucleic Acids",
"Journal of Medicinal Chemistry",
"Pharmacology and Therapeutics",
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"Journal of the American Medical Association",
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"Journal of Biological Chemistry",
"Journal of Nutritional Science and Vitaminology",
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"American Journal of Physiology",
"American Journal of Clinical Nutrition",
"Nucleosides, Nucleotides and Nucleic Acids",
"Journal of Medicinal Chemistry",
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"\nFigure 1 :\n1Role of AdoHcy and Hcy in AdoMet-dependent methylation in yeast and mammals. The enzymes involved in yeast and mammalian metabolism are shown in grey circles. AdoMet: S-adenosyl-L-methionine; AdoHcy: S-adenosyl-L-homocysteine; Hcy: homocysteine; Met: methionine; CTT: cystathionine; in yeast: Sah1: S-adenosyl-L-homocysteine hydrolase; Sam1 and Sam2: Sadenosyl-L-methionine synthetases; Met6: methionine synthase; Str4: cystathionine β-synthase; Str1: cystathionine γ-lyase; Gsh1: γ-glutamylcysteine synthetase; in mammals: AHCY: S-adenosyl-L-homocysteine hydrolase; MAT: methionine adenosyltransferase; MFMT: 5-methyltetrahydrofolate homocysteine methyltransferase; BHMT: betaine homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; GSH: glutathione synthase.",
"\n6\nJournal of LipidsUpregulation of fatty acid, TAG and sterol biosynthes sUPR \n\nER stress \n\nPE \nPC \n\nCho2 \nOpi3 \nPEMT \nAdoMet \nAdoHcy \n\nSam1/Sam2 \nMAT \n\nMET \nHcy \n\nSah1/AHCY \n\nMet6 \nMFMT/BHMT \n\nCTT \n\nCysteine \n\nGlutathione \n\nGsh1/GCS \n\nStr1/CTH \n\nStr4/CBS \n\nAccumulation of SFA \nin membrane phospholipids \n\ni \n\n",
"\nTable 1 :\n1Experimental evidence on deregulation of lipid metabolism and UPR induction under elevated homocysteine levels in yeast and mammals * .Hcy/AdoHcy levels are inversely correlated to the levels of unsaturated fatty acids + * The absence of a plus sign in some columns implies lack of data or nonapplicability.Experimental evidence \nYeast \nMammals \nAdoHcy is formed in vivo in \nresponse to elevated Hcy levels \n+ \n+ \n\nAdoHcy is more toxic than Hcy \nto cells deficient in Hcy \ncatabolism \n\n+ \n\nAdoHcy represents a better \nmarker of cardiovascular risk \nthan Hcy \n\n+ \n\nPhospholipid methylation is \nquantitatively the major \nconsumer of AdoMet \n\n+ \n+ \n\nPhospholipid methylation is \ninhibited in response to Hcy \nsupplementation \n\n+ \n\nPhospholipid methylation is \ninhibited by AdoHcy \n+ \n+ \n\nTAG is accumulating in response \nto Hcy supplementation \n+ \n+ \n\nTAG is accumulating in response \nto deficiency in AdoHcy \nhydrolysis \n\n+ \n\nTAG is accumulating in response \nto deficiency in phospholipid \nmethylation \n\n+ \n+ \n\nUPR is inducted in response to \nHcy supplementation \n+ \n+ \n\nThe de novo phospholipid \nmethylation pathway produces \nphospholipids enriched in \nunsaturated fatty acids \n\n+ \n+ \n\nER stress is inducted by \naccumulation of saturated fatty \nacids in membrane \nphospholipids \n\n+ \n+ \n\n",
"\nFigure 1 :\n1Role of AdoHcy and Hcy in AdoMet-dependent methylation in yeast and mammals. The enzymes involved in yeast and mammalian metabolism are shown in grey circles. AdoMet: S-adenosyl-L-methionine; AdoHcy: S-adenosyl-L-homocysteine; Hcy: homocysteine; Met: methionine; CTT: cystathionine; in yeast: Sah1: S-adenosyl-L-homocysteine hydrolase; Sam1 and Sam2: Sadenosyl-L-methionine synthetases; Met6: methionine synthase; Str4: cystathionine β-synthase; Str1: cystathionine γ-lyase; Gsh1: γ-glutamylcysteine synthetase; in mammals: AHCY: S-adenosyl-L-homocysteine hydrolase; MAT: methionine adenosyltransferase; MFMT: 5-methyltetrahydrofolate homocysteine methyltransferase; BHMT: betaine homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; GSH: glutathione synthase.",
"\n6\nJournal of LipidsUpregulation of fatty acid, TAG and sterol biosynthes sUPR \n\nER stress \n\nPE \nPC \n\nCho2 \nOpi3 \nPEMT \nAdoMet \nAdoHcy \n\nSam1/Sam2 \nMAT \n\nMET \nHcy \n\nSah1/AHCY \n\nMet6 \nMFMT/BHMT \n\nCTT \n\nCysteine \n\nGlutathione \n\nGsh1/GCS \n\nStr1/CTH \n\nStr4/CBS \n\nAccumulation of SFA \nin membrane phospholipids \n\ni \n\n",
"\nTable 1 :\n1Experimental evidence on deregulation of lipid metabolism and UPR induction under elevated homocysteine levels in yeast and mammals * .Hcy/AdoHcy levels are inversely correlated to the levels of unsaturated fatty acids + * The absence of a plus sign in some columns implies lack of data or nonapplicability.Experimental evidence \nYeast \nMammals \nAdoHcy is formed in vivo in \nresponse to elevated Hcy levels \n+ \n+ \n\nAdoHcy is more toxic than Hcy \nto cells deficient in Hcy \ncatabolism \n\n+ \n\nAdoHcy represents a better \nmarker of cardiovascular risk \nthan Hcy \n\n+ \n\nPhospholipid methylation is \nquantitatively the major \nconsumer of AdoMet \n\n+ \n+ \n\nPhospholipid methylation is \ninhibited in response to Hcy \nsupplementation \n\n+ \n\nPhospholipid methylation is \ninhibited by AdoHcy \n+ \n+ \n\nTAG is accumulating in response \nto Hcy supplementation \n+ \n+ \n\nTAG is accumulating in response \nto deficiency in AdoHcy \nhydrolysis \n\n+ \n\nTAG is accumulating in response \nto deficiency in phospholipid \nmethylation \n\n+ \n+ \n\nUPR is inducted in response to \nHcy supplementation \n+ \n+ \n\nThe de novo phospholipid \nmethylation pathway produces \nphospholipids enriched in \nunsaturated fatty acids \n\n+ \n+ \n\nER stress is inducted by \naccumulation of saturated fatty \nacids in membrane \nphospholipids \n\n+ \n+ \n\n"
] | [
"Role of AdoHcy and Hcy in AdoMet-dependent methylation in yeast and mammals. The enzymes involved in yeast and mammalian metabolism are shown in grey circles. AdoMet: S-adenosyl-L-methionine; AdoHcy: S-adenosyl-L-homocysteine; Hcy: homocysteine; Met: methionine; CTT: cystathionine; in yeast: Sah1: S-adenosyl-L-homocysteine hydrolase; Sam1 and Sam2: Sadenosyl-L-methionine synthetases; Met6: methionine synthase; Str4: cystathionine β-synthase; Str1: cystathionine γ-lyase; Gsh1: γ-glutamylcysteine synthetase; in mammals: AHCY: S-adenosyl-L-homocysteine hydrolase; MAT: methionine adenosyltransferase; MFMT: 5-methyltetrahydrofolate homocysteine methyltransferase; BHMT: betaine homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; GSH: glutathione synthase.",
"Journal of LipidsUpregulation of fatty acid, TAG and sterol biosynthes s",
"Experimental evidence on deregulation of lipid metabolism and UPR induction under elevated homocysteine levels in yeast and mammals * .Hcy/AdoHcy levels are inversely correlated to the levels of unsaturated fatty acids + * The absence of a plus sign in some columns implies lack of data or nonapplicability.",
"Role of AdoHcy and Hcy in AdoMet-dependent methylation in yeast and mammals. The enzymes involved in yeast and mammalian metabolism are shown in grey circles. AdoMet: S-adenosyl-L-methionine; AdoHcy: S-adenosyl-L-homocysteine; Hcy: homocysteine; Met: methionine; CTT: cystathionine; in yeast: Sah1: S-adenosyl-L-homocysteine hydrolase; Sam1 and Sam2: Sadenosyl-L-methionine synthetases; Met6: methionine synthase; Str4: cystathionine β-synthase; Str1: cystathionine γ-lyase; Gsh1: γ-glutamylcysteine synthetase; in mammals: AHCY: S-adenosyl-L-homocysteine hydrolase; MAT: methionine adenosyltransferase; MFMT: 5-methyltetrahydrofolate homocysteine methyltransferase; BHMT: betaine homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; GSH: glutathione synthase.",
"Journal of LipidsUpregulation of fatty acid, TAG and sterol biosynthes s",
"Experimental evidence on deregulation of lipid metabolism and UPR induction under elevated homocysteine levels in yeast and mammals * .Hcy/AdoHcy levels are inversely correlated to the levels of unsaturated fatty acids + * The absence of a plus sign in some columns implies lack of data or nonapplicability."
] | [
"(Figure 1",
"Figure 1",
"Figure 1",
"Figure 1",
"(Figure 1",
"Figure 2",
"Figure 2",
"Figure 1",
"(Figure 1",
"Figure 1",
"Figure 1",
"Figure 1",
"(Figure 1",
"Figure 2",
"Figure 2",
"Figure 1"
] | [] | [
"The first indication that sulfur amino acid metabolism is linked to atherosclerosis came from observations in 1953 demonstrating that pathogenic cholesterol concentrations and experimental atherogenesis in monkeys can be inhibited by dietary methionine [1]. Since the early 60s elevated Hcy levels in blood (hyperhomocysteinemia) caused by different deficiencies of sulfur amino acid metabolism were reported to be associated with vascular disease and, in particular, with atherosclerotic plaque formation [2,3]. Today, Hcy is recognized by many studies as a strong, independent and causal risk factor for atherosclerosis [4][5][6][7][8], although there is still controversy on the underlying metabolic connections [9]. In addition to its association with vascular diseases, Hcy is also linked to neurological disorders [10], aging [11], and all-cause mortality [12]. Understanding the pathological mechanisms triggered by Hcy is, therefore, essential for understanding its role in several disease states.",
"Numerous mechanisms have been proposed that explain pathological changes associated with elevated Hcy levels (reviewed in [3]). Several of them, for example, protein homocysteinylation and oxidative stress, are directly triggered by Hcy. However, not Hcy, but rather AdoHcy, an immediate precursor of Hcy (Figure 1), emerged as a more sensitive indicator of cardiovascular disease during the last decade [13,14]. Supporting the potentially pathogenic role of AdoHcy, studies in yeast showed that indeed AdoHcy is more toxic than Hcy to cells that are deficient in Hcy catabolism [15].",
"AdoHcy is synthesized as a universal byproduct of AdoMet-dependent methyltransferase reactions ( Figure 1). It is a strong competitive inhibitor of many AdoMetdependent methyltransferases [16] and, therefore, has to be removed to sustain these reactions. The only eukaryotic enzyme capable of AdoHcy catabolism, S-adenosyl-Lhomocysteine hydrolase (Sah1 in yeast, AHCY in mammals), catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. The equilibrium of S-adenosyl-L-homocysteine hydrolase-catalyzed reaction lies far in the direction of synthesis, and both Hcy and adenosine have to be quickly metabolized in order to drive the net hydrolysis of AdoHcy [17]. Therefore, accumulation of hydrolytic products of the S-adenosyl-L-homocysteine hydrolase-catalyzed reaction, in particular Hcy, results in AdoHcy synthesis and accumulation showing that AdoHcy is not only the precursor, but also the product of Hcy metabolism in vivo [18][19][20].",
"Changes at the epigenetic level are the most extensively studied consequences of methylation deficiency [21][22][23][24]. However, phospholipid methylation that requires three sequential AdoMet-dependent methylation steps to synthesize one molecule of phosphatidylcholine (PC) from phosphatidylethanolamine (PE), the predominant way for PC synthesis in yeast, in particular, in the absence of choline and ethanolamine in the culture medium, is the major consumer of AdoMet. Phospholipid methylation is also the major consumer of AdoMet in mice, since the loss of phosphatidylethanolamine N-methyltransferase (PEMT) in PEMT −/− knockout mice leads to a 50% decrease in plasma Hcy levels [25]. Reexamination of methylation metabolism in humans also revealed that phospholipid methylation, but not creatine synthesis, as was assumed previously, accounts for the major part of AdoMet being utilized in the human body [26].",
"While PE methylation is the predominant way to synthesize phospholipids in yeast, phospholipid synthesis by the de novo methylation pathway is primarily present in the liver in mammals, where it constitutes 30% of PC production and account for estimated 10 μmol and 1,65 mmol PEMTderived PC secreted into bile per day in mice and humans, respectively [26,27]. However, other mammalian tissues and cells are also capable of phospholipid methylation including brain, skeletal muscle, adipose tissues, fibroblasts, arterial smooth muscle cells, endothelial cells, macrophages, and erythrocytes [28][29][30][31][32][33][34][35][36][37]. The evolutionary conservation of phospholipid methylation suggests its essential role in some specific functions in different cell types. For instance, phospholipid methylation is enhanced in hypertrophied myocardium, correlates with the level of β-adrenergic receptors [38,39] and is stimulated by isoproterenol, a potent cardiac stimulant [40]. In contrast, phospholipid methylation is inhibited by quinidine, an antiarrhythmic drug that causes repression of myocardial contractility [41]. Phospholipid methylation was also observed in microsome preparations from aorta [42,43] and was suggested to affect membrane fluidity and function of membrane calcium channels in aorta [42,43] as well as in heart [40]. Moreover, phospholipid methylation appears to be coupled to Ca 2+ influx and von Willebrand factor release in endothelial cells [35]. In accordance, it was shown that increased methylation of phospholipids is required for an influx of Ca 2+ and subsequent release of histamine in mast cells [44]. Furthermore, Ca 2+ influx was correlated with the release of arachidonic acid in rabbit neutrophils and human fibroblasts, which also appears to require phospholipid methylation [32,45]. Requirement of phospholipid methylation for polyunsaturated fatty acid metabolism was also observed in the brain [46]. It was reported that developing, remyelinating, and diabetic brain exhibits increased synthesis of PC by the de novo methylation pathway in comparison with normal adult brain [47,48]. Phospholipid methylation was shown to be linked to diabetes [49][50][51] and neurological disorders [52,53] also in other studies.",
"PEMT mRNA and protein levels increase substantially in differentiating adipocytes [30]. It was shown very recently that phospholipid methylation is required for lipid droplet formation and stability in 3T3-L1 adipocytes, and high-fat challenge induces PEMT expression in adipose tissue [54]. Moreover, PEMT and the CDP-choline pathway for PC synthesis are both required for the secretion of very-low-density lipoproteins [55][56][57]. While cells lacking the rate-limiting enzyme of the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase, do not survive [57], deficiency of phospholipid methylation in PEMT −/− mice under choline deprivation results in development of hepatic steatosis followed by steatohepatitis and hyperacute liver failure [58] and is lethal within 5 days [59]. Moreover, deficiency in phospholipid methylation, but not in the synthesis of PC by the CDP-choline pathway, protects from diet-induced obesity in mice due to increased energy utilization suggesting that PEMT plays a role in whole energy metabolism and is linked to insulin signaling [60]. Finally, an isoform of phosphatidylethanolamine N-methyltransferase, PEMT2, appears to be involved in the control of hepatocyte cell division, since its inactivation is associated with several types of liver cell proliferation including tumorigenesis [61]. Vice versa, rat hepatoma cell growth is suppressed by PEMT2 expression [62].",
"Sensitivity of phospholipid methylation to AdoHcy accumulation [16,18,63] as well as numerous correlations reported for phospholipid methylation pathway suggests that interference with this reaction in Hcy-associated pathology may lead to widespread defects, what indeed seems to be the case. In particular, elevated Hcy levels were found to trigger deregulation of lipid metabolism in yeast and mammalian cells [18,64]. A mechanism of deregulation of lipid metabolism and lipid-associated cellular functions in hyperhomocysteinemia mediated by AdoHcy accumulation and subsequent inhibition of phospholipid methylation is proposed in this paper.",
"Homocysteine is a sulfur-containing amino acid, which does not occur in proteins, but is found at the intersection of methylation and transsulfuration metabolism ( Figure 1, reviewed in [65]). Hcy is formed during methionine metabolism by S-adenosyl-L-homocysteine hydrolase that catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. To be kept in the methylation cycle, Hcy has to be remethylated to methionine, which can be further activated to AdoMet and used by over 50 AdoMet-dependent methyltransferases that release AdoHcy as a by-product after the methyl transfer reaction. The ratio of AdoMet to AdoHcy, that is, the ratio of the substrate versus the specific inhibitor of AdoMet-dependent methyltransferases, is indicative of the cellular methylation potential [66].",
"In addition to its remethylation to methionine, Hcy can be subjected to transsulfuration leading to the synthesis of cysteine, which is also a precursor of glutathione, an essential cellular defense molecule in oxidative stress response [65]. This pathway irreversibly withdraws Hcy from the methylation cycle. An alternative way for Hcy metabolism is the reversal of the reaction catalyzed by S-adenosyl-Lhomocysteine hydrolase. This occurs upon accumulation of the hydrolytic products of the reaction, in particular Hcy, and leads to AdoHcy synthesis and accumulation [19,[67][68][69]. Thus, elevated Hcy levels via accumulation of AdoHcy lead to the disruption of the methylation cycle and, potentially, to methylation deficiency.",
"Deficiency in cystathionine β-synthase (CBS), the first and rate-limiting enzyme of the transsulfuration pathway ( Figure 1), is the major cause of severe hyperhomocysteinemia followed by genetic defects of folate and cobalamin metabolism that is involved in Hcy remethylation [65]. These pathological conditions lead to the plasma Hcy levels of more than 100 μmol/L [65] and are rare in comparison with mild hyperhomocysteinemia that is caused by dietary deficiencies of the vitamin cofactors required for Hcy catabolism -folic acid, vitamins B 6 and B 12 , and characterized by the plasma Hcy levels of 15-25 μmol/L [70]. Vitamin B 6 is required for the activity of CBS. Folic acid and vitamin B 6 are required for the activity of methionine synthase catalyzing 5-methyltetrahydrofolate-dependent remethylation of Hcy to methionine (Figure 1). While vitamin supplementation appeared to be a straightforward strategy to reduce/prevent cardiovascular events, this possibility was studied in several large trials. However, it was observed that vitamins, while capable of lowering elevated plasma Hcy levels, do not reduce the rates of vascular events [71]. Several potential mechanisms that might explain this result by offsetting the positive effect of Hcy-lowering therapy were subsequently proposed. These include promotion of cell proliferation by folic acid through its role in the synthesis of thymidine, increase of the methylation potential leading to changes in gene expression, and increase in the levels of asymmetric dimethylarginine that inhibit the activity of nitric oxide synthase [71]. An additional possibility is that, not Hcy, but rather a related metabolite could be a trigger of some pathological changes associated with elevated Hcy levels. Possibly explaining the failure of Hcy-lowering vitamins to reduce vascular events, it was recently reported that supplementation with B-vitamins including folate does not efficiently lower plasma AdoHcy levels [72], presumably due to elevation of AdoMet-dependent methylation.",
"In yeast, the synthesis of PC from PE by the de novo phospholipid methylation pathway is particularly sensitive to AdoHcy accumulation [18,63]. Both inhibition of S-adenosyl-Lhomocysteine hydrolase and Hcy supplementation results in AdoHcy accumulation and inhibition of phospholipid methylation in yeast [18]. However, not only phospholipid methylation, but also a methylation-independent branch of lipid metabolism, namely, TAG synthesis, is affected by Ado-Hcy accumulation in yeast: yeast cells deficient in AdoHcy catabolism or supplemented with Hcy massively accumulate TAG [18]. Supporting the causal role of impaired phospholipid methylation in the deregulation of TAG metabolism in response to AdoHcy accumulation, it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of PE to PC, cho2 and opi3, also accumulate TAG [18]. TAG is known to play an important role in buffering excess fatty acids [73]. Therefore, TAG accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to TAG synthesis in methylation deficiency in yeast. Another observation as well supports accumulation of fatty acids under these conditions. In addition to TAG metabolism, transcriptional regulation of phospholipid biosynthesis is also affected in yeast mutants deficient in AdoHcy catabolism. Impaired phospholipid methylation in Sah1-depleted cells unable to hydrolyze AdoHcy or in cho2 and opi3 mutants leads to upregulation of genes, which have an inositol-sensitive upstream regulatory sequence (UAS INO ) in their promoter regions, indicating accumulation of the phospholipid precursor, phosphatidic acid, in the ER [18]. ACC1 encoding acetyl-CoA carboxylase, the first and ratelimiting enzyme of fatty acid biosynthesis, is also a subject to UAS INO -mediated regulation, suggesting upregulation of the de novo fatty acid biosynthesis in response to AdoHcy accumulation. Moreover, Sah1 depletion also affects sterol synthesis in yeast, leading to 4-fold elevated squalene levels and suggesting accumulation of early precursors of ergosterol biosynthesis under these conditions (Tehlivets, Kohlwein, unpublished). Taken together, inhibition of phospholipid methylation induced by AdoHcy accumulation appears to lead to upregulation of fatty acid, TAG, and sterol biosynthetic pathways in yeast.",
"AdoHcy inhibits phosphatidylethanolamine N-methyltransferase in vitro and in vivo also in mammals [28,37,74]. Similarly as in yeast, deficiency of phospholipid methylation in PEMT −/− knockout mice leads to a rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in the liver, in the absence of choline supplementation [75]. However, TAG accumulation in the livers of these animals appears to be at least in part due to decreased TAG secretion from hepatocytes [55]. Elevated levels of Hcy are as well linked to deregulation of lipid metabolism in mammals. CBS −/− knockout mice exhibit severe hyperhomocysteinemia and accumulate Ado-Hcy in all tissues tested [68,69]. These mutant animals show elevated TAG and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [76,77]. Another genetic disorder that results in moderately elevated Hcy levels, methylenetetrahydrofolate reductase (MTHFR) deficiency, leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [78,79]. Dietary-induced hyperhomocysteinemia in mice also causes fatty liver, further supporting the role of Hcy in deregulation of lipid metabolism in mammals [64]. In these mice as well as in the CBS −/− knockout mice lipid accumulates in liver rather than in serum [64,76].",
"Preferable accumulation of lipids in the liver and, possibly, other tissues in hyperhomocysteinemia suggests that other mechanisms than those associated with elevation of circulating lipids are responsible for the development of cardiovascular disease under these conditions. Indeed, conventional risk factors including hypercholesterolemia accounts only for approximately 50% of all cases of cardiovascular disease, while 40% of patiens diagnosed with premature coronary artery disease, peripheral vascular disease or venous thrombosis exhibit hyperhomocysteinemia [80]. In accordance, unlike typical lipid-rich atherosclerotic plagues, vascular lesions associated with hyperhomocysteinemia are lipid-poor, fibrous plaques [81,82], greatly outnumbering fatty atherosclerotic lesions [80].",
"In animal models of hyperhomocysteinemia atherosclerotic lesions are rare. They are found only in the MTHFR −/− knockout mice that exhibit aortic lipid accumulation reminiscent of early atherosclerotic lesions [2,78,83], however, not in, for example, CBS −/− knockout mice. This discrepancy might be due to disruption of two different Hcy utilizing pathways in these animals. While 5-methyltetrahydrofolatedependent Hcy remethylation occurs in all mammalian cells, transsulfuration of Hcy occurs primarily in the liver and kidney [65]. Thus, impairment of 5-methyltetrahydrofolatedependent Hcy remethylation in MTHFR −/− knockout mice may differently affect Hcy metabolism in comparison to the deficiency in the first step of Hcy transsulfuration in CBS −/− knockout mice. The observation that dietary (methionine or Hcy supplementation) or genetically (CBS gene deletion) induced hyperhomocysteinemia in apoE-deficient (apoE −/− ) mice leads to development of larger and more advanced atherosclerotic lesions clearly demonstrates a causal relationship between elevated Hcy levels and atherosclerosis [83]. In contrast, lack of PEMT was shown to reduce significantly plasma VLDL and to attenuate atherosclerosis in both PEMT −/− /Ldlr −/− mice deficient in PEMT and LDL receptors as well as in PEMT −/− /ApoE −/− mice [84,85].",
"Elevated Hcy levels induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in a variety of mammalian cells. These include cultured human hepatocytes, vascular endothelial and aortic smooth muscle cells as well as liver cells of the CBS −/− knockout mice [64,[86][87][88]. Furthermore, elevated Hcy levels lead to activation of the sterol regulatory element-binding proteins (SREBPs), which function to activate genes encoding enzymes in cholesterol, fatty acid, and TAG metabolism and uptake, both in cultured mammalian cell lines as well as in the livers of the CBS −/− knockout mice [64,86]. ER stress appears to play a direct role in the activation of TAG and cholesterol biosynthesis, since overexpression of the ER chaperone GRP78/BiP was reported to inhibit Hcy-induced SREBP-1 gene expression in mammalian cell cultures [64] as well as in mice [89] and lead to reduction of the hepatic steatosis in leptin-deficient (ob/ob) mice [89]. SREBP-1 overcomes translation inhibition induced by UPR through an internal ribosome entry site (IRS), similarly to GRP78 [90].",
"Confirming the causal role of Hcy in UPR induction and deregulation of lipid metabolism, a decrease of elevated plasma Hcy levels is accompanied by a decrease in hepatic lipids and ER stress response [91]. A strong correlation between lipid metabolism, ER stress response and elevated Hcy levels is also evident form a literature mining approach [92]. Further demonstration of close relationship between ER stress and Hcy metabolism came from the observation that MTHFR involved in Hcy remethylation is induced in response to ER stress [93]. Evolutionary conservation of the relationship between Hcy and UPR is shown by the induction of ER stress and activation of UPR in response to Hcy supplementation in yeast [94]. Finally, demonstrating its pathophysiological role, ER stress was shown to be strongly associated with accelerated atherosclerosis in hyperhomocysteinemic apoE-deficient mice [95], liver diseases [96] as well as hyperglycemia-induced atherosclerosis [97].",
"The UPR, as a conserved cellular stress response pathway, is aimed at restoring normal ER and secretory function as well as membrane trafficking upon impaired protein folding in the ER. The de novo methylation and the CDP-choline phospholipid biosynthetic pathways produce phospholipid species with distinct fatty acyl chain composition in yeast: the de novo phospholipid methylation pathway produces more unsaturated phospholipids [98,99]. Similarly, the PEMT-generated PC pool in mammals is also enriched in unsaturated fatty acids [100,101]. Supporting the role of PEMT in metabolism of unsaturated fatty acids PEM −/− knockout mice were reported to accumulate more saturated PC molecular species in the liver compared with the control littermates [60] and to exhibit dramatically reduced concentrations of polyunsaturated fatty acids in the plasma and in hepatic PC, independently of choline status [102]. Thus, beyond its role as a compensatory pathway for PC biosynthesis under conditions of choline deprivation, phospholipid methylation plays a crucial role in unsaturated fatty acid metabolism both in yeast and in mammals. The observation that deficiency of phospholipid methylation in cho2 and opi3 yeast mutants is synthetically lethal in the absence of a functional UPR [103] suggests an essential requirement of UPR in response to impaired phospholipid methylation. Thus, Hcy accumulation, which was shown to lead to AdoHcy-mediated inhibition of phospholipid methylation in yeast, may lead to accumulation of saturated fatty acids in membrane phospholipids-a potential pathological mechanism that might be shared by both yeast and mammals. Indeed, accumulation of saturated fatty acids in membrane phospholipids interferes with ER structure and integrity, induces ER stress and leads to cell death in mammalian cell cultures [104,105]. Accordingly, decreased membrane phospholipid desaturation due to stearoyl-CoA desaturase 1 knockdown induces UPR in HeLa cells [106]. Vice versa, overexpression of stearoyl-CoA desaturase attenuates palmitate-induced ER stress and protects from lipoapoptosis [107][108][109]. Treatment with the molecular chaperone 4phenyl butyrate, which is capable of stabilization of protein conformation, improvement of ER folding capacity and facilitation of protein trafficking, leads to abolishment of UPR induction in yeast subjected to lipid-induced ER stress [105]. This finding suggests that accumulation of saturated fatty acids in membrane phospholipids first leads to changes in the membrane environment followed by induction of ER stress and accumulation of misfolded protein(s) that, in turn, activate UPR [105]. Potential mechanisms involved in saturated fatty acid-induced UPR include depletion of ER Ca 2+ stores leading to decreased ER chaperone activity and protein misfolding, as well as interference with ER-to-Golgi trafficking [110]. Recently, proteomic studies showed that carboxypeptidase E, a key enzyme involved in processing [111] and sorting of insulin [112], is involved in induction of ER stress in β-cells in response to palmitate treatment [113]. Degradation of carboxypeptidase E in palmitateinduced ER stress is mediated by palmitate metabolism and Ca 2+ flux [113]. Alternatively, changes of the ER membrane environment may directly activate ER sensors IRE1, ATF6, and PERK or modulate the binding of the ER sensors to the ER chaperone GRP78 causing its dissociation and activation of UPR pathways.",
"Supporting the hypothesis that Hcy interferes with phospholipid acyl chain composition it was observed in humans that elevated plasma AdoHcy levels are negatively correlated with both PC content and the level of polyunsaturated fatty acids in PC, but not in PE, in red blood cells in Alzheimer's patients [114]. Elevated plasma Hcy levels were also shown to be associated with a decrease in polyunsaturated (docosahexaenoic) fatty acids in the plasma of healthy humans [115] and in the plasma and erythrocytes of cystic fibrosis patients; these individuals exhibit increased Hcy and AdoHcy levels as well as altered PE and PC metabolism [116].",
"Taken together, deficiency of phospholipid methylation caused by AdoHcy accumulation in Hcy-associated pathology appears to lead to an increase in saturated PC molecular species in ER membranes followed by ER stress, protein misfolding, induction of UPR, and activation of lipid metabolism ( Figure 2). Upregulation of lipid biosynthesis, which apparently should serve to compensate for suboptimal composition of membrane lipids, leads, however, to accumulation of fatty acids, TAG and sterols in the absence of functional phospholipid methylation. In addition to a role in formation of a specific pool of PC molecular species, phospholipid methylation is crucial for maintenance of a distinct PC/PE ratio important for cell integrity when dietary choline-supply is blunted. Decrease of the PC/PE ratio was reported to result in increased cell permeability of hepatocytes from PEMT −/− mice fed choline deficient diet leading to liver damage [75]. Similarly, decrease of PC or increase in PE was shown to lead to cell damage and/or death in several other mammalian cell types [117][118][119]. Finally, observation of different outcomes in hyperhomocysteinemic apoE −/− mice and both in PEMT −/− /Ldlr −/− and PEMT −/− /ApoE −/− mice suggests mechanisms besides inhibition of phospholipid methylation, for example, AdoHcy-dependent modulation of gene expression that may also contribute to the development of Hcy-dependent atherosclerosis.",
"The metabolism of homocysteine, consequences of its accumulation as well as associated AdoHcy-triggered inhibition of AdoMet-dependent methylation are complex. In this paper a novel mechanism of Hcy-triggered deregulation Figure 2: A model of activation of UPR and upregulation of fatty acid, TAG, and sterol biosynthesis in response to inhibition of phospholipid methylation in hyperhomocysteinemia. Elevated Hcy levels via AdoHcy accumulation and inhibition of phospholipid methylation lead to accumulation of saturated PC molecular species in ER membranes followed by ER stress, UPR activation, and upregulation of fatty acid, TAG, and sterol biosynthesis. The enzymes involved in yeast and mammalian metabolism are shown in grey circles (see Figure 1). PE: phosphatidylethanolamine; PC: phosphatidylcholine; PEMT: phosphatidylethanolamine N-methyltransferase in mammals; Cho2 and Opi3: phosphatidylethanolamine N-methyltransferases in yeast. of lipid metabolism and UPR induction that is mediated through an AdoHcy-dependent inhibition of phospholipid methylation and based on experimental evidence derived from both yeast and mammalian systems is proposed. The observations made in yeast and mammals are summarized in Table 1.",
"S-adenosyl-L-homocysteine hydrolase is recognized since many years as a target for antiviral drug design [120]. Inhibitors that block AdoHcy hydrolysis are efficient against many types of viruses including Ebola and show other effects of pharmacological importance [120][121][122]. However, they are associated with high cytotoxicity due to interference with central metabolic pathways [121,122]. While using these inhibitors to study the effects of AdoHcy accumulation appears to be straightforward, ability of some of nucleoside inhibitors of S-adenosyl-L-homocysteine hydrolase to undergo metabolic phosphorylation to nucleotides may account for a part of their biological activities [121,122]. Inhibitors that would be able to block selectively homocysteine and adenosine conversion to AdoHcy are not available. However, based on current understanding of regulation of homocysteine and methionine metabolism (i) selective blockage of AdoHcy synthesis from homocysteine and adenosine that will relieve not only inhibition of phospholipid methylation but also many other AdoMetdependent methyltransferase reactions, combined with (ii) vitamin B 6 supplementation in order to accelerate homocystein catabolism by transsulfuration pathway, may serve as a way to reduce AdoHcy in hyperhomocysteinemia without elevation of AdoMet.",
"Many questions in the model presented in this paper are still unanswered. Is AdoHcy-mediated accumulation of saturated fatty acids in membrane lipids indeed the way by which elevated Hcy levels induce ER stress? What role do specific lipid precursors have in regulation of lipid metabolism in UPR? What is the impact of AdoHcy accumulation on other methylation reactions unrelated to lipid metabolism? What is the role of deficient phospholipid methylation in homocysteine-associated pathology beyond deregulation of fatty acid, TAG, sterol metabolism, and UPR induction? Elucidation of the molecular mechanisms triggered by elevated Hcy levels will undoubtfully improve our understanding of its pathological role in numerous diseases.",
"AdoMet:",
"S-adenosyl-L-methionine AdoHcy:",
"S-adenosyl-L-homocysteine Hcy:",
"Homocysteine PC:",
"Phosphatidylcholine PE:",
"Phosphatidylethanolamine TAG:",
"Triacylglycerol UPR:",
"Unfolded protein response Sah1:",
"S-adenosyl-L-homocysteine hydrolase in yeast AHCY:",
"S-adenosyl-L-homocysteine hydrolase in mammals Cho2 and Opi3: Phosphatidylethanolamine N-methyltransferases in yeast PEMT:",
"Phosphatidylethanolamine N-methyltransferase in mammals CBS:",
"Cystathionine β-synthase MTHFR:",
"Methylenetetrahydrofolate reductase.",
"The first indication that sulfur amino acid metabolism is linked to atherosclerosis came from observations in 1953 demonstrating that pathogenic cholesterol concentrations and experimental atherogenesis in monkeys can be inhibited by dietary methionine [1]. Since the early 60s elevated Hcy levels in blood (hyperhomocysteinemia) caused by different deficiencies of sulfur amino acid metabolism were reported to be associated with vascular disease and, in particular, with atherosclerotic plaque formation [2,3]. Today, Hcy is recognized by many studies as a strong, independent and causal risk factor for atherosclerosis [4][5][6][7][8], although there is still controversy on the underlying metabolic connections [9]. In addition to its association with vascular diseases, Hcy is also linked to neurological disorders [10], aging [11], and all-cause mortality [12]. Understanding the pathological mechanisms triggered by Hcy is, therefore, essential for understanding its role in several disease states.",
"Numerous mechanisms have been proposed that explain pathological changes associated with elevated Hcy levels (reviewed in [3]). Several of them, for example, protein homocysteinylation and oxidative stress, are directly triggered by Hcy. However, not Hcy, but rather AdoHcy, an immediate precursor of Hcy (Figure 1), emerged as a more sensitive indicator of cardiovascular disease during the last decade [13,14]. Supporting the potentially pathogenic role of AdoHcy, studies in yeast showed that indeed AdoHcy is more toxic than Hcy to cells that are deficient in Hcy catabolism [15].",
"AdoHcy is synthesized as a universal byproduct of AdoMet-dependent methyltransferase reactions ( Figure 1). It is a strong competitive inhibitor of many AdoMetdependent methyltransferases [16] and, therefore, has to be removed to sustain these reactions. The only eukaryotic enzyme capable of AdoHcy catabolism, S-adenosyl-Lhomocysteine hydrolase (Sah1 in yeast, AHCY in mammals), catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. The equilibrium of S-adenosyl-L-homocysteine hydrolase-catalyzed reaction lies far in the direction of synthesis, and both Hcy and adenosine have to be quickly metabolized in order to drive the net hydrolysis of AdoHcy [17]. Therefore, accumulation of hydrolytic products of the S-adenosyl-L-homocysteine hydrolase-catalyzed reaction, in particular Hcy, results in AdoHcy synthesis and accumulation showing that AdoHcy is not only the precursor, but also the product of Hcy metabolism in vivo [18][19][20].",
"Changes at the epigenetic level are the most extensively studied consequences of methylation deficiency [21][22][23][24]. However, phospholipid methylation that requires three sequential AdoMet-dependent methylation steps to synthesize one molecule of phosphatidylcholine (PC) from phosphatidylethanolamine (PE), the predominant way for PC synthesis in yeast, in particular, in the absence of choline and ethanolamine in the culture medium, is the major consumer of AdoMet. Phospholipid methylation is also the major consumer of AdoMet in mice, since the loss of phosphatidylethanolamine N-methyltransferase (PEMT) in PEMT −/− knockout mice leads to a 50% decrease in plasma Hcy levels [25]. Reexamination of methylation metabolism in humans also revealed that phospholipid methylation, but not creatine synthesis, as was assumed previously, accounts for the major part of AdoMet being utilized in the human body [26].",
"While PE methylation is the predominant way to synthesize phospholipids in yeast, phospholipid synthesis by the de novo methylation pathway is primarily present in the liver in mammals, where it constitutes 30% of PC production and account for estimated 10 μmol and 1,65 mmol PEMTderived PC secreted into bile per day in mice and humans, respectively [26,27]. However, other mammalian tissues and cells are also capable of phospholipid methylation including brain, skeletal muscle, adipose tissues, fibroblasts, arterial smooth muscle cells, endothelial cells, macrophages, and erythrocytes [28][29][30][31][32][33][34][35][36][37]. The evolutionary conservation of phospholipid methylation suggests its essential role in some specific functions in different cell types. For instance, phospholipid methylation is enhanced in hypertrophied myocardium, correlates with the level of β-adrenergic receptors [38,39] and is stimulated by isoproterenol, a potent cardiac stimulant [40]. In contrast, phospholipid methylation is inhibited by quinidine, an antiarrhythmic drug that causes repression of myocardial contractility [41]. Phospholipid methylation was also observed in microsome preparations from aorta [42,43] and was suggested to affect membrane fluidity and function of membrane calcium channels in aorta [42,43] as well as in heart [40]. Moreover, phospholipid methylation appears to be coupled to Ca 2+ influx and von Willebrand factor release in endothelial cells [35]. In accordance, it was shown that increased methylation of phospholipids is required for an influx of Ca 2+ and subsequent release of histamine in mast cells [44]. Furthermore, Ca 2+ influx was correlated with the release of arachidonic acid in rabbit neutrophils and human fibroblasts, which also appears to require phospholipid methylation [32,45]. Requirement of phospholipid methylation for polyunsaturated fatty acid metabolism was also observed in the brain [46]. It was reported that developing, remyelinating, and diabetic brain exhibits increased synthesis of PC by the de novo methylation pathway in comparison with normal adult brain [47,48]. Phospholipid methylation was shown to be linked to diabetes [49][50][51] and neurological disorders [52,53] also in other studies.",
"PEMT mRNA and protein levels increase substantially in differentiating adipocytes [30]. It was shown very recently that phospholipid methylation is required for lipid droplet formation and stability in 3T3-L1 adipocytes, and high-fat challenge induces PEMT expression in adipose tissue [54]. Moreover, PEMT and the CDP-choline pathway for PC synthesis are both required for the secretion of very-low-density lipoproteins [55][56][57]. While cells lacking the rate-limiting enzyme of the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase, do not survive [57], deficiency of phospholipid methylation in PEMT −/− mice under choline deprivation results in development of hepatic steatosis followed by steatohepatitis and hyperacute liver failure [58] and is lethal within 5 days [59]. Moreover, deficiency in phospholipid methylation, but not in the synthesis of PC by the CDP-choline pathway, protects from diet-induced obesity in mice due to increased energy utilization suggesting that PEMT plays a role in whole energy metabolism and is linked to insulin signaling [60]. Finally, an isoform of phosphatidylethanolamine N-methyltransferase, PEMT2, appears to be involved in the control of hepatocyte cell division, since its inactivation is associated with several types of liver cell proliferation including tumorigenesis [61]. Vice versa, rat hepatoma cell growth is suppressed by PEMT2 expression [62].",
"Sensitivity of phospholipid methylation to AdoHcy accumulation [16,18,63] as well as numerous correlations reported for phospholipid methylation pathway suggests that interference with this reaction in Hcy-associated pathology may lead to widespread defects, what indeed seems to be the case. In particular, elevated Hcy levels were found to trigger deregulation of lipid metabolism in yeast and mammalian cells [18,64]. A mechanism of deregulation of lipid metabolism and lipid-associated cellular functions in hyperhomocysteinemia mediated by AdoHcy accumulation and subsequent inhibition of phospholipid methylation is proposed in this paper.",
"Homocysteine is a sulfur-containing amino acid, which does not occur in proteins, but is found at the intersection of methylation and transsulfuration metabolism ( Figure 1, reviewed in [65]). Hcy is formed during methionine metabolism by S-adenosyl-L-homocysteine hydrolase that catalyzes the reversible hydrolysis of AdoHcy to Hcy and adenosine. To be kept in the methylation cycle, Hcy has to be remethylated to methionine, which can be further activated to AdoMet and used by over 50 AdoMet-dependent methyltransferases that release AdoHcy as a by-product after the methyl transfer reaction. The ratio of AdoMet to AdoHcy, that is, the ratio of the substrate versus the specific inhibitor of AdoMet-dependent methyltransferases, is indicative of the cellular methylation potential [66].",
"In addition to its remethylation to methionine, Hcy can be subjected to transsulfuration leading to the synthesis of cysteine, which is also a precursor of glutathione, an essential cellular defense molecule in oxidative stress response [65]. This pathway irreversibly withdraws Hcy from the methylation cycle. An alternative way for Hcy metabolism is the reversal of the reaction catalyzed by S-adenosyl-Lhomocysteine hydrolase. This occurs upon accumulation of the hydrolytic products of the reaction, in particular Hcy, and leads to AdoHcy synthesis and accumulation [19,[67][68][69]. Thus, elevated Hcy levels via accumulation of AdoHcy lead to the disruption of the methylation cycle and, potentially, to methylation deficiency.",
"Deficiency in cystathionine β-synthase (CBS), the first and rate-limiting enzyme of the transsulfuration pathway ( Figure 1), is the major cause of severe hyperhomocysteinemia followed by genetic defects of folate and cobalamin metabolism that is involved in Hcy remethylation [65]. These pathological conditions lead to the plasma Hcy levels of more than 100 μmol/L [65] and are rare in comparison with mild hyperhomocysteinemia that is caused by dietary deficiencies of the vitamin cofactors required for Hcy catabolism -folic acid, vitamins B 6 and B 12 , and characterized by the plasma Hcy levels of 15-25 μmol/L [70]. Vitamin B 6 is required for the activity of CBS. Folic acid and vitamin B 6 are required for the activity of methionine synthase catalyzing 5-methyltetrahydrofolate-dependent remethylation of Hcy to methionine (Figure 1). While vitamin supplementation appeared to be a straightforward strategy to reduce/prevent cardiovascular events, this possibility was studied in several large trials. However, it was observed that vitamins, while capable of lowering elevated plasma Hcy levels, do not reduce the rates of vascular events [71]. Several potential mechanisms that might explain this result by offsetting the positive effect of Hcy-lowering therapy were subsequently proposed. These include promotion of cell proliferation by folic acid through its role in the synthesis of thymidine, increase of the methylation potential leading to changes in gene expression, and increase in the levels of asymmetric dimethylarginine that inhibit the activity of nitric oxide synthase [71]. An additional possibility is that, not Hcy, but rather a related metabolite could be a trigger of some pathological changes associated with elevated Hcy levels. Possibly explaining the failure of Hcy-lowering vitamins to reduce vascular events, it was recently reported that supplementation with B-vitamins including folate does not efficiently lower plasma AdoHcy levels [72], presumably due to elevation of AdoMet-dependent methylation.",
"In yeast, the synthesis of PC from PE by the de novo phospholipid methylation pathway is particularly sensitive to AdoHcy accumulation [18,63]. Both inhibition of S-adenosyl-Lhomocysteine hydrolase and Hcy supplementation results in AdoHcy accumulation and inhibition of phospholipid methylation in yeast [18]. However, not only phospholipid methylation, but also a methylation-independent branch of lipid metabolism, namely, TAG synthesis, is affected by Ado-Hcy accumulation in yeast: yeast cells deficient in AdoHcy catabolism or supplemented with Hcy massively accumulate TAG [18]. Supporting the causal role of impaired phospholipid methylation in the deregulation of TAG metabolism in response to AdoHcy accumulation, it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of PE to PC, cho2 and opi3, also accumulate TAG [18]. TAG is known to play an important role in buffering excess fatty acids [73]. Therefore, TAG accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to TAG synthesis in methylation deficiency in yeast. Another observation as well supports accumulation of fatty acids under these conditions. In addition to TAG metabolism, transcriptional regulation of phospholipid biosynthesis is also affected in yeast mutants deficient in AdoHcy catabolism. Impaired phospholipid methylation in Sah1-depleted cells unable to hydrolyze AdoHcy or in cho2 and opi3 mutants leads to upregulation of genes, which have an inositol-sensitive upstream regulatory sequence (UAS INO ) in their promoter regions, indicating accumulation of the phospholipid precursor, phosphatidic acid, in the ER [18]. ACC1 encoding acetyl-CoA carboxylase, the first and ratelimiting enzyme of fatty acid biosynthesis, is also a subject to UAS INO -mediated regulation, suggesting upregulation of the de novo fatty acid biosynthesis in response to AdoHcy accumulation. Moreover, Sah1 depletion also affects sterol synthesis in yeast, leading to 4-fold elevated squalene levels and suggesting accumulation of early precursors of ergosterol biosynthesis under these conditions (Tehlivets, Kohlwein, unpublished). Taken together, inhibition of phospholipid methylation induced by AdoHcy accumulation appears to lead to upregulation of fatty acid, TAG, and sterol biosynthetic pathways in yeast.",
"AdoHcy inhibits phosphatidylethanolamine N-methyltransferase in vitro and in vivo also in mammals [28,37,74]. Similarly as in yeast, deficiency of phospholipid methylation in PEMT −/− knockout mice leads to a rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in the liver, in the absence of choline supplementation [75]. However, TAG accumulation in the livers of these animals appears to be at least in part due to decreased TAG secretion from hepatocytes [55]. Elevated levels of Hcy are as well linked to deregulation of lipid metabolism in mammals. CBS −/− knockout mice exhibit severe hyperhomocysteinemia and accumulate Ado-Hcy in all tissues tested [68,69]. These mutant animals show elevated TAG and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [76,77]. Another genetic disorder that results in moderately elevated Hcy levels, methylenetetrahydrofolate reductase (MTHFR) deficiency, leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [78,79]. Dietary-induced hyperhomocysteinemia in mice also causes fatty liver, further supporting the role of Hcy in deregulation of lipid metabolism in mammals [64]. In these mice as well as in the CBS −/− knockout mice lipid accumulates in liver rather than in serum [64,76].",
"Preferable accumulation of lipids in the liver and, possibly, other tissues in hyperhomocysteinemia suggests that other mechanisms than those associated with elevation of circulating lipids are responsible for the development of cardiovascular disease under these conditions. Indeed, conventional risk factors including hypercholesterolemia accounts only for approximately 50% of all cases of cardiovascular disease, while 40% of patiens diagnosed with premature coronary artery disease, peripheral vascular disease or venous thrombosis exhibit hyperhomocysteinemia [80]. In accordance, unlike typical lipid-rich atherosclerotic plagues, vascular lesions associated with hyperhomocysteinemia are lipid-poor, fibrous plaques [81,82], greatly outnumbering fatty atherosclerotic lesions [80].",
"In animal models of hyperhomocysteinemia atherosclerotic lesions are rare. They are found only in the MTHFR −/− knockout mice that exhibit aortic lipid accumulation reminiscent of early atherosclerotic lesions [2,78,83], however, not in, for example, CBS −/− knockout mice. This discrepancy might be due to disruption of two different Hcy utilizing pathways in these animals. While 5-methyltetrahydrofolatedependent Hcy remethylation occurs in all mammalian cells, transsulfuration of Hcy occurs primarily in the liver and kidney [65]. Thus, impairment of 5-methyltetrahydrofolatedependent Hcy remethylation in MTHFR −/− knockout mice may differently affect Hcy metabolism in comparison to the deficiency in the first step of Hcy transsulfuration in CBS −/− knockout mice. The observation that dietary (methionine or Hcy supplementation) or genetically (CBS gene deletion) induced hyperhomocysteinemia in apoE-deficient (apoE −/− ) mice leads to development of larger and more advanced atherosclerotic lesions clearly demonstrates a causal relationship between elevated Hcy levels and atherosclerosis [83]. In contrast, lack of PEMT was shown to reduce significantly plasma VLDL and to attenuate atherosclerosis in both PEMT −/− /Ldlr −/− mice deficient in PEMT and LDL receptors as well as in PEMT −/− /ApoE −/− mice [84,85].",
"Elevated Hcy levels induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in a variety of mammalian cells. These include cultured human hepatocytes, vascular endothelial and aortic smooth muscle cells as well as liver cells of the CBS −/− knockout mice [64,[86][87][88]. Furthermore, elevated Hcy levels lead to activation of the sterol regulatory element-binding proteins (SREBPs), which function to activate genes encoding enzymes in cholesterol, fatty acid, and TAG metabolism and uptake, both in cultured mammalian cell lines as well as in the livers of the CBS −/− knockout mice [64,86]. ER stress appears to play a direct role in the activation of TAG and cholesterol biosynthesis, since overexpression of the ER chaperone GRP78/BiP was reported to inhibit Hcy-induced SREBP-1 gene expression in mammalian cell cultures [64] as well as in mice [89] and lead to reduction of the hepatic steatosis in leptin-deficient (ob/ob) mice [89]. SREBP-1 overcomes translation inhibition induced by UPR through an internal ribosome entry site (IRS), similarly to GRP78 [90].",
"Confirming the causal role of Hcy in UPR induction and deregulation of lipid metabolism, a decrease of elevated plasma Hcy levels is accompanied by a decrease in hepatic lipids and ER stress response [91]. A strong correlation between lipid metabolism, ER stress response and elevated Hcy levels is also evident form a literature mining approach [92]. Further demonstration of close relationship between ER stress and Hcy metabolism came from the observation that MTHFR involved in Hcy remethylation is induced in response to ER stress [93]. Evolutionary conservation of the relationship between Hcy and UPR is shown by the induction of ER stress and activation of UPR in response to Hcy supplementation in yeast [94]. Finally, demonstrating its pathophysiological role, ER stress was shown to be strongly associated with accelerated atherosclerosis in hyperhomocysteinemic apoE-deficient mice [95], liver diseases [96] as well as hyperglycemia-induced atherosclerosis [97].",
"The UPR, as a conserved cellular stress response pathway, is aimed at restoring normal ER and secretory function as well as membrane trafficking upon impaired protein folding in the ER. The de novo methylation and the CDP-choline phospholipid biosynthetic pathways produce phospholipid species with distinct fatty acyl chain composition in yeast: the de novo phospholipid methylation pathway produces more unsaturated phospholipids [98,99]. Similarly, the PEMT-generated PC pool in mammals is also enriched in unsaturated fatty acids [100,101]. Supporting the role of PEMT in metabolism of unsaturated fatty acids PEM −/− knockout mice were reported to accumulate more saturated PC molecular species in the liver compared with the control littermates [60] and to exhibit dramatically reduced concentrations of polyunsaturated fatty acids in the plasma and in hepatic PC, independently of choline status [102]. Thus, beyond its role as a compensatory pathway for PC biosynthesis under conditions of choline deprivation, phospholipid methylation plays a crucial role in unsaturated fatty acid metabolism both in yeast and in mammals. The observation that deficiency of phospholipid methylation in cho2 and opi3 yeast mutants is synthetically lethal in the absence of a functional UPR [103] suggests an essential requirement of UPR in response to impaired phospholipid methylation. Thus, Hcy accumulation, which was shown to lead to AdoHcy-mediated inhibition of phospholipid methylation in yeast, may lead to accumulation of saturated fatty acids in membrane phospholipids-a potential pathological mechanism that might be shared by both yeast and mammals. Indeed, accumulation of saturated fatty acids in membrane phospholipids interferes with ER structure and integrity, induces ER stress and leads to cell death in mammalian cell cultures [104,105]. Accordingly, decreased membrane phospholipid desaturation due to stearoyl-CoA desaturase 1 knockdown induces UPR in HeLa cells [106]. Vice versa, overexpression of stearoyl-CoA desaturase attenuates palmitate-induced ER stress and protects from lipoapoptosis [107][108][109]. Treatment with the molecular chaperone 4phenyl butyrate, which is capable of stabilization of protein conformation, improvement of ER folding capacity and facilitation of protein trafficking, leads to abolishment of UPR induction in yeast subjected to lipid-induced ER stress [105]. This finding suggests that accumulation of saturated fatty acids in membrane phospholipids first leads to changes in the membrane environment followed by induction of ER stress and accumulation of misfolded protein(s) that, in turn, activate UPR [105]. Potential mechanisms involved in saturated fatty acid-induced UPR include depletion of ER Ca 2+ stores leading to decreased ER chaperone activity and protein misfolding, as well as interference with ER-to-Golgi trafficking [110]. Recently, proteomic studies showed that carboxypeptidase E, a key enzyme involved in processing [111] and sorting of insulin [112], is involved in induction of ER stress in β-cells in response to palmitate treatment [113]. Degradation of carboxypeptidase E in palmitateinduced ER stress is mediated by palmitate metabolism and Ca 2+ flux [113]. Alternatively, changes of the ER membrane environment may directly activate ER sensors IRE1, ATF6, and PERK or modulate the binding of the ER sensors to the ER chaperone GRP78 causing its dissociation and activation of UPR pathways.",
"Supporting the hypothesis that Hcy interferes with phospholipid acyl chain composition it was observed in humans that elevated plasma AdoHcy levels are negatively correlated with both PC content and the level of polyunsaturated fatty acids in PC, but not in PE, in red blood cells in Alzheimer's patients [114]. Elevated plasma Hcy levels were also shown to be associated with a decrease in polyunsaturated (docosahexaenoic) fatty acids in the plasma of healthy humans [115] and in the plasma and erythrocytes of cystic fibrosis patients; these individuals exhibit increased Hcy and AdoHcy levels as well as altered PE and PC metabolism [116].",
"Taken together, deficiency of phospholipid methylation caused by AdoHcy accumulation in Hcy-associated pathology appears to lead to an increase in saturated PC molecular species in ER membranes followed by ER stress, protein misfolding, induction of UPR, and activation of lipid metabolism ( Figure 2). Upregulation of lipid biosynthesis, which apparently should serve to compensate for suboptimal composition of membrane lipids, leads, however, to accumulation of fatty acids, TAG and sterols in the absence of functional phospholipid methylation. In addition to a role in formation of a specific pool of PC molecular species, phospholipid methylation is crucial for maintenance of a distinct PC/PE ratio important for cell integrity when dietary choline-supply is blunted. Decrease of the PC/PE ratio was reported to result in increased cell permeability of hepatocytes from PEMT −/− mice fed choline deficient diet leading to liver damage [75]. Similarly, decrease of PC or increase in PE was shown to lead to cell damage and/or death in several other mammalian cell types [117][118][119]. Finally, observation of different outcomes in hyperhomocysteinemic apoE −/− mice and both in PEMT −/− /Ldlr −/− and PEMT −/− /ApoE −/− mice suggests mechanisms besides inhibition of phospholipid methylation, for example, AdoHcy-dependent modulation of gene expression that may also contribute to the development of Hcy-dependent atherosclerosis.",
"The metabolism of homocysteine, consequences of its accumulation as well as associated AdoHcy-triggered inhibition of AdoMet-dependent methylation are complex. In this paper a novel mechanism of Hcy-triggered deregulation Figure 2: A model of activation of UPR and upregulation of fatty acid, TAG, and sterol biosynthesis in response to inhibition of phospholipid methylation in hyperhomocysteinemia. Elevated Hcy levels via AdoHcy accumulation and inhibition of phospholipid methylation lead to accumulation of saturated PC molecular species in ER membranes followed by ER stress, UPR activation, and upregulation of fatty acid, TAG, and sterol biosynthesis. The enzymes involved in yeast and mammalian metabolism are shown in grey circles (see Figure 1). PE: phosphatidylethanolamine; PC: phosphatidylcholine; PEMT: phosphatidylethanolamine N-methyltransferase in mammals; Cho2 and Opi3: phosphatidylethanolamine N-methyltransferases in yeast. of lipid metabolism and UPR induction that is mediated through an AdoHcy-dependent inhibition of phospholipid methylation and based on experimental evidence derived from both yeast and mammalian systems is proposed. The observations made in yeast and mammals are summarized in Table 1.",
"S-adenosyl-L-homocysteine hydrolase is recognized since many years as a target for antiviral drug design [120]. Inhibitors that block AdoHcy hydrolysis are efficient against many types of viruses including Ebola and show other effects of pharmacological importance [120][121][122]. However, they are associated with high cytotoxicity due to interference with central metabolic pathways [121,122]. While using these inhibitors to study the effects of AdoHcy accumulation appears to be straightforward, ability of some of nucleoside inhibitors of S-adenosyl-L-homocysteine hydrolase to undergo metabolic phosphorylation to nucleotides may account for a part of their biological activities [121,122]. Inhibitors that would be able to block selectively homocysteine and adenosine conversion to AdoHcy are not available. However, based on current understanding of regulation of homocysteine and methionine metabolism (i) selective blockage of AdoHcy synthesis from homocysteine and adenosine that will relieve not only inhibition of phospholipid methylation but also many other AdoMetdependent methyltransferase reactions, combined with (ii) vitamin B 6 supplementation in order to accelerate homocystein catabolism by transsulfuration pathway, may serve as a way to reduce AdoHcy in hyperhomocysteinemia without elevation of AdoMet.",
"Many questions in the model presented in this paper are still unanswered. Is AdoHcy-mediated accumulation of saturated fatty acids in membrane lipids indeed the way by which elevated Hcy levels induce ER stress? What role do specific lipid precursors have in regulation of lipid metabolism in UPR? What is the impact of AdoHcy accumulation on other methylation reactions unrelated to lipid metabolism? What is the role of deficient phospholipid methylation in homocysteine-associated pathology beyond deregulation of fatty acid, TAG, sterol metabolism, and UPR induction? Elucidation of the molecular mechanisms triggered by elevated Hcy levels will undoubtfully improve our understanding of its pathological role in numerous diseases.",
"AdoMet:",
"S-adenosyl-L-methionine AdoHcy:",
"S-adenosyl-L-homocysteine Hcy:",
"Homocysteine PC:",
"Phosphatidylcholine PE:",
"Phosphatidylethanolamine TAG:",
"Triacylglycerol UPR:",
"Unfolded protein response Sah1:",
"S-adenosyl-L-homocysteine hydrolase in yeast AHCY:",
"S-adenosyl-L-homocysteine hydrolase in mammals Cho2 and Opi3: Phosphatidylethanolamine N-methyltransferases in yeast PEMT:",
"Phosphatidylethanolamine N-methyltransferase in mammals CBS:",
"Cystathionine β-synthase MTHFR:",
"Methylenetetrahydrofolate reductase."
] | [
"Hindawi Publishing Corporation",
"Hindawi Publishing Corporation",
"Hindawi Publishing Corporation",
"Hindawi Publishing Corporation"
] | [
"Introduction",
"Role of Homocysteine in the Methylation Cycle",
"AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast",
"Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals",
"Role of the Unfolded Protein Response in Hyperhomocysteinemia and Atherosclerosis",
"Deregulation of Fatty Acid Metabolism in Response to AdoHcy Accumulation",
"Concluding Remarks",
"Abbreviations",
"Figure 1 :",
"6",
"Table 1 :",
"Introduction",
"Role of Homocysteine in the Methylation Cycle",
"AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast",
"Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals",
"Role of the Unfolded Protein Response in Hyperhomocysteinemia and Atherosclerosis",
"Deregulation of Fatty Acid Metabolism in Response to AdoHcy Accumulation",
"Concluding Remarks",
"Abbreviations",
"Figure 1 :",
"6",
"Table 1 :"
] | [
"UPR \n\nER stress \n\nPE \nPC \n\nCho2 \nOpi3 \nPEMT \nAdoMet \nAdoHcy \n\nSam1/Sam2 \nMAT \n\nMET \nHcy \n\nSah1/AHCY \n\nMet6 \nMFMT/BHMT \n\nCTT \n\nCysteine \n\nGlutathione \n\nGsh1/GCS \n\nStr1/CTH \n\nStr4/CBS \n\nAccumulation of SFA \nin membrane phospholipids \n\ni \n\n",
"Experimental evidence \nYeast \nMammals \nAdoHcy is formed in vivo in \nresponse to elevated Hcy levels \n+ \n+ \n\nAdoHcy is more toxic than Hcy \nto cells deficient in Hcy \ncatabolism \n\n+ \n\nAdoHcy represents a better \nmarker of cardiovascular risk \nthan Hcy \n\n+ \n\nPhospholipid methylation is \nquantitatively the major \nconsumer of AdoMet \n\n+ \n+ \n\nPhospholipid methylation is \ninhibited in response to Hcy \nsupplementation \n\n+ \n\nPhospholipid methylation is \ninhibited by AdoHcy \n+ \n+ \n\nTAG is accumulating in response \nto Hcy supplementation \n+ \n+ \n\nTAG is accumulating in response \nto deficiency in AdoHcy \nhydrolysis \n\n+ \n\nTAG is accumulating in response \nto deficiency in phospholipid \nmethylation \n\n+ \n+ \n\nUPR is inducted in response to \nHcy supplementation \n+ \n+ \n\nThe de novo phospholipid \nmethylation pathway produces \nphospholipids enriched in \nunsaturated fatty acids \n\n+ \n+ \n\nER stress is inducted by \naccumulation of saturated fatty \nacids in membrane \nphospholipids \n\n+ \n+ \n\n",
"UPR \n\nER stress \n\nPE \nPC \n\nCho2 \nOpi3 \nPEMT \nAdoMet \nAdoHcy \n\nSam1/Sam2 \nMAT \n\nMET \nHcy \n\nSah1/AHCY \n\nMet6 \nMFMT/BHMT \n\nCTT \n\nCysteine \n\nGlutathione \n\nGsh1/GCS \n\nStr1/CTH \n\nStr4/CBS \n\nAccumulation of SFA \nin membrane phospholipids \n\ni \n\n",
"Experimental evidence \nYeast \nMammals \nAdoHcy is formed in vivo in \nresponse to elevated Hcy levels \n+ \n+ \n\nAdoHcy is more toxic than Hcy \nto cells deficient in Hcy \ncatabolism \n\n+ \n\nAdoHcy represents a better \nmarker of cardiovascular risk \nthan Hcy \n\n+ \n\nPhospholipid methylation is \nquantitatively the major \nconsumer of AdoMet \n\n+ \n+ \n\nPhospholipid methylation is \ninhibited in response to Hcy \nsupplementation \n\n+ \n\nPhospholipid methylation is \ninhibited by AdoHcy \n+ \n+ \n\nTAG is accumulating in response \nto Hcy supplementation \n+ \n+ \n\nTAG is accumulating in response \nto deficiency in AdoHcy \nhydrolysis \n\n+ \n\nTAG is accumulating in response \nto deficiency in phospholipid \nmethylation \n\n+ \n+ \n\nUPR is inducted in response to \nHcy supplementation \n+ \n+ \n\nThe de novo phospholipid \nmethylation pathway produces \nphospholipids enriched in \nunsaturated fatty acids \n\n+ \n+ \n\nER stress is inducted by \naccumulation of saturated fatty \nacids in membrane \nphospholipids \n\n+ \n+ \n\n"
] | [
"Table 1",
"Table 1"
] | [
"Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?",
"Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?",
"Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?",
"Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S -Adenosyl-L -Homocysteine the Key to Deregulated Lipid Metabolism?"
] | [
"Journal of Lipids",
"Journal of Lipids"
] |
15,325,974 | 2022-03-17T09:55:31Z | CCBY | https://doi.org/10.5339/gcsp.2015.47 | GOLD | 484a30cc5be2ae4823c40dd06e78bdffca3a596e | null | null | null | null | 10.5339/gcsp.2015.47 | 2249989708 | 26779522 | 4710869 |
The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension
Mark L Ormiston
The Department of Medicine
School of Clinical Medicine
University of Cambridge
Addenbrooke's and Papworth Hospitals
CambridgeUnited Kingdom
Paul D Upton
The Department of Medicine
School of Clinical Medicine
University of Cambridge
Addenbrooke's and Papworth Hospitals
CambridgeUnited Kingdom
Wei Li
The Department of Medicine
School of Clinical Medicine
University of Cambridge
Addenbrooke's and Papworth Hospitals
CambridgeUnited Kingdom
Nicholas W Morrell
The Department of Medicine
School of Clinical Medicine
University of Cambridge
Addenbrooke's and Papworth Hospitals
CambridgeUnited Kingdom
The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension
10.5339/gcsp.2015.47Submitted: 14 June 2015 Accepted: 27 August 2015 ª 2015 Ormiston, Upton,O P E N A C C E S S Mechanisms of disease
Human genetic discoveries offer a powerful method to implicate pathways of major importance to disease pathobiology and hence provide targets for pharmacological intervention. The genetics of pulmonary arterial hypertension (PAH) strongly implicates loss-of-function of the bone morphogenetic protein type II receptor (BMPR-II) signalling pathway and moreover implicates the endothelial cell as a central cell type involved in disease initiation. We and others have described several approaches to restore BMPR-II function in genetic and non-genetic forms of PAH. Of these, supplementation of endothelial BMP9/10 signalling with exogenous recombinant ligand has been shown to hold considerable promise as a novel large molecule biopharmaceutical therapy. Here, we describe the mechanism of action and discuss potential additional effects of BMP ligand therapy.Cite this article as: Ormiston ML, Upton PD, Li W, Morrell NW. The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension, Global Cardiology Science and Practice 2015:47 http://dx.
INTRODUCTION
Pulmonary arterial hypertension (PAH) is a disease of pathological vascular remodeling, characterized by medial thickening and the formation of occlusive vascular lesions that obstruct the pulmonary circulation, predominantly at the level of the pre-capillary arterioles. This loss of the pulmonary distal circulation, and the resultant increase in pulmonary vascular resistance (PVR), leads to an elevation of pulmonary arterial pressure and right ventricular hypertrophy. PAH is a rare disease, with a prevalence of 15 to 26 patients per million individuals and an incidence of 2.4 to 7.6 cases per million annually. 1,2 It can arise as a primary disease, either in an idiopathic (IPAH) or heritable (HPAH) form, or as a condition associated with immune disorders such as HIV, connective tissues diseases, or exposure to particular drugs or toxins. In its idiopathic and heritable forms, PAH presents in a relatively young patient population, aged 30 -50 years, and preferentially in women at a rate of roughly 2.3:1. If left untreated, PAH can lead to death from right-sided heart failure within 3 -5 years of diagnosis.
Existing treatments for PAH were developed for this indication because of their effects on vascular tone. The regulation of vascular tone by endothelial cells and vascular smooth muscle cells (SMCs) is mediated by a balance of vasodilators, such as prostacyclin and nitric oxide (NO), and vasoconstrictive agents, including endothelin-1. Established PAH is associated with a shift in this balance towards excessive pulmonary vasoconstriction. Recognition of this imbalance was the driving force behind the development and approval of a range of vasodilatory therapies for PAH. 3 These therapies can be divided into three main classes: (i) prostanoids, including epoprostenol and more stable prostacyclin analogues such as iloprost, beraprost and treprostinil; (ii) endothelin receptor antagonists, including bosentan, ambrisentan and macitentan; and (iii) phosphodiesterase 5 inhibitors, such as sildenafil and tadalafil. While these treatments have been successful in improving the haemodynamic parameters and functional status of patients, the three-year survival rate for PAH patients remains poor. 4 Although these therapies may also have modest effects on vascular SMC proliferation, available evidence suggests a minimal impact on the process of vascular remodeling in the lungs of patients with PAH. 5 Here we propose that more effective therapies for PAH will derive from a greater understanding of the molecular basis of pathological pulmonary vascular remodeling, particularly by targeting pathways identified by human genetics in PAH patients.
THE GENETIC BASIS OF PAH
Heritable PAH (HPAH) is an autosomal dominant disease, marked by a low penetrance (average 20 -30%) in at-risk individuals. 6 While the existence of this familial form of PAH has been recognized since the first description of the disease, it was only 15 years ago that mutations in BMPR2, the gene encoding the bone morphogenetic protein (BMP) type II receptor (BMPR-II), was identified as the cause of approximately 75% of HPAH cases. 7,8 BMPR2 mutations also account for 15 -26% of seemingly idiopathic or sporadic cases of PAH, including cases of de novo mutations and parental transmission with no record of a previous family history of disease. As a result of these findings, the definition of HPAH was recently updated to include not only patients in a family with two or more documented cases of PAH, but also includes any PAH patient possessing a mutation in BMPR2. 9 A range of mutations in BMPR2 have been reported in PAH patients. The majority of these mutations lead to a state of haploinsufficiency, 10 where the mutant allele leads to no production of a protein product. The protein expression from the wild type allele is normal but overall protein expression is reduced by at least 50%. Patients bearing BMPR2 mutations develop PAH earlier, have more severe disease and die sooner than those without mutations. 6 Interestingly, PAH patients with BMPR2 mutations exhibit reductions in BMPR-II protein levels of greater than 75% when compared to control subjects, suggesting that the development of PAH can suppress receptor levels to a greater extent than what can be accounted for by haploinsufficiency alone. 11 Reduced BMPR-II protein levels and impaired downstream signaling have also been identified in idiopathic PAH patients lacking mutations in BMPR2, as well as in common, non-genetic rodent models of disease, 12 further supporting a central role for reduced BMPR-II signaling in most forms of the disease, independent of etiology.
When taken together, these factors make the BMPR-II signaling pathway an extremely attractive target for next-generation therapeutic intervention. However, the translation of this approach into in vivo pre-clinical studies has been limited by uncertainty regarding which cell type or types are critically affected by the loss of BMPR-II signaling, and the complexity of the BMP signalling family. Fortunately, genetic studies describing disease phenotypes associated with mutations in other components of the BMP signaling pathway, coupled with knowledge of the tissue-specific distribution of these proteins, can be used to inform decisions on which cell types, and which BMPs, play important roles in the initiation of PAH.
BMPs are members of the transforming growth factor-b (TGFb) superfamily, a highly complex family of proteins including over 30 ligands that signal through heteromeric complexes of type I and type II receptors. As a type II receptor of this superfamily, BMPR-II can form complexes with several type I receptors, including the activin like receptor kinases ALK1, ALK2, ALK3 or ALK6, with each receptor complex recognizing a specific subset of BMP ligands. 13 Canonical signaling generally involves phosphorylation of the Smad-1, 5 or -8 transcription factors, which complex with the co-smad, Smad-4, and translocate to the nucleus to induce gene expression. The tissue-specific nature of BMP responses is highly dependent upon the components making up the ligand-receptor complex. Numerous accessory receptors, such as endoglin (ENG), also modify signaling, further contributing to the tissue-specificity of BMP responses.
Following the initial identification of BMPR2 mutations in PAH, much of the work investigating the role of deficiency of this receptor in disease pathogenesis focused on the impact of these mutations on pulmonary arterial SMC (PASMC) proliferation and migration. 14 In smooth muscle cells, BMP ligands, including BMP2 and BMP4, block serum-induced proliferation via complexes of BMPR-II with ALK3 or ALK6. 14 However, mutations in ALK3 and ALK6 do not cause pulmonary vascular disease, but are instead associated with juvenile gastrointestinal polyposis 15 and hereditary brachydactyly, 16 respectively. This fact calls into question the importance of these receptors, and the signaling complexes that they form, in the pathogenesis of PAH.
More recently, several studies have demonstrated a role for impaired endothelial BMP signaling in the pathogenesis of PAH. In addition to increased endothelial cell proliferation and enhanced susceptibility to apoptosis, 17,18 loss of BMPR-II has also been shown to influence endothelial cell barrier integrity and pulmonary vascular permeability. 19 Genetic evidence also supports a role for the pulmonary endothelium as the critical initiating cell type in PAH. Mutations in Endoglin and ALK1 (gene symbol ACVRL1), two proteins found almost exclusively on the endothelium, are primarily associated with hereditary haemorrhagic telangectasia (HHT), a disease that causes vascular abnormalities, including telangiectases in the skin and mucosal regions, and arteriovenous malformations in the lung, liver, gastrointestinal tract and brain. In addition to HHT, ACVRL1 mutations are occasionally associated with PAH, demonstrating a role for these molecules, and the receptor complexes they form, in the maintenance of pulmonary vascular homeostasis. 20 To date, mutations in eight genes have been found to play a causal role in idiopathic and heritable PAH (Fig. 1). Notably, of all the known mutations identified to date, six out of eight of these directly or indirectly implicate the BMP signalling pathway, and particularly endothelial BMP signaling, as central to pathobiology. Taken together, these findings strongly support the development of therapeutic strategies that restore expression or function of BMPR-II, particularly in pulmonary vascular endothelial cells.
TARGETING BMPR-II DEFICIENCY FOR THE TREATMENT OF PAH
Considering the genetic evidence indicating an important role for BMPR-II in the pathobiology of PAH, several groups have begun to use this information to test the potential of next generation therapies that directly target BMPR-II deficiency. Although not limited to the endothelium, a number of these studies directly target pulmonary endothelial BMPR-II for their mode of action. These therapies are directed towards various stages of BMPR-II signaling, including gene expression, translation, surface expression and receptor degradation, as well as enhancing receptor activity through the delivery of BMPR-II agonists. The various strategies can be grouped into three main approaches: (i) rescue of mutant receptor function, (ii) enhancing the expression or longevity of the wild-type receptor or (iii) enhancing BMP signaling through small molecules or recombinant BMP ligands. The published studies examining these three approaches are summarized below and in Table 1.
Rescuing mutant BMPR-II receptor function: Over 400 different PAH associated BMPR2 mutations have been identified in all functional domains of the BMPR-II protein. 6 These include missense mutations within the extracellular domain, transmembrane region, and kinase region, as well as nonsense mutations that encode premature termination codons (PTCs). Strategies designed to enhance BMP signaling by rescuing the mutated copy of the BMPR2 gene can take one of two forms, depending on the type of mutation present.
Chemical or pharmacological chaperones: Missense mutations can lead to the production of misfolded BMPR-II through the replacement of critical cysteine residues that are essential to the folding of the extracellular domain within the endoplasmic reticulum (ER). These mutants can be targeted with chemical chaperones that enhance the transport of the mutant protein from the ER to the cell surface. Some chemical chaperones, such as glycerol, non-selectively stabilize the tertiary structure of the mutant proteins and facilitate folding. Other compounds, including thapsigargin and sodium 4-phenylbutyrate (4-PBA), inhibit the interactions of the mutant protein with the chaperone proteins responsible for their recognition, retention and degradation. In PAH, a panel of chemical chaperones, including 4-PBA, glycerol and thapsigargin, were shown to promote folding and trafficking of mutant BMPR-II protein to the cell surface. Since, in this case, the mutant receptor was still capable of normal signalling, downstream Smad signalling was restored. 21 While these results are promising, there are several factors limiting the clinical application of these compounds for PAH. The PAH patient population contains a large variety of missense mutations, only a subset of which encode mutations that lead to the misfolding a receptor that is otherwise functional. Nevertheless, 4-PBA has been used for other indications in man and may be a personalized approach in patients with these specific mutations.
Suppression of nonsense mutations: In contrast to missense mutations, where the mutant protein is produced and can be targeted for rescue, the mRNA transcripts produced by alleles bearing nonsense mutations are typically subject to nonsense-mediated decay (NMD), resulting in reduced BMPR2 mRNA and decreased protein levels. The rescue of these mutations requires the use of compounds that suppress or silence PTCs, allowing for the translation of mutant mRNA into full-length, functional protein. 22 A number of compounds belonging to the aminoglycoside family of antibiotics, including gentamicin, have been shown to suppress disease-causing PTCs and partially restore protein function in nonsense mutation-bearing cell culture models of multiple diseases, including cystic fibrosis, Duchenne muscular dystrophy and PAH. 23 However, the high doses required to achieve these beneficial effects in vivo are also associated with renal toxicity and hearing loss, thus preventing the widespread use of aminoglycosides in clinical applications.
More recently, high throughput screens have identified other compounds distinct from aminoglycosides that possess the ability to effectively suppress PTCs without affecting the recognition Figure 1. Simplified schematic summarizing the BMP signalling pathway and genes that have to date been shown to be mutated in PAH and demonstrating that many of these mutations encode protein involved in BMP signalling. Known mutations are numbered and in bold.
Rescue of Mutant BMPR-II Receptor
Chemical Chaperones
4-PBA, glycerol and thapsigargin
In vitro: HeLa cells [21] Suppression of Nonsense Mutations
Gentamycin
In vitro: cell-based reporter assay [23] Ataluren (PTC124) In vitro: PAH endothelial and smooth muscle cells [24] Enhancement of BMPR-II Expression and Longevity
Inhibition of lysosomal degradation
Chloroquine, hydroxychloroquine In vitro: PAH endothelial cells In vivo: Monocrotaline rat model [26,28] BMPR2 Gene Therapy Endothelial-targeted adenoviral vector In vivo: chronic hypoxia and monocrotaline rat models [29,30] Enhancement of BMPR-II-mediated Signaling bmpr2-R899X mouse,
Monocrotaline rat model, Sugen-Hypoxia rat and mouse models [41] of normal stop signals. One such compound, the small molecule Ataluren (PTC124), is currently in phase III clinical trials for Duchenne muscular dystrophy. In PAH, Ataluren was shown to increase BMPR-II protein levels and downstream signaling in primary cells from patients with nonsense mutations in BMPR2 and SMAD9. 24 These effects were accompanied by a restoration of normal proliferation rates in hyperproliferative, patient-derived PAECs and PASMCs.
Enhancing BMPR-II surface expression Inhibiting BMPR-II degradation: An alternative approach that has been explored for enhancing BMP signalling involves extending the longevity of BMPR-II at the cell surface. BMPR-II is subject to constitutive endocytosis by both clathrin and caveolae mediated pathways 25 and is rapidly turned over in endothelial cells. Notable protein loss is observed in these cells as early as 1 hour following the blockade of new protein synthesis. 26 BMPR-II can also be targeted for ubiquitination by Kaposi's sarcoma herpes virus K5 E3 ligase, and directed for lysosomal degradation. 27 Each of these findings highlights the lysosome as the primary route for BMPR-II turnover and points to lysosomal inhibition as a promising target for preserving BMPR-II longevity and enhancing receptor-mediated signaling. The 4-aminoquinolone, chloroquine, achieves lysosomal inactivation through the prevention of vesicular acidification. Used for decades as a prophylactic antimalarial drug, chloroquine, and its less toxic derivative hydroxychloroquine, have recently been repurposed for use in the treatment of rheumatoid arthritis and cancer. In vitro, chloroquine was shown to enhance BMPR-II protein levels, surface expression and downstream signaling in BMPR2 mutation-bearing endothelial cells. 26 In vivo, both chloroquine and hydroxychloroquine inhibited the development of pulmonary hypertension and blocked the progression of established disease in the monocrotaline rat model. 28 Although this therapeutic effect was associated with restoration of BMPR-II protein levels, which are decreased in monocrotaline-treated rats, it is likely that the beneficial effect of chloroquine administration may also be linked to the inhibition of excessive autophagy, which has been linked to the aberrant proliferation of vascular cells in PAH. Translation of hydroxychloroquine into a clinical therapy is facilitated by the fact that the compound is inexpensive and presents acceptable toxicity or side effects at therapeutically effective doses. Long-term treatment with hydroxychloroquine has been associated with retinopathy in 0.5 -1% of patients. However, this can be avoided with proper monitoring of patients undergoing treatment. BMPR2 gene therapy: Gene therapy techniques have also been explored in animal models of PAH as a means to restore BMPR-II receptor levels. The targeted delivery of an adenoviral vector containing the BMPR2 gene to the pulmonary vascular endothelium of rats substantially reduced the severity of pulmonary hypertension in the chronic hypoxia and monocrotaline models of PAH. 29,30 For these studies, targeting of the adenoviral vectors to the pulmonary endothelium appeared to be a key feature of therapeutic efficacy, as similar studies using aerosol vector delivery and a non-specific promoter failed to demonstrate any measurable benefit in the monocrotaline rat model. 31 Enhancement of BMPR-II downstream signalling: One alternative to enhancing BMPR-II receptor expression or longevity involves enhancing signaling through the action of small molecules. Existing PAH therapies, including prostanoids and sildenafil, have been shown to partially restore BMP signaling in PASMCs bearing BMPR2 mutations and prevent the development of PAH in animal models of disease. 32,33 More recently, a high throughput screening approach demonstrated that the calcineurin inhibitor, tacrolimus (FK506), potentiates BMPR-II-mediated signaling in endothelial cells, rescues dysfunctional BMP signaling in endothelial cells from PAH patients with BMPR2 mutations and reverses established disease in multiple rodent models of PAH. 34 Early clinical trials are currently underway to establish the viability of this strategy as a treatment for PAH. However, this approach is likely to lead to widespread and non-specific activation of BMP receptors, as well as having off target effects independent of BMP signalling.
BMP9 AND BMP10 AS POTENTIAL THERAPIES FOR PAH
One of the most direct strategies for targeting BMPR-II deficiency in PAH involves the delivery of exogenous BMP ligand to enhance signaling via the remaining functional receptor. Proof-of-concept studies using patient-derived pulmonary arterial smooth muscle cells (PASMCs) have shown that the addition of increasing concentrations of BMP ligand can overcome the functional defects associated with BMPR2 mutation in vitro. 14 However, identifying the appropriate BMP ligand or ligands to selectively target the endothelium in vivo presents a significant challenge. The selective expression of ALK1 on the endothelium makes the ALK1 /BMPR-II complex an ideal target for exogenous ligand therapy. Although ALK1 was originally thought to be an orphan receptor, two studies in 2007 identified BMP9 and BMP10 as ligands that signal via complexes of ALK1 with either BMPR-II or type II activin receptors (ActRII). 35,36 Further examination of the receptor selectivity of these ligands has primarily focused on BMP9. Endogenously, BMP9 activates ALK1 with high affinity (EC50 ¼ 50 pg/ml). BMP9 also activates ALK2 but with much a lower affinity in the ng/ml range. 35 -39 Biochemical analysis has also revealed discrete differences in the type II receptor selectivity of BMP9 and BMP10. Although BMP9 and BMP10 both bind to BMPRII, ActRIIA and ActRIIB, BMP9 exhibits a preference towards ActRIIB compared to BMPRII and ActRIIA while BMP-10 binds to all type II receptors with similar affinities. 40 In endothelial cells, type II receptor redundancy means that loss of either BMPR-II or ActRIIA has a minor impact on the stimulation of Smad1/5 phosphorylation due to compensation by the remaining type II receptor. 37 Loss of both receptors, however, abolished the Smad phopshorylation responses. Of note, some transcriptional responses to BMP9, such as E-selectin and IL8 induction are more reliant on BMPR-II signaling as ActRIIA does not compensate. 37 This implies that there are BMPR-II-specific signals that may contribute more than others to the pathology of PAH. Another feature of the BMPR-II selectivity of BMP9 relates to the transcriptional regulation of the receptors themselves. BMP9 induces the expression of BMPR-II, but not ALK1 or ActRIIA, suggesting that this ligand switches the balance of signaling in the endothelial cell towards BMPR-II. 35,37 Furthermore, this induction is ALK1-dependent and Smad1-dependent, suggesting a feed forward signaling mechanism. 37,41 It was recently shown that the therapeutic effects of BMP9 include restoration of BMPR-II expression and signaling of BMPR-II in animal models of PAH. 41 Therefore, BMP9 represents a therapy that promotes its key downstream signalling pathways partly via rescue of the deficient BMPR-II expression that underlies the pathogenesis of PAH. A proposed mechanism for this upregulation is shown in Fig. 2.
Based on our studies of BMPR-II dysfunction in PAH and the emerging role of BMP9 as a key circulating regulator of endothelial function and positive regulator of BMPR-II expression, we recently examined the therapeutic delivery of BMP9 to target endothelial dysfunction in PAH. 41 In vitro, BMP9 prevents the enhanced apoptosis observed in BMPR2 mutation-bearing endothelial cells and promotes monolayer integrity through the formation of tight junctions. In vivo, BMP9 prevents and reverses established disease in a range of rodent models, including spontaneous disease in a mouse model bearing a knock-in of the PAH-associated R899X-Bmpr2 mutation. From these studies, we propose that BMP9 constitutes a potential therapy that overcomes the underlying endothelial dysfunction that causes PAH and can overcome the deficient BMPR-II signaling that is a consequence of the major genetic defect in most HPAH patients (Fig. 3). Our anticipation is that, by restoring the balance in BMP signaling, this approach will redress the imbalance of other dysregulated pathways that are targeted by current therapies.
Another theoretical approach to activate the endothelial ALK1/BMPR-II pathway would be to develop a peptide mimetic that possesses BMP9/10-like function. An example of such an approach was recently demonstrated for a peptide derived from BMP7. 42 Although it is considered unlikely by some that a small peptide mimetic would be able to substitute for a BMP dimer in the assembly of two type I and two type II receptors, all of which are required for signalling, 43 -45 the BMP7 peptide mimetic was shown to have similar effects to full-length BMP7 ligand in reversing established fibrosis in five mouse models of acute and chronic renal injury. 42 Although the findings of this study have been challenged by researchers from several groups, synthetic peptides that specifically activate the endothelial ALK1/BMPR-II pathway still represent promising potential drug candidates for treating PAH.
Since BMP9 exerts its beneficial effect on the endothelium in an ALK1-dependent manner, recombinant BMP10 protein, which is the only other BMP ligand that signals through ALK1, could also have a similar therapeutic potential for treating PAH. There are several advantages of BMP10-based therapy. Firstly, unlike BMP9, it does not show osteogenic activity either in vitro signalling assays, or in an in vivo bone-forming screen, 46 and may therefore be potentially safer than BMP9 for treating cardiovascular disease. Secondly, BMP10 binds to ALK1 and BMPR-II with higher affinities than BMP9, 40 making it likely to be more potent and more specific for ALK1 and BMPR-II.
In spite of these advantages, there are still several hurdles to overcome before BMP10 can be developed into a therapy for PAH. For example, the activity of circulating BMP10 in humans is still under investigation. BMP10 is bound tightly by its prodomain, which inhibits BMP10 signalling activity when applied in molar excess. 47 It has therefore been proposed that BMP10, similar to TGFb, exists in a latent form in the circulation and requires an additional activation step to achieve bioactivity, such as cleavage of the prodomain by BMP1. 47 This hypothesis is further supported by the fact that, although circulating BMP10 could be consistently detected and measured by ELISA 48,49 and by pull-down coupled with proteomics, 50 early studies could not detect BMP10 activity in the circulation. 48,51 However, one recent report was able to demonstrate BMP10 activity in mouse serum. 49 A further question to be addressed is whether the presence of the prodomain on therapeutically administered BMP10 is important for the stability and half-life of the ligand. A better understanding of the physiology of BMP10 is required in vivo, since BMP10 is a much less well-studied BMP ligand and most of the data are from embryos of mouse and zebrafish. The data on the role of BMP10 in adult physiology and in PAH are limited, and such information will inform the development and testing of BMP10-based drugs.
PHYSIOLOGICAL ROLES OF BMP9 AND BMP10
During embryonic development and into adult life, BMP9 and BMP10 are both expressed in a highly tissue-specific pattern. BMP9 is expressed in the mouse fetal liver from E9.75 -10 49 and expression remains high in the adult liver. 52,53 The highest levels of expression are observed in mRNA from intrahepatic biliary epithelial cells and hepatocytes. 53 In contrast, BMP10 is highly expressed in the 1 This promotes BMPR-II mRNA transcription and synthesis 2 and trafficking of newly-synthesised BMPR-II to the cell surface where it complexes with ALK1, which has been recycled via the endosomal pathway. 3 In the presence of BMP9, this feed forward pathway continues in an autoregulatory loop. 4 Middle panel: in patients with a heterozygous mutation in BMPR2 leading to haploinsufficiency, cell surface BMPR-II is reduced and in its place, ActRII-A can form a complex with available ALK1 but this does not promote autoregulatory BMPR-II production in response to endogenous concentrations of BMP9, which remain unchanged. The reduced signalling through BMPR-II leads to reduced BMPR-II levels of the receptor at the cell surface. Right Panel: Administration of exogenous BMP9 to PAH patients with a heterozygous mutation in BMPR2, increases the circulating concentration of BMP9 which increases signalling via the Smad1/4 complex to induce BMPR-II protein expression. This shifts the equilibrium of the BMPR-II:ActR-IIA ratio in favour of BMPR-II associating with the available ALK1 and thus restores the autoregulatory production of BMPR-II in response to BMP9, thus restoring normal endothelial BMP9 signalling. developing embryonic mouse heart, with much lower levels of expression detected in lung and liver. 54 The expression of BMP10 is high in the trabecular myocardium between days E9.5 -E13.5, a period that coincides with cardiac growth and chamber maturation. 54 The expression of BMP10 becomes restricted to the atria by day E18.5 and in adults, only the right atrium expresses BMP10. 49,55 BMP9 is secreted and circulates at levels of 2 -12 ng/ml in human plasma 56 and 1.18 -1.84 ng/ml in human serum, 51 based on cell-based luciferase bioassays. Plasma BMP9 levels in mice increase just before birth and peak postnatally at levels of 6 ng/ml on Day 15 before declining to a steady state of approximately 1.5 -2 ng/ml. 53 Similar to other BMPs, BMP9 is synthesised as a 429 amino acid precursor (pre-pro-BMP9) comprising a 22 amino acid signal peptide, a 297 amino acid prodomain and a 110 amino acid mature protein. 53,57 The precursor is then cleaved by serine endoproteases to produce a mature protein dimer (25 kDa) which can remain non-covalently associated with two pro-domains (33 kDa each) forming a 100 kDa complex. 53 Unlike the pro-regions of TGFbs and GDF8, which whilst bound, inhibit their ligands in vitro and in vivo, 58 -61 the mature BMP9 dimer retains its biological activity when in complex with the pro-domain. 62,63 The bound pro-domain is proposed to enhance the stability of BMP9 in vivo. 62 Approximately 60% of the 100 kDa circulating pro-BMP9 complex is cleaved and active, whereas the remaining 40% is unprocessed and may be activated through a local furin cleavage. 53 The role of BMP10 as a secreted ligand is more controversial, since some studies suggest that BMP9 is solely responsible for the ALK1-activating BMP activity in plasma. 48,56 However, a recent study has reported that BMP10 is present in mouse (0.5-2 ng/ml) and human (1 -3 ng/ml) serum, suggesting that BMP10 may be present in the circulation at physiologically important levels. 49 Whether BMP10 in the circulation is processed and circulates in a similar manner to BMP9 is not yet clear. However, evidence supporting a functional role for circulating BMP10 has been revealed in zebrafish embryos, showing that BMP10 is necessary for maintaining vascular stability and endothelial Smad signalling in the vessels proximal to the heart. 64 Loss of BMP10 phenocopies the cranial vascular abnormalities of the Alk1 zebrafish mutants, 65 although as discussed later, this differs from the phenotype of the Bmp10 knockout mouse.
The overlapping expression of BMP10 and Alk1 at about E8.5 49, 66 compared to the expression of BMP9 in the liver at E9.75-10 has been argued as evidence for the developmental regulation of BMP10 signalling through Alk1. 49 As BMP9 and BMP10 both activate ALK1, this may represent temporally important roles of the ligands. For example, Alk1-/-mice die at E10.5 due to defects in angiogenesis, 67 which may represent a failure of both BMP9 and BMP10 signalling, as discussed below. In comparison, Alk1 þ /2 mice develop normally, but exhibit nosebleeds and vascular anomalies. This reflects the pathology of ALK1 mutations causing HHT in man, although the disease penetrance is lower in heterozygous mouse models.
Intriguingly, BMP9 knockout mice develop to adulthood without any overt phenotype, 48 although further analysis has revealed lymphatic vessel defects 68 and delayed closure of the ductus arteriosus. 69 The lack of a severe vascular phenotype in BMP9-/-mice appears to be due to functional redundancy with BMP10, confirmed by defective retinal vascularisation in BMP9-/-mice when BMP10 is also neutralised. 48,49 Of note, ALK1-Fc administration, which inhibits both BMP9 and BMP10, phenocopies this sprouting defect. 49 As circulating BMP10 levels are elevated slightly in BMP9-/-mice, one might speculate that BMP10 may compensate for the absence of BMP9 in the circulation. 48 Consistent with the restricted cardiac expression, BMP10-/-embryos die around E9.5 -E10.5 due to hypoplastic cardiac development, probably as a result of reduced myocyte proliferation. 55 Although original reports reported an absence of vascular defects, 55 recent data indicate that the dorsal aorta and cardinal veins are fused in BMP10-/-mice. 49 Furthermore, cloning of the BMP9 coding region into the BMP10 knockout mouse does not rescue the cardiac defect whereas early vascular development appears normal, suggesting that BMP10 mediates cardiac development via a mechanism that is independent of the signalling capacity of BMP9. 49 This could be due to differences in the selectivity for type II receptors. 49 Intriguingly, ectopic expression of BMP10 driven by the alpha-myosin heavy chain promoter in the mouse myocardium leads to cardiac hypotrophy by 6 weeks of age. 70 The level of BMP10 expression achieved in the hearts was high, so it is not clear whether this represents a physiologically relevant effect of BMP10. 70 Table 2 summarizes the similarity and the difference between BMP9 and BMP10 in physiology.
POTENTIAL SIDE EFFECTS OF BMP9/10 THERAPY One potential concern associated with the delivery of exogenous BMP ligands is the activation of other BMP receptors in non-endothelial cells, much of which is a dose-related phenomenon. It is therefore important to consider the roles of BMP9 in tissue ossification/calcification, hepatic function and tumour regulation.
Osteogenesis, chrondrogenesis and adipogenesis: The potent orthotopic bone-forming activity of BMP9 may be a potential complication of BMP9 therapy in PAH. BMP9 induces the differentiation of mesenchymal stem cells (MSCs -adult stem cells found in bone marrow) into osteocytes, chondrocytes and adipocytes 46 and is one of the most potent osteogenesis-inducing BMP ligands in MSCs. 46,71 -74 The osteogenic induction of bone marrow MSCs is mediated via both ALK1 and ALK2 75 and is reported to involve BMP9 dependent induction of angiogenic HIF1a signaling. 76 The osteogenic response to BMP9 in skeletal muscle has been achieved through several modes of local ligand expression. Delivery of BMP9 into skeletal muscle via direct sonoporation, 77 injection of transfected MSCs 78 or C2C12 cells 46,79 or injection of an adenovirus engineered to express BMP9 46 all elicited ectopic bone formation in muscle tissue. These processes are attributed to the differentiation of local multipotent MSCs or osteoblastic progenitor cells. 80 -82 Ad-BMP9 stimulates lamellar bone in mice by 3 months, 83 although studies have reported evidence of calcification after 9 days of exposure. 84 It is notable that the majority of these studies involve a local inflammatory stimulus and it appears that the heterotopic ossification response to BMP9 requires injury to skeletal muscle. 85 Another important consideration would be the concentration of BMP9 achieved during therapeutic administration. All of the above studies involved high concentrations of BMP9 or uncontrolled local overexpression. Our recent study employed intraperitoneal dosing of BMP9 injections, and we observed no evidence of heterotopic calcification after 4 weeks of daily intraperitoneal BMP9 injection, or indeed 3 weeks of intramuscular injection. 41 The concentration of BMP9 is likely to be important to achieve therapeutic activation of BMPR-II/ALK1, but avoidance of ALK2 activation at higher concentrations. A recent study demonstrated that BMP9 promotes calcification of vascular smooth muscle cells in the context of high phosphate levels. 86 In this study, the authors propose ALK1 as the receptor mediating this calcification, although this conclusion was derived from the use of an ALK1-Fc ligand trap to inhibit BMP9 rather than molecular dissection of the receptor signalling in VSMCs. BMP9 stimulated calcification at concentrations of 5 ng/ml or greater, representing concentrations at which BMP9 can stimulate both ALK1 and ALK2. In contrast, no calcification was observed at 0.5 ng/ml BMP9, a concentration that would exclusively activate ALK1. Indeed, BMP9 and BMP10 might be expected to exert endothelial-mediated vascular protective effects in atherosclerosis, since endothelial-specific knockout of BMPR-II led to enhanced inflammation and atherosclerosis in ApoE-knockout mice. 87 As BMP10 appears to lack osteogenic activity in muscle tissue, at least when transduced with an adenovirus, 46 BMP10 may represent an attractive therapeutic alternative to BMP9, providing that BMP10 proves an effective therapy in animal models of PAH.
Liver function: The ability of BMP9 to stimulate hepatocyte proliferation is long established, being one of the earliest observations of BMP9 function. 88 Early studies demonstrated that BMP9 binds to specific receptors in HepG2 cells, 89 consistent with the expression of the low affinity type I receptor, ALK2 and the type II receptors, BMPRII, ActRIIA and ActRIIB. 90 Furthermore, both the HLE (human, non-differentiated hepatoma) cell line and hepatic stellate cells express ALK1, indicating these cells may exhibit high sensitivity to BMP9, though this has yet to be established. 91,92 Of note, BMP9 stimulates canonical Smad signalling and proliferation in HepG2, Hep3B and HuH7 cells. 93 The lowest BMP9 concentration examined in this report was 1 ng/ml and the responses were completely inhibited by low concentrations of the ALK2/3/6 inhibitor, LDN193189, implying these responses are not through ALK1. 93 The data from normal hepatocytes suggest that BMP9 may participate in liver repair but in conditions of liver carcinogenesis, BMP9 may promote tumour growth as discussed below.
In vivo studies have highlighted a positive role for hepatic BMP9 in energy metabolism. BMP9 inhibits hepatic glucose production and activates the expression of key enzymes of lipid metabolism. 63 Furthermore, recombinant BMP9 improved glucose homeostasis in vivo in diabetic and non-diabetic rodents. 63 However, these effects were observed at doses of BMP9 that were at least 1000 times higher than those employed in our PAH models. 41 Conversely, BMP9 is reduced in the livers of insulin-resistant rats and administration of an anti-BMP9 antibody induced glucose intolerance and insulin resistance in fasted rats. 94 Consistent with this observation, administration of insulin in combination with high glucose induced BMP9 expression in the livers of 12h-fasted rats. 94 This promotion of hepatic insulin sensing is associated with reduced expression of a rate-limiting enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes. 63 In addition to effects on the liver, BMP9 may regulate glucose utilization by skeletal muscle as it activates of Akt2 kinase in differentiated myotubes. 63 Akt2 is essential for the activation of insulin-induced glucose uptake by muscle and Akt2 signalling is impaired in insulin resistance. 95 -97 In differentiated L6 myotubes, Akt2 is activated by Smad5 and this has been proposed as the mechanism for BMP9 action, 98 though a direct effect of BMP9 signalling through Smad5 was not confirmed. Overall, the positive effect of BMP9 on hepatic glucose sensing may be of benefit in patients given the perspective that metabolic dysfunction is a component of the pathophysiology of PAH. 99,100 Tumour regulation: Variable effects of BMP9 on tumours and tumour cell growth have been reported. For example BMP9 promotes proliferation of tumour-derived cell lines from ovary 101 and liver 93 but induces apoptosis in prostate cancer cells 102 and restricts osteosarcoma cell proliferation and migration. 103,104 Furthermore, neutralisation of BMP9 with Endoglin-Fc restricts colonic tumours in mice. 105 The key receptor promoting the proliferative response is ALK2 in ovarian and liver cancer cell lines, shown through siRNA transfection and inhibition by low concentrations of the ALK2/3/6 inhibitor, LDN193189. 93,101 The tumour promoting and inhibitory responses are unlikely to be due to ALK1 activation, as prostate cancer cells do not express ALK1 yet BMP9 promotes apoptosis. 102 In some instances, the proliferation of tumour cell lines is promoted by autocrine BMP9 production. 101 Thus the impact of BMP9 on tumour cells appears to predominantly via ALK2 and at high local concentrations of BMP9.
In addition to effects on tumour cells, it is proposed that BMP9, and possibly BMP10, promote tumour angiogenesis. 106,107 The proangiogenic response to BMP9 appears to be context dependent, as BMP9 is reported to promote proliferation and angiogenic processes in embryonic mouse endothelial cells and transformed endothelial cell lines, 106,107 but inhibits angiogenesis in primary adult endothelial cell lines. 35,36,41 The possible role of BMP9 in tumour angiogenesis has led to the development of Dalantercept, a soluble ALK1 ligand trap, as an anti-tumour angiogenesis therapy. When applied clinically, the potential capacity of BMP9/10 to sustain tumour angiogenesis would need to be balanced against the possible transformational effect of BMP9 in treating patients with life-limiting PAH.
In summary, despite the potential challenges of enhancing signalling downstream of loss-of-function mutations in BMPR-II there are now several approaches that could be taken forwards into the clinic as novel therapeutic approaches in PAH. Of these, the direct enhancement of endothelial BMPR-II/ALK1 signalling with BMP9/10 offers an immediate solution that could be rapidly tested with the appropriate cautions in patients with this life-limiting disease.
Figure 2 .
2Proposed mechanism for the restoration of cell surface BMPR-II expression and signalling using exogenous BMP9 therapy. Left panel: Under normal conditions an individual possesses two wild-type alleles for BMPR2. Under these circumstances BMP9 signalling involves signalling via the ALK1:BMPR-II ligand receptor complex and activation of the Smad1/4 transcriptional complex.
Figure 3 .
3Impact of BMP9 therapy on pulmonary endothelial cell function. In the lungs of PAH patients, loss of BMPR-II leads to endothelial dysfunction, including increased vascular permeability, apoptosis and aberrant angioproliferation. Therapeutic delivery of recombinant BMP9 promotes endothelial quiescence, survival and vascular integrity, while simultaneously enhancing BMPR-II expression.
Table 1 .
1Summary of experimental PAH therapies targeting BMPR-IITherapeutic Strategy
Agent
Model
Table 2 .
2Summary of similarities and differences between BMP9 and BMP10 in physiologyBMP9
BMP10
-/-mice phenotype
Normal, lymphatic vessel defect
Lethal, impaired cardiac development
Adult expression
Liver, into circulation
Right atrium
Circulating form and levels
2 -10 ng/ml (by activity)
Presence shown by ELISA and proteomics;
, 300 pg/ml (ELISA) 108
Only 1 in 3 reports can detect activity
Function
Vascular quiescence factor
Flow-dependent arterial quiescence
Bone-forming activity
Highest among 14 BMPs
Undetected
Endothelial cell signalling
Controlling a similar set of target
genes with similar potency 48
Affinity for ALK1/BMPRII
Higher
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| [
"Human genetic discoveries offer a powerful method to implicate pathways of major importance to disease pathobiology and hence provide targets for pharmacological intervention. The genetics of pulmonary arterial hypertension (PAH) strongly implicates loss-of-function of the bone morphogenetic protein type II receptor (BMPR-II) signalling pathway and moreover implicates the endothelial cell as a central cell type involved in disease initiation. We and others have described several approaches to restore BMPR-II function in genetic and non-genetic forms of PAH. Of these, supplementation of endothelial BMP9/10 signalling with exogenous recombinant ligand has been shown to hold considerable promise as a novel large molecule biopharmaceutical therapy. Here, we describe the mechanism of action and discuss potential additional effects of BMP ligand therapy.Cite this article as: Ormiston ML, Upton PD, Li W, Morrell NW. The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension, Global Cardiology Science and Practice 2015:47 http://dx."
] | [
"Mark L Ormiston \nThe Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom\n",
"Paul D Upton \nThe Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom\n",
"Wei Li \nThe Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom\n",
"Nicholas W Morrell \nThe Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom\n"
] | [
"The Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom",
"The Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom",
"The Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom",
"The Department of Medicine\nSchool of Clinical Medicine\nUniversity of Cambridge\nAddenbrooke's and Papworth Hospitals\nCambridgeUnited Kingdom"
] | [
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"J Z Li, ",
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"E J Beres, ",
"S Das, ",
"J Engh, ",
"J Z Li, ",
"G R Hankins, ",
"C Kao, ",
"H Li, ",
"J Kammauff, ",
"G A Helm, ",
"E Leblanc, ",
"F Trensz, ",
"S Haroun, ",
"G Drouin, ",
"E Bergeron, ",
"C M Penton, ",
"S F Zhu, ",
"H B Hu, ",
"H Y Xu, ",
"X F Fu, ",
"D X Peng, ",
"W Y Su, ",
"C W Kim, ",
"H Song, ",
"S Kumar, ",
"D Nam, ",
"H S Kwon, ",
"K H Chang, ",
"J J Song, ",
"A J Celeste, ",
"F M Kong, ",
"R L Jirtle, ",
"V Rosen, ",
"R S Thies, ",
"A F Miller, ",
"S A Harvey, ",
"R S Thies, ",
"M S Olson, ",
"Y Xia, ",
"J L Babitt, ",
"Y Sidis, ",
"R T Chung, ",
"H Y Lin, ",
"Q Li, ",
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"H Weng, ",
"S Ghafoory, ",
"Y Liu, ",
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"E Wiercinska, ",
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"H M Said, ",
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"S Cruz, ",
"P Walsh, ",
"M Fernandez, ",
"C Roncero, ",
"L C Caperuto, ",
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"E H Akamine, ",
"Carmo Do, ",
"D Buonfiglio, ",
"J Cipolla-Neto, ",
"S George, ",
"J J Rochford, ",
"C Wolfrum, ",
"S L Gray, ",
"S Schinner, ",
"J C Wilson, ",
"K Hussain, ",
"B Challis, ",
"N Rocha, ",
"F Payne, ",
"M Minic, ",
"A Thompson, ",
"H Cho, ",
"J Mu, ",
"J K Kim, ",
"J L Thorvaldsen, ",
"Q Chu, ",
"Crenshaw Eb 3rd, ",
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"C Lellis-Santos, ",
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"L Farkas, ",
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"J Abbate, ",
"A , ",
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"P Inman, ",
"G J , ",
"L Ye, ",
"H Kynaston, ",
"W G Jiang, ",
"B Li, ",
"Y Yang, ",
"S Jiang, ",
"B Ni, ",
"K Chen, ",
"L Jiang, ",
"Z Lv, ",
"D Yang, ",
"J Li, ",
"M Hu, ",
"M Luo, ",
"X Zhan, ",
"R Castonguay, ",
"E D Werner, ",
"R G Matthews, ",
"E Presman, ",
"A W Mulivor, ",
"N Solban, ",
"S I Cunha, ",
"E Pardali, ",
"M Thorikay, ",
"C Anderberg, ",
"L Hawinkels, ",
"M J Goumans, ",
"Y Suzuki, ",
"N Ohga, ",
"Y Morishita, ",
"K Hida, ",
"K Miyazono, ",
"T Watabe, ",
"L J Van Baardewijk, ",
"J Van Der Ende, ",
"S Lissenberg-Thunnissen, ",
"L M Romijn, ",
"L J Hawinkels, ",
"C F Sier, "
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"Arterial endothelium-specific activin receptor-like kinase 1 expression suggests its role in arterialization and vascular remodeling. T Seki, J Yun, S P Oh, Circ Res. 937Seki T, Yun J, Oh SP. Arterial endothelium-specific activin receptor-like kinase 1 expression suggests its role in arterialization and vascular remodeling. Circ Res. 2003;93(7):682-689.",
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"BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus. S Levet, M Ouarne, D Ciais, C Coutton, M Subileau, C Mallet, Proc Natl Acad Sci. 11225Levet S, Ouarne M, Ciais D, Coutton C, Subileau M, Mallet C, et al., BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus. Proc Natl Acad Sci U S A. 2015;112(25):E3207-E3215.",
"Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth. H Chen, W Yong, S Ren, W Shen, Y He, K A Cox, J Biol Chem. 28137Chen H, Yong W, Ren S, Shen W, He Y, Cox KA, et al., Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth. J Biol Chem. 2006;281(37):27481-27491.",
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"Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. Q Luo, Q Kang, W Si, W Jiang, J K Park, Y Peng, J Biol Chem. 27953Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, et al., Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem. 2004;279(53):55958-55968.",
"Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. Y Peng, Q Kang, H Cheng, X Li, M H Sun, W Jiang, Journal of cellular biochemistry. 906Peng Y, Kang Q, Cheng H, Li X, Sun MH, Jiang W, et al., Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. Journal of cellular biochemistry. 2003;90(6):1149-1165.",
"TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells. J Luo, M Tang, J Huang, B C He, J L Gao, L Chen, J Biol Chem. 28538Luo J, Tang M, Huang J, He BC, Gao JL, Chen L, et al., TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells. J Biol Chem. 2010;285(38):29588-29598.",
"BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. N Hu, D Jiang, E Huang, X Liu, R Li, X Liang, J Cell Sci. 1262Hu N, Jiang D, Huang E, Liu X, Li R, Liang X, et al., BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. J Cell Sci. 2013;126(Pt 2):532-541.",
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"The efficiency of muscle-derived cell-mediated bone formation. P Bosch, D Musgrave, S Ghivizzani, C Latterman, C S Day, J Huard, Cell transplantation. 94Bosch P, Musgrave D, Ghivizzani S, Latterman C, Day CS, Huard J. The efficiency of muscle-derived cell-mediated bone formation. Cell transplantation. 2000;9(4):463-470.",
"Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice. J Y Lee, D Musgrave, D Pelinkovic, K Fukushima, J Cummins, A Usas, 83Lee JY, Musgrave D, Pelinkovic D, Fukushima K, Cummins J, Usas A, et al., Effect of bone morphogenetic protein-2- expressing muscle-derived cells on healing of critical-sized bone defects in mice. The Journal of bone and joint surgery American volume. 2001;83-A(7):1032-1039.",
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"BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment. E Leblanc, F Trensz, S Haroun, G Drouin, E Bergeron, C M Penton, American Society for Bone and Mineral Research26Journal of bone and mineral research: the official journal of theLeblanc E, Trensz F, Haroun S, Drouin G, Bergeron E, Penton CM, et al., BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 2011;26(6):1166-1177.",
"Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries. S F Zhu, H B Hu, H Y Xu, X F Fu, D X Peng, W Y Su, J Cell Mol Med. Zhu SF, Hu HB, Xu HY, Fu XF, Peng DX, Su WY, et al., Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries. J Cell Mol Med. 2015.",
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"Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation. J J Song, A J Celeste, F M Kong, R L Jirtle, V Rosen, R S Thies, Endocrinology. 13610Song JJ, Celeste AJ, Kong FM, Jirtle RL, Rosen V, Thies RS. Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation. Endocrinology. 1995;136(10):4293-4297.",
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"Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin. Y Xia, J L Babitt, Y Sidis, R T Chung, H Y Lin, Blood. 11110Xia Y, Babitt JL, Sidis Y, Chung RT, Lin HY. Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin. Blood. 2008;111(10):5195 -5204.",
"Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells. Q Li, X Gu, H Weng, S Ghafoory, Y Liu, T Feng, Cancer Sci. 1043Li Q, Gu X, Weng H, Ghafoory S, Liu Y, Feng T, et al., Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells. Cancer Sci. 2013;104(3):398-408.",
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"Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/AKT pathway. B Li, Y Yang, S Jiang, B Ni, K Chen, L Jiang, Int J Oncol. 415Li B, Yang Y, Jiang S, Ni B, Chen K, Jiang L. Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/AKT pathway. Int J Oncol. 2012;41(5):1809-1819.",
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] | [
"Pulmonary arterial hypertension in France: results from a national registry",
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"Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The International PPH Consortium",
"Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene",
"Genetics and genomics of pulmonary arterial hypertension",
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"Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor",
"Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease",
"TGF-beta and BMPR-II pharmacology-implications for pulmonary vascular diseases",
"Mutations in bone morphogenetic protein type II receptor cause dysregulation of Id gene expression in pulmonary artery smooth muscle cells: implications for familial pulmonary arterial hypertension",
"Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis",
"Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2",
"Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension",
"Proteomic analysis implicates translationally controlled tumor protein as a novel mediator of occlusive vascular remodeling in pulmonary arterial hypertension",
"Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function",
"Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia",
"Failure of bone morphogenetic protein receptor trafficking in pulmonary arterial hypertension: potential for rescue",
"Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases",
"Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension",
"Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension",
"Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling",
"The lysosomal inhibitor, chloroquine, increases cell surface BMPR-II levels and restores BMP9 signalling in endothelial cells harbouring BMPR-II mutations",
"Identification of a lysosomal pathway regulating degradation of the bone morphogenetic protein receptor type II",
"Chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type II receptor degradation",
"Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary hypertension",
"Targeted gene delivery of BMPR2 attenuates pulmonary hypertension",
"Overexpression of human bone morphogenetic protein receptor 2 does not ameliorate monocrotaline pulmonary arterial hypertension",
"Smad-dependent and smad-independent induction of id1 by prostacyclin analogues inhibits proliferation of pulmonary artery smooth muscle cells in vitro and in vivo",
"Sildenafil potentiates bone morphogenetic protein signaling in pulmonary arterial smooth muscle cells and in experimental pulmonary hypertension",
"FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension",
"Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells",
"BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis",
"Bone morphogenetic protein (BMP) and activin type II receptors balance BMP9 signals mediated by activin receptor-like kinase-1 in human pulmonary artery endothelial cells",
"Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redoxdependent Proteolysis",
"BMP9 mutations cause a vascular-anomaly syndrome with phenotypic overlap with hereditary hemorrhagic telangiectasia",
"Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex",
"Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension",
"Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis",
"Regarding the mechanism of action of a proposed peptide agonist of the bone morphogenetic protein receptor activin-like kinase 3",
"Cooperativity of binding epitopes and receptor chains in the BMP/TGFbeta superfamily",
"Bone morphogenetic protein-2 and -6 heterodimer illustrates the nature of ligand-receptor assembly",
"Characterization of the distinct orthotopic boneforming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery",
"Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability",
"BMP9 and BMP10 are critical for postnatal retinal vascular remodeling",
"Context-dependent signaling defines roles of BMP9 and BMP10 in embryonic and postnatal development",
"Proteomic identification and functional validation of activins and bone morphogenetic protein 11 as candidate novel muscle mass regulators",
"A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins. Identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum",
"Identification, cloning, expression, and biochemical characterization of the testis-specific RNA polymerase II elongation factor ELL3",
"BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cellular and molecular life sciences",
"Heart specific expression of mouse BMP-10 a novel member of the TGF-beta superfamily",
"BMP10 is essential for maintaining cardiac growth during murine cardiogenesis",
"Bone morphogenetic protein-9 is a circulating vascular quiescence factor",
"Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases",
"Regulation of myostatin activity and muscle growth",
"GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding",
"Characterization and identification of the inhibitory domain of GDF-8 propeptide",
"Crystal structure of BMP-9 and functional interactions with pro-region and receptors",
"An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis",
"Circulating Bmp10 acts through endothelial Alk1 to mediate flow-dependent arterial quiescence",
"Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels",
"Arterial endothelium-specific activin receptor-like kinase 1 expression suggests its role in arterialization and vascular remodeling",
"Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis",
"Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation",
"BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus",
"Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth",
"Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells",
"Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells",
"Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling",
"TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells",
"BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells",
"Ultrasound-based nonviral gene delivery induces bone formation in vivo",
"Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration",
"BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential",
"The efficiency of muscle-derived cell-mediated bone formation",
"Enhancement of bone healing based on ex vivo gene therapy using human muscle-derived cells expressing bone morphogenetic protein 2",
"Morphologic analysis of BMP-9 gene therapy-induced osteogenesis",
"Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries",
"Anti-inflammatory and antiatherogenic role of BMP receptor II in endothelial cells",
"Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation",
"Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver",
"Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin",
"Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells",
"Id1 is a critical mediator in TGF-betainduced transdifferentiation of rat hepatic stellate cells",
"BMP9 is a proliferative and survival factor for human hepatocellular carcinoma cells",
"Modulation of bone morphogenetic protein-9 expression and processing by insulin, glucose, and glucocorticoids: possible candidate for hepatic insulin-sensitizing substance",
"A family with severe insulin resistance and diabetes due to a mutation in AKT2",
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"Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta)",
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"Severe pulmonary hypertension: The role of metabolic and endocrine disorders",
"Metabolic dysfunction in pulmonary hypertension: the expanding relevance of the Warburg effect",
"Autocrine bone morphogenetic protein-9 signals through activin receptor-like kinase-2/Smad1/Smad4 to promote ovarian cancer cell proliferation",
"Bone morphogenetic protein-9 induces apoptosis in prostate cancer cells, the role of prostate apoptosis response-4",
"Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/AKT pathway",
"Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion",
"Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth",
"Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis",
"BMP-9 induces proliferation of multiple types of endothelial cells in vitro and in vivo",
"Circulating bone morphogenetic protein levels and delayed fracture healing"
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"\nFigure 2 .\n2Proposed mechanism for the restoration of cell surface BMPR-II expression and signalling using exogenous BMP9 therapy. Left panel: Under normal conditions an individual possesses two wild-type alleles for BMPR2. Under these circumstances BMP9 signalling involves signalling via the ALK1:BMPR-II ligand receptor complex and activation of the Smad1/4 transcriptional complex.",
"\nFigure 3 .\n3Impact of BMP9 therapy on pulmonary endothelial cell function. In the lungs of PAH patients, loss of BMPR-II leads to endothelial dysfunction, including increased vascular permeability, apoptosis and aberrant angioproliferation. Therapeutic delivery of recombinant BMP9 promotes endothelial quiescence, survival and vascular integrity, while simultaneously enhancing BMPR-II expression.",
"\nTable 1 .\n1Summary of experimental PAH therapies targeting BMPR-IITherapeutic Strategy \n\nAgent \n\nModel \n\n",
"\nTable 2 .\n2Summary of similarities and differences between BMP9 and BMP10 in physiologyBMP9 \nBMP10 \n\n-/-mice phenotype \nNormal, lymphatic vessel defect \nLethal, impaired cardiac development \nAdult expression \nLiver, into circulation \nRight atrium \nCirculating form and levels \n2 -10 ng/ml (by activity) \nPresence shown by ELISA and proteomics; \n, 300 pg/ml (ELISA) 108 \nOnly 1 in 3 reports can detect activity \nFunction \nVascular quiescence factor \nFlow-dependent arterial quiescence \nBone-forming activity \nHighest among 14 BMPs \nUndetected \nEndothelial cell signalling \nControlling a similar set of target \ngenes with similar potency 48 \nAffinity for ALK1/BMPRII \nHigher \n"
] | [
"Proposed mechanism for the restoration of cell surface BMPR-II expression and signalling using exogenous BMP9 therapy. Left panel: Under normal conditions an individual possesses two wild-type alleles for BMPR2. Under these circumstances BMP9 signalling involves signalling via the ALK1:BMPR-II ligand receptor complex and activation of the Smad1/4 transcriptional complex.",
"Impact of BMP9 therapy on pulmonary endothelial cell function. In the lungs of PAH patients, loss of BMPR-II leads to endothelial dysfunction, including increased vascular permeability, apoptosis and aberrant angioproliferation. Therapeutic delivery of recombinant BMP9 promotes endothelial quiescence, survival and vascular integrity, while simultaneously enhancing BMPR-II expression.",
"Summary of experimental PAH therapies targeting BMPR-II",
"Summary of similarities and differences between BMP9 and BMP10 in physiology"
] | [
"(Fig. 1)",
"Figure 1",
"Fig. 2",
"(Fig. 3)"
] | [] | [
"Pulmonary arterial hypertension (PAH) is a disease of pathological vascular remodeling, characterized by medial thickening and the formation of occlusive vascular lesions that obstruct the pulmonary circulation, predominantly at the level of the pre-capillary arterioles. This loss of the pulmonary distal circulation, and the resultant increase in pulmonary vascular resistance (PVR), leads to an elevation of pulmonary arterial pressure and right ventricular hypertrophy. PAH is a rare disease, with a prevalence of 15 to 26 patients per million individuals and an incidence of 2.4 to 7.6 cases per million annually. 1,2 It can arise as a primary disease, either in an idiopathic (IPAH) or heritable (HPAH) form, or as a condition associated with immune disorders such as HIV, connective tissues diseases, or exposure to particular drugs or toxins. In its idiopathic and heritable forms, PAH presents in a relatively young patient population, aged 30 -50 years, and preferentially in women at a rate of roughly 2.3:1. If left untreated, PAH can lead to death from right-sided heart failure within 3 -5 years of diagnosis.",
"Existing treatments for PAH were developed for this indication because of their effects on vascular tone. The regulation of vascular tone by endothelial cells and vascular smooth muscle cells (SMCs) is mediated by a balance of vasodilators, such as prostacyclin and nitric oxide (NO), and vasoconstrictive agents, including endothelin-1. Established PAH is associated with a shift in this balance towards excessive pulmonary vasoconstriction. Recognition of this imbalance was the driving force behind the development and approval of a range of vasodilatory therapies for PAH. 3 These therapies can be divided into three main classes: (i) prostanoids, including epoprostenol and more stable prostacyclin analogues such as iloprost, beraprost and treprostinil; (ii) endothelin receptor antagonists, including bosentan, ambrisentan and macitentan; and (iii) phosphodiesterase 5 inhibitors, such as sildenafil and tadalafil. While these treatments have been successful in improving the haemodynamic parameters and functional status of patients, the three-year survival rate for PAH patients remains poor. 4 Although these therapies may also have modest effects on vascular SMC proliferation, available evidence suggests a minimal impact on the process of vascular remodeling in the lungs of patients with PAH. 5 Here we propose that more effective therapies for PAH will derive from a greater understanding of the molecular basis of pathological pulmonary vascular remodeling, particularly by targeting pathways identified by human genetics in PAH patients.",
"Heritable PAH (HPAH) is an autosomal dominant disease, marked by a low penetrance (average 20 -30%) in at-risk individuals. 6 While the existence of this familial form of PAH has been recognized since the first description of the disease, it was only 15 years ago that mutations in BMPR2, the gene encoding the bone morphogenetic protein (BMP) type II receptor (BMPR-II), was identified as the cause of approximately 75% of HPAH cases. 7,8 BMPR2 mutations also account for 15 -26% of seemingly idiopathic or sporadic cases of PAH, including cases of de novo mutations and parental transmission with no record of a previous family history of disease. As a result of these findings, the definition of HPAH was recently updated to include not only patients in a family with two or more documented cases of PAH, but also includes any PAH patient possessing a mutation in BMPR2. 9 A range of mutations in BMPR2 have been reported in PAH patients. The majority of these mutations lead to a state of haploinsufficiency, 10 where the mutant allele leads to no production of a protein product. The protein expression from the wild type allele is normal but overall protein expression is reduced by at least 50%. Patients bearing BMPR2 mutations develop PAH earlier, have more severe disease and die sooner than those without mutations. 6 Interestingly, PAH patients with BMPR2 mutations exhibit reductions in BMPR-II protein levels of greater than 75% when compared to control subjects, suggesting that the development of PAH can suppress receptor levels to a greater extent than what can be accounted for by haploinsufficiency alone. 11 Reduced BMPR-II protein levels and impaired downstream signaling have also been identified in idiopathic PAH patients lacking mutations in BMPR2, as well as in common, non-genetic rodent models of disease, 12 further supporting a central role for reduced BMPR-II signaling in most forms of the disease, independent of etiology.",
"When taken together, these factors make the BMPR-II signaling pathway an extremely attractive target for next-generation therapeutic intervention. However, the translation of this approach into in vivo pre-clinical studies has been limited by uncertainty regarding which cell type or types are critically affected by the loss of BMPR-II signaling, and the complexity of the BMP signalling family. Fortunately, genetic studies describing disease phenotypes associated with mutations in other components of the BMP signaling pathway, coupled with knowledge of the tissue-specific distribution of these proteins, can be used to inform decisions on which cell types, and which BMPs, play important roles in the initiation of PAH.",
"BMPs are members of the transforming growth factor-b (TGFb) superfamily, a highly complex family of proteins including over 30 ligands that signal through heteromeric complexes of type I and type II receptors. As a type II receptor of this superfamily, BMPR-II can form complexes with several type I receptors, including the activin like receptor kinases ALK1, ALK2, ALK3 or ALK6, with each receptor complex recognizing a specific subset of BMP ligands. 13 Canonical signaling generally involves phosphorylation of the Smad-1, 5 or -8 transcription factors, which complex with the co-smad, Smad-4, and translocate to the nucleus to induce gene expression. The tissue-specific nature of BMP responses is highly dependent upon the components making up the ligand-receptor complex. Numerous accessory receptors, such as endoglin (ENG), also modify signaling, further contributing to the tissue-specificity of BMP responses.",
"Following the initial identification of BMPR2 mutations in PAH, much of the work investigating the role of deficiency of this receptor in disease pathogenesis focused on the impact of these mutations on pulmonary arterial SMC (PASMC) proliferation and migration. 14 In smooth muscle cells, BMP ligands, including BMP2 and BMP4, block serum-induced proliferation via complexes of BMPR-II with ALK3 or ALK6. 14 However, mutations in ALK3 and ALK6 do not cause pulmonary vascular disease, but are instead associated with juvenile gastrointestinal polyposis 15 and hereditary brachydactyly, 16 respectively. This fact calls into question the importance of these receptors, and the signaling complexes that they form, in the pathogenesis of PAH.",
"More recently, several studies have demonstrated a role for impaired endothelial BMP signaling in the pathogenesis of PAH. In addition to increased endothelial cell proliferation and enhanced susceptibility to apoptosis, 17,18 loss of BMPR-II has also been shown to influence endothelial cell barrier integrity and pulmonary vascular permeability. 19 Genetic evidence also supports a role for the pulmonary endothelium as the critical initiating cell type in PAH. Mutations in Endoglin and ALK1 (gene symbol ACVRL1), two proteins found almost exclusively on the endothelium, are primarily associated with hereditary haemorrhagic telangectasia (HHT), a disease that causes vascular abnormalities, including telangiectases in the skin and mucosal regions, and arteriovenous malformations in the lung, liver, gastrointestinal tract and brain. In addition to HHT, ACVRL1 mutations are occasionally associated with PAH, demonstrating a role for these molecules, and the receptor complexes they form, in the maintenance of pulmonary vascular homeostasis. 20 To date, mutations in eight genes have been found to play a causal role in idiopathic and heritable PAH (Fig. 1). Notably, of all the known mutations identified to date, six out of eight of these directly or indirectly implicate the BMP signalling pathway, and particularly endothelial BMP signaling, as central to pathobiology. Taken together, these findings strongly support the development of therapeutic strategies that restore expression or function of BMPR-II, particularly in pulmonary vascular endothelial cells.",
"Considering the genetic evidence indicating an important role for BMPR-II in the pathobiology of PAH, several groups have begun to use this information to test the potential of next generation therapies that directly target BMPR-II deficiency. Although not limited to the endothelium, a number of these studies directly target pulmonary endothelial BMPR-II for their mode of action. These therapies are directed towards various stages of BMPR-II signaling, including gene expression, translation, surface expression and receptor degradation, as well as enhancing receptor activity through the delivery of BMPR-II agonists. The various strategies can be grouped into three main approaches: (i) rescue of mutant receptor function, (ii) enhancing the expression or longevity of the wild-type receptor or (iii) enhancing BMP signaling through small molecules or recombinant BMP ligands. The published studies examining these three approaches are summarized below and in Table 1.",
"Rescuing mutant BMPR-II receptor function: Over 400 different PAH associated BMPR2 mutations have been identified in all functional domains of the BMPR-II protein. 6 These include missense mutations within the extracellular domain, transmembrane region, and kinase region, as well as nonsense mutations that encode premature termination codons (PTCs). Strategies designed to enhance BMP signaling by rescuing the mutated copy of the BMPR2 gene can take one of two forms, depending on the type of mutation present.",
"Chemical or pharmacological chaperones: Missense mutations can lead to the production of misfolded BMPR-II through the replacement of critical cysteine residues that are essential to the folding of the extracellular domain within the endoplasmic reticulum (ER). These mutants can be targeted with chemical chaperones that enhance the transport of the mutant protein from the ER to the cell surface. Some chemical chaperones, such as glycerol, non-selectively stabilize the tertiary structure of the mutant proteins and facilitate folding. Other compounds, including thapsigargin and sodium 4-phenylbutyrate (4-PBA), inhibit the interactions of the mutant protein with the chaperone proteins responsible for their recognition, retention and degradation. In PAH, a panel of chemical chaperones, including 4-PBA, glycerol and thapsigargin, were shown to promote folding and trafficking of mutant BMPR-II protein to the cell surface. Since, in this case, the mutant receptor was still capable of normal signalling, downstream Smad signalling was restored. 21 While these results are promising, there are several factors limiting the clinical application of these compounds for PAH. The PAH patient population contains a large variety of missense mutations, only a subset of which encode mutations that lead to the misfolding a receptor that is otherwise functional. Nevertheless, 4-PBA has been used for other indications in man and may be a personalized approach in patients with these specific mutations.",
"Suppression of nonsense mutations: In contrast to missense mutations, where the mutant protein is produced and can be targeted for rescue, the mRNA transcripts produced by alleles bearing nonsense mutations are typically subject to nonsense-mediated decay (NMD), resulting in reduced BMPR2 mRNA and decreased protein levels. The rescue of these mutations requires the use of compounds that suppress or silence PTCs, allowing for the translation of mutant mRNA into full-length, functional protein. 22 A number of compounds belonging to the aminoglycoside family of antibiotics, including gentamicin, have been shown to suppress disease-causing PTCs and partially restore protein function in nonsense mutation-bearing cell culture models of multiple diseases, including cystic fibrosis, Duchenne muscular dystrophy and PAH. 23 However, the high doses required to achieve these beneficial effects in vivo are also associated with renal toxicity and hearing loss, thus preventing the widespread use of aminoglycosides in clinical applications.",
"More recently, high throughput screens have identified other compounds distinct from aminoglycosides that possess the ability to effectively suppress PTCs without affecting the recognition Figure 1. Simplified schematic summarizing the BMP signalling pathway and genes that have to date been shown to be mutated in PAH and demonstrating that many of these mutations encode protein involved in BMP signalling. Known mutations are numbered and in bold. ",
"Chemical Chaperones",
"In vitro: HeLa cells [21] Suppression of Nonsense Mutations",
"In vitro: cell-based reporter assay [23] Ataluren (PTC124) In vitro: PAH endothelial and smooth muscle cells [24] Enhancement of BMPR-II Expression and Longevity",
"Chloroquine, hydroxychloroquine In vitro: PAH endothelial cells In vivo: Monocrotaline rat model [26,28] BMPR2 Gene Therapy Endothelial-targeted adenoviral vector In vivo: chronic hypoxia and monocrotaline rat models [29,30] Enhancement of BMPR-II-mediated Signaling bmpr2-R899X mouse,",
"Monocrotaline rat model, Sugen-Hypoxia rat and mouse models [41] of normal stop signals. One such compound, the small molecule Ataluren (PTC124), is currently in phase III clinical trials for Duchenne muscular dystrophy. In PAH, Ataluren was shown to increase BMPR-II protein levels and downstream signaling in primary cells from patients with nonsense mutations in BMPR2 and SMAD9. 24 These effects were accompanied by a restoration of normal proliferation rates in hyperproliferative, patient-derived PAECs and PASMCs.",
"Enhancing BMPR-II surface expression Inhibiting BMPR-II degradation: An alternative approach that has been explored for enhancing BMP signalling involves extending the longevity of BMPR-II at the cell surface. BMPR-II is subject to constitutive endocytosis by both clathrin and caveolae mediated pathways 25 and is rapidly turned over in endothelial cells. Notable protein loss is observed in these cells as early as 1 hour following the blockade of new protein synthesis. 26 BMPR-II can also be targeted for ubiquitination by Kaposi's sarcoma herpes virus K5 E3 ligase, and directed for lysosomal degradation. 27 Each of these findings highlights the lysosome as the primary route for BMPR-II turnover and points to lysosomal inhibition as a promising target for preserving BMPR-II longevity and enhancing receptor-mediated signaling. The 4-aminoquinolone, chloroquine, achieves lysosomal inactivation through the prevention of vesicular acidification. Used for decades as a prophylactic antimalarial drug, chloroquine, and its less toxic derivative hydroxychloroquine, have recently been repurposed for use in the treatment of rheumatoid arthritis and cancer. In vitro, chloroquine was shown to enhance BMPR-II protein levels, surface expression and downstream signaling in BMPR2 mutation-bearing endothelial cells. 26 In vivo, both chloroquine and hydroxychloroquine inhibited the development of pulmonary hypertension and blocked the progression of established disease in the monocrotaline rat model. 28 Although this therapeutic effect was associated with restoration of BMPR-II protein levels, which are decreased in monocrotaline-treated rats, it is likely that the beneficial effect of chloroquine administration may also be linked to the inhibition of excessive autophagy, which has been linked to the aberrant proliferation of vascular cells in PAH. Translation of hydroxychloroquine into a clinical therapy is facilitated by the fact that the compound is inexpensive and presents acceptable toxicity or side effects at therapeutically effective doses. Long-term treatment with hydroxychloroquine has been associated with retinopathy in 0.5 -1% of patients. However, this can be avoided with proper monitoring of patients undergoing treatment. BMPR2 gene therapy: Gene therapy techniques have also been explored in animal models of PAH as a means to restore BMPR-II receptor levels. The targeted delivery of an adenoviral vector containing the BMPR2 gene to the pulmonary vascular endothelium of rats substantially reduced the severity of pulmonary hypertension in the chronic hypoxia and monocrotaline models of PAH. 29,30 For these studies, targeting of the adenoviral vectors to the pulmonary endothelium appeared to be a key feature of therapeutic efficacy, as similar studies using aerosol vector delivery and a non-specific promoter failed to demonstrate any measurable benefit in the monocrotaline rat model. 31 Enhancement of BMPR-II downstream signalling: One alternative to enhancing BMPR-II receptor expression or longevity involves enhancing signaling through the action of small molecules. Existing PAH therapies, including prostanoids and sildenafil, have been shown to partially restore BMP signaling in PASMCs bearing BMPR2 mutations and prevent the development of PAH in animal models of disease. 32,33 More recently, a high throughput screening approach demonstrated that the calcineurin inhibitor, tacrolimus (FK506), potentiates BMPR-II-mediated signaling in endothelial cells, rescues dysfunctional BMP signaling in endothelial cells from PAH patients with BMPR2 mutations and reverses established disease in multiple rodent models of PAH. 34 Early clinical trials are currently underway to establish the viability of this strategy as a treatment for PAH. However, this approach is likely to lead to widespread and non-specific activation of BMP receptors, as well as having off target effects independent of BMP signalling.",
"One of the most direct strategies for targeting BMPR-II deficiency in PAH involves the delivery of exogenous BMP ligand to enhance signaling via the remaining functional receptor. Proof-of-concept studies using patient-derived pulmonary arterial smooth muscle cells (PASMCs) have shown that the addition of increasing concentrations of BMP ligand can overcome the functional defects associated with BMPR2 mutation in vitro. 14 However, identifying the appropriate BMP ligand or ligands to selectively target the endothelium in vivo presents a significant challenge. The selective expression of ALK1 on the endothelium makes the ALK1 /BMPR-II complex an ideal target for exogenous ligand therapy. Although ALK1 was originally thought to be an orphan receptor, two studies in 2007 identified BMP9 and BMP10 as ligands that signal via complexes of ALK1 with either BMPR-II or type II activin receptors (ActRII). 35,36 Further examination of the receptor selectivity of these ligands has primarily focused on BMP9. Endogenously, BMP9 activates ALK1 with high affinity (EC50 ¼ 50 pg/ml). BMP9 also activates ALK2 but with much a lower affinity in the ng/ml range. 35 -39 Biochemical analysis has also revealed discrete differences in the type II receptor selectivity of BMP9 and BMP10. Although BMP9 and BMP10 both bind to BMPRII, ActRIIA and ActRIIB, BMP9 exhibits a preference towards ActRIIB compared to BMPRII and ActRIIA while BMP-10 binds to all type II receptors with similar affinities. 40 In endothelial cells, type II receptor redundancy means that loss of either BMPR-II or ActRIIA has a minor impact on the stimulation of Smad1/5 phosphorylation due to compensation by the remaining type II receptor. 37 Loss of both receptors, however, abolished the Smad phopshorylation responses. Of note, some transcriptional responses to BMP9, such as E-selectin and IL8 induction are more reliant on BMPR-II signaling as ActRIIA does not compensate. 37 This implies that there are BMPR-II-specific signals that may contribute more than others to the pathology of PAH. Another feature of the BMPR-II selectivity of BMP9 relates to the transcriptional regulation of the receptors themselves. BMP9 induces the expression of BMPR-II, but not ALK1 or ActRIIA, suggesting that this ligand switches the balance of signaling in the endothelial cell towards BMPR-II. 35,37 Furthermore, this induction is ALK1-dependent and Smad1-dependent, suggesting a feed forward signaling mechanism. 37,41 It was recently shown that the therapeutic effects of BMP9 include restoration of BMPR-II expression and signaling of BMPR-II in animal models of PAH. 41 Therefore, BMP9 represents a therapy that promotes its key downstream signalling pathways partly via rescue of the deficient BMPR-II expression that underlies the pathogenesis of PAH. A proposed mechanism for this upregulation is shown in Fig. 2.",
"Based on our studies of BMPR-II dysfunction in PAH and the emerging role of BMP9 as a key circulating regulator of endothelial function and positive regulator of BMPR-II expression, we recently examined the therapeutic delivery of BMP9 to target endothelial dysfunction in PAH. 41 In vitro, BMP9 prevents the enhanced apoptosis observed in BMPR2 mutation-bearing endothelial cells and promotes monolayer integrity through the formation of tight junctions. In vivo, BMP9 prevents and reverses established disease in a range of rodent models, including spontaneous disease in a mouse model bearing a knock-in of the PAH-associated R899X-Bmpr2 mutation. From these studies, we propose that BMP9 constitutes a potential therapy that overcomes the underlying endothelial dysfunction that causes PAH and can overcome the deficient BMPR-II signaling that is a consequence of the major genetic defect in most HPAH patients (Fig. 3). Our anticipation is that, by restoring the balance in BMP signaling, this approach will redress the imbalance of other dysregulated pathways that are targeted by current therapies.",
"Another theoretical approach to activate the endothelial ALK1/BMPR-II pathway would be to develop a peptide mimetic that possesses BMP9/10-like function. An example of such an approach was recently demonstrated for a peptide derived from BMP7. 42 Although it is considered unlikely by some that a small peptide mimetic would be able to substitute for a BMP dimer in the assembly of two type I and two type II receptors, all of which are required for signalling, 43 -45 the BMP7 peptide mimetic was shown to have similar effects to full-length BMP7 ligand in reversing established fibrosis in five mouse models of acute and chronic renal injury. 42 Although the findings of this study have been challenged by researchers from several groups, synthetic peptides that specifically activate the endothelial ALK1/BMPR-II pathway still represent promising potential drug candidates for treating PAH.",
"Since BMP9 exerts its beneficial effect on the endothelium in an ALK1-dependent manner, recombinant BMP10 protein, which is the only other BMP ligand that signals through ALK1, could also have a similar therapeutic potential for treating PAH. There are several advantages of BMP10-based therapy. Firstly, unlike BMP9, it does not show osteogenic activity either in vitro signalling assays, or in an in vivo bone-forming screen, 46 and may therefore be potentially safer than BMP9 for treating cardiovascular disease. Secondly, BMP10 binds to ALK1 and BMPR-II with higher affinities than BMP9, 40 making it likely to be more potent and more specific for ALK1 and BMPR-II.",
"In spite of these advantages, there are still several hurdles to overcome before BMP10 can be developed into a therapy for PAH. For example, the activity of circulating BMP10 in humans is still under investigation. BMP10 is bound tightly by its prodomain, which inhibits BMP10 signalling activity when applied in molar excess. 47 It has therefore been proposed that BMP10, similar to TGFb, exists in a latent form in the circulation and requires an additional activation step to achieve bioactivity, such as cleavage of the prodomain by BMP1. 47 This hypothesis is further supported by the fact that, although circulating BMP10 could be consistently detected and measured by ELISA 48,49 and by pull-down coupled with proteomics, 50 early studies could not detect BMP10 activity in the circulation. 48,51 However, one recent report was able to demonstrate BMP10 activity in mouse serum. 49 A further question to be addressed is whether the presence of the prodomain on therapeutically administered BMP10 is important for the stability and half-life of the ligand. A better understanding of the physiology of BMP10 is required in vivo, since BMP10 is a much less well-studied BMP ligand and most of the data are from embryos of mouse and zebrafish. The data on the role of BMP10 in adult physiology and in PAH are limited, and such information will inform the development and testing of BMP10-based drugs.",
"During embryonic development and into adult life, BMP9 and BMP10 are both expressed in a highly tissue-specific pattern. BMP9 is expressed in the mouse fetal liver from E9.75 -10 49 and expression remains high in the adult liver. 52,53 The highest levels of expression are observed in mRNA from intrahepatic biliary epithelial cells and hepatocytes. 53 In contrast, BMP10 is highly expressed in the 1 This promotes BMPR-II mRNA transcription and synthesis 2 and trafficking of newly-synthesised BMPR-II to the cell surface where it complexes with ALK1, which has been recycled via the endosomal pathway. 3 In the presence of BMP9, this feed forward pathway continues in an autoregulatory loop. 4 Middle panel: in patients with a heterozygous mutation in BMPR2 leading to haploinsufficiency, cell surface BMPR-II is reduced and in its place, ActRII-A can form a complex with available ALK1 but this does not promote autoregulatory BMPR-II production in response to endogenous concentrations of BMP9, which remain unchanged. The reduced signalling through BMPR-II leads to reduced BMPR-II levels of the receptor at the cell surface. Right Panel: Administration of exogenous BMP9 to PAH patients with a heterozygous mutation in BMPR2, increases the circulating concentration of BMP9 which increases signalling via the Smad1/4 complex to induce BMPR-II protein expression. This shifts the equilibrium of the BMPR-II:ActR-IIA ratio in favour of BMPR-II associating with the available ALK1 and thus restores the autoregulatory production of BMPR-II in response to BMP9, thus restoring normal endothelial BMP9 signalling. developing embryonic mouse heart, with much lower levels of expression detected in lung and liver. 54 The expression of BMP10 is high in the trabecular myocardium between days E9.5 -E13.5, a period that coincides with cardiac growth and chamber maturation. 54 The expression of BMP10 becomes restricted to the atria by day E18.5 and in adults, only the right atrium expresses BMP10. 49,55 BMP9 is secreted and circulates at levels of 2 -12 ng/ml in human plasma 56 and 1.18 -1.84 ng/ml in human serum, 51 based on cell-based luciferase bioassays. Plasma BMP9 levels in mice increase just before birth and peak postnatally at levels of 6 ng/ml on Day 15 before declining to a steady state of approximately 1.5 -2 ng/ml. 53 Similar to other BMPs, BMP9 is synthesised as a 429 amino acid precursor (pre-pro-BMP9) comprising a 22 amino acid signal peptide, a 297 amino acid prodomain and a 110 amino acid mature protein. 53,57 The precursor is then cleaved by serine endoproteases to produce a mature protein dimer (25 kDa) which can remain non-covalently associated with two pro-domains (33 kDa each) forming a 100 kDa complex. 53 Unlike the pro-regions of TGFbs and GDF8, which whilst bound, inhibit their ligands in vitro and in vivo, 58 -61 the mature BMP9 dimer retains its biological activity when in complex with the pro-domain. 62,63 The bound pro-domain is proposed to enhance the stability of BMP9 in vivo. 62 Approximately 60% of the 100 kDa circulating pro-BMP9 complex is cleaved and active, whereas the remaining 40% is unprocessed and may be activated through a local furin cleavage. 53 The role of BMP10 as a secreted ligand is more controversial, since some studies suggest that BMP9 is solely responsible for the ALK1-activating BMP activity in plasma. 48,56 However, a recent study has reported that BMP10 is present in mouse (0.5-2 ng/ml) and human (1 -3 ng/ml) serum, suggesting that BMP10 may be present in the circulation at physiologically important levels. 49 Whether BMP10 in the circulation is processed and circulates in a similar manner to BMP9 is not yet clear. However, evidence supporting a functional role for circulating BMP10 has been revealed in zebrafish embryos, showing that BMP10 is necessary for maintaining vascular stability and endothelial Smad signalling in the vessels proximal to the heart. 64 Loss of BMP10 phenocopies the cranial vascular abnormalities of the Alk1 zebrafish mutants, 65 although as discussed later, this differs from the phenotype of the Bmp10 knockout mouse.",
"The overlapping expression of BMP10 and Alk1 at about E8.5 49, 66 compared to the expression of BMP9 in the liver at E9.75-10 has been argued as evidence for the developmental regulation of BMP10 signalling through Alk1. 49 As BMP9 and BMP10 both activate ALK1, this may represent temporally important roles of the ligands. For example, Alk1-/-mice die at E10.5 due to defects in angiogenesis, 67 which may represent a failure of both BMP9 and BMP10 signalling, as discussed below. In comparison, Alk1 þ /2 mice develop normally, but exhibit nosebleeds and vascular anomalies. This reflects the pathology of ALK1 mutations causing HHT in man, although the disease penetrance is lower in heterozygous mouse models.",
"Intriguingly, BMP9 knockout mice develop to adulthood without any overt phenotype, 48 although further analysis has revealed lymphatic vessel defects 68 and delayed closure of the ductus arteriosus. 69 The lack of a severe vascular phenotype in BMP9-/-mice appears to be due to functional redundancy with BMP10, confirmed by defective retinal vascularisation in BMP9-/-mice when BMP10 is also neutralised. 48,49 Of note, ALK1-Fc administration, which inhibits both BMP9 and BMP10, phenocopies this sprouting defect. 49 As circulating BMP10 levels are elevated slightly in BMP9-/-mice, one might speculate that BMP10 may compensate for the absence of BMP9 in the circulation. 48 Consistent with the restricted cardiac expression, BMP10-/-embryos die around E9.5 -E10.5 due to hypoplastic cardiac development, probably as a result of reduced myocyte proliferation. 55 Although original reports reported an absence of vascular defects, 55 recent data indicate that the dorsal aorta and cardinal veins are fused in BMP10-/-mice. 49 Furthermore, cloning of the BMP9 coding region into the BMP10 knockout mouse does not rescue the cardiac defect whereas early vascular development appears normal, suggesting that BMP10 mediates cardiac development via a mechanism that is independent of the signalling capacity of BMP9. 49 This could be due to differences in the selectivity for type II receptors. 49 Intriguingly, ectopic expression of BMP10 driven by the alpha-myosin heavy chain promoter in the mouse myocardium leads to cardiac hypotrophy by 6 weeks of age. 70 The level of BMP10 expression achieved in the hearts was high, so it is not clear whether this represents a physiologically relevant effect of BMP10. 70 Table 2 summarizes the similarity and the difference between BMP9 and BMP10 in physiology.",
"POTENTIAL SIDE EFFECTS OF BMP9/10 THERAPY One potential concern associated with the delivery of exogenous BMP ligands is the activation of other BMP receptors in non-endothelial cells, much of which is a dose-related phenomenon. It is therefore important to consider the roles of BMP9 in tissue ossification/calcification, hepatic function and tumour regulation.",
"Osteogenesis, chrondrogenesis and adipogenesis: The potent orthotopic bone-forming activity of BMP9 may be a potential complication of BMP9 therapy in PAH. BMP9 induces the differentiation of mesenchymal stem cells (MSCs -adult stem cells found in bone marrow) into osteocytes, chondrocytes and adipocytes 46 and is one of the most potent osteogenesis-inducing BMP ligands in MSCs. 46,71 -74 The osteogenic induction of bone marrow MSCs is mediated via both ALK1 and ALK2 75 and is reported to involve BMP9 dependent induction of angiogenic HIF1a signaling. 76 The osteogenic response to BMP9 in skeletal muscle has been achieved through several modes of local ligand expression. Delivery of BMP9 into skeletal muscle via direct sonoporation, 77 injection of transfected MSCs 78 or C2C12 cells 46,79 or injection of an adenovirus engineered to express BMP9 46 all elicited ectopic bone formation in muscle tissue. These processes are attributed to the differentiation of local multipotent MSCs or osteoblastic progenitor cells. 80 -82 Ad-BMP9 stimulates lamellar bone in mice by 3 months, 83 although studies have reported evidence of calcification after 9 days of exposure. 84 It is notable that the majority of these studies involve a local inflammatory stimulus and it appears that the heterotopic ossification response to BMP9 requires injury to skeletal muscle. 85 Another important consideration would be the concentration of BMP9 achieved during therapeutic administration. All of the above studies involved high concentrations of BMP9 or uncontrolled local overexpression. Our recent study employed intraperitoneal dosing of BMP9 injections, and we observed no evidence of heterotopic calcification after 4 weeks of daily intraperitoneal BMP9 injection, or indeed 3 weeks of intramuscular injection. 41 The concentration of BMP9 is likely to be important to achieve therapeutic activation of BMPR-II/ALK1, but avoidance of ALK2 activation at higher concentrations. A recent study demonstrated that BMP9 promotes calcification of vascular smooth muscle cells in the context of high phosphate levels. 86 In this study, the authors propose ALK1 as the receptor mediating this calcification, although this conclusion was derived from the use of an ALK1-Fc ligand trap to inhibit BMP9 rather than molecular dissection of the receptor signalling in VSMCs. BMP9 stimulated calcification at concentrations of 5 ng/ml or greater, representing concentrations at which BMP9 can stimulate both ALK1 and ALK2. In contrast, no calcification was observed at 0.5 ng/ml BMP9, a concentration that would exclusively activate ALK1. Indeed, BMP9 and BMP10 might be expected to exert endothelial-mediated vascular protective effects in atherosclerosis, since endothelial-specific knockout of BMPR-II led to enhanced inflammation and atherosclerosis in ApoE-knockout mice. 87 As BMP10 appears to lack osteogenic activity in muscle tissue, at least when transduced with an adenovirus, 46 BMP10 may represent an attractive therapeutic alternative to BMP9, providing that BMP10 proves an effective therapy in animal models of PAH.",
"Liver function: The ability of BMP9 to stimulate hepatocyte proliferation is long established, being one of the earliest observations of BMP9 function. 88 Early studies demonstrated that BMP9 binds to specific receptors in HepG2 cells, 89 consistent with the expression of the low affinity type I receptor, ALK2 and the type II receptors, BMPRII, ActRIIA and ActRIIB. 90 Furthermore, both the HLE (human, non-differentiated hepatoma) cell line and hepatic stellate cells express ALK1, indicating these cells may exhibit high sensitivity to BMP9, though this has yet to be established. 91,92 Of note, BMP9 stimulates canonical Smad signalling and proliferation in HepG2, Hep3B and HuH7 cells. 93 The lowest BMP9 concentration examined in this report was 1 ng/ml and the responses were completely inhibited by low concentrations of the ALK2/3/6 inhibitor, LDN193189, implying these responses are not through ALK1. 93 The data from normal hepatocytes suggest that BMP9 may participate in liver repair but in conditions of liver carcinogenesis, BMP9 may promote tumour growth as discussed below.",
"In vivo studies have highlighted a positive role for hepatic BMP9 in energy metabolism. BMP9 inhibits hepatic glucose production and activates the expression of key enzymes of lipid metabolism. 63 Furthermore, recombinant BMP9 improved glucose homeostasis in vivo in diabetic and non-diabetic rodents. 63 However, these effects were observed at doses of BMP9 that were at least 1000 times higher than those employed in our PAH models. 41 Conversely, BMP9 is reduced in the livers of insulin-resistant rats and administration of an anti-BMP9 antibody induced glucose intolerance and insulin resistance in fasted rats. 94 Consistent with this observation, administration of insulin in combination with high glucose induced BMP9 expression in the livers of 12h-fasted rats. 94 This promotion of hepatic insulin sensing is associated with reduced expression of a rate-limiting enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes. 63 In addition to effects on the liver, BMP9 may regulate glucose utilization by skeletal muscle as it activates of Akt2 kinase in differentiated myotubes. 63 Akt2 is essential for the activation of insulin-induced glucose uptake by muscle and Akt2 signalling is impaired in insulin resistance. 95 -97 In differentiated L6 myotubes, Akt2 is activated by Smad5 and this has been proposed as the mechanism for BMP9 action, 98 though a direct effect of BMP9 signalling through Smad5 was not confirmed. Overall, the positive effect of BMP9 on hepatic glucose sensing may be of benefit in patients given the perspective that metabolic dysfunction is a component of the pathophysiology of PAH. 99,100 Tumour regulation: Variable effects of BMP9 on tumours and tumour cell growth have been reported. For example BMP9 promotes proliferation of tumour-derived cell lines from ovary 101 and liver 93 but induces apoptosis in prostate cancer cells 102 and restricts osteosarcoma cell proliferation and migration. 103,104 Furthermore, neutralisation of BMP9 with Endoglin-Fc restricts colonic tumours in mice. 105 The key receptor promoting the proliferative response is ALK2 in ovarian and liver cancer cell lines, shown through siRNA transfection and inhibition by low concentrations of the ALK2/3/6 inhibitor, LDN193189. 93,101 The tumour promoting and inhibitory responses are unlikely to be due to ALK1 activation, as prostate cancer cells do not express ALK1 yet BMP9 promotes apoptosis. 102 In some instances, the proliferation of tumour cell lines is promoted by autocrine BMP9 production. 101 Thus the impact of BMP9 on tumour cells appears to predominantly via ALK2 and at high local concentrations of BMP9.",
"In addition to effects on tumour cells, it is proposed that BMP9, and possibly BMP10, promote tumour angiogenesis. 106,107 The proangiogenic response to BMP9 appears to be context dependent, as BMP9 is reported to promote proliferation and angiogenic processes in embryonic mouse endothelial cells and transformed endothelial cell lines, 106,107 but inhibits angiogenesis in primary adult endothelial cell lines. 35,36,41 The possible role of BMP9 in tumour angiogenesis has led to the development of Dalantercept, a soluble ALK1 ligand trap, as an anti-tumour angiogenesis therapy. When applied clinically, the potential capacity of BMP9/10 to sustain tumour angiogenesis would need to be balanced against the possible transformational effect of BMP9 in treating patients with life-limiting PAH.",
"In summary, despite the potential challenges of enhancing signalling downstream of loss-of-function mutations in BMPR-II there are now several approaches that could be taken forwards into the clinic as novel therapeutic approaches in PAH. Of these, the direct enhancement of endothelial BMPR-II/ALK1 signalling with BMP9/10 offers an immediate solution that could be rapidly tested with the appropriate cautions in patients with this life-limiting disease."
] | [] | [
"INTRODUCTION",
"THE GENETIC BASIS OF PAH",
"TARGETING BMPR-II DEFICIENCY FOR THE TREATMENT OF PAH",
"Rescue of Mutant BMPR-II Receptor",
"4-PBA, glycerol and thapsigargin",
"Gentamycin",
"Inhibition of lysosomal degradation",
"BMP9 AND BMP10 AS POTENTIAL THERAPIES FOR PAH",
"PHYSIOLOGICAL ROLES OF BMP9 AND BMP10",
"Figure 2 .",
"Figure 3 .",
"Table 1 .",
"Table 2 ."
] | [
"Therapeutic Strategy \n\nAgent \n\nModel \n\n",
"BMP9 \nBMP10 \n\n-/-mice phenotype \nNormal, lymphatic vessel defect \nLethal, impaired cardiac development \nAdult expression \nLiver, into circulation \nRight atrium \nCirculating form and levels \n2 -10 ng/ml (by activity) \nPresence shown by ELISA and proteomics; \n, 300 pg/ml (ELISA) 108 \nOnly 1 in 3 reports can detect activity \nFunction \nVascular quiescence factor \nFlow-dependent arterial quiescence \nBone-forming activity \nHighest among 14 BMPs \nUndetected \nEndothelial cell signalling \nControlling a similar set of target \ngenes with similar potency 48 \nAffinity for ALK1/BMPRII \nHigher \n"
] | [
"Table 1",
"Table 2"
] | [
"The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension",
"The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension"
] | [] |
12,172,348 | 2022-03-20T07:52:25Z | CCBY | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0037540&type=printable | GOLD | 47c5768d7992729eb5d0ed8453cdf9e7c05a193e | null | null | null | null | 10.1371/journal.pone.0037540 | 2087374965 | 22629415 | 3358258 |
Soy Isoflavones Genistein and Daidzein Exert Anti- Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat 1-86 Exposure
Sheila M Adams
Department of Psychology
University of South Carolina
ColumbiaSouth CarolinaUnited States of America
Marina V Aksenova
Department of Psychology
University of South Carolina
ColumbiaSouth CarolinaUnited States of America
Michael Y Aksenov
Department of Psychology
University of South Carolina
ColumbiaSouth CarolinaUnited States of America
Charles F Mactutus
Department of Psychology
University of South Carolina
ColumbiaSouth CarolinaUnited States of America
Rosemarie M Booze
Department of Psychology
University of South Carolina
ColumbiaSouth CarolinaUnited States of America
Soy Isoflavones Genistein and Daidzein Exert Anti- Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat 1-86 Exposure
Background: HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.Methodology/Principal Findings: We demonstrate in primary cortical cell cultures that 17b-estradiol or isoflavones (genistein or daidzein) attenuate Tat 1-86 -induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERa or ERb specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERb selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.Conclusions/Significance: Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons.
Introduction
HIV-1 infection of the central nervous system (CNS) causes several neurological disorders, known as HIV-associated neurocognitive disorders (HAND) [1]. Although the incidence of severe dementia has significantly decreased since the advent of combination anti-retroviral therapy (cART), cognitive and motor impairments persist in up to 50% of HIV-1 positive patients due to longer life expectancy, the lack of patient compliance with cART therapy and the low penetrability of cART into the CNS [2]. The continued prevalence of neurological dysfunction suggests cART fails to provide complete protection from the development of HAND [1,3,4] and there currently are no pharmacotherapies targeted to HAND.
HIV-1 enters the brain early after infection and, although, HIV-1 does not infect neurons, neuronal dysfunction is key in HIV pathogenesis [5][6][7]. The early viral proteins such as Tat are continually produced despite the presence of cART [7][8][9][10]. Accordingly, Tat is rapidly taken up by neuronal cells and has been shown to have direct toxic effects on neurons though various mechanisms. Studies have shown Tat to mediate excitotoxicity via NMDA receptors [11][12][13], synaptic damage and dendritic pruning [14], induce apoptotic cascades [15,16], calcium dysregulation [17], oxidative stress [18], and dopaminergic system dysfunction [19,20]. Tat exposure has been shown to negatively affect cognitive processes in animal models [21,22]. The observations that the viral regulatory protein Tat is actively secreted by infected cells, and that Tat mRNA is elevated in patients with HIV-1 suggest a possible role of extracellular Tat in the progression of HIV-1-induced neurodegeneration [23][24][25].
Phytoestrogens, such as the soy isoflavones genistein and daidzein, mimic the neuroprotective actions and functions of estrogen in the brain, as they bind to the estrogen receptor (ER) and affect estrogen-mediated processes [26][27][28][29]. Several studies have found that soy isoflavones can improve cognitive functions in both humans and rats, but underlying mechanisms remain unknown [30][31][32][33][34][35]. Additional studies have shown that isoflavones have neuroprotective effects against various neurodegenerative insults. Genistein and daidzein have demonstrated neuroprotective efficacy against glutamate excitotoxicity and Ab [25][26][27][28][29][30][31][32][33][34][35] induced loss of cell viability, oxidative stress and initiation of apoptosis in hippocampal neurons [36,37]. As the dopaminergic system is sensitive to HIV in the brain, isoflavones, similar to estradiol, may interact with dopamine to preserve motor and cognitive functions [35,[38][39][40].
Estrogen therapy is met with resistance due to its association with reproductive and breast cancers [41,42]. However, dietary consumption and supplementation with soy isoflavones is widespread. Consuming a typical Western diet yields low nanomolar concentrations of circulating isoflavones [43,44]. In people consuming modest amount of soy products yielding ,50 mg/day of total isoflavones, plasma levels of 50-800 ng/ml have been achieved for daidzein and genistein [43], which is comparable to concentrations observed in a traditional Japanese diet [44]. However, consuming a western vegetarian diet and taking supplements has been shown to achieve increased isoflavone consumption comparable to or higher than Asian levels [44][45][46]. Furthermore, human studies that have reported improved cognitive function with soy isoflavone consumption have used ,60-100 mg/day doses of isoflavones [31,34]. The cognitive improvements seen with high doses of soy in vivo were not associated with abnormalities in reproductive health of humans, including men [31,[47][48][49]. This broadens the use and benefits of these estrogenic compounds to not only women but also men.
Soy isoflavones preferentially binding to ERb is of significant consideration in neuroprotection as this ER subtype is highly expressed in the brain compared to ERa, which due to its high expression in the reproductive tissue, has been associated with the proliferative effects of estrogen. Elucidating whether isoflavone protection is mediated by ER selectivity is a central focus in developing neuroprotective strategies. In the current experiments, we investigated whether treatment with soy isoflavones, genistein or daidzein, could attenuate HIV-1 Tat-induced mitochondria associated apoptosis in cortical cell cultures. Further, we explored whether isoflavone neuroprotection against HIV-1 viral protein Tat-induced neural toxicity involves ER-mediated attenuation of apoptotic signaling. We demonstrated that isoflavones maintained neuronal cell viability in the presence of prolonged Tat exposure. We also observed that isoflavones prevented Tat-induced upregulation of mitochondrial apoptotic cascade regulators. Moreover, we determined that the protective actions of isoflavones were mediated by estrogen receptors.
Results
Physiological Doses of Genistein and Daidzein Prevent Cell Death Following Tat 1-86 Exposure
We have previously shown that 17b-estradiol attenuated Tatinduced cell death [50]. As shown in Figure 1, the cell viability decrease (<25% of control) induced by prolonged (up to 5 days) exposure to the toxic dose of Tat 1-86 B was abrogated by 0.1-10 nM of 17b-estradiol. Similar alleviation of Tat-induced neuronal cell death was observed when isoflavones GEN or DAI were used at doses 0.05, 0.2, and 1 mM (Figure 1). Our levels of 50 nM to 1.0 mM of genistein and daidzein are within the range of observed plasma levels of isoflavones following consumption of soy products which reflect ,200 nM-3 mM. Results indicate that physiologically relevant concentrations of isoflavones are able to effectively protect cortical neurons against Tat toxicity in vitro.
Genistein and Daidzein Attenuate Tat-induced Caspase Activation in Primary Cortical Cell Cultures
In the present experiments, we evaluated if the protective potential of GEN and DAI against Tat involves downregulation of caspase activity. Figure 2A shows significant caspase 9 activation following only 4 hr of Tat exposure (p#0.05). Preincubation with 10 nM 17b-estradiol or 1 mM of isoflavones (GEN or DAI) prevented the increase in Tat-induced caspase 9 activation. Cultures pretreated with GEN or DAI displayed caspase 9 activity similar to that of estradiol treated cultures (p#0.05). Moreover, analysis revealed that caspase 9 activity was not statistically different between the 17b-estradiol, GEN and DAI pretreated cultures.
In regard to effector caspase 3 activity, a similar effect was observed with phytoestrogen pretreatment prior to Tat exposure. These results demonstrated a significant increase in caspase 3 activation in cortical cultures following 4 hr exposure to Tat (p#0.05). The current experiments show that the addition of GEN or DAI prior to incubation with 50 nM Tat significantly attenuated the upregulation of active caspase 3 expression (p#0.05, Figure 2B). The level of activation of caspase 3 in GEN and DAI pretreatment groups was not significantly different from that of vehicle-treated controls; furthermore, these levels were very similar to that of the 17b-estradiol treated cultures. These results indicate that isoflavones GEN and DAI downregulate Tat-induced caspase activation to a level comparable to that of 17b-estradiol, suggesting that isoflavones and estradiol may share a common neuroprotective mechanism.
Genistein and Daidzein Sustain Levels of Mitochondrial Proteins Bax and Bcl-2 Expression Following Tat Exposure
The protection of cortical cell cultures with 17b-estradiol against Tat toxicity is associated with regulating the effects of apoptotic proteins linked to the mitochondrial apoptotic pathway [50]. Therefore, we compared effects of neuroprotective concentrations of 17b-estradiol and isoflavones that completely eliminate Tat-induced death of cortical cells on the alterations in Bcl-2 and Bax protein levels. Results of the Bcl-2 ELISA presented in Figure 3A demonstrate that, similar to estradiol (10 nM), neuroprotective doses of GEN and DAI (1 mM) added to the cell culture medium 24 hr in advance of 50 nM Tat significantly (p#0.05) attenuated the increase of Bcl-2 expression; an effect shown to occur within the first 16-24 hr of Tat exposure in cortical cell cultures [50].
We also evaluated the efficacy of isoflavones against Tatinduced Bax expression. Results of the Bax ELISA ( Figure 3B) demonstrate that pretreatment with 1 mM GEN or DAI, or pretreatment with 10 nM estrogen, significantly (p#0.05) blocks the induction of Bax expression in Tat-exposed cortical cells ( Figure 3B). Effects of all the compounds on Tat-induced changes in Bcl-2/Bax protein expression were specific, since neither the exposure to 10 nM 17b-estradiol, nor the exposure to 1 mM isoflavones caused statistically significant changes in Bcl-2 or Bax immunoreactivities compared to non-treated control cell cultures.
Estrogen Receptor Antagonists Block Anti-apoptotic Actions of Soy Isoflavones
Plant isoflavones, such as GEN and DAI, are similar to 17bestradiol in chemical structure, which allows them to interact with estrogen receptors (ER). Neuroprotective effects of 17b-estradiol against Tat-induced apoptosis are mediated by two subtypes of estrogen receptors, ERa and ERb. ER expression in primary cortical neurons used in the current experiments demonstrates the presence of both ER a and b immunoreactivity in our cultures (data not shown). In the present experiments, we sought to determine if the protective actions of isoflavones were ERmediated. The ER antagonist, ICI 182,780 (100 nM), was added to cultures 1 hr prior to incubation with 17b-estradiol, GEN or DAI. Following 24 hr pretreatment, 50 nM Tat was added to cultures and its effects on the active caspase 3 ( Figure 4A) and Bax ( Figure 4B) protein levels have been analyzed.
As shown in Figure 4A, ICI prevented the downregulation of active caspase 3 by 17b-estradiol (10 nM), GEN or DAI (1 mM) pretreatment. Consistent with our previous study, ICI 182,780 averted estrogen-mediated activation of caspase 3 by Tat treatment of cortical cultures. Activated caspase 3 immunoreactivity in ICI+GEN+Tat-treated cultures was significantly higher than GEN+Tat treated cortical cultures (p#0.05) and protein levels of activated caspase 3 were similar to that in Tat-only treated cultures. Similarly, the DAI effects on caspase 3 were sensitive to ICI inhibition of ERs. There was a significant increase in active caspase 3 expression with the addition of ICI compared to DAI+Tat treated cultures (p#0.05). Results of the experiments indicate that GEN and DAI, similar to 17b-estradiol, inhibit the Tat-induced caspase 3 activation via an ER-mediated mechanism, as the addition of ICI 182,780 prior to estradiol returned caspase 3 activation levels to that of Tat only treated cultures. Moreover, caspase 3 activity did not differ statistically from that of Tat only treated cultures.
The results in Figure 4B show that GEN and DAI effects on Tatinduced Bax expression in cortical cultures are ICI-sensitive. A significant 25% increase in Bax expression was seen with ICI treatment prior to incubation with GEN or DAI (p#0.05) ( Figure 4B). Although, GEN and DAI possess estrogen-like activity, their affinity to ERa-or ERb-subtypes is significantly different from 17b-estradiol. Therefore, we used specific antagonists of a or b ERs to evaluate selectivity of the effects of 17b-estradiol, GEN and DAI on Tatinduced changes in activated caspase 3 or Bax protein levels. The ERsubtype specific antagonists MPP (ERa) and PHTPP (ERb) (1 mM) were added to cortical cultures prior to incubation with GEN or DAI and subsequent exposure to 50 nM Tat.
Neither anor b-selective ER antagonists were able to completely block the inhibitory effects of 10 nM 17b-estradiol ( Figure 5A), or those of 1 mM GEN or DAI, on Tat-induced caspase 3 activation ( Figure 5B, C). Although the ER subtype selective antagonists diminished the ability of all the compounds to decrease active caspase 3 protein levels in Tat-treated cultures, their effects were not statistically significant. Overall, selective antagonists of ERa-or ERb-subtypes similarly affected the ability of both 17b-estradiol and isoflavones to downregulate Tat-induced caspase 3 activation.
The addition of ERa antagonist MPP did not significantly attenuate 17b-estradiol effects on Bax expression ( Figure 5D), but was able to significantly decrease the effect of GEN and DAI. The ERb-specific antagonist PHTPP completely blocked the effect of 17b-estradiol and GEN ( Figure 5E, F). PHTPP also caused a partial, but significant, decrease in the ability of DAI to inhibit the Tat-dependent increase in Bax expression.
Discussion
Neurocognitive deficits associated with HIV infection persist even with effective cART [51]. Targeting ERb function may be a potential therapeutic option since it is highly expressed in the brain, specifically in cortical regions responsible for executive functions significantly affected in HIV-1 associated neurocognitive disorders (HAND) [35,[52][53][54]. In this study, we evaluated whether soy isoflavones, acting as ER selective compounds, were able to mimic the neuroprotective effects of estrogen in HIV-1 Tat 1-86exposed primary neuronal cultures. GEN and DAI represent soy isoflavones with ERa/b binding profiles showing much higher binding selectivity for ERb than 17b-estradiol. Among soy phytoestrogens, GEN exhibits maximum ERa/b binding affinity with approximately 60-fold preference for ERb over ERa. DAI has lower ERa/b binding affinity than GEN with 14-fold selectivity for ERb binding. For comparison with these two soy isoflavones, 17b-estradiol's preference for the ERb binding is 0.78fold [55]. GEN and DAI have very close chemical structures and are known to induce neuroprotective responses but at a much lower magnitude than 17b-estradiol.
Studies have demonstrated that oxidative stress and mitochondrial dysfunction coincide with Tat activation of apoptotic cascades [16,38,50,56]. Moreover, we have recently reported that 17b-estradiol attenuated Tat-induced apoptotic signaling in an estrogen-receptor dependent manner [50]. We now demonstrate that the soy isoflavones genistein and daidzein prevent the upregulation of caspase activity in Tat-exposed cultures. In addition, upstream of caspase activation, we show that Tat exposure significantly increased expression of pro-and antiapoptotic proteins Bax and Bcl-2 respectively, which regulate mitochondrial membrane permeability and thus, the release of apoptogenic substances. Our results indicate that treatment with GEN or DAI also markedly reduces the expression of Bax and Bcl-2 in Tat-exposed cortical cultures. A major finding of our study is that both soy isoflavones GEN and DAI exhibited protective effects similar to that exhibited by 17b-estradiol. GEN and DAI increased cell viability and attenuated the upregulation of apoptotic proteins in a manner comparable to that observed in estradiol treated cultures. At low micromolar doses we observed that isoflavones were able to maintain cell survival following prolonged exposure to Tat. These results suggested a similar neuroprotective action of isoflavones and 17b-estradiol involving inhibition of apoptotic pathways. Our experiments also demonstrated that selective ERb agonists induce an anti-apoptotic effect in primary cultures exposed to HIV-1 Tat. Such observations support the findings that isoflavones are protective against oxidative stress-mediated apoptosis in HIV-1 infection.
Since isoflavones bind estrogen receptors (ERs), the neuroprotective actions of isoflavones may be produced through activation of the ER. We found that the addition of the ER antagonist ICI 182,780, which blocks both the ERa and ERb subtypes, reversed GEN and DAI downregulation of caspase 3 activity and Bax expression with Tat exposure, suggesting that these effects of GEN and DAI were ER-dependent. The addition of ICI 182,780 had a more robust effect against genistein actions on caspase 3 activity, and sustained caspase levels similar to cultures treated with Tat. Furthermore, an isoflavone effect on Bax expression was also shown to be ER-dependent, as ICI 182,780 blocked genistein and daidzein inhibition of Tat-induced Bax expression. Collectively, our results suggest that genistein and daidzein act as estrogen receptor agonists in primary cortical neurons and activate estrogenic neural defense mechanisms.
Another major finding is that isoflavone anti-apoptotic effects are selective relative to estrogen receptor isoform. There are conflicting reports of which ER subtype, ERa or ERb, specifically mediates the protective actions of estrogen [57,58]. We observed in our previous studies with estradiol [50], inhibition of ERs with ICI 182,780 blocked the downregulation of apoptotic proteins in cultures pretreated with isoflavones. Further experiments sought to determine if these receptor-mediated effects were specific to a particular ER-subtype. Cultures in the presence of ERa-specific antagonist MPP or ERb-specific antagonist PHTPP did not show specific attenuation of genistein and daidzein downregulation of caspase 3 activity. Similar to previous results observed with estradiol, both ER subtypes seem to play a role in genistein and daidzein inhibition of Tat activation of caspase 3. However, a more pronounced attenuation of genistein effects on Bax expression was observed in the presence of ERb antagonist PHTPP. Thus, genistein effects on Bax expression may be preferential for ERb-mediated signaling. Our results also demonstrated that the addition of both ER subtype antagonists significantly inhibited daidzein actions on Bax expression, suggesting involvement of both ERa and ERb in daidzein downregulation of Bax. It is possible that daidzein's lower binding affinity for ERs compared to that of genistein and estradiol [55] may explain the absence of the preferential mediation of a specific ER subtype. Another point of consideration is that ERa and ERb may act simultaneously and thus counteract the function of the other receptor subtype [59,60]. Both genistein and daidzein were shown to activate binding to ERb at nanomolar concentrations (30 nM and 350 nM, respectively), which are easily achievable levels in humans consuming soy products or supplements. At the concentrations used in these experiments, it is plausible that both receptor subtypes were activated and as such ER subtype specific effects may be diminished. Caspase 3 has a pivotal role in the apoptotic process. Multiple pro-apoptotic pathways converge on caspase 3 activation in the cell death cascade. Caspase 3 activation may occur through caspase 9 from the mitochondria or from death receptor signaling via caspase 8 as well as through other proapoptotic pathways [61][62][63]. More upstream in the apoptotic cascade, the upregulation of Bax is associated with mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria, leading to caspase activation. The ERb specific effects on Bax may be related to the recent discovery of ERb localization in mitochondria [64], suggesting a direct estrogenic effect on mitochondria function via ERb activation and signaling. The ER-mediated reduction of caspase 3 activity and Bax expression by estradiol and isoflavones suggested that these compounds disrupt apoptotic signaling by downregulating key pro-apoptotic factors in the cell death cascade. As multiple apoptotic pathways converge on mitochondria functioning and caspase 3 activation, Bax and caspase 3 represent potential upstream and downstream receptorsensitive check points for estrogenic compounds to disrupt apoptotic processing in response to neurodegenerative insults.
As isoflavones affect the viability of neurons and cognitive function by acting as an estrogenic agonist, they can also utilize differential distribution and regulation of the ER subtypes, ERa and ERb in the brain. Microarray experiments have shown that ERa and ERb regulate different genes [65,66]. Differences in conformation that occur upon ER binding affects the recruitment of coregulatory proteins, and thus produces differential gene regulation in specific cell types. In addition to tissue or region specific localization of ERs, intracellular localization of ERs may contribute to some of their different mechanisms of action. ERb has been localized to a greater extent at extranuclear sites and in the cytoplasm for trafficking to the plasma membrane [64,67,68]. The extranuclear and membrane localization of ERb enables its interaction with intracellular signaling cascades to integrate rapid signaling events and classical transcriptional mechanisms [69,70]. Given the timing of treatment in our studies, both genomic and nongenomic molecular actions may be utilized by isoflavones to confer ERmediated neuroprotection against Tat.
Despite the success of cART on peripheral viral suppression, protected viral reservoirs in the brain may allow continued release and exposure to toxic viral proteins [10]. The inability of anti-retroviral therapy to prevent the development of neurocognitive dysfunction indicates the need for adjunctive therapies to address the neurodegenerative and subsequent neurological disturbances associated with HAND. Findings in the present study demonstrate that soy isoflavones offer a similar protective effect as endogenous estradiol via a selective estrogen receptormediated mechanism against HIV-1 Tat-induced cell death. Isoflavones, acting as selective ER agonists targeting the neuroprotective effects associated with estradiol, may represent a safe and viable neuroprotectant along with cART to improve the neurological health of both men and women with HAND.
Materials and Methods
Ethics Statement
All of the experimental procedures using animals were performed in accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The relevant animal use protocols were approved by the University of South Carolina Animal Care and Use Committee under the auspices of Animal Assurance Number A3049-01.
Primary Neuronal Cell Culture
Primary cultured cortical neurons were prepared from 18-dayold Sprague-Dawley rat fetuses [50]. Rat cortices were dissected and incubated for 15 min in a solution of 2 mg/mL trypsin in Ca 2+ -and Mg 2+ -free Hanks' balanced salt solution (HBSS) buffered with 10 mM HEPES (Invitrogen, Carlsbad, CA). The tissue was then exposed for 2 min to soybean trypsin inhibitor (1 mg/mL, in HBSS) and then rinsed 3 times in HBSS. Cells were dissociated by trituration and distributed to poly-L-lysine coated culture plates with wells containing DMEM/F12 medium (Invitrogen) supplemented with 100 mL/L fetal bovine serum (Sigma Chemicals, St. Louis, MO). After a 24-hr period, the DMEM/F12 medium was replaced with 2% v/v B-27 Neurobasal medium supplemented with 2 mM GlutaMAX and 0.5% w/v D-(+) glucose (Invitrogen). Two-thirds of the neurobasal medium was replaced with fresh medium of the same composition once a week. Cultures were used for experiments after 12 days in culture in serum-free medium and were .95% neuronal as observed by anti-MAP-2 immunostaining.
Tat 1-86 Exposure and Experimental Treatment of Cultures
Recombinant Tat 1-86 (LAI/Bru strain of HIV-1 clade B, GenBank accession no. K02013, Diatheva, Fano, Italy) was added to cell culture medium. Groups of cultures in 24-well plates were exposed to 50 nM Tat. Cell cultures were treated with 17bestradiol (0.1 nM, 2 nM or10 nM, Sigma) or soy isoflavones (0.05 mM, 0.2 mM or1 mM, genistein (GEN) or daidzein (DAI), Indofine Chemical) for 24 hr prior to Tat exposure and remained present in medium throughout experiments. 17b-estradiol was dissolved in sterile water and diluted in D-PBS. Isoflavones were dissolved in DMSO and diluted in D-PBS. To assess neurotoxicity, the cultures were exposed to Tat for 4, 16, or 24 hr before harvesting. After treatment, medium was removed, cells were washed and lysates collected for ELISA experiments. Cells were also treated with the estrogen receptor antagonist ICI 182,780, the ERa specific antagonist, MPP dihydrocloride, or the ERb specific antagonist PHTPP (100 nM, Tocris Cookson Inc, Ellisville, MO) 1 hr before estradiol or isoflavone treatment to determine if the effects against Tat toxicity were receptor mediated.
Cell Viability Assay
Neuronal survival was determined using a Live/Dead viability/ cytotoxicity kit (Molecular Probes, Eugene, OR) in rat fetal cortical cell cultures prepared in 96-well plates. In accordance with the manufacturer's protocol, neurons were exposed to cellpermeate calcein AM (2 mM), which is hydrolyzed by intracellular esterases, and to ethidium homodimer-1 (4 mM), which binds to nucleic acids. The cleavage product of calcein AM produces a green fluorescence (F 530 nm ) when exposed to 494-nm light and is used to identify live cells. Bound ethidium homodimer-1 produces a red fluorescence (F 645 nm ) when exposed to 528-nm light, allowing the identification of dead cells. Fluorescence was measured using a Bio-Tek Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Each individual F 530 nm and F 645 nm value on a plate was corrected for background fluorescence (readings obtained from cell cultures (wells) that were not exposed to calcein AM and ethidium bromide) by the microplate reader KC4 software package (Bio-Tek Instruments, Inc., Winooski, VT). For each individual cell culture (well) on a plate, ratios between corrected green and red fluorescence (F 530 nm /F 645 nm , Live/Dead ratios) were calculated. All individual relative numbers of live and dead cells were expressed in terms of percentages of average maximum Live/Dead ratio determined for the set of non-treated control cell cultures (8-16 wells) from the same plate: (F 530 nm /F 645 nm ) well n /(F 530 nm /F 645 nm ) average max 6 100%.
Detection of Apoptotic Proteins (ELISA)
Expression of apoptotic signaling proteins in cell lysates was determined by ELISA [16,50]. Cell lysates were prepared from cultures grown in 24-well plates. At the time of harvesting, medium was removed and cells were washed 3 times with Dulbecco phosphate-buffered saline, D-PBS, (8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 0.137 M NaCl and 2.7 mM KCL at pH 7.4) and lysed with CellLytic TM -M mammalian cell lysis buffer (Sigma Chemicals) containing protease inhibitors (protease inhibitors cocktail, Sigma Chemicals). All samples in a group (6 culture wells) were pooled together and protein concentration was determined by the BCA method (Pierce, Rockford, Ill.). Each well of Costar 96-well ELISA plates (Corning Inc, PA) was coated overnight at 4uC using 100 mL of 20 mM carbonate coating buffer, pH 9.6. Cortical cell lysate samples were diluted 1:10 with D-PBS and 20 mg of each sample were added to the plate wells. After overnight incubation at 4uC, plates were rinsed 5 times with PBST (0.05% Tween 20 in PBS, pH 7.4) and blocked with 1% BSA in PBS for 2 hr at room temperature. After blocking, plates were washed again, as described above, and primary anti-Bax, anti-Bcl-2, anti-active Caspase 9 and anti-active Caspase 3 antibodies (all primary antibodies, Abcam, Cambridge, MA) diluted 1:5000 or 1:7500 (caspase 3) in 0.1% BSA-PBST were added to each well except for blanks and no-primary antibody control wells. Plates were kept overnight at 4uC. When the incubation with primary antibodies was completed, plates were again washed 5 times with PBST and secondary antibodies [goat anti-rabbit alkaline phosphatase conjugated, Sigma] diluted 1:2000 in 0.1% BSA-PBST were added to each well, except for blank and no-secondary antibody control wells. After 2 hr of incubation, the secondary antibody solution was removed, plates were washed 5 times with PBST and 100 mL of BluePhos phosphatase substrate mixture (KPL Research, Gaithersburg, MD) was added to the plate wells. After 30 min of incubation, the absorbance at 650 nm was determined using a Bio-Tek Synergy HT microplate reader. Multiple readings were taken within a 1-hr time period.
Statistical Analysis
Statistical comparisons were made using one-way ANOVA and Tukey's multiple comparison tests were used to determine specific treatment effects. Significant differences were set at p#0.05. Data represents mean values 6 standard error of the mean (SEM).
Author Contributions
Figure 1 .
1Soy isoflavones genistein and daidzein protect primary cortical cultures from Tat neurotoxicity. Primary cortical neurons were exposed to estrogen (0.1, 2.0 and 10 nM), or isoflavones (0.05, 0.2 and 1 mM) 24 hr prior to the start of Tat 1-86 B (50 nM) treatment. Cell viability was assessed by Live/Dead assay. Live/Dead ratios were determined after 48 hr (A-C) or 5 days (D-F) of the continuous exposure to Tat or equal volume of vehicle in cell culture groups that were treated or not treated with estrogen, GEN, or DAI. Data represents mean values 6 SEM, n of cultures analyzed = 7-12 per each group. *-indicates significant (p#0.05) protective effects of the selected compounds against Tat neurotoxicity (cell viability decrease) in cortical cell cultures. Repeated (2-3) trials using cell culture preparations from different litters were carried out to ensure the reproducibility of the results. doi:10.1371/journal.pone.0037540.g001
Figure 2 .
2Genistein and daidzein attenuate Tat-induced caspase activation in primary cortical cultures. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of activated apoptotic proteins A. Caspase 9 (4 hr of Tat exposure) and B. caspase 3 (4 hr Tat exposure) was assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tat-treated cultures. doi:10.1371/journal.pone.0037540.g002
Figure 3 .
3Isoflavones prevent Tat-induced expression of Bcl-2 and Bax protein levels. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of apoptotic proteins A. Bcl-2 (16 hr of Tat exposure) and B. Bax (4 hr Tat exposure) were assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tattreated cultures. doi:10.1371/journal.pone.0037540.g003
Figure 4 .
4Estrogen receptors mediate isoflavone effects on Caspase 3 and Bax expression following Tat exposure. A. GEN or DAI effects on caspase 3 expression were blocked in the presence of ER antagonist, ICI 182,780. B. GEN or DAI effects on Tat-induced expression of Bax were reversed by ICI 182,780, suggesting that estrogenic actions on caspase 3 and Bax are ER mediated. Data represents mean values 6 SEM, *p#0.05 vs. GEN/DAI+T treated cultures. doi:10.1371/journal.pone.0037540.g004
Figure 5 .
5ER subtype specific effects against caspase activity and Bax expression. Similar to 17b-estradiol (A), GEN (B) and DAI (C) effects against Tat-induced caspase 3 activity were maintained in the presence of specific antagonists for ERa (MPP) and ERb (PHTPP). ER subtype antagonists reveal that ERb signaling was preferential for GEN (E) effects on Bax. DAI (F) effects on Bax were blocked in the presence of both ER subtype antagonists. Data represents mean values 6 SEM, *p#0.05 as compared to GEN/DAI+Tat treated cultures. doi:10.1371/journal.pone.0037540.g005
Conceived and designed the experiments: SMA CFM RMB. Performed the experiments: SMA MVA MYA. Analyzed the data: SMA MYA. Wrote the paper: SMA CFM RMB. Critical appraisal and approval of final manuscript: SMA MVA MYA CFM RMB.
May 2012 | Volume 7 | Issue 5 | e37540
PLoS ONE | www.plosone.org
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| [
"Background: HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.Methodology/Principal Findings: We demonstrate in primary cortical cell cultures that 17b-estradiol or isoflavones (genistein or daidzein) attenuate Tat 1-86 -induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERa or ERb specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERb selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.Conclusions/Significance: Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons."
] | [
"Sheila M Adams \nDepartment of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America\n",
"Marina V Aksenova \nDepartment of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America\n",
"Michael Y Aksenov \nDepartment of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America\n",
"Charles F Mactutus \nDepartment of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America\n",
"Rosemarie M Booze \nDepartment of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America\n"
] | [
"Department of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America",
"Department of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America",
"Department of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America",
"Department of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America",
"Department of Psychology\nUniversity of South Carolina\nColumbiaSouth CarolinaUnited States of America"
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] | [
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"Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta. M K Tee, I Rogatsky, C Tzagarakis-Foster, A Cvoro, J An, Mol Biol Cell. 153Tee MK, Rogatsky I, Tzagarakis-Foster C, Cvoro A, An J, et al. (2004) Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta. Mol Biol Cell 15(3): 1262-1272.",
"Drug and cell typespecific regulation of genes with different classes of estrogen receptor b-selective agonists. S Paruthiyil, A Cvoro, X Zhao, Z Wu, Y Sui, PLoS One. 476271Paruthiyil S, Cvoro A, Zhao X, Wu Z, Sui Y, et al. (2009) Drug and cell type- specific regulation of genes with different classes of estrogen receptor b-selective agonists. PLoS One 4(7): e6271.",
"Estrogen induces rapid translocation of estrogen receptor beta, but no estrogen receptor alpha, to the neuronal plasma membrane. L C Sheldahl, R A Shapiro, D N Bryant, I P Koerner, D M Dorsa, Neurosci. 1533Sheldahl LC, Shapiro RA, Bryant DN, Koerner IP, Dorsa DM (2008) Estrogen induces rapid translocation of estrogen receptor beta, but no estrogen receptor alpha, to the neuronal plasma membrane. Neurosci 153(3): 751-761.",
"Extranuclear estrogen receptor beta immunoreactivity is on doublecortincontaining cells in the adult and neonatal rat dentate gyrus. S P Herrick, E M Waters, C T Drake, B S Mcewen, T A Milner, Brain Res. 11211Herrick SP, Waters EM, Drake CT, McEwen BS, Milner TA (2006) Extranuclear estrogen receptor beta immunoreactivity is on doublecortin- containing cells in the adult and neonatal rat dentate gyrus. Brain Res 1121(1): 46-58.",
"Multiple pathways transmit neuroprotective effects of gonadal steroids. D N Bryant, L C Sheldahl, L K Marriot, R A Shapiro, D M Dorsa, Endocrine. 292Bryant DN, Sheldahl LC, Marriot LK, Shapiro RA, Dorsa DM (2006) Multiple pathways transmit neuroprotective effects of gonadal steroids. Endocrine 29(2): 199-207.",
"Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. L Björnström, M Sjöberg, Björnström L, Sjöberg M (2005) Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes."
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] | [
"Updated research nosology for HIV-associated neurocognitive disorders",
"HIV-associated neurocognitive disorder: pathogenesis and therapeutic opportunities",
"Effects of antiretroviral therapy on cognitive impairment",
"HIV infection of the central nervous system: clinical features and neuropathogenesis",
"HIV infection and dementia",
"HIV-1 infection and AIDS: consequences for the central nervous system",
"Mechanisms of neuroimmunity and neurodegeneration associated with HIV-1 infection and AIDS",
"Developing neuroprotective strategies for treatment of HIV-associated neurocognitive dysfunction",
"Role of Tat protein in HIV neuropathogenesis",
"Antiretroviral therapy does not block the secretion of the human immodeficiency virus tat protein",
"Differential induction of rat neuronal excitotoxic cell death by human immunodeficiency virus type 1 clade B and C tat proteins",
"Mechanisms of HIV-tat-induced phosphorylation of N-methyl-D-aspartate receptor subunit 2A in human primary neurons: implications for neuroAIDS pathogenesis",
"Human immunodeficiency virus protein Tat induces synapse loss via a reversible process that is distinct from cell death",
"Attenuated neurotoxicity of the transactivation-defective HIV-1 Tat protein in hippocampal cell cultures",
"Neuronal survival and resistance to HIV-1 Tat toxicity in the primary culture of rat fetal neurons",
"The human immunodeficiency virus type-1 transcription factor Tat produces elevations in intracellular Ca 2+ that require function of an N-methyl-D-aspartate receptor polyamine-sensitive site",
"HIV-1 Tat protein induced rapid and reversible decrease in (3H)dopamine uptake: dissociation of (3H) dopamine uptake and (3H)2beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (WIN 35,428) binding in rat striatal synaptosomes",
"Hyperdopaminergic tone in HIV-1 protein treated rats and cocaine sensitization",
"Intrahippocampal injections of Tat: effects on prepulse inhibition of the auditory startle response in adult male rats",
"Neonatal intrahippocampal injection of the HIV-1 proteins gp120 and Tat: differential effects on behavior and the relationship to stereological hippocampal measures",
"Human immunodeficiency virus (HIV) proteins in neuropathogenesis of HIV dementia",
"Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues",
"Neurobiology of HIV",
"Assaying estrogenicity by quantitating the expression levels of endogenous estrogenregulated genes",
"Production and actions of estrogens",
"Phytoestrogens modulate binding response of estrogen receptors a and b to the estrogen response element",
"Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro",
"Soy phytoestrogens improve spatial arm maze performance in ovariectomized retired breeder rats and do not attenuate benefits of 17 b-estradiol treatment",
"Eating soya improves human memory",
"Dietary phytoestrogens enhance spatial memory and spine density in the hippocampus and prefrontal cortex of ovariectomized rats",
"Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens",
"Psychological assessment of the effects of treatment with phytoestrogens on postmenopausal women: a randomized, double-blind, crossover, placebo controlled study",
"Impact of dietary genistein and aging on executive function in rats",
"Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons",
"Genistein ameliorates beta-amyloid peptide (25-35)-induced hippocampal neuronal apoptosis",
"HIV-1 Tat neurotoxicity in primary cultures of rat midbrain fetal neurons: changes in dopamine transporter binding and immunoreactivity",
"Neurotoxicity of HIV-1 Tat protein: involvement of D1 dopamine receptor",
"Estradiol enhances learning and memory in a spatial memory task and effects levels of monoaminergic neurotransmitters",
"Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial",
"Million Women Study Collaborators (2006) Hormonal therapy for menopause and breastcancer risk by histological type: a cohort study and meta-analysis",
"Dietary isoflavones: biological effects and relevance to human health",
"Estimated Asian soy protein and isoflavone intakes",
"The pros and cons of phytoestrogens",
"Analytical and compositional aspects of isoflavones in food and their biological effects",
"Soy isoflavones and cognitive function",
"Clinical studies show no effects of soy protein or isoflavones on reproductive hormones in men: results of a meta-analysis",
"Clinical characteristics and pharmacokinetics of purified soy isoflavones: single dose administration to healthy men",
"ERb mediates 17 b-estradiol attenuation of HIV-1 Tat-induced apoptotic signaling",
"HIV-associated neurocognitive disease continues in the antiretroviral era",
"Cortical and subcortical neurodegeneration is associated with HIV neurocognitive impairment",
"Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system",
"Distribution of estrogen receptor beta immunoreactivity in the rat central nervous system",
"A select combination of clinically relevant phytoestrogens enhances estrogen receptor beta-binding selectivity and neuroprotective activities in vitro and in vivo",
"HIV-1 tat causes apoptotic death and calcium homeostasis alterations in rat neurons",
"The multifaceted mechanisms of estradiol and estrogen receptor signaling",
"Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons",
"Roles of estrogen receptors alpha and beta in sexually dimorphic neuroprotection against glutamate toxicity",
"Neuronal estrogen receptor-alpha mediates neuroprotection by 17 beta-estradiol",
"Caspases: the executioners of apoptosis",
"Emerging roles of caspase-3 in apoptosis",
"Caspases -controlling intracellular signals by protease zymogen activation",
"Mitochondrial localization of estrogen receptor b",
"Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta",
"Drug and cell typespecific regulation of genes with different classes of estrogen receptor b-selective agonists",
"Estrogen induces rapid translocation of estrogen receptor beta, but no estrogen receptor alpha, to the neuronal plasma membrane",
"Extranuclear estrogen receptor beta immunoreactivity is on doublecortincontaining cells in the adult and neonatal rat dentate gyrus",
"Multiple pathways transmit neuroprotective effects of gonadal steroids"
] | [
"Neurology",
"J Neuroimmune Pharmacol",
"Curr HIV/AIDS Rep",
"Neurol Clin",
"Science",
"Cell Death Differ",
"J Neuroimmune Pharmacol",
"Futur HIV Ther",
"Neurotox Res",
"Infect Disord Drug Targets",
"AIDS Res Hum Retroviruses",
"HIV tat and neurotoxicity. Microbes Infect",
"Am J Pathol",
"J Neurosci",
"Exp Neurol",
"Exp Neurol",
"Brain Res",
"Temporal relationships between HIV-1 Tat-induced neuronal degeneration, OX-42 immunoreactivity, reactive astrocytosis, and protein oxidation in the rat striatum",
"J Pharmacol Exp Ther",
"J Neurochem",
"Pharmacol Biochem Behav",
"Brain Res",
"J Infect Dis",
"J Neurovirol",
"Int Rev Psychiatry",
"Environ Health Perspect",
"N Engl J Med",
"J Agric Food Chem",
"Toxicol Sci",
"Menopause",
"Psychopharm (Berl)",
"Brain Res",
"BMC Neurosci",
"Fertil Steril",
"Neurotoxicol Teratol",
"Exp Biol Med (Maywood)",
"Free Radic Biol Med",
"Neurosci Lett",
"Neurotoxicology",
"Horm Behav",
"JAMA",
"Lancet Oncol",
"J Nutr",
"Nutr Cancer",
"Front Neuroendocrin",
"Mol Nutr Food Res",
"J Nutr Biochem",
"Fertil Steril",
"Am J Clin Nutr",
"Synapse",
"Top HIV Med",
"AIDS",
"J Comp Neurol",
"J Comp Neurol",
"Endocrin",
"Biochem. Biophys. Res. Commun",
"J Biol Chem",
"Brain Res",
"Neurosci",
"J Cereb Blood Flow Metab",
"Biochem J",
"Cell Death Differ",
"Biochim. Biophys. Acta",
"Proc Natl Acad Sci",
"Mol Biol Cell",
"PLoS One",
"Neurosci",
"Brain Res",
"Endocrine",
"Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes"
] | [
"\nFigure 1 .\n1Soy isoflavones genistein and daidzein protect primary cortical cultures from Tat neurotoxicity. Primary cortical neurons were exposed to estrogen (0.1, 2.0 and 10 nM), or isoflavones (0.05, 0.2 and 1 mM) 24 hr prior to the start of Tat 1-86 B (50 nM) treatment. Cell viability was assessed by Live/Dead assay. Live/Dead ratios were determined after 48 hr (A-C) or 5 days (D-F) of the continuous exposure to Tat or equal volume of vehicle in cell culture groups that were treated or not treated with estrogen, GEN, or DAI. Data represents mean values 6 SEM, n of cultures analyzed = 7-12 per each group. *-indicates significant (p#0.05) protective effects of the selected compounds against Tat neurotoxicity (cell viability decrease) in cortical cell cultures. Repeated (2-3) trials using cell culture preparations from different litters were carried out to ensure the reproducibility of the results. doi:10.1371/journal.pone.0037540.g001",
"\nFigure 2 .\n2Genistein and daidzein attenuate Tat-induced caspase activation in primary cortical cultures. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of activated apoptotic proteins A. Caspase 9 (4 hr of Tat exposure) and B. caspase 3 (4 hr Tat exposure) was assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tat-treated cultures. doi:10.1371/journal.pone.0037540.g002",
"\nFigure 3 .\n3Isoflavones prevent Tat-induced expression of Bcl-2 and Bax protein levels. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of apoptotic proteins A. Bcl-2 (16 hr of Tat exposure) and B. Bax (4 hr Tat exposure) were assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tattreated cultures. doi:10.1371/journal.pone.0037540.g003",
"\nFigure 4 .\n4Estrogen receptors mediate isoflavone effects on Caspase 3 and Bax expression following Tat exposure. A. GEN or DAI effects on caspase 3 expression were blocked in the presence of ER antagonist, ICI 182,780. B. GEN or DAI effects on Tat-induced expression of Bax were reversed by ICI 182,780, suggesting that estrogenic actions on caspase 3 and Bax are ER mediated. Data represents mean values 6 SEM, *p#0.05 vs. GEN/DAI+T treated cultures. doi:10.1371/journal.pone.0037540.g004",
"\nFigure 5 .\n5ER subtype specific effects against caspase activity and Bax expression. Similar to 17b-estradiol (A), GEN (B) and DAI (C) effects against Tat-induced caspase 3 activity were maintained in the presence of specific antagonists for ERa (MPP) and ERb (PHTPP). ER subtype antagonists reveal that ERb signaling was preferential for GEN (E) effects on Bax. DAI (F) effects on Bax were blocked in the presence of both ER subtype antagonists. Data represents mean values 6 SEM, *p#0.05 as compared to GEN/DAI+Tat treated cultures. doi:10.1371/journal.pone.0037540.g005",
"\n\nConceived and designed the experiments: SMA CFM RMB. Performed the experiments: SMA MVA MYA. Analyzed the data: SMA MYA. Wrote the paper: SMA CFM RMB. Critical appraisal and approval of final manuscript: SMA MVA MYA CFM RMB."
] | [
"Soy isoflavones genistein and daidzein protect primary cortical cultures from Tat neurotoxicity. Primary cortical neurons were exposed to estrogen (0.1, 2.0 and 10 nM), or isoflavones (0.05, 0.2 and 1 mM) 24 hr prior to the start of Tat 1-86 B (50 nM) treatment. Cell viability was assessed by Live/Dead assay. Live/Dead ratios were determined after 48 hr (A-C) or 5 days (D-F) of the continuous exposure to Tat or equal volume of vehicle in cell culture groups that were treated or not treated with estrogen, GEN, or DAI. Data represents mean values 6 SEM, n of cultures analyzed = 7-12 per each group. *-indicates significant (p#0.05) protective effects of the selected compounds against Tat neurotoxicity (cell viability decrease) in cortical cell cultures. Repeated (2-3) trials using cell culture preparations from different litters were carried out to ensure the reproducibility of the results. doi:10.1371/journal.pone.0037540.g001",
"Genistein and daidzein attenuate Tat-induced caspase activation in primary cortical cultures. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of activated apoptotic proteins A. Caspase 9 (4 hr of Tat exposure) and B. caspase 3 (4 hr Tat exposure) was assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tat-treated cultures. doi:10.1371/journal.pone.0037540.g002",
"Isoflavones prevent Tat-induced expression of Bcl-2 and Bax protein levels. Cortical cultures were treated with 1 mM GEN or DAI 24 hr prior to Tat exposure. Expression of apoptotic proteins A. Bcl-2 (16 hr of Tat exposure) and B. Bax (4 hr Tat exposure) were assessed by ELISA experiments. Data represents mean values 6 SEM, with experiments performed in triplicate, *p#0.05 as compared to Tattreated cultures. doi:10.1371/journal.pone.0037540.g003",
"Estrogen receptors mediate isoflavone effects on Caspase 3 and Bax expression following Tat exposure. A. GEN or DAI effects on caspase 3 expression were blocked in the presence of ER antagonist, ICI 182,780. B. GEN or DAI effects on Tat-induced expression of Bax were reversed by ICI 182,780, suggesting that estrogenic actions on caspase 3 and Bax are ER mediated. Data represents mean values 6 SEM, *p#0.05 vs. GEN/DAI+T treated cultures. doi:10.1371/journal.pone.0037540.g004",
"ER subtype specific effects against caspase activity and Bax expression. Similar to 17b-estradiol (A), GEN (B) and DAI (C) effects against Tat-induced caspase 3 activity were maintained in the presence of specific antagonists for ERa (MPP) and ERb (PHTPP). ER subtype antagonists reveal that ERb signaling was preferential for GEN (E) effects on Bax. DAI (F) effects on Bax were blocked in the presence of both ER subtype antagonists. Data represents mean values 6 SEM, *p#0.05 as compared to GEN/DAI+Tat treated cultures. doi:10.1371/journal.pone.0037540.g005",
"Conceived and designed the experiments: SMA CFM RMB. Performed the experiments: SMA MVA MYA. Analyzed the data: SMA MYA. Wrote the paper: SMA CFM RMB. Critical appraisal and approval of final manuscript: SMA MVA MYA CFM RMB."
] | [
"Figure 1",
"(Figure 1",
"Figure 2A",
"Figure 2B",
"Figure 3A",
"Figure 3B",
"Figure 3B",
"Figure 4A",
"Figure 4B",
"Figure 4A",
"Figure 4B",
"Figure 4B",
"Figure 5A",
"Figure 5B",
"Figure 5D",
"Figure 5E, F)"
] | [] | [
"HIV-1 infection of the central nervous system (CNS) causes several neurological disorders, known as HIV-associated neurocognitive disorders (HAND) [1]. Although the incidence of severe dementia has significantly decreased since the advent of combination anti-retroviral therapy (cART), cognitive and motor impairments persist in up to 50% of HIV-1 positive patients due to longer life expectancy, the lack of patient compliance with cART therapy and the low penetrability of cART into the CNS [2]. The continued prevalence of neurological dysfunction suggests cART fails to provide complete protection from the development of HAND [1,3,4] and there currently are no pharmacotherapies targeted to HAND.",
"HIV-1 enters the brain early after infection and, although, HIV-1 does not infect neurons, neuronal dysfunction is key in HIV pathogenesis [5][6][7]. The early viral proteins such as Tat are continually produced despite the presence of cART [7][8][9][10]. Accordingly, Tat is rapidly taken up by neuronal cells and has been shown to have direct toxic effects on neurons though various mechanisms. Studies have shown Tat to mediate excitotoxicity via NMDA receptors [11][12][13], synaptic damage and dendritic pruning [14], induce apoptotic cascades [15,16], calcium dysregulation [17], oxidative stress [18], and dopaminergic system dysfunction [19,20]. Tat exposure has been shown to negatively affect cognitive processes in animal models [21,22]. The observations that the viral regulatory protein Tat is actively secreted by infected cells, and that Tat mRNA is elevated in patients with HIV-1 suggest a possible role of extracellular Tat in the progression of HIV-1-induced neurodegeneration [23][24][25].",
"Phytoestrogens, such as the soy isoflavones genistein and daidzein, mimic the neuroprotective actions and functions of estrogen in the brain, as they bind to the estrogen receptor (ER) and affect estrogen-mediated processes [26][27][28][29]. Several studies have found that soy isoflavones can improve cognitive functions in both humans and rats, but underlying mechanisms remain unknown [30][31][32][33][34][35]. Additional studies have shown that isoflavones have neuroprotective effects against various neurodegenerative insults. Genistein and daidzein have demonstrated neuroprotective efficacy against glutamate excitotoxicity and Ab [25][26][27][28][29][30][31][32][33][34][35] induced loss of cell viability, oxidative stress and initiation of apoptosis in hippocampal neurons [36,37]. As the dopaminergic system is sensitive to HIV in the brain, isoflavones, similar to estradiol, may interact with dopamine to preserve motor and cognitive functions [35,[38][39][40].",
"Estrogen therapy is met with resistance due to its association with reproductive and breast cancers [41,42]. However, dietary consumption and supplementation with soy isoflavones is widespread. Consuming a typical Western diet yields low nanomolar concentrations of circulating isoflavones [43,44]. In people consuming modest amount of soy products yielding ,50 mg/day of total isoflavones, plasma levels of 50-800 ng/ml have been achieved for daidzein and genistein [43], which is comparable to concentrations observed in a traditional Japanese diet [44]. However, consuming a western vegetarian diet and taking supplements has been shown to achieve increased isoflavone consumption comparable to or higher than Asian levels [44][45][46]. Furthermore, human studies that have reported improved cognitive function with soy isoflavone consumption have used ,60-100 mg/day doses of isoflavones [31,34]. The cognitive improvements seen with high doses of soy in vivo were not associated with abnormalities in reproductive health of humans, including men [31,[47][48][49]. This broadens the use and benefits of these estrogenic compounds to not only women but also men.",
"Soy isoflavones preferentially binding to ERb is of significant consideration in neuroprotection as this ER subtype is highly expressed in the brain compared to ERa, which due to its high expression in the reproductive tissue, has been associated with the proliferative effects of estrogen. Elucidating whether isoflavone protection is mediated by ER selectivity is a central focus in developing neuroprotective strategies. In the current experiments, we investigated whether treatment with soy isoflavones, genistein or daidzein, could attenuate HIV-1 Tat-induced mitochondria associated apoptosis in cortical cell cultures. Further, we explored whether isoflavone neuroprotection against HIV-1 viral protein Tat-induced neural toxicity involves ER-mediated attenuation of apoptotic signaling. We demonstrated that isoflavones maintained neuronal cell viability in the presence of prolonged Tat exposure. We also observed that isoflavones prevented Tat-induced upregulation of mitochondrial apoptotic cascade regulators. Moreover, we determined that the protective actions of isoflavones were mediated by estrogen receptors.",
"We have previously shown that 17b-estradiol attenuated Tatinduced cell death [50]. As shown in Figure 1, the cell viability decrease (<25% of control) induced by prolonged (up to 5 days) exposure to the toxic dose of Tat 1-86 B was abrogated by 0.1-10 nM of 17b-estradiol. Similar alleviation of Tat-induced neuronal cell death was observed when isoflavones GEN or DAI were used at doses 0.05, 0.2, and 1 mM (Figure 1). Our levels of 50 nM to 1.0 mM of genistein and daidzein are within the range of observed plasma levels of isoflavones following consumption of soy products which reflect ,200 nM-3 mM. Results indicate that physiologically relevant concentrations of isoflavones are able to effectively protect cortical neurons against Tat toxicity in vitro.",
"In the present experiments, we evaluated if the protective potential of GEN and DAI against Tat involves downregulation of caspase activity. Figure 2A shows significant caspase 9 activation following only 4 hr of Tat exposure (p#0.05). Preincubation with 10 nM 17b-estradiol or 1 mM of isoflavones (GEN or DAI) prevented the increase in Tat-induced caspase 9 activation. Cultures pretreated with GEN or DAI displayed caspase 9 activity similar to that of estradiol treated cultures (p#0.05). Moreover, analysis revealed that caspase 9 activity was not statistically different between the 17b-estradiol, GEN and DAI pretreated cultures.",
"In regard to effector caspase 3 activity, a similar effect was observed with phytoestrogen pretreatment prior to Tat exposure. These results demonstrated a significant increase in caspase 3 activation in cortical cultures following 4 hr exposure to Tat (p#0.05). The current experiments show that the addition of GEN or DAI prior to incubation with 50 nM Tat significantly attenuated the upregulation of active caspase 3 expression (p#0.05, Figure 2B). The level of activation of caspase 3 in GEN and DAI pretreatment groups was not significantly different from that of vehicle-treated controls; furthermore, these levels were very similar to that of the 17b-estradiol treated cultures. These results indicate that isoflavones GEN and DAI downregulate Tat-induced caspase activation to a level comparable to that of 17b-estradiol, suggesting that isoflavones and estradiol may share a common neuroprotective mechanism.",
"The protection of cortical cell cultures with 17b-estradiol against Tat toxicity is associated with regulating the effects of apoptotic proteins linked to the mitochondrial apoptotic pathway [50]. Therefore, we compared effects of neuroprotective concentrations of 17b-estradiol and isoflavones that completely eliminate Tat-induced death of cortical cells on the alterations in Bcl-2 and Bax protein levels. Results of the Bcl-2 ELISA presented in Figure 3A demonstrate that, similar to estradiol (10 nM), neuroprotective doses of GEN and DAI (1 mM) added to the cell culture medium 24 hr in advance of 50 nM Tat significantly (p#0.05) attenuated the increase of Bcl-2 expression; an effect shown to occur within the first 16-24 hr of Tat exposure in cortical cell cultures [50].",
"We also evaluated the efficacy of isoflavones against Tatinduced Bax expression. Results of the Bax ELISA ( Figure 3B) demonstrate that pretreatment with 1 mM GEN or DAI, or pretreatment with 10 nM estrogen, significantly (p#0.05) blocks the induction of Bax expression in Tat-exposed cortical cells ( Figure 3B). Effects of all the compounds on Tat-induced changes in Bcl-2/Bax protein expression were specific, since neither the exposure to 10 nM 17b-estradiol, nor the exposure to 1 mM isoflavones caused statistically significant changes in Bcl-2 or Bax immunoreactivities compared to non-treated control cell cultures.",
"Plant isoflavones, such as GEN and DAI, are similar to 17bestradiol in chemical structure, which allows them to interact with estrogen receptors (ER). Neuroprotective effects of 17b-estradiol against Tat-induced apoptosis are mediated by two subtypes of estrogen receptors, ERa and ERb. ER expression in primary cortical neurons used in the current experiments demonstrates the presence of both ER a and b immunoreactivity in our cultures (data not shown). In the present experiments, we sought to determine if the protective actions of isoflavones were ERmediated. The ER antagonist, ICI 182,780 (100 nM), was added to cultures 1 hr prior to incubation with 17b-estradiol, GEN or DAI. Following 24 hr pretreatment, 50 nM Tat was added to cultures and its effects on the active caspase 3 ( Figure 4A) and Bax ( Figure 4B) protein levels have been analyzed.",
"As shown in Figure 4A, ICI prevented the downregulation of active caspase 3 by 17b-estradiol (10 nM), GEN or DAI (1 mM) pretreatment. Consistent with our previous study, ICI 182,780 averted estrogen-mediated activation of caspase 3 by Tat treatment of cortical cultures. Activated caspase 3 immunoreactivity in ICI+GEN+Tat-treated cultures was significantly higher than GEN+Tat treated cortical cultures (p#0.05) and protein levels of activated caspase 3 were similar to that in Tat-only treated cultures. Similarly, the DAI effects on caspase 3 were sensitive to ICI inhibition of ERs. There was a significant increase in active caspase 3 expression with the addition of ICI compared to DAI+Tat treated cultures (p#0.05). Results of the experiments indicate that GEN and DAI, similar to 17b-estradiol, inhibit the Tat-induced caspase 3 activation via an ER-mediated mechanism, as the addition of ICI 182,780 prior to estradiol returned caspase 3 activation levels to that of Tat only treated cultures. Moreover, caspase 3 activity did not differ statistically from that of Tat only treated cultures.",
"The results in Figure 4B show that GEN and DAI effects on Tatinduced Bax expression in cortical cultures are ICI-sensitive. A significant 25% increase in Bax expression was seen with ICI treatment prior to incubation with GEN or DAI (p#0.05) ( Figure 4B). Although, GEN and DAI possess estrogen-like activity, their affinity to ERa-or ERb-subtypes is significantly different from 17b-estradiol. Therefore, we used specific antagonists of a or b ERs to evaluate selectivity of the effects of 17b-estradiol, GEN and DAI on Tatinduced changes in activated caspase 3 or Bax protein levels. The ERsubtype specific antagonists MPP (ERa) and PHTPP (ERb) (1 mM) were added to cortical cultures prior to incubation with GEN or DAI and subsequent exposure to 50 nM Tat.",
"Neither anor b-selective ER antagonists were able to completely block the inhibitory effects of 10 nM 17b-estradiol ( Figure 5A), or those of 1 mM GEN or DAI, on Tat-induced caspase 3 activation ( Figure 5B, C). Although the ER subtype selective antagonists diminished the ability of all the compounds to decrease active caspase 3 protein levels in Tat-treated cultures, their effects were not statistically significant. Overall, selective antagonists of ERa-or ERb-subtypes similarly affected the ability of both 17b-estradiol and isoflavones to downregulate Tat-induced caspase 3 activation.",
"The addition of ERa antagonist MPP did not significantly attenuate 17b-estradiol effects on Bax expression ( Figure 5D), but was able to significantly decrease the effect of GEN and DAI. The ERb-specific antagonist PHTPP completely blocked the effect of 17b-estradiol and GEN ( Figure 5E, F). PHTPP also caused a partial, but significant, decrease in the ability of DAI to inhibit the Tat-dependent increase in Bax expression.",
"Neurocognitive deficits associated with HIV infection persist even with effective cART [51]. Targeting ERb function may be a potential therapeutic option since it is highly expressed in the brain, specifically in cortical regions responsible for executive functions significantly affected in HIV-1 associated neurocognitive disorders (HAND) [35,[52][53][54]. In this study, we evaluated whether soy isoflavones, acting as ER selective compounds, were able to mimic the neuroprotective effects of estrogen in HIV-1 Tat 1-86exposed primary neuronal cultures. GEN and DAI represent soy isoflavones with ERa/b binding profiles showing much higher binding selectivity for ERb than 17b-estradiol. Among soy phytoestrogens, GEN exhibits maximum ERa/b binding affinity with approximately 60-fold preference for ERb over ERa. DAI has lower ERa/b binding affinity than GEN with 14-fold selectivity for ERb binding. For comparison with these two soy isoflavones, 17b-estradiol's preference for the ERb binding is 0.78fold [55]. GEN and DAI have very close chemical structures and are known to induce neuroprotective responses but at a much lower magnitude than 17b-estradiol.",
"Studies have demonstrated that oxidative stress and mitochondrial dysfunction coincide with Tat activation of apoptotic cascades [16,38,50,56]. Moreover, we have recently reported that 17b-estradiol attenuated Tat-induced apoptotic signaling in an estrogen-receptor dependent manner [50]. We now demonstrate that the soy isoflavones genistein and daidzein prevent the upregulation of caspase activity in Tat-exposed cultures. In addition, upstream of caspase activation, we show that Tat exposure significantly increased expression of pro-and antiapoptotic proteins Bax and Bcl-2 respectively, which regulate mitochondrial membrane permeability and thus, the release of apoptogenic substances. Our results indicate that treatment with GEN or DAI also markedly reduces the expression of Bax and Bcl-2 in Tat-exposed cortical cultures. A major finding of our study is that both soy isoflavones GEN and DAI exhibited protective effects similar to that exhibited by 17b-estradiol. GEN and DAI increased cell viability and attenuated the upregulation of apoptotic proteins in a manner comparable to that observed in estradiol treated cultures. At low micromolar doses we observed that isoflavones were able to maintain cell survival following prolonged exposure to Tat. These results suggested a similar neuroprotective action of isoflavones and 17b-estradiol involving inhibition of apoptotic pathways. Our experiments also demonstrated that selective ERb agonists induce an anti-apoptotic effect in primary cultures exposed to HIV-1 Tat. Such observations support the findings that isoflavones are protective against oxidative stress-mediated apoptosis in HIV-1 infection.",
"Since isoflavones bind estrogen receptors (ERs), the neuroprotective actions of isoflavones may be produced through activation of the ER. We found that the addition of the ER antagonist ICI 182,780, which blocks both the ERa and ERb subtypes, reversed GEN and DAI downregulation of caspase 3 activity and Bax expression with Tat exposure, suggesting that these effects of GEN and DAI were ER-dependent. The addition of ICI 182,780 had a more robust effect against genistein actions on caspase 3 activity, and sustained caspase levels similar to cultures treated with Tat. Furthermore, an isoflavone effect on Bax expression was also shown to be ER-dependent, as ICI 182,780 blocked genistein and daidzein inhibition of Tat-induced Bax expression. Collectively, our results suggest that genistein and daidzein act as estrogen receptor agonists in primary cortical neurons and activate estrogenic neural defense mechanisms.",
"Another major finding is that isoflavone anti-apoptotic effects are selective relative to estrogen receptor isoform. There are conflicting reports of which ER subtype, ERa or ERb, specifically mediates the protective actions of estrogen [57,58]. We observed in our previous studies with estradiol [50], inhibition of ERs with ICI 182,780 blocked the downregulation of apoptotic proteins in cultures pretreated with isoflavones. Further experiments sought to determine if these receptor-mediated effects were specific to a particular ER-subtype. Cultures in the presence of ERa-specific antagonist MPP or ERb-specific antagonist PHTPP did not show specific attenuation of genistein and daidzein downregulation of caspase 3 activity. Similar to previous results observed with estradiol, both ER subtypes seem to play a role in genistein and daidzein inhibition of Tat activation of caspase 3. However, a more pronounced attenuation of genistein effects on Bax expression was observed in the presence of ERb antagonist PHTPP. Thus, genistein effects on Bax expression may be preferential for ERb-mediated signaling. Our results also demonstrated that the addition of both ER subtype antagonists significantly inhibited daidzein actions on Bax expression, suggesting involvement of both ERa and ERb in daidzein downregulation of Bax. It is possible that daidzein's lower binding affinity for ERs compared to that of genistein and estradiol [55] may explain the absence of the preferential mediation of a specific ER subtype. Another point of consideration is that ERa and ERb may act simultaneously and thus counteract the function of the other receptor subtype [59,60]. Both genistein and daidzein were shown to activate binding to ERb at nanomolar concentrations (30 nM and 350 nM, respectively), which are easily achievable levels in humans consuming soy products or supplements. At the concentrations used in these experiments, it is plausible that both receptor subtypes were activated and as such ER subtype specific effects may be diminished. Caspase 3 has a pivotal role in the apoptotic process. Multiple pro-apoptotic pathways converge on caspase 3 activation in the cell death cascade. Caspase 3 activation may occur through caspase 9 from the mitochondria or from death receptor signaling via caspase 8 as well as through other proapoptotic pathways [61][62][63]. More upstream in the apoptotic cascade, the upregulation of Bax is associated with mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria, leading to caspase activation. The ERb specific effects on Bax may be related to the recent discovery of ERb localization in mitochondria [64], suggesting a direct estrogenic effect on mitochondria function via ERb activation and signaling. The ER-mediated reduction of caspase 3 activity and Bax expression by estradiol and isoflavones suggested that these compounds disrupt apoptotic signaling by downregulating key pro-apoptotic factors in the cell death cascade. As multiple apoptotic pathways converge on mitochondria functioning and caspase 3 activation, Bax and caspase 3 represent potential upstream and downstream receptorsensitive check points for estrogenic compounds to disrupt apoptotic processing in response to neurodegenerative insults.",
"As isoflavones affect the viability of neurons and cognitive function by acting as an estrogenic agonist, they can also utilize differential distribution and regulation of the ER subtypes, ERa and ERb in the brain. Microarray experiments have shown that ERa and ERb regulate different genes [65,66]. Differences in conformation that occur upon ER binding affects the recruitment of coregulatory proteins, and thus produces differential gene regulation in specific cell types. In addition to tissue or region specific localization of ERs, intracellular localization of ERs may contribute to some of their different mechanisms of action. ERb has been localized to a greater extent at extranuclear sites and in the cytoplasm for trafficking to the plasma membrane [64,67,68]. The extranuclear and membrane localization of ERb enables its interaction with intracellular signaling cascades to integrate rapid signaling events and classical transcriptional mechanisms [69,70]. Given the timing of treatment in our studies, both genomic and nongenomic molecular actions may be utilized by isoflavones to confer ERmediated neuroprotection against Tat.",
"Despite the success of cART on peripheral viral suppression, protected viral reservoirs in the brain may allow continued release and exposure to toxic viral proteins [10]. The inability of anti-retroviral therapy to prevent the development of neurocognitive dysfunction indicates the need for adjunctive therapies to address the neurodegenerative and subsequent neurological disturbances associated with HAND. Findings in the present study demonstrate that soy isoflavones offer a similar protective effect as endogenous estradiol via a selective estrogen receptormediated mechanism against HIV-1 Tat-induced cell death. Isoflavones, acting as selective ER agonists targeting the neuroprotective effects associated with estradiol, may represent a safe and viable neuroprotectant along with cART to improve the neurological health of both men and women with HAND.",
"All of the experimental procedures using animals were performed in accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The relevant animal use protocols were approved by the University of South Carolina Animal Care and Use Committee under the auspices of Animal Assurance Number A3049-01.",
"Primary cultured cortical neurons were prepared from 18-dayold Sprague-Dawley rat fetuses [50]. Rat cortices were dissected and incubated for 15 min in a solution of 2 mg/mL trypsin in Ca 2+ -and Mg 2+ -free Hanks' balanced salt solution (HBSS) buffered with 10 mM HEPES (Invitrogen, Carlsbad, CA). The tissue was then exposed for 2 min to soybean trypsin inhibitor (1 mg/mL, in HBSS) and then rinsed 3 times in HBSS. Cells were dissociated by trituration and distributed to poly-L-lysine coated culture plates with wells containing DMEM/F12 medium (Invitrogen) supplemented with 100 mL/L fetal bovine serum (Sigma Chemicals, St. Louis, MO). After a 24-hr period, the DMEM/F12 medium was replaced with 2% v/v B-27 Neurobasal medium supplemented with 2 mM GlutaMAX and 0.5% w/v D-(+) glucose (Invitrogen). Two-thirds of the neurobasal medium was replaced with fresh medium of the same composition once a week. Cultures were used for experiments after 12 days in culture in serum-free medium and were .95% neuronal as observed by anti-MAP-2 immunostaining.",
"Recombinant Tat 1-86 (LAI/Bru strain of HIV-1 clade B, GenBank accession no. K02013, Diatheva, Fano, Italy) was added to cell culture medium. Groups of cultures in 24-well plates were exposed to 50 nM Tat. Cell cultures were treated with 17bestradiol (0.1 nM, 2 nM or10 nM, Sigma) or soy isoflavones (0.05 mM, 0.2 mM or1 mM, genistein (GEN) or daidzein (DAI), Indofine Chemical) for 24 hr prior to Tat exposure and remained present in medium throughout experiments. 17b-estradiol was dissolved in sterile water and diluted in D-PBS. Isoflavones were dissolved in DMSO and diluted in D-PBS. To assess neurotoxicity, the cultures were exposed to Tat for 4, 16, or 24 hr before harvesting. After treatment, medium was removed, cells were washed and lysates collected for ELISA experiments. Cells were also treated with the estrogen receptor antagonist ICI 182,780, the ERa specific antagonist, MPP dihydrocloride, or the ERb specific antagonist PHTPP (100 nM, Tocris Cookson Inc, Ellisville, MO) 1 hr before estradiol or isoflavone treatment to determine if the effects against Tat toxicity were receptor mediated.",
"Neuronal survival was determined using a Live/Dead viability/ cytotoxicity kit (Molecular Probes, Eugene, OR) in rat fetal cortical cell cultures prepared in 96-well plates. In accordance with the manufacturer's protocol, neurons were exposed to cellpermeate calcein AM (2 mM), which is hydrolyzed by intracellular esterases, and to ethidium homodimer-1 (4 mM), which binds to nucleic acids. The cleavage product of calcein AM produces a green fluorescence (F 530 nm ) when exposed to 494-nm light and is used to identify live cells. Bound ethidium homodimer-1 produces a red fluorescence (F 645 nm ) when exposed to 528-nm light, allowing the identification of dead cells. Fluorescence was measured using a Bio-Tek Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Each individual F 530 nm and F 645 nm value on a plate was corrected for background fluorescence (readings obtained from cell cultures (wells) that were not exposed to calcein AM and ethidium bromide) by the microplate reader KC4 software package (Bio-Tek Instruments, Inc., Winooski, VT). For each individual cell culture (well) on a plate, ratios between corrected green and red fluorescence (F 530 nm /F 645 nm , Live/Dead ratios) were calculated. All individual relative numbers of live and dead cells were expressed in terms of percentages of average maximum Live/Dead ratio determined for the set of non-treated control cell cultures (8-16 wells) from the same plate: (F 530 nm /F 645 nm ) well n /(F 530 nm /F 645 nm ) average max 6 100%.",
"Expression of apoptotic signaling proteins in cell lysates was determined by ELISA [16,50]. Cell lysates were prepared from cultures grown in 24-well plates. At the time of harvesting, medium was removed and cells were washed 3 times with Dulbecco phosphate-buffered saline, D-PBS, (8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 0.137 M NaCl and 2.7 mM KCL at pH 7.4) and lysed with CellLytic TM -M mammalian cell lysis buffer (Sigma Chemicals) containing protease inhibitors (protease inhibitors cocktail, Sigma Chemicals). All samples in a group (6 culture wells) were pooled together and protein concentration was determined by the BCA method (Pierce, Rockford, Ill.). Each well of Costar 96-well ELISA plates (Corning Inc, PA) was coated overnight at 4uC using 100 mL of 20 mM carbonate coating buffer, pH 9.6. Cortical cell lysate samples were diluted 1:10 with D-PBS and 20 mg of each sample were added to the plate wells. After overnight incubation at 4uC, plates were rinsed 5 times with PBST (0.05% Tween 20 in PBS, pH 7.4) and blocked with 1% BSA in PBS for 2 hr at room temperature. After blocking, plates were washed again, as described above, and primary anti-Bax, anti-Bcl-2, anti-active Caspase 9 and anti-active Caspase 3 antibodies (all primary antibodies, Abcam, Cambridge, MA) diluted 1:5000 or 1:7500 (caspase 3) in 0.1% BSA-PBST were added to each well except for blanks and no-primary antibody control wells. Plates were kept overnight at 4uC. When the incubation with primary antibodies was completed, plates were again washed 5 times with PBST and secondary antibodies [goat anti-rabbit alkaline phosphatase conjugated, Sigma] diluted 1:2000 in 0.1% BSA-PBST were added to each well, except for blank and no-secondary antibody control wells. After 2 hr of incubation, the secondary antibody solution was removed, plates were washed 5 times with PBST and 100 mL of BluePhos phosphatase substrate mixture (KPL Research, Gaithersburg, MD) was added to the plate wells. After 30 min of incubation, the absorbance at 650 nm was determined using a Bio-Tek Synergy HT microplate reader. Multiple readings were taken within a 1-hr time period.",
"Statistical comparisons were made using one-way ANOVA and Tukey's multiple comparison tests were used to determine specific treatment effects. Significant differences were set at p#0.05. Data represents mean values 6 standard error of the mean (SEM). "
] | [] | [
"Introduction",
"Results",
"Physiological Doses of Genistein and Daidzein Prevent Cell Death Following Tat 1-86 Exposure",
"Genistein and Daidzein Attenuate Tat-induced Caspase Activation in Primary Cortical Cell Cultures",
"Genistein and Daidzein Sustain Levels of Mitochondrial Proteins Bax and Bcl-2 Expression Following Tat Exposure",
"Estrogen Receptor Antagonists Block Anti-apoptotic Actions of Soy Isoflavones",
"Discussion",
"Materials and Methods",
"Ethics Statement",
"Primary Neuronal Cell Culture",
"Tat 1-86 Exposure and Experimental Treatment of Cultures",
"Cell Viability Assay",
"Detection of Apoptotic Proteins (ELISA)",
"Statistical Analysis",
"Author Contributions",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 ."
] | [] | [] | [
"Soy Isoflavones Genistein and Daidzein Exert Anti- Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat 1-86 Exposure",
"Soy Isoflavones Genistein and Daidzein Exert Anti- Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat 1-86 Exposure"
] | [] |
497,917 | 2022-03-31T19:37:28Z | CCBY | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0129965&type=printable | GOLD | c2a7e20d8ca88a91d2ace11cdc238eee1de77fa0 | null | null | null | null | 10.1371/journal.pone.0129965 | 2135627065 | 26090800 | 4474976 |
Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin
June 19, 2015 Published: June 19, 2015
Adelma Escobar-Ramirez
Anne-Sophie Vercoutter-Edouart
Marlène Mortuaire
Isabelle Huvent
Stephan Hardivillé
Esthelle Hoedt ¤bTony Lefebvre
Annick Pierce *[email protected]
¤a Current address: Department of Biological Chemistry
UMR 8576
Unité de Glycobiologie Structurale et Fonctionnelle
CNRS
Université des Sciences et Technologies de Lille
CNRS FRABio
FR3688Villeneuve d'AscqFrance
School of Medicine
¤b Current address: Department of Biochemistry and Molecular Pharmacology
Skirball Institute of Biomolecular Medicine
Johns Hopkins University
Baltimore, New YorkMaryland, New YorkUnited States of America, United States of America
Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin
June 19, 2015 Published: June 19, 201510.1371/journal.pone.0129965Received: October 14, 2014 Accepted: May 14, 2015RESEARCH ARTICLE 1 / 21 OPEN ACCESS Citation: Escobar-Ramirez A, Vercoutter-Edouart A-S, Mortuaire M, Huvent I, Hardivillé S, Hoedt E, et al. (2015) Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin. PLoS ONE 10(6): e0129965. Academic Editor: Sumitra Deb, Virginia Commonwealth University, UNITED STATES Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This investigation was supported in part by the Centre National de la Recherche Scientifique, the Unité Mixte de Recherche 8576, the Institut Fédératif de Recherche n°147, the Université des Sciences et Technologies de Lille I, the Comité du Nord de la Ligue Nationale contre le Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.
Introduction
Lactoferrins exist as different variants due to gene polymorphisms, post-transcriptional and post-translational modifications [1]. The two main isoforms are secreted lactoferrin (Lf) and its nucleocytoplasmic counterpart, delta-lactoferrin (ΔLf) [2,3,4]. Their expression is downregulated or silenced in cancer cells [3,5]. In breast cancers, significantly lower levels of Lf and/or ΔLf correlated with more advanced disease and an unfavorable prognosis [5,6]. This downregulation is mainly due to genetic and epigenetic modifications that have been found on the Lf gene in some forms of cancer [7,8]. ΔLf mRNAs derive from the transcription of the Lf gene at the alternative P2 promoter leading, after translation, to a 73 kDa intracellular protein [4]. Although its subcellular distribution is mainly cytoplasmic, confocal microscopy analyses have clearly shown that ΔLf targets the nucleus [4]. Thus, we showed that ΔLf possesses a functional bipartite NLS motif in the C-terminal lobe [9]. ΔLf is capable of binding DNA but the location of its DNA binding domain is not known. Two regions of Lf in which a strong concentration of positive charges were found could be good candidates [10]. ΔLf exhibits antitumoral activities and we previously showed that overexpression of ΔLf leads to cell cycle arrest at the G1/S transition and apoptosis [11,12]. ΔLf mainly exerts its anti-proliferative and pro-apoptotic activities via its role as a transcription factor. Indeed, ΔLf transactivates different target genes such as Skp1, DcpS, Bax, SelH, GTF2F2 and UBE2E1 [9,[12][13][14]. A genome-wide pathway analysis and our quantitative proteomic analysis showed that the re-introduction of Lf isoforms in cancerous cells modified essential genes and/or signaling networks responsible mainly for cell survival, apoptosis and RNA processing [14,15].
Since ΔLf has a variety of target genes and is involved in the control of cell homeostasis, modifications in its activity or concentration may have profound consequences. Its transcriptional activity is controlled by posttranslational modifications (PTM) among which O-GlcNAcylation is a key link between nutrient sensing and signaling. It notably regulates gene activation due to O-GlcNAc cycling on gene-specific transcription factors and components of the basal transcriptional machinery (reviewed in [16]). The targeting of serine S10 by O-GlcNAcylation negatively regulates ΔLf transcriptional activity whereas phosphorylation increases it [17]. Deglycosylation leads to DNA binding and a basal transactivation level which was markedly enhanced when phosphorylation was present at S10. ΔLf possesses a functional PEST sequence which drives the protein to its proteasomal degradation after polyubiquitination of the K379 and/or K391 lysine residues. ΔLf stability is also under the control of O-GlcNAcylation. Indeed, O-GlcNAcylation at S10 protects ΔLf from polyubiquitination increasing its half-life, whereas phosphorylation favours its proteasomal degradation [17].
Recently, we discovered that ΔLf is also modified by SUMOylation. The small ubiquitinrelated modifier (SUMO) is involved in many aspects of cell function and affects pathways as diverse as DNA repair, cell cycle, transcriptional regulation, RNA processing, and cell signaling [18,19]. At a molecular level, SUMOylation of target proteins alters their protein-protein interactions, localization, stability or/and activity [20]. Many transcription factors are targeted by SUMOylation and in most cases SUMOylation triggers transcriptional repression by recruiting transcriptional co-repressors, such as histone deacetylases [21][22][23]. Four different SUMO isoforms (SUMO-1-4) have been identified in higher eukaryotes, although only SUMO-1-3 seem to be covalently attached to proteins. SUMO-2 and SUMO-3 share 96% identity, and both have approximately 46% identity with SUMO-1. The attachment of SUMO is a multi-step process analogous to that of ubiquitin. Thus, the SUMO pathway is mediated by SUMO-activating enzymes (E1), a unique SUMO-conjugating enzyme (E2) called Ubc9 and SUMO-ligases (E3). SUMOylation is a highly dynamic process which can be reversed by the activity of SUMO-specific isopeptidases (SENPs) [24].
SUMOs are conjugated to lysine residues in a CKXE/D sequence where C is a large hydrophobic amino acid residue and X represents any amino acid [25]. This motif is sufficient by itself to mediate a direct interaction with Ubc9 [25,26]. Extended SUMO consensus motifs such as the negatively charged amino acid-dependent SUMO motif (NDSM) constituted by an acidic patch downstream of the CKXE/D motif, the phosphorylation-dependent SUMO motif (PDSM) that includes a phosphorylation site downstream of the consensus core motif and the hydrophobic cluster SUMOylation motif (HCSM) that contains several hydrophobic residues located N-terminal to the core motif have been described to promote substrate SUMOylation via additional interaction with Ubc9 [27][28][29]. Moreover, several proteins are also modified at other sites and until now it is not known how these non-consensus sites are recognized. However, substrates with a SUMO-interacting motif (SIM) could be SUMOylated within a nonconsensus SUMO motif [30] and, as shown for the Death domain-associated protein 6 Daxx, phosphorylation of SIMs enhances SUMO-1 binding and conjugation [31]. SUMO-1 can be attached either to a single or to multiple lysine residues within a target protein leading either to mono-or multi-SUMOylation respectively, whereas chain formation is attributed to SUMO-2/ 3 [32]. However, [27] identified the human Topoisomerase I as a poly-SUMO-1 target. On the other hand, SUMO-1 may be attached to lysine residues within SUMO-2/3 chains, thereby preventing their elongation and acting therefore as a SUMO chain terminator [32,33]. Recently, mixed SUMO/ubiquitin chains have been reported [34].
Crosstalk between the SUMOylation, ubiquitination, and acetylation pathways is crucial for the regulation of protein activity and/or stability since these modifications may have different, sometimes opposing consequences [35]. Thus, SUMOylation can stabilize proteins by competing with ubiquitin [36,37]. However, heterogeneous SUMO2/3-ubiquitin chains were found on IκBα and PLM (promyelocytic leukemia) protein, contributing to their optimal proteosomal degradation [38]. Switches between SUMOylation and acetylation have also been reported for several proteins. SUMOylation of the myocyte-specific enhancer factor 2A (MEF2A) inhibits its transcriptional activity whereas acetylation increases it [39]. A similar SUMO to acetyl switch has also been described for the hypermethylated in cancer 1 protein (HIC1) [40].
Here we demonstrate that the stability and transcriptional activity of ΔLf are regulated by SUMOylation, which provides a novel regulatory mechanism for controlling ΔLf function. We identified the major SUMO and acetylation acceptor sites and we evaluated the impact of the SUMOylation/ubiquitin and the SUMOylation/acetylation interplays.
Experimental Section
Cell culture, transfection, and reagents HEK-293 cells (ATC CRL-1573) were grown in monolayers and transfected (1 μg of DNA for 1 x 10 6 cells) using DreamFect (OZ Biosciences, Marseille, France) as described [17].The amounts of ΔLf expression vectors were adjusted to maintain ΔLf amounts similar to those found in normal breast epithelial NBEC cells [5,6]. Transfections were done in triplicate (n ! 5). Cell viability was assessed by counting using Trypan blue 0.4% (v/v). To measure the ΔLf turnover rate indirectly, we performed incubations with cycloheximide, a potent inhibitor of de novo protein synthesis [41,42]. Cells were transfected with either ΔLf (WT), the SUMO mutant constructs or null vector (NV) then incubated with fresh medium supplemented by 10 μg/mL cycloheximide (CHX) for 0-150 min 24 h post transfection as described [17]. Inhibition of proteasome was performed by incubating cells with a 10 μM concentration of the proteasomal inhibitor MG132 for 2 h prior to lysis as described [17]. Inhibition of histone deacetylases was performed by incubating cells with Trichostatin A (TSA) at 15 ng/mL (TSA treated cells) or not overnight. Cell culture reagents were from Lonza. Other reagents were from Sigma.
Plasmid preparation
pGL3-S1 Skp1 -Luc [9] and p3xFLAG-CMV10-ΔLf (WT) [17] were constructed as described. p3xFLAG-CMV10 (Sigma, St Louis, MO, USA) was used as a null vector (NV). The hemagglutinin A-Ubiquitin (HA-Ub) expression vector was a gift from Dr. C. Couturier (UMR-CNRS 8161, IBL, Lille, France). The psG5-His-SUMO-1 (His-SUMO-1), the pcDNA3.1-His-SUMO-2/3 (SUMO2/3) and the pcDNA3-SENP2-SV5 (SENP2) expression vectors were kind gifts from Dr. D. Leprince (UMR-CNRS 8161, IBL, Lille, France). All plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen Germantown, MD) according to the manufacturer's specifications.
Site-directed mutagenesis
Mutants were generated using the QuikChange Site-directed Mutagenesis Kit (Stratagene, Garden Grove, CA) according to the manufacturer's instructions with p3xFLAG-CMV10-ΔLf as template and primer pairs listed in S1 Table. The constructs in which several sites were mutated were done sequentially. Following sequence verification, positive clones were used directly in transfection.
Ubc9 knockdown
HEK-293 cells (2 x 10 6 cells in 100-mm dish) were transfected with RNAiMax (Life Technologies), according to the manufacturer's instructions, using 5 nM of siRNAs targeting Ubc9 (Hs_UBE2I_8 FlexiTube siRNA, Qiagen) or a scrambled control sequence (siCtrl) (Qiagen). Cells were harvested 48 h post-transfection and lysed. Cell extracts were assayed for Ubc9 content and SUMOylation levels.
Reporter gene assays
Reporter gene assays were performed using the pGL3-S1 Skp1 -Luc reporter vector containing a single ΔLfRE and the ΔLf-expression vector (WT), different ΔLf SUMO mutant constructs or a null vector (NV). HEK-293 cells were synchronized overnight in medium containing 1% FCS before being transfected (250 ng of DNA for 2 x 10 5 cells: 50 ng of reporter vector and 200 ng of ΔLf, SUMO mutants or null vector) using DreamFect (OZ Biosciences, Marseille, France) as described in [17]. Reporter gene assays were also performed in the presence of psG5-His-SUMO-1 (200 ng of DNA) or either in the presence of pcDNA3-SENP2-SV5 expression vectors (200 ng of DNA) or TSA (15 ng/mL, overnight) with their respective controls. Reporter gene assays on Ubc9 knockdown cells were performed in two steps. Cells were siUbc9/siCtrl (5 nM) transfected in serum-free medium which was supplemented 4 h post transfection with 1% FCS. Twenty hours later cells were transfected with WT or mutant constructs and the reporter gene. Cell lysates were assayed using a luciferase assay kit (Promega) in a Tristar multimode microplate reader LB 941 (Berthold Technologies, Bad Wildbab, Germany). Basal luciferase expression was assayed using a null vector and was determined for each condition. Relative luciferase activities were normalized to basal luciferase expression and ΔLf content as in [12] and expressed as a percentage; 100% corresponds to the relative luciferase activity of WT. Each experiment represents at least three sets of independent triplicates.
Western blotting and immunodetection
Proteins were extracted from frozen cell pellets in RIPA buffer as described [9]. In order to inhibit de-SUMOylation of proteins, N-Ethylmaleimide (NEM) was added at 20 mM to lysis, Western blot (WB) and immunoprecipitation (IP) buffers. For direct immunoblotting, samples mixed with 4x Laemmli buffer were boiled for 5 min. Otherwise 10 μg of protein from each sample or immunocomplexes were submitted to 6% SDS-PAGE for IP, 7.5% SDS-PAGE for input and 12.5% SDS-PAGE for Ubc9 Western blot prior to immunoblotting. For immunoprecipitation experiments, 1 or 1.5 mg of total protein were preabsorbed with 20 μL protein G Sepharose 4 Fast Flow (GE Healthcare). Anti-3XFLAG M2 (1/500), anti-acetyllysine (1/1000) or anti-SUMO-1 (1/100) antibodies were mixed with 40 μL Protein G Sepharose beads for 1 h prior to an overnight incubation with the preabsorbed lysate supernatant at 4°C. The beads were then washed five times with lysis buffer (4 washings with RIPA, 1 washing with RIPA/ NaCl 5M: 9/1, v/v) and finally 1 washing in NET-2 (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Triton X-100) buffer. Proteins bound to the beads were eluted with 4X Laemmli buffer and analyzed by immunoblotting as above. Blots were first blocked in 5% non-fat milk for 1 h at room temperature prior being probed with primary antibodies (anti-3XFLAG M2, 1/2000; HA.11, 1/1000; anti-Ubc9, 1/1000; anti-His, 1/1000; anti-SUMO-1, 1/1000; anti-SUMO-2/3, 1/ 500; anti-acetyllysine, 1/1000; anti-GAPDH, 1/3000) overnight at 4°C and then probed with secondary anti-IgG antibodies conjugated to horseradish peroxidase (1/10000) for 1 h at room temperature before detection by chemiluminescence (ECL Advance or ECL Select, GE Healthcare Life Sciences). Each result in which immunoblots are presented corresponds to one representative experiment among at least three.
Antibodies against the 3xFLAG epitope (mouse monoclonal anti-FLAG M2 antibody, Sigma), HA epitope (mouse monoclonal HA.11 antibody 16B12, Covance Research Products), 6XHis epitope (mouse monoclonal anti-6XHis P5A11 antibody for WB, Biolegend; mouse monoclonal anti-His AD1.10 antibody for IP, Santa Cruz Biotechnology), SUMO-1 (rabbit monoclonal anti-SUMO-1, Millipore), SUMO-2/3 (rabbit polyclonal anti-SUMO-2/3, Millipore), Ubc9 (rabbit monoclonal antibody anti-Ubc9, Cell Signaling), acetyllysine (rabbit polyclonal anti-acetyllysine, ABCAM), GAPDH (rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody, Santa Cruz Biotechnologies) were used for immunoprecipitation and/or immunoblotting. Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare Life Sciences. All the antibodies were used according to the manufacturer's instructions.
Densitometric and statistical analyses
The densitometric analysis was performed using the Quantity One v4.1 software (Bio-Rad, Hercules, CA) or ImageJ and statistical analyses were performed with PRISM 5 software (Graphpad, USA). M2 densitometric values were normalized to GAPDH and expressed as D M2 /D GAPDH . Means were statistically analysed using the t-test or ANOVA and differences assessed at p<0.05 ( Ã ) or p<0.01 ( ÃÃ ). SUMO-1 and acetyllysine densitometric values were expressed as D Ac /D SUMO with the WT ratio in the siCtrl condition arbitrarily set as 100%.
Results
ΔLf possesses putative SUMO and acetylation sites and a putative SIMr motif
In silico analysis of the ΔLf sequence with SUMOsp (http://sumosp.biocuckoo.org/) and SUMOplot (htpp://www.abgent.com/tools/sumoplot/) softwares revealed four lysine residues to be putative SUMO acceptors: K13 and K361 that are in the canonical CKXE/D motifs and K308 and K391 in non-canonical SUMO sequences ( Table 1). The K13 consensus motif is of the PDSM-like type and the K308 motif of the NDSM-like type. K13 is within the first putative DBD and next to S10, the main O-GlcNAcylation/Phosphorylation site we previously demonstrated to control ΔLf transcriptional activity and stability ( Fig 1A) [17]. Interestingly, K13 is The single-letter amino acid code is used. c The numbering of the amino acid residues corresponds to human ΔLf. ΔLf is modified by SUMOylation. A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by posttranslational modifications are shown, S10 as the main O-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ! 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ! 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ! 3).
doi:10.1371/journal.pone.0129965.g001 also a putative acetylation site predicted using PAIL (prediction of acetylation on internal lysine [43] (http://bdmpail.biocuckoo.org/prediction.php). The other putative sites are concentrated in the central part of the protein, before the second DBD for K308, between this DBD and the PEST sequence for K361, and within the PEST sequence for K391 ( Fig 1A).
In the case of a SUMO consensus motif, the target lysine is directly recognized by the conjugating enzyme Ubc9 whereas in the case of a non-consensus sequence, the recruitment of SUMO-loaded Ubc9 is realized via interactions with a SIM motif, which increases the modification of proximal lysine residues [30,44,45]. Thus, the prediction of SIM motifs using the GPS-SBM 1.0 User Interface software [46] reveals the presence of one SIM motif within the central region of ΔLf, near to the K361 site ( Fig 1A). This motif is in a reversed orientation (SIMr) ( Table 2) with four hydrophobic positions preceded by an acidic cluster and by a serine residue. Phosphorylation of SIM-associated serine residues is known to favor efficient recognition of SUMO [37,47]. Comparison of this motif with homologs from a number of vertebrate species reveals that it is conserved in Lf/ΔLf ( Table 2). The functional significance of this motif and its role in ΔLf SUMOylation remain to be investigated.
The low abundance of ΔLf and the feeble percentage of SUMOylated conjugates rendered the detection of SUMOylated ΔLf and the subsequent mapping of its SUMO sites extremely difficult. Therefore we used a 3xFLAG-tagged ΔLf in order to detect it and we constructed a first series of SUMOylation mutants in which only one lysine residue was replaced at a time (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R ; S1 Table and S1 Fig left panel). Incubation of ΔLf and mutants with His-SUMO peptides caused the appearance of multiple higher molecular weight species indicative of SUMOylation events ( Fig 1B). Moreover, mutation of each individual lysine residue did not abolish SUMOylation of the entire molecule ( Fig 1B). Since competition between SUMO and ubiquitin ligases often occurs at ubiquitin sites, K379 which is the main ubiquitinated target on ΔLf [17] was also investigated. Thus, we produced a second series of mutant constructs in which only one putative SUMO site was preserved (S1 Table and S1 Fig right panel). We obtained five SUMO mutants named K13, K308, K361, K379 (which in fact corresponds to the M4S mutant) and K391, respectively and the M5S mutant in which all putative SUMOylation sites were abolished. ΔLf and its SUMOylation mutants were then expressed in HEK-293 cells which do not produce ΔLf endogeneously. We detected 3xFLAG-tagged ΔLf isoforms as a single band of the expected 75 kDa predicted molecular weight. The level of their 3
V/C L/V I/V V-E [37].
ΔLf/Lf
SUMO Interacting Motif a Accession number
Human ΔLf b 350 S T T E D C I A L V L K G E 363 Q5EKS1 Bovine Lf c 375 S T T D D C I V L V L K G E 388 P24627 Goat Lf 375 S T T D D C I A L V L K G E 388 Q29477 Mouse Lf 373 P T T E D C I V A I M K G D 386 P08071 Pig Lf 371 S T T E D C I V Q V L K G E 384 P14632 Horse Lf 375 S T T E E C I A L V L K G E 388 O77811 Sheep Lf 375 S T T D E C I A L V L K G E 388 AY792499 Camel Lf 375 S T T E D C I A L V L K G E 388 AJ131674 a
The single-letter amino acid code is used; bold letters indicate the hydrophobic positions of the putative SUMO interacting motif, the acidic cluster is in italics, the SIM-associated serine residues are underlined.
b
The numbering of the amino acid residues corresponds to human ΔLf. c The numbering of the amino acid residues corresponds to Lfs. expression was compared and Fig 1C shows that they were expressed at least at the same level as WT. K361 and notably K379 were expressed at a higher level than the other mutants but statistical analyses showed that these differences were not significant.
SUMOylation was first investigated on WT and M5S which were co-transfected with or without the SUMO-1 expression vector. An immunoprecipitation was then performed using the anti-FLAG antibody in order to specifically immunoprecipitate ΔLf or its SUMO variants. SUMOylation was then investigated using anti-SUMO-1 antibodies. Fig 1D shows that ΔLf was effectively SUMOylated and that SUMOylation was slightly increased when it was incubated with components of the SUMO pathway such as SUMO-1 (lane 1). Multiple higher molecular weight bands which may correspond to multi-or poly-ΔLf-SUMOylated forms were observed. Taking into account the in silico studies, this SUMO pattern (lanes 1-2) suggested that at least four SUMOylation sites are occupied (corresponding to 86, 97, 108 and 119 kDa, as shown by asterisks) for WT.
The feeble amount of SUMO-conjugates ( Fig 1D, upper panel) compared to unmodified ΔLf (Fig 1D, middle panel) is in accordance with the literature. Thus, for most SUMOylated proteins, the levels of the SUMO forms are low relative to the unmodified form due to an efficient SUMOylation/deSUMOylation balance in cells [35].
M5S appeared not to be SUMOylated even when SUMO-1 was overexpressed suggesting that no other SUMO sites are present on the protein (Fig 1D, upper panel). Moreover, overexposure of this film failed to show additional bands that could suggest SUMOylation of the M5S construct (data not shown). Surprisingly, the M5S M2 immunoprecipitation signal is poor compared to the M5S M2 western blot signal. This could be due to the mutation of five lysine residues which may impair ΔLf conformation, resulting in poor immunoprecipitation even if IP was directed against 3XFLAG and not ΔLf itself.
Mapping the main SUMO sites
In order to identify SUMO acceptor sites, SUMO mutants were co-transfected with the His-SUMO-1 expression vector. A band corresponding to ΔLf-SUMO-1 forms is visible in each lane confirming that the five predicted sites were effectively SUMOylated (Fig 2A, left panel). K13, K308 and K379 are the three main acceptor sites. Surprisingly, the K361 mutant which possesses a predictive SUMO sequence that perfectly fits the optimal CKXE consensus sequence was poorly modified, as was K391. Overexpression or not of SUMO-1 did not modify the SUMO profile of the latter site confirming that it is not preferentially targeted by the SUMO machinery (Fig 2A and 2B). K391 is also ubiquitinated but it was not the main ubiquitin target site [17]. Interestingly, K379 which is the main ubiquitination site is also a good acceptor of SUMO, even though this lysine residue does not belong to a SUMO consensus sequence.
In order to investigate whether some of the SUMO-1 target sites might also be SUMO-2/3 acceptor sites, SUMO mutants were co-transfected with the His-SUMO-2/3 expression vector. Fig 2A (right panel) shows that SUMO-2/3 peptides might also bind K13 but the amount of the K13-SUMO2/3 form is feeble. Further work has to be done in order to confirm that this modification occurs as well in vivo. Control of specificity of His antibodies has been added since these antibodies revealed bands above 100 kDa even when His-SUMO-1 was not overexpressed (Fig 2A, upper left panel, lanes 7-10).
We next tried to evaluate whether ΔLf could be SUMOylated in vivo. The presence of SUMO proteases (SENPs) within the cells and also in cell extracts poses a significant problem for the detection of SUMOylated proteins. Therefore, we used N-Ethylmaleimide (NEM), which blocks cysteine proteases, as a SENPs inhibitor. Since NEM is not cell permeable we used it during cell lysis and immunoprecipitation steps. ΔLf-expressing cell lysates were immunoprecipitated with M2 and SUMO forms immunodetected with anti-SUMO-1 antibodies (Fig 2B, right panel). Slow migrating bands of strong intensity were detected for WT (upper panel) which might be due to the fact that WT is multi-SUMOylated. SUMO forms were also observed for K13, K308, K361 and K379 mutants (Fig 2B, upper right panel). Mutants that are unable to be ubiquitinated such as K13, K308 and K361 seem better in promoting SUMOylation of ΔLf (lanes 2-4). The K391 mutant seems poorly or even not modified, like the M5S mutant, which strongly suggests that only four of the five sites are modified by SUMO peptides.
Immunodetection by M2 showed the presence of a ladder of bands for WT, K13 and K379, confirming possible multi-and/or poly-modification (Fig 2B, middle right panel). A slow migrating band around 90 kDa was visible for K308 and K361 confirming the existence of a mono-SUMO-conjugated form of ΔLf. On the other hand, the strong ladder pattern (Fig 2B, middle right panel) observed for WT and K379 may also be partially due to ubiquitination as already described [17], suggesting that a competition between SUMO and ubiquitin modifications may occur. Since K13 is unable to be ubiquitinated, ladder bars might correspond to polySUMOylation. Therefore, M2 immunoprecipitation followed by SUMO-2/3 immunodetection of WT and SUMO mutant cell extracts was performed, but we were unable to observe The data presented correspond to one representative experiment of at least three conducted (n ! 3). B-C) WT and its mutants are SUMOylated in vivo. WT and the SUMO mutant constructs were transfected for 24 h prior to lysis. Whole cell extracts were immunoprecipitated with M2 and immunoblotted with either anti-SUMO-1 antibodies or M2 (B). Reverse immunoprecipitation was also performed (C). 1% of the cell extract (input) was immunoblotted with either M2 or anti-GAPDH antibodies used as loading control. Inhibition of proteasomal degradation was performed by incubating cells with MG132 for 2 h prior to lysis and inhibition of de-SUMOylation was performed by adding NEM to lysis, IP and WB buffers as in Material and Methods (Fig 2A and 2B right panels, and C). Data presented in Fig 2B (left panel) were obtained in the absence of proteasome and SENP inhibitors. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa), the arrow corresponds to ΔLf. All the data presented correspond to one representative experiment of at least three conducted (n ! 3).
doi:10.1371/journal.pone.0129965.g002 SUMO-2/3 modifications on WT or its SUMO mutants (data not shown). Since K13 is also modified by SUMO-2/3 in vitro (Fig 2A, right panel), we will further investigate whether modification by SUMO-2/3 is relevant in vivo, then what impact a mixed SUMO chain could have on ΔLf activity and/or stability.
In order to confirm that endogeneous SUMOylation occurs on ΔLf we performed the same experiment in the absence of SENP and proteasome inhibitors. Fig 2A (left upper panel) effectively showed that no SUMOylation pattern was visible in those conditions. Fig 2C corresponds to the reverse immunoprecipitation of K13, K308, K361 and K379 cell lysates with anti-SUMO-1 antibodies followed by M2 immunodetection. A faint band corresponding to ΔLf-SUMO-1 is visible and a poly-SUMO pattern is observed for K379. Since mixed SUMO/ubiquitin chains could be formed we will further investigate whether ΔLf might be modified at K379 by such a complex.
Collectively these results indicated that ΔLf could be SUMOylated at multiple lysine residues and that the band shift of ΔLf was indeed due to the covalent attachment of SUMO-1 with K13 as hotspot of SUMOylation.
The SUMOylation/ubiquitination interplay at K379 controls ΔLf stability
Since, as for many transcription factors, ΔLf is rapidly degraded, we previously demonstrated that its turnover was dependent on both the Ub-proteasome pathway and the O-GlcNAc/phosphate interplay [17]. We also characterized K379 as the major site for ΔLf poly-ubiquitination. Since K379 is also targeted by SUMO ligases we next investigated whether a crosstalk exists between the ubiquitin and SUMO pathways. Fig 3A shows that a ladder of polyubiquitinated K379 forms is visible in the presence of recombinant HA- Ubiquitin (upper panel, lane 3). The intensity of the polyubiquitination signal decreases when SUMO-1 peptides are overexpressed (Fig 3A upper panel, lane 4). After stripping, the immunoblot was revealed using anti-SUMO-1 antibodies (middle panel). Increased SUMOylation could be observed for the K379 mutant when SUMO-1 was overexpressed (Fig 3A, middle panel, lane 2) as already shown for WT in Fig 1D. We also observed a decrease in this SUMO signal in the presence of recombinant ubiquitin ( Fig 3A, middle panel, lanes 2 and 4). Loadings of K379 (input) confirmed that in the presence of recombinant ubiquitin the expression level of K379 is lower than in the untreated condition or when SUMO-1 is overexpressed. These data support the view that K379 is indeed the target of an ubiquitin/SUMO switch.
We next investigated whether this interplay acts on K379 stability. To measure the K379 turnover rate indirectly, we performed incubations (0-150 min) with cycloheximide (CHX), a potent inhibitor of de novo protein synthesis. The K379 (left panels 1, 3 and 5) and GAPDH (left panels 2, 4 and 6) contents of HEK-293 cells were analysed following addition of CHX (Fig 3B). GAPDH was used as an internal control. Differences in the steady state levels of the K379 mutant were readily apparent after 30 min, which may correspond to the delay necessary for observing the first effects of treatment. HA-Ubiquitin (HA-Ub) or His-SUMO-1 (SUMO-1) expression vectors were co-transfected or not in HEK-293 cells with the K379 construct ( Fig 3B). Densitometric data are expressed as D K379 /D GAPDH ratio as described in Materials and Methods. HA-Ub overexpression led to an overall 3-fold decrease in K379 stability compared to His-SUMO-1 treated cells (Fig 3C) confirming that increasing ubiquitination drives ΔLf to degradation as we showed previously [17]. When K379-expressing cells overexpressed the SUMO-1 peptide, the stability of the mutant was comparable to that of untreated K379 mutant. Nevertheless, the fact that we did not observe a strong protection as may be expected against proteosomal degradation may be due to the fact that the modified pool of K379 is very feeble (less than 10%) even when SUMO-1 is overexpressed (Fig 2A). The same experiment was conducted with MG132 (right panels) and densitometric results showed that K379 is very stable when the degradation of ubiquitin-conjugated proteins is reduced, whatever the applied treatments ( Fig 3D). Taken together these results confirmed, as already described for other substrates, that SUMOylation may antagonize ubiquitination at K379 and hence positively affect the proteolytic stability of ΔLf.
SUMOylation of ΔLf represses its transcriptional activity
To study the physiological consequences of ΔLf SUMOylation, we next assayed the transcriptional activity of the mutants compared to wild type (Fig 4A). SUMOylation usually triggers recruitment of corepressors such as HDACs, which condense chromatin and prevent transcription. We used a luciferase reporter construct driven by a basal promoter and one ΔLf response element present in a fragment of the Skp1 promoter [9]. In this experiment, the cells were not co-transfected with His-SUMO-1, so the status of ΔLf SUMOylation completely relied on endogenous SUMO activity. The 2.5-fold increased transcriptional activity of the SUMOylation-null mutant confirmed that SUMOylation negatively regulates ΔLf transcriptional activity. Since M5S could not be SUMOylated, over-expression of this mutant without co- expression of SUMO-1 justified the conclusion drawn here that the SUMOylation of ΔLf is part of a regulatory event that governs its activity. The small amount of ΔLf-SUMO forms present in cells could not account for the 2.5 fold increment observed in the transcriptional activity induced by the M5S mutant compared to WT. This has been already described for numerous transcription factors and suggests that SUMOylation is required to initiate transcriptional repression but not to maintain it [19,48]. We then compared the activity of mutants in which only one SUMOylation site was preserved to that of M5S in order to evaluate the impact of adding only one regulatory site at a time. The K391 mutant, which is poorly modified, showed transcriptional activity nearly comparable to that of M5S (Fig 4A) suggesting that the presence of SUMO on this site does not crucially regulate ΔLf transcriptional activity. In contrast, the transcriptional activities of the K308, K361 and K379 mutants were strongly inhibited, by 10-fold for K308 and by nearly 6 fold for the other sites compared to M5S, and by 4 fold for K308 and by around 2-2.5 fold for the other two sites compared to WT, suggesting that these three sites are important for regulation. K361, which is poorly SUMOylated, is nevertheless strongly involved in the repression process. The transcriptional activity of the K13 mutant also SUMOylation of ΔLf represses its transcriptional activity. A) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ! 6) (B) and to one representative experiment of at least two conducted (n ! 2) (C, D). E) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*), p < 0.01 (**)).
doi:10.1371/journal.pone.0129965.g004 decreases but to a lesser extent, by 3.5-fold compared to M5S and by 1.5-fold compared to WT. This may be due to the SUMO/acetylation switch discussed below (Fig 5). The transcriptional activity of the SUMO mutants K13, K361, K379 and notably K308, is lower than that of WT. This may be due to the fact that ΔLf is multi-SUMOylated and that the distribution of SUMO conjugates at each site leads to a "dilution" of the effect in the WT compared to the mutants with only one SUMO acceptor site, which may be more heavily SUMOylated.
Since SUMO modifications on the K308 site led to the highest inhibitory impact on ΔLf transcriptional activity, we next focused our attention on K308 and investigated the impact of altering its SUMO pattern. Therefore we increased SUMOylation by using the His-SUMO-1 expression vector and decreased it by performing either de-SUMOylation using recombinant SENP2 protease or knockdown using specific short interfering RNA against Ubc9 (siUbc9). Prior to performing transcriptional activity assays, we first showed that siUbc9 efficiently invalidated Ubc9 expression (Fig 4B) leading to a decrease in the level of SUMOylation of both ΔLf 3). B) Relative transcriptional activity of K13 and K379 mutants compared to WT. Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, K13 or K379. His-SUMO-1 expression vector and/or the deacetylase inhibitor Trichostatin A (TSA, 15 ng/mL) were used to modulate the acetylation/SUMOylation ratio. Relative luciferase activities are expressed as described in Materials and Methods (n!3; p < 0.05 (*)). C-D) Modulation of the SUMOylation level was performed either by knocking down Ubc9 using siUbc9 or by overexpressing His-SUMO-1 peptides (SUMO-1). Cells were reverse transfected or not with RNAiMax using 5 nM of siUbc9/siCtrl for 24 h before being transfected for 24 h with WT or K13 plasmid with or without His-SUMO-1 plasmid. Before cell lysis, the acetylation level was altered or not by an overnight treatment with TSA (15 ng/mL). Cells were then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. (C) Input was immunoblotted with anti-Ubc9 (upper panel) or anti-GAPDH (lower panel) antibodies. (D) Samples were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 (upper panel), then with anti-acetyllysine (middle panel) or finally with M2 (lower panel) antibodies. The acetylation/SUMOylation ratio (R Ac/SUMO ) was assayed as described in Material and Methods. The data presented correspond to one representative experiment of two conducted.
doi:10.1371/journal.pone.0129965.g005 (Fig 4C, IP, lane 3) and other protein substrates (Fig 4C, input, lane 3). Immunoprecipitation of ΔLf or K13-and K308-expressing cell lysates with M2 followed by immunodetection of SUMO forms using anti-His antibodies (Fig 4D) showed that effective de-SUMOylation was produced in the presence of recombinant SENP2 and is visible in the second, fourth and sixth lanes compared to the untreated condition. Fig 4E shows that overexpression of the His-SUMO-1 peptide strongly decreased the transcriptional activity of ΔLf and its K308 mutant whereas overexpression of SENP2 protease significantly increased it. Moreover, siRNA-mediated depletion of endogenous Ubc9 abolished the repressive potential of SUMOylation and led to a drastic activation of the reporter gene activity (Fig 4E). This combination of overexpression and knockdown experiments demonstrate that SUMOylation plays an important role in ΔLfmediated transcriptional activation. Collectively, these results showed that effectively SUMOylation negatively controls ΔLf transcriptional activity and may convert ΔLf into a transcriptional repressor.
Acetylation attenuates SUMO-mediated transcriptional repression
Acetylation regulates numerous cellular processes, including the regulation of transcription [49,50]. Acetylation of internal lysine residues is a reversible PTM which strongly alters the electrostatic properties of its targets, modulating their functions, such as protein-protein interactions, DNA binding, activity, stability and subcellular localization [22]. Since competition between SUMOylation and acetylation occurs on many substrates and a putative acetylation site was found on ΔLf at K13, we next investigated whether the SUMO sites might also be acceptors for acetyltransferases. We performed mapping of ΔLf acetylation sites by western blotting of the different expressed mutant ΔLfs using an anti-acetyllysine antibody. Among the five SUMOylated lysine residues, K13 and K379 were the only acetylation acceptor sites with K13 being the major acetylated residue (Fig 5A, lanes 2 and 5, respectively). Mutation of these two lysine residues in the other SUMO mutants (Fig 5A, lanes 3, 4 and 6) and in M5S (Fig 5A, lane 7) resulted in a complete loss of the acetylation signal suggesting that only two acetylation sites are present on ΔLf. These data also confirmed the existence of a possible interplay between acetylation and SUMOylation for K13 and suggested an acetylation/SUMOylation/ubiquitination crosstalk for K379.
We next assayed the impact of the SUMO/acetylation interplay on ΔLf-mediated transactivation ( Fig 5B). We increased either SUMOylation by overexpressing SUMO-1 peptide or raised the acetylation level by using the HDAC inhibitor Trichostatin A (TSA). TSA-induced acetylation was able to promote ΔLfand K13-mediated activation by nearly 1.5 fold compared to the untreated condition and 4-fold compared to the condition when SUMO forms were overexpressed (SUMO-1). These data suggested that dynamic interactions between these two posttranslational modifications may occur. The fact that the enhanced WT and K13 transcriptional activities due to TSA-induced acetylation were not modified when SUMO-1 peptides were overexpressed (TSA+SUMO-1) may be due to the fact that acetylation is a less labile PTM than SUMOylation. Indeed, SENPs have to be inhibited in order to observe SUMO forms whereas we do not need to inhibit HDACs in order to visualize acetylated forms. That modulation of the SUMO or acetylation pattern on the K379 mutant had less impact on ΔLf transcriptional activity may be due to the fact that ubiquitination also targets this site.
In order to confirm these results we further investigated whether an increase in the levels of acetylation may result in a reduction in the levels of ΔLf SUMOylation and conversely (Fig 5C and 5D). Therefore, we invalidated Ubc9 or inhibited deacetylases with TSA in order to increase acetylation levels, or raised the levels of SUMOylation by overexpressing His-SUMO-1 peptides (SUMO-1), and assessed the impact on ΔLf acetylation. Results shown in Fig 5C confirmed that siUbc9 were functional. More than 80% of the Ubc9 protein disappeared 48 h post-transfection as compared to untreated (NT) or siCtrl treated cells. Moreover, knockdown was not sensitive to treatment with TSA or to the overexpression of His-SUMO-1. Immunoprecipitation of ΔLf-expressing cell lysates with M2 was followed by immunodetection of SUMO-1, acetylated forms or ΔLf (Fig 5D). SUMO profiles of WT and its K13 mutant, when Ubc9 was invalidated, confirmed a decrease in SUMOylation compared to controls (Fig 5D, lane 3 left panel and 2 right panel, respectively) which was more pronounced after TSA treatment (lanes 6 and 12 left panel and lanes 5 and 11 right panel). Overexpression of SUMO-1 peptides together with siUbc9 treatment did not lead to increased SUMOylation of WT and K13 as expected (Fig 5D, lane 9 left panel and 8 right panel, respectively) compared to their respective controls (Fig 5D, lanes 8 and 10 left panel and 7 and 9 right panel, respectively). Thus, ΔLf SUMOylation levels were downregulated following Ubc9 knockdown and when inhibition of HDACs was achieved with TSA. We also analyzed the modification of the acetylation profile of WT and K13 in the above conditions but we did not observe visible variations. Therefore we assayed the acetylation/SUMOylation ratio. This ratio varied slightly when WT was expressed in siUbc9 cells but increased by nearly 2-fold when these cells were grown overnight in the presence of TSA and 3-fold in siUbc9-TSA-treated HEK-293 cells. These data are in accordance with the literature and confirmed increased acetylation when HDACs are inhibited by TSA. Overexpression of SUMO-1 peptides in siCtrl versus siUbc9 cells leads to a comparable acetylation/SUMO ratio. When cells overexpressing SUMO-1 were treated with TSA, acetylation was favoured. The same experiment was conducted with the K13 mutant. The acetylation/SUMOylation ratio was 2-fold higher than WT and rose 4-fold in Ubc9-null cells suggesting that the K13 mutant with only one acetylation/SUMOylation site may preferentially exist as an acetylated form. TSA treatment led to an increased acetylation/SUMOylation ratio as expected. Taken together these results suggest that acetylation antagonizes SUMOylation and may downregulate SUMO effects at K13 (Fig 5B and 5D). The crosstalk between these sites could constitute part of the «ΔLf code » responsible for the control of the transactivation of ΔLf target genes.
Discussion
Transient PTMs like acetylation, phosphorylation, O-GlcNAcylation, ubiquitination and SUMOylation are fast and efficient ways for the cell to respond to different stimuli. Transcription factors are often regulated by combinations of these different PTMs which might act as a molecular barcode [51]. In this report, we demonstrated that ΔLf, known to be modified by O-GlcNAcylation, phosphorylation at S10 and ubiquitination at K379 and K391 [17], can also be modified by SUMOylation and acetylation. We provide experimental evidence that SUMOylation represses ΔLf transcriptional activity whereas acetylation increases it. Moreover, by competing with ubiquitination, SUMOylation influences positively ΔLf stability.
Considering the fact that, for a given protein, only a small fraction is commonly found in the SUMOylated state and that this modification is transient, it was difficult to visualize or isolate endogeneous SUMO forms. Nevertheless we were able to observe SUMO-1 isoforms of ΔLf in situ. The ΔLf SUMOylation pattern, manifested as multiple bands, is consistent with the presence of multiple SUMOylation sites on the protein. We identified five SUMOylation sites among which the K13 and K361 sites conform to the consensus sequence of CKXE/D. Furthermore, SUMOylation was also mapped at K308, K379 and K391, which are non-canonical sites. Among these, K13, K308 and K379 are the three SUMO hotspots.
SUMOylation of transcription factors generally antagonizes their activation potential or mediates repression, although in a few cases SUMOylation has been associated with reciprocal effects resulting in activation. Whereas SUMOylation decreases the transactivation potential of c-Jun and the androgen receptor [52,53], it increases heat shock factor-2 (HSF2) transactivation capacity [54]. SUMOylation can both positively and negatively modulate p53 transcriptional activity depending on the target promoter [55]. Here we demonstrated that SUMOylation of ΔLf represses its transcriptional activity using a fragment of the Skp1 promoter containing one ΔLfRE. ΔLf binds to and transactivates DcpS, Skp1, Bax, SelH, GTF2F2 and UBE2E1 genes [9,[12][13][14] through similar consensus response elements. Nevertheless it may be interesting to investigate whether ΔLf differentially transactivates its target genes depending on its SUMOylation status.
Since ΔLf possesses five SUMO target sites it is difficult to determine whether each of the individual sites has a specific role. Moreover, three of them are targeted by other PTMs, rendering this study even more complex. In order to establish whether any single SUMOylation site was important for the transactivation capacity we compared the activities of mutants disabled specifically at each individual consensus SUMOylation site. The transactivation capacity of each single-site lysine mutant was similar or slightly increased compared to WT (data not shown). Since multiple sites contribute to the control of ΔLf transactivation capacity, the loss of only one SUMO site has only small effects on the activity of ΔLf suggesting either that individual sites act in a redundant manner or that SUMOylation at multiple sites is necessary. Therefore we next studied the transcriptional activity of each mutant in which only one SUMO site was preserved. The K308 mutant strongly inhibited ΔLf transcriptional activity. Moreover when SUMOylation was impaired either after Ubc9 knockdown or in the presence of increased expression of the SENP2 protease, the transcriptional activity of K308 was increased 5-fold compared to that of the K308 mutant expressed in untreated cells. This activity strongly decreased by nearly 2-fold when SUMO-1 peptides were overexpressed. These results demonstrated that SUMOylation at K308 strongly controls ΔLf activity, which may be due to the fact that the region downstream from its SUMO motif is rich in acidic residues as in NDSM. NDSM interacts twice with Ubc9, first between the consensus motif and the active site of the enzyme and also between the acidic tail of the consensus and the basic patch of Ubc9 [27]. Thus, the NDSM acidic patch plays an important role in determining the efficiency of substrate SUMOylation which consequently results in enhanced transcriptional repressive properties.
Our in silico studies led us to discover a reverse putative consensus SIMr motif in the vicinity of the K361 SUMO site which is conserved among mammalian species (Table 1). SIMs, which mediate non-covalent interactions between SUMO and SIM-containing proteins [56], can mediate SUMO modification of numerous proteins, resulting in changes in their activity. Moreover a serine residue that is proximal to this SIMr might be the target of kinases as described for non-histone proteins such as PML, EXO9 and PIAS proteins [47]. The presence of a SIMr and/or a phospho-SIMr might be essential to enhance interactions with a SUMO protein and mediate SUMO conjugation. Therefore, the functionality of such a motif has to be established for ΔLf.
SUMOylation usually competes with ubiquitination, phosphorylation and acetylation. Ubiquitination/SUMOylation and SUMOylation/acetylation are mutually exclusive whereas SUMOylation/phosphorylation can be agonistic or antagonistic depending on the substrates. The dialogue between SUMO and the other modifications is emerging as a common mechanism that allows control of the transcriptional activity of transcription factors [21]. Two of the SUMO sites are targeted by acetyltransferases. Acetylation is also a dynamic process which mainly contributes to activation of transcription factors [57,58]. Thus, K13 and K379 are acetylated with K13 as the major acetylation site. Modulation of the SUMO/acetylation status has a strong impact on K13 transcriptional activity. In this way, SUMO/acetylation modification of ΔLf could act as a form of switch for the selective interaction with corepressor or coactivator partners, thus modulating ΔLf activity from a transcriptional repressor/corepressor to a coactivator. This is consistent with literature data. Thus, it was shown that SUMOylation inhibits MEF2, HIC1 and KLF8 transcriptional activities whereas acetylation blocks these inhibitory effects [39,40,59,60]. This acetylation/SUMOylation switch is regulated by phosphorylation for MEF2 [39] and it will be interesting to investigate whether ΔLf acetylation/SUMOylation interplay is also controlled by phosphorylation events. The K13 site has a SUMOylation motif close to PDSM motifs [28]. Phosphorylation of the SP motif within this consensus sequence plays an important role in promoting SUMOylation of several substrates including MEF2A [28,39]. Therefore we will have to investigate whether S16 might be of potential functional importance in the regulation of SUMOylation at K13. Moreover, since at the N-terminus, the K13 SUMO/ acetylation site is adjacent to the S10 O-GlcNAcylation/phosphorylation site we will further investigate whether the O-GlcNAc/Phosphate interplay interferes with the SUMOylation/acetylation switch or acts in parallel. The crosstalk between these sites may constitute the ΔLf code responsible for the control of the transactivation of ΔLf target genes. We know from our results [17] and from the literature that acetylation and phosphorylation both lead to transcriptional activation whereas O-GlcNAcylation and SUMOylation repress it. So we hypothesize that this region might be part of the ΔLf transactivation domain which has never been identified. On the other hand, Ubc9 itself is acetylated and its acetylation leads to its decreased binding to NDSM substrates, causing a reduction in their SUMOylation status. Therefore Ubc9 acetylation/deacetylation may serve as a dynamic switch for NDSM substrates such as the K308 site in order to control their SUMOylation [61].
K379 and K391 could be both SUMOylated and ubiquitinated. SUMOylation competes with ubiquitination and positively regulates ΔLf stability. Indeed, SUMO frequently influences protein stability by blocking ubiquitin attachment sites [19,36]. K379, which is the main target of both the SUMO and ubiquitin machineries, does not possess a SUMO consensus sequence but is located in the vicinity of the PEST region. It has been shown that Ubc9 could directly interact with the PEST region of SUMO-1 target proteins such as HIPK2 [62] but this is not always the case. It will be interesting to determine the Ubc9-interacting region of ΔLf and investigate whether it overlaps the PEST region. The ubiquitination/SUMOylation interplay exerts a critical role in the maintenance of cellular homeostasis by controlling the turnover of numerous of proteins and notably transcription factors. The switch between these two PTMs needs to be tightly regulated in a spatiotemporal manner and other PTMs, such as phosphorylation, contribute to regulate the ubiquitin/SUMO pathways. PEST sequences are rich in S/TP motifs and are often recognized and phosphorylated by proline-directed S/T protein kinases [63]. Phosphorylation can prevent or favor SUMO-1 conjugation as previously shown for IκBα [36], c-Jun [52] and p53 [52]. We already showed that the ΔLf PEST motif contains three serine residues (S392, S395 and S396) which are phosphorylated prior to ubiquitination of the targets K379 and K391 in their vicinity. Mutation of these two lysine residues or of the three serine residues (S392, S395 and S396) within the PEST motif strongly increased the half-life of ΔLf [17]. Moreover, we showed that they were equivalent phosphorylation targets due to their proximity. Therefore, at the PEST motif, phosphorylation and ubiquitination work in synergy [17], while SUMOylation and ubiquitination are antagonistic PTMs. Therefore this crosstalk could constitute the ΔLf code responsible for the control of ΔLf stability. This regulation is driven by the O-GlcNAc/phosphate interplay at S10. O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity. The pool of ΔLf may exist under a stable but not functional O-GlcNAc isoform. Since the level of O-GlcNAc changes during the cell cycle or is altered, such as in tumorigenesis, deglycosylated ΔLf will become the target of kinases leading to its activation and polyubiquination [1,17]. ΔLf is at the crossroads between cell survival and cell death. It triggers cell cycle arrest and apoptosis via the transactivation of several crucial target genes. Therefore, modifications of their expression may have marked consequences and, depending on cell homeostasis, their transactivation by ΔLf should be transiently suspended. In this context, the SUMOylation/acetylation switch at K13 acts as a second level of control. The activation of the SUMO pathway leads to repression of ΔLf transcriptional activity whereas acetylation, by counteracting SUMOylation at gene promoters, restores it. Increasing evidence shows that O-GlcNAcylation not only interferes with phosphorylation but also crosstalks with other PTMs including acetylation [64], methylation [64,65], ubiquitination [66,67] and poly-ubiquitination [68,69]. However crosstalk with SUMOylation has not yet been reported and we are currently investigated the O-GlcNAcylation/SUMOylation interrelationship.
In conclusion, we showed that SUMO modification provides subtle, context-dependent, regulatory input to modulate ΔLf target gene expression. Moreover, we confirmed that ΔLf, like many transcription factors, is regulated by combinations of different PTMs which act as a molecular barcode. Thus, cooperation and/or competition between SUMOylation, ubiquitination, acetylation, phosphorylation and O-GlcNAcylation may contribute to the establishment of a fine regulation of ΔLf transcriptional activity depending on the type of target gene and cellular homeostasis. In this paper, we have focused on the role of SUMOylation but it has not escaped our attention that lysine residues can also be methylated and that such modifications can also affect the activity and stability of proteins such as p53 [70]. Further studies of the roles of PTMs in the molecular mechanisms of ΔLf functions are warranted.
Supporting Information S1 Table. Name of mutant constructs, location of amino acid modifications and oligonucleotides used for mutagenesis.
doi:10.1371/journal.pone.0129965.t001
Fig 1 .
1Fig 1. ΔLf is modified by SUMOylation. A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by posttranslational modifications are shown, S10 as the main O-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ! 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ! 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ! 3).
doi:10.1371/journal.pone.0129965.t002
Fig 2 .
2Mapping of the SUMO modification sites of ΔLf. A) K13, K308 and K379 are SUMO-1 acceptor sites. Cells were co-transfected by WT or the mutant constructs and pSG5-His-SUMO-1 or pcDNA3.1-His-SUMO-2/3 plasmids and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control.
Fig 3 .
3Competition between SUMOylation and ubiquitination at K379 controls ΔLf turnover. A) HEK-293 cells were co-transfected with K379 or NV constructs, His-SUMO-1 or/and HA-Ub-expression vectors for 24 h and then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. Total cell extracts were immunoprecipitated with M2 or used as input. Samples were immunoblotted with anti-HA (upper panel) or with anti-SUMO-1 (lower panel) antibodies. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control. NS: non-specific. The data presented correspond to one representative experiment of at least three conducted (n ! 3). Lane 6 corresponds to non-transfected cells. B) Cells were transfected with K379, either with the His-SUMO-1 or the HA-Ub expression vector and then incubated with fresh medium supplemented by 10 μg.mL -1 CHX for the indicated time 24 h after transfection. K379 transfected cells were incubated without (left panel) or with (right panel) 10 μM MG132 for 2 h prior to lysis. Total protein extracts were immunoblotted with either M2 or anti-GAPDH antibodies. Detection was carried out using a Fusion SOLO camera (Vilbert Lourmat). The data presented (B) correspond to one representative experiment of at least five conducted. C-D) The M2 densitometric analyses are normalized for the matching GAPDH immunoblots and expressed as ratio D K379 / D GAPDH as described in Materials and Methods. Data are shown as the means ± SD (n = 5).doi:10.1371/journal.pone.0129965.g003
Fig 4 .
4Fig 4. SUMOylation of ΔLf represses its transcriptional activity. A) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ! 6) (B) and to one representative experiment of at least two conducted (n ! 2) (C, D). E) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*), p < 0.01 (**)).
Fig 5 .
5A SUMOylation/acetylation switch at K13 controls ΔLf transcriptional activity. (A) K13 is the main acetylation site. Cells were co-transfected by WT, the mutant constructs or the null vector and then lysed 24 h later. Lysates were immunoprecipitated with anti-acetyllysine antibodies and immunoblotted with M2. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control (n =
. Schematic representation of the two series of ΔLf SUMO mutant constructs. (TIF)
Table 1 .
1SUMO predictive motifs in human ΔLf.SUMO motif a
Table 2 .
2SUMO Interacting Motifs in human ΔLf and Lf from different species compared to the SIMr consensus (D/E)
PLOS ONE | DOI:10.1371/journal.pone.0129965 June 19, 2015
PLOS ONE | DOI:10.1371/journal.pone.0129965 June 19, 2015 9 / 21
AcknowledgmentsWe would like to thank Dr. R. J. Pierce (CIIL, Institut Pasteur de Lille, France) for critical reading of this manuscript.Author ContributionsConceived and designed the experiments: AER ASVE AP. Performed the experiments: AER ASVE MM IH EH. Analyzed the data: AER ASVE SH AP. Contributed reagents/materials/ analysis tools: TL AP. Wrote the paper: AP.
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Nuclear and unclear functions of SUMO. J S Seeler, A Dejean, 14506472Nat Rev Mol Cell Biol. 4Seeler JS, Dejean A. Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol. 2003; 4: 690-699. PMID: 14506472
Protein modification by SUMO. E S Johnson, 15189146Annu Rev Biochem. 73Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004; 73: 355-382. PMID: 15189146
The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. J R Gareau, C D Lima, 10.1038/nrm301121102611Nat Rev Mol Cell Biol. 11Gareau JR, Lima CD. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol. 2010; 11: 861-871. doi: 10.1038/nrm3011 PMID: 21102611
Modification with SUMO. A role in transcriptional regulation. A Verger, J Perdomo, M Crossley, 12612601EMBO Rep. 4Verger A, Perdomo J, Crossley M. Modification with SUMO. A role in transcriptional regulation. EMBO Rep. 2003; 4: 137-142. PMID: 12612601
SUMO promotes HDAC-mediated transcriptional repression. S H Yang, A D Sharrocks, 14992729Mol Cell. 13Yang SH, Sharrocks AD. SUMO promotes HDAC-mediated transcriptional repression. Mol Cell. 2004; 13: 611-617. PMID: 14992729
Something about SUMO inhibits transcription. G Gill, 16095902Curr Opin Genet Dev. 15Gill G. Something about SUMO inhibits transcription. Curr Opin Genet Dev. 2005; 15: 536-541. PMID: 16095902
SUMOylation and De-SUMOylation: wrestling with life's processes. E T Yeh, 10.1074/jbc.R80005020019008217J Biol Chem. 284Yeh ET. SUMOylation and De-SUMOylation: wrestling with life's processes. J Biol Chem. 2009; 284: 8223-8227. doi: 10.1074/jbc.R800050200 PMID: 19008217
SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. M S Rodriguez, C Dargemont, R T Hay, 11124955J Biol Chem. 276Rodriguez MS, Dargemont C, Hay RT. SUMO-1 conjugation in vivo requires both a consensus modifi- cation motif and nuclear targeting. J Biol Chem. 2001; 276: 12654-12659. PMID: 11124955
The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. D A Sampson, M Wang, M J Matunis, 11259410J Biol Chem. 276Sampson DA, Wang M, Matunis MJ. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem. 2001; 276: 21664-21669. PMID: 11259410
An extended consensus motif enhances the specificity of substrate modification by SUMO. S H Yang, A Galanis, J Witty, A D Sharrocks, 17036045EMBO J. 25Yang SH, Galanis A, Witty J, Sharrocks AD. An extended consensus motif enhances the specificity of substrate modification by SUMO. EMBO J. 2006; 25: 5083-5093. PMID: 17036045
PDSM, a motif for phosphorylation-dependent SUMO modification. V Hietakangas, J Anckar, H A Blomster, M Fujimoto, J J Palvimo, A Nakai, 16371476Proc Natl Acad Sci USA. 103Hietakangas V, Anckar J, Blomster HA, Fujimoto M, Palvimo JJ, Nakai A, et al. PDSM, a motif for phos- phorylation-dependent SUMO modification. Proc Natl Acad Sci USA. 2006; 103: 45-50. PMID: 16371476
Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. I Matic, J Schimmel, I A Hendriks, M A Van Santen, F Van De Rijke, H Van Dam, 10.1016/j.molcel.2010.07.02620797634Mol Cell. 39Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, et al. Site-specific identi- fication of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell. 2010; 39: 641-652. doi: 10.1016/j.molcel.2010.07.026 PMID: 20797634
Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of SUMOylated transcription factors. D Y Lin, Y S Huang, J C Jeng, H Y Kuo, C C Chang, T T Chao, 17081986Mol Cell. 24Lin DY, Huang YS, Jeng JC, Kuo HY, Chang CC, Chao TT, et al. Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of SUMOylated transcription factors. Mol Cell. 2006; 24: 341-354. PMID: 17081986
Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation. C C Chang, M T Naik, Y S Huang, J C Jeng, P H Liao, H Y Kuo, 10.1016/j.molcel.2011.02.02221474068Mol Cell. 42Chang CC, Naik MT, Huang YS, Jeng JC, Liao PH, Kuo HY, et al. Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation. Mol Cell. 2011; 42: 62-74. doi: 10.1016/j.molcel.2011.02.022 PMID: 21474068
Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. M H Tatham, E Jaffray, O A Vaughan, Jmp Desterro, C H Botting, J H Naismith, 11451954J Biol Chem. 276Tatham MH, Jaffray E, Vaughan OA, Desterro JMP, Botting CH, Naismith JH, et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem. 2001; 276: 35368-35374. PMID: 11451954
In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. I Matic, M Van Hagen, J Schimmel, B Macek, S C Ogg, M H Tatham, Mol Cell Proteom. 7Matic I, van Hagen M, Schimmel J, Macek B, Ogg SC, Tatham MH, et al. In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol Cell Proteom. 2008; 7: 132-144.
RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. M H Tatham, M-C Geoffroy, L Shen, A Plechanovova, N Hattersley, E G Jaffray, 10.1038/ncb171618408734Nature Cell Biology. 10Tatham MH, Geoffroy M-C, Shen L, Plechanovova A, Hattersley N, Jaffray EG, et al. RNF4 is a poly- SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nature Cell Biol- ogy.2008; 10: 538-546. doi: 10.1038/ncb1716 PMID: 18408734
Starting and stopping SUMOylation. F Z Watts, 10.1007/s00412-013-0422-0Chromosoma. 122What regulates the regulator?Watts FZ. Starting and stopping SUMOylation. What regulates the regulator? Chromosoma. 2013; 122: 451-463. doi: 10.1007/s00412-013-0422-0 PMID: 23812602
SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. J M Desterro, M S Rodriguez, R T Hay, 9734360Mol Cell. 2Desterro JM, Rodriguez MS, Hay RT. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB acti- vation. Mol Cell. 1998; 2: 233-239. PMID: 9734360
SUMOylation as a signal for polyubiquitylation and proteasomal degradation Conjugation and Deconjugation of Ubiquitin Family Modifiers. M Miteva, K Keusekotten, K Hofmann, J K Gerrit, Praefcke Rdohmen, R J , Groettrup MLandes Bioscience and Springer Science+Business MediaMiteva M, Keusekotten K, Hofmann K, Gerrit JK, Praefcke RDohmen RJ. SUMOylation as a signal for polyubiquitylation and proteasomal degradation Conjugation and Deconjugation of Ubiquitin Family Modifiers.In: Groettrup M, editor. Landes Bioscience and Springer Science+Business Media; 2010. pp 195-214.
Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity. F Aillet, F Lopitz-Otsoa, I Egaña, R Hjerpe, P Fraser, R T Hay, 10.1371/journal.pone.005167223284737PLOS ONE. 751672Aillet F, Lopitz-Otsoa F, Egaña I, Hjerpe R, Fraser P, Hay RT, et al. Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity. PLOS ONE. 2012; 7:e51672. doi: 10.1371/ journal.pone.0051672 PMID: 23284737
A calcium-regulated MEF2 SUMOylation switch controls postsynaptic differentiation. A Shalizi, B Gaudillière, Z Yuan, J Stegmüller, T Shirogane, Q Ge, 16484498Science. 311Shalizi A, Gaudillière B, Yuan Z, Stegmüller J, Shirogane T, Ge Q, et al. A calcium-regulated MEF2 SUMOylation switch controls postsynaptic differentiation. Science. 2006; 311: 1012-1017. PMID: 16484498
An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity. N Stankovic-Valentin, S Deltour, J Seeler, S Pinte, G Vergoten, C Guérardel, 17283066Mol Cell Biol. 27Stankovic-Valentin N, Deltour S, Seeler J, Pinte S, Vergoten G, Guérardel C, et al. An acetylation/dea- cetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor sup- pressor HIC1 regulates transcriptional repression activity. Mol Cell Biol. 2007; 27: 2661-2675. PMID: 17283066
Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway. Y Fu, Z Fang, Y Liang, X Zhu, P Prins, Z Li, 17407140J Cell Biochem. 102Fu Y, Fang Z, Liang Y, Zhu X, Prins P, Li Z, et al. Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway. J Cell Biochem. 2007; 102: 704-718. PMID: 17407140
Mitochondrial ubiquitin ligase MITOL ubiquitinates mutant SOD1 and attenuates mutant SOD1-induced reactive oxygen species generation. R Yonashiro, A Sugiura, M Miyachi, T Fukuda, N Matsushita, Y Ogata, 10.1091/mbc.E09-02-0112Mol Biol Cell. 20Yonashiro R, Sugiura A, Miyachi M, Fukuda T, Matsushita N, Ogata Y, et al. Mitochondrial ubiquitin ligase MITOL ubiquitinates mutant SOD1 and attenuates mutant SOD1-induced reactive oxygen spe- cies generation. Mol Biol Cell. 2009; 20: 4524-4530. doi: 10.1091/mbc.E09-02-0112 PMID: 19741096
PAIL: Prediction of Acetylation on Internal Lysines. Y Xue, A Li, X Yao, Xue Y, Li A, Yao X. PAIL: Prediction of Acetylation on Internal Lysines. 2006. Available: http://bdmpail. biocuckoo.org/prediction.php.
Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J Song, Z Zhang, W Hu, Y Chen, 16204249J Biol Chem. 280Song J, Zhang Z, Hu W, Chen Y. Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J Biol Chem. 2005; 280: 40122-40129. PMID: 16204249
SUMOylation: A Regulatory Protein Modification in Health and Disease. A Flotho, F Melchior, 10.1146/annurev-biochem-061909-093311Annu Rev Biochem. 82Flotho A, Melchior F. SUMOylation: A Regulatory Protein Modification in Health and Disease. Annu Rev Biochem. 2013; 82: 357-385. doi: 10.1146/annurev-biochem-061909-093311 PMID: 23746258
GPS-SBM 1.0: a stand-alone program for prediction of SUMO-binding motifs. M Qian, G Xinjiao, C Jun, L Zexian, J Changjiang, R Jian, X Yu, 10.1371/journal.pone.0019001PLOS ONE. 2009. 6419001Qian M, Xinjiao G, Jun C, Zexian L, Changjiang J, Jian R, Yu X. GPS-SBM 1.0: a stand-alone program for prediction of SUMO-binding motifs. PLOS ONE. 2009. 6(4): e19001. doi: 10.1371/journal.pone. 0019001
Phospho-regulated SUMO interaction modules connect the SUMO system to CK2 signaling. P Stehmeier, S Muller, 10.1016/j.molcel.2009.01.01319217413Mol Cell. 33Stehmeier P, Muller S. Phospho-regulated SUMO interaction modules connect the SUMO system to CK2 signaling. Mol Cell. 2009; 33: 400-409. doi: 10.1016/j.molcel.2009.01.013 PMID: 19217413
Concepts in SUMOylation: a decade on. R Geiss-Friedlander, F Melchior, 18000527Nat Rev Mol Cell Biol. 8Geiss-Friedlander R, Melchior F. Concepts in SUMOylation: a decade on. Nat Rev Mol Cell Biol. 2007; 8: 947-956. PMID: 18000527
Dual regulation of c-Myc by p300 via acetylationdependent control of Myc protein turnover and coactivation of Myc-induced transcription. F Faiola, X Liu, S Lo, S Pan, K Zhang, E Lymar, 16287840Mol Cell Biol. 25Faiola F, Liu X, Lo S, Pan S, Zhang K, Lymar E, et al. Dual regulation of c-Myc by p300 via acetylation- dependent control of Myc protein turnover and coactivation of Myc-induced transcription. Mol Cell Biol. 2005; 25: 10220-10234. PMID: 16287840
Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. A Brunet, L B Sweeney, J F Sturgill, K F Chua, P L Greer, Y Lin, 14976264Science. 303Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science. 2004; 303: 2011-2015. PMID: 14976264
A post-translational modification code for transcription factors: sorting through a sea of signals. B A Benayoun, R A Veitia, 10.1016/j.tcb.2009.02.00319328693Trends Cell Biol. 19Benayoun BA, Veitia RA. A post-translational modification code for transcription factors: sorting through a sea of signals. Trends Cell Biol. 2009; 19: 189-197. doi: 10.1016/j.tcb.2009.02.003 PMID: 19328693
Jun and p53 are modulated by SUMO-1 modification. S Muller, M Berger, F Lehembre, J S Seeler, Y Haupt, A Dejean, 10788439J Biol Chem. 275Muller S, Berger M, Lehembre F, Seeler JS, Haupt Y, Dejean A. c-Jun and p53 are modulated by SUMO-1 modification. J Biol Chem. 2000; 275: 13321-13329. PMID: 10788439
Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). H Poukka, U Karvonen, O A Janne, J J Palvimo, 11121022Proc Natl Acad Sci USA. 97Poukka H, Karvonen U, Janne OA, Palvimo JJ. Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). Proc Natl Acad Sci USA. 2000; 97: 14145-14150. PMID: 11121022
Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor. M L Goodson, Y Hong, R Rogers, M J Matunis, O K Park-Sarge, K D Sarge, 11278381J Biol Chem. 276Goodson ML, Hong Y, Rogers R, Matunis MJ, Park-Sarge OK, Sarge KD. Sumo-1 modification regu- lates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor. J Biol Chem.2001; 276: 18513-18518. PMID: 11278381
Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. M Gostissa, A Hengstermann, V Fogal, P Sandy, S E Schwarz, Del Sal, G , 10562558EMBO J. 18Gostissa M, Hengstermann A, Fogal V, Sandy P, Schwarz SE, Del Sal G. Activation of p53 by conjuga- tion to the ubiquitin-like protein SUMO-1. EMBO J. 1999; 18: 6462-6471. PMID: 10562558
SUMO junction-what's your function? New insights through SUMO-interacting motifs. O Kerscher, 17545995EMBO Rep. 8Kerscher O. SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep. 2007; 8: 550-555. PMID: 17545995
Acetylation and deacetylation of non-histone proteins. M A Glozak, N Sengupta, X Zhang, E Seto, 16289629Gene. 363Glozak MA, Sengupta N, Zhang X, Seto E. Acetylation and deacetylation of non-histone proteins. Gene. 2005; 363:15-23. PMID: 16289629
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Regulation of MEF2 by histone deacetylase 4-and SIRT1 deacetylase-mediated lysine modifications. X Zhao, T Sternsdorf, T A Bolger, R M Evans, T P Yao, 16166628Mol Cell Biol. 25Zhao X, Sternsdorf T, Bolger TA, Evans RM, Yao TP. Regulation of MEF2 by histone deacetylase 4- and SIRT1 deacetylase-mediated lysine modifications. Mol Cell Biol. 2005; 25: 8456-8464. PMID: 16166628
Regulation of the oncoprotein KLF8 by a switch between acetylation and SUMOylation. A M Urvalek, H Lu, X Wang, T Li, L Yu, J Zhu, 21416054Am J Transl Res. 3Urvalek AM, Lu H, Wang X, Li T, Yu L, Zhu J. Regulation of the oncoprotein KLF8 by a switch between acetylation and SUMOylation. Am J Transl Res. 2011; 3: 121-132. PMID: 21416054
Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response. Y-L Hsieh, H-Y Kuo, C-C Chang, M T Naik, P-H Liao, C C Ho, 10.1038/emboj.2013.523395904EMBO J. 325Hsieh Y-L, Kuo H-Y, Chang C-C, Naik MT, Liao P-H, Ho CC, et al. Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response. EMBO J. 2013; 32: 791-804. doi: 10.1038/emboj. 2013.5 PMID: 23395904
Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1. Y H Kim, C Y Choi, Y Kim, 10535925Proc Natl Acad Sci USA. 96Kim YH, Choi CY, Kim Y. Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1. Proc Natl Acad Sci USA. 1999; 96: 12350-12355. PMID: 10535925
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| [
"Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability."
] | [
"Adelma Escobar-Ramirez ",
"Anne-Sophie Vercoutter-Edouart ",
"Marlène Mortuaire ",
"Isabelle Huvent ",
"Stephan Hardivillé ",
"Esthelle Hoedt ¤bTony Lefebvre ",
"Annick Pierce *[email protected] ",
"\n¤a Current address: Department of Biological Chemistry\nUMR 8576\nUnité de Glycobiologie Structurale et Fonctionnelle\nCNRS\nUniversité des Sciences et Technologies de Lille\nCNRS FRABio\nFR3688Villeneuve d'AscqFrance\n",
"\nSchool of Medicine\n¤b Current address: Department of Biochemistry and Molecular Pharmacology\nSkirball Institute of Biomolecular Medicine\nJohns Hopkins University\nBaltimore, New YorkMaryland, New YorkUnited States of America, United States of America\n"
] | [
"¤a Current address: Department of Biological Chemistry\nUMR 8576\nUnité de Glycobiologie Structurale et Fonctionnelle\nCNRS\nUniversité des Sciences et Technologies de Lille\nCNRS FRABio\nFR3688Villeneuve d'AscqFrance",
"School of Medicine\n¤b Current address: Department of Biochemistry and Molecular Pharmacology\nSkirball Institute of Biomolecular Medicine\nJohns Hopkins University\nBaltimore, New YorkMaryland, New YorkUnited States of America, United States of America"
] | [
"Adelma",
"Anne-Sophie",
"Marlène",
"Isabelle",
"Stephan",
"Tony",
"Annick"
] | [
"Escobar-Ramirez",
"Vercoutter-Edouart",
"Mortuaire",
"Huvent",
"Hardivillé",
"Lefebvre",
"Pierce"
] | [
"C Mariller, ",
"S Hardivillé, ",
"E Hoedt, ",
"I Huvent, ",
"S Pina-Canseco, ",
"A Pierce, ",
"P L Masson, ",
"J F Heremans, ",
"E Schonne, ",
"P D Siebert, ",
"B C Huang, ",
"D Liu, ",
"X Wang, ",
"Z Zhang, ",
"C T Teng, ",
"M Benaïssa, ",
"J Peyrat, ",
"L Hornez, ",
"C Mariller, ",
"J Mazurier, ",
"A Pierce, ",
"E Hoedt, ",
"S Hardivillé, ",
"C Mariller, ",
"E Elass, ",
"J P Perraudin, ",
"A Pierce, ",
"T J Panella, ",
"Y H Liu, ",
"A T Huang, ",
"C T Teng, ",
"C T Teng, ",
"W Gladwell, ",
"I Raphiou, ",
"E Liu, ",
"C Mariller, ",
"M Benaïssa, ",
"S Hardivillx, ",
"M Breton, ",
"G Pradelle, ",
"J Mazurier, ",
"H M Baker, ",
"E N Baker, ",
"M Breton, ",
"C Mariller, ",
"M Benaïssa, ",
"K Caillaux, ",
"E Browaeys, ",
"M Masson, ",
"S Hardivillé, ",
"A Escobar-Ramirez, ",
"S Pina-Canceco, ",
"E Elass, ",
"A Pierce, ",
"C Mariller, ",
"S Hardivillé, ",
"E Hoedt, ",
"M Benaïssa, ",
"J Mazurier, ",
"A Pierce, ",
"E Hoedt, ",
"K Chaoui, ",
"I Huvent, ",
"C Mariller, ",
"B Monsarrat, ",
"O Burlet-Schiltz, ",
"B Kim, ",
"S Kang, ",
"S J Kim, ",
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"E Hoedt, ",
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"M Benaïssa, ",
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"J S Seeler, ",
"A Dejean, ",
"E S Johnson, ",
"J R Gareau, ",
"C D Lima, ",
"A Verger, ",
"J Perdomo, ",
"M Crossley, ",
"S H Yang, ",
"A D Sharrocks, ",
"G Gill, ",
"E T Yeh, ",
"M S Rodriguez, ",
"C Dargemont, ",
"R T Hay, ",
"D A Sampson, ",
"M Wang, ",
"M J Matunis, ",
"S H Yang, ",
"A Galanis, ",
"J Witty, ",
"A D Sharrocks, ",
"V Hietakangas, ",
"J Anckar, ",
"H A Blomster, ",
"M Fujimoto, ",
"J J Palvimo, ",
"A Nakai, ",
"I Matic, ",
"J Schimmel, ",
"I A Hendriks, ",
"M A Van Santen, ",
"F Van De Rijke, ",
"H Van Dam, ",
"D Y Lin, ",
"Y S Huang, ",
"J C Jeng, ",
"H Y Kuo, ",
"C C Chang, ",
"T T Chao, ",
"C C Chang, ",
"M T Naik, ",
"Y S Huang, ",
"J C Jeng, ",
"P H Liao, ",
"H Y Kuo, ",
"M H Tatham, ",
"E Jaffray, ",
"O A Vaughan, ",
"Jmp Desterro, ",
"C H Botting, ",
"J H Naismith, ",
"I Matic, ",
"M Van Hagen, ",
"J Schimmel, ",
"B Macek, ",
"S C Ogg, ",
"M H Tatham, ",
"M H Tatham, ",
"M-C Geoffroy, ",
"L Shen, ",
"A Plechanovova, ",
"N Hattersley, ",
"E G Jaffray, ",
"F Z Watts, ",
"J M Desterro, ",
"M S Rodriguez, ",
"R T Hay, ",
"M Miteva, ",
"K Keusekotten, ",
"K Hofmann, ",
"J K Gerrit, ",
"Praefcke Rdohmen, ",
"R J , ",
"F Aillet, ",
"F Lopitz-Otsoa, ",
"I Egaña, ",
"R Hjerpe, ",
"P Fraser, ",
"R T Hay, ",
"A Shalizi, ",
"B Gaudillière, ",
"Z Yuan, ",
"J Stegmüller, ",
"T Shirogane, ",
"Q Ge, ",
"N Stankovic-Valentin, ",
"S Deltour, ",
"J Seeler, ",
"S Pinte, ",
"G Vergoten, ",
"C Guérardel, ",
"Y Fu, ",
"Z Fang, ",
"Y Liang, ",
"X Zhu, ",
"P Prins, ",
"Z Li, ",
"R Yonashiro, ",
"A Sugiura, ",
"M Miyachi, ",
"T Fukuda, ",
"N Matsushita, ",
"Y Ogata, ",
"Y Xue, ",
"A Li, ",
"X Yao, ",
"J Song, ",
"Z Zhang, ",
"W Hu, ",
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"F Melchior, ",
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"M Gostissa, ",
"A Hengstermann, ",
"V Fogal, ",
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"S E Schwarz, ",
"Del Sal, ",
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"M A Glozak, ",
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"X Zhang, ",
"E Seto, ",
"X J Yang, ",
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"S W Rogers, ",
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"Z Wang, ",
"G W Hart, ",
"R Deplus, ",
"B Delatte, ",
"M K Schwinn, ",
"M Defrance, ",
"J Mendez, ",
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"C Guinez, ",
"A M Mir, ",
"V Dehennaut, ",
"R Cacan, ",
"A Harduin-Lepers, ",
"J C Michalski, ",
"R Fujiki, ",
"W Hashiba, ",
"H Sekine, ",
"A Yokoyama, ",
"T Chikanishi, ",
"S Ito, ",
"F Capotosti, ",
"S Guernier, ",
"F Lammers, ",
"P Waridel, ",
"Y Cai, ",
"J Jin, ",
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"H B Ruan, ",
"M E Hughes, ",
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"G S , "
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] | [
"Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor. C Mariller, S Hardivillé, E Hoedt, I Huvent, S Pina-Canseco, A Pierce, 10.1139/o11-07022320386Biochem Cell Biol. 90Mariller C, Hardivillé S, Hoedt E, Huvent I, Pina-Canseco S, Pierce A. Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor. Biochem Cell Biol. 2012; 90: 307-319. doi: 10. 1139/o11-070 PMID: 22320386",
"Lactoferrin, an iron-binding protein in neutrophilic leukocytes. P L Masson, J F Heremans, E Schonne, 4979954J Exp Med. 130Masson PL, Heremans JF, Schonne E. Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J Exp Med. 1969; 130: 643-658. PMID: 4979954",
"Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines. P D Siebert, B C Huang, 9122171Proc Natl Acad Sci. 94Siebert PD, Huang BC. Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines. Proc Natl Acad Sci USA 1997; 94: 2198-2203. PMID: 9122171",
"An intronic alternative promoter of the human lactoferrin gene is activated by Ets. D Liu, X Wang, Z Zhang, C T Teng, 12565886Biochem Biophys Res Commun. 301Liu D, Wang X, Zhang Z, Teng CT. An intronic alternative promoter of the human lactoferrin gene is acti- vated by Ets. Biochem Biophys Res Commun. 2003; 301: 472-479. PMID: 12565886",
"Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer. M Benaïssa, J Peyrat, L Hornez, C Mariller, J Mazurier, A Pierce, 15543612Int J Cancer. 114Benaïssa M, Peyrat J, Hornez L, Mariller C, Mazurier J, Pierce A. Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer. Int J Cancer. 2005; 114: 299-306. PMID: 15543612",
"Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR. E Hoedt, S Hardivillé, C Mariller, E Elass, J P Perraudin, A Pierce, 10.1007/s10534-010-9305-5Biometals. 23Hoedt E, Hardivillé S, Mariller C, Elass E, Perraudin JP, Pierce A.. Discrimination and evaluation of lac- toferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR. Biometals. 2010; 23: 441-452. doi: 10.1007/s10534-010-9305-5 PMID: 20155437",
"Polymorphism and altered methylation of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer Cancer Res. T J Panella, Y H Liu, A T Huang, C T Teng, 167444851Panella TJ, Liu YH, Huang AT, Teng CT. Polymorphism and altered methylation of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer Cancer Res. 1991; 51: 3037-3043. PMID: 1674448",
"Methylation and expression of the lactoferrin gene in human tissues and cancer cells. C T Teng, W Gladwell, I Raphiou, E Liu, 15222484Biometals. 17Teng CT, Gladwell W, Raphiou I, Liu E. Methylation and expression of the lactoferrin gene in human tis- sues and cancer cells. Biometals. 2004; 17: 317-323. PMID: 15222484",
"Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression. C Mariller, M Benaïssa, S Hardivillx, M Breton, G Pradelle, J Mazurier, 17371504FEBS J. 274Mariller C, Benaïssa M, Hardivillx S, Breton M, Pradelle G, Mazurier J, et al. Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression. FEBS J. 2007; 274: 2038-2053. PMID: 17371504",
"A structural perspective on lactoferrin function. H M Baker, E N Baker, 10.1139/o11-071Biochem Cell Biol. 90Baker HM, Baker EN. A structural perspective on lactoferrin function. Biochem Cell Biol. 2012; 90: 320- 328. doi: 10.1139/o11-071 PMID: 22292559",
"Expression of delta-lactoferrin induces cell cycle arrest. M Breton, C Mariller, M Benaïssa, K Caillaux, E Browaeys, M Masson, 15222485Biometals. 17Breton M, Mariller C, Benaïssa M, Caillaux K, Browaeys E, Masson M, et al. Expression of delta-lacto- ferrin induces cell cycle arrest. Biometals. 2004; 17: 325-329. PMID: 15222485",
"Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating Bax expression. S Hardivillé, A Escobar-Ramirez, S Pina-Canceco, E Elass, A Pierce, 10.1007/s10534-014-9744-5Biometals. 27Hardivillé S, Escobar-Ramirez A, Pina-Canceco S, Elass E, Pierce A. Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating Bax expression, Biometals. 2014; 27: 875-889. doi: 10.1007/s10534-014-9744-5 PMID: 24824995",
"Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme. C Mariller, S Hardivillé, E Hoedt, M Benaïssa, J Mazurier, A Pierce, 10.1016/j.biochi.2008.07.00918725266Biochimie. 91Mariller C, Hardivillé S, Hoedt E, Benaïssa M, Mazurier J, Pierce A. Proteomic approach to the identifi- cation of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decap- ping enzyme. Biochimie. 2009; 91: 109-122. doi: 10.1016/j.biochi.2008.07.009 PMID: 18725266",
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] | [
"Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor",
"Lactoferrin, an iron-binding protein in neutrophilic leukocytes",
"Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines",
"An intronic alternative promoter of the human lactoferrin gene is activated by Ets",
"Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer",
"Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR",
"Methylation and expression of the lactoferrin gene in human tissues and cancer cells",
"Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression",
"A structural perspective on lactoferrin function",
"Expression of delta-lactoferrin induces cell cycle arrest",
"Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating Bax expression",
"Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme",
"SILAC-based proteomic profiling of the human MDA-MB-231 metastatic breast cancer cell line in response to the two antitumoral lactoferrin isoforms: the secreted lactoferrin and the intracellular delta-lactoferrin",
"Genome-wide pathway analysis reveals different signaling pathways between secreted lactoferrin and intracellular delta-lactoferrin",
"Nutrient Regulation of Signaling, Transcription, and Cell Physiology by O-GlcNAcylation",
"O-GlcNAcylation/ phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin",
"Nuclear and unclear functions of SUMO",
"Protein modification by SUMO",
"The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition",
"Modification with SUMO. A role in transcriptional regulation",
"SUMO promotes HDAC-mediated transcriptional repression",
"Something about SUMO inhibits transcription",
"SUMOylation and De-SUMOylation: wrestling with life's processes",
"SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting",
"The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification",
"An extended consensus motif enhances the specificity of substrate modification by SUMO",
"PDSM, a motif for phosphorylation-dependent SUMO modification",
"Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif",
"Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of SUMOylated transcription factors",
"Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation",
"Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9",
"In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy",
"RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation",
"Starting and stopping SUMOylation",
"SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation",
"Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity",
"A calcium-regulated MEF2 SUMOylation switch controls postsynaptic differentiation",
"An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity",
"Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway",
"Mitochondrial ubiquitin ligase MITOL ubiquitinates mutant SOD1 and attenuates mutant SOD1-induced reactive oxygen species generation",
"Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation",
"SUMOylation: A Regulatory Protein Modification in Health and Disease",
"GPS-SBM 1.0: a stand-alone program for prediction of SUMO-binding motifs",
"Phospho-regulated SUMO interaction modules connect the SUMO system to CK2 signaling",
"Concepts in SUMOylation: a decade on",
"Dual regulation of c-Myc by p300 via acetylationdependent control of Myc protein turnover and coactivation of Myc-induced transcription",
"Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase",
"A post-translational modification code for transcription factors: sorting through a sea of signals",
"Jun and p53 are modulated by SUMO-1 modification",
"Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1)",
"Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor",
"Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1",
"SUMO junction-what's your function? New insights through SUMO-interacting motifs",
"Acetylation and deacetylation of non-histone proteins",
"Lysine acetylation: codified crosstalk with other posttranslational modifications",
"Regulation of MEF2 by histone deacetylase 4-and SIRT1 deacetylase-mediated lysine modifications",
"Regulation of the oncoprotein KLF8 by a switch between acetylation and SUMOylation",
"Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response",
"Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1",
"PEST sequences and regulation by proteolysis",
"Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code",
"TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS",
"Protein ubiquitination is modulated by O-GlcNAc glycosylation",
"GlcNAcylation of histone H2B facilitates its monoubiquitination",
"O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1",
"O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination",
"Regulation of p53 activity through lysine methylation"
] | [
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"Polymorphism and altered methylation of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer Cancer Res",
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"Nature"
] | [
"\n\ndoi:10.1371/journal.pone.0129965.t001",
"\nFig 1 .\n1Fig 1. ΔLf is modified by SUMOylation. A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by posttranslational modifications are shown, S10 as the main O-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ! 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ! 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ! 3).",
"\n\ndoi:10.1371/journal.pone.0129965.t002",
"\nFig 2 .\n2Mapping of the SUMO modification sites of ΔLf. A) K13, K308 and K379 are SUMO-1 acceptor sites. Cells were co-transfected by WT or the mutant constructs and pSG5-His-SUMO-1 or pcDNA3.1-His-SUMO-2/3 plasmids and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control.",
"\nFig 3 .\n3Competition between SUMOylation and ubiquitination at K379 controls ΔLf turnover. A) HEK-293 cells were co-transfected with K379 or NV constructs, His-SUMO-1 or/and HA-Ub-expression vectors for 24 h and then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. Total cell extracts were immunoprecipitated with M2 or used as input. Samples were immunoblotted with anti-HA (upper panel) or with anti-SUMO-1 (lower panel) antibodies. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control. NS: non-specific. The data presented correspond to one representative experiment of at least three conducted (n ! 3). Lane 6 corresponds to non-transfected cells. B) Cells were transfected with K379, either with the His-SUMO-1 or the HA-Ub expression vector and then incubated with fresh medium supplemented by 10 μg.mL -1 CHX for the indicated time 24 h after transfection. K379 transfected cells were incubated without (left panel) or with (right panel) 10 μM MG132 for 2 h prior to lysis. Total protein extracts were immunoblotted with either M2 or anti-GAPDH antibodies. Detection was carried out using a Fusion SOLO camera (Vilbert Lourmat). The data presented (B) correspond to one representative experiment of at least five conducted. C-D) The M2 densitometric analyses are normalized for the matching GAPDH immunoblots and expressed as ratio D K379 / D GAPDH as described in Materials and Methods. Data are shown as the means ± SD (n = 5).doi:10.1371/journal.pone.0129965.g003",
"\nFig 4 .\n4Fig 4. SUMOylation of ΔLf represses its transcriptional activity. A) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ! 6) (B) and to one representative experiment of at least two conducted (n ! 2) (C, D). E) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*), p < 0.01 (**)).",
"\nFig 5 .\n5A SUMOylation/acetylation switch at K13 controls ΔLf transcriptional activity. (A) K13 is the main acetylation site. Cells were co-transfected by WT, the mutant constructs or the null vector and then lysed 24 h later. Lysates were immunoprecipitated with anti-acetyllysine antibodies and immunoblotted with M2. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control (n =",
"\n\n. Schematic representation of the two series of ΔLf SUMO mutant constructs. (TIF)",
"\nTable 1 .\n1SUMO predictive motifs in human ΔLf.SUMO motif a \n",
"\nTable 2 .\n2SUMO Interacting Motifs in human ΔLf and Lf from different species compared to the SIMr consensus (D/E)"
] | [
"doi:10.1371/journal.pone.0129965.t001",
"Fig 1. ΔLf is modified by SUMOylation. A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by posttranslational modifications are shown, S10 as the main O-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ! 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ! 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ! 3).",
"doi:10.1371/journal.pone.0129965.t002",
"Mapping of the SUMO modification sites of ΔLf. A) K13, K308 and K379 are SUMO-1 acceptor sites. Cells were co-transfected by WT or the mutant constructs and pSG5-His-SUMO-1 or pcDNA3.1-His-SUMO-2/3 plasmids and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control.",
"Competition between SUMOylation and ubiquitination at K379 controls ΔLf turnover. A) HEK-293 cells were co-transfected with K379 or NV constructs, His-SUMO-1 or/and HA-Ub-expression vectors for 24 h and then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. Total cell extracts were immunoprecipitated with M2 or used as input. Samples were immunoblotted with anti-HA (upper panel) or with anti-SUMO-1 (lower panel) antibodies. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control. NS: non-specific. The data presented correspond to one representative experiment of at least three conducted (n ! 3). Lane 6 corresponds to non-transfected cells. B) Cells were transfected with K379, either with the His-SUMO-1 or the HA-Ub expression vector and then incubated with fresh medium supplemented by 10 μg.mL -1 CHX for the indicated time 24 h after transfection. K379 transfected cells were incubated without (left panel) or with (right panel) 10 μM MG132 for 2 h prior to lysis. Total protein extracts were immunoblotted with either M2 or anti-GAPDH antibodies. Detection was carried out using a Fusion SOLO camera (Vilbert Lourmat). The data presented (B) correspond to one representative experiment of at least five conducted. C-D) The M2 densitometric analyses are normalized for the matching GAPDH immunoblots and expressed as ratio D K379 / D GAPDH as described in Materials and Methods. Data are shown as the means ± SD (n = 5).doi:10.1371/journal.pone.0129965.g003",
"Fig 4. SUMOylation of ΔLf represses its transcriptional activity. A) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ! 6) (B) and to one representative experiment of at least two conducted (n ! 2) (C, D). E) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*), p < 0.01 (**)).",
"A SUMOylation/acetylation switch at K13 controls ΔLf transcriptional activity. (A) K13 is the main acetylation site. Cells were co-transfected by WT, the mutant constructs or the null vector and then lysed 24 h later. Lysates were immunoprecipitated with anti-acetyllysine antibodies and immunoblotted with M2. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control (n =",
". Schematic representation of the two series of ΔLf SUMO mutant constructs. (TIF)",
"SUMO predictive motifs in human ΔLf.",
"SUMO Interacting Motifs in human ΔLf and Lf from different species compared to the SIMr consensus (D/E)"
] | [
"Fig 1A)",
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"(Fig 5B and 5D"
] | [
"V/C L/V I/V V-E [37].",
"Human ΔLf b 350 S T T E D C I A L V L K G E 363 Q5EKS1 Bovine Lf c 375 S T T D D C I V L V L K G E 388 P24627 Goat Lf 375 S T T D D C I A L V L K G E 388 Q29477 Mouse Lf 373 P T T E D C I V A I M K G D 386 P08071 Pig Lf 371 S T T E D C I V Q V L K G E 384 P14632 Horse Lf 375 S T T E E C I A L V L K G E 388 O77811 Sheep Lf 375 S T T D E C I A L V L K G E 388 AY792499 Camel Lf 375 S T T E D C I A L V L K G E 388 AJ131674 a"
] | [
"Lactoferrins exist as different variants due to gene polymorphisms, post-transcriptional and post-translational modifications [1]. The two main isoforms are secreted lactoferrin (Lf) and its nucleocytoplasmic counterpart, delta-lactoferrin (ΔLf) [2,3,4]. Their expression is downregulated or silenced in cancer cells [3,5]. In breast cancers, significantly lower levels of Lf and/or ΔLf correlated with more advanced disease and an unfavorable prognosis [5,6]. This downregulation is mainly due to genetic and epigenetic modifications that have been found on the Lf gene in some forms of cancer [7,8]. ΔLf mRNAs derive from the transcription of the Lf gene at the alternative P2 promoter leading, after translation, to a 73 kDa intracellular protein [4]. Although its subcellular distribution is mainly cytoplasmic, confocal microscopy analyses have clearly shown that ΔLf targets the nucleus [4]. Thus, we showed that ΔLf possesses a functional bipartite NLS motif in the C-terminal lobe [9]. ΔLf is capable of binding DNA but the location of its DNA binding domain is not known. Two regions of Lf in which a strong concentration of positive charges were found could be good candidates [10]. ΔLf exhibits antitumoral activities and we previously showed that overexpression of ΔLf leads to cell cycle arrest at the G1/S transition and apoptosis [11,12]. ΔLf mainly exerts its anti-proliferative and pro-apoptotic activities via its role as a transcription factor. Indeed, ΔLf transactivates different target genes such as Skp1, DcpS, Bax, SelH, GTF2F2 and UBE2E1 [9,[12][13][14]. A genome-wide pathway analysis and our quantitative proteomic analysis showed that the re-introduction of Lf isoforms in cancerous cells modified essential genes and/or signaling networks responsible mainly for cell survival, apoptosis and RNA processing [14,15].",
"Since ΔLf has a variety of target genes and is involved in the control of cell homeostasis, modifications in its activity or concentration may have profound consequences. Its transcriptional activity is controlled by posttranslational modifications (PTM) among which O-GlcNAcylation is a key link between nutrient sensing and signaling. It notably regulates gene activation due to O-GlcNAc cycling on gene-specific transcription factors and components of the basal transcriptional machinery (reviewed in [16]). The targeting of serine S10 by O-GlcNAcylation negatively regulates ΔLf transcriptional activity whereas phosphorylation increases it [17]. Deglycosylation leads to DNA binding and a basal transactivation level which was markedly enhanced when phosphorylation was present at S10. ΔLf possesses a functional PEST sequence which drives the protein to its proteasomal degradation after polyubiquitination of the K379 and/or K391 lysine residues. ΔLf stability is also under the control of O-GlcNAcylation. Indeed, O-GlcNAcylation at S10 protects ΔLf from polyubiquitination increasing its half-life, whereas phosphorylation favours its proteasomal degradation [17].",
"Recently, we discovered that ΔLf is also modified by SUMOylation. The small ubiquitinrelated modifier (SUMO) is involved in many aspects of cell function and affects pathways as diverse as DNA repair, cell cycle, transcriptional regulation, RNA processing, and cell signaling [18,19]. At a molecular level, SUMOylation of target proteins alters their protein-protein interactions, localization, stability or/and activity [20]. Many transcription factors are targeted by SUMOylation and in most cases SUMOylation triggers transcriptional repression by recruiting transcriptional co-repressors, such as histone deacetylases [21][22][23]. Four different SUMO isoforms (SUMO-1-4) have been identified in higher eukaryotes, although only SUMO-1-3 seem to be covalently attached to proteins. SUMO-2 and SUMO-3 share 96% identity, and both have approximately 46% identity with SUMO-1. The attachment of SUMO is a multi-step process analogous to that of ubiquitin. Thus, the SUMO pathway is mediated by SUMO-activating enzymes (E1), a unique SUMO-conjugating enzyme (E2) called Ubc9 and SUMO-ligases (E3). SUMOylation is a highly dynamic process which can be reversed by the activity of SUMO-specific isopeptidases (SENPs) [24].",
"SUMOs are conjugated to lysine residues in a CKXE/D sequence where C is a large hydrophobic amino acid residue and X represents any amino acid [25]. This motif is sufficient by itself to mediate a direct interaction with Ubc9 [25,26]. Extended SUMO consensus motifs such as the negatively charged amino acid-dependent SUMO motif (NDSM) constituted by an acidic patch downstream of the CKXE/D motif, the phosphorylation-dependent SUMO motif (PDSM) that includes a phosphorylation site downstream of the consensus core motif and the hydrophobic cluster SUMOylation motif (HCSM) that contains several hydrophobic residues located N-terminal to the core motif have been described to promote substrate SUMOylation via additional interaction with Ubc9 [27][28][29]. Moreover, several proteins are also modified at other sites and until now it is not known how these non-consensus sites are recognized. However, substrates with a SUMO-interacting motif (SIM) could be SUMOylated within a nonconsensus SUMO motif [30] and, as shown for the Death domain-associated protein 6 Daxx, phosphorylation of SIMs enhances SUMO-1 binding and conjugation [31]. SUMO-1 can be attached either to a single or to multiple lysine residues within a target protein leading either to mono-or multi-SUMOylation respectively, whereas chain formation is attributed to SUMO-2/ 3 [32]. However, [27] identified the human Topoisomerase I as a poly-SUMO-1 target. On the other hand, SUMO-1 may be attached to lysine residues within SUMO-2/3 chains, thereby preventing their elongation and acting therefore as a SUMO chain terminator [32,33]. Recently, mixed SUMO/ubiquitin chains have been reported [34].",
"Crosstalk between the SUMOylation, ubiquitination, and acetylation pathways is crucial for the regulation of protein activity and/or stability since these modifications may have different, sometimes opposing consequences [35]. Thus, SUMOylation can stabilize proteins by competing with ubiquitin [36,37]. However, heterogeneous SUMO2/3-ubiquitin chains were found on IκBα and PLM (promyelocytic leukemia) protein, contributing to their optimal proteosomal degradation [38]. Switches between SUMOylation and acetylation have also been reported for several proteins. SUMOylation of the myocyte-specific enhancer factor 2A (MEF2A) inhibits its transcriptional activity whereas acetylation increases it [39]. A similar SUMO to acetyl switch has also been described for the hypermethylated in cancer 1 protein (HIC1) [40].",
"Here we demonstrate that the stability and transcriptional activity of ΔLf are regulated by SUMOylation, which provides a novel regulatory mechanism for controlling ΔLf function. We identified the major SUMO and acetylation acceptor sites and we evaluated the impact of the SUMOylation/ubiquitin and the SUMOylation/acetylation interplays.",
"Cell culture, transfection, and reagents HEK-293 cells (ATC CRL-1573) were grown in monolayers and transfected (1 μg of DNA for 1 x 10 6 cells) using DreamFect (OZ Biosciences, Marseille, France) as described [17].The amounts of ΔLf expression vectors were adjusted to maintain ΔLf amounts similar to those found in normal breast epithelial NBEC cells [5,6]. Transfections were done in triplicate (n ! 5). Cell viability was assessed by counting using Trypan blue 0.4% (v/v). To measure the ΔLf turnover rate indirectly, we performed incubations with cycloheximide, a potent inhibitor of de novo protein synthesis [41,42]. Cells were transfected with either ΔLf (WT), the SUMO mutant constructs or null vector (NV) then incubated with fresh medium supplemented by 10 μg/mL cycloheximide (CHX) for 0-150 min 24 h post transfection as described [17]. Inhibition of proteasome was performed by incubating cells with a 10 μM concentration of the proteasomal inhibitor MG132 for 2 h prior to lysis as described [17]. Inhibition of histone deacetylases was performed by incubating cells with Trichostatin A (TSA) at 15 ng/mL (TSA treated cells) or not overnight. Cell culture reagents were from Lonza. Other reagents were from Sigma.",
"pGL3-S1 Skp1 -Luc [9] and p3xFLAG-CMV10-ΔLf (WT) [17] were constructed as described. p3xFLAG-CMV10 (Sigma, St Louis, MO, USA) was used as a null vector (NV). The hemagglutinin A-Ubiquitin (HA-Ub) expression vector was a gift from Dr. C. Couturier (UMR-CNRS 8161, IBL, Lille, France). The psG5-His-SUMO-1 (His-SUMO-1), the pcDNA3.1-His-SUMO-2/3 (SUMO2/3) and the pcDNA3-SENP2-SV5 (SENP2) expression vectors were kind gifts from Dr. D. Leprince (UMR-CNRS 8161, IBL, Lille, France). All plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen Germantown, MD) according to the manufacturer's specifications.",
"Mutants were generated using the QuikChange Site-directed Mutagenesis Kit (Stratagene, Garden Grove, CA) according to the manufacturer's instructions with p3xFLAG-CMV10-ΔLf as template and primer pairs listed in S1 Table. The constructs in which several sites were mutated were done sequentially. Following sequence verification, positive clones were used directly in transfection.",
"HEK-293 cells (2 x 10 6 cells in 100-mm dish) were transfected with RNAiMax (Life Technologies), according to the manufacturer's instructions, using 5 nM of siRNAs targeting Ubc9 (Hs_UBE2I_8 FlexiTube siRNA, Qiagen) or a scrambled control sequence (siCtrl) (Qiagen). Cells were harvested 48 h post-transfection and lysed. Cell extracts were assayed for Ubc9 content and SUMOylation levels.",
"Reporter gene assays were performed using the pGL3-S1 Skp1 -Luc reporter vector containing a single ΔLfRE and the ΔLf-expression vector (WT), different ΔLf SUMO mutant constructs or a null vector (NV). HEK-293 cells were synchronized overnight in medium containing 1% FCS before being transfected (250 ng of DNA for 2 x 10 5 cells: 50 ng of reporter vector and 200 ng of ΔLf, SUMO mutants or null vector) using DreamFect (OZ Biosciences, Marseille, France) as described in [17]. Reporter gene assays were also performed in the presence of psG5-His-SUMO-1 (200 ng of DNA) or either in the presence of pcDNA3-SENP2-SV5 expression vectors (200 ng of DNA) or TSA (15 ng/mL, overnight) with their respective controls. Reporter gene assays on Ubc9 knockdown cells were performed in two steps. Cells were siUbc9/siCtrl (5 nM) transfected in serum-free medium which was supplemented 4 h post transfection with 1% FCS. Twenty hours later cells were transfected with WT or mutant constructs and the reporter gene. Cell lysates were assayed using a luciferase assay kit (Promega) in a Tristar multimode microplate reader LB 941 (Berthold Technologies, Bad Wildbab, Germany). Basal luciferase expression was assayed using a null vector and was determined for each condition. Relative luciferase activities were normalized to basal luciferase expression and ΔLf content as in [12] and expressed as a percentage; 100% corresponds to the relative luciferase activity of WT. Each experiment represents at least three sets of independent triplicates.",
"Proteins were extracted from frozen cell pellets in RIPA buffer as described [9]. In order to inhibit de-SUMOylation of proteins, N-Ethylmaleimide (NEM) was added at 20 mM to lysis, Western blot (WB) and immunoprecipitation (IP) buffers. For direct immunoblotting, samples mixed with 4x Laemmli buffer were boiled for 5 min. Otherwise 10 μg of protein from each sample or immunocomplexes were submitted to 6% SDS-PAGE for IP, 7.5% SDS-PAGE for input and 12.5% SDS-PAGE for Ubc9 Western blot prior to immunoblotting. For immunoprecipitation experiments, 1 or 1.5 mg of total protein were preabsorbed with 20 μL protein G Sepharose 4 Fast Flow (GE Healthcare). Anti-3XFLAG M2 (1/500), anti-acetyllysine (1/1000) or anti-SUMO-1 (1/100) antibodies were mixed with 40 μL Protein G Sepharose beads for 1 h prior to an overnight incubation with the preabsorbed lysate supernatant at 4°C. The beads were then washed five times with lysis buffer (4 washings with RIPA, 1 washing with RIPA/ NaCl 5M: 9/1, v/v) and finally 1 washing in NET-2 (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Triton X-100) buffer. Proteins bound to the beads were eluted with 4X Laemmli buffer and analyzed by immunoblotting as above. Blots were first blocked in 5% non-fat milk for 1 h at room temperature prior being probed with primary antibodies (anti-3XFLAG M2, 1/2000; HA.11, 1/1000; anti-Ubc9, 1/1000; anti-His, 1/1000; anti-SUMO-1, 1/1000; anti-SUMO-2/3, 1/ 500; anti-acetyllysine, 1/1000; anti-GAPDH, 1/3000) overnight at 4°C and then probed with secondary anti-IgG antibodies conjugated to horseradish peroxidase (1/10000) for 1 h at room temperature before detection by chemiluminescence (ECL Advance or ECL Select, GE Healthcare Life Sciences). Each result in which immunoblots are presented corresponds to one representative experiment among at least three.",
"Antibodies against the 3xFLAG epitope (mouse monoclonal anti-FLAG M2 antibody, Sigma), HA epitope (mouse monoclonal HA.11 antibody 16B12, Covance Research Products), 6XHis epitope (mouse monoclonal anti-6XHis P5A11 antibody for WB, Biolegend; mouse monoclonal anti-His AD1.10 antibody for IP, Santa Cruz Biotechnology), SUMO-1 (rabbit monoclonal anti-SUMO-1, Millipore), SUMO-2/3 (rabbit polyclonal anti-SUMO-2/3, Millipore), Ubc9 (rabbit monoclonal antibody anti-Ubc9, Cell Signaling), acetyllysine (rabbit polyclonal anti-acetyllysine, ABCAM), GAPDH (rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody, Santa Cruz Biotechnologies) were used for immunoprecipitation and/or immunoblotting. Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare Life Sciences. All the antibodies were used according to the manufacturer's instructions.",
"The densitometric analysis was performed using the Quantity One v4.1 software (Bio-Rad, Hercules, CA) or ImageJ and statistical analyses were performed with PRISM 5 software (Graphpad, USA). M2 densitometric values were normalized to GAPDH and expressed as D M2 /D GAPDH . Means were statistically analysed using the t-test or ANOVA and differences assessed at p<0.05 ( Ã ) or p<0.01 ( ÃÃ ). SUMO-1 and acetyllysine densitometric values were expressed as D Ac /D SUMO with the WT ratio in the siCtrl condition arbitrarily set as 100%.",
"In silico analysis of the ΔLf sequence with SUMOsp (http://sumosp.biocuckoo.org/) and SUMOplot (htpp://www.abgent.com/tools/sumoplot/) softwares revealed four lysine residues to be putative SUMO acceptors: K13 and K361 that are in the canonical CKXE/D motifs and K308 and K391 in non-canonical SUMO sequences ( Table 1). The K13 consensus motif is of the PDSM-like type and the K308 motif of the NDSM-like type. K13 is within the first putative DBD and next to S10, the main O-GlcNAcylation/Phosphorylation site we previously demonstrated to control ΔLf transcriptional activity and stability ( Fig 1A) [17]. Interestingly, K13 is The single-letter amino acid code is used. c The numbering of the amino acid residues corresponds to human ΔLf. ΔLf is modified by SUMOylation. A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by posttranslational modifications are shown, S10 as the main O-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ! 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ! 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ! 3).",
"doi:10.1371/journal.pone.0129965.g001 also a putative acetylation site predicted using PAIL (prediction of acetylation on internal lysine [43] (http://bdmpail.biocuckoo.org/prediction.php). The other putative sites are concentrated in the central part of the protein, before the second DBD for K308, between this DBD and the PEST sequence for K361, and within the PEST sequence for K391 ( Fig 1A).",
"In the case of a SUMO consensus motif, the target lysine is directly recognized by the conjugating enzyme Ubc9 whereas in the case of a non-consensus sequence, the recruitment of SUMO-loaded Ubc9 is realized via interactions with a SIM motif, which increases the modification of proximal lysine residues [30,44,45]. Thus, the prediction of SIM motifs using the GPS-SBM 1.0 User Interface software [46] reveals the presence of one SIM motif within the central region of ΔLf, near to the K361 site ( Fig 1A). This motif is in a reversed orientation (SIMr) ( Table 2) with four hydrophobic positions preceded by an acidic cluster and by a serine residue. Phosphorylation of SIM-associated serine residues is known to favor efficient recognition of SUMO [37,47]. Comparison of this motif with homologs from a number of vertebrate species reveals that it is conserved in Lf/ΔLf ( Table 2). The functional significance of this motif and its role in ΔLf SUMOylation remain to be investigated.",
"The low abundance of ΔLf and the feeble percentage of SUMOylated conjugates rendered the detection of SUMOylated ΔLf and the subsequent mapping of its SUMO sites extremely difficult. Therefore we used a 3xFLAG-tagged ΔLf in order to detect it and we constructed a first series of SUMOylation mutants in which only one lysine residue was replaced at a time (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R ; S1 Table and S1 Fig left panel). Incubation of ΔLf and mutants with His-SUMO peptides caused the appearance of multiple higher molecular weight species indicative of SUMOylation events ( Fig 1B). Moreover, mutation of each individual lysine residue did not abolish SUMOylation of the entire molecule ( Fig 1B). Since competition between SUMO and ubiquitin ligases often occurs at ubiquitin sites, K379 which is the main ubiquitinated target on ΔLf [17] was also investigated. Thus, we produced a second series of mutant constructs in which only one putative SUMO site was preserved (S1 Table and S1 Fig right panel). We obtained five SUMO mutants named K13, K308, K361, K379 (which in fact corresponds to the M4S mutant) and K391, respectively and the M5S mutant in which all putative SUMOylation sites were abolished. ΔLf and its SUMOylation mutants were then expressed in HEK-293 cells which do not produce ΔLf endogeneously. We detected 3xFLAG-tagged ΔLf isoforms as a single band of the expected 75 kDa predicted molecular weight. The level of their 3 ",
"SUMO Interacting Motif a Accession number",
"The single-letter amino acid code is used; bold letters indicate the hydrophobic positions of the putative SUMO interacting motif, the acidic cluster is in italics, the SIM-associated serine residues are underlined.",
"b",
"The numbering of the amino acid residues corresponds to human ΔLf. c The numbering of the amino acid residues corresponds to Lfs. expression was compared and Fig 1C shows that they were expressed at least at the same level as WT. K361 and notably K379 were expressed at a higher level than the other mutants but statistical analyses showed that these differences were not significant.",
"SUMOylation was first investigated on WT and M5S which were co-transfected with or without the SUMO-1 expression vector. An immunoprecipitation was then performed using the anti-FLAG antibody in order to specifically immunoprecipitate ΔLf or its SUMO variants. SUMOylation was then investigated using anti-SUMO-1 antibodies. Fig 1D shows that ΔLf was effectively SUMOylated and that SUMOylation was slightly increased when it was incubated with components of the SUMO pathway such as SUMO-1 (lane 1). Multiple higher molecular weight bands which may correspond to multi-or poly-ΔLf-SUMOylated forms were observed. Taking into account the in silico studies, this SUMO pattern (lanes 1-2) suggested that at least four SUMOylation sites are occupied (corresponding to 86, 97, 108 and 119 kDa, as shown by asterisks) for WT.",
"The feeble amount of SUMO-conjugates ( Fig 1D, upper panel) compared to unmodified ΔLf (Fig 1D, middle panel) is in accordance with the literature. Thus, for most SUMOylated proteins, the levels of the SUMO forms are low relative to the unmodified form due to an efficient SUMOylation/deSUMOylation balance in cells [35].",
"M5S appeared not to be SUMOylated even when SUMO-1 was overexpressed suggesting that no other SUMO sites are present on the protein (Fig 1D, upper panel). Moreover, overexposure of this film failed to show additional bands that could suggest SUMOylation of the M5S construct (data not shown). Surprisingly, the M5S M2 immunoprecipitation signal is poor compared to the M5S M2 western blot signal. This could be due to the mutation of five lysine residues which may impair ΔLf conformation, resulting in poor immunoprecipitation even if IP was directed against 3XFLAG and not ΔLf itself.",
"In order to identify SUMO acceptor sites, SUMO mutants were co-transfected with the His-SUMO-1 expression vector. A band corresponding to ΔLf-SUMO-1 forms is visible in each lane confirming that the five predicted sites were effectively SUMOylated (Fig 2A, left panel). K13, K308 and K379 are the three main acceptor sites. Surprisingly, the K361 mutant which possesses a predictive SUMO sequence that perfectly fits the optimal CKXE consensus sequence was poorly modified, as was K391. Overexpression or not of SUMO-1 did not modify the SUMO profile of the latter site confirming that it is not preferentially targeted by the SUMO machinery (Fig 2A and 2B). K391 is also ubiquitinated but it was not the main ubiquitin target site [17]. Interestingly, K379 which is the main ubiquitination site is also a good acceptor of SUMO, even though this lysine residue does not belong to a SUMO consensus sequence.",
"In order to investigate whether some of the SUMO-1 target sites might also be SUMO-2/3 acceptor sites, SUMO mutants were co-transfected with the His-SUMO-2/3 expression vector. Fig 2A (right panel) shows that SUMO-2/3 peptides might also bind K13 but the amount of the K13-SUMO2/3 form is feeble. Further work has to be done in order to confirm that this modification occurs as well in vivo. Control of specificity of His antibodies has been added since these antibodies revealed bands above 100 kDa even when His-SUMO-1 was not overexpressed (Fig 2A, upper left panel, lanes 7-10).",
"We next tried to evaluate whether ΔLf could be SUMOylated in vivo. The presence of SUMO proteases (SENPs) within the cells and also in cell extracts poses a significant problem for the detection of SUMOylated proteins. Therefore, we used N-Ethylmaleimide (NEM), which blocks cysteine proteases, as a SENPs inhibitor. Since NEM is not cell permeable we used it during cell lysis and immunoprecipitation steps. ΔLf-expressing cell lysates were immunoprecipitated with M2 and SUMO forms immunodetected with anti-SUMO-1 antibodies (Fig 2B, right panel). Slow migrating bands of strong intensity were detected for WT (upper panel) which might be due to the fact that WT is multi-SUMOylated. SUMO forms were also observed for K13, K308, K361 and K379 mutants (Fig 2B, upper right panel). Mutants that are unable to be ubiquitinated such as K13, K308 and K361 seem better in promoting SUMOylation of ΔLf (lanes 2-4). The K391 mutant seems poorly or even not modified, like the M5S mutant, which strongly suggests that only four of the five sites are modified by SUMO peptides.",
"Immunodetection by M2 showed the presence of a ladder of bands for WT, K13 and K379, confirming possible multi-and/or poly-modification (Fig 2B, middle right panel). A slow migrating band around 90 kDa was visible for K308 and K361 confirming the existence of a mono-SUMO-conjugated form of ΔLf. On the other hand, the strong ladder pattern (Fig 2B, middle right panel) observed for WT and K379 may also be partially due to ubiquitination as already described [17], suggesting that a competition between SUMO and ubiquitin modifications may occur. Since K13 is unable to be ubiquitinated, ladder bars might correspond to polySUMOylation. Therefore, M2 immunoprecipitation followed by SUMO-2/3 immunodetection of WT and SUMO mutant cell extracts was performed, but we were unable to observe The data presented correspond to one representative experiment of at least three conducted (n ! 3). B-C) WT and its mutants are SUMOylated in vivo. WT and the SUMO mutant constructs were transfected for 24 h prior to lysis. Whole cell extracts were immunoprecipitated with M2 and immunoblotted with either anti-SUMO-1 antibodies or M2 (B). Reverse immunoprecipitation was also performed (C). 1% of the cell extract (input) was immunoblotted with either M2 or anti-GAPDH antibodies used as loading control. Inhibition of proteasomal degradation was performed by incubating cells with MG132 for 2 h prior to lysis and inhibition of de-SUMOylation was performed by adding NEM to lysis, IP and WB buffers as in Material and Methods (Fig 2A and 2B right panels, and C). Data presented in Fig 2B (left panel) were obtained in the absence of proteasome and SENP inhibitors. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa), the arrow corresponds to ΔLf. All the data presented correspond to one representative experiment of at least three conducted (n ! 3).",
"doi:10.1371/journal.pone.0129965.g002 SUMO-2/3 modifications on WT or its SUMO mutants (data not shown). Since K13 is also modified by SUMO-2/3 in vitro (Fig 2A, right panel), we will further investigate whether modification by SUMO-2/3 is relevant in vivo, then what impact a mixed SUMO chain could have on ΔLf activity and/or stability.",
"In order to confirm that endogeneous SUMOylation occurs on ΔLf we performed the same experiment in the absence of SENP and proteasome inhibitors. Fig 2A (left upper panel) effectively showed that no SUMOylation pattern was visible in those conditions. Fig 2C corresponds to the reverse immunoprecipitation of K13, K308, K361 and K379 cell lysates with anti-SUMO-1 antibodies followed by M2 immunodetection. A faint band corresponding to ΔLf-SUMO-1 is visible and a poly-SUMO pattern is observed for K379. Since mixed SUMO/ubiquitin chains could be formed we will further investigate whether ΔLf might be modified at K379 by such a complex.",
"Collectively these results indicated that ΔLf could be SUMOylated at multiple lysine residues and that the band shift of ΔLf was indeed due to the covalent attachment of SUMO-1 with K13 as hotspot of SUMOylation.",
"Since, as for many transcription factors, ΔLf is rapidly degraded, we previously demonstrated that its turnover was dependent on both the Ub-proteasome pathway and the O-GlcNAc/phosphate interplay [17]. We also characterized K379 as the major site for ΔLf poly-ubiquitination. Since K379 is also targeted by SUMO ligases we next investigated whether a crosstalk exists between the ubiquitin and SUMO pathways. Fig 3A shows that a ladder of polyubiquitinated K379 forms is visible in the presence of recombinant HA- Ubiquitin (upper panel, lane 3). The intensity of the polyubiquitination signal decreases when SUMO-1 peptides are overexpressed (Fig 3A upper panel, lane 4). After stripping, the immunoblot was revealed using anti-SUMO-1 antibodies (middle panel). Increased SUMOylation could be observed for the K379 mutant when SUMO-1 was overexpressed (Fig 3A, middle panel, lane 2) as already shown for WT in Fig 1D. We also observed a decrease in this SUMO signal in the presence of recombinant ubiquitin ( Fig 3A, middle panel, lanes 2 and 4). Loadings of K379 (input) confirmed that in the presence of recombinant ubiquitin the expression level of K379 is lower than in the untreated condition or when SUMO-1 is overexpressed. These data support the view that K379 is indeed the target of an ubiquitin/SUMO switch.",
"We next investigated whether this interplay acts on K379 stability. To measure the K379 turnover rate indirectly, we performed incubations (0-150 min) with cycloheximide (CHX), a potent inhibitor of de novo protein synthesis. The K379 (left panels 1, 3 and 5) and GAPDH (left panels 2, 4 and 6) contents of HEK-293 cells were analysed following addition of CHX (Fig 3B). GAPDH was used as an internal control. Differences in the steady state levels of the K379 mutant were readily apparent after 30 min, which may correspond to the delay necessary for observing the first effects of treatment. HA-Ubiquitin (HA-Ub) or His-SUMO-1 (SUMO-1) expression vectors were co-transfected or not in HEK-293 cells with the K379 construct ( Fig 3B). Densitometric data are expressed as D K379 /D GAPDH ratio as described in Materials and Methods. HA-Ub overexpression led to an overall 3-fold decrease in K379 stability compared to His-SUMO-1 treated cells (Fig 3C) confirming that increasing ubiquitination drives ΔLf to degradation as we showed previously [17]. When K379-expressing cells overexpressed the SUMO-1 peptide, the stability of the mutant was comparable to that of untreated K379 mutant. Nevertheless, the fact that we did not observe a strong protection as may be expected against proteosomal degradation may be due to the fact that the modified pool of K379 is very feeble (less than 10%) even when SUMO-1 is overexpressed (Fig 2A). The same experiment was conducted with MG132 (right panels) and densitometric results showed that K379 is very stable when the degradation of ubiquitin-conjugated proteins is reduced, whatever the applied treatments ( Fig 3D). Taken together these results confirmed, as already described for other substrates, that SUMOylation may antagonize ubiquitination at K379 and hence positively affect the proteolytic stability of ΔLf.",
"To study the physiological consequences of ΔLf SUMOylation, we next assayed the transcriptional activity of the mutants compared to wild type (Fig 4A). SUMOylation usually triggers recruitment of corepressors such as HDACs, which condense chromatin and prevent transcription. We used a luciferase reporter construct driven by a basal promoter and one ΔLf response element present in a fragment of the Skp1 promoter [9]. In this experiment, the cells were not co-transfected with His-SUMO-1, so the status of ΔLf SUMOylation completely relied on endogenous SUMO activity. The 2.5-fold increased transcriptional activity of the SUMOylation-null mutant confirmed that SUMOylation negatively regulates ΔLf transcriptional activity. Since M5S could not be SUMOylated, over-expression of this mutant without co- expression of SUMO-1 justified the conclusion drawn here that the SUMOylation of ΔLf is part of a regulatory event that governs its activity. The small amount of ΔLf-SUMO forms present in cells could not account for the 2.5 fold increment observed in the transcriptional activity induced by the M5S mutant compared to WT. This has been already described for numerous transcription factors and suggests that SUMOylation is required to initiate transcriptional repression but not to maintain it [19,48]. We then compared the activity of mutants in which only one SUMOylation site was preserved to that of M5S in order to evaluate the impact of adding only one regulatory site at a time. The K391 mutant, which is poorly modified, showed transcriptional activity nearly comparable to that of M5S (Fig 4A) suggesting that the presence of SUMO on this site does not crucially regulate ΔLf transcriptional activity. In contrast, the transcriptional activities of the K308, K361 and K379 mutants were strongly inhibited, by 10-fold for K308 and by nearly 6 fold for the other sites compared to M5S, and by 4 fold for K308 and by around 2-2.5 fold for the other two sites compared to WT, suggesting that these three sites are important for regulation. K361, which is poorly SUMOylated, is nevertheless strongly involved in the repression process. The transcriptional activity of the K13 mutant also SUMOylation of ΔLf represses its transcriptional activity. A) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ! 6) (B) and to one representative experiment of at least two conducted (n ! 2) (C, D). E) Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n!5; p < 0.05 (*), p < 0.01 (**)).",
"doi:10.1371/journal.pone.0129965.g004 decreases but to a lesser extent, by 3.5-fold compared to M5S and by 1.5-fold compared to WT. This may be due to the SUMO/acetylation switch discussed below (Fig 5). The transcriptional activity of the SUMO mutants K13, K361, K379 and notably K308, is lower than that of WT. This may be due to the fact that ΔLf is multi-SUMOylated and that the distribution of SUMO conjugates at each site leads to a \"dilution\" of the effect in the WT compared to the mutants with only one SUMO acceptor site, which may be more heavily SUMOylated.",
"Since SUMO modifications on the K308 site led to the highest inhibitory impact on ΔLf transcriptional activity, we next focused our attention on K308 and investigated the impact of altering its SUMO pattern. Therefore we increased SUMOylation by using the His-SUMO-1 expression vector and decreased it by performing either de-SUMOylation using recombinant SENP2 protease or knockdown using specific short interfering RNA against Ubc9 (siUbc9). Prior to performing transcriptional activity assays, we first showed that siUbc9 efficiently invalidated Ubc9 expression (Fig 4B) leading to a decrease in the level of SUMOylation of both ΔLf 3). B) Relative transcriptional activity of K13 and K379 mutants compared to WT. Cells were co-transfected with pGL3-S1 Skp1 -Luc reporter vector and WT, K13 or K379. His-SUMO-1 expression vector and/or the deacetylase inhibitor Trichostatin A (TSA, 15 ng/mL) were used to modulate the acetylation/SUMOylation ratio. Relative luciferase activities are expressed as described in Materials and Methods (n!3; p < 0.05 (*)). C-D) Modulation of the SUMOylation level was performed either by knocking down Ubc9 using siUbc9 or by overexpressing His-SUMO-1 peptides (SUMO-1). Cells were reverse transfected or not with RNAiMax using 5 nM of siUbc9/siCtrl for 24 h before being transfected for 24 h with WT or K13 plasmid with or without His-SUMO-1 plasmid. Before cell lysis, the acetylation level was altered or not by an overnight treatment with TSA (15 ng/mL). Cells were then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. (C) Input was immunoblotted with anti-Ubc9 (upper panel) or anti-GAPDH (lower panel) antibodies. (D) Samples were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 (upper panel), then with anti-acetyllysine (middle panel) or finally with M2 (lower panel) antibodies. The acetylation/SUMOylation ratio (R Ac/SUMO ) was assayed as described in Material and Methods. The data presented correspond to one representative experiment of two conducted.",
"doi:10.1371/journal.pone.0129965.g005 (Fig 4C, IP, lane 3) and other protein substrates (Fig 4C, input, lane 3). Immunoprecipitation of ΔLf or K13-and K308-expressing cell lysates with M2 followed by immunodetection of SUMO forms using anti-His antibodies (Fig 4D) showed that effective de-SUMOylation was produced in the presence of recombinant SENP2 and is visible in the second, fourth and sixth lanes compared to the untreated condition. Fig 4E shows that overexpression of the His-SUMO-1 peptide strongly decreased the transcriptional activity of ΔLf and its K308 mutant whereas overexpression of SENP2 protease significantly increased it. Moreover, siRNA-mediated depletion of endogenous Ubc9 abolished the repressive potential of SUMOylation and led to a drastic activation of the reporter gene activity (Fig 4E). This combination of overexpression and knockdown experiments demonstrate that SUMOylation plays an important role in ΔLfmediated transcriptional activation. Collectively, these results showed that effectively SUMOylation negatively controls ΔLf transcriptional activity and may convert ΔLf into a transcriptional repressor.",
"Acetylation regulates numerous cellular processes, including the regulation of transcription [49,50]. Acetylation of internal lysine residues is a reversible PTM which strongly alters the electrostatic properties of its targets, modulating their functions, such as protein-protein interactions, DNA binding, activity, stability and subcellular localization [22]. Since competition between SUMOylation and acetylation occurs on many substrates and a putative acetylation site was found on ΔLf at K13, we next investigated whether the SUMO sites might also be acceptors for acetyltransferases. We performed mapping of ΔLf acetylation sites by western blotting of the different expressed mutant ΔLfs using an anti-acetyllysine antibody. Among the five SUMOylated lysine residues, K13 and K379 were the only acetylation acceptor sites with K13 being the major acetylated residue (Fig 5A, lanes 2 and 5, respectively). Mutation of these two lysine residues in the other SUMO mutants (Fig 5A, lanes 3, 4 and 6) and in M5S (Fig 5A, lane 7) resulted in a complete loss of the acetylation signal suggesting that only two acetylation sites are present on ΔLf. These data also confirmed the existence of a possible interplay between acetylation and SUMOylation for K13 and suggested an acetylation/SUMOylation/ubiquitination crosstalk for K379.",
"We next assayed the impact of the SUMO/acetylation interplay on ΔLf-mediated transactivation ( Fig 5B). We increased either SUMOylation by overexpressing SUMO-1 peptide or raised the acetylation level by using the HDAC inhibitor Trichostatin A (TSA). TSA-induced acetylation was able to promote ΔLfand K13-mediated activation by nearly 1.5 fold compared to the untreated condition and 4-fold compared to the condition when SUMO forms were overexpressed (SUMO-1). These data suggested that dynamic interactions between these two posttranslational modifications may occur. The fact that the enhanced WT and K13 transcriptional activities due to TSA-induced acetylation were not modified when SUMO-1 peptides were overexpressed (TSA+SUMO-1) may be due to the fact that acetylation is a less labile PTM than SUMOylation. Indeed, SENPs have to be inhibited in order to observe SUMO forms whereas we do not need to inhibit HDACs in order to visualize acetylated forms. That modulation of the SUMO or acetylation pattern on the K379 mutant had less impact on ΔLf transcriptional activity may be due to the fact that ubiquitination also targets this site.",
"In order to confirm these results we further investigated whether an increase in the levels of acetylation may result in a reduction in the levels of ΔLf SUMOylation and conversely (Fig 5C and 5D). Therefore, we invalidated Ubc9 or inhibited deacetylases with TSA in order to increase acetylation levels, or raised the levels of SUMOylation by overexpressing His-SUMO-1 peptides (SUMO-1), and assessed the impact on ΔLf acetylation. Results shown in Fig 5C confirmed that siUbc9 were functional. More than 80% of the Ubc9 protein disappeared 48 h post-transfection as compared to untreated (NT) or siCtrl treated cells. Moreover, knockdown was not sensitive to treatment with TSA or to the overexpression of His-SUMO-1. Immunoprecipitation of ΔLf-expressing cell lysates with M2 was followed by immunodetection of SUMO-1, acetylated forms or ΔLf (Fig 5D). SUMO profiles of WT and its K13 mutant, when Ubc9 was invalidated, confirmed a decrease in SUMOylation compared to controls (Fig 5D, lane 3 left panel and 2 right panel, respectively) which was more pronounced after TSA treatment (lanes 6 and 12 left panel and lanes 5 and 11 right panel). Overexpression of SUMO-1 peptides together with siUbc9 treatment did not lead to increased SUMOylation of WT and K13 as expected (Fig 5D, lane 9 left panel and 8 right panel, respectively) compared to their respective controls (Fig 5D, lanes 8 and 10 left panel and 7 and 9 right panel, respectively). Thus, ΔLf SUMOylation levels were downregulated following Ubc9 knockdown and when inhibition of HDACs was achieved with TSA. We also analyzed the modification of the acetylation profile of WT and K13 in the above conditions but we did not observe visible variations. Therefore we assayed the acetylation/SUMOylation ratio. This ratio varied slightly when WT was expressed in siUbc9 cells but increased by nearly 2-fold when these cells were grown overnight in the presence of TSA and 3-fold in siUbc9-TSA-treated HEK-293 cells. These data are in accordance with the literature and confirmed increased acetylation when HDACs are inhibited by TSA. Overexpression of SUMO-1 peptides in siCtrl versus siUbc9 cells leads to a comparable acetylation/SUMO ratio. When cells overexpressing SUMO-1 were treated with TSA, acetylation was favoured. The same experiment was conducted with the K13 mutant. The acetylation/SUMOylation ratio was 2-fold higher than WT and rose 4-fold in Ubc9-null cells suggesting that the K13 mutant with only one acetylation/SUMOylation site may preferentially exist as an acetylated form. TSA treatment led to an increased acetylation/SUMOylation ratio as expected. Taken together these results suggest that acetylation antagonizes SUMOylation and may downregulate SUMO effects at K13 (Fig 5B and 5D). The crosstalk between these sites could constitute part of the «ΔLf code » responsible for the control of the transactivation of ΔLf target genes.",
"Transient PTMs like acetylation, phosphorylation, O-GlcNAcylation, ubiquitination and SUMOylation are fast and efficient ways for the cell to respond to different stimuli. Transcription factors are often regulated by combinations of these different PTMs which might act as a molecular barcode [51]. In this report, we demonstrated that ΔLf, known to be modified by O-GlcNAcylation, phosphorylation at S10 and ubiquitination at K379 and K391 [17], can also be modified by SUMOylation and acetylation. We provide experimental evidence that SUMOylation represses ΔLf transcriptional activity whereas acetylation increases it. Moreover, by competing with ubiquitination, SUMOylation influences positively ΔLf stability.",
"Considering the fact that, for a given protein, only a small fraction is commonly found in the SUMOylated state and that this modification is transient, it was difficult to visualize or isolate endogeneous SUMO forms. Nevertheless we were able to observe SUMO-1 isoforms of ΔLf in situ. The ΔLf SUMOylation pattern, manifested as multiple bands, is consistent with the presence of multiple SUMOylation sites on the protein. We identified five SUMOylation sites among which the K13 and K361 sites conform to the consensus sequence of CKXE/D. Furthermore, SUMOylation was also mapped at K308, K379 and K391, which are non-canonical sites. Among these, K13, K308 and K379 are the three SUMO hotspots.",
"SUMOylation of transcription factors generally antagonizes their activation potential or mediates repression, although in a few cases SUMOylation has been associated with reciprocal effects resulting in activation. Whereas SUMOylation decreases the transactivation potential of c-Jun and the androgen receptor [52,53], it increases heat shock factor-2 (HSF2) transactivation capacity [54]. SUMOylation can both positively and negatively modulate p53 transcriptional activity depending on the target promoter [55]. Here we demonstrated that SUMOylation of ΔLf represses its transcriptional activity using a fragment of the Skp1 promoter containing one ΔLfRE. ΔLf binds to and transactivates DcpS, Skp1, Bax, SelH, GTF2F2 and UBE2E1 genes [9,[12][13][14] through similar consensus response elements. Nevertheless it may be interesting to investigate whether ΔLf differentially transactivates its target genes depending on its SUMOylation status.",
"Since ΔLf possesses five SUMO target sites it is difficult to determine whether each of the individual sites has a specific role. Moreover, three of them are targeted by other PTMs, rendering this study even more complex. In order to establish whether any single SUMOylation site was important for the transactivation capacity we compared the activities of mutants disabled specifically at each individual consensus SUMOylation site. The transactivation capacity of each single-site lysine mutant was similar or slightly increased compared to WT (data not shown). Since multiple sites contribute to the control of ΔLf transactivation capacity, the loss of only one SUMO site has only small effects on the activity of ΔLf suggesting either that individual sites act in a redundant manner or that SUMOylation at multiple sites is necessary. Therefore we next studied the transcriptional activity of each mutant in which only one SUMO site was preserved. The K308 mutant strongly inhibited ΔLf transcriptional activity. Moreover when SUMOylation was impaired either after Ubc9 knockdown or in the presence of increased expression of the SENP2 protease, the transcriptional activity of K308 was increased 5-fold compared to that of the K308 mutant expressed in untreated cells. This activity strongly decreased by nearly 2-fold when SUMO-1 peptides were overexpressed. These results demonstrated that SUMOylation at K308 strongly controls ΔLf activity, which may be due to the fact that the region downstream from its SUMO motif is rich in acidic residues as in NDSM. NDSM interacts twice with Ubc9, first between the consensus motif and the active site of the enzyme and also between the acidic tail of the consensus and the basic patch of Ubc9 [27]. Thus, the NDSM acidic patch plays an important role in determining the efficiency of substrate SUMOylation which consequently results in enhanced transcriptional repressive properties.",
"Our in silico studies led us to discover a reverse putative consensus SIMr motif in the vicinity of the K361 SUMO site which is conserved among mammalian species (Table 1). SIMs, which mediate non-covalent interactions between SUMO and SIM-containing proteins [56], can mediate SUMO modification of numerous proteins, resulting in changes in their activity. Moreover a serine residue that is proximal to this SIMr might be the target of kinases as described for non-histone proteins such as PML, EXO9 and PIAS proteins [47]. The presence of a SIMr and/or a phospho-SIMr might be essential to enhance interactions with a SUMO protein and mediate SUMO conjugation. Therefore, the functionality of such a motif has to be established for ΔLf.",
"SUMOylation usually competes with ubiquitination, phosphorylation and acetylation. Ubiquitination/SUMOylation and SUMOylation/acetylation are mutually exclusive whereas SUMOylation/phosphorylation can be agonistic or antagonistic depending on the substrates. The dialogue between SUMO and the other modifications is emerging as a common mechanism that allows control of the transcriptional activity of transcription factors [21]. Two of the SUMO sites are targeted by acetyltransferases. Acetylation is also a dynamic process which mainly contributes to activation of transcription factors [57,58]. Thus, K13 and K379 are acetylated with K13 as the major acetylation site. Modulation of the SUMO/acetylation status has a strong impact on K13 transcriptional activity. In this way, SUMO/acetylation modification of ΔLf could act as a form of switch for the selective interaction with corepressor or coactivator partners, thus modulating ΔLf activity from a transcriptional repressor/corepressor to a coactivator. This is consistent with literature data. Thus, it was shown that SUMOylation inhibits MEF2, HIC1 and KLF8 transcriptional activities whereas acetylation blocks these inhibitory effects [39,40,59,60]. This acetylation/SUMOylation switch is regulated by phosphorylation for MEF2 [39] and it will be interesting to investigate whether ΔLf acetylation/SUMOylation interplay is also controlled by phosphorylation events. The K13 site has a SUMOylation motif close to PDSM motifs [28]. Phosphorylation of the SP motif within this consensus sequence plays an important role in promoting SUMOylation of several substrates including MEF2A [28,39]. Therefore we will have to investigate whether S16 might be of potential functional importance in the regulation of SUMOylation at K13. Moreover, since at the N-terminus, the K13 SUMO/ acetylation site is adjacent to the S10 O-GlcNAcylation/phosphorylation site we will further investigate whether the O-GlcNAc/Phosphate interplay interferes with the SUMOylation/acetylation switch or acts in parallel. The crosstalk between these sites may constitute the ΔLf code responsible for the control of the transactivation of ΔLf target genes. We know from our results [17] and from the literature that acetylation and phosphorylation both lead to transcriptional activation whereas O-GlcNAcylation and SUMOylation repress it. So we hypothesize that this region might be part of the ΔLf transactivation domain which has never been identified. On the other hand, Ubc9 itself is acetylated and its acetylation leads to its decreased binding to NDSM substrates, causing a reduction in their SUMOylation status. Therefore Ubc9 acetylation/deacetylation may serve as a dynamic switch for NDSM substrates such as the K308 site in order to control their SUMOylation [61].",
"K379 and K391 could be both SUMOylated and ubiquitinated. SUMOylation competes with ubiquitination and positively regulates ΔLf stability. Indeed, SUMO frequently influences protein stability by blocking ubiquitin attachment sites [19,36]. K379, which is the main target of both the SUMO and ubiquitin machineries, does not possess a SUMO consensus sequence but is located in the vicinity of the PEST region. It has been shown that Ubc9 could directly interact with the PEST region of SUMO-1 target proteins such as HIPK2 [62] but this is not always the case. It will be interesting to determine the Ubc9-interacting region of ΔLf and investigate whether it overlaps the PEST region. The ubiquitination/SUMOylation interplay exerts a critical role in the maintenance of cellular homeostasis by controlling the turnover of numerous of proteins and notably transcription factors. The switch between these two PTMs needs to be tightly regulated in a spatiotemporal manner and other PTMs, such as phosphorylation, contribute to regulate the ubiquitin/SUMO pathways. PEST sequences are rich in S/TP motifs and are often recognized and phosphorylated by proline-directed S/T protein kinases [63]. Phosphorylation can prevent or favor SUMO-1 conjugation as previously shown for IκBα [36], c-Jun [52] and p53 [52]. We already showed that the ΔLf PEST motif contains three serine residues (S392, S395 and S396) which are phosphorylated prior to ubiquitination of the targets K379 and K391 in their vicinity. Mutation of these two lysine residues or of the three serine residues (S392, S395 and S396) within the PEST motif strongly increased the half-life of ΔLf [17]. Moreover, we showed that they were equivalent phosphorylation targets due to their proximity. Therefore, at the PEST motif, phosphorylation and ubiquitination work in synergy [17], while SUMOylation and ubiquitination are antagonistic PTMs. Therefore this crosstalk could constitute the ΔLf code responsible for the control of ΔLf stability. This regulation is driven by the O-GlcNAc/phosphate interplay at S10. O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity. The pool of ΔLf may exist under a stable but not functional O-GlcNAc isoform. Since the level of O-GlcNAc changes during the cell cycle or is altered, such as in tumorigenesis, deglycosylated ΔLf will become the target of kinases leading to its activation and polyubiquination [1,17]. ΔLf is at the crossroads between cell survival and cell death. It triggers cell cycle arrest and apoptosis via the transactivation of several crucial target genes. Therefore, modifications of their expression may have marked consequences and, depending on cell homeostasis, their transactivation by ΔLf should be transiently suspended. In this context, the SUMOylation/acetylation switch at K13 acts as a second level of control. The activation of the SUMO pathway leads to repression of ΔLf transcriptional activity whereas acetylation, by counteracting SUMOylation at gene promoters, restores it. Increasing evidence shows that O-GlcNAcylation not only interferes with phosphorylation but also crosstalks with other PTMs including acetylation [64], methylation [64,65], ubiquitination [66,67] and poly-ubiquitination [68,69]. However crosstalk with SUMOylation has not yet been reported and we are currently investigated the O-GlcNAcylation/SUMOylation interrelationship.",
"In conclusion, we showed that SUMO modification provides subtle, context-dependent, regulatory input to modulate ΔLf target gene expression. Moreover, we confirmed that ΔLf, like many transcription factors, is regulated by combinations of different PTMs which act as a molecular barcode. Thus, cooperation and/or competition between SUMOylation, ubiquitination, acetylation, phosphorylation and O-GlcNAcylation may contribute to the establishment of a fine regulation of ΔLf transcriptional activity depending on the type of target gene and cellular homeostasis. In this paper, we have focused on the role of SUMOylation but it has not escaped our attention that lysine residues can also be methylated and that such modifications can also affect the activity and stability of proteins such as p53 [70]. Further studies of the roles of PTMs in the molecular mechanisms of ΔLf functions are warranted.",
"Supporting Information S1 Table. Name of mutant constructs, location of amino acid modifications and oligonucleotides used for mutagenesis. "
] | [] | [
"Introduction",
"Experimental Section",
"Plasmid preparation",
"Site-directed mutagenesis",
"Ubc9 knockdown",
"Reporter gene assays",
"Western blotting and immunodetection",
"Densitometric and statistical analyses",
"Results",
"ΔLf possesses putative SUMO and acetylation sites and a putative SIMr motif",
"ΔLf/Lf",
"Mapping the main SUMO sites",
"The SUMOylation/ubiquitination interplay at K379 controls ΔLf stability",
"SUMOylation of ΔLf represses its transcriptional activity",
"Acetylation attenuates SUMO-mediated transcriptional repression",
"Discussion",
"Fig 1 .",
"Fig 2 .",
"Fig 3 .",
"Fig 4 .",
"Fig 5 .",
"Table 1 .",
"Table 2 ."
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"Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin",
"Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin"
] | [] |
10,072,896 | 2022-03-22T00:45:40Z | CCBYNC | https://europepmc.org/articles/pmc3047627?pdf=render | GREEN | d91b10be406f4a52826884866246f9ab911a6bc7 | null | null | null | null | 10.1007/s12307-010-0061-4 | 2064374238 | 21505567 | 3047627 |
Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells
Laura E Pascal [email protected]
Junkui Ai
Ricardo Z N Vêncio [email protected]:[email protected]
Eneida F Vêncio [email protected]
Yong Zhou [email protected]
Laura S Page
Lawrence D True [email protected]
Zhou Wang [email protected]
Alvin Y Liu [email protected]
L E Pascal
E F Vêncio
L S Page
A Y Liu
E F Vêncio
A Y Liu
L E Pascal
R Z N Vêncio
E F Vêncio
Y Zhou
L S Page
A Y Liu
R Z N Vêncio
Y Zhou
L E Pascal
J Ai
Z Wang
J Ai
Z Wang
R Z N Vêncio
L D True
Z Wang
E F Vêncio
Department of Urology
Institute for Stem Cell and Regenerative Medicine
Institute for Systems Biology
Department of Urology
University of Washington
98195, 98103Seattle, SeattleWA, WAUSA, USA
Cancer Institute
School of Medicine
Department of Genetics
University of Pittsburgh
University of Pittsburgh
15232PittsburghPAUSA
Paulo's Medical School
Department of Pathology
University of São
Ribeirão PretoBrazil
Department of Pharmacology and Chemical Biology
University of Washington
98195SeattleWAUSA
School of Medicine
Department of Pathology
School of Dentistry
University of Pittsburgh
University of Pittsburgh Cancer Institute
15232PittsburghPAUSA
Federal University of Goias
GoianiaGOBrazil
Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells
Received: 24 August 2010 / Accepted: 16 December 2010 / Published online: 7 January 2011ORIGINAL PAPER4) contains supplementary material, which is available to authorized users. Present Address: Cancer Microenvironment (2011) 4:51-59
Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. These CD90 + cancer-associated stromal fibroblastic cells differ in gene expression from their normal counterpart, CD49a + CD90 lo stromal smooth muscle cells; and were postulated to represent a less differentiated cell type with altered inductive properties. CD90 + stromal cells were isolated from tumor tissue specimens and co-cultured with the pluripotent embryonal carcinoma cell line NCCIT in order to elucidate the impact of tumor-associated stroma on stem cells, and the 'cancer stem cell.' Transcriptome analysis identified a notable decreased induction of smooth muscle and prostate stromal genes such as PENK, BMP2 and ChGn compared to previously determined NCCIT response to normal prostate stromal cell induction. CD90 + stromal cell secreted factors Electronic supplementary material The online version of this article (
Introduction
The prostate is a glandular organ composed of secretory luminal cell-lined acini and an underlying layer of basal cells supported within a fibromuscular stroma. In urologic organ development, the stroma provides the inductive signaling to stem/progenitor cells to produce the resultant tissue [1]. Increasingly, it is thought that the stromal environment might play a significant role in disease development and progression as well. An altered stroma has been identified in association with both prostate cancer [2][3][4] as well as premalignant prostate diseases including prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) [3,5]. In prostate cancer, the stroma is also affected in that gene expression in cancer-associated fibroblastic (CAF) stromal cells differs significantly from that of normal tissue stromal cells [6][7][8][9][10]. Prostate CAFs are capable of inducing proliferation and malignant transformation [1] and have been postulated to drive tumor progression [11]. These studies strongly suggest that the prostate stroma plays a significant role in facilitating disease progression.
CAFs immediately surrounding prostate cancer can be identified by strong CD90 immunostaining [12] and CD90 hi cancer-associated fibroblasts (CAFs) were postulated to have greater tumor-promoting effects than CD90 lo CAFs [10]. Comparative transcriptome analysis of isolated CD90 + CAFs and CD49a + normal tissue stromal cells revealed a decrease in the expression of genes involved in smooth muscle cell differentiation and those specific or restricted to the prostate [5,7]. Genes that encode secreted proteins or hormones are likely candidates responsible for organspecific stromal induction, and dysregulation of these genes might contribute to disease progression through stromal-stem cell signaling.
To examine the molecular mechanisms involved in prostate stromal-stem cell interaction, we previously developed an in vitro co-culture system with stromal cells isolated from normal prostate (referred to as NP) and a cancer stem cell type, the embryonal carcinoma (EC) cell line, NCCIT [13]. NCCIT is a pluripotent cell line that can be readily maintained in cell culture in an undifferentiated state [14]. When co-cultured without direct cell contact, NCCIT cells were induced by NP stromal cells to differentiate into a cell population that expressed predominantly prostate stromal cell genes (e.g., PENK). Induced NCCIT cells also lost expression of stem cell genes and underwent a change in morphology with reduced proliferation [13]. Bladder stromal cells isolated from human specimens induced a bladder stromal-like expression (e.g., absent PENK) [13], thus demonstrating plasticity-a property of stem cells-in NCCIT response. This cellto-cell signaling was presumably mediated by diffusible factors. Interestingly, NP prostate stromal cells were also significantly altered by co-culture with NCCIT, whereas NCCIT had no significant effect on the gene expression of CP stromal cells [15]. In this study, NCCIT induced an increase in expression for CD90, MIRN21, HGF, SFRP1, BGN, and decreased expression of IGFBP5, HSD11B1a in NP stromal cells. These findings suggest that alterations in prostate stroma could be induced by stem or cancer stem cell influence. In this study, we examined the inductive functioning of cultured CD90 + CAFs (referred to here as CP) stromal cells for comparison to that of NP stromal cells.
Materials and Methods
Cell Lines and Tissue Specimens
NCCIT and prostate cancer cell line PC3 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 (Cambrex BioScience, Walkersville, MD) media supplemented with 10% heat-inactivated fetal bovine serum (FBS). In this culture condition, NCCIT cells maintain expression of stem cell genes and microRNA, and are alkaline phosphatase positive [13,15]. The tissue samples used in this study consisted of cancer-enriched CP tissue specimens, characterized as CD90 hi TIMP1 lo by Western blot analysis of tissue digestion media [7], obtained from 2 radical prostatectomy surgery patients. Note 'pure' CP specimens had increased CD90 expression and minimal reactivity to TIMP1, which is secreted by luminal cells and not by cancer cells [16], whereas NP specimens were CD90 lo TIMP1 hi . All tissue samples were obtained under approval by the University of Washington IRB and were collected following a standard protocol. Upon receipt of a resected specimen, 3-mm thick transverse sections were made of the gland after inking the exterior surface. Frozen sections of tissue blocks were histologically prepared to locate the tumor foci for dissection. Pathology characteristics of the 2 tumors were 08-021CP: Gleason 5+4, T3a, tumor volume 4.5 cc; 08-028CP: Gleason 3+4, T2c, tumor volume 2.5 cc. NP specimens were obtained and analyzed as described previously [13]. Briefly, between 1 and 10 g of tissue from the anterior aspect of the prostate (transition zone) were excised; corresponding frozen sections of the tissue blocks were histologically assessed to confirm specimens were free of cancer.
Tissue samples were minced and digested by overnight incubation at room temperature in 0.2% collagenase type I (Invitrogen, Carlsbad, CA) in RPMI-1640 media supplemented with 5% FBS and 10 −8 M dihydrotestosterone on a magnetic stirrer. The resultant cell suspension was filtered with a 70-μm Falcon cell strainer to remove any nondigested tissue, diluted with an equal volume of Hanks balanced salt solution (HBSS), and aspirated with an 18gauge needle. The single cell preparation was partitioned into stromal and epithelial fractions on a discontinuous Percoll density gradient (Amersham Pharmacia, Piscataway, NJ) as described [17,18]. Cells banding at a density of ρ= 1.035 were collected as the stromal fraction for magnetic cell sorting (MACS). The cell-free tissue digestion media supernatant was analyzed by Western blotting to verify that the specimens were of cancer.
CP stromal cells were sorted using anti-CD90. Briefly, the gradient-purified stromal cell fraction was resuspended in 100 μl 0.1% bovine serum albumin (BSA)-HBSS, and CD90-Phycoerythrin (PE) mouse monoclonal antibody (clone 5E10, BD-PharMingen, San Diego, CA) at 1:20 was added for 15 min at room temperature in the dark. The reaction was stopped by 1 ml 0.1% BSA-HBSS and centrifugation. The labeled cells were resuspended in BSA-HBSS, and 15 μl paramagnetic microbead conjugated anti-PE (Miltenyi Biotec, Auburn, CA) was added for 15 min. After incubation, the positive and negative cells were separated by AutoMACS (Miltenyi Biotec) using the double positive sort program. Aliquots of positive and negative cell fractions were analyzed by fluorescenceactivated cell sorting (FACS, Becton Dickinson, Mountain View, CA) to gauge the sort efficiency; only >85% CD90 + fractions were used for experiments.
Cell Culture
The sorted CD90 + stromal cells were adapted to culture for 3-5 passages in RPMI-1640 media supplemented with 10% FBS, and their identity verified by RT-PCR analysis of gene expression as described [5]. For co-culture experiments, 0.4 μm polycarbonate membrane trans-well inserts (Corning, Corning, NY) to preclude cell contact were employed. NCCIT cells were seeded at 10 4 /ml in RPMI-1640, 10% FBS on 6-well plates, and CP stromal cells were seeded at 10 4 /ml on the insert. Controls were NCCIT and CP stromal cells alone. Cultures were maintained for 3 d. The 3 d-time point was chosen based on our previous time course study of NP stromal cell-induced differentiation of NCCIT [13]. In that experiment, gene expression changes in NCCIT were detected as early as 6 h in co-culture, and by the third day, nearly the entire stromal gene repertoire was induced as shown by a principal components analysis of time-point transcriptomes. Cells were trypsinized and lysed in RLT Buffer (Qiagen, Valencia, CA).
Transcriptome Analysis
RNA was isolated from cultures of CP stromal, NCCIT, and NCCIT + CP stromal cells at 3 d. Transcriptomes of untreated NCCIT, NP stromal cells and NCCIT at 3 d of co-culture with NP stromal cells were determined previously [13], as were sorted CD90 + CP stromal cells, i.e., not cultured [7]. These datasets were made available online (http://scgap.systemsbiology.net/data/). Quality and concentration of RNA were determined using an Agilent 2100 Bioanalyzer and RNA Nano Labchip (Agilent Technologies, Santa Clara, CA). Between 2 and 7 replicates of each experimental condition or control were assayed with the Human Genome U133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA). The U133 Plus 2.0 array contains probesets representing 54,675 genes, splice variants, and ESTs. The GeneChips were prepared, hybridized, and scanned according to the protocols provided by Affymetrix (P/N 702232 Rev. 2). Briefly, 200 ng of RNA was reverse transcribed with poly (dT) primer containing a T7 promoter, and the cDNA was made double-stranded. In vitro transcription was performed to produce unlabeled cRNA. Next, first-strand cDNA was produced with random primers, and the cDNA was made double-stranded with poly (dT) primer/T7 promoter. A final in vitro transcription was done with biotinylated ribonucleotides. The biotin-labeled cRNA was hybridized to the GeneChips. The chips were washed and stained with streptavidin-PE using an Affymetrix FS-450 fluidics station. Data was collected with an Affymetrix GeneChip Scanner 3000.
Bioinformatic Analysis
A probabilistic comparative analysis between transcriptomes of treated NCCIT was used to highlight differentially expressed genes with respect to that of untreated NCCIT [7]. Gene expression level was defined as the normalized and summarized intensities of each GeneChip probeset, and was presented as its logarithmic value: X=log 2 (Normalized intensity). This step was carried out using the standard robust multi-array average (RMA) method [19], implemented in the in-house analysis pipeline SBEAMS [20]. Data were presented on a grayscale indicating RMAnormalized Affymetrix signal intensity [21]. Signals of 10 or less were represented as white and signals greater than or equal to 10,000 as black. Higher Affymetrix signal (more black) indicated higher levels of gene expression.
The strength of differential expression between any pair of experiments was estimated by M i ¼ log 2 ratio ð Þ¼ X 3d À X 0h , where 0 h represented the untreated NCCIT and 3d represented treated NCCIT at 3 d. Reliability of the differential expression was estimated by calculating the probability P=P(X 3d >X 0h ) or P=P(X 3d <X 0h ) according to a statistical model that assumed a normal distribution X j ∼N (m j ,s j ), where m j and s j were the mean and maximum difference, respectively, among the replicates of group j. Consistently, P= P(X 3d > X 0h ) or P= P(X 3d < X 0h ) was reported if m 3d >m 0h or m 3d <m 0h . Functional and ontology enrichment analysis was performed using the DAVID webbased tool [22]. Freely available prediction software for determination of signal peptides and likely cell membranespanning sequences was also used. Signal peptides were predicted using SignalP 3.0 [23], and transmembrane (TM) regions were predicted using TMHMM 2.0 [24] for protein topology and the number of TM helices. Information from both SignalP and TMHMM were combined to identify proteins that contained predicted cleavable signal peptides and no predicted TM segments as reported previously [25].
Results
Gene expression changes induced in NCCIT by secreted factors from CP stromal cells were determined by Affymetrix DNA microarray analysis. Following 3-days co-culture with CP stromal cells, the induced expression in NCCIT cells (CP-NCCIT) of smooth muscle genes ACTA2, CALD1, CNN1, prostate stromal genes PENK, CNTN1, ChGn, BMP2 [5,7], androgen receptor (AR) and a stromal gene GFRA1 was significantly less than that previously shown by NP stromal cells (NP-NCCIT) [13]. The CP-NCCIT transcriptome dataset contained minimal signal levels for CALD1, and CNN1 compared to that of NP-NCCIT ( Fig. 1a and b). Other queried prostate genes such as tenascin C (TNC) [5] were also lacking. The similar induction of stromal gene stanniocalcin (STC1) and increased expression of CD90/THY1 in CP-NCCIT showed that gene expression changes did occur in NCCIT cocultured with CP stromal cells. Induction of CD90 was notably higher in CP-NCCIT than in NP-NCCIT (NCCIT cells are also positive for CD90, a stem cell marker). This reflected the increased CD90 expression in CP stromal cells.
A comparison of stem cell gene expression in treated NCCIT showed higher signal levels of NANOG, POU5F1, TDGF1, and SOX2 in CP-NCCIT than in NP-NCCIT ( Fig. 1c and d). Induction of NCCIT by NP stromal cells was found to lead to almost complete down-regulation of these stem cell genes [13], whereas CP stromal cells had apparently little effect. Of note was the detection of ABCG2 (a prostate progenitor cell marker) expression in NP-NCCIT but not CP-NCCIT (Fig. 1d). NCCIT is negative for ABCG2 expression.
To identify genes encoding secreted proteins that might function in cell-cell signaling, the most differentially expressed genes in NP-NCCIT vs. CP-NCCIT (or NP stromal vs. CP stromal) were analyzed using the DAVID annotation tool. CP stromal induction led to upregulation of several such genes including LEFTY2 and TAC1, while NP stromal induction led to up-regulation of ADAMTS1, IGFBP5, WNT5A and others (Table S1). Overall, there were many more such genes induced in NP-NCCIT than CP-NCCIT. This showed a smaller pool of candidate signaling molecules produced by CP stromal cells.
Differentially expressed genes were also analyzed for significant enrichment with respect to functional categories using DAVID. The top KEGG pathways identified were cytokine-cytokine receptor interaction, chemokine signaling pathway, extracellular matrix (ECM)-receptor interaction, cell adhesion and focal adhesion. Enrichment of these functional categories was prominent in the CP-NCCIT vs. NP-NCCIT datasets. Of particular interest were the genes that contribute to the functioning and maintenance of ECM. Matrix metalloproteinases (MMPs) are involved in the degradation of ECM proteins and have been associated with tumor cell invasion. The membrane-anchored reversioninducing cysteine-rich protein with Kazal motifs (RECK) is a potent inhibitor of MMP activity. RECK down-regulation has been identified in many cancers, and it has been reported that high RECK expression levels were associated with favorable prognosis in prostate cancer [26,27]. In comparing the CP-NCCIT and NP-NCCIT expression profiles, several genes associated with RECK were differentially expressed (Fig. 2a). For example, induction of MMP9, a potential prostate cancer urine biomarker [28], was greater in CP-NCCIT than in NP-NCCIT (Fig. 2b). MMP9 expression was also higher in sorted CP vs. NP stromal cell transcriptomes. In contrast, RECK was more up-regulated in NP-NCCIT than CP-NCCIT, as was tissue inhibitor of metalloproteinase TIMP1, an antagonist of MMPs. With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. Figure 3 illustrates the RECK pathway network in which MMPs synthesized by CP stromal cells could lead to ECM degradation, which would in turn promote tumor cell escape. The MMP effect is amplified by the decrease in TIMP expression in cancer cells. Increase in MMPs is due to down-regulation of RECK. Fig. 1 Expression profiles of stromal and stem cell genes in treated NCCIT. a Increased expression of prostate stromal cell-specific genes relative to untreated NCCIT was detected in co-cultures of NP stromal + NCCIT cells (labeled NP-NCCIT). Expression of these stromal genes was less pronounced in co-culture of CP stromal + NCCIT cells (CP-NCCIT). For example, PENK was not induced. Note the increase in tumor-associated stromal marker CD90/THY1. b The CP-NCCIT transcriptome dataset (first column) contains minimal signal for smooth muscle differentiation genes (CALD1, CNN1) present in the NP-NCCIT transcriptome (third column) compared to untreated NCCIT transcriptome (second column) in virtual Northern blot format (darker shades of boxes indicate higher mRNA levels with background ≤50 RMA units). c Higher expression of several stem cell genes was detected in CP-NCCIT relative to NP-NCCIT as well as in cultured CP stromal cells relative to NP stromal cells. d Virtual Northern blot format shows that for stem cell genes NANOG, SOX2, CD9 and THY1, expression was increased in CP-NCCIT compared to untreated NCCIT, whereas expression was decreased in NP-NCCIT
Discussion
NCCIT response to CD90 + CAFs tumor-associated was significantly altered from the gene expression changes induced by normal stromal cell factors. Previously, NP stromal cells induced a loss of embryonic stem cell markers and an up-regulation of genes characteristic of stromal mesenchyme, some epithelial genes and cancer stem cell genes. NCCIT response to CP stromal cells was characterized by an absence or decreased induction of genes involved in smooth muscle cell differentiation and those expressed by the prostate but not the bladder, i.e., organ restricted. At the same time, the decrease in stem cell gene expression was not as pronounced. This altered differenti-ation response could be due to differences in signaling factors secreted from tumor-associated stromal cells. These differences could be the result of either a reduction or loss of certain proteins such as the hormone PENK. Stromal cells are important in tissue repair and renewal as suggested by their demonstrated role in prostate and bladder formation. Organ specificity in this process could be due to the differentially expressed genes between the stromal cell types. Indeed, we previously showed that bladder stromal cells induced a different response from NCCIT than prostate stromal cells. Thus, if tumor-associated stromal cells were unable to provide the appropriate signaling, then normal histodevelopment would not occur, instead cancer development takes place. Although only two cases of CP stromal cells were tested, they did provide a demonstration that stem cell induction was markedly different from that by NP stromal cells. These differences, including reduced induction of smooth muscle cell genes and increased induction of genes involved in ECM remodeling, are consistent with alterations to the prostate tumor microenvironment. When we can model epithelial cytodifferentiation, i.e., PSA secretion, with cell contact and ECM, then a study using multiple samples of CP stromal cells can be carried out. For example, one could contrast the effect of CP stromal cells isolated from Gleason 3 +3 vs. Gleason 4+4 tumors.
The differentially induced expression pattern of genes involved in the ECM RECK pathway in CP-NCCIT vs. NP-NCCIT appears to mimic that in primary tumors as inferred from the transcriptomes of sorted stromal and epithelial cell types. NP stromal induction produced upregulation of TIMP1 and RECK whereas CP stromal induction produced up-regulation of MMP9 and comparatively less of TIMP1 and RECK. In the sorted cells, expression of MMP9 (and HRAS, which inhibits RECK) is higher in CP stromal than NP stromal, whereas that of RECK is higher in NP stromal. TIMP1 protein in cancer is absent [16]. Thus, our in vitro model of CP stromal induction of NCCIT recapitulated to some degree a major pathway important in cancer development. In response to NP stromal influence, NCCIT cells were induced to express a transcriptome with a predominant but incomplete stromal mesenchyme profile. However, the response to CP stromal cell influence with regard to induction of stromal mesenchyme genes and loss of stem cell genes was significantly less. This difference could simply reflect a reduction in secreted factors from CP stromal cells compared to NP stromal cells and therefore a lesser degree of influence on NCCIT cells, or it could represent a shift in the heterogeneity of the treated NCCIT cell population.
With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. In summary, these experimental results showed that in induction of stem cells CP stromal cells were very different from NP stromal cells. The abnormal gene expression of CP stromal cells may well be the cause. Whether this would lead to cancer cell differentiation is still unknown since heterotypic cell contact and ECM were not provided for in the co-culture. Also unknown is whether other cell types beside stromal (e.g., epithelial) were induced in this system. For example, some ABCG2 expression was detected in NP-NCCIT, and this may suggest a small subpopulation with this marker. ABCG2 expression was identified in a putative prostate progenitor cell population localized to the basal epithelium [30]. It is therefore possible that more than one cell lineage, stromal and epithelial, could result from stromal induction of stem cells.
Fig. 3
3Schematic of RECK pathway in stromal-epithelial interaction in prostate cancer. Decreased RECK expression leads to activation of MMPs and degradation of ECM proteins allowing dissemination of tumor cells. Virtual Northern blot format shows array signals for MMP9, HRAS and RECK in NP stromal vs. CP stromal cells (1 and 2 from two different specimens) and for TIMPs in NP epithelial vs. CP cancer cells
Acknowledgements We thank Drs Paul Lange, William Ellis and Thomas Takayama for providing prostate tissue for this study. We also thank Adam Van Mason for collecting and preparing the tissue samples. This work was supported by grants DK63630 from NIDDK, CA111244 (EDRN) from NCI, and P50 CA097186 (Pacific Northwest Prostate Cancer SPORE) from NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIDDK or NCI. Additional funding came from grant PM50 GMO76547/Center for Systems Biology.Conflicts of interest The authors declare no conflict of interest.Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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| [
"Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. These CD90 + cancer-associated stromal fibroblastic cells differ in gene expression from their normal counterpart, CD49a + CD90 lo stromal smooth muscle cells; and were postulated to represent a less differentiated cell type with altered inductive properties. CD90 + stromal cells were isolated from tumor tissue specimens and co-cultured with the pluripotent embryonal carcinoma cell line NCCIT in order to elucidate the impact of tumor-associated stroma on stem cells, and the 'cancer stem cell.' Transcriptome analysis identified a notable decreased induction of smooth muscle and prostate stromal genes such as PENK, BMP2 and ChGn compared to previously determined NCCIT response to normal prostate stromal cell induction. CD90 + stromal cell secreted factors Electronic supplementary material The online version of this article (",
"Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. These CD90 + cancer-associated stromal fibroblastic cells differ in gene expression from their normal counterpart, CD49a + CD90 lo stromal smooth muscle cells; and were postulated to represent a less differentiated cell type with altered inductive properties. CD90 + stromal cells were isolated from tumor tissue specimens and co-cultured with the pluripotent embryonal carcinoma cell line NCCIT in order to elucidate the impact of tumor-associated stroma on stem cells, and the 'cancer stem cell.' Transcriptome analysis identified a notable decreased induction of smooth muscle and prostate stromal genes such as PENK, BMP2 and ChGn compared to previously determined NCCIT response to normal prostate stromal cell induction. CD90 + stromal cell secreted factors Electronic supplementary material The online version of this article ("
] | [
"Laura E Pascal [email protected] ",
"Junkui Ai ",
"Ricardo Z N Vêncio [email protected]:[email protected] ",
"Eneida F Vêncio [email protected] ",
"Yong Zhou [email protected] ",
"Laura S Page ",
"Lawrence D True [email protected] ",
"Zhou Wang [email protected] ",
"Alvin Y Liu [email protected] ",
"L E Pascal ",
"E F Vêncio ",
"L S Page ",
"A Y Liu ",
"E F Vêncio ",
"A Y Liu ",
"L E Pascal ",
"R Z N Vêncio ",
"E F Vêncio ",
"Y Zhou ",
"L S Page ",
"A Y Liu ",
"R Z N Vêncio ",
"Y Zhou ",
"L E Pascal ",
"J Ai ",
"Z Wang ",
"J Ai ",
"Z Wang ",
"R Z N Vêncio ",
"L D True ",
"Z Wang ",
"E F Vêncio ",
"\nDepartment of Urology\nInstitute for Stem Cell and Regenerative Medicine\nInstitute for Systems Biology\nDepartment of Urology\nUniversity of Washington\n98195, 98103Seattle, SeattleWA, WAUSA, USA\n",
"\nCancer Institute\nSchool of Medicine\nDepartment of Genetics\nUniversity of Pittsburgh\nUniversity of Pittsburgh\n15232PittsburghPAUSA\n",
"\nPaulo's Medical School\nDepartment of Pathology\nUniversity of São\nRibeirão PretoBrazil\n",
"\nDepartment of Pharmacology and Chemical Biology\nUniversity of Washington\n98195SeattleWAUSA\n",
"\nSchool of Medicine\nDepartment of Pathology\nSchool of Dentistry\nUniversity of Pittsburgh\nUniversity of Pittsburgh Cancer Institute\n15232PittsburghPAUSA\n",
"\nFederal University of Goias\nGoianiaGOBrazil\n",
"Laura E Pascal [email protected] ",
"Junkui Ai ",
"Ricardo Z N Vêncio [email protected]:[email protected] ",
"Eneida F Vêncio [email protected] ",
"Yong Zhou [email protected] ",
"Laura S Page ",
"Lawrence D True [email protected] ",
"Zhou Wang [email protected] ",
"Alvin Y Liu [email protected] ",
"L E Pascal ",
"E F Vêncio ",
"L S Page ",
"A Y Liu ",
"E F Vêncio ",
"A Y Liu ",
"L E Pascal ",
"R Z N Vêncio ",
"E F Vêncio ",
"Y Zhou ",
"L S Page ",
"A Y Liu ",
"R Z N Vêncio ",
"Y Zhou ",
"L E Pascal ",
"J Ai ",
"Z Wang ",
"J Ai ",
"Z Wang ",
"R Z N Vêncio ",
"L D True ",
"Z Wang ",
"E F Vêncio ",
"\nDepartment of Urology\nInstitute for Stem Cell and Regenerative Medicine\nInstitute for Systems Biology\nDepartment of Urology\nUniversity of Washington\n98195, 98103Seattle, SeattleWA, WAUSA, USA\n",
"\nCancer Institute\nSchool of Medicine\nDepartment of Genetics\nUniversity of Pittsburgh\nUniversity of Pittsburgh\n15232PittsburghPAUSA\n",
"\nPaulo's Medical School\nDepartment of Pathology\nUniversity of São\nRibeirão PretoBrazil\n",
"\nDepartment of Pharmacology and Chemical Biology\nUniversity of Washington\n98195SeattleWAUSA\n",
"\nSchool of Medicine\nDepartment of Pathology\nSchool of Dentistry\nUniversity of Pittsburgh\nUniversity of Pittsburgh Cancer Institute\n15232PittsburghPAUSA\n",
"\nFederal University of Goias\nGoianiaGOBrazil\n"
] | [
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"L W Chung, ",
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"Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. G R Cunha, W Ricke, A Thomson, P C Marker, G Risbridger, S W Hayward, Y Z Wang, A A Donjacour, T Kurita, J Steroid Biochem Mol Biol. 924Cunha GR, Ricke W, Thomson A, Marker PC, Risbridger G, Hayward SW, Wang YZ, Donjacour AA, Kurita T (2004) Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. J Steroid Biochem Mol Biol 92(4):221-236",
"Reactive stroma in prostate cancer progression. J A Tuxhorn, G E Ayala, D R Rowley, J Urol. 1666Tuxhorn JA, Ayala GE, Rowley DR (2001) Reactive stroma in prostate cancer progression. J Urol 166(6):2472-2483",
"Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling. J A Tuxhorn, G E Ayala, M J Smith, V C Smith, T D Dang, D R Rowley, Clin Cancer Res. 89Tuxhorn JA, Ayala GE, Smith MJ, Smith VC, Dang TD, Rowley DR (2002) Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling. Clin Cancer Res 8(9):2912-2923",
"What might a stromal response mean to prostate cancer progression?. D R Rowley, Cancer Metastasis Rev. 174Rowley DR (1998) What might a stromal response mean to prostate cancer progression? Cancer Metastasis Rev 17(4):411-419",
"Stromal mesenchyme cell genes of the human prostate and bladder. Y A Goo, D R Goodlett, L E Pascal, K D Worthington, R L Vessella, L D True, A Y Liu, BMC Urol. 517Goo YA, Goodlett DR, Pascal LE, Worthington KD, Vessella RL, True LD, Liu AY (2005) Stromal mesenchyme cell genes of the human prostate and bladder. BMC Urol 5:17",
"Global expression analysis of prostate cancer-associated stroma and epithelia. A M Richardson, K Woodson, Y Wang, J Rodriguez-Canales, H S Erickson, M A Tangrea, K Novakovic, S Gonzalez, A Velasco, E S Kawasaki, M R Emmert-Buck, R F Chuaqui, A Player, Diagn Mol Pathol. 164Richardson AM, Woodson K, Wang Y, Rodriguez-Canales J, Erickson HS, Tangrea MA, Novakovic K, Gonzalez S, Velasco A, Kawasaki ES, Emmert-Buck MR, Chuaqui RF, Player A (2007) Global expression analysis of prostate cancer-associated stroma and epithelia. Diagn Mol Pathol 16(4):189-197",
"Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes. L E Pascal, Y A Goo, R Z Vencio, L S Page, A A Chambers, E S Liebeskind, T K Takayama, L D True, A Y Liu, BMC Cancer. 9317Pascal LE, Goo YA, Vencio RZ, Page LS, Chambers AA, Liebeskind ES, Takayama TK, True LD, Liu AY (2009) Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes. BMC Cancer 9:317",
"Global gene expression analysis of reactive stroma in prostate cancer. O Dakhova, M Ozen, C J Creighton, R Li, G Ayala, D Rowley, M Ittmann, Clin Cancer Res. 1512Dakhova O, Ozen M, Creighton CJ, Li R, Ayala G, Rowley D, Ittmann M (2009) Global gene expression analysis of reactive stroma in prostate cancer. Clin Cancer Res 15(12):3979-3989",
"Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. T Ernst, M Hergenhahn, M Kenzelmann, C D Cohen, M Bonrouhi, A Weninger, R Klaren, E F Grone, M Wiesel, C Gudemann, J Kuster, W Schott, G Staehler, M Kretzler, M Hollstein, H J Grone, Am J Pathol. 1606Ernst T, Hergenhahn M, Kenzelmann M, Cohen CD, Bonrouhi M, Weninger A, Klaren R, Grone EF, Wiesel M, Gudemann C, Kuster J, Schott W, Staehler G, Kretzler M, Hollstein M, Grone HJ (2002) Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. Am J Pathol 160(6):2169-2180",
"Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts. H Zhao, D M Peehl, Prostate. 699Zhao H, Peehl DM (2009) Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts. Prostate 69(9):991-1000",
"Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting. S Y Sung, L W Chung, Differentiation. 709Sung SY, Chung LW (2002) Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting. Differentiation 70(9-10):506-521",
"Cd90/thy1 is overexpressed in prostate cancerassociated fibroblasts and could serve as a cancer biomarker. L D True, H Zhang, M Ye, C Y Huang, P S Nelson, Von Haller, P D Tjoelker, L W Kim, J S Qian, W J Smith, R D Ellis, W J Liebeskind, E S Liu, A Y , Mod Pathol. 2310True LD, Zhang H, Ye M, Huang CY, Nelson PS, von Haller PD, Tjoelker LW, Kim JS, Qian WJ, Smith RD, Ellis WJ, Liebeskind ES, Liu AY (2010) Cd90/thy1 is overexpressed in prostate cancer- associated fibroblasts and could serve as a cancer biomarker. Mod Pathol 2010 Oct; 23(10):1346-56",
"Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells. L E Pascal, R Z Vencio, Y A Goo, L S Page, C P Shadle, A Y Liu, Prostate. 6912Pascal LE, Vencio RZ, Goo YA, Page LS, Shadle CP, Liu AY (2009) Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells. Prostate 69(12):1353-1365",
"Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit. I Damjanov, B Horvat, Z Gibas, Lab Invest. 682Damjanov I, Horvat B, Gibas Z (1993) Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit. Lab Invest 68(2):220-232",
"Embryonal carcinoma cell induction of mirna and mrna changes in cocultured prostate stromal fibromuscular cells. E F Vencio, L E Pascal, L S Page, G Denyer, A J Wang, H Ruohola-Baker, S Zhang, K Wang, D J Galas, A Y Liu, doi:1002/jcp.22464J Clin Pathol. Vencio EF, Pascal LE, Page LS, Denyer G, Wang AJ, Ruohola- Baker H, Zhang S, Wang K, Galas DJ, Liu AY (2010) Embryonal carcinoma cell induction of mirna and mrna changes in co- cultured prostate stromal fibromuscular cells. J Clin Pathol doi:1002/jcp.22464",
"Analysis of prostate cancer by proteomics using tissue specimens. A Y Liu, H Zhang, C M Sorensen, D L Diamond, J Urol. 1731Liu AY, Zhang H, Sorensen CM, Diamond DL (2005) Analysis of prostate cancer by proteomics using tissue specimens. J Urol 173 (1):73-78",
"Cell-cell interaction in prostate gene regulation and cytodifferentiation. A Y Liu, L D True, L Latray, P S Nelson, W J Ellis, R L Vessella, P H Lange, L Hood, G Van Den Engh, Proc Natl Acad Sci. 9420Liu AY, True LD, LaTray L, Nelson PS, Ellis WJ, Vessella RL, Lange PH, Hood L, van den Engh G (1997) Cell-cell interaction in prostate gene regulation and cytodifferentiation. Proc Natl Acad Sci USA 94(20):10705-10710",
"Stromal cells of the human prostate: initial isolation and characterization. A Kassen, D M Sutkowski, H Ahn, J A Sensibar, J M Kozlowski, C Lee, Prostate. 282Kassen A, Sutkowski DM, Ahn H, Sensibar JA, Kozlowski JM, Lee C (1996) Stromal cells of the human prostate: initial isolation and characterization. Prostate 28(2):89-97",
"Exploration, normalization, and summaries of high density oligonucleotide array probe level data. R A Irizarry, B Hobbs, Collin F Beazer-Barclay, Y D Antonellis, K J Scherf, U Speed, T P , Biostatistics. 42Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4(2):249-264",
"Sbeams-microarray: database software supporting genomic expression analyses for systems biology. B Marzolf, E W Deutsch, P Moss, D Campbell, M H Johnson, T Galitski, BMC Bioinform. 7286Marzolf B, Deutsch EW, Moss P, Campbell D, Johnson MH, Galitski T (2006) Sbeams-microarray: database software supporting genomic expression analyses for systems biology. BMC Bioinform 7:286",
"The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder. L E Pascal, E W Deutsch, D S Campbell, M Korb, L D True, A Y Liu, BMC Urol. 719Pascal LE, Deutsch EW, Campbell DS, Korb M, True LD, Liu AY (2007) The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder. BMC Urol 7:19",
"David: database for annotation, visualization, and integrated discovery. G DennisJr, B T Sherman, D A Hosack, J Yang, W Gao, H C Lane, R A Lempicki, Genome Biol. 453Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) David: database for annotation, visualization, and integrated discovery. Genome Biol 4(5):P3",
"A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. H Nielsen, J Engelbrecht, S Brunak, G Von Heijne, Int J Neural Syst. 85-6Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8(5-6):581-599",
"Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. A Krogh, B Larsson, G Von Heijne, E L Sonnhammer, J Mol Biol. 3053Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305(3):567-580",
"Mass spectrometric detection of tissue proteins in plasma. H Zhang, A Y Liu, P Loriaux, B Wollscheid, Y Zhou, J D Watts, R Aebersold, Mol Cell Proteomics. 61Zhang H, Liu AY, Loriaux P, Wollscheid B, Zhou Y, Watts JD, Aebersold R (2007) Mass spectrometric detection of tissue proteins in plasma. Mol Cell Proteomics 6(1):64-71",
"Reck-a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer. J C Clark, D M Thomas, P F Choong, C R Dass, Cancer Metastasis Rev. 263-4Clark JC, Thomas DM, Choong PF, Dass CR (2007) Reck-a newly discovered inhibitor of metastasis with prognostic signifi- cance in multiple forms of cancer. Cancer Metastasis Rev 26(3- 4):675-683",
"Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. A C Riddick, C J Shukla, C J Pennington, R Bass, R K Nuttall, A Hogan, K K Sethia, V Ellis, A T Collins, N J Maitland, R Y Ball, D R Edwards, Br J Cancer. 9212Riddick AC, Shukla CJ, Pennington CJ, Bass R, Nuttall RK, Hogan A, Sethia KK, Ellis V, Collins AT, Maitland NJ, Ball RY, Edwards DR (2005) Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. Br J Cancer 92(12):2171-2180",
"Increased incidence of matrix metalloproteinases in urine of cancer patients. M A Moses, D Wiederschain, K R Loughlin, D Zurakowski, C C Lamb, M R Freeman, Cancer Res. 587Moses MA, Wiederschain D, Loughlin KR, Zurakowski D, Lamb CC, Freeman MR (1998) Increased incidence of matrix metal- loproteinases in urine of cancer patients. Cancer Res 58(7):1395- 1399",
"Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. C K Taranger, A Noer, A L Sorensen, A M Hakelien, A C Boquest, P Collas, Mol Biol Cell. 1612Taranger CK, Noer A, Sorensen AL, Hakelien AM, Boquest AC, Collas P (2005) Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Mol Biol Cell 16 (12):5719-5735",
"Molecular and cellular characterization of abcg2 in the prostate. L E Pascal, A J Oudes, T W Petersen, Y A Goo, L S Walashek, L D True, A Y Liu, BMC Urol. 76Pascal LE, Oudes AJ, Petersen TW, Goo YA, Walashek LS, True LD, Liu AY (2007) Molecular and cellular characterization of abcg2 in the prostate. BMC Urol 7:6",
"Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. G R Cunha, W Ricke, A Thomson, P C Marker, G Risbridger, S W Hayward, Y Z Wang, A A Donjacour, T Kurita, J Steroid Biochem Mol Biol. 924Cunha GR, Ricke W, Thomson A, Marker PC, Risbridger G, Hayward SW, Wang YZ, Donjacour AA, Kurita T (2004) Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. J Steroid Biochem Mol Biol 92(4):221-236",
"Reactive stroma in prostate cancer progression. J A Tuxhorn, G E Ayala, D R Rowley, J Urol. 1666Tuxhorn JA, Ayala GE, Rowley DR (2001) Reactive stroma in prostate cancer progression. J Urol 166(6):2472-2483",
"Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling. J A Tuxhorn, G E Ayala, M J Smith, V C Smith, T D Dang, D R Rowley, Clin Cancer Res. 89Tuxhorn JA, Ayala GE, Smith MJ, Smith VC, Dang TD, Rowley DR (2002) Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling. Clin Cancer Res 8(9):2912-2923",
"What might a stromal response mean to prostate cancer progression?. D R Rowley, Cancer Metastasis Rev. 174Rowley DR (1998) What might a stromal response mean to prostate cancer progression? Cancer Metastasis Rev 17(4):411-419",
"Stromal mesenchyme cell genes of the human prostate and bladder. Y A Goo, D R Goodlett, L E Pascal, K D Worthington, R L Vessella, L D True, A Y Liu, BMC Urol. 517Goo YA, Goodlett DR, Pascal LE, Worthington KD, Vessella RL, True LD, Liu AY (2005) Stromal mesenchyme cell genes of the human prostate and bladder. BMC Urol 5:17",
"Global expression analysis of prostate cancer-associated stroma and epithelia. A M Richardson, K Woodson, Y Wang, J Rodriguez-Canales, H S Erickson, M A Tangrea, K Novakovic, S Gonzalez, A Velasco, E S Kawasaki, M R Emmert-Buck, R F Chuaqui, A Player, Diagn Mol Pathol. 164Richardson AM, Woodson K, Wang Y, Rodriguez-Canales J, Erickson HS, Tangrea MA, Novakovic K, Gonzalez S, Velasco A, Kawasaki ES, Emmert-Buck MR, Chuaqui RF, Player A (2007) Global expression analysis of prostate cancer-associated stroma and epithelia. Diagn Mol Pathol 16(4):189-197",
"Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes. L E Pascal, Y A Goo, R Z Vencio, L S Page, A A Chambers, E S Liebeskind, T K Takayama, L D True, A Y Liu, BMC Cancer. 9317Pascal LE, Goo YA, Vencio RZ, Page LS, Chambers AA, Liebeskind ES, Takayama TK, True LD, Liu AY (2009) Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes. BMC Cancer 9:317",
"Global gene expression analysis of reactive stroma in prostate cancer. O Dakhova, M Ozen, C J Creighton, R Li, G Ayala, D Rowley, M Ittmann, Clin Cancer Res. 1512Dakhova O, Ozen M, Creighton CJ, Li R, Ayala G, Rowley D, Ittmann M (2009) Global gene expression analysis of reactive stroma in prostate cancer. Clin Cancer Res 15(12):3979-3989",
"Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. T Ernst, M Hergenhahn, M Kenzelmann, C D Cohen, M Bonrouhi, A Weninger, R Klaren, E F Grone, M Wiesel, C Gudemann, J Kuster, W Schott, G Staehler, M Kretzler, M Hollstein, H J Grone, Am J Pathol. 1606Ernst T, Hergenhahn M, Kenzelmann M, Cohen CD, Bonrouhi M, Weninger A, Klaren R, Grone EF, Wiesel M, Gudemann C, Kuster J, Schott W, Staehler G, Kretzler M, Hollstein M, Grone HJ (2002) Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. Am J Pathol 160(6):2169-2180",
"Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts. H Zhao, D M Peehl, Prostate. 699Zhao H, Peehl DM (2009) Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts. Prostate 69(9):991-1000",
"Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting. S Y Sung, L W Chung, Differentiation. 709Sung SY, Chung LW (2002) Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting. Differentiation 70(9-10):506-521",
"Cd90/thy1 is overexpressed in prostate cancerassociated fibroblasts and could serve as a cancer biomarker. L D True, H Zhang, M Ye, C Y Huang, P S Nelson, Von Haller, P D Tjoelker, L W Kim, J S Qian, W J Smith, R D Ellis, W J Liebeskind, E S Liu, A Y , Mod Pathol. 2310True LD, Zhang H, Ye M, Huang CY, Nelson PS, von Haller PD, Tjoelker LW, Kim JS, Qian WJ, Smith RD, Ellis WJ, Liebeskind ES, Liu AY (2010) Cd90/thy1 is overexpressed in prostate cancer- associated fibroblasts and could serve as a cancer biomarker. Mod Pathol 2010 Oct; 23(10):1346-56",
"Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells. L E Pascal, R Z Vencio, Y A Goo, L S Page, C P Shadle, A Y Liu, Prostate. 6912Pascal LE, Vencio RZ, Goo YA, Page LS, Shadle CP, Liu AY (2009) Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells. Prostate 69(12):1353-1365",
"Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit. I Damjanov, B Horvat, Z Gibas, Lab Invest. 682Damjanov I, Horvat B, Gibas Z (1993) Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit. Lab Invest 68(2):220-232",
"Embryonal carcinoma cell induction of mirna and mrna changes in cocultured prostate stromal fibromuscular cells. E F Vencio, L E Pascal, L S Page, G Denyer, A J Wang, H Ruohola-Baker, S Zhang, K Wang, D J Galas, A Y Liu, doi:1002/jcp.22464J Clin Pathol. Vencio EF, Pascal LE, Page LS, Denyer G, Wang AJ, Ruohola- Baker H, Zhang S, Wang K, Galas DJ, Liu AY (2010) Embryonal carcinoma cell induction of mirna and mrna changes in co- cultured prostate stromal fibromuscular cells. J Clin Pathol doi:1002/jcp.22464",
"Analysis of prostate cancer by proteomics using tissue specimens. A Y Liu, H Zhang, C M Sorensen, D L Diamond, J Urol. 1731Liu AY, Zhang H, Sorensen CM, Diamond DL (2005) Analysis of prostate cancer by proteomics using tissue specimens. J Urol 173 (1):73-78",
"Cell-cell interaction in prostate gene regulation and cytodifferentiation. A Y Liu, L D True, L Latray, P S Nelson, W J Ellis, R L Vessella, P H Lange, L Hood, G Van Den Engh, Proc Natl Acad Sci. 9420Liu AY, True LD, LaTray L, Nelson PS, Ellis WJ, Vessella RL, Lange PH, Hood L, van den Engh G (1997) Cell-cell interaction in prostate gene regulation and cytodifferentiation. Proc Natl Acad Sci USA 94(20):10705-10710",
"Stromal cells of the human prostate: initial isolation and characterization. A Kassen, D M Sutkowski, H Ahn, J A Sensibar, J M Kozlowski, C Lee, Prostate. 282Kassen A, Sutkowski DM, Ahn H, Sensibar JA, Kozlowski JM, Lee C (1996) Stromal cells of the human prostate: initial isolation and characterization. Prostate 28(2):89-97",
"Exploration, normalization, and summaries of high density oligonucleotide array probe level data. R A Irizarry, B Hobbs, Collin F Beazer-Barclay, Y D Antonellis, K J Scherf, U Speed, T P , Biostatistics. 42Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4(2):249-264",
"Sbeams-microarray: database software supporting genomic expression analyses for systems biology. B Marzolf, E W Deutsch, P Moss, D Campbell, M H Johnson, T Galitski, BMC Bioinform. 7286Marzolf B, Deutsch EW, Moss P, Campbell D, Johnson MH, Galitski T (2006) Sbeams-microarray: database software supporting genomic expression analyses for systems biology. BMC Bioinform 7:286",
"The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder. L E Pascal, E W Deutsch, D S Campbell, M Korb, L D True, A Y Liu, BMC Urol. 719Pascal LE, Deutsch EW, Campbell DS, Korb M, True LD, Liu AY (2007) The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder. BMC Urol 7:19",
"David: database for annotation, visualization, and integrated discovery. G DennisJr, B T Sherman, D A Hosack, J Yang, W Gao, H C Lane, R A Lempicki, Genome Biol. 453Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) David: database for annotation, visualization, and integrated discovery. Genome Biol 4(5):P3",
"A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. H Nielsen, J Engelbrecht, S Brunak, G Von Heijne, Int J Neural Syst. 85-6Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8(5-6):581-599",
"Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. A Krogh, B Larsson, G Von Heijne, E L Sonnhammer, J Mol Biol. 3053Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305(3):567-580",
"Mass spectrometric detection of tissue proteins in plasma. H Zhang, A Y Liu, P Loriaux, B Wollscheid, Y Zhou, J D Watts, R Aebersold, Mol Cell Proteomics. 61Zhang H, Liu AY, Loriaux P, Wollscheid B, Zhou Y, Watts JD, Aebersold R (2007) Mass spectrometric detection of tissue proteins in plasma. Mol Cell Proteomics 6(1):64-71",
"Reck-a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer. J C Clark, D M Thomas, P F Choong, C R Dass, Cancer Metastasis Rev. 263-4Clark JC, Thomas DM, Choong PF, Dass CR (2007) Reck-a newly discovered inhibitor of metastasis with prognostic signifi- cance in multiple forms of cancer. Cancer Metastasis Rev 26(3- 4):675-683",
"Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. A C Riddick, C J Shukla, C J Pennington, R Bass, R K Nuttall, A Hogan, K K Sethia, V Ellis, A T Collins, N J Maitland, R Y Ball, D R Edwards, Br J Cancer. 9212Riddick AC, Shukla CJ, Pennington CJ, Bass R, Nuttall RK, Hogan A, Sethia KK, Ellis V, Collins AT, Maitland NJ, Ball RY, Edwards DR (2005) Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. Br J Cancer 92(12):2171-2180",
"Increased incidence of matrix metalloproteinases in urine of cancer patients. M A Moses, D Wiederschain, K R Loughlin, D Zurakowski, C C Lamb, M R Freeman, Cancer Res. 587Moses MA, Wiederschain D, Loughlin KR, Zurakowski D, Lamb CC, Freeman MR (1998) Increased incidence of matrix metal- loproteinases in urine of cancer patients. Cancer Res 58(7):1395- 1399",
"Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. C K Taranger, A Noer, A L Sorensen, A M Hakelien, A C Boquest, P Collas, Mol Biol Cell. 1612Taranger CK, Noer A, Sorensen AL, Hakelien AM, Boquest AC, Collas P (2005) Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Mol Biol Cell 16 (12):5719-5735",
"Molecular and cellular characterization of abcg2 in the prostate. L E Pascal, A J Oudes, T W Petersen, Y A Goo, L S Walashek, L D True, A Y Liu, BMC Urol. 76Pascal LE, Oudes AJ, Petersen TW, Goo YA, Walashek LS, True LD, Liu AY (2007) Molecular and cellular characterization of abcg2 in the prostate. BMC Urol 7:6"
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] | [
"Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development",
"Reactive stroma in prostate cancer progression",
"Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling",
"What might a stromal response mean to prostate cancer progression?",
"Stromal mesenchyme cell genes of the human prostate and bladder",
"Global expression analysis of prostate cancer-associated stroma and epithelia",
"Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes",
"Global gene expression analysis of reactive stroma in prostate cancer",
"Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue",
"Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts",
"Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting",
"Cd90/thy1 is overexpressed in prostate cancerassociated fibroblasts and could serve as a cancer biomarker",
"Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells",
"Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit",
"Embryonal carcinoma cell induction of mirna and mrna changes in cocultured prostate stromal fibromuscular cells",
"Analysis of prostate cancer by proteomics using tissue specimens",
"Cell-cell interaction in prostate gene regulation and cytodifferentiation",
"Stromal cells of the human prostate: initial isolation and characterization",
"Exploration, normalization, and summaries of high density oligonucleotide array probe level data",
"Sbeams-microarray: database software supporting genomic expression analyses for systems biology",
"The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder",
"David: database for annotation, visualization, and integrated discovery",
"A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites",
"Predicting transmembrane protein topology with a hidden markov model: application to complete genomes",
"Mass spectrometric detection of tissue proteins in plasma",
"Reck-a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer",
"Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues",
"Increased incidence of matrix metalloproteinases in urine of cancer patients",
"Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells",
"Molecular and cellular characterization of abcg2 in the prostate",
"Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development",
"Reactive stroma in prostate cancer progression",
"Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling",
"What might a stromal response mean to prostate cancer progression?",
"Stromal mesenchyme cell genes of the human prostate and bladder",
"Global expression analysis of prostate cancer-associated stroma and epithelia",
"Gene expression down-regulation in cd90+ prostate tumor-associated stromal cells involves potential organ-specific genes",
"Global gene expression analysis of reactive stroma in prostate cancer",
"Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue",
"Tumor-promoting phenotype of cd90hi prostate cancer-associated fibroblasts",
"Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting",
"Cd90/thy1 is overexpressed in prostate cancerassociated fibroblasts and could serve as a cancer biomarker",
"Temporal expression profiling of the effects of secreted factors from prostate stromal cells on embryonal carcinoma stem cells",
"Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, nccit",
"Embryonal carcinoma cell induction of mirna and mrna changes in cocultured prostate stromal fibromuscular cells",
"Analysis of prostate cancer by proteomics using tissue specimens",
"Cell-cell interaction in prostate gene regulation and cytodifferentiation",
"Stromal cells of the human prostate: initial isolation and characterization",
"Exploration, normalization, and summaries of high density oligonucleotide array probe level data",
"Sbeams-microarray: database software supporting genomic expression analyses for systems biology",
"The urologic epithelial stem cell database (uesc)-a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder",
"David: database for annotation, visualization, and integrated discovery",
"A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites",
"Predicting transmembrane protein topology with a hidden markov model: application to complete genomes",
"Mass spectrometric detection of tissue proteins in plasma",
"Reck-a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer",
"Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues",
"Increased incidence of matrix metalloproteinases in urine of cancer patients",
"Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells",
"Molecular and cellular characterization of abcg2 in the prostate"
] | [
"J Steroid Biochem Mol Biol",
"J Urol",
"Clin Cancer Res",
"Cancer Metastasis Rev",
"BMC Urol",
"Diagn Mol Pathol",
"BMC Cancer",
"Clin Cancer Res",
"Am J Pathol",
"Prostate",
"Differentiation",
"Mod Pathol",
"Prostate",
"Lab Invest",
"J Clin Pathol",
"J Urol",
"Proc Natl Acad Sci",
"Prostate",
"Biostatistics",
"BMC Bioinform",
"BMC Urol",
"Genome Biol",
"Int J Neural Syst",
"J Mol Biol",
"Mol Cell Proteomics",
"Cancer Metastasis Rev",
"Br J Cancer",
"Cancer Res",
"Mol Biol Cell",
"BMC Urol",
"J Steroid Biochem Mol Biol",
"J Urol",
"Clin Cancer Res",
"Cancer Metastasis Rev",
"BMC Urol",
"Diagn Mol Pathol",
"BMC Cancer",
"Clin Cancer Res",
"Am J Pathol",
"Prostate",
"Differentiation",
"Mod Pathol",
"Prostate",
"Lab Invest",
"J Clin Pathol",
"J Urol",
"Proc Natl Acad Sci",
"Prostate",
"Biostatistics",
"BMC Bioinform",
"BMC Urol",
"Genome Biol",
"Int J Neural Syst",
"J Mol Biol",
"Mol Cell Proteomics",
"Cancer Metastasis Rev",
"Br J Cancer",
"Cancer Res",
"Mol Biol Cell",
"BMC Urol"
] | [
"\nFig. 3\n3Schematic of RECK pathway in stromal-epithelial interaction in prostate cancer. Decreased RECK expression leads to activation of MMPs and degradation of ECM proteins allowing dissemination of tumor cells. Virtual Northern blot format shows array signals for MMP9, HRAS and RECK in NP stromal vs. CP stromal cells (1 and 2 from two different specimens) and for TIMPs in NP epithelial vs. CP cancer cells",
"\nFig. 3\n3Schematic of RECK pathway in stromal-epithelial interaction in prostate cancer. Decreased RECK expression leads to activation of MMPs and degradation of ECM proteins allowing dissemination of tumor cells. Virtual Northern blot format shows array signals for MMP9, HRAS and RECK in NP stromal vs. CP stromal cells (1 and 2 from two different specimens) and for TIMPs in NP epithelial vs. CP cancer cells"
] | [
"Schematic of RECK pathway in stromal-epithelial interaction in prostate cancer. Decreased RECK expression leads to activation of MMPs and degradation of ECM proteins allowing dissemination of tumor cells. Virtual Northern blot format shows array signals for MMP9, HRAS and RECK in NP stromal vs. CP stromal cells (1 and 2 from two different specimens) and for TIMPs in NP epithelial vs. CP cancer cells",
"Schematic of RECK pathway in stromal-epithelial interaction in prostate cancer. Decreased RECK expression leads to activation of MMPs and degradation of ECM proteins allowing dissemination of tumor cells. Virtual Northern blot format shows array signals for MMP9, HRAS and RECK in NP stromal vs. CP stromal cells (1 and 2 from two different specimens) and for TIMPs in NP epithelial vs. CP cancer cells"
] | [
"Fig. 1a and b)",
"Fig. 1c and d)",
"(Fig. 1d)",
"(Fig. 2a)",
"(Fig. 2b)",
"Figure 3",
"Fig. 1",
"Fig. 1a and b)",
"Fig. 1c and d)",
"(Fig. 1d)",
"(Fig. 2a)",
"(Fig. 2b)",
"Figure 3",
"Fig. 1"
] | [] | [
"The prostate is a glandular organ composed of secretory luminal cell-lined acini and an underlying layer of basal cells supported within a fibromuscular stroma. In urologic organ development, the stroma provides the inductive signaling to stem/progenitor cells to produce the resultant tissue [1]. Increasingly, it is thought that the stromal environment might play a significant role in disease development and progression as well. An altered stroma has been identified in association with both prostate cancer [2][3][4] as well as premalignant prostate diseases including prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) [3,5]. In prostate cancer, the stroma is also affected in that gene expression in cancer-associated fibroblastic (CAF) stromal cells differs significantly from that of normal tissue stromal cells [6][7][8][9][10]. Prostate CAFs are capable of inducing proliferation and malignant transformation [1] and have been postulated to drive tumor progression [11]. These studies strongly suggest that the prostate stroma plays a significant role in facilitating disease progression.",
"CAFs immediately surrounding prostate cancer can be identified by strong CD90 immunostaining [12] and CD90 hi cancer-associated fibroblasts (CAFs) were postulated to have greater tumor-promoting effects than CD90 lo CAFs [10]. Comparative transcriptome analysis of isolated CD90 + CAFs and CD49a + normal tissue stromal cells revealed a decrease in the expression of genes involved in smooth muscle cell differentiation and those specific or restricted to the prostate [5,7]. Genes that encode secreted proteins or hormones are likely candidates responsible for organspecific stromal induction, and dysregulation of these genes might contribute to disease progression through stromal-stem cell signaling.",
"To examine the molecular mechanisms involved in prostate stromal-stem cell interaction, we previously developed an in vitro co-culture system with stromal cells isolated from normal prostate (referred to as NP) and a cancer stem cell type, the embryonal carcinoma (EC) cell line, NCCIT [13]. NCCIT is a pluripotent cell line that can be readily maintained in cell culture in an undifferentiated state [14]. When co-cultured without direct cell contact, NCCIT cells were induced by NP stromal cells to differentiate into a cell population that expressed predominantly prostate stromal cell genes (e.g., PENK). Induced NCCIT cells also lost expression of stem cell genes and underwent a change in morphology with reduced proliferation [13]. Bladder stromal cells isolated from human specimens induced a bladder stromal-like expression (e.g., absent PENK) [13], thus demonstrating plasticity-a property of stem cells-in NCCIT response. This cellto-cell signaling was presumably mediated by diffusible factors. Interestingly, NP prostate stromal cells were also significantly altered by co-culture with NCCIT, whereas NCCIT had no significant effect on the gene expression of CP stromal cells [15]. In this study, NCCIT induced an increase in expression for CD90, MIRN21, HGF, SFRP1, BGN, and decreased expression of IGFBP5, HSD11B1a in NP stromal cells. These findings suggest that alterations in prostate stroma could be induced by stem or cancer stem cell influence. In this study, we examined the inductive functioning of cultured CD90 + CAFs (referred to here as CP) stromal cells for comparison to that of NP stromal cells.",
"NCCIT and prostate cancer cell line PC3 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 (Cambrex BioScience, Walkersville, MD) media supplemented with 10% heat-inactivated fetal bovine serum (FBS). In this culture condition, NCCIT cells maintain expression of stem cell genes and microRNA, and are alkaline phosphatase positive [13,15]. The tissue samples used in this study consisted of cancer-enriched CP tissue specimens, characterized as CD90 hi TIMP1 lo by Western blot analysis of tissue digestion media [7], obtained from 2 radical prostatectomy surgery patients. Note 'pure' CP specimens had increased CD90 expression and minimal reactivity to TIMP1, which is secreted by luminal cells and not by cancer cells [16], whereas NP specimens were CD90 lo TIMP1 hi . All tissue samples were obtained under approval by the University of Washington IRB and were collected following a standard protocol. Upon receipt of a resected specimen, 3-mm thick transverse sections were made of the gland after inking the exterior surface. Frozen sections of tissue blocks were histologically prepared to locate the tumor foci for dissection. Pathology characteristics of the 2 tumors were 08-021CP: Gleason 5+4, T3a, tumor volume 4.5 cc; 08-028CP: Gleason 3+4, T2c, tumor volume 2.5 cc. NP specimens were obtained and analyzed as described previously [13]. Briefly, between 1 and 10 g of tissue from the anterior aspect of the prostate (transition zone) were excised; corresponding frozen sections of the tissue blocks were histologically assessed to confirm specimens were free of cancer.",
"Tissue samples were minced and digested by overnight incubation at room temperature in 0.2% collagenase type I (Invitrogen, Carlsbad, CA) in RPMI-1640 media supplemented with 5% FBS and 10 −8 M dihydrotestosterone on a magnetic stirrer. The resultant cell suspension was filtered with a 70-μm Falcon cell strainer to remove any nondigested tissue, diluted with an equal volume of Hanks balanced salt solution (HBSS), and aspirated with an 18gauge needle. The single cell preparation was partitioned into stromal and epithelial fractions on a discontinuous Percoll density gradient (Amersham Pharmacia, Piscataway, NJ) as described [17,18]. Cells banding at a density of ρ= 1.035 were collected as the stromal fraction for magnetic cell sorting (MACS). The cell-free tissue digestion media supernatant was analyzed by Western blotting to verify that the specimens were of cancer.",
"CP stromal cells were sorted using anti-CD90. Briefly, the gradient-purified stromal cell fraction was resuspended in 100 μl 0.1% bovine serum albumin (BSA)-HBSS, and CD90-Phycoerythrin (PE) mouse monoclonal antibody (clone 5E10, BD-PharMingen, San Diego, CA) at 1:20 was added for 15 min at room temperature in the dark. The reaction was stopped by 1 ml 0.1% BSA-HBSS and centrifugation. The labeled cells were resuspended in BSA-HBSS, and 15 μl paramagnetic microbead conjugated anti-PE (Miltenyi Biotec, Auburn, CA) was added for 15 min. After incubation, the positive and negative cells were separated by AutoMACS (Miltenyi Biotec) using the double positive sort program. Aliquots of positive and negative cell fractions were analyzed by fluorescenceactivated cell sorting (FACS, Becton Dickinson, Mountain View, CA) to gauge the sort efficiency; only >85% CD90 + fractions were used for experiments.",
"The sorted CD90 + stromal cells were adapted to culture for 3-5 passages in RPMI-1640 media supplemented with 10% FBS, and their identity verified by RT-PCR analysis of gene expression as described [5]. For co-culture experiments, 0.4 μm polycarbonate membrane trans-well inserts (Corning, Corning, NY) to preclude cell contact were employed. NCCIT cells were seeded at 10 4 /ml in RPMI-1640, 10% FBS on 6-well plates, and CP stromal cells were seeded at 10 4 /ml on the insert. Controls were NCCIT and CP stromal cells alone. Cultures were maintained for 3 d. The 3 d-time point was chosen based on our previous time course study of NP stromal cell-induced differentiation of NCCIT [13]. In that experiment, gene expression changes in NCCIT were detected as early as 6 h in co-culture, and by the third day, nearly the entire stromal gene repertoire was induced as shown by a principal components analysis of time-point transcriptomes. Cells were trypsinized and lysed in RLT Buffer (Qiagen, Valencia, CA).",
"RNA was isolated from cultures of CP stromal, NCCIT, and NCCIT + CP stromal cells at 3 d. Transcriptomes of untreated NCCIT, NP stromal cells and NCCIT at 3 d of co-culture with NP stromal cells were determined previously [13], as were sorted CD90 + CP stromal cells, i.e., not cultured [7]. These datasets were made available online (http://scgap.systemsbiology.net/data/). Quality and concentration of RNA were determined using an Agilent 2100 Bioanalyzer and RNA Nano Labchip (Agilent Technologies, Santa Clara, CA). Between 2 and 7 replicates of each experimental condition or control were assayed with the Human Genome U133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA). The U133 Plus 2.0 array contains probesets representing 54,675 genes, splice variants, and ESTs. The GeneChips were prepared, hybridized, and scanned according to the protocols provided by Affymetrix (P/N 702232 Rev. 2). Briefly, 200 ng of RNA was reverse transcribed with poly (dT) primer containing a T7 promoter, and the cDNA was made double-stranded. In vitro transcription was performed to produce unlabeled cRNA. Next, first-strand cDNA was produced with random primers, and the cDNA was made double-stranded with poly (dT) primer/T7 promoter. A final in vitro transcription was done with biotinylated ribonucleotides. The biotin-labeled cRNA was hybridized to the GeneChips. The chips were washed and stained with streptavidin-PE using an Affymetrix FS-450 fluidics station. Data was collected with an Affymetrix GeneChip Scanner 3000.",
"A probabilistic comparative analysis between transcriptomes of treated NCCIT was used to highlight differentially expressed genes with respect to that of untreated NCCIT [7]. Gene expression level was defined as the normalized and summarized intensities of each GeneChip probeset, and was presented as its logarithmic value: X=log 2 (Normalized intensity). This step was carried out using the standard robust multi-array average (RMA) method [19], implemented in the in-house analysis pipeline SBEAMS [20]. Data were presented on a grayscale indicating RMAnormalized Affymetrix signal intensity [21]. Signals of 10 or less were represented as white and signals greater than or equal to 10,000 as black. Higher Affymetrix signal (more black) indicated higher levels of gene expression.",
"The strength of differential expression between any pair of experiments was estimated by M i ¼ log 2 ratio ð Þ¼ X 3d À X 0h , where 0 h represented the untreated NCCIT and 3d represented treated NCCIT at 3 d. Reliability of the differential expression was estimated by calculating the probability P=P(X 3d >X 0h ) or P=P(X 3d <X 0h ) according to a statistical model that assumed a normal distribution X j ∼N (m j ,s j ), where m j and s j were the mean and maximum difference, respectively, among the replicates of group j. Consistently, P= P(X 3d > X 0h ) or P= P(X 3d < X 0h ) was reported if m 3d >m 0h or m 3d <m 0h . Functional and ontology enrichment analysis was performed using the DAVID webbased tool [22]. Freely available prediction software for determination of signal peptides and likely cell membranespanning sequences was also used. Signal peptides were predicted using SignalP 3.0 [23], and transmembrane (TM) regions were predicted using TMHMM 2.0 [24] for protein topology and the number of TM helices. Information from both SignalP and TMHMM were combined to identify proteins that contained predicted cleavable signal peptides and no predicted TM segments as reported previously [25].",
"Gene expression changes induced in NCCIT by secreted factors from CP stromal cells were determined by Affymetrix DNA microarray analysis. Following 3-days co-culture with CP stromal cells, the induced expression in NCCIT cells (CP-NCCIT) of smooth muscle genes ACTA2, CALD1, CNN1, prostate stromal genes PENK, CNTN1, ChGn, BMP2 [5,7], androgen receptor (AR) and a stromal gene GFRA1 was significantly less than that previously shown by NP stromal cells (NP-NCCIT) [13]. The CP-NCCIT transcriptome dataset contained minimal signal levels for CALD1, and CNN1 compared to that of NP-NCCIT ( Fig. 1a and b). Other queried prostate genes such as tenascin C (TNC) [5] were also lacking. The similar induction of stromal gene stanniocalcin (STC1) and increased expression of CD90/THY1 in CP-NCCIT showed that gene expression changes did occur in NCCIT cocultured with CP stromal cells. Induction of CD90 was notably higher in CP-NCCIT than in NP-NCCIT (NCCIT cells are also positive for CD90, a stem cell marker). This reflected the increased CD90 expression in CP stromal cells.",
"A comparison of stem cell gene expression in treated NCCIT showed higher signal levels of NANOG, POU5F1, TDGF1, and SOX2 in CP-NCCIT than in NP-NCCIT ( Fig. 1c and d). Induction of NCCIT by NP stromal cells was found to lead to almost complete down-regulation of these stem cell genes [13], whereas CP stromal cells had apparently little effect. Of note was the detection of ABCG2 (a prostate progenitor cell marker) expression in NP-NCCIT but not CP-NCCIT (Fig. 1d). NCCIT is negative for ABCG2 expression.",
"To identify genes encoding secreted proteins that might function in cell-cell signaling, the most differentially expressed genes in NP-NCCIT vs. CP-NCCIT (or NP stromal vs. CP stromal) were analyzed using the DAVID annotation tool. CP stromal induction led to upregulation of several such genes including LEFTY2 and TAC1, while NP stromal induction led to up-regulation of ADAMTS1, IGFBP5, WNT5A and others (Table S1). Overall, there were many more such genes induced in NP-NCCIT than CP-NCCIT. This showed a smaller pool of candidate signaling molecules produced by CP stromal cells.",
"Differentially expressed genes were also analyzed for significant enrichment with respect to functional categories using DAVID. The top KEGG pathways identified were cytokine-cytokine receptor interaction, chemokine signaling pathway, extracellular matrix (ECM)-receptor interaction, cell adhesion and focal adhesion. Enrichment of these functional categories was prominent in the CP-NCCIT vs. NP-NCCIT datasets. Of particular interest were the genes that contribute to the functioning and maintenance of ECM. Matrix metalloproteinases (MMPs) are involved in the degradation of ECM proteins and have been associated with tumor cell invasion. The membrane-anchored reversioninducing cysteine-rich protein with Kazal motifs (RECK) is a potent inhibitor of MMP activity. RECK down-regulation has been identified in many cancers, and it has been reported that high RECK expression levels were associated with favorable prognosis in prostate cancer [26,27]. In comparing the CP-NCCIT and NP-NCCIT expression profiles, several genes associated with RECK were differentially expressed (Fig. 2a). For example, induction of MMP9, a potential prostate cancer urine biomarker [28], was greater in CP-NCCIT than in NP-NCCIT (Fig. 2b). MMP9 expression was also higher in sorted CP vs. NP stromal cell transcriptomes. In contrast, RECK was more up-regulated in NP-NCCIT than CP-NCCIT, as was tissue inhibitor of metalloproteinase TIMP1, an antagonist of MMPs. With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. Figure 3 illustrates the RECK pathway network in which MMPs synthesized by CP stromal cells could lead to ECM degradation, which would in turn promote tumor cell escape. The MMP effect is amplified by the decrease in TIMP expression in cancer cells. Increase in MMPs is due to down-regulation of RECK. Fig. 1 Expression profiles of stromal and stem cell genes in treated NCCIT. a Increased expression of prostate stromal cell-specific genes relative to untreated NCCIT was detected in co-cultures of NP stromal + NCCIT cells (labeled NP-NCCIT). Expression of these stromal genes was less pronounced in co-culture of CP stromal + NCCIT cells (CP-NCCIT). For example, PENK was not induced. Note the increase in tumor-associated stromal marker CD90/THY1. b The CP-NCCIT transcriptome dataset (first column) contains minimal signal for smooth muscle differentiation genes (CALD1, CNN1) present in the NP-NCCIT transcriptome (third column) compared to untreated NCCIT transcriptome (second column) in virtual Northern blot format (darker shades of boxes indicate higher mRNA levels with background ≤50 RMA units). c Higher expression of several stem cell genes was detected in CP-NCCIT relative to NP-NCCIT as well as in cultured CP stromal cells relative to NP stromal cells. d Virtual Northern blot format shows that for stem cell genes NANOG, SOX2, CD9 and THY1, expression was increased in CP-NCCIT compared to untreated NCCIT, whereas expression was decreased in NP-NCCIT",
"NCCIT response to CD90 + CAFs tumor-associated was significantly altered from the gene expression changes induced by normal stromal cell factors. Previously, NP stromal cells induced a loss of embryonic stem cell markers and an up-regulation of genes characteristic of stromal mesenchyme, some epithelial genes and cancer stem cell genes. NCCIT response to CP stromal cells was characterized by an absence or decreased induction of genes involved in smooth muscle cell differentiation and those expressed by the prostate but not the bladder, i.e., organ restricted. At the same time, the decrease in stem cell gene expression was not as pronounced. This altered differenti-ation response could be due to differences in signaling factors secreted from tumor-associated stromal cells. These differences could be the result of either a reduction or loss of certain proteins such as the hormone PENK. Stromal cells are important in tissue repair and renewal as suggested by their demonstrated role in prostate and bladder formation. Organ specificity in this process could be due to the differentially expressed genes between the stromal cell types. Indeed, we previously showed that bladder stromal cells induced a different response from NCCIT than prostate stromal cells. Thus, if tumor-associated stromal cells were unable to provide the appropriate signaling, then normal histodevelopment would not occur, instead cancer development takes place. Although only two cases of CP stromal cells were tested, they did provide a demonstration that stem cell induction was markedly different from that by NP stromal cells. These differences, including reduced induction of smooth muscle cell genes and increased induction of genes involved in ECM remodeling, are consistent with alterations to the prostate tumor microenvironment. When we can model epithelial cytodifferentiation, i.e., PSA secretion, with cell contact and ECM, then a study using multiple samples of CP stromal cells can be carried out. For example, one could contrast the effect of CP stromal cells isolated from Gleason 3 +3 vs. Gleason 4+4 tumors.",
"The differentially induced expression pattern of genes involved in the ECM RECK pathway in CP-NCCIT vs. NP-NCCIT appears to mimic that in primary tumors as inferred from the transcriptomes of sorted stromal and epithelial cell types. NP stromal induction produced upregulation of TIMP1 and RECK whereas CP stromal induction produced up-regulation of MMP9 and comparatively less of TIMP1 and RECK. In the sorted cells, expression of MMP9 (and HRAS, which inhibits RECK) is higher in CP stromal than NP stromal, whereas that of RECK is higher in NP stromal. TIMP1 protein in cancer is absent [16]. Thus, our in vitro model of CP stromal induction of NCCIT recapitulated to some degree a major pathway important in cancer development. In response to NP stromal influence, NCCIT cells were induced to express a transcriptome with a predominant but incomplete stromal mesenchyme profile. However, the response to CP stromal cell influence with regard to induction of stromal mesenchyme genes and loss of stem cell genes was significantly less. This difference could simply reflect a reduction in secreted factors from CP stromal cells compared to NP stromal cells and therefore a lesser degree of influence on NCCIT cells, or it could represent a shift in the heterogeneity of the treated NCCIT cell population.",
"With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. In summary, these experimental results showed that in induction of stem cells CP stromal cells were very different from NP stromal cells. The abnormal gene expression of CP stromal cells may well be the cause. Whether this would lead to cancer cell differentiation is still unknown since heterotypic cell contact and ECM were not provided for in the co-culture. Also unknown is whether other cell types beside stromal (e.g., epithelial) were induced in this system. For example, some ABCG2 expression was detected in NP-NCCIT, and this may suggest a small subpopulation with this marker. ABCG2 expression was identified in a putative prostate progenitor cell population localized to the basal epithelium [30]. It is therefore possible that more than one cell lineage, stromal and epithelial, could result from stromal induction of stem cells.",
"The prostate is a glandular organ composed of secretory luminal cell-lined acini and an underlying layer of basal cells supported within a fibromuscular stroma. In urologic organ development, the stroma provides the inductive signaling to stem/progenitor cells to produce the resultant tissue [1]. Increasingly, it is thought that the stromal environment might play a significant role in disease development and progression as well. An altered stroma has been identified in association with both prostate cancer [2][3][4] as well as premalignant prostate diseases including prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) [3,5]. In prostate cancer, the stroma is also affected in that gene expression in cancer-associated fibroblastic (CAF) stromal cells differs significantly from that of normal tissue stromal cells [6][7][8][9][10]. Prostate CAFs are capable of inducing proliferation and malignant transformation [1] and have been postulated to drive tumor progression [11]. These studies strongly suggest that the prostate stroma plays a significant role in facilitating disease progression.",
"CAFs immediately surrounding prostate cancer can be identified by strong CD90 immunostaining [12] and CD90 hi cancer-associated fibroblasts (CAFs) were postulated to have greater tumor-promoting effects than CD90 lo CAFs [10]. Comparative transcriptome analysis of isolated CD90 + CAFs and CD49a + normal tissue stromal cells revealed a decrease in the expression of genes involved in smooth muscle cell differentiation and those specific or restricted to the prostate [5,7]. Genes that encode secreted proteins or hormones are likely candidates responsible for organspecific stromal induction, and dysregulation of these genes might contribute to disease progression through stromal-stem cell signaling.",
"To examine the molecular mechanisms involved in prostate stromal-stem cell interaction, we previously developed an in vitro co-culture system with stromal cells isolated from normal prostate (referred to as NP) and a cancer stem cell type, the embryonal carcinoma (EC) cell line, NCCIT [13]. NCCIT is a pluripotent cell line that can be readily maintained in cell culture in an undifferentiated state [14]. When co-cultured without direct cell contact, NCCIT cells were induced by NP stromal cells to differentiate into a cell population that expressed predominantly prostate stromal cell genes (e.g., PENK). Induced NCCIT cells also lost expression of stem cell genes and underwent a change in morphology with reduced proliferation [13]. Bladder stromal cells isolated from human specimens induced a bladder stromal-like expression (e.g., absent PENK) [13], thus demonstrating plasticity-a property of stem cells-in NCCIT response. This cellto-cell signaling was presumably mediated by diffusible factors. Interestingly, NP prostate stromal cells were also significantly altered by co-culture with NCCIT, whereas NCCIT had no significant effect on the gene expression of CP stromal cells [15]. In this study, NCCIT induced an increase in expression for CD90, MIRN21, HGF, SFRP1, BGN, and decreased expression of IGFBP5, HSD11B1a in NP stromal cells. These findings suggest that alterations in prostate stroma could be induced by stem or cancer stem cell influence. In this study, we examined the inductive functioning of cultured CD90 + CAFs (referred to here as CP) stromal cells for comparison to that of NP stromal cells.",
"NCCIT and prostate cancer cell line PC3 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 (Cambrex BioScience, Walkersville, MD) media supplemented with 10% heat-inactivated fetal bovine serum (FBS). In this culture condition, NCCIT cells maintain expression of stem cell genes and microRNA, and are alkaline phosphatase positive [13,15]. The tissue samples used in this study consisted of cancer-enriched CP tissue specimens, characterized as CD90 hi TIMP1 lo by Western blot analysis of tissue digestion media [7], obtained from 2 radical prostatectomy surgery patients. Note 'pure' CP specimens had increased CD90 expression and minimal reactivity to TIMP1, which is secreted by luminal cells and not by cancer cells [16], whereas NP specimens were CD90 lo TIMP1 hi . All tissue samples were obtained under approval by the University of Washington IRB and were collected following a standard protocol. Upon receipt of a resected specimen, 3-mm thick transverse sections were made of the gland after inking the exterior surface. Frozen sections of tissue blocks were histologically prepared to locate the tumor foci for dissection. Pathology characteristics of the 2 tumors were 08-021CP: Gleason 5+4, T3a, tumor volume 4.5 cc; 08-028CP: Gleason 3+4, T2c, tumor volume 2.5 cc. NP specimens were obtained and analyzed as described previously [13]. Briefly, between 1 and 10 g of tissue from the anterior aspect of the prostate (transition zone) were excised; corresponding frozen sections of the tissue blocks were histologically assessed to confirm specimens were free of cancer.",
"Tissue samples were minced and digested by overnight incubation at room temperature in 0.2% collagenase type I (Invitrogen, Carlsbad, CA) in RPMI-1640 media supplemented with 5% FBS and 10 −8 M dihydrotestosterone on a magnetic stirrer. The resultant cell suspension was filtered with a 70-μm Falcon cell strainer to remove any nondigested tissue, diluted with an equal volume of Hanks balanced salt solution (HBSS), and aspirated with an 18gauge needle. The single cell preparation was partitioned into stromal and epithelial fractions on a discontinuous Percoll density gradient (Amersham Pharmacia, Piscataway, NJ) as described [17,18]. Cells banding at a density of ρ= 1.035 were collected as the stromal fraction for magnetic cell sorting (MACS). The cell-free tissue digestion media supernatant was analyzed by Western blotting to verify that the specimens were of cancer.",
"CP stromal cells were sorted using anti-CD90. Briefly, the gradient-purified stromal cell fraction was resuspended in 100 μl 0.1% bovine serum albumin (BSA)-HBSS, and CD90-Phycoerythrin (PE) mouse monoclonal antibody (clone 5E10, BD-PharMingen, San Diego, CA) at 1:20 was added for 15 min at room temperature in the dark. The reaction was stopped by 1 ml 0.1% BSA-HBSS and centrifugation. The labeled cells were resuspended in BSA-HBSS, and 15 μl paramagnetic microbead conjugated anti-PE (Miltenyi Biotec, Auburn, CA) was added for 15 min. After incubation, the positive and negative cells were separated by AutoMACS (Miltenyi Biotec) using the double positive sort program. Aliquots of positive and negative cell fractions were analyzed by fluorescenceactivated cell sorting (FACS, Becton Dickinson, Mountain View, CA) to gauge the sort efficiency; only >85% CD90 + fractions were used for experiments.",
"The sorted CD90 + stromal cells were adapted to culture for 3-5 passages in RPMI-1640 media supplemented with 10% FBS, and their identity verified by RT-PCR analysis of gene expression as described [5]. For co-culture experiments, 0.4 μm polycarbonate membrane trans-well inserts (Corning, Corning, NY) to preclude cell contact were employed. NCCIT cells were seeded at 10 4 /ml in RPMI-1640, 10% FBS on 6-well plates, and CP stromal cells were seeded at 10 4 /ml on the insert. Controls were NCCIT and CP stromal cells alone. Cultures were maintained for 3 d. The 3 d-time point was chosen based on our previous time course study of NP stromal cell-induced differentiation of NCCIT [13]. In that experiment, gene expression changes in NCCIT were detected as early as 6 h in co-culture, and by the third day, nearly the entire stromal gene repertoire was induced as shown by a principal components analysis of time-point transcriptomes. Cells were trypsinized and lysed in RLT Buffer (Qiagen, Valencia, CA).",
"RNA was isolated from cultures of CP stromal, NCCIT, and NCCIT + CP stromal cells at 3 d. Transcriptomes of untreated NCCIT, NP stromal cells and NCCIT at 3 d of co-culture with NP stromal cells were determined previously [13], as were sorted CD90 + CP stromal cells, i.e., not cultured [7]. These datasets were made available online (http://scgap.systemsbiology.net/data/). Quality and concentration of RNA were determined using an Agilent 2100 Bioanalyzer and RNA Nano Labchip (Agilent Technologies, Santa Clara, CA). Between 2 and 7 replicates of each experimental condition or control were assayed with the Human Genome U133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA). The U133 Plus 2.0 array contains probesets representing 54,675 genes, splice variants, and ESTs. The GeneChips were prepared, hybridized, and scanned according to the protocols provided by Affymetrix (P/N 702232 Rev. 2). Briefly, 200 ng of RNA was reverse transcribed with poly (dT) primer containing a T7 promoter, and the cDNA was made double-stranded. In vitro transcription was performed to produce unlabeled cRNA. Next, first-strand cDNA was produced with random primers, and the cDNA was made double-stranded with poly (dT) primer/T7 promoter. A final in vitro transcription was done with biotinylated ribonucleotides. The biotin-labeled cRNA was hybridized to the GeneChips. The chips were washed and stained with streptavidin-PE using an Affymetrix FS-450 fluidics station. Data was collected with an Affymetrix GeneChip Scanner 3000.",
"A probabilistic comparative analysis between transcriptomes of treated NCCIT was used to highlight differentially expressed genes with respect to that of untreated NCCIT [7]. Gene expression level was defined as the normalized and summarized intensities of each GeneChip probeset, and was presented as its logarithmic value: X=log 2 (Normalized intensity). This step was carried out using the standard robust multi-array average (RMA) method [19], implemented in the in-house analysis pipeline SBEAMS [20]. Data were presented on a grayscale indicating RMAnormalized Affymetrix signal intensity [21]. Signals of 10 or less were represented as white and signals greater than or equal to 10,000 as black. Higher Affymetrix signal (more black) indicated higher levels of gene expression.",
"The strength of differential expression between any pair of experiments was estimated by M i ¼ log 2 ratio ð Þ¼ X 3d À X 0h , where 0 h represented the untreated NCCIT and 3d represented treated NCCIT at 3 d. Reliability of the differential expression was estimated by calculating the probability P=P(X 3d >X 0h ) or P=P(X 3d <X 0h ) according to a statistical model that assumed a normal distribution X j ∼N (m j ,s j ), where m j and s j were the mean and maximum difference, respectively, among the replicates of group j. Consistently, P= P(X 3d > X 0h ) or P= P(X 3d < X 0h ) was reported if m 3d >m 0h or m 3d <m 0h . Functional and ontology enrichment analysis was performed using the DAVID webbased tool [22]. Freely available prediction software for determination of signal peptides and likely cell membranespanning sequences was also used. Signal peptides were predicted using SignalP 3.0 [23], and transmembrane (TM) regions were predicted using TMHMM 2.0 [24] for protein topology and the number of TM helices. Information from both SignalP and TMHMM were combined to identify proteins that contained predicted cleavable signal peptides and no predicted TM segments as reported previously [25].",
"Gene expression changes induced in NCCIT by secreted factors from CP stromal cells were determined by Affymetrix DNA microarray analysis. Following 3-days co-culture with CP stromal cells, the induced expression in NCCIT cells (CP-NCCIT) of smooth muscle genes ACTA2, CALD1, CNN1, prostate stromal genes PENK, CNTN1, ChGn, BMP2 [5,7], androgen receptor (AR) and a stromal gene GFRA1 was significantly less than that previously shown by NP stromal cells (NP-NCCIT) [13]. The CP-NCCIT transcriptome dataset contained minimal signal levels for CALD1, and CNN1 compared to that of NP-NCCIT ( Fig. 1a and b). Other queried prostate genes such as tenascin C (TNC) [5] were also lacking. The similar induction of stromal gene stanniocalcin (STC1) and increased expression of CD90/THY1 in CP-NCCIT showed that gene expression changes did occur in NCCIT cocultured with CP stromal cells. Induction of CD90 was notably higher in CP-NCCIT than in NP-NCCIT (NCCIT cells are also positive for CD90, a stem cell marker). This reflected the increased CD90 expression in CP stromal cells.",
"A comparison of stem cell gene expression in treated NCCIT showed higher signal levels of NANOG, POU5F1, TDGF1, and SOX2 in CP-NCCIT than in NP-NCCIT ( Fig. 1c and d). Induction of NCCIT by NP stromal cells was found to lead to almost complete down-regulation of these stem cell genes [13], whereas CP stromal cells had apparently little effect. Of note was the detection of ABCG2 (a prostate progenitor cell marker) expression in NP-NCCIT but not CP-NCCIT (Fig. 1d). NCCIT is negative for ABCG2 expression.",
"To identify genes encoding secreted proteins that might function in cell-cell signaling, the most differentially expressed genes in NP-NCCIT vs. CP-NCCIT (or NP stromal vs. CP stromal) were analyzed using the DAVID annotation tool. CP stromal induction led to upregulation of several such genes including LEFTY2 and TAC1, while NP stromal induction led to up-regulation of ADAMTS1, IGFBP5, WNT5A and others (Table S1). Overall, there were many more such genes induced in NP-NCCIT than CP-NCCIT. This showed a smaller pool of candidate signaling molecules produced by CP stromal cells.",
"Differentially expressed genes were also analyzed for significant enrichment with respect to functional categories using DAVID. The top KEGG pathways identified were cytokine-cytokine receptor interaction, chemokine signaling pathway, extracellular matrix (ECM)-receptor interaction, cell adhesion and focal adhesion. Enrichment of these functional categories was prominent in the CP-NCCIT vs. NP-NCCIT datasets. Of particular interest were the genes that contribute to the functioning and maintenance of ECM. Matrix metalloproteinases (MMPs) are involved in the degradation of ECM proteins and have been associated with tumor cell invasion. The membrane-anchored reversioninducing cysteine-rich protein with Kazal motifs (RECK) is a potent inhibitor of MMP activity. RECK down-regulation has been identified in many cancers, and it has been reported that high RECK expression levels were associated with favorable prognosis in prostate cancer [26,27]. In comparing the CP-NCCIT and NP-NCCIT expression profiles, several genes associated with RECK were differentially expressed (Fig. 2a). For example, induction of MMP9, a potential prostate cancer urine biomarker [28], was greater in CP-NCCIT than in NP-NCCIT (Fig. 2b). MMP9 expression was also higher in sorted CP vs. NP stromal cell transcriptomes. In contrast, RECK was more up-regulated in NP-NCCIT than CP-NCCIT, as was tissue inhibitor of metalloproteinase TIMP1, an antagonist of MMPs. With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. Figure 3 illustrates the RECK pathway network in which MMPs synthesized by CP stromal cells could lead to ECM degradation, which would in turn promote tumor cell escape. The MMP effect is amplified by the decrease in TIMP expression in cancer cells. Increase in MMPs is due to down-regulation of RECK. Fig. 1 Expression profiles of stromal and stem cell genes in treated NCCIT. a Increased expression of prostate stromal cell-specific genes relative to untreated NCCIT was detected in co-cultures of NP stromal + NCCIT cells (labeled NP-NCCIT). Expression of these stromal genes was less pronounced in co-culture of CP stromal + NCCIT cells (CP-NCCIT). For example, PENK was not induced. Note the increase in tumor-associated stromal marker CD90/THY1. b The CP-NCCIT transcriptome dataset (first column) contains minimal signal for smooth muscle differentiation genes (CALD1, CNN1) present in the NP-NCCIT transcriptome (third column) compared to untreated NCCIT transcriptome (second column) in virtual Northern blot format (darker shades of boxes indicate higher mRNA levels with background ≤50 RMA units). c Higher expression of several stem cell genes was detected in CP-NCCIT relative to NP-NCCIT as well as in cultured CP stromal cells relative to NP stromal cells. d Virtual Northern blot format shows that for stem cell genes NANOG, SOX2, CD9 and THY1, expression was increased in CP-NCCIT compared to untreated NCCIT, whereas expression was decreased in NP-NCCIT",
"NCCIT response to CD90 + CAFs tumor-associated was significantly altered from the gene expression changes induced by normal stromal cell factors. Previously, NP stromal cells induced a loss of embryonic stem cell markers and an up-regulation of genes characteristic of stromal mesenchyme, some epithelial genes and cancer stem cell genes. NCCIT response to CP stromal cells was characterized by an absence or decreased induction of genes involved in smooth muscle cell differentiation and those expressed by the prostate but not the bladder, i.e., organ restricted. At the same time, the decrease in stem cell gene expression was not as pronounced. This altered differenti-ation response could be due to differences in signaling factors secreted from tumor-associated stromal cells. These differences could be the result of either a reduction or loss of certain proteins such as the hormone PENK. Stromal cells are important in tissue repair and renewal as suggested by their demonstrated role in prostate and bladder formation. Organ specificity in this process could be due to the differentially expressed genes between the stromal cell types. Indeed, we previously showed that bladder stromal cells induced a different response from NCCIT than prostate stromal cells. Thus, if tumor-associated stromal cells were unable to provide the appropriate signaling, then normal histodevelopment would not occur, instead cancer development takes place. Although only two cases of CP stromal cells were tested, they did provide a demonstration that stem cell induction was markedly different from that by NP stromal cells. These differences, including reduced induction of smooth muscle cell genes and increased induction of genes involved in ECM remodeling, are consistent with alterations to the prostate tumor microenvironment. When we can model epithelial cytodifferentiation, i.e., PSA secretion, with cell contact and ECM, then a study using multiple samples of CP stromal cells can be carried out. For example, one could contrast the effect of CP stromal cells isolated from Gleason 3 +3 vs. Gleason 4+4 tumors.",
"The differentially induced expression pattern of genes involved in the ECM RECK pathway in CP-NCCIT vs. NP-NCCIT appears to mimic that in primary tumors as inferred from the transcriptomes of sorted stromal and epithelial cell types. NP stromal induction produced upregulation of TIMP1 and RECK whereas CP stromal induction produced up-regulation of MMP9 and comparatively less of TIMP1 and RECK. In the sorted cells, expression of MMP9 (and HRAS, which inhibits RECK) is higher in CP stromal than NP stromal, whereas that of RECK is higher in NP stromal. TIMP1 protein in cancer is absent [16]. Thus, our in vitro model of CP stromal induction of NCCIT recapitulated to some degree a major pathway important in cancer development. In response to NP stromal influence, NCCIT cells were induced to express a transcriptome with a predominant but incomplete stromal mesenchyme profile. However, the response to CP stromal cell influence with regard to induction of stromal mesenchyme genes and loss of stem cell genes was significantly less. This difference could simply reflect a reduction in secreted factors from CP stromal cells compared to NP stromal cells and therefore a lesser degree of influence on NCCIT cells, or it could represent a shift in the heterogeneity of the treated NCCIT cell population.",
"With regard to the possible genesis of CP stromal cells, we also examined the effect of NCCIT factors on NP stromal cells [15]. NCCIT extracts, when injected into differentiated cells, can activate expression of stem cell genes [29]. Instead of extract injection, NCCIT factors were examined in the co-culture format with NP stromal cells. We found that at day 3, the co-cultured NP stromal cells showed a gene expression profile for both mRNA and microRNA resembling that of CP stromal cells. Thus, CP stromal cells appear to represent a more primitive cell type in the stromal lineage. This is certainly in line with their lower expression of smooth muscle cell genes, and CP stromal cells are characterized by a loss of smooth muscle differentiation [3]. The basal epithelium also contains the progenitor cell population, which could affect stromal cell differentiation. To isolate enough CD90 + NP stromal cells for study presents a technical challenge because of their low number, which necessitates the need to obtain large tissue specimens for sorting. In summary, these experimental results showed that in induction of stem cells CP stromal cells were very different from NP stromal cells. The abnormal gene expression of CP stromal cells may well be the cause. Whether this would lead to cancer cell differentiation is still unknown since heterotypic cell contact and ECM were not provided for in the co-culture. Also unknown is whether other cell types beside stromal (e.g., epithelial) were induced in this system. For example, some ABCG2 expression was detected in NP-NCCIT, and this may suggest a small subpopulation with this marker. ABCG2 expression was identified in a putative prostate progenitor cell population localized to the basal epithelium [30]. It is therefore possible that more than one cell lineage, stromal and epithelial, could result from stromal induction of stem cells."
] | [] | [
"Introduction",
"Materials and Methods",
"Cell Lines and Tissue Specimens",
"Cell Culture",
"Transcriptome Analysis",
"Bioinformatic Analysis",
"Results",
"Discussion",
"Fig. 3",
"Introduction",
"Materials and Methods",
"Cell Lines and Tissue Specimens",
"Cell Culture",
"Transcriptome Analysis",
"Bioinformatic Analysis",
"Results",
"Discussion",
"Fig. 3"
] | [] | [
"(Table S1)",
"(Table S1)"
] | [
"Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells",
"Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells",
"Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells",
"Differential Inductive Signaling of CD90 + Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells"
] | [] |
238,415,320 | 2022-01-09T16:29:12Z | CCBY | https://www.frontiersin.org/articles/10.3389/fcell.2021.731311/pdf | GOLD | 126815364cd1e315546f0a958cc5a3ee29e9878b | null | null | null | null | 10.3389/fcell.2021.731311 | null | 34692688 | 8529014 |
Citation: Immune-Related LncRNAs Affect the Prognosis of Osteosarcoma, Which Are Related to the Tumor Immune Microenvironment
published: 07 October 2021 Published: 07 October 2021
Zong Sheng Guo
Juliano Andreoli Miyake
Wei Guo
Huang Q
Lin Y
Chen C
Lou J
Ren T Huang
Y
Zhang H
Yu Y
Guo Y
Wang W
Wang B
Niu J
Xu J
Qingshan Huang
Yilin Lin
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Laboratory of Surgical Oncology, Peking University People's Hospital
BeijingChina
Chenglong Chen
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Jingbing Lou
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Tingting Ren
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Yi Huang
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Hongliang Zhang
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Yiyang Yu
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Yu Guo
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Wei Wang
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Boyang Wang
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Jianfang Niu
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Jiuhui Xu
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Lei Guo
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Wei Guo
Musculoskeletal Tumor Center
Peking University People's Hospital
BeijingChina
Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital
BeijingChina
Roswell Park Comprehensive Cancer Center
Ferreira de Vasconcellos
Federal University of Santa Catarina
United States, Brazil Jaira
James Madison University
United States
Citation: Immune-Related LncRNAs Affect the Prognosis of Osteosarcoma, Which Are Related to the Tumor Immune Microenvironment
published: 07 October 2021 Published: 07 October 202110.3389/fcell.2021.731311Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology Received: 26 June 2021 Accepted: 20 September 2021ORIGINAL RESEARCH Edited by: Reviewed by: *Correspondence: Guo L and Guo W (2021) Immune-Related LncRNAs Affect the Prognosis of Osteosarcoma, Which Are Related to the Tumor Immune Microenvironment. Front. Cell Dev. Biol. 9:731311.osteosarcomalncRNAtumor immune microenvironmentimmune escapemetastasis
Background: Abnormal expression of lncRNA is closely related to the occurrence and metastasis of osteosarcoma. The tumor immune microenvironment (TIM) is considered to be an important factor affecting the prognosis and treatment of osteosarcoma. This study aims to explore the effect of immune-related lncRNAs (IRLs) on the prognosis of osteosarcoma and its relationship with the TIM.Methods: Ninety-five osteosarcoma samples from the TARGET database were included. Iterative LASSO regression and multivariate Cox regression analysis were used to screen the IRLs signature with the optimal AUC. The predict function was used to calculate the risk score and divide osteosarcoma into a high-risk group and low-risk group based on the optimal cut-off value of the risk score. The lncRNAs in IRLs signature that affect metastasis were screened for in vitro validation. Single sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms were used to evaluate the role of TIM in the influence of IRLs on osteosarcoma prognosis.Results: Ten IRLs constituted the IRLs signature, with an AUC of 0.96. The recurrence and metastasis rates of osteosarcoma in the high-risk group were higher than those in the low-risk group. In vitro experiments showed that knockdown of lncRNA (AC006033.2) could increase the proliferation, migration, and invasion of osteosarcoma. ssGSEA and ESTIMATE results showed that the immune cell content and immune score in the low-risk group were generally higher than those in the high-risk group. In addition, the expression levels of immune escape-related genes were higher in the high-risk group.Conclusion:The IRLs signature is a reliable biomarker for the prognosis of osteosarcoma, and they alter the prognosis of osteosarcoma. In addition, IRLs signature
and patient prognosis may be related to TIM in osteosarcoma. The higher the content of immune cells in the TIM of osteosarcoma, the lower the risk score of patients and the better the prognosis. The higher the expression of immune escape-related genes, the lower the risk score of patients and the better the prognosis.
INTRODUCTION
Osteosarcoma is the most common primary malignant bone tumor, most commonly occurring in adolescents and children (Huang et al., 2021). Its incidence is about 4.4 per million, accounting for 5% of all childhood malignancies (Han et al., 2021). Nearly 60% of osteosarcomas occur in the femur, tibia, and pelvis (Zhou and Mu, 2021). Osteosarcoma has the characteristics of high malignancy, rapid growth, and easy metastasis. Pulmonary metastasis is one of the major factors leading to the poor prognosis of osteosarcoma, and more than 20% of patients with osteosarcoma have had pulmonary metastasis at the time of diagnosis (Anderson, 2016). Therefore, it is important to clarify the causes and mechanisms affecting the prognosis of osteosarcoma to prolong the survival time of osteosarcoma.
LncRNA regulates gene transcription, translation, editing, and other biological processes (Qian et al., 2019). Its abnormal expression is closely related to the occurrence and metastasis of tumors (Kornfeld and Bruning, 2014;Bartonicek et al., 2016). It has been confirmed that multiple lncRNAs can promote the occurrence and metastasis of osteosarcoma through competitive inhibition of miRNA expression (Pan et al., 2021;Zhou and Mu, 2021). Notably, lncRNAs can regulate the development and activation of a variety of immune cells (Atianand et al., 2017). The tumor microenvironment is composed of extracellular matrix, mesenchymal cells, immune cells, and other components, and plays an important role in the occurrence of tumors (Pitt et al., 2016;De Nola et al., 2019). The immune cells in the tumor microenvironment are closely related to the treatment and prognosis of tumors. Studies have shown that lncRNA can promote tumor-associated macrophage polarization to regulate the proliferation and migration of tumor cells (Zhao et al., 2021). LncRNA NKILA can also act on T cells to promote the immune escape of tumor cells (Huang et al., 2018). LncRNA THRIL has also been shown to regulate TNF-α expression and participate in immune response in osteosarcoma (Xu et al., 2020). Therefore, these lncRNAs mediated tumor immune microenvironment (TIM) regulation may play an important role in the metastasis and prognosis of osteosarcoma.
In this study, immune-related lncRNAs (IRLs) signature was constructed from the publicly available RNA-Seq dataset to evaluate the prognosis of osteosarcoma. In particular, the role of IRLs signature in osteosarcoma metastasis was Abbreviations: EdU, 5-ethynyl-2 -deoxyuridine; FBS, fetal bovine serum; GSEA, gene set enrichment analysis; GSVA, Gene Set Variation Analysis; IRLs, immunerelated lncRNAs; PCA, principal component analysis; ssGSEA, single sample gene set enrichment analysis; TIM, tumor immune microenvironment. evaluated and validated in vitro. Because the metastasis of osteosarcoma is an important factor affecting its poor prognosis. In addition, this study further explored the role of TIM in the influence of IRLs on the progression of osteosarcoma.
MATERIALS AND METHODS
Data Collection and Processing
Osteosarcoma expression spectrum and clinical information from the TARGET database. 1 Samples were selected and data were processed by the following steps: (1) samples with both expression profiles and prognostic information were selected; (2) delete the samples with a survival time of 0 months; (3) according to the human gene annotation file (version GRCH38.p13), the ID of the gene of the osteosarcoma samples was converted into the gene symbol; and (4) there were multiple expression levels of the same gene in the expression profile, and the mean expression level was taken.
Co-expression Analyses of Immune-Related LncRNAs
A gene set named "IMMUNE RESPONSE TO TUMOR CELL" was download from Molecular Signatures Database. 2 Correlation analysis was conducted between all lncRNAs and the gene set, and IRLs with a correlation coefficient greater than 0.6 were obtained.
Construction Immune-Related LncRNAs Prognostic Signature
LncRNAs with expression variance greater than 0.2 in IRLs were screened. Univariate Cox regression analysis was performed on these lncRNAs and those with P-value less than 0.5 were screened (Sveen et al., 2012). Iterative LASSO regression was used to identify high-frequency lncRNA . Through 1000 iterations, a total of 11 lncRNAs with a frequency greater than 300 were screened out. These lncRNAs were incorporated into the Cox regression analysis one by one until the AUC value of IRLs signature reached the maximum. The risk score of each osteosarcoma sample was calculated using the predict function. The patients were divided into a high-risk group and low-risk group using the optimal risk score cut-off value and the Survminer R package was used for survival analysis.
Single Sample Gene Set Enrichment Analysis and Gene Set Enrichment Analysis
Fifty immune cell gene sets (Bindea et al., 2013;Cheng et al., 2013;Newman et al., 2015;Senbabaoglu et al., 2016), stromal cell gene set, and total immune cell gene set included in this study (Yoshihara et al., 2013) came from previous studies. Single sample gene set enrichment analysis (ssGSEA) of these gene sets were performed using Gene Set Variation Analysis (GSVA) R package (Wang et al., 2021), and the results of ssGSEA were normalized. KEGG pathway enrichment and GO function enrichment analysis were performed using GSEA software (version 4.0.1). Gene sets of KEGG and GO (C2.Cp.KEGG.v7.1 and C5.All.V7.1.Symbols) download from Molecular Signatures Database (see text footnote 2).
Construction of the Nomogram
The risk score and osteosarcoma features such as age, gender, recurrence, metastasis, and tumor site were used to construct a FIGURE 2 | The role of IRLs signature in the prognosis of osteosarcoma. (A) Osteosarcoma was divided into high-risk group and low-risk group according to the optimal cut-off value of risk score. (B) Relationship between risk score and survival states in patients with osteosarcoma. (C,D) Osteosarcomas were grouped according to the optimal cut-off value of lncRNAs expression in IRLs signature, and survival analysis was performed.
nomogram (Iasonos et al., 2008;Wang et al., 2013) to intuitively evaluate the prognosis of patients with osteosarcoma. The decision curve was used to verify the accuracy of the nomogram.
Estimation of Tumor Microenvironment Score
ESTIMATE algorithm was used to evaluate stromal score, immune score, and tumor cell purity of osteosarcoma (Hu et al., 2021). Kruskal-Wallis was used to analyze the differences in the stromal score, immune score, Estimate score, and tumor cell purity of different risk score osteosarcomas.
Cell Culture and Transfection
Human osteosarcoma cell lines KHOS and 143B were derived from American Type Culture Collection (ATCC, VA, United States). 143B cells were cultured in DMEM medium (HyClone, UT, United States) containing 10% fetal bovine serum (FBS, Gibco, NY, United States), and KHOS cells were cultured in RPMI-1640 medium (HyClone, UT, United States) containing 10% FBS. Si-AC006033.2 was obtained from Gemma Gene (Suzhou, China) (sequences: 5 -GCAGCUGCUUUGACAGUUUTT-3 ). Lipofectamine 3000 (Invitrogen, CA, United States) was used for transfection. The transfection process was carried out according to the instructions.
Reverse Transcription-Quantitative Polymerase Chain Reaction
RNA extraction from osteosarcoma cell lines was performed using TRIzol (Invitrogen, CA, United States). GAPDH was selected as an endogenous control. The primers of AC006033.2 and GAPDH are shown in Supplementary Table 1.
Cell Proliferation Assay (Cell Counting Kit-8 and 5-Ethynyl-2 -Deoxyuridine)
Osteosarcoma cells were cultured in 96-well plates with a cell density of 3000 cells per well. A total of 10 µL Cell Counting Kit-8 (CCK-8) solution (Beyotime, Shanghai, China) was added at 0, 24, 48, and 72 h, respectively. OD values were measured at 450 nm wavelength. Osteosarcoma cells were inoculated in six-well plates and cultured for 12 h, 5-ethynyl-2 -deoxyuridine (EdU, Beyotime, Shanghai, China), was added. The final concentration of EdU was 10 µM. It was incubated in an incubator at 37 • C for 2 h and fixed with 4% paraformaldehyde. A total of 1 mL of osmotic solution was added to each well and incubate at room temperature for 15 min. A total of 0.5 mL click reaction solution was added to each well and incubate at room temperature in dark for 30 min. Finally, the nuclei were stained with DAPI (Beyotime, Shanghai, China).
Wound Healing Assay and Transwell Invasion Assay
The wound-healing assay was performed in a six-well plate. Scratches were made with the tip of a sterile pipette. The changes of scratches at 24 h were observed under a microscope. Image-Pro Plus 6.0 software (Media Cybernetics, United States) was used to calculate the area change of the scratches. Invasion experiments were performed in transwell chambers (Corning, NY, United States) with an 8 µm pore diameter membrane. The upper layer of the chamber was added with matrigel (BD, NJ, United States). The number of osteosarcoma cells inoculated was 1 × 10 5 pre well. The upper chamber was cultured with serum-free medium, and the lower chamber was cultured with a 700 'L complete medium. After 48 h, the cells in the upper part of the basement membrane were erased, and the cells in the lower part of the basement membrane were fixed and stained.
Statistical Analysis
The statistical software R (version 3.6.1) was used for data analysis and image production. The Chi-square test was used to compare differences in recurrence rates or metastasis rates of osteosarcoma. Spearman correlation analysis was used to evaluate the correlation between the expression level of lncRNAs, the correlation between risk score and lncRNA expression level, or the correlation between lncRNA expression level and immune cell content. The Kruskal-Wallis test was used to analyze the differences in immune cell content, tumor microenvironment score, or gene expression level. Kaplan-Meier survival analysis was used to evaluate prognostic differences between different risk scores or between different lncRNAs expression levels in osteosarcoma. Univariate Cox regression analysis and multivariate Cox regression analysis were used to evaluating the effects of lncRNA, clinical characteristics, or risk score on the prognosis of osteosarcoma. Two-tailed P-values were used, and the statistical significance was set at P < 0.05.
RESULTS
Data Processing and Co-expression Analyses of Immune-Related LncRNAs
Sequencing data and clinical data were downloaded from the TARGET database and expression levels of all samples were combined into an expression profile. The names of samples with both clinical and sequencing data are shown in Supplementary
Construction of Immune-Related LncRNAs Prognostic Signature
There were 4986 lncRNAs with variances greater than 0.2 expressed in IRLs. Univariate Cox regression analysis was performed on these IRLs, among which 1743 IRLs with P-value less than 0.5 were identified. Iterative LASSO regression was used to identify high-frequency lncRNAs. One thousand iterations were executed, and 11 lncRNAs with a frequency greater than 300 were screened out. These lncRNAs were incorporated into the Cox regression analysis one by one until the AUC of ROC reached the maximum. At this point, the number of lncRNAs was 10 and the AUC was 0.96 (Figures 1A,B). The predict function was used to calculate the risk score of each osteosarcoma sample, and the osteosarcoma patients are divided into the high-risk group (n = 48) and low-risk group (n = 47) according to the optimal cut-off value (0.92) of risk score. Kaplan-Meier survival analysis showed that the lower risk score was associated with a better prognosis for osteosarcoma ( Figure 1C). The distribution of 10 IRLs expression levels with the change of risk score was shown in Figure 1D. The results of principal component analysis (PCA) showed that IRLs signature can achieve better dimension reduction (Figure 1E). Correlation analysis results showed that the expression levels of these lncRNAs were not highly correlated (Figure 1F), which further demonstrated the rationality of IRLs signature.
The Role of Immune-Related LncRNAs Signature in the Prognosis of Osteosarcoma
Osteosarcoma patients were divided into high-risk group and low-risk group according to the optimal cut-off value of risk score (Figure 2A). With the increase of
The Role of Clinical Features in the Prognosis of Osteosarcoma
Univariate and multivariate Cox regression analysis results showed that IRLs signature and the clinical features of osteosarcoma including recurrence, metastasis, and tumor location could all be independent prognostic factors for osteosarcoma ( Figure 3A and Supplementary Tables 7, 8).
In the prognostic evaluation of osteosarcoma, the predictive performance of IRLs signature was the highest among these features. In the ROC curve, the 1-, 3-, and 5-year AUC values were 0.802, 0.925, and 0.96, respectively ( Figure 3B). In addition, the Chi-square test confirmed that the recurrence rates and metastasis rates were higher in the high-risk group than in the low-risk group (Figure 3C). Survival analysis showed a poor prognosis for recurrent, metastatic, and non-limb osteosarcomas (Supplementary Figure 3A), and no significant difference in prognosis between sex and age (Supplementary Figure 3B).
The Construction and Verification of the Nomogram
The nomogram is widely used to evaluate the prognosis of tumors. It can reduce the statistical prediction model to a probability value. In this study, risk score, gender, age, recurrence, metastasis, and tumor location were integrated to FIGURE 9 | Relationship between IRLs signature and tumor microenvironment score. (A-C) Analysis of differences in the immune score, stromal score, and Estimate score between high-risk and low-risk osteosarcoma. (D-F) Correlation analysis between risk score and osteosarcoma immune score, matrix score, and Estimate score. (G) Analysis of difference in tumor cell purity between high-risk and low-risk osteosarcoma. (H) Correlation analysis between risk score and tumor cell purity of osteosarcoma.
construct a nomogram to evaluate the prognosis of osteosarcoma ( Figure 4A). The nomogram showed the predicted survival rates for 3 and 5 years, respectively. Calibration curves showed that the nomogram was able to accurately evaluate the prognosis of osteosarcoma ( Figure 4B).
The Role of Immune-Related LncRNAs Signature in Osteosarcoma Metastasis
The metastasis of osteosarcoma is one of the most important factors affecting its prognosis. Therefore, this study focused on the role of the IRLs signature in the metastasis of osteosarcoma. The Chi-square test confirmed that the metastatic rate of osteosarcoma was higher in the high-risk group than in the lowrisk group (Figure 3C). The waterfall chart also shows that the probability of metastasis gradually decreases with the decrease of risk score ( Figure 5A). Therefore, IRLs signature may play an important role in the metastasis of osteosarcoma. The results of the differential analysis showed that only AC006033.2 of the 10 lncRNAs of IRLs signature was different between the metastatic and non-metastatic osteosarcomas, and the expression level of AC006033.2 was low in the metastatic osteosarcomas ( Figure 5B). For this reason, AC006033.2 was knocked down and validated in osteosarcoma cell lines. Knockdown efficacy was confirmed by PCR in osteosarcoma cell line 143B and KHOS (Figure 5C). We first conducted a cell proliferation experiment, and the results showed that knocking down AC006033.2 could increase the proliferation ability of osteosarcoma cells (Figures 5D,E). The wound-healing assay results further showed that knocking down AC006033.2 increased the migration ability of osteosarcoma cells (Figures 6A,B). Transwell invasion assay showed increased invasiveness of osteosarcoma cells after AC006033.2 knockdown (Figures 6C,D).
KEGG Pathway Enrichment and GO Function Enrichment
Gene set enrichment analysis (GSEA) software was used to assess KEGG pathway enrichment in high-risk and lowrisk osteosarcomas. The pathways that enriched in low-risk osteosarcomas were mainly immune-related (Figure 7A), while no associated pathways enriched in high-risk osteosarcomas. The better prognosis of osteosarcoma in the low-risk group may be related to the local immune microenvironment.
To further explore the possible role of IRLs signature in osteosarcoma, GO enrichment analysis was performed using GSEA software. The results showed that osteosarcomas in the low-risk group were mainly enriched in immune-related functions (Figure 7B), while the high-risk group osteosarcomas were also not enriched in any meaningfully related functions.
The Role of Tumor Immune Microenvironment in the Influence of Immune-Related LncRNAs on Osteosarcoma Prognosis
Relationship Between Immune-Related LncRNAs Signature and Immune Cell Infiltration LncRNA can regulate the development and activation of various immune cells (Atianand et al., 2017). Therefore, our study investigated the relationship between the degree of immune cell infiltration and IRLs signature in the immune microenvironment of osteosarcoma. The results of ssGSEA are shown in Supplementary Table 9, and the content of immune cells in osteosarcoma decreased with the increase of risk score (Figure 8A), and the correlation analysis results showed that risk score was negatively correlated with the content of most immune cells (Supplementary Figure 4), which suggested that the low degree of immune cell infiltration might be an important reason for recurrence and metastasis in the high-risk osteosarcoma. In addition, AC006033.2 was also positively correlated with the content of most immune cells (Figure 8B). Osteosarcoma with low AC006033.2 expression had a low level of immune cell infiltration, which was not conducive to the prognosis of osteosarcoma. This was consistent with the conclusion that AC006033.2 knockdown in vitro enhanced the invasiveness of osteosarcoma cells (Figures 6C,D).
Relationship Between Immune-Related LncRNAs
Signature and Tumor Microenvironment Score ESTIMATE algorithm can be used to evaluate the tumor microenvironment score of osteosarcoma to evaluate tumor stroma content, immune cell content, and tumor cell purity (Supplementary Table 10; Hu et al., 2021). Our study calculated the tumor microenvironment score of osteosarcoma by the ESTIMATE algorithm, and analyzed the relationship between them and risk score or the degree of immune cell infiltration (Supplementary Figure 5). Differential analysis results showed that the immunoscore, stromal score, and Estimate score of the low-risk group were higher than those of the high-risk group (Figures 9A-C). Correlation analysis results showed that risk score was negatively correlated with immune score, stromal score, and Estimate score. With the increase of risk score, the immune score, stromal score, and Estimate score of osteosarcoma gradually decreased (Figures 9D-F). Conversely, the purity of tumor cells in the low-risk osteosarcoma group was lower than that in the high-risk group, and the purity of tumor cells was negatively correlated with the risk score (Figures 9G,H).
Relationship Between Immune-Related LncRNAs Signature and Immune Escape in Osteosarcoma
Immune escape is an important reason for the rapid growth of tumor cells in the human body. In this study, we found that the prognosis of osteosarcoma may be related to the mechanism of endogenous immune escape. The molecules involved in endogenous immune escape include MHC-I molecules, MHC-II molecules, costimulatory molecules. Our results showed that the expression levels of MHC-I molecules, MHC-II molecules, and costimulatory molecules decreased with the increase of risk score (Figures 10A-C), and the different analysis results showed that the expression levels of these molecules in the high-risk group were lower than those in the low-risk group (Figures 10D-F). This suggests that immune escape is more likely to occur in the high-risk group osteosarcoma.
DISCUSSION
Compared with previous amputations alone, the survival rate for osteosarcoma has improved from 20% to more than 60% (Koster et al., 2018;Wang et al., 2018) but has not improved further in recent years. The high incidence of pulmonary metastasis is one of the important reasons. Therefore, searching for effective prognostic markers is an important method for early diagnosis of the prognosis of osteosarcoma and early intervention. In recent years, the role of TIM in the treatment of osteosarcoma has become increasingly evident. A growing number of immunotherapy agents are entering clinical trials (NCT04668300, NCT04544995, and NCT02500797). Therefore, we hope to find immune-related biomarkers that can not only better predict patient survival, but also provide potential therapeutic targets for future immunotherapy. LncRNA has been proved to be involved in the immune response of osteosarcoma by regulating the expression of TNF-α (Xu et al., 2020). To this end, we constructed a prognostic signature using IRLs to explore its role in the prognosis of osteosarcoma. Further, we explored the relationship between the IRLs signature and the TIM of osteosarcoma.
In this study, 10 IRLs with good predictive ability were selected as a prognostic marker for osteosarcoma by iterative LSSSO regression and multivariate Cox regression analysis. Compared with the simple univariate and multivariate Cox regression analysis, the IRLs signature has better predictive performance, with an AUC of 0.96. The results of PCA showed that IRLs signature could classify osteosarcoma into two groups. With the increase of risk score, the mortality of patients with osteosarcoma showed a significant upward trend. Recurrence and metastasis are important clinical features for the prognosis of osteosarcoma. Compared with the common clinical features of osteosarcoma, IRLs signature has higher predictive power for the prognosis of osteosarcoma, even higher than the two clinical features of osteosarcoma recurrence and metastasis.
Our study showed that the metastatic rate of osteosarcoma in the high-risk group was significantly higher than that in the low-risk group, and the probability of metastasis tended to increase with the increase of risk score. It is worth noting that the differential expression analysis of lncRNA in IRLs signature between the metastatic group and the non-metastatic group showed that only the expression level of AC006033.2 was different. Therefore, our study further verified the role of AC006033.2 in the biological behavior of osteosarcoma through an in vitro experiment. By interfering with AC006033.2, we found that the proliferation, migration, and invasion of osteosarcoma cell lines were enhanced. Studies have shown that AC006033.2 is also an effective biomarker for anti-tumor immunity in gastric cancer (He and Wang, 2020), which is similar to the results of our study. We found that IRLs signature is closely related to the TIM of osteosarcoma. In addition, the expression level of AC006033.2 was significantly positively correlated with the content of various immune cells. Therefore, AC006033.2 may be an important molecule affecting the prognosis of osteosarcoma.
LncRNA THRIL has been shown to regulate TNF-α expression and participate in immune response in osteosarcoma (Xu et al., 2020). KEGG and GO enrichment analysis results also showed that osteosarcoma in the low-risk group was highly correlated with immune pathways and functions. Therefore, we further explored the relationship between IRLs signature and TIM of osteosarcoma. Firstly, the ssGSEA was used in the study to evaluate the level of immune cell infiltration in the microenvironment of osteosarcoma, and the level of immune cell infiltration was higher in the low-risk group. Results of the ESTIMATE algorithm also indicated a higher immune score in osteosarcoma in the low-risk group. These suggest that IRLs may be associated with the immune cell infiltration in the microenvironment of osteosarcoma.
Studies have shown that lncRNA can also act on T cells to promote the immune escape of tumor cells (Huang et al., 2018). Therefore, we further explored the relationship between the IRLs signature and the TIM of osteosarcoma. The decrease of MHC-I molecular presentation function is considered to be an important reason for the immune escape of tumor cells (Lee et al., 2005). Low expression of MHC-II molecules can also lead to the occurrence of immune escape due to the inability to effectively activate T cells (Zhi et al., 2021). In addition, the activation of T cells also requires the participation of costimulatory molecules (Angulo et al., 2021). We found that MHC-I molecules, MHC-II molecules, and costimulatory molecules, which are associated with endogenous immune escape, were all low expressed in the high-risk group. These results suggest that immune escape may be prevalent in high-risk group osteosarcoma, and IRLs may be associated with the immune escape of osteosarcoma cells.
CONCLUSION
The IRLs signature is a reliable biomarker for the prognosis of osteosarcoma, and they alter the prognosis of osteosarcoma. In addition, IRLs signature and patient prognosis may be related to TIM in osteosarcoma. The higher the content of immune cells in the TIM of osteosarcoma, the lower the risk score of patients and the better the prognosis. The higher the expression of immune escape-related genes, the lower the risk score of patients and the better the prognosis.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repositories and accession numbers can be found in the article material.
AUTHOR CONTRIBUTIONS
WG conceived the project. QH, YL, CC, JL, TR, YH, HZ, YY, YG, WW, BW, JN, JX, and LG performed the literature search and data analysis. QH and WG drafted and critically revised the work. All authors contributed to manuscript revision, read, and approved the submitted version.
FUNDING
This work was supported by the Beijing Science and Technology Planning Project (No. Z161100000116100) and the National Natural Science Foundation of China (Nos. 81572633 and 82072970).
FIGURE 1 |
1Construction of IRLs prognostic signature. (A) Iterative LASSO and Cox regression analysis are used to screen IRLs signature with the best AUC. (B) The ROC curve with the best AUC. (C) Osteosarcomas were grouped according to the optimal cut-off value of risk score, and survival analysis was performed. (D) Expression levels of 10 lncRNA in IRLs signature. (E) PCA analysis was performed with IRLs signature. (F) Correlation analysis among 10 IRLs in IRLs signature.
FIGURE 3 |
3Role of clinical features in the prognosis of osteosarcoma. (A) Univariate and multivariate Cox regression analysis of clinical features of osteosarcoma. (B) ROC curve for predicting the prognosis of osteosarcoma based on risk score and clinical features of osteosarcoma. (C) Differences in metastasis and recurrence rates between the high-risk group and low-risk group.
FIGURE 4 |
4Nomogram for evaluating the prognosis of osteosarcoma at 3 and 5 years. (A) The risk score and clinical characteristics of osteosarcoma were used to construct the nomogram. (B) Calibration curves were used to verify the accuracy of the nomogram.
FIGURE 5 |
5Role of IRLs signature in metastasis and proliferation of osteosarcoma. (A) The probability of metastasis of osteosarcoma gradually decreases with the decrease of risk score. (B) Differential expression of 10 IRLs in metastatic and non-metastatic osteosarcomas. Only the expression level of AC006033.2 was different. (C) Knockdown results of AC006033.2 in osteosarcoma cell lines. (D,E) Effects of AC006033.2 knockdown on proliferation of osteosarcoma cells. *P < 0.05 and ***P < 0.001. EdU, 5-ethynyl-2 -deoxyuridine.
FIGURE 6 |
6Influence of AC006033.2 on migration and invasion ability of osteosarcoma. (A,B) Effects of AC006033.2 knockdown on the migration ability of osteosarcoma cells. (C,D) Effects of AC006033.2 knockdown on the invasion ability of osteosarcoma cells. *P < 0.05 and **P < 0.01.
FIGURE 7 |
7Pathway and functional enrichment analysis of osteosarcoma in the high-risk and low-risk group osteosarcomas. (A) KEGG enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related pathways. (B) GO enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related functions.risk score, the mortality rate of osteosarcoma increased significantly (Figure 2B). Results of survival analysis showed that osteosarcoma with high expression of AC006033.2, LINC02315, AL133523.1, USP30-AS1, or AC079760.2 had a better prognosis(Figure 2Cand SupplementaryFigure 1A). High expression of AP000943.1, AC015795.1, AC016746.1, LINC01976, and SNHG6 was not conducive to the prognosis of osteosarcoma(Figure 2Dand SupplementaryFigure 1B). Cox regression analysis showed that AL133523.1, AC079760.2, and LINC01976 were independent prognostic factors for osteosarcoma(Supplementary Tables 5, 6and SupplementaryFigure 2).
FIGURE 8 |
8Relationship between IRLs signature and the degree of immune cell infiltration. (A) Changes of the degree of immune cell infiltration in osteosarcoma with the change of risk score. (B) Correlation analysis between 10 IRLs and the degree of immune cell infiltration.
FIGURE 10 |
10Relationship between IRLs signature and endogenous immune escape in osteosarcoma. (A-C) The expression of MHC-I molecule, MHC-II molecule, and costimulatory molecule changed with the increase of risk score. (D-F) Differential analysis of the expression of MHC-I, MHC-II, and costimulatory molecules in high-risk and low-risk osteosarcoma. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Table 2 .
2Follow the above method, 95 osteosarcoma samples were included in the study. The expression levels of all lncRNA were shown in Supplementary Table 3. Correlation analysis was conducted between all lncRNAs and the gene set (IMMUNE RESPONSE TO TUMOR CELL), and 4986 IRLs were obtained (Supplementary Table 4).
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October 2021 | Volume 9 | Article 731311
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SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. 731311/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Copyright © 2021Huang, Lin, Chen, Lou, Ren, Huang, Zhang, Yu, Guo, Wang, Wang, Niu, Xu, Guo and Guo.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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| [
"Background: Abnormal expression of lncRNA is closely related to the occurrence and metastasis of osteosarcoma. The tumor immune microenvironment (TIM) is considered to be an important factor affecting the prognosis and treatment of osteosarcoma. This study aims to explore the effect of immune-related lncRNAs (IRLs) on the prognosis of osteosarcoma and its relationship with the TIM.Methods: Ninety-five osteosarcoma samples from the TARGET database were included. Iterative LASSO regression and multivariate Cox regression analysis were used to screen the IRLs signature with the optimal AUC. The predict function was used to calculate the risk score and divide osteosarcoma into a high-risk group and low-risk group based on the optimal cut-off value of the risk score. The lncRNAs in IRLs signature that affect metastasis were screened for in vitro validation. Single sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms were used to evaluate the role of TIM in the influence of IRLs on osteosarcoma prognosis.Results: Ten IRLs constituted the IRLs signature, with an AUC of 0.96. The recurrence and metastasis rates of osteosarcoma in the high-risk group were higher than those in the low-risk group. In vitro experiments showed that knockdown of lncRNA (AC006033.2) could increase the proliferation, migration, and invasion of osteosarcoma. ssGSEA and ESTIMATE results showed that the immune cell content and immune score in the low-risk group were generally higher than those in the high-risk group. In addition, the expression levels of immune escape-related genes were higher in the high-risk group.Conclusion:The IRLs signature is a reliable biomarker for the prognosis of osteosarcoma, and they alter the prognosis of osteosarcoma. In addition, IRLs signature"
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"Chenglong Chen \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Jingbing Lou \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Tingting Ren \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
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"Hongliang Zhang \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Yiyang Yu \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Yu Guo \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Wei Wang \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Boyang Wang \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Jianfang Niu \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Jiuhui Xu \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Lei Guo \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"Wei Guo \nMusculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina\n\nBeijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina\n",
"\nRoswell Park Comprehensive Cancer Center\nFerreira de Vasconcellos\nFederal University of Santa Catarina\nUnited States, Brazil Jaira\n",
"\nJames Madison University\nUnited States\n"
] | [
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Laboratory of Surgical Oncology, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Musculoskeletal Tumor Center\nPeking University People's Hospital\nBeijingChina",
"Beijing Key Laboratory of Musculoskeletal Tumor, Peking University People's Hospital\nBeijingChina",
"Roswell Park Comprehensive Cancer Center\nFerreira de Vasconcellos\nFederal University of Santa Catarina\nUnited States, Brazil Jaira",
"James Madison University\nUnited States"
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"G Angulo, ",
"J Zeleznjak, ",
"P Martinez-Vicente, ",
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"H Hengel, ",
"M Messerle, ",
"M K Atianand, ",
"D R Caffrey, ",
"K A Fitzgerald, ",
"N Bartonicek, ",
"J L Maag, ",
"M E Dinger, ",
"G Bindea, ",
"B Mlecnik, ",
"M Tosolini, ",
"A Kirilovsky, ",
"M Waldner, ",
"A C Obenauf, ",
"W Y Cheng, ",
"T H Yang, ",
"D Anastassiou, ",
"R De Nola, ",
"A Menga, ",
"A Castegna, ",
"V Loizzi, ",
"G Ranieri, ",
"E Cicinelli, ",
"G Han, ",
"Q Guo, ",
"N Ma, ",
"W Bi, ",
"M Xu, ",
"J Jia, ",
"Y He, ",
"Wang , ",
"X , ",
"X Hu, ",
"L Wu, ",
"B Liu, ",
"Chen , ",
"K , ",
"D Huang, ",
"J Chen, ",
"L Yang, ",
"Q Ouyang, ",
"J Li, ",
"L Lao, ",
"Q Huang, ",
"X Liang, ",
"T Ren, ",
"Y Huang, ",
"H Zhang, ",
"Y Yu, ",
"A Iasonos, ",
"D Schrag, ",
"G V Raj, ",
"K S Panageas, ",
"J W Kornfeld, ",
"J C Bruning, ",
"R Koster, ",
"O A Panagiotou, ",
"W A Wheeler, ",
"E Karlins, ",
"J M Gastier-Foster, ",
"F T Lee, ",
"A J Mountain, ",
"M P Kelly, ",
"C Hall, ",
"A Rigopoulos, ",
"T G Johns, ",
"A M Newman, ",
"C L Liu, ",
"M R Green, ",
"A J Gentles, ",
"W Feng, ",
"Y Xu, ",
"X Pan, ",
"J Tan, ",
"T Tao, ",
"X Zhang, ",
"Y Weng, ",
"X Weng, ",
"J M Pitt, ",
"A Marabelle, ",
"A Eggermont, ",
"J C Soria, ",
"G Kroemer, ",
"L Zitvogel, ",
"X Qian, ",
"J Zhao, ",
"P Y Yeung, ",
"Q C Zhang, ",
"C K Kwok, ",
"Y Senbabaoglu, ",
"R S Gejman, ",
"A G Winer, ",
"M Liu, ",
"E M Van Allen, ",
"G De Velasco, ",
"A Sveen, ",
"T H Agesen, ",
"A Nesbakken, ",
"G I Meling, ",
"T O Rognum, ",
"K Liestol, ",
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"C Wang, ",
"S Wang, ",
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"LncRNA BCRT1 facilitates osteosarcoma progression via regulating miR-1303/FGF7 axis. G Han, Q Guo, N Ma, W Bi, M Xu, J Jia, 10.18632/aging.203106Aging. 13Han, G., Guo, Q., Ma, N., Bi, W., Xu, M., Jia, J., et al. (2021). LncRNA BCRT1 facilitates osteosarcoma progression via regulating miR-1303/FGF7 axis. Aging (Albany N. Y.) 13, 15501-15510. doi: 10.18632/aging.203106",
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"Immune infiltration subtypes characterization and identification of prognosis-related lncRNAs in adenocarcinoma of the esophagogastric junction. X Hu, L Wu, B Liu, Chen , K , 10.3389/fimmu.2021.651056Front. Immunol. 12651056Hu, X., Wu, L., Liu, B., and Chen, K. (2021). Immune infiltration subtypes characterization and identification of prognosis-related lncRNAs in adenocarcinoma of the esophagogastric junction. Front. Immunol. 12:651056. doi: 10.3389/fimmu.2021.651056",
"NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activationinduced cell death. D Huang, J Chen, L Yang, Q Ouyang, J Li, L Lao, 10.1038/s41590-018-0207-yNat. Immunol. 19Huang, D., Chen, J., Yang, L., Ouyang, Q., Li, J., Lao, L., et al. (2018). NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activation- induced cell death. Nat. Immunol. 19, 1112-1125. doi: 10.1038/s41590-018- 0207-y",
"The role of tumor-associated macrophages in osteosarcoma progression-therapeutic implications. Q Huang, X Liang, T Ren, Y Huang, H Zhang, Y Yu, 10.1007/s13402-021-00598-wCell. Oncol. (Dordr.). 44Huang, Q., Liang, X., Ren, T., Huang, Y., Zhang, H., Yu, Y., et al. (2021). The role of tumor-associated macrophages in osteosarcoma progression-therapeutic implications. Cell. Oncol. (Dordr.) 44, 525-539. doi: 10.1007/s13402-021- 00598-w",
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"LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma. Y Zhou, T Mu, 10.26355/eurrev_202104_25727Eur. Rev. Med. Pharmacol. Sci. 25Zhou, Y., and Mu, T. (2021). LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma. Eur. Rev. Med. Pharmacol. Sci. 25, 3182-3199. doi: 10.26355/eurrev_202104_25727"
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"Update on Survival in Osteosarcoma",
"Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion",
"Immunobiology of long noncoding RNAs",
"Long noncoding RNAs in cancer: mechanisms of action and technological advancements",
"Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer",
"Biomolecular events in cancer revealed by attractor metagenes",
"The crowded crosstalk between cancer cells and stromal microenvironment in gynecological malignancies: biological pathways and therapeutic implication",
"LncRNA BCRT1 facilitates osteosarcoma progression via regulating miR-1303/FGF7 axis",
"Identification of molecular features correlating with tumor immunity in gastric cancer by multi-omics data analysis",
"Immune infiltration subtypes characterization and identification of prognosis-related lncRNAs in adenocarcinoma of the esophagogastric junction",
"NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activationinduced cell death",
"The role of tumor-associated macrophages in osteosarcoma progression-therapeutic implications",
"How to build and interpret a nomogram for cancer prognosis",
"Regulation of metabolism by long, non-coding RNAs",
"Caminada de Toledo SR, et al. Genome-wide association study identifies the GLDC/IL33 locus associated with survival of osteosarcoma patients",
"Enhanced efficacy of radioimmunotherapy with 90Y-CHX-A\"-DTPA-hu3S193 by inhibition of epidermal growth factor receptor (EGFR) signaling with EGFR tyrosine kinase inhibitor AG1478",
"Robust enumeration of cell subsets from tissue expression profiles",
"LINC01123 enhances osteosarcoma cell growth by activating the Hedgehog pathway via the miR-516b-5p/Gli1 axis",
"Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy",
"Revealing lncRNA structures and interactions by sequencing-based approaches",
"Tumor immune microenvironment characterization in clear cell renal cell carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures",
"ColoGuidePro: a prognostic 7-gene expression signature for stage III colorectal cancer patients",
"HER4 promotes cell survival and chemoresistance in osteosarcoma via interaction with NDRG1",
"Immune cell infiltration-based signature for prognosis and immunogenomic analysis in breast cancer",
"Prognostic nomogram for intrahepatic cholangiocarcinoma after partial hepatectomy",
"Upregulated lncRNA THRIL/TNF-alpha signals promote cell growth and predict poor clinical outcomes of osteosarcoma",
"Inferring tumour purity and stromal and immune cell admixture from expression data",
"Development of a machine learning-based autophagy-related lncRNA signature to improve prognosis prediction in osteosarcoma patients",
"lncRNA-Xist/miR-101-3p/KLF6/C/EBPalpha axis promotes TAM polarization to regulate cancer cell proliferation and migration",
"Inhibition of BRAF sensitizes thyroid carcinoma to immunotherapy by enhancing tsMHCIImediated immune recognition",
"LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma"
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"Nat. Methods",
"Cancer Sci",
"Ann. Oncol",
"Trends Biochem. Sci",
"Genome Biol",
"Clin. Cancer Res",
"Biochim. Biophys. Acta Mol. Basis Dis",
"Brief. Bioinform",
"J. Clin. Oncol",
"Onco Targets Ther",
"Nat. Commun",
"Front. Mol. Biosci",
"Mol. Ther. Nucleic Acids",
"J. Clin. Endocrinol. Metab",
"Eur. Rev. Med. Pharmacol. Sci"
] | [
"\nFIGURE 1 |\n1Construction of IRLs prognostic signature. (A) Iterative LASSO and Cox regression analysis are used to screen IRLs signature with the best AUC. (B) The ROC curve with the best AUC. (C) Osteosarcomas were grouped according to the optimal cut-off value of risk score, and survival analysis was performed. (D) Expression levels of 10 lncRNA in IRLs signature. (E) PCA analysis was performed with IRLs signature. (F) Correlation analysis among 10 IRLs in IRLs signature.",
"\nFIGURE 3 |\n3Role of clinical features in the prognosis of osteosarcoma. (A) Univariate and multivariate Cox regression analysis of clinical features of osteosarcoma. (B) ROC curve for predicting the prognosis of osteosarcoma based on risk score and clinical features of osteosarcoma. (C) Differences in metastasis and recurrence rates between the high-risk group and low-risk group.",
"\nFIGURE 4 |\n4Nomogram for evaluating the prognosis of osteosarcoma at 3 and 5 years. (A) The risk score and clinical characteristics of osteosarcoma were used to construct the nomogram. (B) Calibration curves were used to verify the accuracy of the nomogram.",
"\nFIGURE 5 |\n5Role of IRLs signature in metastasis and proliferation of osteosarcoma. (A) The probability of metastasis of osteosarcoma gradually decreases with the decrease of risk score. (B) Differential expression of 10 IRLs in metastatic and non-metastatic osteosarcomas. Only the expression level of AC006033.2 was different. (C) Knockdown results of AC006033.2 in osteosarcoma cell lines. (D,E) Effects of AC006033.2 knockdown on proliferation of osteosarcoma cells. *P < 0.05 and ***P < 0.001. EdU, 5-ethynyl-2 -deoxyuridine.",
"\nFIGURE 6 |\n6Influence of AC006033.2 on migration and invasion ability of osteosarcoma. (A,B) Effects of AC006033.2 knockdown on the migration ability of osteosarcoma cells. (C,D) Effects of AC006033.2 knockdown on the invasion ability of osteosarcoma cells. *P < 0.05 and **P < 0.01.",
"\nFIGURE 7 |\n7Pathway and functional enrichment analysis of osteosarcoma in the high-risk and low-risk group osteosarcomas. (A) KEGG enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related pathways. (B) GO enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related functions.risk score, the mortality rate of osteosarcoma increased significantly (Figure 2B). Results of survival analysis showed that osteosarcoma with high expression of AC006033.2, LINC02315, AL133523.1, USP30-AS1, or AC079760.2 had a better prognosis(Figure 2Cand SupplementaryFigure 1A). High expression of AP000943.1, AC015795.1, AC016746.1, LINC01976, and SNHG6 was not conducive to the prognosis of osteosarcoma(Figure 2Dand SupplementaryFigure 1B). Cox regression analysis showed that AL133523.1, AC079760.2, and LINC01976 were independent prognostic factors for osteosarcoma(Supplementary Tables 5, 6and SupplementaryFigure 2).",
"\nFIGURE 8 |\n8Relationship between IRLs signature and the degree of immune cell infiltration. (A) Changes of the degree of immune cell infiltration in osteosarcoma with the change of risk score. (B) Correlation analysis between 10 IRLs and the degree of immune cell infiltration.",
"\nFIGURE 10 |\n10Relationship between IRLs signature and endogenous immune escape in osteosarcoma. (A-C) The expression of MHC-I molecule, MHC-II molecule, and costimulatory molecule changed with the increase of risk score. (D-F) Differential analysis of the expression of MHC-I, MHC-II, and costimulatory molecules in high-risk and low-risk osteosarcoma. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.",
"\nTable 2 .\n2Follow the above method, 95 osteosarcoma samples were included in the study. The expression levels of all lncRNA were shown in Supplementary Table 3. Correlation analysis was conducted between all lncRNAs and the gene set (IMMUNE RESPONSE TO TUMOR CELL), and 4986 IRLs were obtained (Supplementary Table 4)."
] | [
"Construction of IRLs prognostic signature. (A) Iterative LASSO and Cox regression analysis are used to screen IRLs signature with the best AUC. (B) The ROC curve with the best AUC. (C) Osteosarcomas were grouped according to the optimal cut-off value of risk score, and survival analysis was performed. (D) Expression levels of 10 lncRNA in IRLs signature. (E) PCA analysis was performed with IRLs signature. (F) Correlation analysis among 10 IRLs in IRLs signature.",
"Role of clinical features in the prognosis of osteosarcoma. (A) Univariate and multivariate Cox regression analysis of clinical features of osteosarcoma. (B) ROC curve for predicting the prognosis of osteosarcoma based on risk score and clinical features of osteosarcoma. (C) Differences in metastasis and recurrence rates between the high-risk group and low-risk group.",
"Nomogram for evaluating the prognosis of osteosarcoma at 3 and 5 years. (A) The risk score and clinical characteristics of osteosarcoma were used to construct the nomogram. (B) Calibration curves were used to verify the accuracy of the nomogram.",
"Role of IRLs signature in metastasis and proliferation of osteosarcoma. (A) The probability of metastasis of osteosarcoma gradually decreases with the decrease of risk score. (B) Differential expression of 10 IRLs in metastatic and non-metastatic osteosarcomas. Only the expression level of AC006033.2 was different. (C) Knockdown results of AC006033.2 in osteosarcoma cell lines. (D,E) Effects of AC006033.2 knockdown on proliferation of osteosarcoma cells. *P < 0.05 and ***P < 0.001. EdU, 5-ethynyl-2 -deoxyuridine.",
"Influence of AC006033.2 on migration and invasion ability of osteosarcoma. (A,B) Effects of AC006033.2 knockdown on the migration ability of osteosarcoma cells. (C,D) Effects of AC006033.2 knockdown on the invasion ability of osteosarcoma cells. *P < 0.05 and **P < 0.01.",
"Pathway and functional enrichment analysis of osteosarcoma in the high-risk and low-risk group osteosarcomas. (A) KEGG enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related pathways. (B) GO enrichment analysis of osteosarcomas showed that osteosarcomas in the low-risk group were mostly enriched in immune-related functions.risk score, the mortality rate of osteosarcoma increased significantly (Figure 2B). Results of survival analysis showed that osteosarcoma with high expression of AC006033.2, LINC02315, AL133523.1, USP30-AS1, or AC079760.2 had a better prognosis(Figure 2Cand SupplementaryFigure 1A). High expression of AP000943.1, AC015795.1, AC016746.1, LINC01976, and SNHG6 was not conducive to the prognosis of osteosarcoma(Figure 2Dand SupplementaryFigure 1B). Cox regression analysis showed that AL133523.1, AC079760.2, and LINC01976 were independent prognostic factors for osteosarcoma(Supplementary Tables 5, 6and SupplementaryFigure 2).",
"Relationship between IRLs signature and the degree of immune cell infiltration. (A) Changes of the degree of immune cell infiltration in osteosarcoma with the change of risk score. (B) Correlation analysis between 10 IRLs and the degree of immune cell infiltration.",
"Relationship between IRLs signature and endogenous immune escape in osteosarcoma. (A-C) The expression of MHC-I molecule, MHC-II molecule, and costimulatory molecule changed with the increase of risk score. (D-F) Differential analysis of the expression of MHC-I, MHC-II, and costimulatory molecules in high-risk and low-risk osteosarcoma. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.",
"Follow the above method, 95 osteosarcoma samples were included in the study. The expression levels of all lncRNA were shown in Supplementary Table 3. Correlation analysis was conducted between all lncRNAs and the gene set (IMMUNE RESPONSE TO TUMOR CELL), and 4986 IRLs were obtained (Supplementary Table 4)."
] | [
"(Figures 1A,B)",
"Figure 1C)",
"Figure 1D",
"(Figure 1E)",
"(Figure 1F)",
"(Figure 2A)",
"Figure 3A and Supplementary Tables 7, 8)",
"Figure 3B",
"(Figure 3C)",
"(Supplementary Figure 3A)",
"Figure 3B)",
"Figure 4A)",
"Figure 4B",
"(Figure 3C)",
"Figure 5A)",
"Figure 5B)",
"(Figure 5C)",
"(Figures 5D,E)",
"(Figures 6A,B)",
"(Figures 6C,D)",
"(Figure 7A)",
"(Figure 7B)",
"(Figure 8A)",
"(Supplementary Figure 4)",
"(Figure 8B",
"(Figures 6C,D)",
"(Supplementary Figure 5)",
"(Figures 9A-C)",
"(Figures 9D-F)",
"(Figures 9G,H)",
"(Figures 10A-C)",
"(Figures 10D-F)"
] | [] | [
"and patient prognosis may be related to TIM in osteosarcoma. The higher the content of immune cells in the TIM of osteosarcoma, the lower the risk score of patients and the better the prognosis. The higher the expression of immune escape-related genes, the lower the risk score of patients and the better the prognosis.",
"Osteosarcoma is the most common primary malignant bone tumor, most commonly occurring in adolescents and children (Huang et al., 2021). Its incidence is about 4.4 per million, accounting for 5% of all childhood malignancies (Han et al., 2021). Nearly 60% of osteosarcomas occur in the femur, tibia, and pelvis (Zhou and Mu, 2021). Osteosarcoma has the characteristics of high malignancy, rapid growth, and easy metastasis. Pulmonary metastasis is one of the major factors leading to the poor prognosis of osteosarcoma, and more than 20% of patients with osteosarcoma have had pulmonary metastasis at the time of diagnosis (Anderson, 2016). Therefore, it is important to clarify the causes and mechanisms affecting the prognosis of osteosarcoma to prolong the survival time of osteosarcoma.",
"LncRNA regulates gene transcription, translation, editing, and other biological processes (Qian et al., 2019). Its abnormal expression is closely related to the occurrence and metastasis of tumors (Kornfeld and Bruning, 2014;Bartonicek et al., 2016). It has been confirmed that multiple lncRNAs can promote the occurrence and metastasis of osteosarcoma through competitive inhibition of miRNA expression (Pan et al., 2021;Zhou and Mu, 2021). Notably, lncRNAs can regulate the development and activation of a variety of immune cells (Atianand et al., 2017). The tumor microenvironment is composed of extracellular matrix, mesenchymal cells, immune cells, and other components, and plays an important role in the occurrence of tumors (Pitt et al., 2016;De Nola et al., 2019). The immune cells in the tumor microenvironment are closely related to the treatment and prognosis of tumors. Studies have shown that lncRNA can promote tumor-associated macrophage polarization to regulate the proliferation and migration of tumor cells (Zhao et al., 2021). LncRNA NKILA can also act on T cells to promote the immune escape of tumor cells (Huang et al., 2018). LncRNA THRIL has also been shown to regulate TNF-α expression and participate in immune response in osteosarcoma (Xu et al., 2020). Therefore, these lncRNAs mediated tumor immune microenvironment (TIM) regulation may play an important role in the metastasis and prognosis of osteosarcoma.",
"In this study, immune-related lncRNAs (IRLs) signature was constructed from the publicly available RNA-Seq dataset to evaluate the prognosis of osteosarcoma. In particular, the role of IRLs signature in osteosarcoma metastasis was Abbreviations: EdU, 5-ethynyl-2 -deoxyuridine; FBS, fetal bovine serum; GSEA, gene set enrichment analysis; GSVA, Gene Set Variation Analysis; IRLs, immunerelated lncRNAs; PCA, principal component analysis; ssGSEA, single sample gene set enrichment analysis; TIM, tumor immune microenvironment. evaluated and validated in vitro. Because the metastasis of osteosarcoma is an important factor affecting its poor prognosis. In addition, this study further explored the role of TIM in the influence of IRLs on the progression of osteosarcoma.",
"Osteosarcoma expression spectrum and clinical information from the TARGET database. 1 Samples were selected and data were processed by the following steps: (1) samples with both expression profiles and prognostic information were selected; (2) delete the samples with a survival time of 0 months; (3) according to the human gene annotation file (version GRCH38.p13), the ID of the gene of the osteosarcoma samples was converted into the gene symbol; and (4) there were multiple expression levels of the same gene in the expression profile, and the mean expression level was taken.",
"A gene set named \"IMMUNE RESPONSE TO TUMOR CELL\" was download from Molecular Signatures Database. 2 Correlation analysis was conducted between all lncRNAs and the gene set, and IRLs with a correlation coefficient greater than 0.6 were obtained.",
"LncRNAs with expression variance greater than 0.2 in IRLs were screened. Univariate Cox regression analysis was performed on these lncRNAs and those with P-value less than 0.5 were screened (Sveen et al., 2012). Iterative LASSO regression was used to identify high-frequency lncRNA . Through 1000 iterations, a total of 11 lncRNAs with a frequency greater than 300 were screened out. These lncRNAs were incorporated into the Cox regression analysis one by one until the AUC value of IRLs signature reached the maximum. The risk score of each osteosarcoma sample was calculated using the predict function. The patients were divided into a high-risk group and low-risk group using the optimal risk score cut-off value and the Survminer R package was used for survival analysis. ",
"Fifty immune cell gene sets (Bindea et al., 2013;Cheng et al., 2013;Newman et al., 2015;Senbabaoglu et al., 2016), stromal cell gene set, and total immune cell gene set included in this study (Yoshihara et al., 2013) came from previous studies. Single sample gene set enrichment analysis (ssGSEA) of these gene sets were performed using Gene Set Variation Analysis (GSVA) R package (Wang et al., 2021), and the results of ssGSEA were normalized. KEGG pathway enrichment and GO function enrichment analysis were performed using GSEA software (version 4.0.1). Gene sets of KEGG and GO (C2.Cp.KEGG.v7.1 and C5.All.V7.1.Symbols) download from Molecular Signatures Database (see text footnote 2).",
"The risk score and osteosarcoma features such as age, gender, recurrence, metastasis, and tumor site were used to construct a FIGURE 2 | The role of IRLs signature in the prognosis of osteosarcoma. (A) Osteosarcoma was divided into high-risk group and low-risk group according to the optimal cut-off value of risk score. (B) Relationship between risk score and survival states in patients with osteosarcoma. (C,D) Osteosarcomas were grouped according to the optimal cut-off value of lncRNAs expression in IRLs signature, and survival analysis was performed.",
"nomogram (Iasonos et al., 2008;Wang et al., 2013) to intuitively evaluate the prognosis of patients with osteosarcoma. The decision curve was used to verify the accuracy of the nomogram.",
"ESTIMATE algorithm was used to evaluate stromal score, immune score, and tumor cell purity of osteosarcoma (Hu et al., 2021). Kruskal-Wallis was used to analyze the differences in the stromal score, immune score, Estimate score, and tumor cell purity of different risk score osteosarcomas.",
"Human osteosarcoma cell lines KHOS and 143B were derived from American Type Culture Collection (ATCC, VA, United States). 143B cells were cultured in DMEM medium (HyClone, UT, United States) containing 10% fetal bovine serum (FBS, Gibco, NY, United States), and KHOS cells were cultured in RPMI-1640 medium (HyClone, UT, United States) containing 10% FBS. Si-AC006033.2 was obtained from Gemma Gene (Suzhou, China) (sequences: 5 -GCAGCUGCUUUGACAGUUUTT-3 ). Lipofectamine 3000 (Invitrogen, CA, United States) was used for transfection. The transfection process was carried out according to the instructions.",
"RNA extraction from osteosarcoma cell lines was performed using TRIzol (Invitrogen, CA, United States). GAPDH was selected as an endogenous control. The primers of AC006033.2 and GAPDH are shown in Supplementary Table 1.",
"Osteosarcoma cells were cultured in 96-well plates with a cell density of 3000 cells per well. A total of 10 µL Cell Counting Kit-8 (CCK-8) solution (Beyotime, Shanghai, China) was added at 0, 24, 48, and 72 h, respectively. OD values were measured at 450 nm wavelength. Osteosarcoma cells were inoculated in six-well plates and cultured for 12 h, 5-ethynyl-2 -deoxyuridine (EdU, Beyotime, Shanghai, China), was added. The final concentration of EdU was 10 µM. It was incubated in an incubator at 37 • C for 2 h and fixed with 4% paraformaldehyde. A total of 1 mL of osmotic solution was added to each well and incubate at room temperature for 15 min. A total of 0.5 mL click reaction solution was added to each well and incubate at room temperature in dark for 30 min. Finally, the nuclei were stained with DAPI (Beyotime, Shanghai, China).",
"The wound-healing assay was performed in a six-well plate. Scratches were made with the tip of a sterile pipette. The changes of scratches at 24 h were observed under a microscope. Image-Pro Plus 6.0 software (Media Cybernetics, United States) was used to calculate the area change of the scratches. Invasion experiments were performed in transwell chambers (Corning, NY, United States) with an 8 µm pore diameter membrane. The upper layer of the chamber was added with matrigel (BD, NJ, United States). The number of osteosarcoma cells inoculated was 1 × 10 5 pre well. The upper chamber was cultured with serum-free medium, and the lower chamber was cultured with a 700 'L complete medium. After 48 h, the cells in the upper part of the basement membrane were erased, and the cells in the lower part of the basement membrane were fixed and stained.",
"The statistical software R (version 3.6.1) was used for data analysis and image production. The Chi-square test was used to compare differences in recurrence rates or metastasis rates of osteosarcoma. Spearman correlation analysis was used to evaluate the correlation between the expression level of lncRNAs, the correlation between risk score and lncRNA expression level, or the correlation between lncRNA expression level and immune cell content. The Kruskal-Wallis test was used to analyze the differences in immune cell content, tumor microenvironment score, or gene expression level. Kaplan-Meier survival analysis was used to evaluate prognostic differences between different risk scores or between different lncRNAs expression levels in osteosarcoma. Univariate Cox regression analysis and multivariate Cox regression analysis were used to evaluating the effects of lncRNA, clinical characteristics, or risk score on the prognosis of osteosarcoma. Two-tailed P-values were used, and the statistical significance was set at P < 0.05.",
"Sequencing data and clinical data were downloaded from the TARGET database and expression levels of all samples were combined into an expression profile. The names of samples with both clinical and sequencing data are shown in Supplementary ",
"There were 4986 lncRNAs with variances greater than 0.2 expressed in IRLs. Univariate Cox regression analysis was performed on these IRLs, among which 1743 IRLs with P-value less than 0.5 were identified. Iterative LASSO regression was used to identify high-frequency lncRNAs. One thousand iterations were executed, and 11 lncRNAs with a frequency greater than 300 were screened out. These lncRNAs were incorporated into the Cox regression analysis one by one until the AUC of ROC reached the maximum. At this point, the number of lncRNAs was 10 and the AUC was 0.96 (Figures 1A,B). The predict function was used to calculate the risk score of each osteosarcoma sample, and the osteosarcoma patients are divided into the high-risk group (n = 48) and low-risk group (n = 47) according to the optimal cut-off value (0.92) of risk score. Kaplan-Meier survival analysis showed that the lower risk score was associated with a better prognosis for osteosarcoma ( Figure 1C). The distribution of 10 IRLs expression levels with the change of risk score was shown in Figure 1D. The results of principal component analysis (PCA) showed that IRLs signature can achieve better dimension reduction (Figure 1E). Correlation analysis results showed that the expression levels of these lncRNAs were not highly correlated (Figure 1F), which further demonstrated the rationality of IRLs signature.",
"Osteosarcoma patients were divided into high-risk group and low-risk group according to the optimal cut-off value of risk score (Figure 2A). With the increase of ",
"Univariate and multivariate Cox regression analysis results showed that IRLs signature and the clinical features of osteosarcoma including recurrence, metastasis, and tumor location could all be independent prognostic factors for osteosarcoma ( Figure 3A and Supplementary Tables 7, 8).",
"In the prognostic evaluation of osteosarcoma, the predictive performance of IRLs signature was the highest among these features. In the ROC curve, the 1-, 3-, and 5-year AUC values were 0.802, 0.925, and 0.96, respectively ( Figure 3B). In addition, the Chi-square test confirmed that the recurrence rates and metastasis rates were higher in the high-risk group than in the low-risk group (Figure 3C). Survival analysis showed a poor prognosis for recurrent, metastatic, and non-limb osteosarcomas (Supplementary Figure 3A), and no significant difference in prognosis between sex and age (Supplementary Figure 3B).",
"The nomogram is widely used to evaluate the prognosis of tumors. It can reduce the statistical prediction model to a probability value. In this study, risk score, gender, age, recurrence, metastasis, and tumor location were integrated to FIGURE 9 | Relationship between IRLs signature and tumor microenvironment score. (A-C) Analysis of differences in the immune score, stromal score, and Estimate score between high-risk and low-risk osteosarcoma. (D-F) Correlation analysis between risk score and osteosarcoma immune score, matrix score, and Estimate score. (G) Analysis of difference in tumor cell purity between high-risk and low-risk osteosarcoma. (H) Correlation analysis between risk score and tumor cell purity of osteosarcoma.",
"construct a nomogram to evaluate the prognosis of osteosarcoma ( Figure 4A). The nomogram showed the predicted survival rates for 3 and 5 years, respectively. Calibration curves showed that the nomogram was able to accurately evaluate the prognosis of osteosarcoma ( Figure 4B).",
"The metastasis of osteosarcoma is one of the most important factors affecting its prognosis. Therefore, this study focused on the role of the IRLs signature in the metastasis of osteosarcoma. The Chi-square test confirmed that the metastatic rate of osteosarcoma was higher in the high-risk group than in the lowrisk group (Figure 3C). The waterfall chart also shows that the probability of metastasis gradually decreases with the decrease of risk score ( Figure 5A). Therefore, IRLs signature may play an important role in the metastasis of osteosarcoma. The results of the differential analysis showed that only AC006033.2 of the 10 lncRNAs of IRLs signature was different between the metastatic and non-metastatic osteosarcomas, and the expression level of AC006033.2 was low in the metastatic osteosarcomas ( Figure 5B). For this reason, AC006033.2 was knocked down and validated in osteosarcoma cell lines. Knockdown efficacy was confirmed by PCR in osteosarcoma cell line 143B and KHOS (Figure 5C). We first conducted a cell proliferation experiment, and the results showed that knocking down AC006033.2 could increase the proliferation ability of osteosarcoma cells (Figures 5D,E). The wound-healing assay results further showed that knocking down AC006033.2 increased the migration ability of osteosarcoma cells (Figures 6A,B). Transwell invasion assay showed increased invasiveness of osteosarcoma cells after AC006033.2 knockdown (Figures 6C,D).",
"Gene set enrichment analysis (GSEA) software was used to assess KEGG pathway enrichment in high-risk and lowrisk osteosarcomas. The pathways that enriched in low-risk osteosarcomas were mainly immune-related (Figure 7A), while no associated pathways enriched in high-risk osteosarcomas. The better prognosis of osteosarcoma in the low-risk group may be related to the local immune microenvironment.",
"To further explore the possible role of IRLs signature in osteosarcoma, GO enrichment analysis was performed using GSEA software. The results showed that osteosarcomas in the low-risk group were mainly enriched in immune-related functions (Figure 7B), while the high-risk group osteosarcomas were also not enriched in any meaningfully related functions.",
"The Role of Tumor Immune Microenvironment in the Influence of Immune-Related LncRNAs on Osteosarcoma Prognosis",
"Relationship Between Immune-Related LncRNAs Signature and Immune Cell Infiltration LncRNA can regulate the development and activation of various immune cells (Atianand et al., 2017). Therefore, our study investigated the relationship between the degree of immune cell infiltration and IRLs signature in the immune microenvironment of osteosarcoma. The results of ssGSEA are shown in Supplementary Table 9, and the content of immune cells in osteosarcoma decreased with the increase of risk score (Figure 8A), and the correlation analysis results showed that risk score was negatively correlated with the content of most immune cells (Supplementary Figure 4), which suggested that the low degree of immune cell infiltration might be an important reason for recurrence and metastasis in the high-risk osteosarcoma. In addition, AC006033.2 was also positively correlated with the content of most immune cells (Figure 8B). Osteosarcoma with low AC006033.2 expression had a low level of immune cell infiltration, which was not conducive to the prognosis of osteosarcoma. This was consistent with the conclusion that AC006033.2 knockdown in vitro enhanced the invasiveness of osteosarcoma cells (Figures 6C,D).",
"Signature and Tumor Microenvironment Score ESTIMATE algorithm can be used to evaluate the tumor microenvironment score of osteosarcoma to evaluate tumor stroma content, immune cell content, and tumor cell purity (Supplementary Table 10; Hu et al., 2021). Our study calculated the tumor microenvironment score of osteosarcoma by the ESTIMATE algorithm, and analyzed the relationship between them and risk score or the degree of immune cell infiltration (Supplementary Figure 5). Differential analysis results showed that the immunoscore, stromal score, and Estimate score of the low-risk group were higher than those of the high-risk group (Figures 9A-C). Correlation analysis results showed that risk score was negatively correlated with immune score, stromal score, and Estimate score. With the increase of risk score, the immune score, stromal score, and Estimate score of osteosarcoma gradually decreased (Figures 9D-F). Conversely, the purity of tumor cells in the low-risk osteosarcoma group was lower than that in the high-risk group, and the purity of tumor cells was negatively correlated with the risk score (Figures 9G,H).",
"Immune escape is an important reason for the rapid growth of tumor cells in the human body. In this study, we found that the prognosis of osteosarcoma may be related to the mechanism of endogenous immune escape. The molecules involved in endogenous immune escape include MHC-I molecules, MHC-II molecules, costimulatory molecules. Our results showed that the expression levels of MHC-I molecules, MHC-II molecules, and costimulatory molecules decreased with the increase of risk score (Figures 10A-C), and the different analysis results showed that the expression levels of these molecules in the high-risk group were lower than those in the low-risk group (Figures 10D-F). This suggests that immune escape is more likely to occur in the high-risk group osteosarcoma.",
"Compared with previous amputations alone, the survival rate for osteosarcoma has improved from 20% to more than 60% (Koster et al., 2018;Wang et al., 2018) but has not improved further in recent years. The high incidence of pulmonary metastasis is one of the important reasons. Therefore, searching for effective prognostic markers is an important method for early diagnosis of the prognosis of osteosarcoma and early intervention. In recent years, the role of TIM in the treatment of osteosarcoma has become increasingly evident. A growing number of immunotherapy agents are entering clinical trials (NCT04668300, NCT04544995, and NCT02500797). Therefore, we hope to find immune-related biomarkers that can not only better predict patient survival, but also provide potential therapeutic targets for future immunotherapy. LncRNA has been proved to be involved in the immune response of osteosarcoma by regulating the expression of TNF-α (Xu et al., 2020). To this end, we constructed a prognostic signature using IRLs to explore its role in the prognosis of osteosarcoma. Further, we explored the relationship between the IRLs signature and the TIM of osteosarcoma.",
"In this study, 10 IRLs with good predictive ability were selected as a prognostic marker for osteosarcoma by iterative LSSSO regression and multivariate Cox regression analysis. Compared with the simple univariate and multivariate Cox regression analysis, the IRLs signature has better predictive performance, with an AUC of 0.96. The results of PCA showed that IRLs signature could classify osteosarcoma into two groups. With the increase of risk score, the mortality of patients with osteosarcoma showed a significant upward trend. Recurrence and metastasis are important clinical features for the prognosis of osteosarcoma. Compared with the common clinical features of osteosarcoma, IRLs signature has higher predictive power for the prognosis of osteosarcoma, even higher than the two clinical features of osteosarcoma recurrence and metastasis.",
"Our study showed that the metastatic rate of osteosarcoma in the high-risk group was significantly higher than that in the low-risk group, and the probability of metastasis tended to increase with the increase of risk score. It is worth noting that the differential expression analysis of lncRNA in IRLs signature between the metastatic group and the non-metastatic group showed that only the expression level of AC006033.2 was different. Therefore, our study further verified the role of AC006033.2 in the biological behavior of osteosarcoma through an in vitro experiment. By interfering with AC006033.2, we found that the proliferation, migration, and invasion of osteosarcoma cell lines were enhanced. Studies have shown that AC006033.2 is also an effective biomarker for anti-tumor immunity in gastric cancer (He and Wang, 2020), which is similar to the results of our study. We found that IRLs signature is closely related to the TIM of osteosarcoma. In addition, the expression level of AC006033.2 was significantly positively correlated with the content of various immune cells. Therefore, AC006033.2 may be an important molecule affecting the prognosis of osteosarcoma.",
"LncRNA THRIL has been shown to regulate TNF-α expression and participate in immune response in osteosarcoma (Xu et al., 2020). KEGG and GO enrichment analysis results also showed that osteosarcoma in the low-risk group was highly correlated with immune pathways and functions. Therefore, we further explored the relationship between IRLs signature and TIM of osteosarcoma. Firstly, the ssGSEA was used in the study to evaluate the level of immune cell infiltration in the microenvironment of osteosarcoma, and the level of immune cell infiltration was higher in the low-risk group. Results of the ESTIMATE algorithm also indicated a higher immune score in osteosarcoma in the low-risk group. These suggest that IRLs may be associated with the immune cell infiltration in the microenvironment of osteosarcoma.",
"Studies have shown that lncRNA can also act on T cells to promote the immune escape of tumor cells (Huang et al., 2018). Therefore, we further explored the relationship between the IRLs signature and the TIM of osteosarcoma. The decrease of MHC-I molecular presentation function is considered to be an important reason for the immune escape of tumor cells (Lee et al., 2005). Low expression of MHC-II molecules can also lead to the occurrence of immune escape due to the inability to effectively activate T cells (Zhi et al., 2021). In addition, the activation of T cells also requires the participation of costimulatory molecules (Angulo et al., 2021). We found that MHC-I molecules, MHC-II molecules, and costimulatory molecules, which are associated with endogenous immune escape, were all low expressed in the high-risk group. These results suggest that immune escape may be prevalent in high-risk group osteosarcoma, and IRLs may be associated with the immune escape of osteosarcoma cells.",
"The IRLs signature is a reliable biomarker for the prognosis of osteosarcoma, and they alter the prognosis of osteosarcoma. In addition, IRLs signature and patient prognosis may be related to TIM in osteosarcoma. The higher the content of immune cells in the TIM of osteosarcoma, the lower the risk score of patients and the better the prognosis. The higher the expression of immune escape-related genes, the lower the risk score of patients and the better the prognosis.",
"The datasets presented in this study can be found in online repositories. The names of the repositories and accession numbers can be found in the article material.",
"WG conceived the project. QH, YL, CC, JL, TR, YH, HZ, YY, YG, WW, BW, JN, JX, and LG performed the literature search and data analysis. QH and WG drafted and critically revised the work. All authors contributed to manuscript revision, read, and approved the submitted version.",
"This work was supported by the Beijing Science and Technology Planning Project (No. Z161100000116100) and the National Natural Science Foundation of China (Nos. 81572633 and 82072970)."
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Data Collection and Processing",
"Co-expression Analyses of Immune-Related LncRNAs",
"Construction Immune-Related LncRNAs Prognostic Signature",
"Single Sample Gene Set Enrichment Analysis and Gene Set Enrichment Analysis",
"Construction of the Nomogram",
"Estimation of Tumor Microenvironment Score",
"Cell Culture and Transfection",
"Reverse Transcription-Quantitative Polymerase Chain Reaction",
"Cell Proliferation Assay (Cell Counting Kit-8 and 5-Ethynyl-2 -Deoxyuridine)",
"Wound Healing Assay and Transwell Invasion Assay",
"Statistical Analysis",
"RESULTS",
"Data Processing and Co-expression Analyses of Immune-Related LncRNAs",
"Construction of Immune-Related LncRNAs Prognostic Signature",
"The Role of Immune-Related LncRNAs Signature in the Prognosis of Osteosarcoma",
"The Role of Clinical Features in the Prognosis of Osteosarcoma",
"The Construction and Verification of the Nomogram",
"The Role of Immune-Related LncRNAs Signature in Osteosarcoma Metastasis",
"KEGG Pathway Enrichment and GO Function Enrichment",
"Relationship Between Immune-Related LncRNAs",
"Relationship Between Immune-Related LncRNAs Signature and Immune Escape in Osteosarcoma",
"DISCUSSION",
"CONCLUSION",
"DATA AVAILABILITY STATEMENT",
"AUTHOR CONTRIBUTIONS",
"FUNDING",
"FIGURE 1 |",
"FIGURE 3 |",
"FIGURE 4 |",
"FIGURE 5 |",
"FIGURE 6 |",
"FIGURE 7 |",
"FIGURE 8 |",
"FIGURE 10 |",
"Table 2 ."
] | [] | [
"Supplementary Table 9",
"Table 10"
] | [
"Citation: Immune-Related LncRNAs Affect the Prognosis of Osteosarcoma, Which Are Related to the Tumor Immune Microenvironment",
"Citation: Immune-Related LncRNAs Affect the Prognosis of Osteosarcoma, Which Are Related to the Tumor Immune Microenvironment"
] | [] |
198,062,659 | 2022-02-05T22:49:19Z | CCBYNC | https://academic.oup.com/biomethods/article-pdf/3/1/bpy011/26024439/bpy011.pdf | GOLD | 0496eb3acb57c87ac09a340cd2d3141b653609ca | null | null | null | null | 10.1093/biomethods/bpy011 | 2950364254 | 32161804 | 6994031 |
Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools
Juan Cruz Landoni
Research Programs Unit, Molecular Neurology
University of Helsinki
00290HelsinkiFinland
Liya Wang
Department of Anatomy, Physiology, and Biochemistry
Swedish University of Agricultural Sciences
75007UppsalaSweden
Anu Suomalainen
Research Programs Unit, Molecular Neurology
University of Helsinki
00290HelsinkiFinland
Department of Neurosciences
Helsinki University Hospital
00290HelsinkiFinland;
Neuroscience Center
HiLife
University of Helsinki
00790HelsinkiFinland
Correspondence address. Biomedicum-Helsinki
Molecular Neurology Research Program
University of Helsinki
Haartmaninkatu 800290HelsinkiFinland
Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools
10.1093/biomethods/bpy011M E T H O D S M A N U S C R I P TdNTPanalysissolid-phasemethodnucleotide
deoxynucleoside triphosphate (dNTPs) are the reduced nucleotides used as the building blocks and energy source for deoxyribonucleic acid (DNA) replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways that are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understanding the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilizing the current state-of-theart polymerase method, modified to a solid-phase setting and optimized for larger scale measurements.
Introduction
Deoxyribonucleic acid (DNA) carries the genetic information of all known living organisms, and its synthesis utilizes deoxynucleoside triphosphates (dNTPs), both as building blocks and as energy source for the polymerization reaction. dNTP pools depend on cell cycle and cell and tissue type, with proliferating cells typically having higher pools than post-mitotic cells [1]. dNTPs are typically present at very low concentrations, which are tightly regulated via the expression, regulation and localization of their biosynthetic and catabolic enzymes [2][3][4]. These enzymes form a complex, highly regulated network in animal cells, involving the nuclear, mitochondrial and cytoplasmic compartments. These interlinked pathways include the biosynthetic de novo pathway and the parallel cytoplasmic and mitochondrial salvage pathways [5,6].
The absolute amounts and ratios of the different dNTPs in time and space have been shown to be critical for a wide range of cellular processes, including DNA replication fidelity and mutagenesis [7,8], DNA repair, cell cycle progression and regulation, mitochondrial DNA (mtDNA) maintenance function and oncogenic and apoptotic processes [9]. Because of the mutagenic effects of imbalanced dNTP pools, they have been studied in detail in the context of cancer biology. However, accumulating data points to tight co-regulation of the mitochondrial and cytoplasmic pools, evidenced by defects in the core synthetic enzymes causing depletion or mutagenesis of mtDNA [10,11]. These disorders present varying tissue specificity and different This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] ages of onset, typically with multi-systemic devastating consequences [12][13][14]. Furthermore, disorders of mitochondrial DNA maintenance can secondarily affect the cytoplasmic dNTP pool balance [15,16], underscoring the intimate crosstalk of the different compartmentalized dNTP pools. Preclinical evidence suggest that some mtDNA instability disorders could be treated with nucleosides [17][18][19]. However, knowledge of the regulation of the dNTP pools in pathological metabolic states or upon supplementation therapies is still scarce, and sensitive methodology is needed.
The most widely used approach to measure dNTP concentrations from biological samples is based on a polymerase assay using radioactively labelled substrate, first proposed in the late 1960s [20] and further applied and developed two decades later [21]. This traditional method is laborious and multistep, challenging its sensitivity. After different modifications and the replacement of the Klenow fragment for a thermostable polymerase to improve ribonucleotide discrimination [22], the most recent update for this method was developed for the measurement of mitochondrial dNTP pools [23]. The assay relies on the specificity of a polymerase to utilize the dNTPs present in the sample, and bind them on a designed template, together with a different radiolabelled dNTP which will be incorporated proportionally. The amount of the radioactive labelled products (newly synthesized oligonucleotides) together with a standard series of known dNTP concentrations, provides a quantitative result. However, the field has been hampered by the recently restricted commercial availability of specific materials needed for this protocol, motivating method development.
We present here the adaptation of the dNTP measurement into a solid-phase format, utilizing combined knowledge from the conventional polymerase-based dNTP measurement and the 'solid-phase mini-sequencing' principle of single-nucleotide detection [24,25]. This allows a microtitre-plate-based, automatable approach, with an accurate measurement of a large number of samples, improving efficiency, safety and accuracy of the methodology. Figure 1A shows an example of the reaction in the setting of the present method, being used to measure deoxycytidine triphosphate (dCTP). Table 1 below shows the sequences of the primer and the oligonucleotides used in the reaction. The templates are labelled 'Oligo'þN, with N indicating the base of the specific dNTP to be measured with that template and the primer being common to all reactions.
Materials and methods
Biotinylated oligonucleotides and primer
The biotin-labelled oligonucleotides should be High-performance liquid chromatography (HPLC)-purified, and the primer diluted to 5 lM concentration for the reaction, aliquoted and stored at À20 C.
Reagents and equipment
The content in parenthesis indicates the name of the product/ company used in the set-up • 60% methanol in water, kept at À20 C.
• 50 mM NaOH (made fresh every 4 weeks).
• Thermostable DNA polymerase (DyNAzyme II DNA Polymerase by ThermoFisher Scientific), with its 10x optimized Enzyme Buffer, stored at À20 C.
• 0.5 M dithiothreitol (DTT) in small aliquots stored at À20 C (single use because of instability).
• dNTP mix stock, 40 mM dNTPs (10 mM each) (Bioline), and series dilutions until 1 mM stored at À20 C. • Microplate washer (Wellwash TM by Thermo Labsystems).
• Speed vacuum concentrator.
• Countess automated cell counter (Invitrogen).
dNTP isolation
1. Culture cells in Petri dishes to an ideal minimum of 1 x 10 6 cells. As dNTPs vary greatly between dividing and nondividing cells and throughout the cell cycle, plan to harvest the cells in similar confluency and cell cycle stage. Wash the cells carefully with cold PBS and detach by trypsinization. 2. For normalization, count the cell concentration in the suspension. Then pellet the cells in a microcentrifuge at 250Ág, and store the pellet at À80 C. 3. For the isolation, add 1.5 ml of cold (À20 C) 60% methanol in water and vortex thoroughly. Then incubate for at least 1 h at À80 C. 4. Pellet the insoluble cell contents for 15 min at maximum speed (20 000Ág at 4 C); incubate the samples at 95 C for 3 min, cool down on ice and pellet again similarly. 5. Collect the supernatant and transfer it to a new tube.
Desiccate the supernatant with a speed vacuum concentrator until fully dry. 6. Store the solid extract at À80 C until analysis.
Solid-phase radioactive polymerase reaction Dissolve the frozen solid nucleotide extract in cold sterile water (normally 100 ml, modify according to the cell number), vortex thoroughly and keep on ice for 10 min. 5. Prepare replicates of the different dilution coefficients (normally 1:5 and 1:10) for each sample to be measured, in a volume sufficient for four reactions per dilution (minimum 50 ml).
Reaction:
Prepare master-mixes for each nucleotide to be measured according to Table 3. [ 3 H]-dATP is used for measuring dTTP, dCTP and dGTP (the wells with OligoT, OligoC and OligoG) in the sample, while [ 3 H]-dTTP is used for the dATP (OligoA) reaction mixture. Since the radiolabelled dNTPs are stored in ethanol:water mixture, the molar concentration of each batch should be calculated from the activity per volume and specific radioactivity (usually 1mCi/ml and 10-25 Ci/mmol), and the solvent of the required amount evaporated with a speed-vacuum concentrator and dissolved in water, as recommended by [23]. A 1:3 dilution of the radiolabel with cold dATP or dTTP is also described by [23] to lower the specific radioactivity and thus the costs without appreciable loss of sensitivity. 7. Incubate the plate at 55 C for 1 h, with a flotation device in a water bath. 8. Discard the contents of the wells and perform the washing process as in step 2. 9. Release the newly synthesized labelled strand by adding 60 ml of 50 mM NaOH to each well and incubate for 3 min at room temperature. 10. Transfer the eluted DNA to scintillation vials into 3 ml of scintillation cocktail and measure the radioactivity in a beta counter, with 1 min counting time per sample.
Data analysis:
After exporting the data, analyse the counts-per-minute (CPM) values for each nucleotide independently. Subtract the blank value from all samples measuring that nucleotide. Next, generate a linear standard curve from the origin (Fig. 1B), and use the regression slope to calculate the concentration of the measured replicates based on their CPM values. Values outside the range of standards should be omitted and re-measured at a different dilution. Then use the cell number and dilution factors utilized to calculate the amount of dNTP per number of cells (Fig. 1C).
Results
The assay was performed on an array of different cell lines. The standard curves show consistently strong linearity within the measurement range, with R 2 values for the regression $0.99 (Fig. 1B). Each line or cell type presented a particular dNTP pool profile ( Fig. 2A). The measurements show good reproducibility with same samples measured on different days (SH-SY5Y 1 vs. 2 and HEK293 1 vs. 2), as well as with independently cultured (and differentiated in case of myotubes) samples from a same cell line (SH-SY5Y 1-2 vs. 3 and myotubes 1-2) (Fig. 2B). Finally, in order to compare the performance of the methodology with the traditional one from in vivo samples, instead of cell lines, we measured identical aliquots of freshly isolated mouse bone marrow extracts with a modified version of the protocol in [23] for whole cell lysates, and with the present method. The resulting values (Fig. 2C-D) show very similar dNTP concentration landscapes, with a tendency of increased absolute value, particularly strong, concerning the measurement of dTTP.
Discussion
We report here a development to the current state-of-the-art methodology for quantitative measurement of the four canonical dNTPs from biological samples. The method allows the performance of the measurement in a solid-phase multiwell setting, and the microtitre-plate format allows automation of the pipetting and washing steps, as well as circumventing timeconsuming primer preparation and filter-based steps from the previous methods [23]. Furthermore, it allows utilization of optimized reaction buffers and template sequences. All these features dramatically reduce the required analysis steps, time and manual work required, diminishing the potential sources of error. Importantly, it also increases method safety, reducing direct contact with hazardous compounds.
The main observable difference in the results obtained with the new methodology compared to the conventional filterbased method is the tendency to higher absolute concentrations, particularly for dTTP. The consistent standard curve linearity, the nature of dTTP as the most concentrated canonical dNTP in the cell and the reduction of steps that could potentially lead to loss of sample in the method, as well as the unlikely possibility of consistent contamination or signal acquisition specific for dTTP, prompts us to propose that the higher values may be a true improvement in the sensitivity of the method.
The current shortcoming of the methodology is the increase of the reaction volume due to the nature of the well, leading to a larger requirement of sample and reagents. Moreover, the normalization strategy is a challenge in the field [1], due to the inaccuracy of cell counting and difficulties comparing results from different publications. The most widely used normalization method is cell number, although other strategies are possible, such as utilizing the absorbance of an alkaline lysate, proportional to cell mass [26]. The consistency of the results obtained by cell number normalization lead us to utilize it due to its simplicity, but a more sensitive approach should be considered for more challenging biological samples, such as protein or DNA content measurement, or evaluation of cell volume for molar concentration presentation.
The methanol-based nucleotide isolation is commonly used for dNTP measurements and hence was chosen for this methodology. However, methanol extracts have been described to present residual levels of enzymes that could interfere with the polymerase reaction [27]. The boiling step between precipitations should aid the inactivation of the remaining enzymatic activities [28], but in further development of assay sensitivity, other alternatives excluding this possibility could be considered, such as perchlorate/trioctylamine extraction/neutralization [21,29].
Nucleotide metabolism is one of the main targets of chemotherapeutic drugs in cancer, despite the fact that current understanding on nucleotide metabolism and tissue-specific features of dNTP regulation is superficial. Furthermore, mitochondrial dysfunction has turned out to be a common cause of inherited degenerative conditions, also causing rare and devastating mitochondrial disorders in children, where nucleoside therapies are emerging as a potential therapeutic option [12]. Our sensitive, quantitative and efficient method should be a highly useful tool to provide evidence of dNTP metabolism in different biological samples, in pathological states and in treatment of cancer and metabolic diseases.
Funding
This work was supported by grants from the Academy of Finland, Sigrid Jusé lius Foundation, European Research Council and University of Helsinki.
Received: 30 May 2018; Revised: 9 August 2018. Accepted: 15 August 2018 V C The Author(s) 2018. Published by Oxford University Press.
:water mixture (PerkinElmer), stored at À20 C.• [Methyl-3 H]-Deoxythymidine 5 0 -Triphosphate Tetrasodium Salt in 1:1 ethanol:water mixture (PerkinElmer), stored at À20 C. • Phosphate buffered Saline (PBS). • TWEEN V R 20 (Amresco).
Figure 1 :
1(A) Schematic description of the reaction principle using dCTP measurement as an example. (A1) Affinity capture of biotinylated oligonucleotide OligoC to streptavidin-coated plate. (A2) Polymerization: priming of the template, followed by the proportional incorporation of the measured nucleotide (dCTP) and the radiolabelled nucleotide (dATP*). (A3) Alkaline release of the labelled chain and radioactive count. (B) Example of standard curves and slopes to be used in the data analysis, with the regression's calculated R 2 for each nucleotide measurement. (C) Equation for the analysis of the CPM obtained from the procedure, utilizing the resulting slope from the standards and the blank, as well as the recorded dilution factors and cell number to obtain the concentration of the nucleotide per million cells. • TENT solution (40 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 0.1% TWEEN V R 20, pH 8.0-8.8). • Biotinylated oligonucleotides and primer (Sigma Aldrich), stock and dilutions in À20 C. • Streptavidin-coated 96-well plate (BioBind Streptavidin Strip Assembled Solid by Thermo Scientific). • Ultima Gold TM liquid scintillation cocktail for aqueous and nonaqueous samples (PerkinElmer). • Scintillation vials and beta counter (MicroBeta 2 by PerkinElmer).
Figure 2 :
2(A) Illustrative dNTP concentration profiles from an array of cell lines. Error bars represent the standard deviation of two technical replicates. (B) Concentration values obtained from the measurement of the same sample on different days (SH-SY5Y 1 vs. 2 and HEK293 1 vs. 2) and from independently cultured samples of the same cell line (SH-SY5Y 1-2 vs. 3 and Myotubes 1 vs. 2), as well as the values for the cell lines plotted above, 6 standard deviation between two technical replicates. The SEM is also calculated to assess the variability between the replicates. (C-D) Comparison between the dNTP concentration values of identical isolates of mouse bone marrow cells (n ¼ 6) measured with the traditional filter-based protocol or with the present solid-phase methodology in technical duplicates. Data in (C) is represented as means of the values of each bone marrow isolate from a separate mouse. Each data point in (D) represents the value for a single mouse individual obtained from technical duplicates. Error bars represent standard deviation. iPSC, induced pluripotent stem cells; NSP, neurospheres.
Table 1 :
1Detail of the primers and biotinylated oligonucleotides used for the reactions.The initial after 'Oligo' indicates the base of the dNTP to
Table 2 :
2Composition of the affinity capture reaction mixtureVolume (ll)
Final concentration
PBS/TWEEN
V R solution
47.5
5 lM oligonucleotide
2.5
0.25 lM
Total volume
50.0
Table 3 :
3Composition of the polymerase reaction mixtureVolume (ll)
Final concentration
| Landoni et al.
Conflict of interest statement. None declared.
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| [
"deoxynucleoside triphosphate (dNTPs) are the reduced nucleotides used as the building blocks and energy source for deoxyribonucleic acid (DNA) replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways that are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understanding the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilizing the current state-of-theart polymerase method, modified to a solid-phase setting and optimized for larger scale measurements."
] | [
"Juan Cruz Landoni \nResearch Programs Unit, Molecular Neurology\nUniversity of Helsinki\n00290HelsinkiFinland\n",
"Liya Wang \nDepartment of Anatomy, Physiology, and Biochemistry\nSwedish University of Agricultural Sciences\n75007UppsalaSweden\n",
"Anu Suomalainen \nResearch Programs Unit, Molecular Neurology\nUniversity of Helsinki\n00290HelsinkiFinland\n\nDepartment of Neurosciences\nHelsinki University Hospital\n00290HelsinkiFinland;\n\nNeuroscience Center\nHiLife\nUniversity of Helsinki\n00790HelsinkiFinland\n\nCorrespondence address. Biomedicum-Helsinki\nMolecular Neurology Research Program\nUniversity of Helsinki\nHaartmaninkatu 800290HelsinkiFinland\n"
] | [
"Research Programs Unit, Molecular Neurology\nUniversity of Helsinki\n00290HelsinkiFinland",
"Department of Anatomy, Physiology, and Biochemistry\nSwedish University of Agricultural Sciences\n75007UppsalaSweden",
"Research Programs Unit, Molecular Neurology\nUniversity of Helsinki\n00290HelsinkiFinland",
"Department of Neurosciences\nHelsinki University Hospital\n00290HelsinkiFinland;",
"Neuroscience Center\nHiLife\nUniversity of Helsinki\n00790HelsinkiFinland",
"Correspondence address. Biomedicum-Helsinki\nMolecular Neurology Research Program\nUniversity of Helsinki\nHaartmaninkatu 800290HelsinkiFinland"
] | [
"Juan",
"Cruz",
"Liya",
"Anu"
] | [
"Landoni",
"Wang",
"Suomalainen"
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"Gonzá Lez-Vioque",
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"Ferraro",
"Khym"
] | [
"A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells. V V Gandhi, D C Samuels, Nucleosides, Nucleotides and Nucleic Acids. 30Gandhi VV, Samuels DC. A review comparing deoxyribonu- cleoside triphosphate (dNTP) concentrations in the mito- chondrial and cytoplasmic compartments of normal and transformed cells. Nucleosides, Nucleotides and Nucleic Acids 2011;30:317-39.",
"DNA precursor metabolism and genomic stability. C K Mathews, FASEB J. 20Mathews CK. DNA precursor metabolism and genomic sta- bility. FASEB J 2006;20:1300-14.",
"DNA building blocks: keeping control of manufacture. A Hofer, M Crona, D T Logan, Crit Rev Biochem Mol Biol. 47Hofer A, Crona M, Logan DT et al. DNA building blocks: keep- ing control of manufacture. Crit Rev Biochem Mol Biol 2012;47: 50-63.",
"Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. S An, R Kumar, E D Sheets, Science. 320An S, Kumar R, Sheets ED et al. Reversible compartmentali- zation of de novo purine biosynthetic complexes in living cells. Science 2008;320:103-06.",
"Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage. G Pontarin, A Fijolek, P Pizzo, Proc Natl Acad Sci. 105Pontarin G, Fijolek A, Pizzo P et al. Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage. Proc Natl Acad Sci USA 2008;105:17801-06.",
"Mitochondrial purine and pyrimidine metabolism and beyond. L Wang, Nucleosides, Nucleotides and Nucleic Acids. 35Wang L. Mitochondrial purine and pyrimidine metabolism and beyond. Nucleosides, Nucleotides and Nucleic Acids 2016;35: 578-94.",
"Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype. M B Davidson, Y Katou, A Keszthelyi, EMBO J. 31Davidson MB, Katou Y, Keszthelyi A et al. Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype. EMBO J 2012;31:895-907.",
"Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity. R J Buckland, D L Watt, B Chittoor, PLoS Genet. 101004846Buckland RJ, Watt DL, Chittoor B et al. Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity. PLoS Genet 2014;10:e1004846.",
"Deoxyribonucleotides as genetic and metabolic regulators. C K Mathews, FASEB J. 28Mathews CK. Deoxyribonucleotides as genetic and meta- bolic regulators. FASEB J 2014;28:3832-40.",
"Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. A Bourdon, L Minai, V Serre, Nat Genet. 39Bourdon A, Minai L, Serre V et al. Mutation of RRM2B, encod- ing p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 2007;39: 776-80.",
"A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions. H Tyynismaa, E Ylikallio, M Patel, Am J Hum Genet. 85Tyynismaa H, Ylikallio E, Patel M et al. A heterozygous trun- cating mutation in RRM2B causes autosomal-dominant pro- gressive external ophthalmoplegia with multiple mtDNA deletions. Am J Hum Genet 2009;85:290-95.",
"Mitochondrial diseases. G S Gorman, P F Chinnery, S Dimauro, Nat Rev Dis Primers. 216080Gorman GS, Chinnery PF, DiMauro S et al. Mitochondrial dis- eases. Nat Rev Dis Primers 2016;2:16080.",
"Mechanisms of mitochondrial diseases. E Ylikallio, A Suomalainen, Ann Med. 44Ylikallio E, Suomalainen A. Mechanisms of mitochondrial diseases. Ann Med 2012;44:41-59.",
"Mitochondria: in sickness and in health. J Nunnari, A Suomalainen, Cell. 148Nunnari J, Suomalainen A. Mitochondria: in sickness and in health. Cell 2012;148:1145-59.",
"Mitochondrial DNA replication defects disturb cellular dNTP pools and remodel one-carbon metabolism. J Nikkanen, S Forsströ M, L Euro, Cell Metab. 23Nikkanen J, Forsströ m S, Euro L et al. Mitochondrial DNA replication defects disturb cellular dNTP pools and re- model one-carbon metabolism. Cell Metab 2016;23: 635-48.",
"MPV17 loss causes deoxynucleotide insufficiency and slow DNA replication in mitochondria. Dalla Rosa, I Cá Mara, Y Durigon, R , PLoS Genet. 121005779Dalla Rosa I, Cá mara Y, Durigon R et al. MPV17 loss causes deoxynucleotide insufficiency and slow DNA replication in mitochondria. PLoS Genet 2016;12:e1005779.",
"Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome. Y Cá Mara, E Gonzá Lez-Vioque, M Scarpelli, 10.1093/hmg/ddt641Hum Mol Genet. Cá mara Y, Gonzá lez-Vioque E, Scarpelli M et al. Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome. Hum Mol Genet 2014, 10.1093/hmg/ddt641.",
"Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency. C Garone, B Garcia-Diaz, V Emmanuele, EMBO Mol Med. 6Garone C, Garcia-Diaz B, Emmanuele V et al. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency. EMBO Mol Med 2014;6: 1016-27.",
"Deoxycytidine and deoxythymidine treatment for thymidine kinase 2 deficiency. C Lopez-Gomez, R J Levy, M J Sanchez-Quintero, Ann Neurol. 81Lopez-Gomez C, Levy RJ, Sanchez-Quintero MJ et al. Deoxycytidine and deoxythymidine treatment for thymi- dine kinase 2 deficiency. Ann Neurol 2017;81:641-52.",
"A rapid quantitative determination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA. A W Solter, R E Handschumacher, BBA Sect Nucleic Acids Protein Synth. 174Solter AW, Handschumacher RE. A rapid quantitative deter- mination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA. BBA Sect Nucleic Acids Protein Synth 1969;174:585-90.",
"Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers. P A Sherman, J A Fyfe, Anal Biochem. 180Sherman PA, Fyfe JA. Enzymatic assay for deoxyribonucleo- side triphosphates using synthetic oligonucleotides as tem- plate primers. Anal Biochem 1989;180:222-26.",
"Quantitation of cellular deoxynucleoside triphosphates. P Ferraro, E Franzolin, G Pontarin, Nucleic Acids Res. 3885Ferraro P, Franzolin E, Pontarin G et al. Quantitation of cellu- lar deoxynucleoside triphosphates. Nucleic Acids Res 2010;38: e85.",
"Measurement of mitochondrial dNTP pools. R Martí, B Dorado, M Hirano, Methods Mol Biol. 837Martí R, Dorado B, Hirano M. Measurement of mitochondrial dNTP pools. Methods Mol Biol 2012;837:135-48.",
"Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing. A C Syvä Nen, T Hultman, K Aalto-Setä Lä, Genet Anal Biomol Eng. 8Syvä nen AC, Hultman T, Aalto-Setä lä K et al. Genetic analy- sis of the polymorphism of the human apolipoprotein E us- ing automated solid-phase sequencing. Genet Anal Biomol Eng 1991;8:117-23.",
"Quantitative analysis of DNA sequences by PCR and solid-phase minisequencing. A Suomalainen, Syvä Nen A-C, Molecular Biomethods Handbook. Walker JM and Rapley R.Totowa, NJHumana Press Inc2nd ednSuomalainen A, Syvä nen A-C. Quantitative analysis of DNA sequences by PCR and solid-phase minisequencing. In: Walker JM and Rapley R. (eds), Molecular Biomethods Handbook. 2nd edn. Totowa, NJ: Humana Press Inc., 2007, 169-78.",
"Synthesis of mitochondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes. M Frangini, E Franzolin, F Chemello, J Biol Chem. 288Frangini M, Franzolin E, Chemello F et al. Synthesis of mito- chondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes. J Biol Chem 2013;288:5624-35.",
"Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates. T W North, R K Bestwick, C K Mathews, J Biol Chem. 465North TW, Bestwick RK, Mathews CK. Detection of activities that interfere with the enzymatic assay of deoxyribonucleo- side 5'-triphosphates. J Biol Chem 1980;465:6640-6645.",
"Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools. G Pontarin, L Gallinaro, P Ferraro, Proc Natl Acad Sci. 10012159Pontarin G, Gallinaro L, Ferraro P et al. Origins of mitochon- drial thymidine triphosphate: dynamic relations to cytosolic pools. Proc Natl Acad Sci USA 2003;100:12159.",
"An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. J X Khym, Clin Chem. 21Khym JX. An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin Chem 1975;21:1245-52."
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"A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells",
"DNA precursor metabolism and genomic stability",
"DNA building blocks: keeping control of manufacture",
"Reversible compartmentalization of de novo purine biosynthetic complexes in living cells",
"Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage",
"Mitochondrial purine and pyrimidine metabolism and beyond",
"Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype",
"Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity",
"Deoxyribonucleotides as genetic and metabolic regulators",
"Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion",
"A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions",
"Mitochondrial diseases",
"Mechanisms of mitochondrial diseases",
"Mitochondria: in sickness and in health",
"Mitochondrial DNA replication defects disturb cellular dNTP pools and remodel one-carbon metabolism",
"MPV17 loss causes deoxynucleotide insufficiency and slow DNA replication in mitochondria",
"Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome",
"Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency",
"Deoxycytidine and deoxythymidine treatment for thymidine kinase 2 deficiency",
"A rapid quantitative determination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA",
"Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers",
"Quantitation of cellular deoxynucleoside triphosphates",
"Measurement of mitochondrial dNTP pools",
"Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing",
"Quantitative analysis of DNA sequences by PCR and solid-phase minisequencing",
"Synthesis of mitochondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes",
"Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates",
"Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools",
"An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers"
] | [
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"\n\nReceived: 30 May 2018; Revised: 9 August 2018. Accepted: 15 August 2018 V C The Author(s) 2018. Published by Oxford University Press.",
"\n\n:water mixture (PerkinElmer), stored at À20 C.• [Methyl-3 H]-Deoxythymidine 5 0 -Triphosphate Tetrasodium Salt in 1:1 ethanol:water mixture (PerkinElmer), stored at À20 C. • Phosphate buffered Saline (PBS). • TWEEN V R 20 (Amresco).",
"\nFigure 1 :\n1(A) Schematic description of the reaction principle using dCTP measurement as an example. (A1) Affinity capture of biotinylated oligonucleotide OligoC to streptavidin-coated plate. (A2) Polymerization: priming of the template, followed by the proportional incorporation of the measured nucleotide (dCTP) and the radiolabelled nucleotide (dATP*). (A3) Alkaline release of the labelled chain and radioactive count. (B) Example of standard curves and slopes to be used in the data analysis, with the regression's calculated R 2 for each nucleotide measurement. (C) Equation for the analysis of the CPM obtained from the procedure, utilizing the resulting slope from the standards and the blank, as well as the recorded dilution factors and cell number to obtain the concentration of the nucleotide per million cells. • TENT solution (40 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 0.1% TWEEN V R 20, pH 8.0-8.8). • Biotinylated oligonucleotides and primer (Sigma Aldrich), stock and dilutions in À20 C. • Streptavidin-coated 96-well plate (BioBind Streptavidin Strip Assembled Solid by Thermo Scientific). • Ultima Gold TM liquid scintillation cocktail for aqueous and nonaqueous samples (PerkinElmer). • Scintillation vials and beta counter (MicroBeta 2 by PerkinElmer).",
"\nFigure 2 :\n2(A) Illustrative dNTP concentration profiles from an array of cell lines. Error bars represent the standard deviation of two technical replicates. (B) Concentration values obtained from the measurement of the same sample on different days (SH-SY5Y 1 vs. 2 and HEK293 1 vs. 2) and from independently cultured samples of the same cell line (SH-SY5Y 1-2 vs. 3 and Myotubes 1 vs. 2), as well as the values for the cell lines plotted above, 6 standard deviation between two technical replicates. The SEM is also calculated to assess the variability between the replicates. (C-D) Comparison between the dNTP concentration values of identical isolates of mouse bone marrow cells (n ¼ 6) measured with the traditional filter-based protocol or with the present solid-phase methodology in technical duplicates. Data in (C) is represented as means of the values of each bone marrow isolate from a separate mouse. Each data point in (D) represents the value for a single mouse individual obtained from technical duplicates. Error bars represent standard deviation. iPSC, induced pluripotent stem cells; NSP, neurospheres.",
"\nTable 1 :\n1Detail of the primers and biotinylated oligonucleotides used for the reactions.The initial after 'Oligo' indicates the base of the dNTP to \n",
"\nTable 2 :\n2Composition of the affinity capture reaction mixtureVolume (ll) \nFinal concentration \n\nPBS/TWEEN \nV R solution \n47.5 \n5 lM oligonucleotide \n2.5 \n0.25 lM \nTotal volume \n50.0 \n\n",
"\nTable 3 :\n3Composition of the polymerase reaction mixtureVolume (ll) \nFinal concentration \n\n"
] | [
"Received: 30 May 2018; Revised: 9 August 2018. Accepted: 15 August 2018 V C The Author(s) 2018. Published by Oxford University Press.",
":water mixture (PerkinElmer), stored at À20 C.• [Methyl-3 H]-Deoxythymidine 5 0 -Triphosphate Tetrasodium Salt in 1:1 ethanol:water mixture (PerkinElmer), stored at À20 C. • Phosphate buffered Saline (PBS). • TWEEN V R 20 (Amresco).",
"(A) Schematic description of the reaction principle using dCTP measurement as an example. (A1) Affinity capture of biotinylated oligonucleotide OligoC to streptavidin-coated plate. (A2) Polymerization: priming of the template, followed by the proportional incorporation of the measured nucleotide (dCTP) and the radiolabelled nucleotide (dATP*). (A3) Alkaline release of the labelled chain and radioactive count. (B) Example of standard curves and slopes to be used in the data analysis, with the regression's calculated R 2 for each nucleotide measurement. (C) Equation for the analysis of the CPM obtained from the procedure, utilizing the resulting slope from the standards and the blank, as well as the recorded dilution factors and cell number to obtain the concentration of the nucleotide per million cells. • TENT solution (40 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 0.1% TWEEN V R 20, pH 8.0-8.8). • Biotinylated oligonucleotides and primer (Sigma Aldrich), stock and dilutions in À20 C. • Streptavidin-coated 96-well plate (BioBind Streptavidin Strip Assembled Solid by Thermo Scientific). • Ultima Gold TM liquid scintillation cocktail for aqueous and nonaqueous samples (PerkinElmer). • Scintillation vials and beta counter (MicroBeta 2 by PerkinElmer).",
"(A) Illustrative dNTP concentration profiles from an array of cell lines. Error bars represent the standard deviation of two technical replicates. (B) Concentration values obtained from the measurement of the same sample on different days (SH-SY5Y 1 vs. 2 and HEK293 1 vs. 2) and from independently cultured samples of the same cell line (SH-SY5Y 1-2 vs. 3 and Myotubes 1 vs. 2), as well as the values for the cell lines plotted above, 6 standard deviation between two technical replicates. The SEM is also calculated to assess the variability between the replicates. (C-D) Comparison between the dNTP concentration values of identical isolates of mouse bone marrow cells (n ¼ 6) measured with the traditional filter-based protocol or with the present solid-phase methodology in technical duplicates. Data in (C) is represented as means of the values of each bone marrow isolate from a separate mouse. Each data point in (D) represents the value for a single mouse individual obtained from technical duplicates. Error bars represent standard deviation. iPSC, induced pluripotent stem cells; NSP, neurospheres.",
"Detail of the primers and biotinylated oligonucleotides used for the reactions.",
"Composition of the affinity capture reaction mixture",
"Composition of the polymerase reaction mixture"
] | [
"Figure 1A",
"(Fig. 1B)",
"(Fig. 1C)",
"(Fig. 1B)",
"Fig. 2A)",
"(Fig. 2B)",
"(Fig. 2C-D)"
] | [] | [
"Deoxyribonucleic acid (DNA) carries the genetic information of all known living organisms, and its synthesis utilizes deoxynucleoside triphosphates (dNTPs), both as building blocks and as energy source for the polymerization reaction. dNTP pools depend on cell cycle and cell and tissue type, with proliferating cells typically having higher pools than post-mitotic cells [1]. dNTPs are typically present at very low concentrations, which are tightly regulated via the expression, regulation and localization of their biosynthetic and catabolic enzymes [2][3][4]. These enzymes form a complex, highly regulated network in animal cells, involving the nuclear, mitochondrial and cytoplasmic compartments. These interlinked pathways include the biosynthetic de novo pathway and the parallel cytoplasmic and mitochondrial salvage pathways [5,6].",
"The absolute amounts and ratios of the different dNTPs in time and space have been shown to be critical for a wide range of cellular processes, including DNA replication fidelity and mutagenesis [7,8], DNA repair, cell cycle progression and regulation, mitochondrial DNA (mtDNA) maintenance function and oncogenic and apoptotic processes [9]. Because of the mutagenic effects of imbalanced dNTP pools, they have been studied in detail in the context of cancer biology. However, accumulating data points to tight co-regulation of the mitochondrial and cytoplasmic pools, evidenced by defects in the core synthetic enzymes causing depletion or mutagenesis of mtDNA [10,11]. These disorders present varying tissue specificity and different This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] ages of onset, typically with multi-systemic devastating consequences [12][13][14]. Furthermore, disorders of mitochondrial DNA maintenance can secondarily affect the cytoplasmic dNTP pool balance [15,16], underscoring the intimate crosstalk of the different compartmentalized dNTP pools. Preclinical evidence suggest that some mtDNA instability disorders could be treated with nucleosides [17][18][19]. However, knowledge of the regulation of the dNTP pools in pathological metabolic states or upon supplementation therapies is still scarce, and sensitive methodology is needed.",
"The most widely used approach to measure dNTP concentrations from biological samples is based on a polymerase assay using radioactively labelled substrate, first proposed in the late 1960s [20] and further applied and developed two decades later [21]. This traditional method is laborious and multistep, challenging its sensitivity. After different modifications and the replacement of the Klenow fragment for a thermostable polymerase to improve ribonucleotide discrimination [22], the most recent update for this method was developed for the measurement of mitochondrial dNTP pools [23]. The assay relies on the specificity of a polymerase to utilize the dNTPs present in the sample, and bind them on a designed template, together with a different radiolabelled dNTP which will be incorporated proportionally. The amount of the radioactive labelled products (newly synthesized oligonucleotides) together with a standard series of known dNTP concentrations, provides a quantitative result. However, the field has been hampered by the recently restricted commercial availability of specific materials needed for this protocol, motivating method development.",
"We present here the adaptation of the dNTP measurement into a solid-phase format, utilizing combined knowledge from the conventional polymerase-based dNTP measurement and the 'solid-phase mini-sequencing' principle of single-nucleotide detection [24,25]. This allows a microtitre-plate-based, automatable approach, with an accurate measurement of a large number of samples, improving efficiency, safety and accuracy of the methodology. Figure 1A shows an example of the reaction in the setting of the present method, being used to measure deoxycytidine triphosphate (dCTP). Table 1 below shows the sequences of the primer and the oligonucleotides used in the reaction. The templates are labelled 'Oligo'þN, with N indicating the base of the specific dNTP to be measured with that template and the primer being common to all reactions.",
"The biotin-labelled oligonucleotides should be High-performance liquid chromatography (HPLC)-purified, and the primer diluted to 5 lM concentration for the reaction, aliquoted and stored at À20 C.",
"The content in parenthesis indicates the name of the product/ company used in the set-up • 60% methanol in water, kept at À20 C.",
"• 50 mM NaOH (made fresh every 4 weeks).",
"• Thermostable DNA polymerase (DyNAzyme II DNA Polymerase by ThermoFisher Scientific), with its 10x optimized Enzyme Buffer, stored at À20 C.",
"• 0.5 M dithiothreitol (DTT) in small aliquots stored at À20 C (single use because of instability).",
"• dNTP mix stock, 40 mM dNTPs (10 mM each) (Bioline), and series dilutions until 1 mM stored at À20 C. • Microplate washer (Wellwash TM by Thermo Labsystems).",
"• Speed vacuum concentrator.",
"• Countess automated cell counter (Invitrogen).",
"1. Culture cells in Petri dishes to an ideal minimum of 1 x 10 6 cells. As dNTPs vary greatly between dividing and nondividing cells and throughout the cell cycle, plan to harvest the cells in similar confluency and cell cycle stage. Wash the cells carefully with cold PBS and detach by trypsinization. 2. For normalization, count the cell concentration in the suspension. Then pellet the cells in a microcentrifuge at 250Ág, and store the pellet at À80 C. 3. For the isolation, add 1.5 ml of cold (À20 C) 60% methanol in water and vortex thoroughly. Then incubate for at least 1 h at À80 C. 4. Pellet the insoluble cell contents for 15 min at maximum speed (20 000Ág at 4 C); incubate the samples at 95 C for 3 min, cool down on ice and pellet again similarly. 5. Collect the supernatant and transfer it to a new tube.",
"Desiccate the supernatant with a speed vacuum concentrator until fully dry. 6. Store the solid extract at À80 C until analysis.",
"Solid-phase radioactive polymerase reaction Dissolve the frozen solid nucleotide extract in cold sterile water (normally 100 ml, modify according to the cell number), vortex thoroughly and keep on ice for 10 min. 5. Prepare replicates of the different dilution coefficients (normally 1:5 and 1:10) for each sample to be measured, in a volume sufficient for four reactions per dilution (minimum 50 ml).",
"Prepare master-mixes for each nucleotide to be measured according to Table 3. [ 3 H]-dATP is used for measuring dTTP, dCTP and dGTP (the wells with OligoT, OligoC and OligoG) in the sample, while [ 3 H]-dTTP is used for the dATP (OligoA) reaction mixture. Since the radiolabelled dNTPs are stored in ethanol:water mixture, the molar concentration of each batch should be calculated from the activity per volume and specific radioactivity (usually 1mCi/ml and 10-25 Ci/mmol), and the solvent of the required amount evaporated with a speed-vacuum concentrator and dissolved in water, as recommended by [23]. A 1:3 dilution of the radiolabel with cold dATP or dTTP is also described by [23] to lower the specific radioactivity and thus the costs without appreciable loss of sensitivity. 7. Incubate the plate at 55 C for 1 h, with a flotation device in a water bath. 8. Discard the contents of the wells and perform the washing process as in step 2. 9. Release the newly synthesized labelled strand by adding 60 ml of 50 mM NaOH to each well and incubate for 3 min at room temperature. 10. Transfer the eluted DNA to scintillation vials into 3 ml of scintillation cocktail and measure the radioactivity in a beta counter, with 1 min counting time per sample.",
"After exporting the data, analyse the counts-per-minute (CPM) values for each nucleotide independently. Subtract the blank value from all samples measuring that nucleotide. Next, generate a linear standard curve from the origin (Fig. 1B), and use the regression slope to calculate the concentration of the measured replicates based on their CPM values. Values outside the range of standards should be omitted and re-measured at a different dilution. Then use the cell number and dilution factors utilized to calculate the amount of dNTP per number of cells (Fig. 1C).",
"The assay was performed on an array of different cell lines. The standard curves show consistently strong linearity within the measurement range, with R 2 values for the regression $0.99 (Fig. 1B). Each line or cell type presented a particular dNTP pool profile ( Fig. 2A). The measurements show good reproducibility with same samples measured on different days (SH-SY5Y 1 vs. 2 and HEK293 1 vs. 2), as well as with independently cultured (and differentiated in case of myotubes) samples from a same cell line (SH-SY5Y 1-2 vs. 3 and myotubes 1-2) (Fig. 2B). Finally, in order to compare the performance of the methodology with the traditional one from in vivo samples, instead of cell lines, we measured identical aliquots of freshly isolated mouse bone marrow extracts with a modified version of the protocol in [23] for whole cell lysates, and with the present method. The resulting values (Fig. 2C-D) show very similar dNTP concentration landscapes, with a tendency of increased absolute value, particularly strong, concerning the measurement of dTTP.",
"We report here a development to the current state-of-the-art methodology for quantitative measurement of the four canonical dNTPs from biological samples. The method allows the performance of the measurement in a solid-phase multiwell setting, and the microtitre-plate format allows automation of the pipetting and washing steps, as well as circumventing timeconsuming primer preparation and filter-based steps from the previous methods [23]. Furthermore, it allows utilization of optimized reaction buffers and template sequences. All these features dramatically reduce the required analysis steps, time and manual work required, diminishing the potential sources of error. Importantly, it also increases method safety, reducing direct contact with hazardous compounds.",
"The main observable difference in the results obtained with the new methodology compared to the conventional filterbased method is the tendency to higher absolute concentrations, particularly for dTTP. The consistent standard curve linearity, the nature of dTTP as the most concentrated canonical dNTP in the cell and the reduction of steps that could potentially lead to loss of sample in the method, as well as the unlikely possibility of consistent contamination or signal acquisition specific for dTTP, prompts us to propose that the higher values may be a true improvement in the sensitivity of the method.",
"The current shortcoming of the methodology is the increase of the reaction volume due to the nature of the well, leading to a larger requirement of sample and reagents. Moreover, the normalization strategy is a challenge in the field [1], due to the inaccuracy of cell counting and difficulties comparing results from different publications. The most widely used normalization method is cell number, although other strategies are possible, such as utilizing the absorbance of an alkaline lysate, proportional to cell mass [26]. The consistency of the results obtained by cell number normalization lead us to utilize it due to its simplicity, but a more sensitive approach should be considered for more challenging biological samples, such as protein or DNA content measurement, or evaluation of cell volume for molar concentration presentation.",
"The methanol-based nucleotide isolation is commonly used for dNTP measurements and hence was chosen for this methodology. However, methanol extracts have been described to present residual levels of enzymes that could interfere with the polymerase reaction [27]. The boiling step between precipitations should aid the inactivation of the remaining enzymatic activities [28], but in further development of assay sensitivity, other alternatives excluding this possibility could be considered, such as perchlorate/trioctylamine extraction/neutralization [21,29].",
"Nucleotide metabolism is one of the main targets of chemotherapeutic drugs in cancer, despite the fact that current understanding on nucleotide metabolism and tissue-specific features of dNTP regulation is superficial. Furthermore, mitochondrial dysfunction has turned out to be a common cause of inherited degenerative conditions, also causing rare and devastating mitochondrial disorders in children, where nucleoside therapies are emerging as a potential therapeutic option [12]. Our sensitive, quantitative and efficient method should be a highly useful tool to provide evidence of dNTP metabolism in different biological samples, in pathological states and in treatment of cancer and metabolic diseases.",
"This work was supported by grants from the Academy of Finland, Sigrid Jusé lius Foundation, European Research Council and University of Helsinki."
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"Biotinylated oligonucleotides and primer",
"Reagents and equipment",
"dNTP isolation",
"Reaction:",
"Data analysis:",
"Results",
"Discussion",
"Funding",
"Figure 1 :",
"Figure 2 :",
"Table 1 :",
"Table 2 :",
"Table 3 :"
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"The initial after 'Oligo' indicates the base of the dNTP to \n",
"Volume (ll) \nFinal concentration \n\nPBS/TWEEN \nV R solution \n47.5 \n5 lM oligonucleotide \n2.5 \n0.25 lM \nTotal volume \n50.0 \n\n",
"Volume (ll) \nFinal concentration \n\n"
] | [
"Table 1",
"Table 3"
] | [
"Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools",
"Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools"
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122,552,501 | 2022-02-20T15:38:22Z | CCBY | https://doi.org/10.1371/journal.pone.0215489 | GOLD | 6d2171fdf1d24f9ede8a0a934fc88e49e912f708 | null | null | null | null | 10.1371/journal.pone.0215489 | 2937056601 | 30998788 | 6472773 |
Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation
April 18, 2019
Mitsunori Miyazaki Id
Department of Physical Therapy
School of Rehabilitation Sciences
Health Sciences University of Hokkaido
HokkaidoJapan
Michito Shimozuru
Graduate School of Veterinary Medicine
Laboratory of Wildlife Biology and Medicine
Hokkaido University
HokkaidoJapan
Medical Center
University of Minnesota
UNITED STATES
Toshio Tsubota
Graduate School of Veterinary Medicine
Laboratory of Wildlife Biology and Medicine
Hokkaido University
HokkaidoJapan
Medical Center
University of Minnesota
UNITED STATES
Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation
April 18, 201910.1371/journal.pone.0215489RESEARCH ARTICLE 1 / 13
Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to "muscle atrophy resistance" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/ mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization.Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.
Introduction
Skeletal muscle mass is generally determined by the net dynamic balance of protein synthesis and degradation [1]. Under catabolic conditions, muscle protein degradation is enhanced through ubiquitin-proteasome and autophagy-lysosome systems [2]. The muscle-specific E3 ubiquitin ligases atrogin1 and murf1 (muscle RING finger 1) are highly expressed in response to unloading/inactivity and contribute to protein ubiquitination and proteasome-dependent degradation [3,4]. Autophagosome formation and lysosomal degradation of cytoplasmic components/organelles are also enhanced under catabolic conditions in skeletal muscle [5,6]. In contrast, decreases in muscle protein synthesis have been observed in several animal and human models of disuse atrophy [7,8]. Metabolic dysfunction is also induced in skeletal muscle following long-term disuse [9]. Consequently, prolonged periods of disuse lead to skeletal muscle atrophy/weakness and metabolic dysfunction, which then can cause impaired locomotive function and increased risk of morbidity/mortality.
Physiological characteristics during hibernation are highly variable between animal species. In general, small hibernators, such as ground squirrels, repeat torpor and arousal cycles every 1-2 weeks during hibernation periods. The thermal and energy metabolism are robustly down-regulated during torpor phase (body temperature: 3˚C -5˚C, heart rate: less than 10 bpm), whereas they return to the normothermic state (body temperature: 37˚C, heart rate: 300-400 bpm) during short arousal phases. Eating, drinking, and excretion (defecation/urination) behaviors are observed during hibernation periods when the animals are awaking [10][11][12]. In contrast, larger hibernators, such as bears, experience no arousal (i.e., remain in a relatively shallow torpor) and do not eat, drink, urinate, or defecate during the entire period of hibernation even though they maintain thermal and energy metabolism at relatively high levels (i.e., body temperature is slightly decreased but maintained to 30˚C -36˚C) [13][14][15].
Although hibernating animals experience long-term inactivity and fasting during winter survival, they can successfully maintain their muscle mass and locomotive function until arousal in spring. Previous reports have indicated that skeletal muscle mass and strength are well preserved during hibernation. Fiber size in skeletal muscle was almost perfectly maintained during hibernation in small mammals (e.g., ground squirrels) [16,17]. In the case of bears (e.g., American black bears or brown bears), protein content slightly decreased but loss of muscle mass and decline in contractile function were limited during hibernation [18][19][20][21][22]. These physiological features in hibernating animals allow us to hypothesize that the skeletal muscle of hibernators possesses a potential resistance to muscle atrophy during continued physical inactivity and malnutrition. In this study, alteration of signaling pathways that govern protein and energy metabolism was examined in skeletal muscle of the Japanese black bear.
Materials and methods
Antibodies
Mouse anti-dystrophin, developed by Morris, G.E. (DSHB Hybridoma Product MANDRA1 (7A10)), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA 52242. Phospho-S6K1 (T389, Cat#: 9205) and phospho-S6K1 (T421/S424, Cat#: 9204) were obtained from Cell Signaling Technology (Danvers, MA, USA). S6K1 (Cat#: sc-230) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 680LT Goat anti-Rabbit IgG (Cat#: 926-68021) was from LI-COR Biosciences (Lincoln, NE, USA). Alexa Fluor 568-conjugated Goat anti-Mouse IgG (Cat#: A11031) was from Thermo Fisher Scientific (Rockford, IL, USA).
Animal care and use
All experimental procedures and animal care performed in this study were conducted in accordance with institutional Guidelines for Animal Care and Use, as approved by the Animal Care and Use Committee of the Graduate School of Veterinary Medicine, Hokkaido University (Permit Number: 9124). A total of six non-pregnant female Japanese black bears (Ursus thibetanus japonicus), between 5 to 15 years of age, kept in Ani Mataginosato Bear Park (Akita Prefecture, Japan, N40˚E140.4˚) were used in this study. All animal care and handling procedures were followed as previously described [14,23]. Briefly, animals were fed with dried corn (360 kcal/100 g, approximately 1.5 kg/head) combined with fruits and vegetables as supplements once a day at 16:00 h during the active period (i.e., from late April to late November). During the two weeks before or after the fasting period (i.e., late November/early December to early/mid-April) as a transition phase to/from torpor status, the amount of feeding was reduced to one-third (0.5 kg corn meal/head) compared to the active period. All animals were kept isolated in the indoor dark rooms for denning and had no access to the food during the torpor period. Access to drinking water was allowed ad libitum throughout the year.
Muscle sample collection
Skeletal muscle samples (sartorius muscle) were collected from bear legs during both prehibernation state (i.e., late November, just prior to the beginning of food deprivation) and post-hibernation state (i.e., early April, one week following the onset of re-feeding). Immediate post-hibernation period (approximately 3 weeks following emerged from denning) was known as "walking hibernation" [15]. During this period, the biochemical stage of hibernation was likely persisted in bears, even though physical activity and food/water intake were minimal. Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). Animals were anesthetized with an intramuscular administration of 3.0 mg/kg zolazepam HCL and tiletamine HCL cocktail and 40 μg/kg medetomidine HCL using a blow dart shot. After immobilization, small pieces of sartorius muscle were excised directly and quickly frozen in liquid nitrogen. Feeding was restricted overnight (about 15-16 h) until the anesthesia and sample collection surgery were completed the next morning. Following the sample collection surgery, meloxicam (subcutaneously at 0.2 mg/kg for analgesia) and atipamezole HCl (intramuscularly at 200 μg/kg as an antagonist to medetomidine HCL) were administered to aid recovery.
Histochemistry/immunohistochemistry
Muscle samples for histochemical or immunohistochemical analyses were frozen in liquid nitrogen-cooled isopentane. Cross sections (8 μm) were cut in a cryostat (Leica CM 1860, Leica Biosystems, Eisfeld, Germany) and stored at −80˚C until analysis. For hematoxylin/eosin (HE) staining, sections were fixed in 4% paraformaldehyde (PFA), stained with Mayer's hematoxylin solution for 5 min and then with eosin solution for 5 min. For the NADH-tetrazolium reductase (NADH-TR) staining, sections were incubated for 30 min at 37˚C in a reaction mixture containing 1.5 mM NADH and 1.5 mM nitrotetrazolium blue in 200 mM Tris (pH 7.4). For the immunohistochemical analysis, sections were fixed in 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. Mouse anti-dystrophin and Alexa Fluor 568-conjugated goat anti-mouse IgG antibodies were used for detecting dystrophin localization. Sections were mounted with Vectashield mounting medium for microscopic observation. All images were captured using the OLYMPUS IX73 system and cellSens imaging software (OLYMPUS, Tokyo, Japan). Cross-sectional areas (CSA) of muscle fibers were measured using dystrophin-stained 20X magnification images and WinROOF image analysis software (MITANI corporation, Tokyo, Japan).
RNA isolation and real-time PCR
Total RNA was prepared using the TRIzol Reagent (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's directions. RNA samples were treated with TURBO DNA-free (Thermo Fisher Scientific, Rockford, IL, USA) to remove genomic DNA contamination. Isolated RNA was quantified using spectrophotometry (λ = 260 nm). First-strand cDNA synthesis from total RNA was performed using the PrimeScript RT Reagent Kit. SYBR Premix Ex Taq II and TaKaRa Thermal Cycler Dice Real Time System TP850 (Takara Bio, Shiga, Japan) were used for PCR amplification and quantification of each studied gene. Primer sequences were designed based on partial sequencing of each gene obtained from the Japanese black bear and/or the American black bear [24] using Primer3 software. Expression levels of each studied gene were determined by the 2 -ΔΔCT method with referencing ribosomal protein L26 as an internal control.
Protein extraction and western blotting
For protein extraction, tissue samples were lysed in ice-cold RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaCl, 20 mM Tris-HCl [pH, 7.6], 1 mM PMSF, 5 mM benzamidine, 1 mM EDTA, 5 mM N-ethylmaleimide, 50 mM NaF, 25 mM B-glycerophosphate, 1 mM sodium orthovanadate, and 1X protease inhibitor cocktail [Nacalai Tesque, Kyoto, Japan]). Lysed samples were then centrifuged at 16,000 × g for 10 min at 4˚C, and supernatants were collected for analysis. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples were separated using a precast polyacrylamide gel system (e-PAGEL; ATTO, Tokyo, Japan) and transferred to PVDF membranes. Membranes were then blocked in Odyssey Blocking Buffer and incubated with dilutions of each primary antibody. IRDye 680LT goat anti-rabbit IgG was used as secondary antibody. Bound antibody complexes were scanned and quantified using the Odyssey CLx Imaging System operated with Image Studio Version 3.1 software (LI-COR Biosciences, Lincoln, NE, USA).
Statistical analysis
All results are reported as means ± standard error. Statistical differences between pre-hibernation (November) and post-hibernation (April) were determined by the Student's t-test. For all comparisons, the level of statistical significance was set at p < 0.05.
Results and discussion
The "Use It or Lose It" phenomenon is a well-accepted physiological principle for skeletal muscles. Skeletal muscle is highly plastic in response to functional demands, such that a reduced level of contractile activity (i.e., disuse) typically leads to skeletal muscle loss and metabolic dysfunction in many animal species, including humans. However, hibernating animals are likely better described under the principle "Limited Use, but Limited Loss" phenomenon, in that there is a potential resistance to muscle atrophy during continued disuse conditions. In the study presented herein, we analyzed alteration of signaling pathways regulating protein and energy metabolism in skeletal muscle of hibernating bears.
Alteration of muscle fiber cross-sectional areas: Pre-hibernation vs. posthibernation
HE staining revealed no seasonal differences in the gross morphology of muscle fibers in bears between pre-hibernation (late November) and post-hibernation (early April). We originally speculated that there is a very small reduction of fiber size in bear skeletal muscle following hibernation. In practice, however, muscle fiber size following hibernation decreased significantly (pre-hibernation, 4207.0 ± 194.6 μm 2 ; post-hibernation, 3111.6 ± 87.2 μm 2 ; 26% decrease). This result was consistent with previous reports demonstrating that force generation capacity [21] or protein concentration [18] in bear skeletal muscle was significantly decreased following hibernation. These observations clearly indicate that muscle atrophy is essentially induced following hibernation in bears. Although skeletal muscle atrophy had happened, this decrease in muscle fiber size following hibernation in bears seems to be limited. Daily loss of muscle size or muscle protein content was predicted at about 0.5%-1.0% under disuse conditions in human subjects [25,26]. If loss of muscle fiber size were to occur at 0.5%, an approximate 53% decrease would be expected after five months (150 days) of hibernation. Therefore, while we observed a significant decrease in muscle fiber size following hibernation, it is possible that over 27% of predicted muscle loss was protected during hibernation (Fig 1).
Regulation of muscle protein catabolism
Next, we evaluated the activation status of protein breakdown regulation. In skeletal muscle, there are two major pathways of protein breakdown which are activated during muscle atrophy. The ubiquitin-proteasome pathway is generally activated under disuse conditions in skeletal muscle. Autophagy-dependent protein breakdown is activated under poor nutrition states such as long-term fasting or malnutrition. Although we could not detect significant alteration in atrogin1 expression, transcript level of murf1, a muscle-specific E3 ubiquitin ligase, was significantly increased. In addition, expression levels of autophagy-related genes, including atg7, beclin1, and microtubule-associated protein 1 light chain 3 (lc3), were significantly upregulated following hibernation. These results suggest that protein degradation pathways through ubiquitin-proteasome-dependent and autophagy-dependent systems were both likely activated in skeletal muscle in response to the long-term disuse and malnutrition environment of hibernation (Fig 2). In contrast, a previous report investigating muscle protein metabolism directly by using radio-isotope tracers showed that protein synthesis and breakdown were both lowered during winter-denning period in American black bears [22]. The reason for this contradicted observation is still unknown. However, increased expression of autophagyrelated genes in skeletal muscle following hibernation revealed the possibility that autophagydependent proteolysis could contribute to amino acid production as an alternative energy resource in response to long-term fasting. One other possibility could be related to the seasonal effects of sample collection periods. In this study, the muscle samples were collected at the immediate post-hibernation state (i.e., 1 week following the onset of re-feeding), as opposed to collection during denning periods as in previous report [22]. Although the biochemical stage of hibernation persisted in bears during the immediate post-hibernation period (known as "walking hibernation") [15], protein metabolism in skeletal muscle could have been potentially affected by arousal and physical activity status.
Regulation of muscle protein anabolism
Together with the changes in muscle fiber size, the activation status of the mechanistic target of rapamycin (mTOR)-dependent signaling was examined in skeletal muscle. The mTOR complex 1 (mTORC1) has been suggested as a central regulator of protein synthesis through modulating translational efficiency from mRNA to amino acid, which could lead to increased activity of mTORC1 signaling resulting in enhanced protein synthesis. [1]. Interestingly, S6K1 phosphorylation that shows functional activity of mTORC1-dependent signaling was significantly increased following hibernation. We also observed that expression levels of myostatin mRNA was significantly reduced following hibernation. Myostatin is a negative regulator of skeletal muscle growth that reduces mTORC1 activity via Smad2/3 signaling [27]. Previous reports have indicated that skeletal muscle with decreased levels of myostatin show bulky musculature phenotypes in many animal species, including cattle, dogs, and humans [28,29]. In bear skeletal muscle, myostatin mRNA expression was significantly down-regulated following hibernation (Fig 3). Measurements of the absolute rate of protein synthesis were not collected in this study. However, activation of mTORC1-dependent signaling and suppression of myostatin expression suggests that the growth response of bear skeletal muscle may be enhanced following hibernation. Conversely, previous reports have pointed out that protein synthesis rates in skeletal muscle were essentially diminished during hibernation compared with that of summer periods in American black bears [22]. We postulate that increased molecular responses (i.e., mTOR activation and myostatin down regulation), which can contribute to skeletal muscle growth, likely counteract a corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Other potential explanations could be related to the effects of nutrient availability and the energy status of muscle cells, as the post-hibernation muscle samples were collected at one week following the onset of re-feeding. Although the acute effect was minimized (i.e., feeding was restricted overnight until sample collection), potential amino acid availability in muscle cells could possibly reflect the nutrient-induced activation of the mTORC1 pathway.
Phenotype shifting and mitochondrial biogenesis are enhanced in skeletal muscle following hibernation
We also examined the muscle fiber phenotypes in bear skeletal muscle. Long-term disuse generally results in a profound shifting from slow-oxidative to fast-glycolytic muscle fiber phenotypes [30]. However, histochemistry analyses demonstrated increased staining intensity of the mitochondrial enzyme NADH following hibernation. To confirm that the fiber type shifted toward the more oxidative/mitochondrial phenotype following hibernation, expression levels of mitochondrial genes in skeletal muscle were examined. Although the master regulators of mitochondrial biogenesis peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (pgc1a) and pgc1b were not altered, expression levels of mitochondria-related genes were significantly up-regulated following hibernation, including the mitochondrial uncoupling protein ucp3, regulators of electron transport and complex formation (cytochrome c: cycs and cytochrome c oxidase subunit 4: cox4), and a regulator of fatty acid beta-oxidation (carnitine palmitoyltransferase 1b: cpt1b). These data support the idea that mitochondrial biogenesis had occurred following hibernation in bear skeletal muscle (Fig 4). This observation was also consistent with previous reports in other hibernating mammals showing that skeletal muscle fiber type shifted from the fast/glycolytic to slow-oxidative phenotype during hibernation [31][32][33]. Although muscle contractile activity was essentially minimal during hibernation, previous reports have indicated that peaks in average electromyogram amplitude were observed when shivering bursts occurred [13,34]. We postulate that increased mitochondrial biogenesis in skeletal muscle could contribute to more efficient utilization of nutrients (e.g., fatty acids and/ or glucose) as energy resources for muscle contraction during winter survival, particularly for shivering-induced thermogenesis through mitochondrial oxidative phosphorylation. Up-regulation of UCP3 mRNA also explains that increased mitochondrial biogenesis in skeletal muscle may contribute toward more efficient thermogenesis during hibernation [35,36]. An alternative explanation for the observed increases in mitochondria-related genes in skeletal muscle may be attributed to the physiological alterations that accompany hibernation rather than an enhanced number/content of mitochondria. The fasted state could up-regulate UCP3 expression as a mediator of fatty acid metabolism in skeletal muscle [35]. Expression of CPT1, a regulating enzyme for fatty acid beta-oxidation, was also increased in skeletal muscle when the animals were exposed to cold ambient temperature [37]. Therefore, it will be important in future studies to evaluate whether enhanced mitochondrial biogenesis is induced and whether this contributes to improved mitochondrial respiratory function in skeletal muscle during hibernation.
Common disadvantages of studies using non-model organisms include limited information and research resources, including availability of sequenced genomes and mutants, and a lack of gene or protein expression profiles. Recent advances in sequencing technology offer unprecedented opportunities for research in non-model organisms. In hibernating animals, proteomic and transcriptomic analyses in skeletal muscle have been extensively explored with ground squirrels, mammals that exhibit typical physiological extremes in small hibernators [32,33,[38][39][40][41]. Comprehensive works by Dr. Andrews' lab, which investigated gene expression profiles in skeletal muscle of thirteen-lined ground squirrels, have identified seasonal alteration of differentially-expressed genes that may contribute to the regulation of muscle contractile function, protein catabolism, and energy homeostasis throughout the circannual cycle [32,[38][39][40]. The other study using proteomic analysis also indicated that physiological transitions for the hibernator skeletal muscle, such as changes in metabolic preference and fast-to-slow fiber type shifting have been induced [33]. Although availability of detailed omics datasets of larger hibernators is still very limited, some studies have attempted to identify the potential adaptive mechanisms in skeletal muscle during hibernation. A gene expression screening using cDNA microarray specifically developed for the American black bear identified 12 genes, mostly involved in protein biosynthesis, that were elevated in skeletal muscle during hibernation compared with summer active periods [24]. Elevated expression of genes involved in protein biosynthesis and ribosome biogenesis during hibernation was also observed in skeletal muscle of both small (arctic ground squirrels) and large (American black bear) hibernators [41]. These previous reports, along with our observations in this study, suggest that translational regulation of muscle protein possibly contributes to a common mechanism for "muscle atrophy resistance" in hibernating animals. Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears
Conclusion
According to our results, muscle protein anabolism was potentially altered by mTOR activation and down regulation of myostatin expression following hibernation. This anabolic response of bear skeletal muscle may be a counteraction to enhanced muscle catabolism (i.e., increased ubiquitin-proteasome-dependent and autophagy-dependent systems) for preventing excessive loss of muscle mass following hibernation. We also observed muscle phenotypes shifting toward oxidative fiber and mitochondrial biogenesis. These alterations of energy metabolisms in skeletal muscle may contribute to the more efficient utilization of fatty acids and/or glucose as energy resources for muscle contraction during winter survival (Fig 5). Overall, these physiological adaptations in bear skeletal muscle could contribute to muscle atrophy/weakness resistance against long-term disuse and malnutrition during hibernation.
Author Contributions
Conceptualization: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota.
Miyazaki M, Shimozuru M, Tsubota T (2019) Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation. PLoS ONE 14(4): e0215489. https://doi. org/10.1371/journal.pone.0215489
Fig 1 .
1Alteration of Muscle Fiber Cross-Sectional Areas: Pre-Hibernation vs. Post-Hibernation. Fiber crosssectional areas (CSAs) were significantly decreased following hibernation. The sartorius muscle was collected from bear legs both at pre-hibernation (late November) and post-hibernation (early April). Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). (A) Typical images of cross sections with Hematoxylin-Eosin staining and immunohistochemistry (dystrophin localization) are shown. The scale bar shows 100 μm. (B) The mean size of the fibers was obtained for each individual sample followed by the calculation of group data. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g001 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019
Fig 2 .
2Gene expression of protein breakdown pathways. Gene expression levels of the ubiquitin-proteasome system (atrogin1 and murf1) and autophagy-lysosome system (atg7, beclin1, and map1lc3) were quantified by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.https://doi.org/10.1371/journal.pone.0215489.g002 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019
Fig 3 .
3Enhanced mTORC1 signaling and suppression of myostatin expression following hibernation. (A) Representative images of Western blotting for phosphorylated (Thr389 and Thr421/Ser424) and total expression of S6K1. (B) Phosphorylation status of S6K1 at Thr421/Ser424 sites were quantified. (C) Gene expression levels of myostatin were determined by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g003
Fig 4 .
4Expression of mitochondria-related genes following hibernation. (A) Typical images of cross sections with NADH-tetrazolium reductase (NADH-TR) staining are shown. The scale bar shows 200 μm. (B) Expression levels of mitochondria-related gene transcripts (pgc1a, pgc1b, ucp3, cycs, cox4, and cpt1b) were quantified by real-time RT-PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g004
Fig 5 .
5Hypothetical schema of skeletal muscle adaptation in hibernating bear. Protein anabolism in skeletal muscle of hibernating bears is potentially activated by mTORC1 activation and down regulation of myostatin expression for counteracting to the corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Muscle phenotypes shifting toward slow-oxidative fiber and mitochondrial biogenesis are also induced and likely contribute to the more efficient utilization of energy resources for muscle contraction during winter survival.https://doi.org/10.1371/journal.pone.0215489.g005
Data curation: Mitsunori Miyazaki, Toshio Tsubota. Formal analysis: Mitsunori Miyazaki.
Funding acquisition :
acquisitionMitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Investigation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Methodology: Mitsunori Miyazaki. Project administration: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Supervision: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Validation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Visualization: Mitsunori Miyazaki. Writing -original draft: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Writing -review & editing: Mitsunori Miyazaki.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019
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| [
"Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to \"muscle atrophy resistance\" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/ mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization.Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi."
] | [
"Mitsunori Miyazaki Id \nDepartment of Physical Therapy\nSchool of Rehabilitation Sciences\nHealth Sciences University of Hokkaido\nHokkaidoJapan\n",
"Michito Shimozuru \nGraduate School of Veterinary Medicine\nLaboratory of Wildlife Biology and Medicine\nHokkaido University\nHokkaidoJapan\n\nMedical Center\nUniversity of Minnesota\nUNITED STATES\n",
"Toshio Tsubota \nGraduate School of Veterinary Medicine\nLaboratory of Wildlife Biology and Medicine\nHokkaido University\nHokkaidoJapan\n\nMedical Center\nUniversity of Minnesota\nUNITED STATES\n"
] | [
"Department of Physical Therapy\nSchool of Rehabilitation Sciences\nHealth Sciences University of Hokkaido\nHokkaidoJapan",
"Graduate School of Veterinary Medicine\nLaboratory of Wildlife Biology and Medicine\nHokkaido University\nHokkaidoJapan",
"Medical Center\nUniversity of Minnesota\nUNITED STATES",
"Graduate School of Veterinary Medicine\nLaboratory of Wildlife Biology and Medicine\nHokkaido University\nHokkaidoJapan",
"Medical Center\nUniversity of Minnesota\nUNITED STATES"
] | [
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"K Smith, ",
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"E M Macdonald, ",
"L A Leinwand, ",
"D K Merriman, ",
"J L Barger, ",
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"C Erlanson-Albertsson, ",
"I G Shabalina, ",
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"T V Kramarova, ",
"P Schrauwen, ",
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"J Nedergaard, ",
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"N C Stewart, ",
"O Toien, ",
"C Chang, ",
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"Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals",
"Protein breakdown in muscle wasting: role of autophagy-lysosome and ubiquitin-proteasome",
"Identification of ubiquitin ligases required for skeletal muscle atrophy",
"Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy",
"FoxO3 controls autophagy in skeletal muscle in vivo",
"The time course of the adaptations of human muscle proteome to bed rest and the underlying mechanisms",
"Disuse-induced muscle wasting",
"Alterations of protein turnover underlying disuse atrophy in human skeletal muscle",
"The Role of Exercise and TFAM in Preventing Skeletal Muscle Atrophy",
"CNS regulation of body temperature during hibernation",
"Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator",
"Intrinsic circannual regulation of brown adipose tissue form and function in tune with hibernation",
"Hibernation in black bears: independence of metabolic suppression from body temperature",
"Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy",
"Impaired skeletal muscle regeneration in the absence of fibrosis during hibernation in 13-lined ground squirrels",
"Protein use and muscle-fiber changes in free-ranging, hibernating black bears",
"Minimal seasonal alterations in the skeletal muscle of captive brown bears",
"Skeletal muscles of hibernating brown bears are unusually resistant to effects of denervation",
"Muscle strength in overwintering bears",
"Hibernating black bears (Ursus americanus) experience skeletal muscle protein balance during winter anorexia",
"Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)",
"Facts, noise and wishful thinking: muscle protein turnover in aging and human disuse atrophy",
"The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse",
"Myostatin reduces Akt/ TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size",
"Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member",
"Myostatin and the skeletal muscle atrophy and hypertrophy signaling pathways",
"Muscle type and fiber type specificity in muscle wasting",
"Proteomic analysis reveals the distinct energy and protein metabolism characteristics involved in myofiber type conversion and resistance of atrophy in the extensor digitorum longus muscle of hibernating Daurian ground squirrels",
"Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal",
"Proteogenomic Analysis of a Hibernating Mammal Indicates Contribution of Skeletal Muscle Physiology to the Hibernation Phenotype",
"Hibernating squirrel muscle activates the endurance exercise pathway despite prolonged immobilization",
"Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak",
"The role of uncoupling proteins in the regulation of metabolism",
"Cold tolerance of UCP1-ablated mice: a skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid-or ROS-induced uncoupling or enhanced GDP effects",
"Nature's fat-burning machine: brown adipose tissue in a hibernating mammal",
"Turning down the heat: Down-regulation of sarcolipin in a hibernating mammal",
"Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal",
"Comparative functional genomics of adaptation to muscular disuse in hibernating mammals"
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"Changes in expression of hepatic genes involved in energy metabolism during hibernation in captive, adult",
"Comp Biochem Physiol B Biochem Mol Biol",
"Biochemistry, and Hibernation in Black, Grizzly, and Polar Bears",
"EMBO Mol Med",
"PLoS One",
"Physiol Zool",
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"Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears",
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"Comp Biochem Physiol Part D Genomics Proteomics",
"Physiol Genomics",
"J Proteome Res",
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] | [
"\n\nMiyazaki M, Shimozuru M, Tsubota T (2019) Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation. PLoS ONE 14(4): e0215489. https://doi. org/10.1371/journal.pone.0215489",
"\nFig 1 .\n1Alteration of Muscle Fiber Cross-Sectional Areas: Pre-Hibernation vs. Post-Hibernation. Fiber crosssectional areas (CSAs) were significantly decreased following hibernation. The sartorius muscle was collected from bear legs both at pre-hibernation (late November) and post-hibernation (early April). Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). (A) Typical images of cross sections with Hematoxylin-Eosin staining and immunohistochemistry (dystrophin localization) are shown. The scale bar shows 100 μm. (B) The mean size of the fibers was obtained for each individual sample followed by the calculation of group data. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g001 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019",
"\nFig 2 .\n2Gene expression of protein breakdown pathways. Gene expression levels of the ubiquitin-proteasome system (atrogin1 and murf1) and autophagy-lysosome system (atg7, beclin1, and map1lc3) were quantified by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.https://doi.org/10.1371/journal.pone.0215489.g002 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019",
"\nFig 3 .\n3Enhanced mTORC1 signaling and suppression of myostatin expression following hibernation. (A) Representative images of Western blotting for phosphorylated (Thr389 and Thr421/Ser424) and total expression of S6K1. (B) Phosphorylation status of S6K1 at Thr421/Ser424 sites were quantified. (C) Gene expression levels of myostatin were determined by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g003",
"\nFig 4 .\n4Expression of mitochondria-related genes following hibernation. (A) Typical images of cross sections with NADH-tetrazolium reductase (NADH-TR) staining are shown. The scale bar shows 200 μm. (B) Expression levels of mitochondria-related gene transcripts (pgc1a, pgc1b, ucp3, cycs, cox4, and cpt1b) were quantified by real-time RT-PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g004",
"\nFig 5 .\n5Hypothetical schema of skeletal muscle adaptation in hibernating bear. Protein anabolism in skeletal muscle of hibernating bears is potentially activated by mTORC1 activation and down regulation of myostatin expression for counteracting to the corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Muscle phenotypes shifting toward slow-oxidative fiber and mitochondrial biogenesis are also induced and likely contribute to the more efficient utilization of energy resources for muscle contraction during winter survival.https://doi.org/10.1371/journal.pone.0215489.g005",
"\n\nData curation: Mitsunori Miyazaki, Toshio Tsubota. Formal analysis: Mitsunori Miyazaki.",
"\nFunding acquisition :\nacquisitionMitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Investigation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Methodology: Mitsunori Miyazaki. Project administration: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Supervision: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Validation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Visualization: Mitsunori Miyazaki. Writing -original draft: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Writing -review & editing: Mitsunori Miyazaki."
] | [
"Miyazaki M, Shimozuru M, Tsubota T (2019) Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation. PLoS ONE 14(4): e0215489. https://doi. org/10.1371/journal.pone.0215489",
"Alteration of Muscle Fiber Cross-Sectional Areas: Pre-Hibernation vs. Post-Hibernation. Fiber crosssectional areas (CSAs) were significantly decreased following hibernation. The sartorius muscle was collected from bear legs both at pre-hibernation (late November) and post-hibernation (early April). Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). (A) Typical images of cross sections with Hematoxylin-Eosin staining and immunohistochemistry (dystrophin localization) are shown. The scale bar shows 100 μm. (B) The mean size of the fibers was obtained for each individual sample followed by the calculation of group data. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g001 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019",
"Gene expression of protein breakdown pathways. Gene expression levels of the ubiquitin-proteasome system (atrogin1 and murf1) and autophagy-lysosome system (atg7, beclin1, and map1lc3) were quantified by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05.https://doi.org/10.1371/journal.pone.0215489.g002 Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears PLOS ONE | https://doi.org/10.1371/journal.pone.0215489 April 18, 2019",
"Enhanced mTORC1 signaling and suppression of myostatin expression following hibernation. (A) Representative images of Western blotting for phosphorylated (Thr389 and Thr421/Ser424) and total expression of S6K1. (B) Phosphorylation status of S6K1 at Thr421/Ser424 sites were quantified. (C) Gene expression levels of myostatin were determined by real-time PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g003",
"Expression of mitochondria-related genes following hibernation. (A) Typical images of cross sections with NADH-tetrazolium reductase (NADH-TR) staining are shown. The scale bar shows 200 μm. (B) Expression levels of mitochondria-related gene transcripts (pgc1a, pgc1b, ucp3, cycs, cox4, and cpt1b) were quantified by real-time RT-PCR. RPL26 was used as an internal control for the 2 -ΔΔCT method. N = 3 in each group. Data are expressed as mean ± standard error. Significant differences: #, p < 0.05. https://doi.org/10.1371/journal.pone.0215489.g004",
"Hypothetical schema of skeletal muscle adaptation in hibernating bear. Protein anabolism in skeletal muscle of hibernating bears is potentially activated by mTORC1 activation and down regulation of myostatin expression for counteracting to the corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Muscle phenotypes shifting toward slow-oxidative fiber and mitochondrial biogenesis are also induced and likely contribute to the more efficient utilization of energy resources for muscle contraction during winter survival.https://doi.org/10.1371/journal.pone.0215489.g005",
"Data curation: Mitsunori Miyazaki, Toshio Tsubota. Formal analysis: Mitsunori Miyazaki.",
"Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Investigation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Methodology: Mitsunori Miyazaki. Project administration: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Supervision: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Validation: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Visualization: Mitsunori Miyazaki. Writing -original draft: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. Writing -review & editing: Mitsunori Miyazaki."
] | [
"(Fig 1)",
"(Fig 2)",
"(Fig 3)",
"(Fig 4)",
"(Fig 5)"
] | [] | [
"Skeletal muscle mass is generally determined by the net dynamic balance of protein synthesis and degradation [1]. Under catabolic conditions, muscle protein degradation is enhanced through ubiquitin-proteasome and autophagy-lysosome systems [2]. The muscle-specific E3 ubiquitin ligases atrogin1 and murf1 (muscle RING finger 1) are highly expressed in response to unloading/inactivity and contribute to protein ubiquitination and proteasome-dependent degradation [3,4]. Autophagosome formation and lysosomal degradation of cytoplasmic components/organelles are also enhanced under catabolic conditions in skeletal muscle [5,6]. In contrast, decreases in muscle protein synthesis have been observed in several animal and human models of disuse atrophy [7,8]. Metabolic dysfunction is also induced in skeletal muscle following long-term disuse [9]. Consequently, prolonged periods of disuse lead to skeletal muscle atrophy/weakness and metabolic dysfunction, which then can cause impaired locomotive function and increased risk of morbidity/mortality.",
"Physiological characteristics during hibernation are highly variable between animal species. In general, small hibernators, such as ground squirrels, repeat torpor and arousal cycles every 1-2 weeks during hibernation periods. The thermal and energy metabolism are robustly down-regulated during torpor phase (body temperature: 3˚C -5˚C, heart rate: less than 10 bpm), whereas they return to the normothermic state (body temperature: 37˚C, heart rate: 300-400 bpm) during short arousal phases. Eating, drinking, and excretion (defecation/urination) behaviors are observed during hibernation periods when the animals are awaking [10][11][12]. In contrast, larger hibernators, such as bears, experience no arousal (i.e., remain in a relatively shallow torpor) and do not eat, drink, urinate, or defecate during the entire period of hibernation even though they maintain thermal and energy metabolism at relatively high levels (i.e., body temperature is slightly decreased but maintained to 30˚C -36˚C) [13][14][15].",
"Although hibernating animals experience long-term inactivity and fasting during winter survival, they can successfully maintain their muscle mass and locomotive function until arousal in spring. Previous reports have indicated that skeletal muscle mass and strength are well preserved during hibernation. Fiber size in skeletal muscle was almost perfectly maintained during hibernation in small mammals (e.g., ground squirrels) [16,17]. In the case of bears (e.g., American black bears or brown bears), protein content slightly decreased but loss of muscle mass and decline in contractile function were limited during hibernation [18][19][20][21][22]. These physiological features in hibernating animals allow us to hypothesize that the skeletal muscle of hibernators possesses a potential resistance to muscle atrophy during continued physical inactivity and malnutrition. In this study, alteration of signaling pathways that govern protein and energy metabolism was examined in skeletal muscle of the Japanese black bear.",
"Mouse anti-dystrophin, developed by Morris, G.E. (DSHB Hybridoma Product MANDRA1 (7A10)), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA 52242. Phospho-S6K1 (T389, Cat#: 9205) and phospho-S6K1 (T421/S424, Cat#: 9204) were obtained from Cell Signaling Technology (Danvers, MA, USA). S6K1 (Cat#: sc-230) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 680LT Goat anti-Rabbit IgG (Cat#: 926-68021) was from LI-COR Biosciences (Lincoln, NE, USA). Alexa Fluor 568-conjugated Goat anti-Mouse IgG (Cat#: A11031) was from Thermo Fisher Scientific (Rockford, IL, USA).",
"All experimental procedures and animal care performed in this study were conducted in accordance with institutional Guidelines for Animal Care and Use, as approved by the Animal Care and Use Committee of the Graduate School of Veterinary Medicine, Hokkaido University (Permit Number: 9124). A total of six non-pregnant female Japanese black bears (Ursus thibetanus japonicus), between 5 to 15 years of age, kept in Ani Mataginosato Bear Park (Akita Prefecture, Japan, N40˚E140.4˚) were used in this study. All animal care and handling procedures were followed as previously described [14,23]. Briefly, animals were fed with dried corn (360 kcal/100 g, approximately 1.5 kg/head) combined with fruits and vegetables as supplements once a day at 16:00 h during the active period (i.e., from late April to late November). During the two weeks before or after the fasting period (i.e., late November/early December to early/mid-April) as a transition phase to/from torpor status, the amount of feeding was reduced to one-third (0.5 kg corn meal/head) compared to the active period. All animals were kept isolated in the indoor dark rooms for denning and had no access to the food during the torpor period. Access to drinking water was allowed ad libitum throughout the year.",
"Skeletal muscle samples (sartorius muscle) were collected from bear legs during both prehibernation state (i.e., late November, just prior to the beginning of food deprivation) and post-hibernation state (i.e., early April, one week following the onset of re-feeding). Immediate post-hibernation period (approximately 3 weeks following emerged from denning) was known as \"walking hibernation\" [15]. During this period, the biochemical stage of hibernation was likely persisted in bears, even though physical activity and food/water intake were minimal. Due to the limitation of animal availability, different individual bears were used at each time point (N = 3 in each group). Animals were anesthetized with an intramuscular administration of 3.0 mg/kg zolazepam HCL and tiletamine HCL cocktail and 40 μg/kg medetomidine HCL using a blow dart shot. After immobilization, small pieces of sartorius muscle were excised directly and quickly frozen in liquid nitrogen. Feeding was restricted overnight (about 15-16 h) until the anesthesia and sample collection surgery were completed the next morning. Following the sample collection surgery, meloxicam (subcutaneously at 0.2 mg/kg for analgesia) and atipamezole HCl (intramuscularly at 200 μg/kg as an antagonist to medetomidine HCL) were administered to aid recovery.",
"Muscle samples for histochemical or immunohistochemical analyses were frozen in liquid nitrogen-cooled isopentane. Cross sections (8 μm) were cut in a cryostat (Leica CM 1860, Leica Biosystems, Eisfeld, Germany) and stored at −80˚C until analysis. For hematoxylin/eosin (HE) staining, sections were fixed in 4% paraformaldehyde (PFA), stained with Mayer's hematoxylin solution for 5 min and then with eosin solution for 5 min. For the NADH-tetrazolium reductase (NADH-TR) staining, sections were incubated for 30 min at 37˚C in a reaction mixture containing 1.5 mM NADH and 1.5 mM nitrotetrazolium blue in 200 mM Tris (pH 7.4). For the immunohistochemical analysis, sections were fixed in 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. Mouse anti-dystrophin and Alexa Fluor 568-conjugated goat anti-mouse IgG antibodies were used for detecting dystrophin localization. Sections were mounted with Vectashield mounting medium for microscopic observation. All images were captured using the OLYMPUS IX73 system and cellSens imaging software (OLYMPUS, Tokyo, Japan). Cross-sectional areas (CSA) of muscle fibers were measured using dystrophin-stained 20X magnification images and WinROOF image analysis software (MITANI corporation, Tokyo, Japan).",
"Total RNA was prepared using the TRIzol Reagent (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's directions. RNA samples were treated with TURBO DNA-free (Thermo Fisher Scientific, Rockford, IL, USA) to remove genomic DNA contamination. Isolated RNA was quantified using spectrophotometry (λ = 260 nm). First-strand cDNA synthesis from total RNA was performed using the PrimeScript RT Reagent Kit. SYBR Premix Ex Taq II and TaKaRa Thermal Cycler Dice Real Time System TP850 (Takara Bio, Shiga, Japan) were used for PCR amplification and quantification of each studied gene. Primer sequences were designed based on partial sequencing of each gene obtained from the Japanese black bear and/or the American black bear [24] using Primer3 software. Expression levels of each studied gene were determined by the 2 -ΔΔCT method with referencing ribosomal protein L26 as an internal control.",
"For protein extraction, tissue samples were lysed in ice-cold RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaCl, 20 mM Tris-HCl [pH, 7.6], 1 mM PMSF, 5 mM benzamidine, 1 mM EDTA, 5 mM N-ethylmaleimide, 50 mM NaF, 25 mM B-glycerophosphate, 1 mM sodium orthovanadate, and 1X protease inhibitor cocktail [Nacalai Tesque, Kyoto, Japan]). Lysed samples were then centrifuged at 16,000 × g for 10 min at 4˚C, and supernatants were collected for analysis. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples were separated using a precast polyacrylamide gel system (e-PAGEL; ATTO, Tokyo, Japan) and transferred to PVDF membranes. Membranes were then blocked in Odyssey Blocking Buffer and incubated with dilutions of each primary antibody. IRDye 680LT goat anti-rabbit IgG was used as secondary antibody. Bound antibody complexes were scanned and quantified using the Odyssey CLx Imaging System operated with Image Studio Version 3.1 software (LI-COR Biosciences, Lincoln, NE, USA).",
"All results are reported as means ± standard error. Statistical differences between pre-hibernation (November) and post-hibernation (April) were determined by the Student's t-test. For all comparisons, the level of statistical significance was set at p < 0.05.",
"The \"Use It or Lose It\" phenomenon is a well-accepted physiological principle for skeletal muscles. Skeletal muscle is highly plastic in response to functional demands, such that a reduced level of contractile activity (i.e., disuse) typically leads to skeletal muscle loss and metabolic dysfunction in many animal species, including humans. However, hibernating animals are likely better described under the principle \"Limited Use, but Limited Loss\" phenomenon, in that there is a potential resistance to muscle atrophy during continued disuse conditions. In the study presented herein, we analyzed alteration of signaling pathways regulating protein and energy metabolism in skeletal muscle of hibernating bears.",
"HE staining revealed no seasonal differences in the gross morphology of muscle fibers in bears between pre-hibernation (late November) and post-hibernation (early April). We originally speculated that there is a very small reduction of fiber size in bear skeletal muscle following hibernation. In practice, however, muscle fiber size following hibernation decreased significantly (pre-hibernation, 4207.0 ± 194.6 μm 2 ; post-hibernation, 3111.6 ± 87.2 μm 2 ; 26% decrease). This result was consistent with previous reports demonstrating that force generation capacity [21] or protein concentration [18] in bear skeletal muscle was significantly decreased following hibernation. These observations clearly indicate that muscle atrophy is essentially induced following hibernation in bears. Although skeletal muscle atrophy had happened, this decrease in muscle fiber size following hibernation in bears seems to be limited. Daily loss of muscle size or muscle protein content was predicted at about 0.5%-1.0% under disuse conditions in human subjects [25,26]. If loss of muscle fiber size were to occur at 0.5%, an approximate 53% decrease would be expected after five months (150 days) of hibernation. Therefore, while we observed a significant decrease in muscle fiber size following hibernation, it is possible that over 27% of predicted muscle loss was protected during hibernation (Fig 1).",
"Next, we evaluated the activation status of protein breakdown regulation. In skeletal muscle, there are two major pathways of protein breakdown which are activated during muscle atrophy. The ubiquitin-proteasome pathway is generally activated under disuse conditions in skeletal muscle. Autophagy-dependent protein breakdown is activated under poor nutrition states such as long-term fasting or malnutrition. Although we could not detect significant alteration in atrogin1 expression, transcript level of murf1, a muscle-specific E3 ubiquitin ligase, was significantly increased. In addition, expression levels of autophagy-related genes, including atg7, beclin1, and microtubule-associated protein 1 light chain 3 (lc3), were significantly upregulated following hibernation. These results suggest that protein degradation pathways through ubiquitin-proteasome-dependent and autophagy-dependent systems were both likely activated in skeletal muscle in response to the long-term disuse and malnutrition environment of hibernation (Fig 2). In contrast, a previous report investigating muscle protein metabolism directly by using radio-isotope tracers showed that protein synthesis and breakdown were both lowered during winter-denning period in American black bears [22]. The reason for this contradicted observation is still unknown. However, increased expression of autophagyrelated genes in skeletal muscle following hibernation revealed the possibility that autophagydependent proteolysis could contribute to amino acid production as an alternative energy resource in response to long-term fasting. One other possibility could be related to the seasonal effects of sample collection periods. In this study, the muscle samples were collected at the immediate post-hibernation state (i.e., 1 week following the onset of re-feeding), as opposed to collection during denning periods as in previous report [22]. Although the biochemical stage of hibernation persisted in bears during the immediate post-hibernation period (known as \"walking hibernation\") [15], protein metabolism in skeletal muscle could have been potentially affected by arousal and physical activity status. ",
"Together with the changes in muscle fiber size, the activation status of the mechanistic target of rapamycin (mTOR)-dependent signaling was examined in skeletal muscle. The mTOR complex 1 (mTORC1) has been suggested as a central regulator of protein synthesis through modulating translational efficiency from mRNA to amino acid, which could lead to increased activity of mTORC1 signaling resulting in enhanced protein synthesis. [1]. Interestingly, S6K1 phosphorylation that shows functional activity of mTORC1-dependent signaling was significantly increased following hibernation. We also observed that expression levels of myostatin mRNA was significantly reduced following hibernation. Myostatin is a negative regulator of skeletal muscle growth that reduces mTORC1 activity via Smad2/3 signaling [27]. Previous reports have indicated that skeletal muscle with decreased levels of myostatin show bulky musculature phenotypes in many animal species, including cattle, dogs, and humans [28,29]. In bear skeletal muscle, myostatin mRNA expression was significantly down-regulated following hibernation (Fig 3). Measurements of the absolute rate of protein synthesis were not collected in this study. However, activation of mTORC1-dependent signaling and suppression of myostatin expression suggests that the growth response of bear skeletal muscle may be enhanced following hibernation. Conversely, previous reports have pointed out that protein synthesis rates in skeletal muscle were essentially diminished during hibernation compared with that of summer periods in American black bears [22]. We postulate that increased molecular responses (i.e., mTOR activation and myostatin down regulation), which can contribute to skeletal muscle growth, likely counteract a corresponding enhancement of protein degradation, which can then lead to a net balance of protein metabolism and prevention of excessive muscle loss. Other potential explanations could be related to the effects of nutrient availability and the energy status of muscle cells, as the post-hibernation muscle samples were collected at one week following the onset of re-feeding. Although the acute effect was minimized (i.e., feeding was restricted overnight until sample collection), potential amino acid availability in muscle cells could possibly reflect the nutrient-induced activation of the mTORC1 pathway.",
"We also examined the muscle fiber phenotypes in bear skeletal muscle. Long-term disuse generally results in a profound shifting from slow-oxidative to fast-glycolytic muscle fiber phenotypes [30]. However, histochemistry analyses demonstrated increased staining intensity of the mitochondrial enzyme NADH following hibernation. To confirm that the fiber type shifted toward the more oxidative/mitochondrial phenotype following hibernation, expression levels of mitochondrial genes in skeletal muscle were examined. Although the master regulators of mitochondrial biogenesis peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (pgc1a) and pgc1b were not altered, expression levels of mitochondria-related genes were significantly up-regulated following hibernation, including the mitochondrial uncoupling protein ucp3, regulators of electron transport and complex formation (cytochrome c: cycs and cytochrome c oxidase subunit 4: cox4), and a regulator of fatty acid beta-oxidation (carnitine palmitoyltransferase 1b: cpt1b). These data support the idea that mitochondrial biogenesis had occurred following hibernation in bear skeletal muscle (Fig 4). This observation was also consistent with previous reports in other hibernating mammals showing that skeletal muscle fiber type shifted from the fast/glycolytic to slow-oxidative phenotype during hibernation [31][32][33]. Although muscle contractile activity was essentially minimal during hibernation, previous reports have indicated that peaks in average electromyogram amplitude were observed when shivering bursts occurred [13,34]. We postulate that increased mitochondrial biogenesis in skeletal muscle could contribute to more efficient utilization of nutrients (e.g., fatty acids and/ or glucose) as energy resources for muscle contraction during winter survival, particularly for shivering-induced thermogenesis through mitochondrial oxidative phosphorylation. Up-regulation of UCP3 mRNA also explains that increased mitochondrial biogenesis in skeletal muscle may contribute toward more efficient thermogenesis during hibernation [35,36]. An alternative explanation for the observed increases in mitochondria-related genes in skeletal muscle may be attributed to the physiological alterations that accompany hibernation rather than an enhanced number/content of mitochondria. The fasted state could up-regulate UCP3 expression as a mediator of fatty acid metabolism in skeletal muscle [35]. Expression of CPT1, a regulating enzyme for fatty acid beta-oxidation, was also increased in skeletal muscle when the animals were exposed to cold ambient temperature [37]. Therefore, it will be important in future studies to evaluate whether enhanced mitochondrial biogenesis is induced and whether this contributes to improved mitochondrial respiratory function in skeletal muscle during hibernation.",
"Common disadvantages of studies using non-model organisms include limited information and research resources, including availability of sequenced genomes and mutants, and a lack of gene or protein expression profiles. Recent advances in sequencing technology offer unprecedented opportunities for research in non-model organisms. In hibernating animals, proteomic and transcriptomic analyses in skeletal muscle have been extensively explored with ground squirrels, mammals that exhibit typical physiological extremes in small hibernators [32,33,[38][39][40][41]. Comprehensive works by Dr. Andrews' lab, which investigated gene expression profiles in skeletal muscle of thirteen-lined ground squirrels, have identified seasonal alteration of differentially-expressed genes that may contribute to the regulation of muscle contractile function, protein catabolism, and energy homeostasis throughout the circannual cycle [32,[38][39][40]. The other study using proteomic analysis also indicated that physiological transitions for the hibernator skeletal muscle, such as changes in metabolic preference and fast-to-slow fiber type shifting have been induced [33]. Although availability of detailed omics datasets of larger hibernators is still very limited, some studies have attempted to identify the potential adaptive mechanisms in skeletal muscle during hibernation. A gene expression screening using cDNA microarray specifically developed for the American black bear identified 12 genes, mostly involved in protein biosynthesis, that were elevated in skeletal muscle during hibernation compared with summer active periods [24]. Elevated expression of genes involved in protein biosynthesis and ribosome biogenesis during hibernation was also observed in skeletal muscle of both small (arctic ground squirrels) and large (American black bear) hibernators [41]. These previous reports, along with our observations in this study, suggest that translational regulation of muscle protein possibly contributes to a common mechanism for \"muscle atrophy resistance\" in hibernating animals. Atrophy resistance and phenotype shifting in skeletal muscle of hibernating bears",
"According to our results, muscle protein anabolism was potentially altered by mTOR activation and down regulation of myostatin expression following hibernation. This anabolic response of bear skeletal muscle may be a counteraction to enhanced muscle catabolism (i.e., increased ubiquitin-proteasome-dependent and autophagy-dependent systems) for preventing excessive loss of muscle mass following hibernation. We also observed muscle phenotypes shifting toward oxidative fiber and mitochondrial biogenesis. These alterations of energy metabolisms in skeletal muscle may contribute to the more efficient utilization of fatty acids and/or glucose as energy resources for muscle contraction during winter survival (Fig 5). Overall, these physiological adaptations in bear skeletal muscle could contribute to muscle atrophy/weakness resistance against long-term disuse and malnutrition during hibernation.",
"Conceptualization: Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota. "
] | [] | [
"Introduction",
"Materials and methods",
"Antibodies",
"Animal care and use",
"Muscle sample collection",
"Histochemistry/immunohistochemistry",
"RNA isolation and real-time PCR",
"Protein extraction and western blotting",
"Statistical analysis",
"Results and discussion",
"Alteration of muscle fiber cross-sectional areas: Pre-hibernation vs. posthibernation",
"Regulation of muscle protein catabolism",
"Regulation of muscle protein anabolism",
"Phenotype shifting and mitochondrial biogenesis are enhanced in skeletal muscle following hibernation",
"Conclusion",
"Author Contributions",
"Fig 1 .",
"Fig 2 .",
"Fig 3 .",
"Fig 4 .",
"Fig 5 .",
"Funding acquisition :"
] | [] | [] | [
"Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation",
"Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation"
] | [] |
155,219,027 | 2022-03-05T13:50:27Z | CCBY | https://downloads.hindawi.com/journals/cmmm/2019/7614850.pdf | GOLD | 12399c42c01f0a73e5f22c91b4b7065217bfe8b0 | null | null | null | journals/cmmm/XuanFYPZZW19 | 10.1155/2019/7614850 | 2943030817 | 31191710 | 6525924 |
A Novel Method for Predicting Disease-Associated LncRNA-MiRNA Pairs Based on the Higher-Order Orthogonal Iteration
Published 2 May 2019
Zhanwei Xuan
College of Computer Engineering & Applied Mathematics
Changsha University
410001Changsha, HunanChina
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Xiang Feng [email protected]
College of Computer Engineering & Applied Mathematics
Changsha University
410001Changsha, HunanChina
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Jingwen Yu
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Pengyao Ping
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Haochen Zhao
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Xianyou Zhu
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Department of Computer Science
Hengyang Normal University
421008HengyangChina
Lei Wang [email protected]
College of Computer Engineering & Applied Mathematics
Changsha University
410001Changsha, HunanChina
Key Laboratory of Intelligent Computing & Information Processing
Xiangtan University
411105XiangtanChina
Emil Alexov
A Novel Method for Predicting Disease-Associated LncRNA-MiRNA Pairs Based on the Higher-Order Orthogonal Iteration
Published 2 May 201910.1155/2019/7614850Received 31 October 2018; Revised 25 January 2019; Accepted 10 February 2019;Research Article Correspondence should be addressed to Xiang Feng; Academic Editor:
A lot of research studies have shown that many complex human diseases are associated not only with microRNAs (miRNAs) but also with long noncoding RNAs (lncRNAs). However, most of the current existing studies focus on the prediction of diseaserelated miRNAs or lncRNAs, and to our knowledge, until now, there are few literature studies reported to pay attention to the study of impact of miRNA-lncRNA pairs on diseases, although more and more studies have shown that both lncRNAs and miRNAs play important roles in cell proliferation and differentiation during the recent years. e identification of disease-related genes provides great insight into the underlying pathogenesis of diseases at a system level. In this study, a novel model called PADLMHOOI was proposed to predict potential associations between diseases and lncRNA-miRNA pairs based on the higherorder orthogonal iteration, and in order to evaluate its prediction performance, the global and local LOOCV were implemented, respectively, and simulation results demonstrated that PADLMHOOI could achieve reliable AUCs of 0.9545 and 0.8874 in global and local LOOCV separately. Moreover, case studies further demonstrated the effectiveness of PADLMHOOI to infer unknown disease-related lncRNA-miRNA pairs.
Introduction
Noncoding RNA, according to its size, can be divided into small and long noncoding RNAs approximately. Generally, small RNAs include tRNAs, miRNAs, piRNAs, and snoR-NAs [1][2][3][4], and miRNAs are widely present in the cytoplasm of eukaryotic cells and are approximately 18-22 nucleotides in length, which can bind to 3′-untranslated region of mRNA (3′-UTR) to inhibit the translation process of mRNA or to degrade mRNA, thereby affecting the expression of related genes [5][6][7]. miRNAs play important roles in a series of life activities such as cell differentiation of living body [8], growth and development [9], and apoptosis [10]. Compared to small-molecule ncRNA, lncRNA has a longer nucleotide chain with more than 200 nucleotides and has a specific and complex secondary space structure inside the molecule and can provide multiple sites for protein binding [11]. In addition, both lncRNAs and miRNAs are key members of noncoding RNAs and play important roles in coding and regulation of many complex human diseases [12][13][14][15][16].
Up to now, there have been many studies on relationships between diseases and miRNAs. For example, some important methods proposed by Xing Chen et al. [17][18][19][20] and Zou et al. [21][22][23][24]. In terms of prediction of potential associations between lncRNAs and diseases, Yu et al. [25] and Xing et al. [26] proposed two kinds of computational models called NBCLAD and LRLSLDA, respectively. Moreover, studies have also shown that there exist relationships between lncRNAs and miRNAs. For example, Gernapudi et al. demonstrated that miRNA 140 can induce the expression of lncRNA NEAT1 [27]. Dey et al. showed that the silencing of lncRNA H19 and knockout of H19 gene in myoblasts significantly decreased skeletal muscle differentiation [28]. Yilong et al. discovered that, after low XIST expression in gliomas, XIST could regulate miR-152 glioma stem cells to inhibit cell proliferation, migration, and invasion [29]. Xinyu et al. demonstrated that lncRNA MALAT1 could achieve posttranscriptional regulation of esophageal squamous cell carcinoma cells through miR-101 and miR-217 [30]. Er-bao et al. proposed that lncRNA ANRIL interacted with miR-99a/miR449a to regulate cell proliferation during gastric cancer formation [31]. You et al. found that the expression of miR-449a and the expression of lncRNA NEAT1 in lung cancer cell L9981 inhibited each other. When miR-449a was overexpressed, NEAT1 expression was decreased, cell proliferation was inhibited, and apoptosis was increased, and vice versa [32]. Emmrich et al. found that the expression of lncRNA MONC and MIR100HG was closely related to the miRNA groups of miR-99a∼125b-2 and miR-100∼125b-1. After silencing of lncRNA MONC and MIR100HG, acute megakaryocytes in the early stage of the disease, the tumor cells of leukemia patients, were severely inhibited [33]. Amy et al. found that lncRNA Ang362 was the host transcriptor of miR-211 and miR-222, and their interactions regulated Ang II and induced proliferation of vascular smooth muscle cells [34]. Miaojun et al. found that the interactions between lncRNA H19 and miRNA-675 play an important role in the metastasis of prostate cancer [35]. Obviously, the exploration of these relationships was conducive to the construction of gene regulatory networks and the identification of the mechanisms of complex human diseases [36][37][38].
From the above description, it is easy to see that more and more studies have shown that lncRNA-miRNA interactions are involved in the development of complex diseases. However, to the best of our knowledge, so far, in addition to the model of PADLMP proposed by Zhou et al. [39], few models have been proposed for large-scale prediction of potential associations between diseases and lncRNA-miRNA interactions. Hence, inspired by state-ofthe-art methods [40][41][42][43][44], which show that the miRNA-miRNA pairs can work cooperatively to regulate a single gene or gene clusters being involved in similar processes [45], and simultaneously, based on the reasonable assumption that functionally similar lncRNA-miRNA pairs tend to be associated with similar diseases, in this paper, a new prediction model called PADLMHOOI was proposed to infer potential associations between diseases and the lncRNA-miRNA pairs. And, as illustrated in Figure 1, our newly proposed prediction model PADLMHOOI consists of the following four major steps:
Step 1 (Data Integration and Network Construction). In this step, first of all, we downloaded known disease-lncRNA associations from three different disease-lncRNA databases such as disease-lncRNA [46], MNDR [47,48], and lnc2cancer [49], respectively, and then, based on these datasets, we constructed a bipartite network of disease-lncRNA. Next, we downloaded known disease-miRNA associations from three different databases such as miR2Disease [50], HMDD [51], and miRCancer [52] separately, and then, based on these datasets, we constructed a bipartite network of disease-miRNA. Moreover, we downloaded the 2015 and 2017 versions of known lncRNA-miRNA associations from the starBasev2.0 database [53] (http:// starbase.sysu.edu.cn/) on Feb 2, 2017, and based on these datasets, we constructed a bipartite network of lncRNA-miRNA. Finally, based on these three kinds of bipartite networks, we constructed an integrated tripartite network of disease-lncRNA-miRNA, which could be denoted as a tensor T.
Step 2 (Similarity Calculation). In this step, we would integrate the disease semantic similarity and Gaussian Interaction Profile Kernel similarity firstly to measure the similarity of diseases. Next, we would integrate the lncRNA functional similarity and miRNA functional similarity in three different ways to measure the functional similarity of lncRNA-miRNA pairs.
Step 3 (Weighted K-Nearest Neighbor Profile). Considering that there may be diseases that are unrelated to all lncRNA-miRNA pairs, which may lead to unsatisfactory prediction results while implementing PADLMHOOI to infer potential associations between diseases and lncRNA-miRNA pairs. Hence, in this step, we would introduce the weighted K-nearest neighbor profile (WKNNP) to add more interaction information between diseases, lncRNAs, and miRNAs to improve the prediction performance of PADLMHOOI.
Step 4 (Tensor Decomposition). In this step, we would perform tensor decomposition on the newly constructed disease-lncRNA-miRNA tensor T. Since the results of tensor decomposition include a core tensor and three matrices, we can define the final predicted association tensor as the modal product between the core tensor and these three matrices. ereafter, we would sort scores of the lncRNA-miRNA pairs associated with each disease in the descending order in the final predicted association tensor, and it is obvious that the higher the ranking of the score, the bigger the possibility that there may exist potential association between the disease and the lncRNA-miRNA pair would be.
Materials and Methods
Construction of the Bipartite Network of Disease-lncRNA.
In order to construct the bipartite network of disease-lncRNA, firstly, known associations between diseases and lncRNAs were downloaded from three different databases such as the LncRNADisease, MNDR, and Lnc2Cancer, respectively, and then, after feature processing (including feature cleaning and data imbalance processing etc.), 2048 different disease-lncRNA associations were finally obtained (Supplementary Table 1). ereafter, based on these newly obtained 2048 known disease-lncRNA associations, we can construct a disease-lncRNA bipartite network G 1 � (V 1 , E 1 ) according to the following steps:
Step 1. Let V l 1 � l i |i ∈ [1, n l 1 ] be the set of all different lncRNAs in these 2048 known disease-lncRNA associations and V d 1 � d i |i ∈ [1, n d 1 ] be the set of all different diseases in these 2048 known disease-lncRNA associations, then we define V 1 � V l 1 ∪ V d 1 as the vertex set in G 1 .
Step 2. ∀l i ∈ V l 1 , d j ∈ V d 1 , if (l i , d j ) belongs to these 2048 downloaded known disease-lncRNA associations, then we define that there is an edge between l i and d j in G 1 ; thereafter, we can obtain the edge set E 1 in G 1 . New tensor T
Data integration and association network construction
lncRNA- disease data lncRNA-disease bipartite network G 1 miRNA-disease bipartite network G 2 Similarity calculation l 4 l 4 l 3 l 3 l 2 l 2 l 1 l 1 l 4 l 3 l 2 l 1 d 4 d 3 d 2 d 1 d 4 d 4 d 3 d 3 d 2 d 2 d 1 d 1 d 4 d 3 d 2
Tensor decomposition
Step 1. Input T , R 1 , R 2 and R 3 and initialize Z 1 , Z 2 , Z 3 , G, and convergence threshold ε
S , Z 1 , Z 2 , Z 3 Z 1 : (N D × R 1 ) Z 2 : (N L × R 2 ) Z 3 : (N M × R 3 )
Step 2. Repeat for n = 1 to 3:
Update Z n according to (31) end for
Update G according to (35) Until Computational and Mathematical Methods in Medicine 3
||T -[[G; Z 1 , Z 2 , Z 3 ]]|| 2 F < ε
Construction of the Bipartite Network of Disease-miRNA.
In order to construct the bipartite network of disease-miRNA, at first, known disease-miRNA associations were downloaded from three different databases such as the miR2Disease, HMDD, and miRCancer separately, and then, after these newly acquired miRNAs and diseases being mapped to the database miRBase v21 [54] and disease ontology (DO) [55], respectively, 4041 different disease-miRNA associations were finally obtained (Supplementary Table 2). Hence, based on these newly obtained 4041 known disease-miRNA associations, we can construct a disease-miRNA bipartite network G 2 � (V 2 , E 2 ) according to the following steps:
Step 1. Let V m 1 � m i |i ∈ [1, n m 1 ] be the set of all different miRNAs in these 4041 known disease-miRNA associations and V d 2 � d i |i ∈ [1, n d 2 ] be the set of all different diseases in these 4041 known disease-miRNA associations, then we define
V 2 � V m 1 ∪ V d 2 as the vertex set in G 2 . Step 2. ∀m i ∈ V m 1 , d j ∈ V d 2 , if (m i , d j )
belongs to these 4041 known disease-miRNA associations, then we define that there is an edge between m i and d j in G 2 ; thereafter, we can obtain the edge set E 2 in G 2 .
Construction of the Bipartite Network of lncRNA-miRNA.
In order to construct the bipartite network of lncRNA-miRNA, at first, two different versions (2015 and 2017) of lncRNA-miRNA dataset were downloaded from the starBa-sev2.0 database separately, and then, after feature processing (including feature cleaning and data imbalance processing), 20324 different lncRNA-miRNA interactions were finally obtained (Supplementary Table 3). ereafter, based on these newly obtained 20324 known lncRNA-miRNA associations, we can construct a lncRNA-miRNA bipartite network G 3 � (V 3 , E 3 ) according to the following steps:
Step 1. Let V l 2 � l i |i ∈ [1, n l 2 ] denote the set of all different lncRNAs in these 20324 known lncRNA-miRNA associations and V m 2 � m i |i ∈ [1, n m 2 ] denote the set of all different miRNAs in these 20324 known lncRNA-miRNA associations, then we define
V 3 � V m 2 ∪ V l 2 as the vertex set in G 3 . Step 2. ∀l i ∈ V l 2 , m j ∈ V m 2 , if (l i , m j )
belongs to these 20324 known lncRNA-miRNA associations, then we define that there is an edge between l i and m j in G 3 ; thereafter, we can obtain the edge set E 3 in G 3 .
Construction of the Tripartite
Network of Disease-lncRNA-miRNA. Based on the above newly obtained networks such as G 1 , G 2 , and G 3 , we can construct a tripartite network G 4 � (V 4 , E 4 ) according to the following steps:
Step
1. Let V d � V d 1 ∪ V d 2 , V m � V m 1 ∪ V m 2 , V l � V l 1 ∪ V l 2 , V 4 � {}, E 4 � {}, and V d′ � {}.
Step 2. While V d is not null, Repeat:
∀d i ∈ V d ,
If ∃l j ∈ V l and m k ∈ V m satisfyies the following three kinds of conditions simultaneously:
(a) (d i , l j ) ∈ E 1 (b) (d i , m k ) ∈ E 2 (c) (l j , m k ) ∈ E 3 en (d i , l j ), (d i , m k )
, and (l j , m k ) will be added into E 4 firstly, and then, d i will be added into V d ′ and removed from V d . Finally, l j and m k will be added into V 4 if l j and m k are not inV 4 . Else, d i will be removed from V d .
Step
3. Let V 4 � V 4 + V d′ .
According to above steps, a tripartite disease-lncRNA-miRNA association network can be obtained finally. And, it is obvious that, in the tripartite network, there are three kinds of different nodes such as disease nodes, lncRNA nodes, and miRNA nodes; moreover, the number of disease nodes, lncRNA nodes, and miRNA nodes is 68, 44, and 211, respectively, and the number of associations between diseases and lncRNA-miRNA pairs is 3,047.
Construction of the Disease-lncRNA-miRNA Tensor.
Based on the newly constructed tripartite network, for any given disease node d i , lncRNA node l j , and miRNA node m k in G 4 , we can define a tensor T as follows:
T(i, j, k) � 1, d i , l j , d i , m k , l j , m k ∈ E 4 , , 0, otherwise. ⎧ ⎨ ⎩(1)(d) � (T(d), E(d)), where T(d)
denotes the set of nodes containing the node d itself and its ancestors and E(d) denotes the set of edges of the respective direct links from parent to child nodes [56]. ereafter, based on the newly constructed directed acyclic graph DAG(d), the semantic contribution of an ancestor node d s to the disease d can be calculated as follows:
D d d s � 1, if d s � d, max Δ * D d d ′ | d ′ ∈ children of d s , otherwise, DV(d) � d i ∈T(d) D d d i ,(2)
where Δ is the semantic contribution decay factor with value between 0 and 1. And, in addition, according to the experimental results of some previous state-of-the-art methods [57,58], the most appropriate value for Δ will be 0.5. Hence, based on the assumption that two diseases with more common ancestor nodes in their DAGs shall have higher semantic similarity, the semantic similarity between two diseases d i and d j can be defined as follows:
4
Computational and Mathematical Methods in Medicine
DisSemSim(i, j) � t∈T d i ( )∩T d j D d i (t) + D d j (t) DV d i + DV d j .
(3)
Calculation of the Gaussian Interaction Profile Kernel
Similarity for Diseases (GIPSim). Based on the hypothesis that functionally similar genes are often associated with similar diseases, in this section, we will adopt the Gaussian Interaction Profile Kernel to calculate the similarity of diseases according to the following steps: Firstly, based on the networks G 1 and G 2 constructed above, for any given lncRNA l i and disease d j , we define that
Y 1 (i, j) � 1, if l i , d j ∈ G 1 , 0, otherwise. ⎧ ⎨ ⎩ (4)
Next, for any given miRNA m i and disease d j , we define that
Y 2 (i, j) � 1, if m i , d j ∈ G 2 , 0, otherwise. ⎧ ⎨ ⎩ (5)
Hence, let IP l (d i ) denote the ith column of the matrix Y 1 , then we can calculate the Gaussian Kernel Similarity between diseases d i and d j based on their interaction profiles as follows:
GIP LDSIM (i, j) � exp −c d1 IP l d i − IP l d j � � � � � � � � � � 2 ,(6)c d1 � 1 1/n d1 n d1 i�1 IP d l (i) 2 � � � � � � � � � � ,(7)
where the parameter n d1 denotes the number of different diseases in G 1 .
In a similar way, let IP m (d i ) denote the ith column of matrix Y 2 , then we can calculate the Gaussian Kernel Similarity between diseases d i and d j based on their interaction profiles as follows:
GIP MDSIM (i, j) � exp −c d2 IP m d i − IP m d j � � � � � � � � � � 2 , c d2 � 1 1/n d2 n d2 i�1 IP m d i � � � � � � � � 2 ,(8)
Here, the parameter n d2 denotes the number of different diseases in G 2 . ereafter, based on these above formulas, we can calculate the Gaussian Interaction Profile Kernel Similarity between diseases d i and d j as follows:
GIPSim(i, j) � GIP LDSIM (i, j) + GIP MDSIM (i, j) 2 .(9)
Calculation of the Similarity of lncRNA Pairs (lncSim)
Calculation of the lncRNA Functional Similarity
(lncfunSim). For any two given lncRNAs such as l i and l j , let DT 1 � dt 11 , dt 12 , . . . , dt 1m be all the diseases related to l i in G 1 and DT 2 � dt 21 , dt 22 , . . . , dt 2n be all the diseases related to l j in G 1 , then we can define the functional similarity between l i and l j as follows:
lncfunSim(i, j) � 1≤k≤m SemSims dt 1k , DT 2 + 1≤k≤n SemSims dt 2k , DT 1 m + n ,(10)
where SemSims dt 1k , DT 2 � max 1≤l≤n DisSemSim dt 1k , dt 2l ,
SemSims dt 2k , DT 1 � max 1≤l≤m DisSemSim dt 2k , dt 1l .(11)
Calculation of the Gaussian Interaction Profile Kernel
Similarity for lncRNAs (GIP lncSim ). For any two given lncRNAs such as l i and l j , similar to the definition of formula (6), let IP(l i ) and IP(l j ) denote the ith and the jth row of the matrix Y 1 , respectively, then we can calculate the Gaussian Kernel Similarity between diseases l i and l j based on their interaction profiles as follows:
GIP lncSim (i, j) � exp −c l IP l i − IP l j � � � � � � � � � � 2 , c l � 1 1/n l n l i�1 IP l i � � � � � � � � 2 ,(12)
where n l1 denotes the number of different lncRNAs in G 1 .
Hence, based on these formulas given above, we can finally define the similarity measurement between lncRNAs l i and l j as follows:
lncSim(i, j) � lncfunSim(i, j) + GIP lncSim (i, j) 2 .(13)
Calculation of the Gaussian Interaction Profile Kernel
Similarity for miRNAs (GIP miRSim ). For any two given miRNAs, such as m i and m j , in a similar way, let IP(m i ) and IP(m j ) represent the ith and jth row in matrix Y 2 , respectively, then we can calculate the Gaussian Kernel Similarity between diseases m i and m j based on their interaction profiles as follows:
GIP miRSim (i, j) � exp −c m IP m i − IP m j � � � � � � � � � � 2 , c m � 1 1/n m n m i�1 IP m i � � � � � � � � 2 ,(15)
where n m2 denotes the number of miRNAs in G 2.
Hence, based on these formulas presented above, we can finally define the similarity measurement between miRNAs m i and m j as follows: en, it is obvious that the values in these three kinds of interaction profiles of any novel diseases, lncRNAs, or miRNAs are all zeros, which may lead to unsatisfactory prediction performance during inferring potential associations between diseases and lncRNA-miRNA pairs. Hence, in this section, we will perform a procedure for the construction of new interaction profiles to address the problem mentioned above. And, in this procedure, for each disease d i , its association with other K nearest known diseases (including at least one experimentally verified association) and corresponding K interaction profiles will be utilized to obtain the following interaction profile:
miRSim(i, j) � miRfunSim(i, j) + GIP miRSim (i, j) 2 .(16T D d i � 1 Q d K t�1 w t T d t , :, : ,(17)
where, d 1 , d 2 , . . . , d K are the diseases sorted in descending order based on their similarity to d i , w t is the weight coefficient, and w t � α t−1 * disSim(d t , d i ), which means that a higher weight will be assigned if d t is more similar to d i . e parameter α is a decay term with values between 0 and 1. e parameter Q d is a normalization term, and there is
Q d � K t�1 disSim(d t , d i ).
In the same manner, the new interaction profile for each l k can be determined as follows:
T L l k � 1 Q l K t�1 w t T :, l t , : ,(18)
where l 1 , l 2 , . . . , l K are the lncRNAs sorted in the descending order based on their similarity to l k , w t is the weight coefficient, and w t � α t−1 * lncSim(l t , l k ), which means that a higher weight will be assigned if l t is more similar to l k . e parameter Q l is a normalization term, and there is Q l � K t�1 lncSim(l t , l k ). Similarly, the new interaction profile for each m p can be determined as follows:
T M m p � 1 Q m K t�1 w t T :, :, m p ,(19)
where m 1 , m 2 , . . . , m K are the miRNAs sorted in the descending order based on their similarity to m p , w t is the weight coefficient, and w t � α t−1 * miRSim(m t , m p ), which means that a higher weight is assigned if m t is more similar to m p . e parameter Q m is a normalization term, and there is Q m � K t�1 miRSim(m t , m p ). ereafter, after combining the above three kinds of tensors T D , T L , and T M obtained from different data spaces and replacing T(i, j, k) � 0 with an associated likelihood score, we can update the original adjacency matrix T as follows:
T � max T, T DLM ,(20)
where T DLM � (a 1 T D + a 2 T L + a 3 T M / a i ), (i � 1, 2, 3).
PADLMHOOI.
Inspired by the successful application of tensor decomposition in the field of link prediction and the application of nonnegative matrix decomposition methods in inferring disease-miRNA associations, in this section, we proposed a novel model called PADLMHOOI to predict new associations between diseases and miRNA-lncRNA pairs. From above descriptions, it is easy to know that a tensor is a multidimensional array. Currently, the most commonly used tensor decomposition techniques include Tucker decomposition [59], HOSVD [60], and HOOI [61]. In this section, we will perform Tucker decomposition on above constructed tensor T. Assuming T ∈ R n 1 ×n 2 ×n 3 , the tucker decomposition aims at finding Z α (α ∈ (1, 2, 3)) and core tensor G ∈ R R 1 ×R 2 ×R 3 that can solve the following optimization problem:
minimize � � � � � � T − T � � � � � � 2 F , s.t. T i,j,k � r 1 ,r 2 ,r 3 Z i,r 1 1 Z j,r 2 2 Z k,r 3 3 G r 1 ,r 2 ,r 3 , ∀ i, j, k.(21)
Computational and Mathematical Methods in Medicine
Hence, based on formula (21), we can further transform this equation to following simple form:
minimize ‖T − T‖ 2 F , s.t. T � GX 1 Z 1 X 2 Z 2 X 3 Z 3 � ⟦G; Z 1 , Z 2 , Z 3 ⟧,(22)
Z 1 ∈ R n 1 ×R 1 , Z 2 ∈ R n 2 ×R 2 , and Z 3 ∈ R n 3 ×R 3 are the factor matrices, which are usually orthogonal and can be considered as the main component of each mode. R 1 , R 2 , and R 3 are the number of columns (max(R 1 , R 2 , R 3 ) ≪ min(n 1 , n 2 , n 3 )) in the factor matrices Z 1 , Z 2 , and Z 3 respectively. e notation X n denotes n-mode product; ⟦G; Z 1 , Z 2 , Z 3 ⟧ is the shorthand introduced by Kolda and Gibson [62] (Supplementary File A).
Based on equation (22), the above optimization problem can be solved according to the following steps:
Considering that the derivation forms of Z 1 , Z 2 , and Z 3 are similar, we will only derive the iterative formula of Z 1 as an example. Firstly, as illustrated in formula (23), the objective function given in formula (22) can be rewritten as a matrix form of T along the first dimension:
T (1) − Z 1 ⟦G; Z 2 , Z 3 ⟧ (1) � � � � � � � � 2 F ,(23)
where T (1) ∈ R n 1 ×(n 2 * n 3 ) is the unfolding of T along the first dimension (Supplementary File A). Assuming that the optimal solution Z 1 satisfies all the constraints in equation (22), we have
T (1) � Z 1 ⟦G; Z 2 , Z 3 ⟧ (1) � Z 1 G (1) Z 2 ⊗ Z 3 T ,(24)
where ⊗ denotes the Kronecker product, and moreover, we have
S 1 � G (1) Z 2 ⊗ Z 3 T .(25)
Hence, formula (24) can be regarded as a nonnegative matrix factorization (NMF) form [63]. en, we can finally obtain the solution of Z 1 by updating NMF as follows:
Z 1 ⟵ Z 1 * T (1) S T (1) Z 1 S (1) S T(1)
.
Hence, we can finally obtain the factor matrices Z 2 and Z 3 in a similar way. ereafter, while fixing the factor matrices Z 1 , Z 2 , and Z 3 , the objective function in formula (22) can be converted to the following form:
� � � � � � T − T � � � � � � 2 F � vec(T) − Z 3 ⊗ Z 2 ⊗ Z 1 vec(G) � � � � � � � � 2 F ,(27)
where vec(·) denotes the vectorization of the tensor. And moreover, based on formula (27), the following linear equation can be obtained:
vec(X) � Z 3 ⊗ Z 2 ⊗ Z 1 vec(G).(28)
Let Q � Z 3 ⊗ Z 2 ⊗ Z 1 , then obviously, formula (28) can also be regarded as a NMF, and thereafter, the core tensor in formula (28) can be obtained as follows [63]:
vec(G) ⟵ vec(G) * Q T vec(T) Q T Qvec(G) � vec G * ⟦T; Z T 1 , Z T 2 , Z T 3 ⟧ ⟦G; Z T 1 , Z T 2 , Z T 3 ⟧ ,(29)G ⟵ G * ⟦T; Z T 1 , Z T 2 , Z T 3 ⟧ ⟦G; Z T 1 , Z T 2 , Z T 3 ⟧ .(30)
Based on above formulas, the pseudocode of our prediction model PADLMHOOI based on tensor decomposition can be described as follows:
Step 1. Input: T, R 1 , R 2 , R 3 , Z 1 , Z 2 , Z 3 , G, and the convergence threshold ε.
Step 2. Repeat
For i � 1 to 3:
Update Z i according to formula (26) End For Update G according to formula (30) Until ‖T − [[G; Z 1 , Z 2 , Z 3 ]]‖ 2 F < ε Step 3. Return Z 1 , Z 2 , Z 3 , G
According to above steps, we can obtain the final predicted disease-lncRNA-miRNA association tensor T * � GX 1 Z 1 X 2 Z 2 X 3 Z 3 , and after prioritizing the disease-related lncRNA-miRNA pairs based on the entities in the tensor T * , obviously, the top-ranked lncRNA-miRNA pairs can be regarded as more likely to be related to the corresponding disease.
Results and Analysis
Leave-One-Out Cross-Validation (LOOCV).
In order to estimate the prediction performance of our newly proposed prediction model, the global leave-one-out cross-validation (LOOCV), 2-fold cross-validation (2-fold CV), and 10-fold cross-validation (10-fold CV) were implemented on PAD-LMHOOI, respectively. In the K-fold cross-validation, the initial sample will be divided into K subsample sets, and a single subsample set is retained as the data for the validation model, while the other K − 1 samples are used to train the model. During simulation, the cross-validation will be performed K times, and each subsampling set will be verified once, and the average results of K times will be utilized to obtain a single estimation. Moreover, in order to reduce the performance deviation caused by the random sample partitioning, we divide the partition 100 times and then obtain the ROC curve and the AUC value in the same way as the LOOCV. And, as a result, from the following Table 1, it is easy to see that PADLMHOOI can achieve reliable AUCs of 0.9545, 0.9730 ± 0.0119, and 0.9626 ± 0.0150 in the frameworks of global LOOCV, 2-fold CV, and 10-fold CV, respectively. Additionally, in order to further estimate the prediction performance of PADLMHOOI, we implemented it under the framework of local LOOCV, and the simulation results of 50 predicted related diseases were illustrated in Supplementary Table 4.
Performance Comparison with Other Methods.
To the best of our knowledge, up to now, PADLMP [39] is the unique model having been proposed for predicting potential associations between disease and lncRNA-miRNA pairs, in which, these three kinds of nodes such as disease nodes, lncRNA nodes, and miRNA nodes are considered simultaneously to construct a triple network. And, the major difference between PADLMP and our model PADLMHOOI is that PADLMP is based on the method of link prediction. erefore, in order to compare PADLMP with our model PADLMHOOI, we implemented LOOCV to verify the prediction performance of these two models based on the 3047 known disease-lncRNA-miRNA associations downloaded above. In the first experiment, we set the parameters in PADLMP to their best values; specifically, the step size K is set to 2 and the attenuation coefficient c is set to 0.01. Meanwhile, for convenience, we set the parameters in PADLMHOOI as follows: the parameters a 1 , a 2 , and a 3 in formula (20) are all set to 1, the parameters r 1 , r 2 , and r 3 in formula (21) are all set to 5, and the parameters K and α in formulas (17)- (19) are all set to 3 and 0.1 separately. And, as illustrated in Figure 2, it is easy to see that PADLMHOOI and PADLMP can achieve the AUCs of 0.9545 and 0.9318 separately, which demonstrate that the prediction performance of PADLMHOOI is superior to that of PADLMP.
As time went by, we found that some databases have been updated. Hence, in order to further demonstrate the advancement of PADLMHOOI, we once again collected the latest disease-lncRNA correlations from the databases lnc2cancer v2.0, lncRNADisease 2.0 [64], and MNDR v2.0 [48], collected the latest disease-miRNA associations from the database HMDD v3.0, and collected the latest lncRNA-miRNA associations from the database RAID v2.0 [65] separately. And thereafter, we reconstructed the triple network based on these newly collected latest datasets. In the newly constructed triple network, the numbers of disease nodes, lncRNA nodes, and miRNA nodes are 42, 234, and 251 respectively; the number of known associations between diseases and lncRNA-miRNA pairs is 3,768; the number of known associations between diseases and lncRNAs is 733; and the number of known associations between diseases and miRNAs is 674. en, based on the new triple network, we compared our model PAD-LMHOOI with PADLMP once more. And, in this second experiment, we set the parameters K and α to 10 and 0.5, respectively, in PADLMHOOI and kept other parameters unchanged as in the first experiment. And, as illustrated in Figure 3, simulation results show that PADLMHOOI and PADLMP can achieve AUCs of 0.9026 and 0.9013, respectively, which demonstrate that the prediction performance of PADLMHOOI outperforms that of PADLMP markedly.
Additionally, the interesting point is that our model can infer potential disease-lncRNA associations and disease-miRNA associations incidentally, while predicting potential associations between diseases and lncRNA-miRNA pairs. Hence, it is reasonable as well to compare our model PADLMHOOI with prediction models for inferring potential disease-lnRNA or disease-miRNA associations. erefore, in this section, we would compare PADLMHOOI with some state-of-the-art computational prediction models such as the LRLSLDA [26], NBCLAD [25], WBSMDA [66], and RLSMDA [67]. Among them, LRLSLDA is a semisupervised learning-based prediction model for inferring potential lncRNA-disease associations; NBCLAD is Figure 2, the reason that the AUCs of our model decline in Figure 3 is that the values of parameters K and α are different. In Figure 2, K � 3 and α � 0.1, while in Figure 3, K � 10 and α � 0.5. a probabilistic model for predicting potential associations between diseases and lncRNAs; WBSMDA is a prediction model for predicting potential associations between diseases and miRNAs; and RLSMDA is a prediction model for predicting disease-related miRNAs based on the framework of regularized least squares. In addition, while comparing with LRSLDA, known disease-lncRNA associations were obtained from the triple disease-lncRNA-miRNA network; however, the parameters in LRSLDA are set to the same values given in the literature. Moreover, while comparing with NBCLDA, considering that there are four kinds of nodes such as diseases, lncRNAs, miRNAs, and genes included in NBCLDA, there are three kinds of nodes such as diseases, lncRNAs, and miRNAs in our model PAD-LMHOOI. Hence, for the sake of fairness, we only compared PADLMHOOI with the submethod NBCLDA-GN1-SD. And, as illustrated in Figure 4, simulation results show that PADLMHOOI, NBCLDA-G1-SD, and LRSLDA can achieve AUCs of 0.9568, 0.7928, and 0.5924 separately, which demonstrate that PADLMHOOI thoroughly defeats both NBCLDA-G1-SD and LRSLDA. In addition, while comparing with WBSMDA and RLSMDA, 674 known disease-miRNA associations were obtained from the triple disease-lncRNA-miRNA network; however, the parameters in both WBSMDA and RLSMDA are set to the same values given in the literatures. And, as illustrated in Figure 5, simulation results show that PADLMHOOI, WBSMDA, and RLSMDA can achieve AUCs of 0.9157, 0.8544, and 0.8991, respectively, which demonstrate that PADLMHOOI outperforms both WBSMDA and RLSMDA thoroughly as well.
Recall Ratio Analysis.
In this section, in order to further evaluate the prediction performance of PADLMHOOI, we compared the recall value of PADLMHOOI and other stateof-the-art models. It is well known that the higher recall ratio of all selected diseases in a top k ranking list means that the more positive testing samples (real disease-related lncRNA-miRNA pairs) have been identified successfully. And, as a result, Figure 6 illustrates the recall rate of all selected diseases in different top k ranking lists. Moreover, we further listed the recall rate of some given diseases associated with at least 80 verified lncRNA-miRNA associations in Supplementary Table 5.
Case Studies.
In this section, case studies of breast neoplasms, colon neoplasms, and prostate neoplasms were conducted to further verify the capability of PADLMHOOI to detect novel associations between diseases and lncRNA-miRNA pairs separately. And, among these three kinds of case studies, breast cancer is the second leading cause of female cancer death and comprises 22% of all cancers in women [68,69]. e related literature has suggested that lncRNAs and miRNAs play an important role in the formation of many diseases, and the formation of breast cancer may be more relevant to them [70,71]. Predicting breast cancer-associated lncRNA-miRNA pairs and identifying lncRNAs and miRNAs as biomarkers may make a significant contribution to better diagnosis and treatment of breast cancer [71]. In Supplementary Table 6, the column of lncRID and miRID denotes lncRNA ID and miRNA ID, respectively. Evi1 and Evi2 denote some authority database or published literature containing verified disease-lncRNA or disease-miRNA associations separately. "#" and " * " stand for databases of lncRNADisease and MNDR v2.0, respectively, which consist of known disease-lncRNA associations or contain published literatures to support the association between predicted lncRNAs and breast cancer. "!," "&," and "+" stand for databases of HMDD, miR2Disease, and miRCancer, respectively, which consist of known disease- Computational and Mathematical Methods in Medicine miRNA associations or contain published literature to support the association between predicted miRNAs and breast cancer. Particularly, "Nan" indicates that there is no database or no published literature to support the predicted results. From Supplementary Table 6, it is easy to see that all candidate disease-lncRNA associations have been verified in databases of the lncRNADisease and MNDR v2.0 or published papers containing these databases. And, in addition, there are 42 out of 50 candidate disease-miRNA associations having been reported by HMDD, miR2Disease, and miR-Cancer or published paper containing these databases. Moreover, we discovered that those novel miRNAs with miRID 35, 51, 73, 164, and 186 are related to some important factors affecting the development of breast neoplasms. Hence, it is obvious that we infer that these lncRNA-miRNA pairs may be associated with breast cancer.
In addition, colonic tumors are a type of malignancy that is common in the rectum and sigmoid borders [72]. Early colon cancer is difficult to detect because of its insignificant symptoms [73]. Unfortunately, the related literature reports that its incidence has been on the rise in recent years [74]. erefore, predicting potential miRNAs and lncRNAs associated with colon tumors is of great significance for the diagnosis of early colon cancer. In Supplementary Table 7, we have listed the top 30 candidate lncRNA-miRNA pairs predicted to be associated with colon tumors. Moreover, all of these candidate lncRNAs and most of these candidate miRNAs have been verified by lncRNADisease database and MNDR v2.0, respectively.
Moreover, prostate neoplasm is one of the most common cancers in white and African-American men, and it is reported that there are about one in six white men and one in five African-American men having prostate cancer in their lifetime. Recent researches have shown that prostate neoplasm is caused by the malignancy of prostate epithelial cells [75], its formation includes many factors such as age, family history, and race [76], and particularly, some miRNAs such as has-let-7a-5p and lncRNAs such as XIST have been found to be involved in the formation of prostate neoplasms successively. Hence, it is interesting to infer potential miRNAs and lncRNAs associated with prostate neoplasms. In Supplementary Table 8, we have listed the top 30 prostate neoplasm-related candidate lncRNA-miRNA pairs. Moreover, all of these candidate lncRNAs and most of these candidate miRNAs have been verified by lncRNADisease and MNDR v2.0, respectively.
Parameter Sensitivity Analysis.
Considering that there are some key parameters such as K and α, which may be significant to the performance of our prediction model PADLMHOOI, in this section, we will further estimate the effects of these key parameters to the prediction performance of PADLMHOOI. Firstly, we varied K from 1 to 10 during simulation. And, as a result, Table 2 illustrates the impacts of parameter K on the performance of PADLMHOOI. By observing Table 2, it is obvious that PADLMHOOI can achieve the maximum AUC value of 0.9708 while K � 8. And additionally, as for the impacts of the parameter α, considering the time costs, we set K � 3 and varied α from 0.1 to 0.9 during simulation. And as a result, Table 3 illustrates the impacts of parameter α on the performance of PAD-LMHOOI. By observing Table 3, it is obvious that PAD-LMHOOI can achieve the maximum AUC value of 0.9591 while α � 0.7.
Discussion and Conclusion
Researches on prediction of potential associations between lncRNA-miRNA pairs and diseases not only are helpful in understanding the disease mechanisms on lncRNA and miRNA levels but also play an important role in the detection of disease biomarkers, diagnosis, prognosis, and prevention. However, to our knowledge, although there are many researches having demonstrated that lncRNA-miRNA interactions are associated with the development of complex diseases, up to now, there are few models having been proposed for large-scale forecasting potential associations between diseases and lncRNA-miRNA pairs. Since traditional biological experiments are quite expensive and timeconsuming, in this paper, based on the existing disease-miRNA associations, disease-lncRNA associations, lncRNA-miRNA interactions, and the assumption that genes with similar functions are often associated with similar diseases; we firstly constructed a three-order tensor T by adopting the method of WKNNP, and then, based on the method of tensor factorization, we further proposed a prediction model called PADLMHOOI to infer potential relations between diseases and lncRNA-miRNA pairs. And thereafter, simulation results under the frameworks of global and local LOOCV, 2-fold CV, and 10-fold CV, all confirmed the superiority of PADLMHOOI. Moreover, case studies of breast neoplasms, colon neoplasms, and prostate neoplasms further demonstrate that our model PADLMHOOI is an effective method for predicting potential disease-associated lncRNA-miRNA pairs. Certainly, there are still some limitations in PADLMHOOI. For example, although a large number of datasets have been integrated in PADLMHOOI, the amount of data available is still not enough; it is obvious that the prediction performance of PADLMHOOI will be better if more datasets can be collected. And in addition, in this paper, we only predicted the association between disease and a single lncRNA-miRNA pair. In the future, we will further modify PADLMHOOI to predict potential associations between diseases and multiple lncRNA-miRNA pairs.
Abbreviations
PADLMHOOI: Prediction of potential associations between diseases and lncRNA-miRNA pairs based on the higher-order orthogonal iteration.
Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
e authors declare no conflicts of interest.
Supplementary 7. Table 6: the candidate lncRNA-miRNA pairs associated with breast cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with breast cancer.
Supplementary 8. Table 7: the candidate lncRNA-miRNA pairs associated with colon cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with colon cancer.
Supplementary 9. Table 8: the candidate lncRNA-miRNA pairs associated with pprostate cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with colon cancer.
T
(d i , l j , m k ) S (d i , l j , m k S: (R 1 × R 2 × R 3 )
Step 3 . 3 Figure 1 :
331Return G , Z 1 , Z 2 , Z 3 miRNA Predicted association tensor T * lncRNA Disease T D (d i ) = (1/Q d ) ∑ K t=1 w t T(d t , : , :) T L (l k ) = (1/Q l ) ∑K t=1 w t T(: , l t , :) T M (m p ) = (1/Q m ) ∑ K t=1 w t T(: , : , m p ) T DLM = (T D + T L + T M )/Flow chart of PADLMHOOI for predicting potential associations between diseases and lncRNA-miRNA pairs.
) 2 . 9 .
29Weighted K Nearest Neighbor Profiles for Diseases, lncRNAs, and miRNAs (WKNNP). Let D � d 1 , d 2 , . . . , d m , L � l 1 , l 2 , . . . , l n , and M � m 1 , m 2 , . . . , m k denote the set of m diseases, n lncRNAs, and k miRNAs, respectively. Let T(d i ) � T(i, :, :) denote the ith horizontal slice matrix in disease axis of the tensor T, hence, T(d i ) also represents the interaction profile for the disease d i . Let T(l j ) � T(:, j, :) denote the jth lateral slice matrix in lncRNA axis of the tensor T, hence, T(l j ) also represents the interaction profile for lncRNA l j . Let T(m p ) � T(:, :, p) denote the pth frontal slice matrix in miRNA axis of the tensor T, hence, T(m p ) also denotes the interaction profile for miRNA m p .
Figure 2 :
2Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the 3047 known disease-lncRNA-miRNA associations.
Figure 3 :
3Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the latest 3678 known disease-lncRNA-miRNA associations. Here, comparing with the AUCs in
Figure 4
4: e comparison results between PADLMHOOI and LRLSLDA and NBCLAD.
Figure 6
6: e recall rate of all the selected diseases in different top k ranking lists.
Table
Table 6 ,
6we have listed the top
Table 2 :
2Impacts of the parameter K on the performance of PADLMHOOI.K
1
2
3
4
5
6
7
8
9
10
AUC
0.9660
0.9649
0.9591
0.9607
0.9666
0.9657
0.9675
0.9708
0.9703
0.9703
Table 3 :
3Impacts of the parameter α on the performance of PADLMHOOI.α
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
AUC
0.9545
00.9565
0.9582
0.9586
0.9583
0.9585
0.9591
0.9587
0.9539
Computational and Mathematical Methods in Medicine
Acknowledgments e project is partly sponsored by the National Natural Science Foundation of China (Nos. 61873221 and 61672447), the Natural Science Foundation of Hunan Province (Nos. 2018JJ4058 and 2017JJ5036), and the CERNET Next Generation Internet Technology Innovation Project (Nos. NGII20160305 and NGII20170109).Supplementary MaterialsSupplementary 1. File 1: introduction to tensor and optimization of objective function and update of factor matrix and core tensor.Table 1: 2048 known disease-lncRNA associations.Supplementary 3.Table 2: 4041 known disease-miRNA associations.Table 3: 20324 known lncRNA-miRNA associations.Table 4: AUC score for 50 diseases in the framework of local LOOCV.Table 5: recall ratio of some important diseases.Supplementary 4.Supplementary 5.Supplementary 6.
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| [
"A lot of research studies have shown that many complex human diseases are associated not only with microRNAs (miRNAs) but also with long noncoding RNAs (lncRNAs). However, most of the current existing studies focus on the prediction of diseaserelated miRNAs or lncRNAs, and to our knowledge, until now, there are few literature studies reported to pay attention to the study of impact of miRNA-lncRNA pairs on diseases, although more and more studies have shown that both lncRNAs and miRNAs play important roles in cell proliferation and differentiation during the recent years. e identification of disease-related genes provides great insight into the underlying pathogenesis of diseases at a system level. In this study, a novel model called PADLMHOOI was proposed to predict potential associations between diseases and lncRNA-miRNA pairs based on the higherorder orthogonal iteration, and in order to evaluate its prediction performance, the global and local LOOCV were implemented, respectively, and simulation results demonstrated that PADLMHOOI could achieve reliable AUCs of 0.9545 and 0.8874 in global and local LOOCV separately. Moreover, case studies further demonstrated the effectiveness of PADLMHOOI to infer unknown disease-related lncRNA-miRNA pairs."
] | [
"Zhanwei Xuan \nCollege of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina\n\nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Xiang Feng [email protected] \nCollege of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina\n\nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Jingwen Yu \nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Pengyao Ping \nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Haochen Zhao \nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Xianyou Zhu \nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n\nDepartment of Computer Science\nHengyang Normal University\n421008HengyangChina\n",
"Lei Wang [email protected] \nCollege of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina\n\nKey Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina\n",
"Emil Alexov "
] | [
"College of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"College of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina",
"Department of Computer Science\nHengyang Normal University\n421008HengyangChina",
"College of Computer Engineering & Applied Mathematics\nChangsha University\n410001Changsha, HunanChina",
"Key Laboratory of Intelligent Computing & Information Processing\nXiangtan University\n411105XiangtanChina"
] | [
"Zhanwei",
"Xiang",
"Jingwen",
"Pengyao",
"Haochen",
"Xianyou",
"Lei",
"Emil"
] | [
"Xuan",
"Feng",
"Yu",
"Ping",
"Zhao",
"Zhu",
"Wang",
"Alexov"
] | [
"Encode Project, ",
"Consortium, ",
"P Consortium, ",
"T Carninci, ",
"Kasukawa, ",
"P Kapranov, ",
"J Cheng, ",
"S Dike, ",
"Y Okazaki, ",
"M Furuno, ",
"T Kasukawa, ",
"T Tuschl, ",
"M Lagos-Quintana, ",
"W Lendeckel, ",
"J Dammann, ",
"R Rauhut, ",
"L J Robin, ",
"T A Yario, ",
"J A Steitz, ",
"D P Bartel, ",
"J P G Sluijter, ",
"A Van Mil, ",
"P Van Vliet, ",
"J Kota, ",
"R R Chivukula, ",
"K A O'donnell, ",
"L , ",
"Cliff Ji-Fan, ",
"G Hong-Yi, ",
"T Hung-Chia, ",
"W Wei-Lun, ",
"W Jen-Leih, ",
"K C Wang, ",
"H Y Chang, ",
"X Chen, ",
"C C Yan, ",
"X Zhang, ",
"Z H You, ",
"X Chen, ",
"D Xie, ",
"Q Zhao, ",
"Z H You, ",
"L Yanhui, ",
"W Changliang, ",
"M Zhengqiang, ",
"W Deng, ",
"H Yan, ",
"K Juanjuan, ",
"T Zhang, ",
"P Tan, ",
"L Wang, ",
"X Chen, ",
"L Wang, ",
"J Qu, ",
"N.-N Guan, ",
"J.-Q Li, ",
"X Chen, ",
"D Xie, ",
"L Wang, ",
"Q Zhao, ",
"Z.-H You, ",
"H Liu, ",
"X Chen, ",
"L Huang, ",
"Z.-H You, ",
"Z.-A Huang, ",
"Z Zhu, ",
"Q Zou, ",
"J Li, ",
"L Song, ",
"X Zeng, ",
"G Wang, ",
"X Zeng, ",
"X Zhang, ",
"Q Zou, ",
"X Zeng, ",
"L Liu, ",
"L Lü, ",
"Q Zou, ",
"W Tang, ",
"S Wan, ",
"Z Yang, ",
"A E Teschendorff, ",
"Q Zou, ",
"J Yu, ",
"P Ping, ",
"L Wang, ",
"L Kuang, ",
"X Li, ",
"Z Wu, ",
"C Xing, ",
"Y Gui-Ying, ",
"R Gernapudi, ",
"B Wolfson, ",
"Y Zhang, ",
"B K Dey, ",
"K Pfeifer, ",
"A Dutta, ",
"Y Yilong, ",
"M Jun, ",
"X Yixue, ",
"W Xinyu, ",
"L Meng, ",
"W Zhiqiong, ",
"Z Er-Bao, ",
"K Rong, ",
"Y Dan-Dan, ",
"J You, ",
"Y Zhang, ",
"B Liu, ",
"S Emmrich, ",
"A Streltsov, ",
"F Schmidt, ",
"V Angapandi, ",
"D Reinhardt, ",
"J.-H Klusmann, ",
"L Amy, ",
"T Candi, ",
"J Wen, ",
"Z Miaojun, ",
"C Qin, ",
"L Xin, ",
"V Huang, ",
"L.-C Li, ",
"L M Vaschetto, ",
"R Ramchandran, ",
"P Chaluvally-Raghavan, ",
"S Zhou, ",
"Z Xuan, ",
"L Wang, ",
"P Ping, ",
"T Pei, ",
"H Peng, ",
"C Lan, ",
"Y Zheng, ",
"G Hutvagner, ",
"D Tao, ",
"J Li, ",
"H Zhao, ",
"L Kuang, ",
"L Wang, ",
"P Ping, ",
"L Wang, ",
"L Kuang, ",
"S Ye, ",
"M F B Iqbal, ",
"T Pei, ",
"W Lei, ",
"P Ping, ",
"L Kuang, ",
"S Ye, ",
"F M B Lqbal, ",
"T Pei, ",
"S Yamaguchi, ",
"S Imoto, ",
"M Satoru, ",
"L Xin, ",
"S Ulf, ",
"S K Gupta, ",
"C Geng, ",
"W Ziyun, ",
"W Dongqing, ",
"Y Wang, ",
"L Chen, ",
"B Chen, ",
"T Cui, ",
"L Zhang, ",
"Y Huang, ",
"S Ning, ",
"Z Jizhou, ",
"P Wang, ",
"Y Jiang, ",
"Q Wang, ",
"Y Hao, ",
"L Yang, ",
"Q Chengxiang, ",
"T Jian, ",
"X Boya, ",
"D Qin, ",
"H Hongjin, ",
"W Di, ",
"L Jun-Hao, ",
"L Shun, ",
"Z Hui, ",
"Q Liang-Hu, ",
"Y Jian-Hua, ",
"K Ana, ",
"G J Sam, ",
"S Lynn Marie, ",
"A Cesar, ",
"N Suvarna, ",
"D Wang, ",
"J Wang, ",
"M Lu, ",
"F Song, ",
"Q Cui, ",
"X Chen, ",
"Z H You, ",
"G Y Yan, ",
"D W Gong, ",
"Y A Huang, ",
"X Chen, ",
"Z H You, ",
"D S Huang, ",
"K C C Chan, ",
"L R Tucker, ",
"L De Lathauwer, ",
"B. De Moor, ",
"L D Lathauwer, ",
"B D Moor, ",
"J Vandewalle, ",
"T G Kolda, ",
"T Gibson, ",
"H S D D Lee, ",
"Z Bao, ",
"Z Yang, ",
"Z Huang, ",
"Y Zhou, ",
"Q Cui, ",
"D Dong, ",
"Y Yi, ",
"Y Zhao, ",
"C Li, ",
"X Chen, ",
"C C Yan, ",
"X Zhang, ",
"X Chen, ",
"G Y Yan, ",
"H J Donahue, ",
"D C Genetos, ",
"K Karagoz, ",
"R Sinha, ",
"K Y Arga, ",
"J Meng, ",
"P Li, ",
"Q Zhang, ",
"Z Yang, ",
"S Fu, ",
"Y Wang, ",
"X Feng, ",
"R Jia, ",
"A I Phipps, ",
"N M Lindor, ",
"M A Jenkins, ",
"S Pita-Fernández, ",
"S Pértega-Díaz, ",
"B López-Calviño, ",
"V H Chong, ",
"M S Abdullah, ",
"P U Telisinghe, ",
"A , ",
"G A Gmyrek, ",
"M Walburg, ",
"C P Webb, ",
"P C Walsh, ",
"A W Partin, "
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"ViRBase: a resource for virus-host ncRNA-associated interactions",
"ncRDeathDB: a comprehensive bioinformatics resource for deciphering network organization of the ncRNA-mediated cell death system",
"RNALocate: a resource for RNA subcellular localizations",
"Predicting miRNA-disease association based on inductive matrix completion",
"BNPMDA: bipartite network projection for MiRNA-disease association prediction",
"LRSSLMDA: laplacian regularized sparse subspace learning for MiRNA-disease association prediction",
"PBMDA: a novel and effective path-based computational model for miRNA-disease association prediction",
"Similarity computation strategies in the microRNA-disease network: a survey",
"Integrative approaches for predicting microRNA function and prioritizing disease-related microRNA using biological interaction networks",
"Prediction of potential disease-associated microRNAs using structural perturbation method",
"Tumor origin detection with tissue-specific miRNA and DNA methylation markers",
"A novel probability model for LncRNA-disease association prediction based on the naïve bayesian classifier",
"Novel human lncRNA-disease association inference based on lncRNA expression profiles",
"miR-140 promotes expression of long non-coding RNA NEAT1 in adipogenesis",
"e h19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration",
"Knockdown of long noncoding RNA xist exerts tumor-suppressive functions in human glioblastoma stem cells by up-regulating miR-152",
"Silencing of long noncoding RNA MALAT1 by miR-101 and miR-217 inhibits proliferation, migration, and invasion of esophageal squamous cell carcinoma cells",
"Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a",
"MicroRNA-449a inhibits cell growth in lung cancer and regulates long noncoding RNA nuclear enriched abundant transcript 1",
"LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia",
"Novel long noncoding RNAs are regulated by angiotensin ii in vascular smooth muscle cells",
"lncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI",
"miRNA goes nuclear",
"miRNA activation is an endogenous gene expression pathway",
"miRNAmediated RNA activation in mammalian cells",
"A novel model for predicting associations between diseases and lncRNA-miRNA pairs based on a newly constructed bipartite network",
"Cross disease analysis of co-functional microRNA pairs on a reconstructed network of disease-gene-microRNA tripartite",
"Prediction of microRNAdisease associations based on distance correlation set",
"A novel approach based on bipartite network to predict human microbe-disease associations",
"Network-based predictions and simulations by biological state space models: search for drug mode of action",
"Computational analysis of target hub gene repression regulated by multiple and cooperative miRNAs",
"LncRNADisease: a database for long-non-coding RNA-associated diseases",
"Mammalian ncRNAdisease repository: a global view of ncRNA-mediated disease network",
"MNDR v2.0: an updated resource of ncRNA-disease associations in mammals",
"Lnc2Cancer: a manually curated database of experimentally supported lncRNAs associated with various human cancers",
"miR2Disease: a manually curated database for microRNA deregulation in human disease",
"HMDD v2.0: a database for experimentally supported human microRNA and disease associations",
"miRCancer: a microRNA-cancer association database constructed by text mining on literature",
"starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data",
"miRBase: annotating high confidence microRNAs using deep sequencing data",
"Disease ontology: a backbone for disease semantic integration",
"Inferring the human microRNA functional similarity and functional network based on microRNA-associated diseases",
"IRWRLDA: improved random walk with restart for lncRNA-disease association prediction",
"ILNCSIM: improved lncRNA functional similarity calculation model",
"Some mathematical notes on three-mode factor analysis",
"A multi-linear singular value decomposition",
"R N ) approximation of higherorder tensor",
"Learning the parts of objects by non-negative matrix factorization",
"LncRNADisease 2.0: an updated database of long noncoding RNA-associated diseases",
"RAID v2.0: an updated resource of RNA-associated interactions across organisms",
"WBSMDA within and between score for miRNA-disease association prediction",
"Semi-supervised learning for potential human microRNA-disease associations inference",
"Genomic approaches in breast cancer research",
"Triple negative breast cancer: a multi-omics network discovery strategy for candidate targets and driving pathways",
"A four-long noncoding RNA signature in predicting breast cancer survival",
"Microarray expression profile analysis of long non-coding RNAs of advanced stage human gastric cardia adenocarcinoma",
"Colon and rectal cancer survival by tumor location and microsatellite instability",
"Diagnostic and treatment delay, quality of life and satisfaction with care in colorectal cancer patients: a study protocol",
"Colorectal cancer: incidence and trend in Brunei Darussalam",
"Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor",
"Family history facilitates the early diagnosis of prostate carcinoma"
] | [
"Nature",
"Science",
"Science",
"Nature",
"Science",
"Proceedings of the National Academy of Sciences",
"Cell",
"Arteriosclerosis, rombosis, and Vascular Biology",
"Cell",
"Biochemical and Biophysical Research Communications",
"Molecular Cell",
"Briefings in Bioinformatics",
"Briefings in Bioinformatics",
"Nucleic Acids Research",
"Autophagy",
"Nucleic Acids Research",
"Bioinformatics",
"Bioinformatics",
"PLoS Computational Biology",
"PLoS Computational Biology",
"Briefings in Functional Genomics",
"Briefings in Bioinformatics",
"Bioinformatics",
"Bioinformatics",
"Genes",
"Bioinformatics",
"Molecular and Cellular Biology",
"Genes & Development",
"Cancer Letters",
"Journal of Biological Chemistry",
"Oncotarget",
"Indian Journal of Cancer",
"Molecular Cancer",
"Circulation Research",
"FEBS Journal",
"RNA Biology",
"RNA Biology",
"Advances in Experimental Medicine and Biology",
"Computational and Mathematical Methods in Medicine",
"BMC Bioinformatics",
"BMC Bioinformatics",
"A novel method for lncRNA-disease association prediction based on an lncRNA-disease association network",
"IEEE/ACM Transactions on Computational Biology and Bioinformatics",
"Current Bioinformatics",
"Journal of Computer Science & Technology",
"Nucleic Acids Research",
"Nucleic Acids Research",
"Cell Death & Disease",
"Nucleic Acids Research",
"Nucleic acids research",
"Nucleic Acids Research",
"Nucleic Acids Research",
"Bioinformatics",
"Nucleic Acids Research",
"Nucleic Acids Research",
"Nucleic Acids Research",
"Bioinformatics",
"Oncotarget",
"Oncotarget",
"Psychometrika",
"SIAM Journal on Matrix Analysis and Applications",
"SIAM Journal on Matrix Analysis and Applications",
"Multilinear operators for higherorder decompositions",
"Nature",
"Nucleic Acids Research",
"Nucleic Acids Research",
"Scientific Reports",
"Scientific Reports",
"Briefings in Functional Genomics",
"OMICS: A Journal of Integrative Biology",
"Journal of Experimental & Clinical Cancer Research",
"Molecular Genetics and Genomics",
"Diseases of the Colon & Rectum",
"Health and Quality of Life Outcomes",
"Singapore Medical Journal",
"American Journal of Pathology",
"Cancer",
"Singapore"
] | [
"\nT\n(d i , l j , m k ) S (d i , l j , m k S: (R 1 × R 2 × R 3 )",
"\nStep 3 . 3 Figure 1 :\n331Return G , Z 1 , Z 2 , Z 3 miRNA Predicted association tensor T * lncRNA Disease T D (d i ) = (1/Q d ) ∑ K t=1 w t T(d t , : , :) T L (l k ) = (1/Q l ) ∑K t=1 w t T(: , l t , :) T M (m p ) = (1/Q m ) ∑ K t=1 w t T(: , : , m p ) T DLM = (T D + T L + T M )/Flow chart of PADLMHOOI for predicting potential associations between diseases and lncRNA-miRNA pairs.",
"\n) 2 . 9 .\n29Weighted K Nearest Neighbor Profiles for Diseases, lncRNAs, and miRNAs (WKNNP). Let D � d 1 , d 2 , . . . , d m , L � l 1 , l 2 , . . . , l n , and M � m 1 , m 2 , . . . , m k denote the set of m diseases, n lncRNAs, and k miRNAs, respectively. Let T(d i ) � T(i, :, :) denote the ith horizontal slice matrix in disease axis of the tensor T, hence, T(d i ) also represents the interaction profile for the disease d i . Let T(l j ) � T(:, j, :) denote the jth lateral slice matrix in lncRNA axis of the tensor T, hence, T(l j ) also represents the interaction profile for lncRNA l j . Let T(m p ) � T(:, :, p) denote the pth frontal slice matrix in miRNA axis of the tensor T, hence, T(m p ) also denotes the interaction profile for miRNA m p .",
"\nFigure 2 :\n2Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the 3047 known disease-lncRNA-miRNA associations.",
"\nFigure 3 :\n3Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the latest 3678 known disease-lncRNA-miRNA associations. Here, comparing with the AUCs in",
"\nFigure 4\n4: e comparison results between PADLMHOOI and LRLSLDA and NBCLAD.",
"\nFigure 6\n6: e recall rate of all the selected diseases in different top k ranking lists.",
"\nTable\n",
"\nTable 6 ,\n6we have listed the top \n",
"\nTable 2 :\n2Impacts of the parameter K on the performance of PADLMHOOI.K \n1 \n2 \n3 \n4 \n5 \n6 \n7 \n8 \n9 \n10 \nAUC \n0.9660 \n0.9649 \n0.9591 \n0.9607 \n0.9666 \n0.9657 \n0.9675 \n0.9708 \n0.9703 \n0.9703 \n\n",
"\nTable 3 :\n3Impacts of the parameter α on the performance of PADLMHOOI.α \n0.1 \n0.2 \n0.3 \n0.4 \n0.5 \n0.6 \n0.7 \n0.8 \n0.9 \nAUC \n0.9545 \n00.9565 \n0.9582 \n0.9586 \n0.9583 \n0.9585 \n0.9591 \n0.9587 \n0.9539 \n"
] | [
"(d i , l j , m k ) S (d i , l j , m k S: (R 1 × R 2 × R 3 )",
"Return G , Z 1 , Z 2 , Z 3 miRNA Predicted association tensor T * lncRNA Disease T D (d i ) = (1/Q d ) ∑ K t=1 w t T(d t , : , :) T L (l k ) = (1/Q l ) ∑K t=1 w t T(: , l t , :) T M (m p ) = (1/Q m ) ∑ K t=1 w t T(: , : , m p ) T DLM = (T D + T L + T M )/Flow chart of PADLMHOOI for predicting potential associations between diseases and lncRNA-miRNA pairs.",
"Weighted K Nearest Neighbor Profiles for Diseases, lncRNAs, and miRNAs (WKNNP). Let D � d 1 , d 2 , . . . , d m , L � l 1 , l 2 , . . . , l n , and M � m 1 , m 2 , . . . , m k denote the set of m diseases, n lncRNAs, and k miRNAs, respectively. Let T(d i ) � T(i, :, :) denote the ith horizontal slice matrix in disease axis of the tensor T, hence, T(d i ) also represents the interaction profile for the disease d i . Let T(l j ) � T(:, j, :) denote the jth lateral slice matrix in lncRNA axis of the tensor T, hence, T(l j ) also represents the interaction profile for lncRNA l j . Let T(m p ) � T(:, :, p) denote the pth frontal slice matrix in miRNA axis of the tensor T, hence, T(m p ) also denotes the interaction profile for miRNA m p .",
"Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the 3047 known disease-lncRNA-miRNA associations.",
"Performance comparison between PADLMHOOI and PADLMP in terms of ROC curves and AUCs based on the latest 3678 known disease-lncRNA-miRNA associations. Here, comparing with the AUCs in",
": e comparison results between PADLMHOOI and LRLSLDA and NBCLAD.",
": e recall rate of all the selected diseases in different top k ranking lists.",
"Impacts of the parameter K on the performance of PADLMHOOI.",
"Impacts of the parameter α on the performance of PADLMHOOI."
] | [
"Figure 1",
"(1, 2, 3)",
"(Supplementary File A)",
"Figure 2",
"Figure 3",
"Figure 2",
"Figure 3",
"Figure 2",
"Figure 3",
"Figure 4",
"Figure 5",
"Figure 6"
] | [
"lncRNA- disease data lncRNA-disease bipartite network G 1 miRNA-disease bipartite network G 2 Similarity calculation l 4 l 4 l 3 l 3 l 2 l 2 l 1 l 1 l 4 l 3 l 2 l 1 d 4 d 3 d 2 d 1 d 4 d 4 d 3 d 3 d 2 d 2 d 1 d 1 d 4 d 3 d 2",
"S , Z 1 , Z 2 , Z 3 Z 1 : (N D × R 1 ) Z 2 : (N L × R 2 ) Z 3 : (N M × R 3 )",
"||T -[[G; Z 1 , Z 2 , Z 3 ]]|| 2 F < ε",
"V 2 � V m 1 ∪ V d 2 as the vertex set in G 2 . Step 2. ∀m i ∈ V m 1 , d j ∈ V d 2 , if (m i , d j )",
"V 3 � V m 2 ∪ V l 2 as the vertex set in G 3 . Step 2. ∀l i ∈ V l 2 , m j ∈ V m 2 , if (l i , m j )",
"1. Let V d � V d 1 ∪ V d 2 , V m � V m 1 ∪ V m 2 , V l � V l 1 ∪ V l 2 , V 4 � {}, E 4 � {}, and V d′ � {}.",
"∀d i ∈ V d ,",
"(a) (d i , l j ) ∈ E 1 (b) (d i , m k ) ∈ E 2 (c) (l j , m k ) ∈ E 3 en (d i , l j ), (d i , m k )",
"3. Let V 4 � V 4 + V d′ .",
"T(i, j, k) � 1, d i , l j , d i , m k , l j , m k ∈ E 4 , , 0, otherwise. ⎧ ⎨ ⎩(1)",
"(d) � (T(d), E(d)), where T(d)",
"D d d s � 1, if d s � d, max Δ * D d d ′ | d ′ ∈ children of d s , otherwise, DV(d) � d i ∈T(d) D d d i ,(2)",
"DisSemSim(i, j) � t∈T d i ( )∩T d j D d i (t) + D d j (t) DV d i + DV d j .",
"Y 1 (i, j) � 1, if l i , d j ∈ G 1 , 0, otherwise. ⎧ ⎨ ⎩ (4)",
"Y 2 (i, j) � 1, if m i , d j ∈ G 2 , 0, otherwise. ⎧ ⎨ ⎩ (5)",
"GIP LDSIM (i, j) � exp −c d1 IP l d i − IP l d j � � � � � � � � � � 2 ,(6)",
"c d1 � 1 1/n d1 n d1 i�1 IP d l (i) 2 � � � � � � � � � � ,(7)",
"GIP MDSIM (i, j) � exp −c d2 IP m d i − IP m d j � � � � � � � � � � 2 , c d2 � 1 1/n d2 n d2 i�1 IP m d i � � � � � � � � 2 ,(8)",
"GIPSim(i, j) � GIP LDSIM (i, j) + GIP MDSIM (i, j) 2 .(9)",
"lncfunSim(i, j) � 1≤k≤m SemSims dt 1k , DT 2 + 1≤k≤n SemSims dt 2k , DT 1 m + n ,(10)",
"SemSims dt 2k , DT 1 � max 1≤l≤m DisSemSim dt 2k , dt 1l .(11)",
"GIP lncSim (i, j) � exp −c l IP l i − IP l j � � � � � � � � � � 2 , c l � 1 1/n l n l i�1 IP l i � � � � � � � � 2 ,(12)",
"lncSim(i, j) � lncfunSim(i, j) + GIP lncSim (i, j) 2 .(13)",
"GIP miRSim (i, j) � exp −c m IP m i − IP m j � � � � � � � � � � 2 , c m � 1 1/n m n m i�1 IP m i � � � � � � � � 2 ,(15)",
"miRSim(i, j) � miRfunSim(i, j) + GIP miRSim (i, j) 2 .(16",
"T D d i � 1 Q d K t�1 w t T d t , :, : ,(17)",
"Q d � K t�1 disSim(d t , d i ).",
"T L l k � 1 Q l K t�1 w t T :, l t , : ,(18)",
"T M m p � 1 Q m K t�1 w t T :, :, m p ,(19)",
"T � max T, T DLM ,(20)",
"minimize � � � � � � T − T � � � � � � 2 F , s.t. T i,j,k � r 1 ,r 2 ,r 3 Z i,r 1 1 Z j,r 2 2 Z k,r 3 3 G r 1 ,r 2 ,r 3 , ∀ i, j, k.(21)",
"minimize ‖T − T‖ 2 F , s.t. T � GX 1 Z 1 X 2 Z 2 X 3 Z 3 � ⟦G; Z 1 , Z 2 , Z 3 ⟧,(22)",
"T (1) − Z 1 ⟦G; Z 2 , Z 3 ⟧ (1) � � � � � � � � 2 F ,(23)",
"T (1) � Z 1 ⟦G; Z 2 , Z 3 ⟧ (1) � Z 1 G (1) Z 2 ⊗ Z 3 T ,(24)",
"S 1 � G (1) Z 2 ⊗ Z 3 T .(25)",
"Z 1 ⟵ Z 1 * T (1) S T (1) Z 1 S (1) S T(1)",
"� � � � � � T − T � � � � � � 2 F � vec(T) − Z 3 ⊗ Z 2 ⊗ Z 1 vec(G) � � � � � � � � 2 F ,(27)",
"vec(X) � Z 3 ⊗ Z 2 ⊗ Z 1 vec(G).(28)",
"vec(G) ⟵ vec(G) * Q T vec(T) Q T Qvec(G) � vec G * ⟦T; Z T 1 , Z T 2 , Z T 3 ⟧ ⟦G; Z T 1 , Z T 2 , Z T 3 ⟧ ,(29)",
"G ⟵ G * ⟦T; Z T 1 , Z T 2 , Z T 3 ⟧ ⟦G; Z T 1 , Z T 2 , Z T 3 ⟧ .(30)",
"Update Z i according to formula (26) End For Update G according to formula (30) Until ‖T − [[G; Z 1 , Z 2 , Z 3 ]]‖ 2 F < ε Step 3. Return Z 1 , Z 2 , Z 3 , G"
] | [
"Noncoding RNA, according to its size, can be divided into small and long noncoding RNAs approximately. Generally, small RNAs include tRNAs, miRNAs, piRNAs, and snoR-NAs [1][2][3][4], and miRNAs are widely present in the cytoplasm of eukaryotic cells and are approximately 18-22 nucleotides in length, which can bind to 3′-untranslated region of mRNA (3′-UTR) to inhibit the translation process of mRNA or to degrade mRNA, thereby affecting the expression of related genes [5][6][7]. miRNAs play important roles in a series of life activities such as cell differentiation of living body [8], growth and development [9], and apoptosis [10]. Compared to small-molecule ncRNA, lncRNA has a longer nucleotide chain with more than 200 nucleotides and has a specific and complex secondary space structure inside the molecule and can provide multiple sites for protein binding [11]. In addition, both lncRNAs and miRNAs are key members of noncoding RNAs and play important roles in coding and regulation of many complex human diseases [12][13][14][15][16].",
"Up to now, there have been many studies on relationships between diseases and miRNAs. For example, some important methods proposed by Xing Chen et al. [17][18][19][20] and Zou et al. [21][22][23][24]. In terms of prediction of potential associations between lncRNAs and diseases, Yu et al. [25] and Xing et al. [26] proposed two kinds of computational models called NBCLAD and LRLSLDA, respectively. Moreover, studies have also shown that there exist relationships between lncRNAs and miRNAs. For example, Gernapudi et al. demonstrated that miRNA 140 can induce the expression of lncRNA NEAT1 [27]. Dey et al. showed that the silencing of lncRNA H19 and knockout of H19 gene in myoblasts significantly decreased skeletal muscle differentiation [28]. Yilong et al. discovered that, after low XIST expression in gliomas, XIST could regulate miR-152 glioma stem cells to inhibit cell proliferation, migration, and invasion [29]. Xinyu et al. demonstrated that lncRNA MALAT1 could achieve posttranscriptional regulation of esophageal squamous cell carcinoma cells through miR-101 and miR-217 [30]. Er-bao et al. proposed that lncRNA ANRIL interacted with miR-99a/miR449a to regulate cell proliferation during gastric cancer formation [31]. You et al. found that the expression of miR-449a and the expression of lncRNA NEAT1 in lung cancer cell L9981 inhibited each other. When miR-449a was overexpressed, NEAT1 expression was decreased, cell proliferation was inhibited, and apoptosis was increased, and vice versa [32]. Emmrich et al. found that the expression of lncRNA MONC and MIR100HG was closely related to the miRNA groups of miR-99a∼125b-2 and miR-100∼125b-1. After silencing of lncRNA MONC and MIR100HG, acute megakaryocytes in the early stage of the disease, the tumor cells of leukemia patients, were severely inhibited [33]. Amy et al. found that lncRNA Ang362 was the host transcriptor of miR-211 and miR-222, and their interactions regulated Ang II and induced proliferation of vascular smooth muscle cells [34]. Miaojun et al. found that the interactions between lncRNA H19 and miRNA-675 play an important role in the metastasis of prostate cancer [35]. Obviously, the exploration of these relationships was conducive to the construction of gene regulatory networks and the identification of the mechanisms of complex human diseases [36][37][38].",
"From the above description, it is easy to see that more and more studies have shown that lncRNA-miRNA interactions are involved in the development of complex diseases. However, to the best of our knowledge, so far, in addition to the model of PADLMP proposed by Zhou et al. [39], few models have been proposed for large-scale prediction of potential associations between diseases and lncRNA-miRNA interactions. Hence, inspired by state-ofthe-art methods [40][41][42][43][44], which show that the miRNA-miRNA pairs can work cooperatively to regulate a single gene or gene clusters being involved in similar processes [45], and simultaneously, based on the reasonable assumption that functionally similar lncRNA-miRNA pairs tend to be associated with similar diseases, in this paper, a new prediction model called PADLMHOOI was proposed to infer potential associations between diseases and the lncRNA-miRNA pairs. And, as illustrated in Figure 1, our newly proposed prediction model PADLMHOOI consists of the following four major steps:",
"Step 1 (Data Integration and Network Construction). In this step, first of all, we downloaded known disease-lncRNA associations from three different disease-lncRNA databases such as disease-lncRNA [46], MNDR [47,48], and lnc2cancer [49], respectively, and then, based on these datasets, we constructed a bipartite network of disease-lncRNA. Next, we downloaded known disease-miRNA associations from three different databases such as miR2Disease [50], HMDD [51], and miRCancer [52] separately, and then, based on these datasets, we constructed a bipartite network of disease-miRNA. Moreover, we downloaded the 2015 and 2017 versions of known lncRNA-miRNA associations from the starBasev2.0 database [53] (http:// starbase.sysu.edu.cn/) on Feb 2, 2017, and based on these datasets, we constructed a bipartite network of lncRNA-miRNA. Finally, based on these three kinds of bipartite networks, we constructed an integrated tripartite network of disease-lncRNA-miRNA, which could be denoted as a tensor T.",
"Step 2 (Similarity Calculation). In this step, we would integrate the disease semantic similarity and Gaussian Interaction Profile Kernel similarity firstly to measure the similarity of diseases. Next, we would integrate the lncRNA functional similarity and miRNA functional similarity in three different ways to measure the functional similarity of lncRNA-miRNA pairs.",
"Step 3 (Weighted K-Nearest Neighbor Profile). Considering that there may be diseases that are unrelated to all lncRNA-miRNA pairs, which may lead to unsatisfactory prediction results while implementing PADLMHOOI to infer potential associations between diseases and lncRNA-miRNA pairs. Hence, in this step, we would introduce the weighted K-nearest neighbor profile (WKNNP) to add more interaction information between diseases, lncRNAs, and miRNAs to improve the prediction performance of PADLMHOOI.",
"Step 4 (Tensor Decomposition). In this step, we would perform tensor decomposition on the newly constructed disease-lncRNA-miRNA tensor T. Since the results of tensor decomposition include a core tensor and three matrices, we can define the final predicted association tensor as the modal product between the core tensor and these three matrices. ereafter, we would sort scores of the lncRNA-miRNA pairs associated with each disease in the descending order in the final predicted association tensor, and it is obvious that the higher the ranking of the score, the bigger the possibility that there may exist potential association between the disease and the lncRNA-miRNA pair would be.",
"In order to construct the bipartite network of disease-lncRNA, firstly, known associations between diseases and lncRNAs were downloaded from three different databases such as the LncRNADisease, MNDR, and Lnc2Cancer, respectively, and then, after feature processing (including feature cleaning and data imbalance processing etc.), 2048 different disease-lncRNA associations were finally obtained (Supplementary Table 1). ereafter, based on these newly obtained 2048 known disease-lncRNA associations, we can construct a disease-lncRNA bipartite network G 1 � (V 1 , E 1 ) according to the following steps:",
"Step 1. Let V l 1 � l i |i ∈ [1, n l 1 ] be the set of all different lncRNAs in these 2048 known disease-lncRNA associations and V d 1 � d i |i ∈ [1, n d 1 ] be the set of all different diseases in these 2048 known disease-lncRNA associations, then we define V 1 � V l 1 ∪ V d 1 as the vertex set in G 1 .",
"Step 2. ∀l i ∈ V l 1 , d j ∈ V d 1 , if (l i , d j ) belongs to these 2048 downloaded known disease-lncRNA associations, then we define that there is an edge between l i and d j in G 1 ; thereafter, we can obtain the edge set E 1 in G 1 . New tensor T",
"Step 1. Input T , R 1 , R 2 and R 3 and initialize Z 1 , Z 2 , Z 3 , G, and convergence threshold ε",
"Step 2. Repeat for n = 1 to 3:",
"Update Z n according to (31) end for",
"Update G according to (35) Until Computational and Mathematical Methods in Medicine 3",
"In order to construct the bipartite network of disease-miRNA, at first, known disease-miRNA associations were downloaded from three different databases such as the miR2Disease, HMDD, and miRCancer separately, and then, after these newly acquired miRNAs and diseases being mapped to the database miRBase v21 [54] and disease ontology (DO) [55], respectively, 4041 different disease-miRNA associations were finally obtained (Supplementary Table 2). Hence, based on these newly obtained 4041 known disease-miRNA associations, we can construct a disease-miRNA bipartite network G 2 � (V 2 , E 2 ) according to the following steps:",
"Step 1. Let V m 1 � m i |i ∈ [1, n m 1 ] be the set of all different miRNAs in these 4041 known disease-miRNA associations and V d 2 � d i |i ∈ [1, n d 2 ] be the set of all different diseases in these 4041 known disease-miRNA associations, then we define",
"belongs to these 4041 known disease-miRNA associations, then we define that there is an edge between m i and d j in G 2 ; thereafter, we can obtain the edge set E 2 in G 2 .",
"In order to construct the bipartite network of lncRNA-miRNA, at first, two different versions (2015 and 2017) of lncRNA-miRNA dataset were downloaded from the starBa-sev2.0 database separately, and then, after feature processing (including feature cleaning and data imbalance processing), 20324 different lncRNA-miRNA interactions were finally obtained (Supplementary Table 3). ereafter, based on these newly obtained 20324 known lncRNA-miRNA associations, we can construct a lncRNA-miRNA bipartite network G 3 � (V 3 , E 3 ) according to the following steps:",
"Step 1. Let V l 2 � l i |i ∈ [1, n l 2 ] denote the set of all different lncRNAs in these 20324 known lncRNA-miRNA associations and V m 2 � m i |i ∈ [1, n m 2 ] denote the set of all different miRNAs in these 20324 known lncRNA-miRNA associations, then we define",
"belongs to these 20324 known lncRNA-miRNA associations, then we define that there is an edge between l i and m j in G 3 ; thereafter, we can obtain the edge set E 3 in G 3 .",
"Network of Disease-lncRNA-miRNA. Based on the above newly obtained networks such as G 1 , G 2 , and G 3 , we can construct a tripartite network G 4 � (V 4 , E 4 ) according to the following steps:",
"Step",
"Step 2. While V d is not null, Repeat:",
"If ∃l j ∈ V l and m k ∈ V m satisfyies the following three kinds of conditions simultaneously:",
", and (l j , m k ) will be added into E 4 firstly, and then, d i will be added into V d ′ and removed from V d . Finally, l j and m k will be added into V 4 if l j and m k are not inV 4 . Else, d i will be removed from V d .",
"Step",
"According to above steps, a tripartite disease-lncRNA-miRNA association network can be obtained finally. And, it is obvious that, in the tripartite network, there are three kinds of different nodes such as disease nodes, lncRNA nodes, and miRNA nodes; moreover, the number of disease nodes, lncRNA nodes, and miRNA nodes is 68, 44, and 211, respectively, and the number of associations between diseases and lncRNA-miRNA pairs is 3,047.",
"Based on the newly constructed tripartite network, for any given disease node d i , lncRNA node l j , and miRNA node m k in G 4 , we can define a tensor T as follows: ",
"denotes the set of nodes containing the node d itself and its ancestors and E(d) denotes the set of edges of the respective direct links from parent to child nodes [56]. ereafter, based on the newly constructed directed acyclic graph DAG(d), the semantic contribution of an ancestor node d s to the disease d can be calculated as follows:",
"where Δ is the semantic contribution decay factor with value between 0 and 1. And, in addition, according to the experimental results of some previous state-of-the-art methods [57,58], the most appropriate value for Δ will be 0.5. Hence, based on the assumption that two diseases with more common ancestor nodes in their DAGs shall have higher semantic similarity, the semantic similarity between two diseases d i and d j can be defined as follows:",
"4",
"Computational and Mathematical Methods in Medicine",
"(3)",
"Similarity for Diseases (GIPSim). Based on the hypothesis that functionally similar genes are often associated with similar diseases, in this section, we will adopt the Gaussian Interaction Profile Kernel to calculate the similarity of diseases according to the following steps: Firstly, based on the networks G 1 and G 2 constructed above, for any given lncRNA l i and disease d j , we define that",
"Next, for any given miRNA m i and disease d j , we define that",
"Hence, let IP l (d i ) denote the ith column of the matrix Y 1 , then we can calculate the Gaussian Kernel Similarity between diseases d i and d j based on their interaction profiles as follows:",
"where the parameter n d1 denotes the number of different diseases in G 1 .",
"In a similar way, let IP m (d i ) denote the ith column of matrix Y 2 , then we can calculate the Gaussian Kernel Similarity between diseases d i and d j based on their interaction profiles as follows:",
"Here, the parameter n d2 denotes the number of different diseases in G 2 . ereafter, based on these above formulas, we can calculate the Gaussian Interaction Profile Kernel Similarity between diseases d i and d j as follows:",
"(lncfunSim). For any two given lncRNAs such as l i and l j , let DT 1 � dt 11 , dt 12 , . . . , dt 1m be all the diseases related to l i in G 1 and DT 2 � dt 21 , dt 22 , . . . , dt 2n be all the diseases related to l j in G 1 , then we can define the functional similarity between l i and l j as follows:",
"where SemSims dt 1k , DT 2 � max 1≤l≤n DisSemSim dt 1k , dt 2l ,",
"Similarity for lncRNAs (GIP lncSim ). For any two given lncRNAs such as l i and l j , similar to the definition of formula (6), let IP(l i ) and IP(l j ) denote the ith and the jth row of the matrix Y 1 , respectively, then we can calculate the Gaussian Kernel Similarity between diseases l i and l j based on their interaction profiles as follows:",
"where n l1 denotes the number of different lncRNAs in G 1 .",
"Hence, based on these formulas given above, we can finally define the similarity measurement between lncRNAs l i and l j as follows: ",
"Similarity for miRNAs (GIP miRSim ). For any two given miRNAs, such as m i and m j , in a similar way, let IP(m i ) and IP(m j ) represent the ith and jth row in matrix Y 2 , respectively, then we can calculate the Gaussian Kernel Similarity between diseases m i and m j based on their interaction profiles as follows:",
"where n m2 denotes the number of miRNAs in G 2.",
"Hence, based on these formulas presented above, we can finally define the similarity measurement between miRNAs m i and m j as follows: en, it is obvious that the values in these three kinds of interaction profiles of any novel diseases, lncRNAs, or miRNAs are all zeros, which may lead to unsatisfactory prediction performance during inferring potential associations between diseases and lncRNA-miRNA pairs. Hence, in this section, we will perform a procedure for the construction of new interaction profiles to address the problem mentioned above. And, in this procedure, for each disease d i , its association with other K nearest known diseases (including at least one experimentally verified association) and corresponding K interaction profiles will be utilized to obtain the following interaction profile:",
"where, d 1 , d 2 , . . . , d K are the diseases sorted in descending order based on their similarity to d i , w t is the weight coefficient, and w t � α t−1 * disSim(d t , d i ), which means that a higher weight will be assigned if d t is more similar to d i . e parameter α is a decay term with values between 0 and 1. e parameter Q d is a normalization term, and there is",
"In the same manner, the new interaction profile for each l k can be determined as follows:",
"where l 1 , l 2 , . . . , l K are the lncRNAs sorted in the descending order based on their similarity to l k , w t is the weight coefficient, and w t � α t−1 * lncSim(l t , l k ), which means that a higher weight will be assigned if l t is more similar to l k . e parameter Q l is a normalization term, and there is Q l � K t�1 lncSim(l t , l k ). Similarly, the new interaction profile for each m p can be determined as follows:",
"where m 1 , m 2 , . . . , m K are the miRNAs sorted in the descending order based on their similarity to m p , w t is the weight coefficient, and w t � α t−1 * miRSim(m t , m p ), which means that a higher weight is assigned if m t is more similar to m p . e parameter Q m is a normalization term, and there is Q m � K t�1 miRSim(m t , m p ). ereafter, after combining the above three kinds of tensors T D , T L , and T M obtained from different data spaces and replacing T(i, j, k) � 0 with an associated likelihood score, we can update the original adjacency matrix T as follows:",
"where T DLM � (a 1 T D + a 2 T L + a 3 T M / a i ), (i � 1, 2, 3).",
"Inspired by the successful application of tensor decomposition in the field of link prediction and the application of nonnegative matrix decomposition methods in inferring disease-miRNA associations, in this section, we proposed a novel model called PADLMHOOI to predict new associations between diseases and miRNA-lncRNA pairs. From above descriptions, it is easy to know that a tensor is a multidimensional array. Currently, the most commonly used tensor decomposition techniques include Tucker decomposition [59], HOSVD [60], and HOOI [61]. In this section, we will perform Tucker decomposition on above constructed tensor T. Assuming T ∈ R n 1 ×n 2 ×n 3 , the tucker decomposition aims at finding Z α (α ∈ (1, 2, 3)) and core tensor G ∈ R R 1 ×R 2 ×R 3 that can solve the following optimization problem:",
"Hence, based on formula (21), we can further transform this equation to following simple form:",
"Z 1 ∈ R n 1 ×R 1 , Z 2 ∈ R n 2 ×R 2 , and Z 3 ∈ R n 3 ×R 3 are the factor matrices, which are usually orthogonal and can be considered as the main component of each mode. R 1 , R 2 , and R 3 are the number of columns (max(R 1 , R 2 , R 3 ) ≪ min(n 1 , n 2 , n 3 )) in the factor matrices Z 1 , Z 2 , and Z 3 respectively. e notation X n denotes n-mode product; ⟦G; Z 1 , Z 2 , Z 3 ⟧ is the shorthand introduced by Kolda and Gibson [62] (Supplementary File A).",
"Based on equation (22), the above optimization problem can be solved according to the following steps:",
"Considering that the derivation forms of Z 1 , Z 2 , and Z 3 are similar, we will only derive the iterative formula of Z 1 as an example. Firstly, as illustrated in formula (23), the objective function given in formula (22) can be rewritten as a matrix form of T along the first dimension:",
"where T (1) ∈ R n 1 ×(n 2 * n 3 ) is the unfolding of T along the first dimension (Supplementary File A). Assuming that the optimal solution Z 1 satisfies all the constraints in equation (22), we have",
"where ⊗ denotes the Kronecker product, and moreover, we have",
"Hence, formula (24) can be regarded as a nonnegative matrix factorization (NMF) form [63]. en, we can finally obtain the solution of Z 1 by updating NMF as follows:",
".",
"Hence, we can finally obtain the factor matrices Z 2 and Z 3 in a similar way. ereafter, while fixing the factor matrices Z 1 , Z 2 , and Z 3 , the objective function in formula (22) can be converted to the following form:",
"where vec(·) denotes the vectorization of the tensor. And moreover, based on formula (27), the following linear equation can be obtained:",
"Let Q � Z 3 ⊗ Z 2 ⊗ Z 1 , then obviously, formula (28) can also be regarded as a NMF, and thereafter, the core tensor in formula (28) can be obtained as follows [63]:",
"Based on above formulas, the pseudocode of our prediction model PADLMHOOI based on tensor decomposition can be described as follows:",
"Step 1. Input: T, R 1 , R 2 , R 3 , Z 1 , Z 2 , Z 3 , G, and the convergence threshold ε.",
"Step 2. Repeat",
"For i � 1 to 3:",
"According to above steps, we can obtain the final predicted disease-lncRNA-miRNA association tensor T * � GX 1 Z 1 X 2 Z 2 X 3 Z 3 , and after prioritizing the disease-related lncRNA-miRNA pairs based on the entities in the tensor T * , obviously, the top-ranked lncRNA-miRNA pairs can be regarded as more likely to be related to the corresponding disease.",
"In order to estimate the prediction performance of our newly proposed prediction model, the global leave-one-out cross-validation (LOOCV), 2-fold cross-validation (2-fold CV), and 10-fold cross-validation (10-fold CV) were implemented on PAD-LMHOOI, respectively. In the K-fold cross-validation, the initial sample will be divided into K subsample sets, and a single subsample set is retained as the data for the validation model, while the other K − 1 samples are used to train the model. During simulation, the cross-validation will be performed K times, and each subsampling set will be verified once, and the average results of K times will be utilized to obtain a single estimation. Moreover, in order to reduce the performance deviation caused by the random sample partitioning, we divide the partition 100 times and then obtain the ROC curve and the AUC value in the same way as the LOOCV. And, as a result, from the following Table 1, it is easy to see that PADLMHOOI can achieve reliable AUCs of 0.9545, 0.9730 ± 0.0119, and 0.9626 ± 0.0150 in the frameworks of global LOOCV, 2-fold CV, and 10-fold CV, respectively. Additionally, in order to further estimate the prediction performance of PADLMHOOI, we implemented it under the framework of local LOOCV, and the simulation results of 50 predicted related diseases were illustrated in Supplementary Table 4.",
"To the best of our knowledge, up to now, PADLMP [39] is the unique model having been proposed for predicting potential associations between disease and lncRNA-miRNA pairs, in which, these three kinds of nodes such as disease nodes, lncRNA nodes, and miRNA nodes are considered simultaneously to construct a triple network. And, the major difference between PADLMP and our model PADLMHOOI is that PADLMP is based on the method of link prediction. erefore, in order to compare PADLMP with our model PADLMHOOI, we implemented LOOCV to verify the prediction performance of these two models based on the 3047 known disease-lncRNA-miRNA associations downloaded above. In the first experiment, we set the parameters in PADLMP to their best values; specifically, the step size K is set to 2 and the attenuation coefficient c is set to 0.01. Meanwhile, for convenience, we set the parameters in PADLMHOOI as follows: the parameters a 1 , a 2 , and a 3 in formula (20) are all set to 1, the parameters r 1 , r 2 , and r 3 in formula (21) are all set to 5, and the parameters K and α in formulas (17)- (19) are all set to 3 and 0.1 separately. And, as illustrated in Figure 2, it is easy to see that PADLMHOOI and PADLMP can achieve the AUCs of 0.9545 and 0.9318 separately, which demonstrate that the prediction performance of PADLMHOOI is superior to that of PADLMP.",
"As time went by, we found that some databases have been updated. Hence, in order to further demonstrate the advancement of PADLMHOOI, we once again collected the latest disease-lncRNA correlations from the databases lnc2cancer v2.0, lncRNADisease 2.0 [64], and MNDR v2.0 [48], collected the latest disease-miRNA associations from the database HMDD v3.0, and collected the latest lncRNA-miRNA associations from the database RAID v2.0 [65] separately. And thereafter, we reconstructed the triple network based on these newly collected latest datasets. In the newly constructed triple network, the numbers of disease nodes, lncRNA nodes, and miRNA nodes are 42, 234, and 251 respectively; the number of known associations between diseases and lncRNA-miRNA pairs is 3,768; the number of known associations between diseases and lncRNAs is 733; and the number of known associations between diseases and miRNAs is 674. en, based on the new triple network, we compared our model PAD-LMHOOI with PADLMP once more. And, in this second experiment, we set the parameters K and α to 10 and 0.5, respectively, in PADLMHOOI and kept other parameters unchanged as in the first experiment. And, as illustrated in Figure 3, simulation results show that PADLMHOOI and PADLMP can achieve AUCs of 0.9026 and 0.9013, respectively, which demonstrate that the prediction performance of PADLMHOOI outperforms that of PADLMP markedly.",
"Additionally, the interesting point is that our model can infer potential disease-lncRNA associations and disease-miRNA associations incidentally, while predicting potential associations between diseases and lncRNA-miRNA pairs. Hence, it is reasonable as well to compare our model PADLMHOOI with prediction models for inferring potential disease-lnRNA or disease-miRNA associations. erefore, in this section, we would compare PADLMHOOI with some state-of-the-art computational prediction models such as the LRLSLDA [26], NBCLAD [25], WBSMDA [66], and RLSMDA [67]. Among them, LRLSLDA is a semisupervised learning-based prediction model for inferring potential lncRNA-disease associations; NBCLAD is Figure 2, the reason that the AUCs of our model decline in Figure 3 is that the values of parameters K and α are different. In Figure 2, K � 3 and α � 0.1, while in Figure 3, K � 10 and α � 0.5. a probabilistic model for predicting potential associations between diseases and lncRNAs; WBSMDA is a prediction model for predicting potential associations between diseases and miRNAs; and RLSMDA is a prediction model for predicting disease-related miRNAs based on the framework of regularized least squares. In addition, while comparing with LRSLDA, known disease-lncRNA associations were obtained from the triple disease-lncRNA-miRNA network; however, the parameters in LRSLDA are set to the same values given in the literature. Moreover, while comparing with NBCLDA, considering that there are four kinds of nodes such as diseases, lncRNAs, miRNAs, and genes included in NBCLDA, there are three kinds of nodes such as diseases, lncRNAs, and miRNAs in our model PAD-LMHOOI. Hence, for the sake of fairness, we only compared PADLMHOOI with the submethod NBCLDA-GN1-SD. And, as illustrated in Figure 4, simulation results show that PADLMHOOI, NBCLDA-G1-SD, and LRSLDA can achieve AUCs of 0.9568, 0.7928, and 0.5924 separately, which demonstrate that PADLMHOOI thoroughly defeats both NBCLDA-G1-SD and LRSLDA. In addition, while comparing with WBSMDA and RLSMDA, 674 known disease-miRNA associations were obtained from the triple disease-lncRNA-miRNA network; however, the parameters in both WBSMDA and RLSMDA are set to the same values given in the literatures. And, as illustrated in Figure 5, simulation results show that PADLMHOOI, WBSMDA, and RLSMDA can achieve AUCs of 0.9157, 0.8544, and 0.8991, respectively, which demonstrate that PADLMHOOI outperforms both WBSMDA and RLSMDA thoroughly as well.",
"In this section, in order to further evaluate the prediction performance of PADLMHOOI, we compared the recall value of PADLMHOOI and other stateof-the-art models. It is well known that the higher recall ratio of all selected diseases in a top k ranking list means that the more positive testing samples (real disease-related lncRNA-miRNA pairs) have been identified successfully. And, as a result, Figure 6 illustrates the recall rate of all selected diseases in different top k ranking lists. Moreover, we further listed the recall rate of some given diseases associated with at least 80 verified lncRNA-miRNA associations in Supplementary Table 5.",
"In this section, case studies of breast neoplasms, colon neoplasms, and prostate neoplasms were conducted to further verify the capability of PADLMHOOI to detect novel associations between diseases and lncRNA-miRNA pairs separately. And, among these three kinds of case studies, breast cancer is the second leading cause of female cancer death and comprises 22% of all cancers in women [68,69]. e related literature has suggested that lncRNAs and miRNAs play an important role in the formation of many diseases, and the formation of breast cancer may be more relevant to them [70,71]. Predicting breast cancer-associated lncRNA-miRNA pairs and identifying lncRNAs and miRNAs as biomarkers may make a significant contribution to better diagnosis and treatment of breast cancer [71]. In Supplementary Table 6, the column of lncRID and miRID denotes lncRNA ID and miRNA ID, respectively. Evi1 and Evi2 denote some authority database or published literature containing verified disease-lncRNA or disease-miRNA associations separately. \"#\" and \" * \" stand for databases of lncRNADisease and MNDR v2.0, respectively, which consist of known disease-lncRNA associations or contain published literatures to support the association between predicted lncRNAs and breast cancer. \"!,\" \"&,\" and \"+\" stand for databases of HMDD, miR2Disease, and miRCancer, respectively, which consist of known disease- Computational and Mathematical Methods in Medicine miRNA associations or contain published literature to support the association between predicted miRNAs and breast cancer. Particularly, \"Nan\" indicates that there is no database or no published literature to support the predicted results. From Supplementary Table 6, it is easy to see that all candidate disease-lncRNA associations have been verified in databases of the lncRNADisease and MNDR v2.0 or published papers containing these databases. And, in addition, there are 42 out of 50 candidate disease-miRNA associations having been reported by HMDD, miR2Disease, and miR-Cancer or published paper containing these databases. Moreover, we discovered that those novel miRNAs with miRID 35, 51, 73, 164, and 186 are related to some important factors affecting the development of breast neoplasms. Hence, it is obvious that we infer that these lncRNA-miRNA pairs may be associated with breast cancer.",
"In addition, colonic tumors are a type of malignancy that is common in the rectum and sigmoid borders [72]. Early colon cancer is difficult to detect because of its insignificant symptoms [73]. Unfortunately, the related literature reports that its incidence has been on the rise in recent years [74]. erefore, predicting potential miRNAs and lncRNAs associated with colon tumors is of great significance for the diagnosis of early colon cancer. In Supplementary Table 7, we have listed the top 30 candidate lncRNA-miRNA pairs predicted to be associated with colon tumors. Moreover, all of these candidate lncRNAs and most of these candidate miRNAs have been verified by lncRNADisease database and MNDR v2.0, respectively.",
"Moreover, prostate neoplasm is one of the most common cancers in white and African-American men, and it is reported that there are about one in six white men and one in five African-American men having prostate cancer in their lifetime. Recent researches have shown that prostate neoplasm is caused by the malignancy of prostate epithelial cells [75], its formation includes many factors such as age, family history, and race [76], and particularly, some miRNAs such as has-let-7a-5p and lncRNAs such as XIST have been found to be involved in the formation of prostate neoplasms successively. Hence, it is interesting to infer potential miRNAs and lncRNAs associated with prostate neoplasms. In Supplementary Table 8, we have listed the top 30 prostate neoplasm-related candidate lncRNA-miRNA pairs. Moreover, all of these candidate lncRNAs and most of these candidate miRNAs have been verified by lncRNADisease and MNDR v2.0, respectively.",
"Considering that there are some key parameters such as K and α, which may be significant to the performance of our prediction model PADLMHOOI, in this section, we will further estimate the effects of these key parameters to the prediction performance of PADLMHOOI. Firstly, we varied K from 1 to 10 during simulation. And, as a result, Table 2 illustrates the impacts of parameter K on the performance of PADLMHOOI. By observing Table 2, it is obvious that PADLMHOOI can achieve the maximum AUC value of 0.9708 while K � 8. And additionally, as for the impacts of the parameter α, considering the time costs, we set K � 3 and varied α from 0.1 to 0.9 during simulation. And as a result, Table 3 illustrates the impacts of parameter α on the performance of PAD-LMHOOI. By observing Table 3, it is obvious that PAD-LMHOOI can achieve the maximum AUC value of 0.9591 while α � 0.7.",
"Researches on prediction of potential associations between lncRNA-miRNA pairs and diseases not only are helpful in understanding the disease mechanisms on lncRNA and miRNA levels but also play an important role in the detection of disease biomarkers, diagnosis, prognosis, and prevention. However, to our knowledge, although there are many researches having demonstrated that lncRNA-miRNA interactions are associated with the development of complex diseases, up to now, there are few models having been proposed for large-scale forecasting potential associations between diseases and lncRNA-miRNA pairs. Since traditional biological experiments are quite expensive and timeconsuming, in this paper, based on the existing disease-miRNA associations, disease-lncRNA associations, lncRNA-miRNA interactions, and the assumption that genes with similar functions are often associated with similar diseases; we firstly constructed a three-order tensor T by adopting the method of WKNNP, and then, based on the method of tensor factorization, we further proposed a prediction model called PADLMHOOI to infer potential relations between diseases and lncRNA-miRNA pairs. And thereafter, simulation results under the frameworks of global and local LOOCV, 2-fold CV, and 10-fold CV, all confirmed the superiority of PADLMHOOI. Moreover, case studies of breast neoplasms, colon neoplasms, and prostate neoplasms further demonstrate that our model PADLMHOOI is an effective method for predicting potential disease-associated lncRNA-miRNA pairs. Certainly, there are still some limitations in PADLMHOOI. For example, although a large number of datasets have been integrated in PADLMHOOI, the amount of data available is still not enough; it is obvious that the prediction performance of PADLMHOOI will be better if more datasets can be collected. And in addition, in this paper, we only predicted the association between disease and a single lncRNA-miRNA pair. In the future, we will further modify PADLMHOOI to predict potential associations between diseases and multiple lncRNA-miRNA pairs.",
"PADLMHOOI: Prediction of potential associations between diseases and lncRNA-miRNA pairs based on the higher-order orthogonal iteration.",
"e data used to support the findings of this study are available from the corresponding author upon request.",
"e authors declare no conflicts of interest.",
"Supplementary 7. Table 6: the candidate lncRNA-miRNA pairs associated with breast cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with breast cancer.",
"Supplementary 8. Table 7: the candidate lncRNA-miRNA pairs associated with colon cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with colon cancer.",
"Supplementary 9. Table 8: the candidate lncRNA-miRNA pairs associated with pprostate cancer. In addition, the LncRNADisease and MNDR v2.0 databases have confirmed that these lncRNAs or miRNAs are associated with colon cancer. "
] | [] | [
"Introduction",
"Materials and Methods",
"Construction of the Bipartite Network of Disease-lncRNA.",
"Data integration and association network construction",
"Tensor decomposition",
"Construction of the Bipartite Network of Disease-miRNA.",
"Construction of the Bipartite Network of lncRNA-miRNA.",
"Construction of the Tripartite",
"Construction of the Disease-lncRNA-miRNA Tensor.",
"Calculation of the Gaussian Interaction Profile Kernel",
"Calculation of the Similarity of lncRNA Pairs (lncSim)",
"Calculation of the lncRNA Functional Similarity",
"Calculation of the Gaussian Interaction Profile Kernel",
"Calculation of the Gaussian Interaction Profile Kernel",
"PADLMHOOI.",
"Computational and Mathematical Methods in Medicine",
"Results and Analysis",
"Leave-One-Out Cross-Validation (LOOCV).",
"Performance Comparison with Other Methods.",
"Recall Ratio Analysis.",
"Case Studies.",
"Parameter Sensitivity Analysis.",
"Discussion and Conclusion",
"Abbreviations",
"Data Availability",
"Conflicts of Interest",
"T",
"Step 3 . 3 Figure 1 :",
") 2 . 9 .",
"Figure 2 :",
"Figure 3 :",
"Figure 4",
"Figure 6",
"Table",
"Table 6 ,",
"Table 2 :",
"Table 3 :"
] | [
"we have listed the top \n",
"K \n1 \n2 \n3 \n4 \n5 \n6 \n7 \n8 \n9 \n10 \nAUC \n0.9660 \n0.9649 \n0.9591 \n0.9607 \n0.9666 \n0.9657 \n0.9675 \n0.9708 \n0.9703 \n0.9703 \n\n",
"α \n0.1 \n0.2 \n0.3 \n0.4 \n0.5 \n0.6 \n0.7 \n0.8 \n0.9 \nAUC \n0.9545 \n00.9565 \n0.9582 \n0.9586 \n0.9583 \n0.9585 \n0.9591 \n0.9587 \n0.9539 \n"
] | [
"(Supplementary Table 1",
"(Supplementary Table 2",
"(Supplementary Table 3",
"Table 1",
"Supplementary Table 4",
"Table 5",
"Table 6",
"Table 6",
"Supplementary Table 7",
"Supplementary Table 8, we have listed the top 30 prostate",
"Table 2",
"Table 2",
"Table 3",
"Table 3",
"Table 6",
"Table 7",
"Table 8"
] | [
"A Novel Method for Predicting Disease-Associated LncRNA-MiRNA Pairs Based on the Higher-Order Orthogonal Iteration",
"A Novel Method for Predicting Disease-Associated LncRNA-MiRNA Pairs Based on the Higher-Order Orthogonal Iteration"
] | [] |
229,686,191 | 2022-06-07T06:59:44Z | CCBY | https://www.mdpi.com/1422-0067/21/24/9661/pdf | GOLD | 116ffb9cea4f970282a2a0df243586504762e89c | null | null | null | null | 10.3390/ijms21249661 | 3108611207 | 33352896 | 7765816 |
Molecular Sciences Mitophagy and the Brain
Natalie S Swerdlow
Alzheimer's Disease Center
University of Kansas
University of Kansas
66160Kansas CityKSUSA
Heather M Wilkins [email protected]
Alzheimer's Disease Center
University of Kansas
University of Kansas
66160Kansas CityKSUSA
Department of Neurology
Medical Center
University of Kansas
66160Kansas CityKSUSA
Department of Biochemistry and Molecular Biology
Medical Center
University of Kansas
66160Kansas CityKSUSA
International Journal
Molecular Sciences Mitophagy and the Brain
10.3390/ijms21249661Received: 2 December 2020; Accepted: 17 December 2020; Published: 18 December 2020Review * Correspondence:mitochondriaAlzheimer's Diseasemitophagyneurodegenerationaging
Stress mechanisms have long been associated with neuronal loss and neurodegenerative diseases. The origin of cell stress and neuronal loss likely stems from multiple pathways. These include (but are not limited to) bioenergetic failure, neuroinflammation, and loss of proteostasis. Cells have adapted compensatory mechanisms to overcome stress and circumvent death. One mechanism is mitophagy. Mitophagy is a form of macroautophagy, were mitochondria and their contents are ubiquitinated, engulfed, and removed through lysosome degradation. Recent studies have implicated mitophagy dysregulation in several neurodegenerative diseases and clinical trials are underway which target mitophagy pathways. Here we review mitophagy pathways, the role of mitophagy in neurodegeneration, potential therapeutics, and the need for further study. Int. J. Mol. Sci. 2020, 21, 9661 2 of 26 neurodegenerative diseases. This could lead to the disruption of mitophagy processes and requires more research efforts to understand. The inner membrane space of mitochondria houses enzymes and allows for proton storage and protein folding. The mitochondrial inner membrane contains the ETC and ATP synthase enzymes and the mitochondrial matrix stores enzymes for the TCA cycle, mitochondrial DNA (mtDNA), and other crucial enzymes for protein folding and maintenance of pH gradients. For more detailed analysis of mitochondrial localized proteins MitoCarta3.0 was recently published[8].Synaptic loss is strongly correlated with cognitive deficits and motor dysfunction[9][10][11][12]. Mitochondria are essential for synaptic function and neurotransmitter synthesis, release, and uptake[13][14][15]. Accumulation of damaged mitochondria could lead to synaptic dysfunction and neurodegeneration. Mitophagy may play a role in ensuring synaptic mitochondrial integrity by degrading damaged mitochondria.Mitochondria evolved from a prokaryotic endosymbiont. As such, mitochondria share characteristics with bacteria including a double membrane, circular DNA, formyl-methionine amino acids, and cardiolipin [1]. In certain contexts failure of mitophagy pathways could lead to the release of mitochondrial components into the extracellular space, activation of a damage-associated molecular response (DAMP), and inflammation[1,5]. Mitochondria are also master regulators of cell death pathways (such as apoptosis and necrosis)[4,5,16]. As a by-product of the respiratory chain function superoxide radicals are produced. These free radicals generate multiple species of reactive oxygen species (ROS) and reactive nitrogen species (RNS). During periods of mitochondrial dysfunction and failure of mitochondrial quality control mechanisms (such as mitophagy) ROS/RNS can induce damage to cellular macromolecules and necrotic cell death[4,5,16]. Proper control and coordination of mitophagy pathways are crucial to prevent cell death and inflammation.Mitophagy is imperative for glial cell function. Signaling between microglia, astrocytes, and neurons are modulated by mitophagy pathways. Novel data show that transcellular mitophagy pathways occur within the brain. Transcellular mitophagy is a process by which cells release mitochondria for engulfment and mitophagy in surrounding cell types. Dysregulation of this process can lead to neuroinflammation and loss of proteostasis[17][18][19].Disruption of mitophagy is observed with aging and in many neurodegenerative diseases. Recent advances have described novel mechanisms of mitophagy within the central nervous system (CNS). Here, we will review current knowledge of mitophagy regulation, its role in neurodegenerative disease, and therapeutic potential.For this review article, we used PubMed, clinicaltrials.gov, and Google Scholar to identify studies related to mitophagy, AD, PD, ALS, and MS. We used search terms including mitophagy, mitophagy and neurodegeneration, mitophagy and AD, mitophagy and PD, mitophagy and MS, mitophagy and ALS, mitochondria and neurodegeneration, and autophagy.Mitophagy: Autophagy for MitochondriaAutophagosomes can be derived from membranes of endoplasmic reticulum (ER), Golgi, mitochondria, or plasma membrane [20][21][22][23][24]. The biogenesis of autophagosomes involves the formation of the isolation membrane (IM), elongation and maturation, closure, and then fusion with the lysosome to form the autolysosome. The first step in autophagosome biogenesis is the activation of the Unc-51-like kinase 1 complex (ULK1; pre-initiation complex), which contains ULK1, autophagy-related proteins 13 and 101 (Atg13, Atg101), and focal adhesion kinase family interacting partner 200 (FIP200) [25][26][27][28]. This complex recruits the class III phosphatidylinositide 3-kinase (PI3K) Vps34 complex (Beclin1, autophagy-related protein 14 (Atg14), autophagy and beclin 1 regulator 1 (Ambra1), and vascular protein sorting 34 and 15 (Vps34 and Vps15)) to produce phosphatidylinositol 3-phosphate (PI3P), also called the initiation complex [25,27,29,30]. PI3P binding proteins, FYVE domain containing proteins (DFCP1), and WD repeat protein interacting with phosphoinositide (WIPIs) localize to the IM [26]. All of this culminates in the formation of the omegasome and IM. Autophagy-related proteins 12, 5, and 16 (Atg12, Atg5, and Atg16) and LC3-phosphatidylethanolamine (PE) facilitate the elongation and closure of the IM, following fusion with the lysosome [25,26,31]. Int. J. Mol. Sci. 2020, 21, 9661 3 of 26Mitochondrial quality control mechanisms include mitochondrial chaperones, the mitochondrial unfolded protein response (UPR mt ), degradation of mitochondrial proteins in the cytoplasm via the proteasome (p97, 26S proteasome), removal of damaged proteins via mitochondrial derived vesicles (MDVs), and mitophagy (Figure 1) [32]. Misfolded mitochondrial proteins can be refolded by mitochondrial chaperone proteins (heat shock proteins 22, 60, and 70) or cleaved/degraded by mitochondrial proteases Lon and Clp through the UPR mt [33][34][35][36][37]. Damaged mitochondrial proteins can also be targeted specifically for proteasome degradation by p97 through the proteasome [38]. MDVs bud off mitochondria after engulfing damaged mitochondrial macromolecules. MDVs are associated with impaired mitochondrial import channels. These MDVs are either degraded by lysosomes, peroxisomes, or exocytosed [39,40]. Mitophagy (reviewed extensively below) functions to remove damaged mitochondria with the goal of preventing cell death. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 27 Int. J. Mol. Sci. 2020, 21, 9661 4 of 26 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 27 Figure 2. Mitophagy. (A) Non-Receptor Mediated Mitophagy or Classical Mitophagy. Activation of PINK1 leads to recruitment of ubiquitin and Parkin. Parkin ubiquitinates and phosphorylates mitochondrial proteins (such as VDAC, MFN1/2, and TOM20) and this initiates receptor adaptor protein recruitment (p62, NDP52, OPTN, TAX1BP1, and NBR1). These adaptor proteins interact with LC3 to form the autophagosome. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50] (B) Receptor Mediated Mitophagy. Mitochondrial receptor proteins (BNIP3, NIX, FUNC1, AMBRA1, PHB2, or cardiolipin) are ubiquitinated and phosphorylated. This facilitates their interaction with LC3 and GABARAP for autophagosome formation. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50]. Created with BioRender.com.
Introduction
Mitochondria are essential organelles that regulate energy homeostasis, cell signaling, and cell death [1][2][3]. During threatened cell death or nutrient starvation, mitochondria can be degraded and recycled through mitophagy. Mitophagy is a specific form of autophagy, a process where cell contents are degraded and recycled. In a broad sense, mitophagy involves tagging mitochondria for removal, engulfment of the organelle by an autophagosome, and degradation in a lysosome. There are several pathways which control, initiate, and facilitate mitophagy. The many facets of mitochondrial function contribute to mitophagy pathways.
Mitochondria coordinate and balance energy production through beta oxidation, the citric acid cycle (TCA cycle), and oxidative phosphorylation at the electron transport chain (ETC). Beta oxidation is a catabolic pathway where free fatty acids are converted to acetyl coA, which enter the TCA cycle and ultimately oxidative phosphorylation. In the TCA cycle, either pyruvate (from glycolysis) or acetyl coA are oxidized to generate the high energy electron carriers, NADH and FADH 2 . NADH and FADH 2 enter the ETC at complex I and complex II, respectively. These high energy electron carriers undergo oxidation/reduction reactions in the ETC in order to pump protons into the matrix. These protons ultimately power ATP synthase (or Complex V) for the generation of ATP from ADP. These bioenergetic reactions maintain the mitochondrial electrochemical gradient, or mitochondrial membrane potential. Mitochondrial membrane potential is the main signal which either inhibits or initiates mitophagy.
Mitochondria are double membrane organelles. The outer mitochondrial membrane is imperative for mitophagy function [4][5][6][7]. Transport and signaling proteins localize to the outer mitochondrial membrane to facilitate protein and metabolite import. As discussed in more detail below, aggregation-prone proteins are known to block these import channels on the outer mitochondrial membrane in some phosphatidylethanolamine (PE) facilitate the elongation and closure of the IM, following fusion with the lysosome [25,26,31].
Mitochondrial quality control mechanisms include mitochondrial chaperones, the mitochondrial unfolded protein response (UPR mt ), degradation of mitochondrial proteins in the cytoplasm via the proteasome (p97, 26S proteasome), removal of damaged proteins via mitochondrial derived vesicles (MDVs), and mitophagy ( Figure 1) [32]. Misfolded mitochondrial proteins can be refolded by mitochondrial chaperone proteins (heat shock proteins 22, 60, and 70) or cleaved/degraded by mitochondrial proteases Lon and Clp through the UPR mt [33][34][35][36][37]. Damaged mitochondrial proteins can also be targeted specifically for proteasome degradation by p97 through the proteasome [38]. MDVs bud off mitochondria after engulfing damaged mitochondrial macromolecules. MDVs are associated with impaired mitochondrial import channels. These MDVs are either degraded by lysosomes, peroxisomes, or exocytosed [39,40]. Mitophagy (reviewed extensively below) functions to remove damaged mitochondria with the goal of preventing cell death.
Non-Receptor Mediated Mitophagy
Non-receptor mediated mitophagy ( Figure 2) or classical mitophagy involves PTEN-induced kinase 1 (PINK1) and Parkin. PINK1 is normally degraded in the inner mitochondrial membrane (IMM) by PARL (presenilin-associated rhomboid-like protease) [41,42]. During mitophagy induction, PINK1 becomes active and accumulates on the outer mitochondrial membrane (OMM), where it recruits Parkin and ubiquitin through phosphorylation [43][44][45][46][47][48][49][50][51]. Parkin accumulates and polyubiquitinates mitochondrial proteins triggering proteasomal degradation. Parkin ubiquitination of OMM proteins leads to more PINK1 activity and phosphorylation of substrates, including the recruitment of additional Parkin proteins at the OMM, creating a positive feedback loop. OMM proteins mitofusins 1 and 2 (MFN1/2), voltage-dependent anion channel 1 (VDAC1), and translocase of the outer mitochondrial membrane 20 (TOM20) are ubiquitinated by Parkin [44,[46][47][48][49][50][51][52][53][54][55]. Ubiquitination of MFN1/2 blocks mitochondrial fusion allowing for the isolation of damaged mitochondria and smaller mitochondria facilitate autophagosome targeting [6,32,49,56].
Non-Receptor Mediated Mitophagy
Non-receptor mediated mitophagy ( Figure 2) or classical mitophagy involves PTEN-induced kinase 1 (PINK1) and Parkin. PINK1 is normally degraded in the inner mitochondrial membrane (IMM) by PARL (presenilin-associated rhomboid-like protease) [41,42]. During mitophagy induction, PINK1 becomes active and accumulates on the outer mitochondrial membrane (OMM), where it recruits Parkin and ubiquitin through phosphorylation [43][44][45][46][47][48][49][50][51]. Parkin accumulates and polyubiquitinates mitochondrial proteins triggering proteasomal degradation. Parkin ubiquitination of OMM proteins leads to more PINK1 activity and phosphorylation of substrates, including the recruitment of additional Parkin proteins at the OMM, creating a positive feedback loop. OMM proteins mitofusins 1 and 2 (MFN1/2), voltage-dependent anion channel 1 (VDAC1), and translocase of the outer mitochondrial membrane 20 (TOM20) are ubiquitinated by Parkin [44,[46][47][48][49][50][51][52][53][54][55]. Ubiquitination of MFN1/2 blocks mitochondrial fusion allowing for the isolation of damaged mitochondria and smaller mitochondria facilitate autophagosome targeting [6,32,49,56].
Autophagy adaptor proteins are recruited to the OMM by ubiquitinated proteins. These adaptors include neighbor BRCA1 gene (NBR1), nuclear dot protein 52 (NDP52), optineurin (OPTN), sequestosome-1 (SQSTM1/p62), and Tax1-binding protein (TAX1BP1) [23,52,[57][58][59][60][61][62][63][64]. Adaptor proteins recruit and interact with autophagosome proteins; gamma-aminobutyric acid receptorassociated protein (GABARAP) or microtubule-associated protein 1A/1B-light chain 3 (LC3) to mediate the formation of the mitophagosome and lysosomal fusion/degradation. These adaptor proteins interact with LC3 and GABARAP through LC3 interacting regions (LIR) motifs (W/F/YxxL/I) [6,7,23,32]. The importance of different adaptor proteins has been up for debate and often their Activation of PINK1 leads to recruitment of ubiquitin and Parkin. Parkin ubiquitinates and phosphorylates mitochondrial proteins (such as VDAC, MFN1/2, and TOM20) and this initiates receptor adaptor protein recruitment (p62, NDP52, OPTN, TAX1BP1, and NBR1). These adaptor proteins interact with LC3 to form the autophagosome. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50] (B) Receptor Mediated Mitophagy. Mitochondrial receptor proteins (BNIP3, NIX, FUNC1, AMBRA1, PHB2, or cardiolipin) are ubiquitinated and phosphorylated. This facilitates their interaction with LC3 and GABARAP for autophagosome formation. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50]. Created with BioRender.com.
Autophagy adaptor proteins are recruited to the OMM by ubiquitinated proteins. These adaptors include neighbor BRCA1 gene (NBR1), nuclear dot protein 52 (NDP52), optineurin (OPTN), sequestosome-1 (SQSTM1/p62), and Tax1-binding protein (TAX1BP1) [23,52,[57][58][59][60][61][62][63][64]. Adaptor proteins recruit and interact with autophagosome proteins; gamma-aminobutyric acid receptor-associated protein (GABARAP) or microtubule-associated protein 1A/1B-light chain 3 (LC3) to mediate the formation of the mitophagosome and lysosomal fusion/degradation. These adaptor proteins interact with LC3 and GABARAP through LC3 interacting regions (LIR) motifs (W/F/YxxL/I) [6,7,23,32]. The importance of different adaptor proteins has been up for debate and often their involvement in mitophagy is context-dependent. The same is true for Parkin, as there are some pathways of mitophagy which are Parkin-independent and these are discussed below [23,44].
Receptor Mediated Mitophagy
Receptor mediated mitophagy ( Figure 2) is driven by mitochondrial receptor proteins which contain LIR motifs (W/F/YxxL/I) [23,50]. Outer mitochondrial membrane proteins, autophagy and Beclin 1 regulator 1 (AMBRA1), Bcl-2 interacting partner 3 (BNIP3), FUN14 domain-containing protein 1 (FUNDC1), Nip3-like protein X (NIX); and inner mitochondrial membrane proteins cardiolipin and prohibitin 2 (PHB2) are the most studied receptors which mediate mitophagy [23,50,[65][66][67][68][69][70].
Receptor mediated mitophagy is activated under specific conditions. For example, during hypoxia BNIP3 and NIX transcription are activated by hypoxia inducible factor 1 alpha (HIF1α) [70][71][72][73]. BNIP3 and NIX activity are regulated by phosphorylation where increased phosphorylation increases their binding affinity for LC3 [74,75]. Hypoxia also promotes FUNDC1 binding to LC3 through dephosphorylation via phosphoglycerate mutase family member 5 phosphatase (PGAM5) [65,67,76,77]. Conversely, FUNDC1 phosphorylation by ULK1 is also a mitophagy activating event. Ultimately ubiquitination of FUNDC1 by E3 ubiquitin protein ligase 5 (UBR5) promotes lysosomal degradation of mitochondria [65,67,78]. The inner mitochondrial membrane receptors, PHB2 and cardiolipin have been shown to interact the LC3, especially during times of mitochondrial permeabilization [79,80]. AMBRA1 is sequestered and inhibited by B-cell lymphoma protein 2 (Bcl-2) on the outer mitochondrial membrane but upon mitophagy activated AMBRA1 binds LC3 in a Parkin-dependent or -independent manner [81].
Receptor mediated mitophagy culminates in the elongation of and closure of phagophore membranes, resulting in engulfment of the mitochondria. The elongation and closure of the phagophore is driven by the mitochondrial receptor binding LCR and/or GABARAP, leading to closure of the phagophore by GABARAP [23,50]. Lastly, the autophagosome fuses with a lysosome for degradation.
Mitoptosis
Separate from mitophagy pathways, damaged mitochondria can be partitioned and removed through mitoptosis. This phenomenon was first proposed in 1992 [82]. Mitoptosis has several proposed definitions. One possible process of mitoptosis occurs when damaged mitochondria gather around the nucleus, are selectively partitioned into lipid membranes and extruded from the cell [83]. A separate definition is when mitochondria undergo condensation with swelling and fragmentation of cristae. This leads to the bursting of the outer mitochondrial membrane and fragmented cristae are extruded into the cytoplasm. In other forms of mitoptosis the outer mitochondrial membrane can remain intact and the cristae deteriorate through refraction and coalescence [6,[83][84][85][86][87]. The main benefit of mitoptosis is to prevent opening of the mitochondrial permeability transition pore (MPTP) and apoptosis. Overall, the method of which cells dispose of damaged mitochondria or part of mitochondria can vary and requires further study. Cell-specific pathways which evolved to provide the least devastating consequences based on cell and tissue function likely exist.
Mitoptosis does not require extramitochondrial signaling or protein complexes according to current knowledge. Some evidence suggests PINK1 and Parkin are involved, but no consensus currently exists [6,[83][84][85][86][87][88]. Situations which likely activate mitoptosis include mitochondrial membrane depolarization, increased ROS production, and degradation of mtDNA.
Transcellular Mitophagy
Recent studies have described exocytosis of mitochondria from cells followed by endocytosis or phagocytosis of these extracellular mitochondria. In the brain neurons release mitochondria at synapses, and these extracellular mitochondria were taken up by glial cells for phagocytosis [17,18]. This phenomenon will be referred to as transcellular mitophagy here ( Figure 3). A recent study showed that retinal ganglion axons shed mitochondria, which were then then degraded by adjacent astrocytes in mice [18]. Mitochondria might undergo degradation in the axoplasm (cytoplasm of the nerve axon) with the assistance of axonal lysosomes [17]. Essentially instead of being degraded in the soma transcellular mitophagy describes the fact that axonal mitochondria are instead enclosed by axoplasmic membranes that are shed and degraded by neighboring cells. showed that retinal ganglion axons shed mitochondria, which were then then degraded by adjacent astrocytes in mice [18]. Mitochondria might undergo degradation in the axoplasm (cytoplasm of the nerve axon) with the assistance of axonal lysosomes [17]. Essentially instead of being degraded in the soma transcellular mitophagy describes the fact that axonal mitochondria are instead enclosed by axoplasmic membranes that are shed and degraded by neighboring cells. [8,9]. Created with BioRender.com.
The notion that transcellular mitophagy occurs is logical given that it is not energetically favorable for neurons to bring mitochondria from dendrites back to the cell body for mitophagy. The process of transcellular mitophagy requires more study and understanding at the basic level and with regards to disease.
In addition to transcellular mitophagy, the observation of glial cells transferring mitochondria to neurons has been documented. In stroke models, glial cells transfer mitochondria to neurons as a likely means to protect neurons from energy stress and hypoxia [89,90]. The specific pathways which facilitate mitochondrial transfer between cells are unknown. However, some studies suggest that in astrocytes, glial acidic fibrillary protein (GFAP), and in neurons, uncoupling protein 2 (UCP2) may play a role [91,92].
In mesenchymal stem cells, connexins (particularly connexin 43) oligomerize to form Gap junctions. These Gap junctions may facilitate the formation of tunneling nanotubules, which allow the exchange of cellular contents, such as mitochondria [93]. Other studies suggest Miro1, a protein which connects cytoskeletal motor proteins to mitochondria is involved in mitochondrial transfer between cells [94]. Finally, S100A4 guides tunneling nanotubule growth [95]. The phenomenon of mitochondrial transfer between cells has been documented in a wide variety of model and tissue types [17,89,90,[92][93][94][95][96][97][98][99][100][101]. An important question remains regarding what initiates mitochondrial transfer and the specific mechanisms which control this process. [8,9]. Created with BioRender.com.
The notion that transcellular mitophagy occurs is logical given that it is not energetically favorable for neurons to bring mitochondria from dendrites back to the cell body for mitophagy. The process of transcellular mitophagy requires more study and understanding at the basic level and with regards to disease.
In addition to transcellular mitophagy, the observation of glial cells transferring mitochondria to neurons has been documented. In stroke models, glial cells transfer mitochondria to neurons as a likely means to protect neurons from energy stress and hypoxia [89,90]. The specific pathways which facilitate mitochondrial transfer between cells are unknown. However, some studies suggest that in astrocytes, glial acidic fibrillary protein (GFAP), and in neurons, uncoupling protein 2 (UCP2) may play a role [91,92].
In mesenchymal stem cells, connexins (particularly connexin 43) oligomerize to form Gap junctions. These Gap junctions may facilitate the formation of tunneling nanotubules, which allow the exchange of cellular contents, such as mitochondria [93]. Other studies suggest Miro1, a protein which connects cytoskeletal motor proteins to mitochondria is involved in mitochondrial transfer between cells [94]. Finally, S100A4 guides tunneling nanotubule growth [95]. The phenomenon of mitochondrial transfer between cells has been documented in a wide variety of model and tissue types [17,89,90,[92][93][94][95][96][97][98][99][100][101]. An important question remains regarding what initiates mitochondrial transfer and the specific mechanisms which control this process.
Mitophagy in Aging and Neurodegeneration
Changes in mitophagy flux, signaling, and mitochondrial function are observed with aging and in neurodegenerative diseases. It is imperative to understand the role mitophagy could contribute to brain aging and neurodegeneration. We discuss these implications below.
Aging
Aging is associated with a loss of proteostasis, mitochondrial dysfunction, genome instability, inflammation, changes to redox balance, and metabolic deficits [32]. Reduced autophagy and mitophagy are observed in models of aging [32,102].
Mitochondrial homeostasis and function are altered in aging. However, the exact mechanisms and findings are not consistent across models and studies. Brain cytochrome oxidase (COX; complex IV) and complex I activity are reduced while ROS production and oxidized proteins are increased in aged rats [103,104]. Calcium homeostasis is changed in aged rat brain mitochondria and synaptosomes; neither were able to take up calcium at a rate equivalent to young rats [105]. Aged mice have altered proteomic expression of glycolytic, TCA, and oxidative phosphorylation pathways in the brain but mitochondrial function is unchanged [106]. This suggests a compensatory mechanism during aging. Altered brain mitochondrial morphology is observed in aged rats and monkeys [107,108]. Other findings suggest a change to mtDNA epigenetic markers and increased mtDNA deletions in aged mouse brain [109,110]. Oxidative damage to brain mtDNA is related to reduced lifespan across numerous species (birds and mammals) [111,112]. mtDNA in the aged human brain shows increased somatic mutation burden and oxidative damage [113][114][115].
In multiple organisms, mitophagy is associated with longevity and lifespan. Pink1 knockout causes a shorter lifespan, while Parkin overexpression in neurons increases lifespan in D. melanogaster [45,116,117]. C. elegans models exposed to mild mitochondrial stress and upregulated mitophagy have extended lifespan [118,119]. Urolithin A (UA), tomatidine, and catechinic acid induce mitophagy and increase the lifespan of C. elegans models [120][121][122]. In an aging mouse model, stimulation of mitophagy with NAD + prolongs lifespan [123].
Mitochondrial quality control emerges as a central theme in most neurodegenerative diseases, including Alzheimer' Disease (AD), Parkinson's Disease (PD, Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS). Mitophagy stimulation has shown positive effects in models of these diseases.
Alzheimer's Disease
AD is the most common form of dementia diagnosed upon autopsy with neuropathological examination [124,125]. The pathological hallmarks which lead to AD diagnosis postmortem are considerable Aβ plaques and tau tangles throughout the brain [124][125][126][127]. Recent advances in neuroimaging have allowed the determination of Aβ plaque and tau tangle load in living subjects, showing these proteins accumulate in the brain decades before clinical signs of cognitive decline [126,[128][129][130].
One of the earlier observations in AD subjects was reduced glucose uptake/utilization in the brain via fluorodeoxyglucose (FDG)-positron emission tomography (PET) [128][129][130][131][132][133]. Accumulation of evidence supports an overall metabolic deficit in AD subjects both within the brain and systemically [134]. The mitochondrial ETC enzyme, COX (or complex IV), has reduced Vmax in the AD brain, fibroblasts, and blood samples [132,[135][136][137][138][139][140][141][142][143][144][145][146]. AD-like changes can be transferred to other cell types when AD patient mitochondria (mtDNA) are transferred [139,[147][148][149][150][151]. This process of creating cytoplasmic hybrid cells (cybrids) allows for the determination of the contribution of mtDNA on disease and cell physiological processes [150].
Mitochondria in AD autopsy brain samples have fragmented cristae and vary widely in size compared to age-matched non-demented brain samples [152]. Mitochondria within dendritic spines and presynaptic terminals show the most fragmented and disorganized cristae. Alterations to mitochondrial ultrastructure were observed in areas of the brain with and without Aβ and tau pathology (cerebellar cortex, hypothalamus, cerebellum, and visual cortex). In addition to altered mitochondrial morphology, presynaptic terminals had reduced synaptic vesicles and fragmentation of golgi cisternae was observed [152]. mtDNA inheritance confers risk to AD. Studies show that offspring of maternal AD subjects have a higher risk of AD diagnosis than offspring of paternal AD subjects. Nearly all mitochondria are inherited maternally. Offspring from maternal AD subjects show metabolic and neuroimaging changes earlier in life than offspring of paternal AD subjects [129,[153][154][155][156]. Furthermore, inherited mtDNA haplogroups are associated with both increased and decreased AD risk [157][158][159][160][161][162][163][164]. These inherited mtDNA haplogroups also interact with the nuclear DNA encoded risk factor, ApoE (apolipoprotein E) to influence AD risk [159,164,165]. Thus, it is important to understand the role of mitochondria, mitophagy, and metabolism in AD.
AD mouse models have disrupted mitophagy [166]. This is observed in tau transgenic mice and AD postmortem human brains with accumulated tau aggregates [167]. Mutant Amyloid Precursor Protein (APP; Swedish mutant) mice show increased mitochondrial fission proteins and decreased mitochondrial fusion and mitophagy protein expression in hippocampal neurons [168]. APPsw/PS1dE9 transgenic mice show increased LC3, PINK1, and Parkin expression [169]. Cortical neurons derived from AD transgenic mice (J20; Swedish and Indiana APP mutations) also show increased mitophagy protein expression with depolarized mitochondrial membrane potential [170].
In AD postmortem brain samples, accumulation of damaged mitochondria and autophagosome vacuoles is observed [171][172][173]. The UPR mt pathway is upregulated at the gene level, with reduced proteasomal activity through the 26S proteasome. Parkin, SQSTM1/p62, and LCR mitochondrial localization are increased [172][173][174][175][176]. Mitophagy pathways are altered in human postmortem AD brain. Cytosolic Parkin is depleted, and lysosomal deficits are observed. Impaired Parkin recruitment to mitochondria is possibly caused by tau-mediated sequestration of Parkin in the cytosol [170,177]. Defects in the activation of ULK1 and TBK1 lead to impaired mitophagy [177]. In AD, mitophagy increases or decreases depending on the part of the cell observed. Although it is increased in lysosomes, other parts of the cell fail at completing mitophagy.
Mechanisms of altered mitophagy and mitochondrial function warrant further study in AD, specifically given the strong association of mitochondria and mitophagy with synapse health and function.
Parkinson's Disease
PD is a neurodegenerative disease with both cognitive and neuromuscular changes. Motor deficits, tremors, rigidity, bradykinesia, dyssomnia, and depression are clinical hallmarks of PD. In some cases, PD can cause cognitive impairment. Within the brain, PD causes degeneration of dopaminergic neurons in the substantia nigra with Lewy body accumulation (composed of aggregating α-synuclein) [178][179][180].
Mitochondrial dysfunction is observed in PD. A complex I deficiency is noted in brain tissue (substantia nigra) but not in skeletal muscle [181,182]. The complex I deficiency might be brain-specific, but some studies suggest deficits in platelets of PD subjects [183,184]. These findings are dependent on the methodology used. Mitochondrial dysfunction is also observed in cybrid cell lines derived from transfer of PD subject mtDNA, suggesting mtDNA may play a role [150,[185][186][187]. Furthermore, PD patients have increased rates of mtDNA deletion in the substantia nigra [188,189].
Familial forms of PD are caused by mutations in genes involved in mitophagy. Mutations in PARK6 (encodes for PINK1), PARK2 (encodes for Parkin), PARK1/4 (α-synuclein), PARK7 (DJ1), PARK8 (LRRK2), PARK17 (Vsp35), and PARK9 (ATP13A2) genes are linked to familial PD [177,[190][191][192][193][194][195]. The role of PINK1, Parkin, and Vsp35 in mitophagy are well known and reviewed above. DJ1 and α-synuclein have been shown to modulate mitophagy either through direct interactions with PINK1 and Parkin or by causing mitochondrial fragmentation. Loss of function of LRRK2 and ATP13A2 have been shown to impair mitochondrial turnover.
Despite these genetic studies most PD cases are sporadic with no known genetic cause. Inhibition of complex I function with rotenone (a pesticide) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces PD in rodent and non-human primates [196][197][198][199][200]. Dopaminergic neurons form many synapses (up to one million per neuron), and thus, have a high bioenergetic demand to maintain these unmyelinated synapses [9,201]. In human postmortem brain, mitophagy markers (phosphorylated S65 ubiquitin) increase across age and with PD diagnosis [202]. This mitophagy marker also associated with Lewy Bodies, showing an increase of mitophagy in early PD stages and a decrease in late PD stages [202]. α-synuclein is also associated with an increase in Miro expression in postmortem human brain tissue, human neurons, and fly models of PD [203]. Reducing Miro in the human neuronal and fly models rescued neurodegeneration and mitophagy [203].
Overall, PD patients and disease models consistently show mitochondrial and mitophagy dysfunction.
Multiple Sclerosis
MS is a neurodegenerative disease marked by an autoimmune response against the myelin sheath. Demyelination of white matter is caused by autoreactive T cells which target the myelin sheath within the central nervous system. This demyelination leads to a secondary loss of neuronal axons and neurodegeneration. MS has no known genetic cause and occurs in young adults with a higher incidence in females (3:1 ratio female to male) [204,205]. Most MS subjects (85%) have a relapse-remission disease course, which includes periods of demyelination followed by neurological recovery. Eventually a secondary progressive course occurs with few remission periods [204,205]. A smaller subset of MS patients (10-15%) show continued progression known as primary progressive MS.
MS pathology begins with the formation of a lesion with acute inflammation, which transitions to a state of chronic inflammation followed by neurodegeneration. Chronic inflammation is believed to allow penetration of the blood brain barrier by activated T cells directed against the myelin sheath.
The inflammation observed in MS shows activation of both innate and adaptive immunity pathways [204,205]. In addition to demyelination, loss of oligodendrocytes is observed. Myelin autoreactive T-cells can induce experimental autoimmune encephalomyelitis (EAE) in animal models and human MS subject genome wide association studies (GWAS) have a high representation of immune genes related to T cell differentiation [204,205]. Gray matter lesions and brain atrophy are present before clinical MS onset. These findings highlight the lack of understanding of what ultimately initiates the autoimmune reaction in MS.
Mitochondria and mitophagy are critical for immune signaling. ROS signals from mitochondria are known to modulate inflammatory responses. ROS signals in MS damage myelin and the blood brain barrier, further exacerbating disease. Oxidation of phospholipids and DNA damage are observed during periods of chronic inflammation with disruption of neuronal axons. Chronic inflammation induces damage to macromolecules including mtDNA, ETC protein, and lipids [204]. Systemic mitochondrial changes are observed in MS. Peripheral lymphocytes (mostly T cells) show increased mitochondrial superoxide production, decreased ETC protein expression, increased lactate, and decreased antioxidant capacity in human MS subjects [206]. These findings suggest a mitochondrial and bioenergetic deficit in MS.
Of interest, MS subjects show decreased expression of COX5B [204]. Active lesions from MS subjects show reductions in COX and its catalytic component COX-I; decreased expression was explicitly observed in oligodendrocytes, astrocytes, and neuronal axons [207]. A separate study showed reduced COX activity in lesions from MS patients; they also observed correlation of this endpoint with neurofilament protein (SMI32) expression and with macrophage/microglial density [208]. An axon-specific protein, syntaphilin, which functions as a mitochondrial docking protein, was increased in chronic lesions [208]. Inactive lesion areas in the same MS subjects had elevated COX activity and increased mitochondrial mass [208]. Gene expression analysis of cortex tissue from MS subjects show overall reductions in nuclear encoded mitochondrial genes and ETC complex expression specifically in neurons [209]. Overall, mitochondrial deficits are observed in MS and the role of these deficits requires further study.
Autophagy and mitophagy pathways are altered in MS human subjects and animal models, which mimic MS pathology. Atg5 modulates T cell survival and its expression correlates with clinical disability in mouse models of EAE. In MS brain samples, encephalitogenic T cells appear to be the major source of Atg5 expression and systemic T cells in human MS subjects showed increased Atg5 expression [210]. In MS brain lesions from human subjects, Lamp2 and LC3II/I ratios are decreased, suggesting impaired autophagy [211]. Further studies have shown serum and CSF concentrations of Atg5 are elevated in MS patients. This study also noted increased expression of Parkin in both serum and CSF with higher serum levels of lactate [212]. In addition, blood from MS subjects show altered expression of several autophagy-related genes (these included ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, and HTT) [213]. Autophagy and mitophagy are imperative for immune cell function, differentiation, and adaptive immunity. The role of these pathways in driving autoreactive T cell differentiation in MS needs to be understood.
Amyotrophic Lateral Sclerosis
ALS is a neurodegenerative disease marked by the loss of alpha motor neurons in the lumbar spinal cord and motor cortex [214,215]. The lifespan post ALS diagnosis is short, often 2-3 years, because progressive muscle wasting leads to lung paralysis. In some cases of ALS, dementia is present [216]. Most ALS cases are sporadic, with a rare subset (less than 5% of total cases) being familial. These familial cases are caused by mutations in genes including Tdp43, Fus, Fig4, Ang, Vapb, and C9orf72 [217][218][219][220]. Mutations in the Optn gene which encodes an autophagy protein, optineurin, were found to be causative of ALS in 2010 [221][222][223]. After the discovery of mutations in Sod1 and Tdp43, transgenic mouse models were developed [224][225][226].
Changes to mitochondrial ultrastructure in human ALS subjects were revealed several decades ago [227][228][229]. Cytoplasmic inclusions that may represent mitochondria-containing autophagic vacuoles are observed in ALS motor neurons [230,231]. Although ALS neurodegeneration is anatomically specific, mitochondrial abnormalities are found systemically [214,215,227,229,232,233]. Mitochondrial dysfunction is present in platelet and muscle mitochondria from ALS subjects [227,229,[234][235][236][237][238]. mtDNA may contribute to ALS pathologies, as cybrid cells harboring mtDNA from ALS subjects often show mitochondrial abnormalities and increased cell death [150,229,239,240].
ALS is modeled using rodents that express mutant SOD1 or mutant TDP43 [224,226]. SOD1 is a cytoplasmic enzyme which was identified within mitochondrial membranes [225,229,[241][242][243]. This is the case for both mutant SOD1 and to a lesser extent wild type SOD1. Mutant SOD1 ALS transgenic mice have altered mitochondrial morphology and mitochondrial SOD1 accumulation [242,243]. This raises the possibility that mutant SOD1 may drive neurodegeneration by damaging mitochondria. TDP43 mutants are also observed within mitochondria and appear to induce mitochondrial dysfunction [242][243][244][245][246]. Both TDP43 and SOD1 are known to aggregate within motor neurons and muscle; TDP43 interacts with proteins critical to mitophagy in an inhibitory manner [219,244,245,[247][248][249][250][251].
Impaired mitophagy was proposed to be involved in the denervation of neuromuscular junctions in an ALS mouse model [252]. Lysosomal dysfunction has also been implicated in ALS. Specifically, lysosomal deficits result in an abnormal accumulation of autophagic vacuoles that engulf damaged mitochondria within the motor neuron axons of G93A SOD1 ALS mice [177]. Impaired mitochondrial turnover along with the accumulation of misfolded proteins and protein aggregates contributes to ALS-linked mitochondrial dysfunction and motor neuron death. Mitochondrial and mitophagy ultrastructure are varied across compartments of motor neurons [253,254]. Parkin, Miro1, and Mfn2 are depleted in an ALS mouse model (G93A SOD1); however, mitochondrial localized p62 is upregulated [255]. Mutant forms of optineurin interfere with Parkin ubiquitin ligase function [256].
In human post-mortem samples, increased autophagic vesicles are observed in lumbar spinal cord motor neurons [257]. Induced pluripotent stem cell (iPSC)-derived motor neurons from familial ALS subjects with C9orf72 mutations or haploinsufficiency have dysfunction of autophagy pathways [258,259].
As discussed above mitochondrial dysfunction and mitophagy alterations are prevalent in human AD, PD, MS, and ALS samples as well as cell and animal models of disease. We discuss below the methods being investigated to modulate mitophagy.
Modulating Mitophagy in Neurodegeneration
Increasing mitophagy in transgenic mouse models of neurodegeneration have shown mostly beneficial effects. In AD models (iPSC derived, transgenic mice, and C. elegans), increasing mitophagy using nicotinamide mononucleotide (NMN), UA, or actinonin (AC) reduced Aβ and tau aggregation. In AD transgenic mice, mitophagy induction benefited cognition [260,261]. These compounds are NAD + precursors, which may drive mitophagy through alterations in redox balance (NAD + /NADH). UA likely drives mitophagy through a PINK1/Parkin/Nix axis.
Broad autophagy induction with Rilmenidine in the G93ASOD1 mouse model of ALS did not change disease progression [262]. The mechanism(s) of Rilmenidine autophagy/mitophagy induction are currently unknown. Rapamaycin (an mTOR inhibitor) treatment of this same mouse model was detrimental unless mature lymphocytes were depleted [263]. These studies highlight the importance of understanding the non-cell autonomous effects of autophagy and mitophagy pathways.
In PD rodent models (MPTP injection), a drug, Salidroside, increased Parkin and PINK1 expression and preserved dopaminergic neurons in the substantia nigra [264]. A cell permeable form of Parkin rescued cells from aggregating α-synuclein, partially restored motor function, and protected dopaminergic neurons in the 6-OHDA PD (6-hydroxydopamine) mouse model [265].
In an MS-related mouse model of EAE administration of rapamycin, an mTOR inhibitor improves outcomes [211]. Further studies of the EAE mouse model show that excessive activation of Drp1 through nitration leads to an overaction of mitophagy [266]. Blocking this pathway alleviated the disease burden in the EAE mouse mode [267]. Genetic ablation of Beclin 1 was also protective in the EAE mouse model [268]. Overall, in MS inhibition of mitophagy specifically in T cells could be beneficial.
UA has been shown to be safe and well-tolerated in elderly adults, with plasma concentrations detectable at a range of doses. Furthermore, UA affected mitochondrial gene expression in muscle [269]. A separate study in healthy adults is registered for UA (NCT04160312), but no results have been posted. Clinical trials for NMN (NCT04228640 safety trial) are recruiting or ongoing (NCT03151239 effects on cardiometabolic health). In Japan, the first human clinical trial of NMN showed no deleterious effects, suggesting NMN is tolerable and safe [270,271]. No clinical trials for these NAD + precursor mitophagy modulators are currently registered for neurodegenerative diseases.
Lifestyle interventions could be useful tools to boost mitophagy. Exercise and diet have been shown to induce mitophagy [272][273][274][275][276]. In both AD animal models and human clinical trials, exercise has shown cognitive benefit [275,[277][278][279][280][281]. The exercise effects in ALS and PD are more controversial, but overall, exercise seems to improve physical and cognitive outcomes [282][283][284][285][286][287]. Intermittent fasting and ketogenic diets have also been shown to induce mitophagy and improve cognition/motor performance [288][289][290][291][292][293][294].
Current clinical trials aimed at increasing autophagy, mitophagy, or mitochondrial function are ongoing or recently completed. For AD, these include treatment with nicotinamide riboside (NR; NCT04430517; NAD + precursor), Dimebon (NCT00675623, NCT00829374; stimulates mTOR-dependent mitophagy), resveratrol (NCT00678431; mTOR inhibitory), ketogenic diets (NCT03860792), and caloric restriction diets (NCT02460783). In PD, these include nicotinamide supplementation (NCT03568968; NAD + precursor), ubiquinol/Coenzyme Q10 (NCT03061513; autophagy mechanism unknown), ketogenic diets, and ketone esters (NCT01364545, NCT04477161). In MS, clinical trials include ketogenic diet, dimethyl fumarate, and MitoQ (NCT03740295, NCT04267926, NCT02461069). In ALS, one clinical trial for ubiquinol/Coenzyme Q10 (NCT00243932; autophagy mechanism unknown) was completed. Overall, the clinical trials directly modulating mitophagy are lacking and require more attention. The majority of mitophagy inducers in clinical trials have unknown mechanisms and pleotropic affects.
Targeting specific pathways and tissues could be advantageous in avoiding deleterious or off-target effects. Designing new therapeutic strategies should focus on modulating specific mitophagy targets while also enhancing mitochondrial function and biogenesis.
Concluding Remarks
Mitophagy and mitochondrial quality control are important mechanisms which should be further studied in the context of brain aging and neurodegeneration. Novel mechanisms of mitochondrial quality control in neurons and glia have illuminated the knowledge gaps in this field of study. Mitochondria are dynamic and multifaceted organelles at the forefront of pathways associated with aging and neurodegeneration (proteostasis, metabolism, inflammation, and synapse loss). Thus, targeting mitochondrial health and mitochondrial quality control will target the most common pathological mechanisms in neurodegeneration. 19
Figure 1 .
1Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.
Figure 1 .
1Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.
Figure 2 .
2Mitophagy. (A) Non-Receptor Mediated Mitophagy or Classical Mitophagy.
Figure 3 .
3Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation
Figure 3 .
3Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation
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| [
"Stress mechanisms have long been associated with neuronal loss and neurodegenerative diseases. The origin of cell stress and neuronal loss likely stems from multiple pathways. These include (but are not limited to) bioenergetic failure, neuroinflammation, and loss of proteostasis. Cells have adapted compensatory mechanisms to overcome stress and circumvent death. One mechanism is mitophagy. Mitophagy is a form of macroautophagy, were mitochondria and their contents are ubiquitinated, engulfed, and removed through lysosome degradation. Recent studies have implicated mitophagy dysregulation in several neurodegenerative diseases and clinical trials are underway which target mitophagy pathways. Here we review mitophagy pathways, the role of mitophagy in neurodegeneration, potential therapeutics, and the need for further study. Int. J. Mol. Sci. 2020, 21, 9661 2 of 26 neurodegenerative diseases. This could lead to the disruption of mitophagy processes and requires more research efforts to understand. The inner membrane space of mitochondria houses enzymes and allows for proton storage and protein folding. The mitochondrial inner membrane contains the ETC and ATP synthase enzymes and the mitochondrial matrix stores enzymes for the TCA cycle, mitochondrial DNA (mtDNA), and other crucial enzymes for protein folding and maintenance of pH gradients. For more detailed analysis of mitochondrial localized proteins MitoCarta3.0 was recently published[8].Synaptic loss is strongly correlated with cognitive deficits and motor dysfunction[9][10][11][12]. Mitochondria are essential for synaptic function and neurotransmitter synthesis, release, and uptake[13][14][15]. Accumulation of damaged mitochondria could lead to synaptic dysfunction and neurodegeneration. Mitophagy may play a role in ensuring synaptic mitochondrial integrity by degrading damaged mitochondria.Mitochondria evolved from a prokaryotic endosymbiont. As such, mitochondria share characteristics with bacteria including a double membrane, circular DNA, formyl-methionine amino acids, and cardiolipin [1]. In certain contexts failure of mitophagy pathways could lead to the release of mitochondrial components into the extracellular space, activation of a damage-associated molecular response (DAMP), and inflammation[1,5]. Mitochondria are also master regulators of cell death pathways (such as apoptosis and necrosis)[4,5,16]. As a by-product of the respiratory chain function superoxide radicals are produced. These free radicals generate multiple species of reactive oxygen species (ROS) and reactive nitrogen species (RNS). During periods of mitochondrial dysfunction and failure of mitochondrial quality control mechanisms (such as mitophagy) ROS/RNS can induce damage to cellular macromolecules and necrotic cell death[4,5,16]. Proper control and coordination of mitophagy pathways are crucial to prevent cell death and inflammation.Mitophagy is imperative for glial cell function. Signaling between microglia, astrocytes, and neurons are modulated by mitophagy pathways. Novel data show that transcellular mitophagy pathways occur within the brain. Transcellular mitophagy is a process by which cells release mitochondria for engulfment and mitophagy in surrounding cell types. Dysregulation of this process can lead to neuroinflammation and loss of proteostasis[17][18][19].Disruption of mitophagy is observed with aging and in many neurodegenerative diseases. Recent advances have described novel mechanisms of mitophagy within the central nervous system (CNS). Here, we will review current knowledge of mitophagy regulation, its role in neurodegenerative disease, and therapeutic potential.For this review article, we used PubMed, clinicaltrials.gov, and Google Scholar to identify studies related to mitophagy, AD, PD, ALS, and MS. We used search terms including mitophagy, mitophagy and neurodegeneration, mitophagy and AD, mitophagy and PD, mitophagy and MS, mitophagy and ALS, mitochondria and neurodegeneration, and autophagy.Mitophagy: Autophagy for MitochondriaAutophagosomes can be derived from membranes of endoplasmic reticulum (ER), Golgi, mitochondria, or plasma membrane [20][21][22][23][24]. The biogenesis of autophagosomes involves the formation of the isolation membrane (IM), elongation and maturation, closure, and then fusion with the lysosome to form the autolysosome. The first step in autophagosome biogenesis is the activation of the Unc-51-like kinase 1 complex (ULK1; pre-initiation complex), which contains ULK1, autophagy-related proteins 13 and 101 (Atg13, Atg101), and focal adhesion kinase family interacting partner 200 (FIP200) [25][26][27][28]. This complex recruits the class III phosphatidylinositide 3-kinase (PI3K) Vps34 complex (Beclin1, autophagy-related protein 14 (Atg14), autophagy and beclin 1 regulator 1 (Ambra1), and vascular protein sorting 34 and 15 (Vps34 and Vps15)) to produce phosphatidylinositol 3-phosphate (PI3P), also called the initiation complex [25,27,29,30]. PI3P binding proteins, FYVE domain containing proteins (DFCP1), and WD repeat protein interacting with phosphoinositide (WIPIs) localize to the IM [26]. All of this culminates in the formation of the omegasome and IM. Autophagy-related proteins 12, 5, and 16 (Atg12, Atg5, and Atg16) and LC3-phosphatidylethanolamine (PE) facilitate the elongation and closure of the IM, following fusion with the lysosome [25,26,31]. Int. J. Mol. Sci. 2020, 21, 9661 3 of 26Mitochondrial quality control mechanisms include mitochondrial chaperones, the mitochondrial unfolded protein response (UPR mt ), degradation of mitochondrial proteins in the cytoplasm via the proteasome (p97, 26S proteasome), removal of damaged proteins via mitochondrial derived vesicles (MDVs), and mitophagy (Figure 1) [32]. Misfolded mitochondrial proteins can be refolded by mitochondrial chaperone proteins (heat shock proteins 22, 60, and 70) or cleaved/degraded by mitochondrial proteases Lon and Clp through the UPR mt [33][34][35][36][37]. Damaged mitochondrial proteins can also be targeted specifically for proteasome degradation by p97 through the proteasome [38]. MDVs bud off mitochondria after engulfing damaged mitochondrial macromolecules. MDVs are associated with impaired mitochondrial import channels. These MDVs are either degraded by lysosomes, peroxisomes, or exocytosed [39,40]. Mitophagy (reviewed extensively below) functions to remove damaged mitochondria with the goal of preventing cell death. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 27 Int. J. Mol. Sci. 2020, 21, 9661 4 of 26 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 27 Figure 2. Mitophagy. (A) Non-Receptor Mediated Mitophagy or Classical Mitophagy. Activation of PINK1 leads to recruitment of ubiquitin and Parkin. Parkin ubiquitinates and phosphorylates mitochondrial proteins (such as VDAC, MFN1/2, and TOM20) and this initiates receptor adaptor protein recruitment (p62, NDP52, OPTN, TAX1BP1, and NBR1). These adaptor proteins interact with LC3 to form the autophagosome. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50] (B) Receptor Mediated Mitophagy. Mitochondrial receptor proteins (BNIP3, NIX, FUNC1, AMBRA1, PHB2, or cardiolipin) are ubiquitinated and phosphorylated. This facilitates their interaction with LC3 and GABARAP for autophagosome formation. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50]. Created with BioRender.com."
] | [
"Natalie S Swerdlow \nAlzheimer's Disease Center\nUniversity of Kansas\nUniversity of Kansas\n66160Kansas CityKSUSA\n",
"Heather M Wilkins [email protected] \nAlzheimer's Disease Center\nUniversity of Kansas\nUniversity of Kansas\n66160Kansas CityKSUSA\n\nDepartment of Neurology\nMedical Center\nUniversity of Kansas\n66160Kansas CityKSUSA\n\nDepartment of Biochemistry and Molecular Biology\nMedical Center\nUniversity of Kansas\n66160Kansas CityKSUSA\n",
"\nInternational Journal\n\n"
] | [
"Alzheimer's Disease Center\nUniversity of Kansas\nUniversity of Kansas\n66160Kansas CityKSUSA",
"Alzheimer's Disease Center\nUniversity of Kansas\nUniversity of Kansas\n66160Kansas CityKSUSA",
"Department of Neurology\nMedical Center\nUniversity of Kansas\n66160Kansas CityKSUSA",
"Department of Biochemistry and Molecular Biology\nMedical Center\nUniversity of Kansas\n66160Kansas CityKSUSA",
"International Journal\n"
] | [
"Natalie",
"S",
"Heather",
"M"
] | [
"Swerdlow",
"Wilkins"
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"D Krolikowski, ",
"M Pagnoni, ",
"C Franks, ",
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"G Bellot, ",
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"P Gounon, ",
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"H Suzuki, ",
"M Marinkovic, ",
"V Lang, ",
"R Kato, ",
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"T Zhao, ",
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"D Feng, ",
"Y Chen, ",
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"H Wu, ",
"L Huang, ",
"C Zhou, ",
"X Cai, ",
"C Fu, ",
"Z Chen, ",
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"Q Cheng, ",
"Y Li, ",
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"Y Wang, ",
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"S Siraj, ",
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"M Tang, ",
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"F Nazio, ",
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"A Romagnoli, ",
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"M Campanella, ",
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"M J Los, ",
"A Tinari, ",
"T Garofalo, ",
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"K Hayakawa, ",
"E Esposito, ",
"X Wang, ",
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"Y Liu, ",
"C Xing, ",
"X Ji, ",
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"Corrigendum, ",
"K Hayakawa, ",
"E Esposito, ",
"X Wang, ",
"Y Terasaki, ",
"Y Liu, ",
"C Xing, ",
"X Ji, ",
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"C J Barnstable, ",
"L Gao, ",
"Z Zhang, ",
"J Lu, ",
"G Pei, ",
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"S R Das, ",
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"M Wei, ",
"L Sun, ",
"K Westphalen, ",
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"S K Quadri, ",
"S Bhattacharya, ",
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"T Ahmad, ",
"S Mukherjee, ",
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"M Kumar, ",
"S Singh, ",
"M Kumar, ",
"R Rehman, ",
"B K Tiwari, ",
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"D Torralba, ",
"F Baixauli, ",
"F Sanchez-Madrid, ",
"Mitochondria, ",
"H Han, ",
"J Hu, ",
"Q Yan, ",
"J Zhu, ",
"Z Zhu, ",
"Y Chen, ",
"J Sun, ",
"R Zhang, ",
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"S H Koh, ",
"H S Ahn, ",
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"X Wang, ",
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"M Hansen, ",
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"D W Walker, ",
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"V De Benedictis, ",
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"R P Farrar, ",
"K L Stauch, ",
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"L M Villeneuve, ",
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"C Bertoni-Freddari, ",
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"C Spagna, ",
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"J Ulrich, ",
"C Bertoni-Freddari, ",
"M Balietti, ",
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"S Dzitoyeva, ",
"H Chen, ",
"H Manev, ",
"A Herrero, ",
"G Barja, ",
"G Barja, ",
"A Herrero, ",
"P Mecocci, ",
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"J M Shoffner, ",
"D C Wallace, ",
"M F Beal, ",
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"A Shaik, ",
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"Zhang",
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"Lohr",
"Popovic",
"Occhipinti",
"Wu",
"Tian",
"Hu",
"Chen",
"Huang",
"Li",
"Zhang",
"Xue",
"Zhou",
"Liu",
"Zhang",
"Ney",
"Sandoval",
"Thiagarajan",
"Dasgupta",
"Schumacher",
"Prchal",
"Chen",
"Wang",
"Zhang",
"Ney",
"Sowter",
"Ratcliffe",
"Watson",
"Greenberg",
"Harris",
"Guo",
"Searfoss",
"Krolikowski",
"Pagnoni",
"Franks",
"Clark",
"Yu",
"Jaye",
"Ivashchenko",
"Bellot",
"Garcia-Medina",
"Gounon",
"Chiche",
"Roux",
"Pouyssegur",
"Mazure",
"Liu",
"Frazier",
"Rogov",
"Suzuki",
"Marinkovic",
"Lang",
"Kato",
"Kawasaki",
"Buljubasic",
"Sprung",
"Rogova",
"Wakatsuki",
"Ma",
"Zhang",
"Chang",
"Cheng",
"Mu",
"Zhao",
"Chen",
"Zhang",
"Luo",
"Lin",
"Chen",
"Han",
"Feng",
"Chen",
"Chen",
"Wu",
"Huang",
"Zhou",
"Cai",
"Fu",
"Chen",
"Liu",
"Cheng",
"Li",
"Wu",
"Zhang",
"Wang",
"Sehgal",
"Siraj",
"Wang",
"Yan",
"Gong",
"Chen",
"Xu",
"Abou-Hamdan",
"Tang",
"Desaubry",
"Song",
"Li",
"Tsoi",
"Li",
"Kurihara",
"He",
"Strappazzon",
"Nazio",
"Corrado",
"Cianfanelli",
"Romagnoli",
"Fimia",
"Campello",
"Nardacci",
"Piacentini",
"Campanella",
"Zorov",
"Kinnally",
"Tedeschi",
"Lyamzaev",
"Nepryakhina",
"Saprunova",
"Bakeeva",
"Pletjushkina",
"Chernyak",
"Skulachev",
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"Skulachev",
"Jangamreddy",
"Los",
"Tinari",
"Garofalo",
"Sorice",
"Esposti",
"Malorni",
"Skulachev",
"Hayakawa",
"Esposito",
"Wang",
"Terasaki",
"Liu",
"Xing",
"Ji",
"Lo",
"Corrigendum",
"Hayakawa",
"Esposito",
"Wang",
"Terasaki",
"Liu",
"Xing",
"Ji",
"Lo",
"Hass",
"Barnstable",
"Gao",
"Zhang",
"Lu",
"Pei",
"Islam",
"Das",
"Emin",
"Wei",
"Sun",
"Westphalen",
"Rowlands",
"Quadri",
"Bhattacharya",
"Bhattacharya",
"Ahmad",
"Mukherjee",
"Pattnaik",
"Kumar",
"Singh",
"Kumar",
"Rehman",
"Tiwari",
"Jha",
"Barhanpurkar",
"Torralba",
"Baixauli",
"Sanchez-Madrid",
"Mitochondria",
"Han",
"Hu",
"Yan",
"Zhu",
"Zhu",
"Chen",
"Sun",
"Zhang",
"Liu",
"Ji",
"Guo",
"Wu",
"Lu",
"Shan",
"Yan",
"Cho",
"Kim",
"Kim",
"Park",
"Koh",
"Ahn",
"Kang",
"Lee",
"Park",
"Lee",
"Konari",
"Nagaishi",
"Kikuchi",
"Fujimiya",
"Spees",
"Olson",
"Whitney",
"Prockop",
"Wang",
"Gerdes",
"Hansen",
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"Walker",
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"Ruggiero",
"Paradies",
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"Matera",
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"Ruggiero",
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"Leslie",
"Chandler",
"Barr",
"Farrar",
"Stauch",
"Purnell",
"Villeneuve",
"Fox",
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"Casoli",
"Spagna",
"Meier-Ruge",
"Ulrich",
"Bertoni-Freddari",
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"Giorgetti",
"Grossi",
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"Tanhauser",
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"Dzitoyeva",
"Chen",
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"Kaufman",
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"Jiang",
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"Huh",
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"Hay",
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"Walker",
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"Maglioni",
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"Shaik",
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"Brinkmann",
"Torgovnick",
"Castelein",
"De Henau",
"Braeckman",
"Wu",
"Al-Amin",
"Zhao",
"An",
"Wang",
"Huang",
"Teng",
"Song",
"Fang",
"Waltz",
"Kassahun",
"Lu",
"Kerr",
"Morevati",
"Fivenson",
"Wollman",
"Marosi",
"Wilson",
"Ryu",
"Mouchiroud",
"Andreux",
"Katsyuba",
"Moullan",
"Nicolet-Dit-Felix",
"Williams",
"Jha",
"Lo Sasso",
"Huzard",
"Fang",
"Hou",
"Lautrup",
"Jensen",
"Yang",
"Sengupta",
"Caponio",
"Khezri",
"Demarest",
"Aman",
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"Braak",
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"Jack",
"Jr",
"Barrio",
"Kepe",
"Dubois",
"Feldman",
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"Dekosky",
"Barberger-Gateau",
"Cummings",
"Delacourte",
"Galasko",
"Gauthier",
"Jicha",
"Marcus",
"Mena",
"Subramaniam",
"Mosconi",
"Mchugh",
"Suppiah",
"Didier",
"Vinjamuri",
"Herholz",
"Salmon",
"Perani",
"Baron",
"Holthoff",
"Frolich",
"Schonknecht",
"Ito",
"Mielke",
"Kalbe",
"Valla",
"Berndt",
"Gonzalez-Lima",
"Messa",
"Perani",
"Lucignani",
"Zenorini",
"Zito",
"Rizzo",
"Grassi",
"Del Sole",
"Franceschi",
"Gilardi",
"Morris",
"Honea",
"Vidoni",
"Swerdlow",
"Burns",
"Bosetti",
"Brizzi",
"Barogi",
"Mancuso",
"Siciliano",
"Tendi",
"Murri",
"Rapoport",
"Solaini",
"Fukui",
"Diaz",
"Garcia",
"Moraes",
"Parker",
"Jr",
"Parks",
"Cardoso",
"Proenca",
"Santos",
"Santana",
"Oliveira",
"Khan",
"Cassarino",
"Abramova",
"Keeney",
"Borland",
"Trimmer",
"Krebs",
"Bennett",
"Parks",
"Swerdlow",
"Kish",
"Bergeron",
"Rajput",
"Dozic",
"Mastrogiacomo",
"Chang",
"Wilson",
"Distefano",
"Nobrega",
"Kish",
"Mutisya",
"Bowling",
"Beal",
"Parker",
"Jr",
"Parker",
"Jr",
"Filley",
"Parks",
"Alberts",
"Ioannou",
"Deucher",
"Gilbert",
"Lee",
"Middleton",
"Roses",
"Curti",
"Rognoni",
"Gasparini",
"Cattaneo",
"Paolillo",
"Racchi",
"Zani",
"Bianchetti",
"Trabucchi",
"Bergamaschi",
"Silva",
"Selfridge",
"Lu",
"Lezi",
"Roy",
"Hutfles",
"Burns",
"Michaelis",
"Yan",
"Cardoso",
"Sheehan",
"Swerdlow",
"Miller",
"Davis",
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"Parker",
"Tuttle",
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"Santana",
"Swerdlow",
"Oliveira",
"Swerdlow",
"Trimmer",
"Keeney",
"Borland",
"Simon",
"Almeida",
"Swerdlow",
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"Parker",
"Jr",
"Bennett",
"Jr",
"Baloyannis",
"Berti",
"Mosconi",
"Glodzik",
"Li",
"Murray",
"De Santi",
"Pupi",
"Tsui",
"De Leon",
"Mosconi",
"Mosconi",
"Mistur",
"Switalski",
"Brys",
"Glodzik",
"Rich",
"Pirraglia",
"Tsui",
"De Santi",
"De Leon",
"Mosconi",
"Rinne",
"Tsui",
"Murray",
"Li",
"Glodzik",
"Mchugh",
"Williams",
"Cummings",
"Pirraglia",
"Swerdlow",
"Hui",
"Chalise",
"Sharma",
"Wang",
"Andrews",
"Pa",
"Mahnken",
"Morris",
"Wilkins",
"Andrews",
"Fulton-Howard",
"Patterson",
"Mcfall",
"Gross",
"Michaelis",
"Goate",
"Swerdlow",
"Pa",
"Carrieri",
"Bonafe",
"De Luca",
"Rose",
"Varcasia",
"Bruni",
"Maletta",
"Nacmias",
"Sorbi",
"Corsonello",
"Coto",
"Gomez",
"Alonso",
"Corao",
"Diaz",
"Menendez",
"Martinez",
"Calatayud",
"Moris",
"Alvarez",
"Fesahat",
"Houshmand",
"Panahi",
"Gharagozli",
"Mirzajani",
"Takasaki",
"Van Der Walt",
"Dementieva",
"Martin",
"Scott",
"Nicodemus",
"Kroner",
"Welsh-Bohmer",
"Saunders",
"Roses",
"Small",
"Zhou",
"Chen",
"Mok",
"Kwok",
"Mok",
"Guo",
"Ip",
"Chen",
"Mullapudi",
"Lakatos",
"Derbeneva",
"Younes",
"Keator",
"Bakken",
"Lvova",
"Brandon",
"Guffanti",
"Reglodi",
"Saykin",
"Reddy",
"Oliver",
"Hu",
"Li",
"Wang",
"Luo",
"Zhang",
"Liu",
"Feng",
"Wang",
"Yue",
"Chen",
"Manczak",
"Kandimalla",
"Yin",
"Reddy",
"Wang",
"Zhao",
"Xu",
"Wang",
"Wei",
"Liu",
"Yang",
"Wang",
"Xie",
"Bi",
"Ye",
"Sun",
"Starovoytov",
"Cai",
"Bordi",
"Berg",
"Mohan",
"Peterhoff",
"Alldred",
"Che",
"Ginsberg",
"Nixon",
"Kerr",
"Adriaanse",
"Greig",
"Mattson",
"Cader",
"Bohr",
"Fang",
"Nixon",
"Kurosawa",
"Matsumoto",
"Sumikura",
"Hatsuta",
"Murayama",
"Sakurai",
"Shimogori",
"Hattori",
"Nukina",
"Bharadwaj",
"Martins",
"Lonskaya",
"Shekoyan",
"Hebron",
"Desforges",
"Algarzae",
"Moussa",
"Yan",
"Wang",
"Hu",
"Wang",
"Zhang",
"Poewe",
"Seppi",
"Tanner",
"Halliday",
"Brundin",
"Volkmann",
"Schrag",
"Lang",
"Parkinson Disease",
"Gibb",
"Lees",
"Clarke",
"Mann",
"Cooper",
"Krige",
"Daniel",
"Schapira",
"Marsden",
"Gatt",
"Duncan",
"Attems",
"Francis",
"Ballard",
"Bateman",
"Blake",
"Spitz",
"Leehey",
"Hoffer",
"Boyson",
"Krige",
"Carroll",
"Cooper",
"Marsden",
"Schapira",
"Arduino",
"Esteves",
"Swerdlow",
"Cardoso",
"Esteves",
"Domingues",
"Ferreira",
"Januario",
"Swerdlow",
"Oliveira",
"Cardoso",
"Esteves",
"Lu",
"Rodova",
"Onyango",
"Lezi",
"Dubinsky",
"Lyons",
"Pahwa",
"Burns",
"Cardoso",
"Dolle",
"Flones",
"Nido",
"Miletic",
"Osuagwu",
"Kristoffersen",
"Lilleng",
"Larsen",
"Tysnes",
"Haugarvoll",
"Gu",
"Reyes",
"Golden",
"Woltjer",
"Hulette",
"Montine",
"Zhang",
"Valente",
"Abou-Sleiman",
"Caputo",
"Muqit",
"Harvey",
"Gispert",
"Ali",
"Del Turco",
"Bentivoglio",
"Healy",
"Polymeropoulos",
"Lavedan",
"Leroy",
"Ide",
"Dehejia",
"Dutra",
"Pike",
"Root",
"Rubenstein",
"Boyer",
"Lee",
"Nagano",
"Taylor",
"Lim",
"Yao",
"Lesage",
"Condroyer",
"Klebe",
"Honore",
"Tison",
"Brefel-Courbon",
"Durr",
"Brice",
"Kitada",
"Asakawa",
"Hattori",
"Matsumine",
"Yamamura",
"Minoshima",
"Yokochi",
"Mizuno",
"Shimizu",
"Chen",
"Chen",
"Song",
"Chen",
"Cao",
"Huang",
"Zhao",
"Guo",
"Burgunder",
"Li",
"Saravanan",
"Sindhu",
"Mohanakumar",
"Duty",
"Jenner",
"Betarbet",
"Sherer",
"Mackenzie",
"Garcia-Osuna",
"Panov",
"Greenamyre",
"Zeng",
"Geng",
"Jia",
"Von Wrangel",
"Schwabe",
"John",
"Krauss",
"Alam",
"Bolam",
"Pissadaki",
"Hou",
"Fiesel",
"Truban",
"Casey",
"Lin",
"Soto",
"Tacik",
"Rousseau",
"Diehl",
"Heckman",
"Hsieh",
"Shaltouki",
"Gonzalez",
"Bettencourt Da Cruz",
"Burbulla",
"St Lawrence",
"Schule",
"Krainc",
"Palmer",
"Wang",
"Barcelos",
"Troxell",
"Graves",
"Vaughn",
"Jakimovski",
"Kavak",
"Ramanathan",
"Benedict",
"Zivadinov",
"Weinstock-Guttman",
"Gonzalo",
"Nogueras",
"Gil-Sanchez",
"Hervas",
"Valcheva",
"Gonzalez-Mingot",
"Martin-Gari",
"Canudes",
"Peralta",
"Solana",
"Mahad",
"Ziabreva",
"Lassmann",
"Turnbull",
"Mahad",
"Ziabreva",
"Campbell",
"Lax",
"White",
"Hanson",
"Lassmann",
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"Dutta",
"Mcdonough",
"Yin",
"Peterson",
"Chang",
"Torres",
"Gudz",
"Macklin",
"Lewis",
"Fox",
"Alirezaei",
"Fox",
"Flynn",
"Moore",
"Hebb",
"Frausto",
"Bhan",
"Kiosses",
"Whitton",
"Robertson",
"Liang",
"Le",
"Castellazzi",
"Patergnani",
"Donadio",
"Giorgi",
"Bonora",
"Fainardi",
"Casetta",
"Granieri",
"Pugliatti",
"Pinton",
"Igci",
"Baysan",
"Yigiter",
"Ulasli",
"Geyik",
"Bayraktar",
"Bozgeyik",
"Bozgeyik",
"Bayram",
"Cakmak",
"Smith",
"Shaw",
"De Vos",
"Swerdlow",
"Parks",
"Pattee",
"Parker",
"Jr",
"Beeldman",
"Raaphorst",
"Klein Twennaar",
"De Visser",
"Schmand",
"De Haan",
"Gubbay",
"Kahana",
"Zilber",
"Cooper",
"Pintov",
"Leibowitz",
"Norris",
"Shepherd",
"Denys",
"Kwei",
"Mukai",
"Elias",
"Holden",
"Norris",
"Rothstein",
"Rowland",
"Shneider",
"Deng",
"Bigio",
"Zhai",
"Fecto",
"Ajroud",
"Shi",
"Yan",
"Mishra",
"Ajroud-Driss",
"Heller",
"Van Blitterswijk",
"Van Vught",
"Van Es",
"Schelhaas",
"Van Der Kooi",
"Visser",
"Veldink",
"Van Den",
"Berg",
"Sakaguchi",
"Irie",
"Kawabata",
"Yoshida",
"Maruyama",
"Kawakami",
"Gurney",
"Pu",
"Chiu",
"Canto",
"Polchow",
"Alexander",
"Caliendo",
"Hentati",
"Kwon",
"Deng",
"Kong",
"Xu",
"Wegorzewska",
"Bell",
"Cairns",
"Miller",
"Baloh",
"Vielhaber",
"Kunz",
"Winkler",
"Wiedemann",
"Kirches",
"Feistner",
"Heinze",
"Elger",
"Schubert",
"Kunz",
"Duffy",
"Chapman",
"Shaw",
"Grierson",
"Swerdlow",
"Parks",
"Cassarino",
"Trimmer",
"Miller",
"Maguire",
"Sheehan",
"Maguire",
"Pattee",
"Juel",
"Hart",
"Cancilla",
"Frommes",
"Hirano",
"Mizuno",
"Amari",
"Takatama",
"Aizawa",
"Mihara",
"Okamoto",
"Rodriguez",
"Gonzalez",
"Monachelli",
"Costa",
"Nicola",
"Sica",
"Swerdlow",
"Catalan-Garcia",
"Garrabou",
"Moren",
"Guitart-Mampel",
"Hernando",
"Diaz-Ramos",
"Gonzalez-Casacuberta",
"Juarez",
"Bano",
"Enrich-Bengoa",
"Horvath",
"Fu",
"Johns",
"Genge",
"Karpati",
"Shoubridge",
"Wiedemann",
"Manfredi",
"Mawrin",
"Beal",
"Schon",
"Borthwick",
"Johnson",
"Ince",
"Shaw",
"Turnbull",
"Fujita",
"Yamauchi",
"Shibayama",
"Ando",
"Honda",
"Nagata",
"Shrivastava",
"Subbiah",
"Wilkins",
"Carl",
"Swerdlow",
"Wong",
"Pardo",
"Borchelt",
"Lee",
"Copeland",
"Jenkins",
"Sisodia",
"Cleveland",
"Price",
"Mattiazzi",
"D'aurelio",
"Gajewski",
"Martushova",
"Kiaei",
"Beal",
"Manfredi",
"Tafuri",
"Ronchi",
"Magri",
"Comi",
"Corti",
"Davis",
"Itaman",
"Khalid-Janney",
"Sherard",
"Dowell",
"Cairns",
"Gitcho",
"Huntley",
"Gao",
"Termsarasab",
"Wang",
"Zeng",
"Thammongkolchai",
"Liu",
"Cohen",
"Wang",
"Wang",
"Deng",
"Dong",
"Liu",
"Bigio",
"Mesulam",
"Wang",
"Sun",
"Wang",
"Lee",
"Blokhuis",
"Groen",
"Koppers",
"Van Den",
"Berg",
"Pasterkamp",
"Johnson",
"Snead",
"Lee",
"Mccaffery",
"Shorter",
"Gitler",
"Nogalska",
"D'agostino",
"Terracciano",
"Engel",
"Askanas",
"Vattemi",
"Nogalska",
"King Engel",
"D'agostino",
"Checler",
"Askanas",
"Watanabe",
"Dykes-Hoberg",
"Culotta",
"Price",
"Wong",
"Rothstein",
"Rogers",
"Tungtur",
"Tanaka",
"Nadeau",
"Badawi",
"Wang",
"Ni",
"Ding",
"Nishimune",
"Natale",
"Lenzi",
"Lazzeri",
"Falleni",
"Biagioni",
"Ryskalin",
"Fornai",
"Ruffoli",
"Bartalucci",
"Frati",
"Fornai",
"Palomo",
"Granatiero",
"Kawamata",
"Konrad",
"Kim",
"Arreguin",
"Zhao",
"Milner",
"Manfredi",
"Wong",
"Holzbaur",
"Sasaki",
"Almeida",
"Gascon",
"Tran",
"Chou",
"Gendron",
"Degroot",
"Tapper",
"Sellier",
"Charlet-Berguerand",
"Karydas",
"Webster",
"Smith",
"Bauer",
"Moller",
"Hautbergue",
"Ferraiuolo",
"Myszczynska",
"Higginbottom",
"Walsh",
"Whitworth",
"Fang",
"Fang",
"Hou",
"Palikaras",
"Adriaanse",
"Kerr",
"Yang",
"Lautrup",
"Hasan-Olive",
"Caponio",
"Dan",
"Perera",
"Sheean",
"Lau",
"Shin",
"Beart",
"Horne",
"Turner",
"Staats",
"Hernandez",
"Schonefeldt",
"Bento-Abreu",
"Dooley",
"Van Damme",
"Liston",
"Robberecht",
"Li",
"Chen",
"Chung",
"Choi",
"Park",
"Nah",
"Park",
"Jung",
"Lee",
"Lee",
"Park",
"Hwang",
"Li",
"Feng",
"Gao",
"Wu",
"Du",
"Tsoi",
"Wang",
"Yang",
"Shen",
"Li",
"Deng",
"Jing",
"Chen",
"Yang",
"Shen",
"Kovacs",
"Li",
"Yang",
"Li",
"Garcia",
"Ju",
"Roodman",
"Windle",
"Zhang",
"Lu",
"Andreux",
"Blanco-Bose",
"Ryu",
"Burdet",
"Ibberson",
"Aebischer",
"Auwerx",
"Singh",
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"Kim",
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"Gordon",
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"Koppel",
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] | [
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"A Ketogenic Diet Improves Cognition and Has Biochemical Effects in Prefrontal Cortex That Are Dissociable From Hippocampus. Front. A R Hernandez, C M Hernandez, K Campos, L Truckenbrod, Q Federico, B Moon, J A Mcquail, A P Maurer, J L Bizon, S N Burke, 10.3389/fnagi.2018.00391Aging Neurosci. 10391Hernandez, A.R.; Hernandez, C.M.; Campos, K.; Truckenbrod, L.; Federico, Q.; Moon, B.; McQuail, J.A.; Maurer, A.P.; Bizon, J.L.; Burke, S.N. A Ketogenic Diet Improves Cognition and Has Biochemical Effects in Prefrontal Cortex That Are Dissociable From Hippocampus. Front. Aging Neurosci. 2018, 10, 391. [CrossRef]",
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"19"
] | [
"Relationships between Mitochondria and Neuroinflammation: Implications for Alzheimer's Disease",
"Mitochondrial dynamics in adaptive and maladaptive cellular stress responses",
"Mitochondria and cell signalling",
"Mitochondria and the autophagy-inflammation-cell death axis in organismal aging",
"Mitophagy and mitoptosis in disease processes",
"A Molecular Approach to Mitophagy and Mitochondrial Dynamics",
"0: An updated mitochondrial proteome now with sub-organelle localization and pathway annotations",
"The energy cost of action potential propagation in dopamine neurons: Clues to susceptibility in Parkinson's disease",
"Perforant path synaptic loss correlates with cognitive impairment and Alzheimer's Disease in the oldest-old",
"Synapse loss and progress of Alzheimer's Disease-A network model",
"Synaptic dysfunction in neurological disorders-A review from students to students",
"Impaired mitochondrial function in psychiatric disorders",
"Mitochondrial transport in neurons: Impact on synaptic homeostasis and neurodegeneration",
"Mitophagy regulates integrity of mitochondria at synapses and is critical for synaptic maintenance",
"Mitochondria and apoptosis",
"Discovery and implications of transcellular mitophagy",
"Transcellular degradation of axonal mitochondria",
"Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL",
"Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)-dependent ubiquitination of endogenous Parkin attenuates mitophagy: Study in human primary fibroblasts and induced pluripotent stem cell-derived neurons",
"PINK1-and Parkin-mediated mitophagy at a glance",
"PINK1 is selectively stabilized on impaired mitochondria to activate Parkin",
"PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy",
"PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1",
"PINK1-dependent recruitment of Parkin to mitochondria in mitophagy",
"PINK1-parkin-dependent mitophagy involves ubiquitination of mitofusins 1 and 2: Implications for Parkinson disease pathogenesis",
"Selective removal of mitochondria via mitophagy: Distinct pathways for different mitochondrial stresses",
"The three 'P's of mitophagy: PARKIN, PINK1, and post-translational modifications",
"p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both",
"Parkin is recruited selectively to impaired mitochondria and promotes their autophagy",
"Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane",
"Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin",
"Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy",
"Autophagy: Renovation of cells and tissues",
"NBR1 is dispensable for PARK2-mediated mitophagy regardless of the presence or absence of SQSTM1",
"Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria",
"Nakagawa, I. Rab35 GTPase recruits NDP52 to autophagy targets",
"Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria",
"From autophagy to mitophagy: The roles of P62 in neurodegenerative diseases",
"SQSTM1/p62 promotes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy",
"Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells",
"Nix is a selective autophagy receptor for mitochondrial clearance",
"ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy",
"NIX induces mitochondrial autophagy in reticulocytes",
"Essential role for Nix in autophagic maturation of erythroid cells",
"Role of BNIP3 and NIX in cell death, autophagy, and mitophagy",
"HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors",
"Hypoxia induces the expression of the pro-apoptotic gene BNIP3",
"Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains",
"Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy",
"Dynamic PGAM5 multimers dephosphorylate BCL-xL or FUNDC1 to regulate mitochondrial and cellular fate",
"A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy",
"Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine-tune hypoxic mitophagy",
"PHB2 (prohibitin 2) promotes PINK1-PRKN/Parkin-dependent mitophagy by the PARL-PGAM5-PINK1 axis",
"Cardiolipin and its different properties in mitophagy and apoptosis",
"AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1",
"Voltage activation of heart inner mitochondrial membrane channels",
"Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): Formation of mitoptotic bodies and extrusion of mitochondrial material from the cell",
"Bioenergetic aspects of apoptosis, necrosis and mitoptosis",
"Mitochondrial physiology and pathology; concepts of programmed death of organelles, cells and organisms",
"Mitoptosis, a novel mitochondrial death mechanism leading predominantly to activation of autophagy",
"Mitoptosis: Different pathways for mitochondrial execution",
"Programmed death phenomena: From organelle to organism",
"Transfer of mitochondria from astrocytes to neurons after stroke",
"Transfer of mitochondria from astrocytes to neurons after stroke",
"Mitochondrial Uncoupling Protein 2 Knock-out Promotes Mitophagy to Decrease Retinal Ganglion Cell Death in a Mouse Model of Glaucoma",
"Mitochondria Are Dynamically Transferring Between Human Neural Cells and Alexander Disease-Associated GFAP Mutations Impair the Astrocytic Transfer",
"Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury",
"Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy",
"Know No Boundaries: Mechanisms and Functions of Intercellular Mitochondrial Transfer. Front",
"Bone marrow-derived mesenchymal stem cells rescue injured H9c2 cells via transferring intact mitochondria through tunneling nanotubes in an in vitro simulated ischemia/reperfusion model",
"Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer",
"Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations",
"Mitochondria transfer from mesenchymal stem cells structurally and functionally repairs renal proximal tubular epithelial cells in diabetic nephropathy in vivo",
"Mitochondrial transfer between cells can rescue aerobic respiration",
"Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells",
"Autophagy as a promoter of longevity: Insights from model organisms",
"Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin",
"Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin",
"Reduced calcium uptake by rat brain mitochondria and synaptosomes in response to aging",
"Proteomic analysis and functional characterization of mouse brain mitochondria during aging reveal alterations in energy metabolism",
"Morphological plasticity of synaptic mitochondria during aging",
"Selective decline of the metabolic competence of oversized synaptic mitochondria in the old monkey cerebellum",
"Multiple deletions are detectable in mitochondrial DNA of aging mice",
"Effect of aging on 5-hydroxymethylcytosine in brain mitochondria",
"8-oxo-deoxyguanosine levels in heart and brain mitochondrial and nuclear DNA of two mammals and three birds in relation to their different rates of aging",
"Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals",
"Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain",
"Mitochondrial DNA in aging and disease",
"A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues",
"Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin",
"Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan",
"Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans",
"Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans",
"Catechinic acid, a natural polyphenol compound, extends the lifespan of Caenorhabditis elegans via mitophagy pathways",
"Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents",
"NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome",
"Diagnostic criteria for neuropathologic assessment of Alzheimer's Disease",
"Diagnostic criteria for the neuropathologic assessment of Alzheimer's Disease",
"Cerebral amyloid PET imaging in Alzheimer's Disease",
"Research criteria for the diagnosis of Alzheimer's Disease: Revising the NINCDS-ADRDA criteria",
"Brain PET in the diagnosis of Alzheimer's Disease",
"FDG-and amyloid-PET in Alzheimer's Disease: Is the whole greater than the sum of the parts?",
"The Who, When, Why, and How of PET Amyloid Imaging in Management of Alzheimer's Disease-Review of Literature and Interesting Images",
"Discrimination between Alzheimer dementia and controls by automated analysis of multicenter FDG PET",
"Energy hypometabolism in posterior cingulate cortex of Alzheimer's patients: Superficial laminar cytochrome oxidase associated with disease duration",
"High-resolution technetium-99m-HMPAO SPECT in patients with probable Alzheimer's Disease: Comparison with fluorine-18-FDG PET",
"Is Alzheimer's Disease a systemic disease?",
"Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer's Disease",
"Cytochrome c oxidase deficiency in neurons decreases both oxidative stress and amyloid formation in a mouse model of Alzheimer's Disease",
"Cytochrome c oxidase in Alzheimer's Disease brain: Purification and characterization",
"Cytochrome c oxidase is decreased in Alzheimer's Disease platelets",
"Alzheimer's Disease cybrids replicate beta-amyloid abnormalities through cell death pathways",
"Brain cytochrome oxidase in Alzheimer's Disease",
"Brain energy metabolizing enzymes in Alzheimer's Disease: Alpha-ketoglutarate dehydrogenase complex and cytochrome oxidase",
"Cortical cytochrome oxidase activity is reduced in Alzheimer's Disease",
"Cytochrome oxidase deficiency in Alzheimer's Disease",
"Cytochrome oxidase deficiency in Alzheimer's Disease",
"Isolation of a cytochrome oxidase gene overexpressed in Alzheimer's Disease brain",
"Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's Disease (AD) patients",
"Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines",
"Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's Disease",
"Mitochondria dysfunction of Alzheimer's Disease cybrids enhances Abeta toxicity",
"Mitochondria in cybrids containing mtDNA from persons with mitochondriopathies",
"Mitochondrial abnormalities in cybrid cell models of sporadic Alzheimer's Disease worsen with passage in culture",
"Structural brain changes in normal individuals with a maternal history of Alzheimer's",
"Glucose metabolism in normal aging and Alzheimer's Disease: Methodological and physiological considerations for PET studies",
"Declining brain glucose metabolism in normal individuals with a maternal history of Alzheimer disease",
"Amyloid and metabolic positron emission tomography imaging of cognitively normal adults with Alzheimer's parents",
"Exploratory analysis of mtDNA haplogroups in two Alzheimer's longitudinal cohorts",
"Mitochondrial haplogroups & a nuclear encoded mitochondrial polygenic risk score interact to influence dementia risk",
"Mitochondrial DNA haplogroups and APOE4 allele are non-independent variables in sporadic Alzheimer's Disease",
"Late-onset Alzheimer's Disease is associated with mitochondrial DNA 7028C/haplogroup H and D310 poly-C tract heteroplasmy",
"Do haplogroups H and U act to increase the penetrance of Alzheimer's Disease?",
"Mitochondrial haplogroups associated with Japanese Alzheimer's patients",
"Analysis of European mitochondrial haplogroups with Alzheimer disease risk",
"Alzheimer's Disease Neuroimaging, I.; et al. Non-coding variability at the APOE locus contributes to the Alzheimer's risk",
"Association between mitochondrial DNA variations and Alzheimer's Disease in the ADNI cohort",
"Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin",
"Hippocampal mutant APP and amyloid beta-induced cognitive decline, dendritic spine loss, defective autophagy, mitophagy and mitochondrial abnormalities in a mouse model of Alzheimer's Disease",
"Mitophagy in APPsw/PS1dE9 transgenic mice and APPsw stably expressing in HEK293 cells",
"Parkin-mediated mitophagy in mutant hAPP neurons and Alzheimer's Disease patient brains",
"Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy",
"Mitophagy and Alzheimer's Disease: Cellular and Molecular Mechanisms",
"The role of autophagy in neurodegenerative disease",
"Serine 403-phosphorylated p62/SQSTM1 immunoreactivity in inclusions of neurodegenerative diseases",
"PRKAG2 Gene Expression Is Elevated and its Protein Levels Are Associated with Increased Amyloid-beta Accumulation in the Alzheimer's Disease Brain",
"Diminished parkin solubility and co-localization with intraneuronal amyloid-beta are associated with autophagic defects in Alzheimer's Disease",
"Abnormal Mitochondrial Quality Control in Neurodegenerative Diseases",
"The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease",
"Parkinson's disease",
"Brain, skeletal muscle and platelet homogenate mitochondrial function in Parkinson's disease",
"Dementia in Parkinson's disease is associated with enhanced mitochondrial complex I deficiency",
"Platelet mitochondrial respiratory chain function in Parkinson's disease",
"Platelet mitochondrial function in Parkinson's disease. The Royal Kings and Queens Parkinson Disease Research Group",
"A cybrid cell model for the assessment of the link between mitochondrial deficits and sporadic Parkinson's disease",
"Mitochondrial respiration and respiration-associated proteins in cell lines created through Parkinson's subject mitochondrial transfer",
"Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease",
"Mitochondrial DNA deletions/rearrangements in parkinson disease and related neurodegenerative disorders",
"Hereditary early-onset Parkinson's disease caused by mutations in PINK1",
"Mutation in the alpha-synuclein gene identified in families with Parkinson's disease",
"Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy",
"French Parkinson's Disease Genetics Study, G. Identification of VPS35 mutations replicated in French families with Parkinson disease",
"Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism",
"VPS35 Asp620Asn and EIF4G1 Arg1205His mutations are rare in Parkinson disease from southwest China",
"Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson's disease",
"Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease",
"Chronic systemic pesticide exposure reproduces features of Parkinson's disease",
"Neurotoxin-Induced Animal Models of Parkinson Disease: Pathogenic Mechanism and Assessment",
"The rotenone-induced rat model of Parkinson's disease: Behavioral and electrophysiological findings",
"Living on the edge with too many mouths to feed: Why dopamine neurons die",
"Age-and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease",
"Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease",
"Mitochondrial Dysfunction and Multiple Sclerosis",
"Epidemiology and treatment of multiple sclerosis in elderly populations",
"Impairment of Mitochondrial Redox Status in Peripheral Lymphocytes of Multiple Sclerosis Patients",
"Mitochondrial defects in acute multiple sclerosis lesions",
"Mitochondrial changes within axons in multiple sclerosis",
"Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients",
"Elevated ATG5 expression in autoimmune demyelination and multiple sclerosis",
"Role of autophagy in the pathogenesis of multiple sclerosis",
"Correlation between auto/mitophagic processes and magnetic resonance imaging activity in multiple sclerosis patients",
"Gene expression profiles of autophagy-related genes in multiple sclerosis",
"The role of mitochondria in amyotrophic lateral sclerosis",
"Role of mitochondria in amyotrophic lateral sclerosis",
"The cognitive profile of ALS: A systematic review and meta-analysis update",
"Amyotrophic lateral sclerosis. A study of its presentation and prognosis",
"Onset, natural history and outcome in idiopathic adult motor neuron disease",
"Current hypotheses for the underlying biology of amyotrophic lateral sclerosis",
"Amyotrophic lateral sclerosis",
"Differential involvement of optineurin in amyotrophic lateral sclerosis with or without SOD1 mutations",
"Novel optineurin mutations in sporadic amyotrophic lateral sclerosis patients",
"Optineurin with amyotrophic lateral sclerosis-related mutations abrogates inhibition of interferon regulatory factor-3 activation",
"Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation",
"Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1",
"TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration",
"Mitochondrial DNA abnormalities in skeletal muscle of patients with sporadic amyotrophic lateral sclerosis",
"Review: The role of mitochondria in the pathogenesis of amyotrophic lateral sclerosis",
"Mitochondria in sporadic amyotrophic lateral sclerosis",
"Anterior horn cell degeneration and Bunina-type inclusions associated with dementia",
"Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis",
"Morphological abnormalities in mitochondria of the skin of patients with sporadic amyotrophic lateral sclerosis",
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"\nFigure 1 .\n1Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.",
"\nFigure 1 .\n1Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.",
"\nFigure 2 .\n2Mitophagy. (A) Non-Receptor Mediated Mitophagy or Classical Mitophagy.",
"\nFigure 3 .\n3Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation",
"\nFigure 3 .\n3Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation",
"\n\n. Joshi, A.U.; Minhas, P.S.; Liddelow, S.A.; Haileselassie, B.; Andreasson, K.I.; Dorn, G.W., 2nd; Mochly-Rosen, D. Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration. Nat. Neurosci. 2019, 22, 1635-1648. [CrossRef] 20. Hailey, D.W.; Rambold, A.S.; Satpute-Krishnan, P.; Mitra, K.; Sougrat, R.; Kim, P.K.; Lippincott-Schwartz, J. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 2010, 141, 656-667. [CrossRef] 21. Ravikumar, B.; Moreau, K.; Jahreiss, L.; Puri, C.; Rubinsztein, D.C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 2010, 12, 747-757. [CrossRef] [PubMed] 22. Hayashi-Nishino, M.; Fujita, N.; Noda, T.; Yamaguchi, A.; Yoshimori, T.; Yamamoto, A. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 2009, 11, 1433-1437. [CrossRef] [PubMed] 23. Feng, D.; Liu, L.; Zhu, Y.; Chen, Q. Molecular signaling toward mitophagy and its physiological significance. Exp. Cell Res. 2013, 319, 1697-1705. [CrossRef] [PubMed] 24. Ge, L.; Zhang, M.; Kenny, S.J.; Liu, D.; Maeda, M.; Saito, K.; Mathur, A.; Xu, K.; Schekman, R. Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 2017, 18, 1586-1603. [CrossRef] 25. Tanida, I. Autophagosome formation and molecular mechanism of autophagy. Antioxid. Redox Signal. 2011, 14, 2201-2214. [CrossRef] 26. Itakura, E.; Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 2010, 6, 764-776. [CrossRef] 27. Hurley, J.H.; Young, L.N. Mechanisms of Autophagy Initiation. Annu. Rev. Biochem. 2017, 86, 225-244. [CrossRef] 28. Hosokawa, N.; Hara, T.; Kaizuka, T.; Kishi, C.; Takamura, A.; Miura, Y.; Iemura, S.; Natsume, T.; Takehana, K.; Yamada, N.; et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 2009, 20, 1981-1991. [CrossRef] 29. Funderburk, S.F.; Wang, Q.J.; Yue, Z. The Beclin 1-VPS34 complex-At the crossroads of autophagy and beyond. Trends Cell Biol. 2010, 20, 355-362. [CrossRef] 30. Tassa, A.; Roux, M.P.; Attaix, D.; Bechet, D.M. Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem. J. 2003, 376, 577-586. [CrossRef] 31. Romanov, J.; Walczak, M.; Ibiricu, I.; Schuchner, S.; Ogris, E.; Kraft, C.; Martens, S. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 2012, 31, 4304-4317. [CrossRef] [PubMed] 32. Fivenson, E.M.; Lautrup, S.; Sun, N.; Scheibye-Knudsen, M.; Stevnsner, T.; Nilsen, H.; Bohr, V.A.; Fang, E.F. Mitophagy in neurodegeneration and aging. Neurochem. Int. 2017, 109, 202-209. [CrossRef] [PubMed] 33. Lin, Y.F.; Haynes, C.M. Metabolism and the UPR(mt). Mol. Cell 2016, 61, 677-682. [CrossRef] [PubMed] 34. Haynes, C.M.; Ron, D. The mitochondrial UPR-Protecting organelle protein homeostasis. J. Cell Sci. 2010, 123, 3849-3855. [CrossRef] 35. Pellegrino, M.W.; Nargund, A.M.; Haynes, C.M. Signaling the mitochondrial unfolded protein response. Biochim. Biophys. Acta 2013, 1833, 410-416. [CrossRef] 36. Voos, W.; Rottgers, K. Molecular chaperones as essential mediators of mitochondrial biogenesis. Biochim. Biophys. Acta 2002, 1592, 51-62. [CrossRef] 37. Ryan, M.T.; Naylor, D.J.; Hoj, P.B.; Clark, M.S.; Hoogenraad, N.J. The role of molecular chaperones in mitochondrial protein import and folding. Int. Rev. Cytol. 1997, 174, 127-193. [CrossRef] 38. Xu, S.; Peng, G.; Wang, Y.; Fang, S.; Karbowski, M. The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover. Mol. Biol. Cell 2011, 22, 291-300. [CrossRef] 39. Cadete, V.J.; Deschenes, S.; Cuillerier, A.; Brisebois, F.; Sugiura, A.; Vincent, A.; Turnbull, D.; Picard, M.; McBride, H.M.; Burelle, Y. Formation of mitochondrial-derived vesicles is an active and physiologically relevant mitochondrial quality control process in the cardiac system. J. Physiol. 2016, 594, 5343-5362. [CrossRef] 40. Sugiura, A.; McLelland, G.L.; Fon, E.A.; McBride, H.M. A new pathway for mitochondrial quality control: Mitochondrial-derived vesicles. EMBO J. 2014, 33, 2142-2156. [CrossRef] 41. Meissner, C.; Lorenz, H.; Weihofen, A.; Selkoe, D.J.; Lemberg, M.K. The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking. J. Neurochem. 2011, 117, 856-867. [CrossRef]"
] | [
"Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.",
"Mitochondrial Quality Control Mechanisms. Unfolded protein response, mitochondrial derived vesicles, and mitophagy [23,33] Created with BioRender.com.",
"Mitophagy. (A) Non-Receptor Mediated Mitophagy or Classical Mitophagy.",
"Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation",
"Transcellular Mitophagy. Mitochondria are released at neuronal synapses where they are taken up by glial cells (astrocytes and/or microglia) for degradation",
". Joshi, A.U.; Minhas, P.S.; Liddelow, S.A.; Haileselassie, B.; Andreasson, K.I.; Dorn, G.W., 2nd; Mochly-Rosen, D. Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration. Nat. Neurosci. 2019, 22, 1635-1648. [CrossRef] 20. Hailey, D.W.; Rambold, A.S.; Satpute-Krishnan, P.; Mitra, K.; Sougrat, R.; Kim, P.K.; Lippincott-Schwartz, J. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 2010, 141, 656-667. [CrossRef] 21. Ravikumar, B.; Moreau, K.; Jahreiss, L.; Puri, C.; Rubinsztein, D.C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 2010, 12, 747-757. [CrossRef] [PubMed] 22. Hayashi-Nishino, M.; Fujita, N.; Noda, T.; Yamaguchi, A.; Yoshimori, T.; Yamamoto, A. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 2009, 11, 1433-1437. [CrossRef] [PubMed] 23. Feng, D.; Liu, L.; Zhu, Y.; Chen, Q. Molecular signaling toward mitophagy and its physiological significance. Exp. Cell Res. 2013, 319, 1697-1705. [CrossRef] [PubMed] 24. Ge, L.; Zhang, M.; Kenny, S.J.; Liu, D.; Maeda, M.; Saito, K.; Mathur, A.; Xu, K.; Schekman, R. Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 2017, 18, 1586-1603. [CrossRef] 25. Tanida, I. Autophagosome formation and molecular mechanism of autophagy. Antioxid. Redox Signal. 2011, 14, 2201-2214. [CrossRef] 26. Itakura, E.; Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 2010, 6, 764-776. [CrossRef] 27. Hurley, J.H.; Young, L.N. Mechanisms of Autophagy Initiation. Annu. Rev. Biochem. 2017, 86, 225-244. [CrossRef] 28. Hosokawa, N.; Hara, T.; Kaizuka, T.; Kishi, C.; Takamura, A.; Miura, Y.; Iemura, S.; Natsume, T.; Takehana, K.; Yamada, N.; et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 2009, 20, 1981-1991. [CrossRef] 29. Funderburk, S.F.; Wang, Q.J.; Yue, Z. The Beclin 1-VPS34 complex-At the crossroads of autophagy and beyond. Trends Cell Biol. 2010, 20, 355-362. [CrossRef] 30. Tassa, A.; Roux, M.P.; Attaix, D.; Bechet, D.M. Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem. J. 2003, 376, 577-586. [CrossRef] 31. Romanov, J.; Walczak, M.; Ibiricu, I.; Schuchner, S.; Ogris, E.; Kraft, C.; Martens, S. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 2012, 31, 4304-4317. [CrossRef] [PubMed] 32. Fivenson, E.M.; Lautrup, S.; Sun, N.; Scheibye-Knudsen, M.; Stevnsner, T.; Nilsen, H.; Bohr, V.A.; Fang, E.F. Mitophagy in neurodegeneration and aging. Neurochem. Int. 2017, 109, 202-209. [CrossRef] [PubMed] 33. Lin, Y.F.; Haynes, C.M. Metabolism and the UPR(mt). Mol. Cell 2016, 61, 677-682. [CrossRef] [PubMed] 34. Haynes, C.M.; Ron, D. The mitochondrial UPR-Protecting organelle protein homeostasis. J. Cell Sci. 2010, 123, 3849-3855. [CrossRef] 35. Pellegrino, M.W.; Nargund, A.M.; Haynes, C.M. Signaling the mitochondrial unfolded protein response. Biochim. Biophys. Acta 2013, 1833, 410-416. [CrossRef] 36. Voos, W.; Rottgers, K. Molecular chaperones as essential mediators of mitochondrial biogenesis. Biochim. Biophys. Acta 2002, 1592, 51-62. [CrossRef] 37. Ryan, M.T.; Naylor, D.J.; Hoj, P.B.; Clark, M.S.; Hoogenraad, N.J. The role of molecular chaperones in mitochondrial protein import and folding. Int. Rev. Cytol. 1997, 174, 127-193. [CrossRef] 38. Xu, S.; Peng, G.; Wang, Y.; Fang, S.; Karbowski, M. The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover. Mol. Biol. Cell 2011, 22, 291-300. [CrossRef] 39. Cadete, V.J.; Deschenes, S.; Cuillerier, A.; Brisebois, F.; Sugiura, A.; Vincent, A.; Turnbull, D.; Picard, M.; McBride, H.M.; Burelle, Y. Formation of mitochondrial-derived vesicles is an active and physiologically relevant mitochondrial quality control process in the cardiac system. J. Physiol. 2016, 594, 5343-5362. [CrossRef] 40. Sugiura, A.; McLelland, G.L.; Fon, E.A.; McBride, H.M. A new pathway for mitochondrial quality control: Mitochondrial-derived vesicles. EMBO J. 2014, 33, 2142-2156. [CrossRef] 41. Meissner, C.; Lorenz, H.; Weihofen, A.; Selkoe, D.J.; Lemberg, M.K. The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking. J. Neurochem. 2011, 117, 856-867. [CrossRef]"
] | [
"Figure 1)",
"Figure 2",
"Figure 2",
"Figure 2",
"Figure 3)"
] | [] | [
"Mitochondria are essential organelles that regulate energy homeostasis, cell signaling, and cell death [1][2][3]. During threatened cell death or nutrient starvation, mitochondria can be degraded and recycled through mitophagy. Mitophagy is a specific form of autophagy, a process where cell contents are degraded and recycled. In a broad sense, mitophagy involves tagging mitochondria for removal, engulfment of the organelle by an autophagosome, and degradation in a lysosome. There are several pathways which control, initiate, and facilitate mitophagy. The many facets of mitochondrial function contribute to mitophagy pathways.",
"Mitochondria coordinate and balance energy production through beta oxidation, the citric acid cycle (TCA cycle), and oxidative phosphorylation at the electron transport chain (ETC). Beta oxidation is a catabolic pathway where free fatty acids are converted to acetyl coA, which enter the TCA cycle and ultimately oxidative phosphorylation. In the TCA cycle, either pyruvate (from glycolysis) or acetyl coA are oxidized to generate the high energy electron carriers, NADH and FADH 2 . NADH and FADH 2 enter the ETC at complex I and complex II, respectively. These high energy electron carriers undergo oxidation/reduction reactions in the ETC in order to pump protons into the matrix. These protons ultimately power ATP synthase (or Complex V) for the generation of ATP from ADP. These bioenergetic reactions maintain the mitochondrial electrochemical gradient, or mitochondrial membrane potential. Mitochondrial membrane potential is the main signal which either inhibits or initiates mitophagy.",
"Mitochondria are double membrane organelles. The outer mitochondrial membrane is imperative for mitophagy function [4][5][6][7]. Transport and signaling proteins localize to the outer mitochondrial membrane to facilitate protein and metabolite import. As discussed in more detail below, aggregation-prone proteins are known to block these import channels on the outer mitochondrial membrane in some phosphatidylethanolamine (PE) facilitate the elongation and closure of the IM, following fusion with the lysosome [25,26,31].",
"Mitochondrial quality control mechanisms include mitochondrial chaperones, the mitochondrial unfolded protein response (UPR mt ), degradation of mitochondrial proteins in the cytoplasm via the proteasome (p97, 26S proteasome), removal of damaged proteins via mitochondrial derived vesicles (MDVs), and mitophagy ( Figure 1) [32]. Misfolded mitochondrial proteins can be refolded by mitochondrial chaperone proteins (heat shock proteins 22, 60, and 70) or cleaved/degraded by mitochondrial proteases Lon and Clp through the UPR mt [33][34][35][36][37]. Damaged mitochondrial proteins can also be targeted specifically for proteasome degradation by p97 through the proteasome [38]. MDVs bud off mitochondria after engulfing damaged mitochondrial macromolecules. MDVs are associated with impaired mitochondrial import channels. These MDVs are either degraded by lysosomes, peroxisomes, or exocytosed [39,40]. Mitophagy (reviewed extensively below) functions to remove damaged mitochondria with the goal of preventing cell death. ",
"Non-receptor mediated mitophagy ( Figure 2) or classical mitophagy involves PTEN-induced kinase 1 (PINK1) and Parkin. PINK1 is normally degraded in the inner mitochondrial membrane (IMM) by PARL (presenilin-associated rhomboid-like protease) [41,42]. During mitophagy induction, PINK1 becomes active and accumulates on the outer mitochondrial membrane (OMM), where it recruits Parkin and ubiquitin through phosphorylation [43][44][45][46][47][48][49][50][51]. Parkin accumulates and polyubiquitinates mitochondrial proteins triggering proteasomal degradation. Parkin ubiquitination of OMM proteins leads to more PINK1 activity and phosphorylation of substrates, including the recruitment of additional Parkin proteins at the OMM, creating a positive feedback loop. OMM proteins mitofusins 1 and 2 (MFN1/2), voltage-dependent anion channel 1 (VDAC1), and translocase of the outer mitochondrial membrane 20 (TOM20) are ubiquitinated by Parkin [44,[46][47][48][49][50][51][52][53][54][55]. Ubiquitination of MFN1/2 blocks mitochondrial fusion allowing for the isolation of damaged mitochondria and smaller mitochondria facilitate autophagosome targeting [6,32,49,56]. ",
"Non-receptor mediated mitophagy ( Figure 2) or classical mitophagy involves PTEN-induced kinase 1 (PINK1) and Parkin. PINK1 is normally degraded in the inner mitochondrial membrane (IMM) by PARL (presenilin-associated rhomboid-like protease) [41,42]. During mitophagy induction, PINK1 becomes active and accumulates on the outer mitochondrial membrane (OMM), where it recruits Parkin and ubiquitin through phosphorylation [43][44][45][46][47][48][49][50][51]. Parkin accumulates and polyubiquitinates mitochondrial proteins triggering proteasomal degradation. Parkin ubiquitination of OMM proteins leads to more PINK1 activity and phosphorylation of substrates, including the recruitment of additional Parkin proteins at the OMM, creating a positive feedback loop. OMM proteins mitofusins 1 and 2 (MFN1/2), voltage-dependent anion channel 1 (VDAC1), and translocase of the outer mitochondrial membrane 20 (TOM20) are ubiquitinated by Parkin [44,[46][47][48][49][50][51][52][53][54][55]. Ubiquitination of MFN1/2 blocks mitochondrial fusion allowing for the isolation of damaged mitochondria and smaller mitochondria facilitate autophagosome targeting [6,32,49,56].",
"Autophagy adaptor proteins are recruited to the OMM by ubiquitinated proteins. These adaptors include neighbor BRCA1 gene (NBR1), nuclear dot protein 52 (NDP52), optineurin (OPTN), sequestosome-1 (SQSTM1/p62), and Tax1-binding protein (TAX1BP1) [23,52,[57][58][59][60][61][62][63][64]. Adaptor proteins recruit and interact with autophagosome proteins; gamma-aminobutyric acid receptorassociated protein (GABARAP) or microtubule-associated protein 1A/1B-light chain 3 (LC3) to mediate the formation of the mitophagosome and lysosomal fusion/degradation. These adaptor proteins interact with LC3 and GABARAP through LC3 interacting regions (LIR) motifs (W/F/YxxL/I) [6,7,23,32]. The importance of different adaptor proteins has been up for debate and often their Activation of PINK1 leads to recruitment of ubiquitin and Parkin. Parkin ubiquitinates and phosphorylates mitochondrial proteins (such as VDAC, MFN1/2, and TOM20) and this initiates receptor adaptor protein recruitment (p62, NDP52, OPTN, TAX1BP1, and NBR1). These adaptor proteins interact with LC3 to form the autophagosome. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50] (B) Receptor Mediated Mitophagy. Mitochondrial receptor proteins (BNIP3, NIX, FUNC1, AMBRA1, PHB2, or cardiolipin) are ubiquitinated and phosphorylated. This facilitates their interaction with LC3 and GABARAP for autophagosome formation. The Vps34 and Atg5/12/16 complex facilitate autophagosome maturation, closure, and lysosome fusion [44,50]. Created with BioRender.com.",
"Autophagy adaptor proteins are recruited to the OMM by ubiquitinated proteins. These adaptors include neighbor BRCA1 gene (NBR1), nuclear dot protein 52 (NDP52), optineurin (OPTN), sequestosome-1 (SQSTM1/p62), and Tax1-binding protein (TAX1BP1) [23,52,[57][58][59][60][61][62][63][64]. Adaptor proteins recruit and interact with autophagosome proteins; gamma-aminobutyric acid receptor-associated protein (GABARAP) or microtubule-associated protein 1A/1B-light chain 3 (LC3) to mediate the formation of the mitophagosome and lysosomal fusion/degradation. These adaptor proteins interact with LC3 and GABARAP through LC3 interacting regions (LIR) motifs (W/F/YxxL/I) [6,7,23,32]. The importance of different adaptor proteins has been up for debate and often their involvement in mitophagy is context-dependent. The same is true for Parkin, as there are some pathways of mitophagy which are Parkin-independent and these are discussed below [23,44].",
"Receptor mediated mitophagy ( Figure 2) is driven by mitochondrial receptor proteins which contain LIR motifs (W/F/YxxL/I) [23,50]. Outer mitochondrial membrane proteins, autophagy and Beclin 1 regulator 1 (AMBRA1), Bcl-2 interacting partner 3 (BNIP3), FUN14 domain-containing protein 1 (FUNDC1), Nip3-like protein X (NIX); and inner mitochondrial membrane proteins cardiolipin and prohibitin 2 (PHB2) are the most studied receptors which mediate mitophagy [23,50,[65][66][67][68][69][70].",
"Receptor mediated mitophagy is activated under specific conditions. For example, during hypoxia BNIP3 and NIX transcription are activated by hypoxia inducible factor 1 alpha (HIF1α) [70][71][72][73]. BNIP3 and NIX activity are regulated by phosphorylation where increased phosphorylation increases their binding affinity for LC3 [74,75]. Hypoxia also promotes FUNDC1 binding to LC3 through dephosphorylation via phosphoglycerate mutase family member 5 phosphatase (PGAM5) [65,67,76,77]. Conversely, FUNDC1 phosphorylation by ULK1 is also a mitophagy activating event. Ultimately ubiquitination of FUNDC1 by E3 ubiquitin protein ligase 5 (UBR5) promotes lysosomal degradation of mitochondria [65,67,78]. The inner mitochondrial membrane receptors, PHB2 and cardiolipin have been shown to interact the LC3, especially during times of mitochondrial permeabilization [79,80]. AMBRA1 is sequestered and inhibited by B-cell lymphoma protein 2 (Bcl-2) on the outer mitochondrial membrane but upon mitophagy activated AMBRA1 binds LC3 in a Parkin-dependent or -independent manner [81].",
"Receptor mediated mitophagy culminates in the elongation of and closure of phagophore membranes, resulting in engulfment of the mitochondria. The elongation and closure of the phagophore is driven by the mitochondrial receptor binding LCR and/or GABARAP, leading to closure of the phagophore by GABARAP [23,50]. Lastly, the autophagosome fuses with a lysosome for degradation.",
"Separate from mitophagy pathways, damaged mitochondria can be partitioned and removed through mitoptosis. This phenomenon was first proposed in 1992 [82]. Mitoptosis has several proposed definitions. One possible process of mitoptosis occurs when damaged mitochondria gather around the nucleus, are selectively partitioned into lipid membranes and extruded from the cell [83]. A separate definition is when mitochondria undergo condensation with swelling and fragmentation of cristae. This leads to the bursting of the outer mitochondrial membrane and fragmented cristae are extruded into the cytoplasm. In other forms of mitoptosis the outer mitochondrial membrane can remain intact and the cristae deteriorate through refraction and coalescence [6,[83][84][85][86][87]. The main benefit of mitoptosis is to prevent opening of the mitochondrial permeability transition pore (MPTP) and apoptosis. Overall, the method of which cells dispose of damaged mitochondria or part of mitochondria can vary and requires further study. Cell-specific pathways which evolved to provide the least devastating consequences based on cell and tissue function likely exist.",
"Mitoptosis does not require extramitochondrial signaling or protein complexes according to current knowledge. Some evidence suggests PINK1 and Parkin are involved, but no consensus currently exists [6,[83][84][85][86][87][88]. Situations which likely activate mitoptosis include mitochondrial membrane depolarization, increased ROS production, and degradation of mtDNA.",
"Recent studies have described exocytosis of mitochondria from cells followed by endocytosis or phagocytosis of these extracellular mitochondria. In the brain neurons release mitochondria at synapses, and these extracellular mitochondria were taken up by glial cells for phagocytosis [17,18]. This phenomenon will be referred to as transcellular mitophagy here ( Figure 3). A recent study showed that retinal ganglion axons shed mitochondria, which were then then degraded by adjacent astrocytes in mice [18]. Mitochondria might undergo degradation in the axoplasm (cytoplasm of the nerve axon) with the assistance of axonal lysosomes [17]. Essentially instead of being degraded in the soma transcellular mitophagy describes the fact that axonal mitochondria are instead enclosed by axoplasmic membranes that are shed and degraded by neighboring cells. showed that retinal ganglion axons shed mitochondria, which were then then degraded by adjacent astrocytes in mice [18]. Mitochondria might undergo degradation in the axoplasm (cytoplasm of the nerve axon) with the assistance of axonal lysosomes [17]. Essentially instead of being degraded in the soma transcellular mitophagy describes the fact that axonal mitochondria are instead enclosed by axoplasmic membranes that are shed and degraded by neighboring cells. [8,9]. Created with BioRender.com.",
"The notion that transcellular mitophagy occurs is logical given that it is not energetically favorable for neurons to bring mitochondria from dendrites back to the cell body for mitophagy. The process of transcellular mitophagy requires more study and understanding at the basic level and with regards to disease.",
"In addition to transcellular mitophagy, the observation of glial cells transferring mitochondria to neurons has been documented. In stroke models, glial cells transfer mitochondria to neurons as a likely means to protect neurons from energy stress and hypoxia [89,90]. The specific pathways which facilitate mitochondrial transfer between cells are unknown. However, some studies suggest that in astrocytes, glial acidic fibrillary protein (GFAP), and in neurons, uncoupling protein 2 (UCP2) may play a role [91,92].",
"In mesenchymal stem cells, connexins (particularly connexin 43) oligomerize to form Gap junctions. These Gap junctions may facilitate the formation of tunneling nanotubules, which allow the exchange of cellular contents, such as mitochondria [93]. Other studies suggest Miro1, a protein which connects cytoskeletal motor proteins to mitochondria is involved in mitochondrial transfer between cells [94]. Finally, S100A4 guides tunneling nanotubule growth [95]. The phenomenon of mitochondrial transfer between cells has been documented in a wide variety of model and tissue types [17,89,90,[92][93][94][95][96][97][98][99][100][101]. An important question remains regarding what initiates mitochondrial transfer and the specific mechanisms which control this process. [8,9]. Created with BioRender.com.",
"The notion that transcellular mitophagy occurs is logical given that it is not energetically favorable for neurons to bring mitochondria from dendrites back to the cell body for mitophagy. The process of transcellular mitophagy requires more study and understanding at the basic level and with regards to disease.",
"In addition to transcellular mitophagy, the observation of glial cells transferring mitochondria to neurons has been documented. In stroke models, glial cells transfer mitochondria to neurons as a likely means to protect neurons from energy stress and hypoxia [89,90]. The specific pathways which facilitate mitochondrial transfer between cells are unknown. However, some studies suggest that in astrocytes, glial acidic fibrillary protein (GFAP), and in neurons, uncoupling protein 2 (UCP2) may play a role [91,92].",
"In mesenchymal stem cells, connexins (particularly connexin 43) oligomerize to form Gap junctions. These Gap junctions may facilitate the formation of tunneling nanotubules, which allow the exchange of cellular contents, such as mitochondria [93]. Other studies suggest Miro1, a protein which connects cytoskeletal motor proteins to mitochondria is involved in mitochondrial transfer between cells [94]. Finally, S100A4 guides tunneling nanotubule growth [95]. The phenomenon of mitochondrial transfer between cells has been documented in a wide variety of model and tissue types [17,89,90,[92][93][94][95][96][97][98][99][100][101]. An important question remains regarding what initiates mitochondrial transfer and the specific mechanisms which control this process.",
"Changes in mitophagy flux, signaling, and mitochondrial function are observed with aging and in neurodegenerative diseases. It is imperative to understand the role mitophagy could contribute to brain aging and neurodegeneration. We discuss these implications below.",
"Aging is associated with a loss of proteostasis, mitochondrial dysfunction, genome instability, inflammation, changes to redox balance, and metabolic deficits [32]. Reduced autophagy and mitophagy are observed in models of aging [32,102].",
"Mitochondrial homeostasis and function are altered in aging. However, the exact mechanisms and findings are not consistent across models and studies. Brain cytochrome oxidase (COX; complex IV) and complex I activity are reduced while ROS production and oxidized proteins are increased in aged rats [103,104]. Calcium homeostasis is changed in aged rat brain mitochondria and synaptosomes; neither were able to take up calcium at a rate equivalent to young rats [105]. Aged mice have altered proteomic expression of glycolytic, TCA, and oxidative phosphorylation pathways in the brain but mitochondrial function is unchanged [106]. This suggests a compensatory mechanism during aging. Altered brain mitochondrial morphology is observed in aged rats and monkeys [107,108]. Other findings suggest a change to mtDNA epigenetic markers and increased mtDNA deletions in aged mouse brain [109,110]. Oxidative damage to brain mtDNA is related to reduced lifespan across numerous species (birds and mammals) [111,112]. mtDNA in the aged human brain shows increased somatic mutation burden and oxidative damage [113][114][115].",
"In multiple organisms, mitophagy is associated with longevity and lifespan. Pink1 knockout causes a shorter lifespan, while Parkin overexpression in neurons increases lifespan in D. melanogaster [45,116,117]. C. elegans models exposed to mild mitochondrial stress and upregulated mitophagy have extended lifespan [118,119]. Urolithin A (UA), tomatidine, and catechinic acid induce mitophagy and increase the lifespan of C. elegans models [120][121][122]. In an aging mouse model, stimulation of mitophagy with NAD + prolongs lifespan [123].",
"Mitochondrial quality control emerges as a central theme in most neurodegenerative diseases, including Alzheimer' Disease (AD), Parkinson's Disease (PD, Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS). Mitophagy stimulation has shown positive effects in models of these diseases.",
"AD is the most common form of dementia diagnosed upon autopsy with neuropathological examination [124,125]. The pathological hallmarks which lead to AD diagnosis postmortem are considerable Aβ plaques and tau tangles throughout the brain [124][125][126][127]. Recent advances in neuroimaging have allowed the determination of Aβ plaque and tau tangle load in living subjects, showing these proteins accumulate in the brain decades before clinical signs of cognitive decline [126,[128][129][130].",
"One of the earlier observations in AD subjects was reduced glucose uptake/utilization in the brain via fluorodeoxyglucose (FDG)-positron emission tomography (PET) [128][129][130][131][132][133]. Accumulation of evidence supports an overall metabolic deficit in AD subjects both within the brain and systemically [134]. The mitochondrial ETC enzyme, COX (or complex IV), has reduced Vmax in the AD brain, fibroblasts, and blood samples [132,[135][136][137][138][139][140][141][142][143][144][145][146]. AD-like changes can be transferred to other cell types when AD patient mitochondria (mtDNA) are transferred [139,[147][148][149][150][151]. This process of creating cytoplasmic hybrid cells (cybrids) allows for the determination of the contribution of mtDNA on disease and cell physiological processes [150].",
"Mitochondria in AD autopsy brain samples have fragmented cristae and vary widely in size compared to age-matched non-demented brain samples [152]. Mitochondria within dendritic spines and presynaptic terminals show the most fragmented and disorganized cristae. Alterations to mitochondrial ultrastructure were observed in areas of the brain with and without Aβ and tau pathology (cerebellar cortex, hypothalamus, cerebellum, and visual cortex). In addition to altered mitochondrial morphology, presynaptic terminals had reduced synaptic vesicles and fragmentation of golgi cisternae was observed [152]. mtDNA inheritance confers risk to AD. Studies show that offspring of maternal AD subjects have a higher risk of AD diagnosis than offspring of paternal AD subjects. Nearly all mitochondria are inherited maternally. Offspring from maternal AD subjects show metabolic and neuroimaging changes earlier in life than offspring of paternal AD subjects [129,[153][154][155][156]. Furthermore, inherited mtDNA haplogroups are associated with both increased and decreased AD risk [157][158][159][160][161][162][163][164]. These inherited mtDNA haplogroups also interact with the nuclear DNA encoded risk factor, ApoE (apolipoprotein E) to influence AD risk [159,164,165]. Thus, it is important to understand the role of mitochondria, mitophagy, and metabolism in AD.",
"AD mouse models have disrupted mitophagy [166]. This is observed in tau transgenic mice and AD postmortem human brains with accumulated tau aggregates [167]. Mutant Amyloid Precursor Protein (APP; Swedish mutant) mice show increased mitochondrial fission proteins and decreased mitochondrial fusion and mitophagy protein expression in hippocampal neurons [168]. APPsw/PS1dE9 transgenic mice show increased LC3, PINK1, and Parkin expression [169]. Cortical neurons derived from AD transgenic mice (J20; Swedish and Indiana APP mutations) also show increased mitophagy protein expression with depolarized mitochondrial membrane potential [170].",
"In AD postmortem brain samples, accumulation of damaged mitochondria and autophagosome vacuoles is observed [171][172][173]. The UPR mt pathway is upregulated at the gene level, with reduced proteasomal activity through the 26S proteasome. Parkin, SQSTM1/p62, and LCR mitochondrial localization are increased [172][173][174][175][176]. Mitophagy pathways are altered in human postmortem AD brain. Cytosolic Parkin is depleted, and lysosomal deficits are observed. Impaired Parkin recruitment to mitochondria is possibly caused by tau-mediated sequestration of Parkin in the cytosol [170,177]. Defects in the activation of ULK1 and TBK1 lead to impaired mitophagy [177]. In AD, mitophagy increases or decreases depending on the part of the cell observed. Although it is increased in lysosomes, other parts of the cell fail at completing mitophagy.",
"Mechanisms of altered mitophagy and mitochondrial function warrant further study in AD, specifically given the strong association of mitochondria and mitophagy with synapse health and function.",
"PD is a neurodegenerative disease with both cognitive and neuromuscular changes. Motor deficits, tremors, rigidity, bradykinesia, dyssomnia, and depression are clinical hallmarks of PD. In some cases, PD can cause cognitive impairment. Within the brain, PD causes degeneration of dopaminergic neurons in the substantia nigra with Lewy body accumulation (composed of aggregating α-synuclein) [178][179][180].",
"Mitochondrial dysfunction is observed in PD. A complex I deficiency is noted in brain tissue (substantia nigra) but not in skeletal muscle [181,182]. The complex I deficiency might be brain-specific, but some studies suggest deficits in platelets of PD subjects [183,184]. These findings are dependent on the methodology used. Mitochondrial dysfunction is also observed in cybrid cell lines derived from transfer of PD subject mtDNA, suggesting mtDNA may play a role [150,[185][186][187]. Furthermore, PD patients have increased rates of mtDNA deletion in the substantia nigra [188,189].",
"Familial forms of PD are caused by mutations in genes involved in mitophagy. Mutations in PARK6 (encodes for PINK1), PARK2 (encodes for Parkin), PARK1/4 (α-synuclein), PARK7 (DJ1), PARK8 (LRRK2), PARK17 (Vsp35), and PARK9 (ATP13A2) genes are linked to familial PD [177,[190][191][192][193][194][195]. The role of PINK1, Parkin, and Vsp35 in mitophagy are well known and reviewed above. DJ1 and α-synuclein have been shown to modulate mitophagy either through direct interactions with PINK1 and Parkin or by causing mitochondrial fragmentation. Loss of function of LRRK2 and ATP13A2 have been shown to impair mitochondrial turnover.",
"Despite these genetic studies most PD cases are sporadic with no known genetic cause. Inhibition of complex I function with rotenone (a pesticide) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces PD in rodent and non-human primates [196][197][198][199][200]. Dopaminergic neurons form many synapses (up to one million per neuron), and thus, have a high bioenergetic demand to maintain these unmyelinated synapses [9,201]. In human postmortem brain, mitophagy markers (phosphorylated S65 ubiquitin) increase across age and with PD diagnosis [202]. This mitophagy marker also associated with Lewy Bodies, showing an increase of mitophagy in early PD stages and a decrease in late PD stages [202]. α-synuclein is also associated with an increase in Miro expression in postmortem human brain tissue, human neurons, and fly models of PD [203]. Reducing Miro in the human neuronal and fly models rescued neurodegeneration and mitophagy [203].",
"Overall, PD patients and disease models consistently show mitochondrial and mitophagy dysfunction.",
"MS is a neurodegenerative disease marked by an autoimmune response against the myelin sheath. Demyelination of white matter is caused by autoreactive T cells which target the myelin sheath within the central nervous system. This demyelination leads to a secondary loss of neuronal axons and neurodegeneration. MS has no known genetic cause and occurs in young adults with a higher incidence in females (3:1 ratio female to male) [204,205]. Most MS subjects (85%) have a relapse-remission disease course, which includes periods of demyelination followed by neurological recovery. Eventually a secondary progressive course occurs with few remission periods [204,205]. A smaller subset of MS patients (10-15%) show continued progression known as primary progressive MS.",
"MS pathology begins with the formation of a lesion with acute inflammation, which transitions to a state of chronic inflammation followed by neurodegeneration. Chronic inflammation is believed to allow penetration of the blood brain barrier by activated T cells directed against the myelin sheath.",
"The inflammation observed in MS shows activation of both innate and adaptive immunity pathways [204,205]. In addition to demyelination, loss of oligodendrocytes is observed. Myelin autoreactive T-cells can induce experimental autoimmune encephalomyelitis (EAE) in animal models and human MS subject genome wide association studies (GWAS) have a high representation of immune genes related to T cell differentiation [204,205]. Gray matter lesions and brain atrophy are present before clinical MS onset. These findings highlight the lack of understanding of what ultimately initiates the autoimmune reaction in MS.",
"Mitochondria and mitophagy are critical for immune signaling. ROS signals from mitochondria are known to modulate inflammatory responses. ROS signals in MS damage myelin and the blood brain barrier, further exacerbating disease. Oxidation of phospholipids and DNA damage are observed during periods of chronic inflammation with disruption of neuronal axons. Chronic inflammation induces damage to macromolecules including mtDNA, ETC protein, and lipids [204]. Systemic mitochondrial changes are observed in MS. Peripheral lymphocytes (mostly T cells) show increased mitochondrial superoxide production, decreased ETC protein expression, increased lactate, and decreased antioxidant capacity in human MS subjects [206]. These findings suggest a mitochondrial and bioenergetic deficit in MS.",
"Of interest, MS subjects show decreased expression of COX5B [204]. Active lesions from MS subjects show reductions in COX and its catalytic component COX-I; decreased expression was explicitly observed in oligodendrocytes, astrocytes, and neuronal axons [207]. A separate study showed reduced COX activity in lesions from MS patients; they also observed correlation of this endpoint with neurofilament protein (SMI32) expression and with macrophage/microglial density [208]. An axon-specific protein, syntaphilin, which functions as a mitochondrial docking protein, was increased in chronic lesions [208]. Inactive lesion areas in the same MS subjects had elevated COX activity and increased mitochondrial mass [208]. Gene expression analysis of cortex tissue from MS subjects show overall reductions in nuclear encoded mitochondrial genes and ETC complex expression specifically in neurons [209]. Overall, mitochondrial deficits are observed in MS and the role of these deficits requires further study.",
"Autophagy and mitophagy pathways are altered in MS human subjects and animal models, which mimic MS pathology. Atg5 modulates T cell survival and its expression correlates with clinical disability in mouse models of EAE. In MS brain samples, encephalitogenic T cells appear to be the major source of Atg5 expression and systemic T cells in human MS subjects showed increased Atg5 expression [210]. In MS brain lesions from human subjects, Lamp2 and LC3II/I ratios are decreased, suggesting impaired autophagy [211]. Further studies have shown serum and CSF concentrations of Atg5 are elevated in MS patients. This study also noted increased expression of Parkin in both serum and CSF with higher serum levels of lactate [212]. In addition, blood from MS subjects show altered expression of several autophagy-related genes (these included ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, and HTT) [213]. Autophagy and mitophagy are imperative for immune cell function, differentiation, and adaptive immunity. The role of these pathways in driving autoreactive T cell differentiation in MS needs to be understood.",
"ALS is a neurodegenerative disease marked by the loss of alpha motor neurons in the lumbar spinal cord and motor cortex [214,215]. The lifespan post ALS diagnosis is short, often 2-3 years, because progressive muscle wasting leads to lung paralysis. In some cases of ALS, dementia is present [216]. Most ALS cases are sporadic, with a rare subset (less than 5% of total cases) being familial. These familial cases are caused by mutations in genes including Tdp43, Fus, Fig4, Ang, Vapb, and C9orf72 [217][218][219][220]. Mutations in the Optn gene which encodes an autophagy protein, optineurin, were found to be causative of ALS in 2010 [221][222][223]. After the discovery of mutations in Sod1 and Tdp43, transgenic mouse models were developed [224][225][226].",
"Changes to mitochondrial ultrastructure in human ALS subjects were revealed several decades ago [227][228][229]. Cytoplasmic inclusions that may represent mitochondria-containing autophagic vacuoles are observed in ALS motor neurons [230,231]. Although ALS neurodegeneration is anatomically specific, mitochondrial abnormalities are found systemically [214,215,227,229,232,233]. Mitochondrial dysfunction is present in platelet and muscle mitochondria from ALS subjects [227,229,[234][235][236][237][238]. mtDNA may contribute to ALS pathologies, as cybrid cells harboring mtDNA from ALS subjects often show mitochondrial abnormalities and increased cell death [150,229,239,240].",
"ALS is modeled using rodents that express mutant SOD1 or mutant TDP43 [224,226]. SOD1 is a cytoplasmic enzyme which was identified within mitochondrial membranes [225,229,[241][242][243]. This is the case for both mutant SOD1 and to a lesser extent wild type SOD1. Mutant SOD1 ALS transgenic mice have altered mitochondrial morphology and mitochondrial SOD1 accumulation [242,243]. This raises the possibility that mutant SOD1 may drive neurodegeneration by damaging mitochondria. TDP43 mutants are also observed within mitochondria and appear to induce mitochondrial dysfunction [242][243][244][245][246]. Both TDP43 and SOD1 are known to aggregate within motor neurons and muscle; TDP43 interacts with proteins critical to mitophagy in an inhibitory manner [219,244,245,[247][248][249][250][251].",
"Impaired mitophagy was proposed to be involved in the denervation of neuromuscular junctions in an ALS mouse model [252]. Lysosomal dysfunction has also been implicated in ALS. Specifically, lysosomal deficits result in an abnormal accumulation of autophagic vacuoles that engulf damaged mitochondria within the motor neuron axons of G93A SOD1 ALS mice [177]. Impaired mitochondrial turnover along with the accumulation of misfolded proteins and protein aggregates contributes to ALS-linked mitochondrial dysfunction and motor neuron death. Mitochondrial and mitophagy ultrastructure are varied across compartments of motor neurons [253,254]. Parkin, Miro1, and Mfn2 are depleted in an ALS mouse model (G93A SOD1); however, mitochondrial localized p62 is upregulated [255]. Mutant forms of optineurin interfere with Parkin ubiquitin ligase function [256].",
"In human post-mortem samples, increased autophagic vesicles are observed in lumbar spinal cord motor neurons [257]. Induced pluripotent stem cell (iPSC)-derived motor neurons from familial ALS subjects with C9orf72 mutations or haploinsufficiency have dysfunction of autophagy pathways [258,259].",
"As discussed above mitochondrial dysfunction and mitophagy alterations are prevalent in human AD, PD, MS, and ALS samples as well as cell and animal models of disease. We discuss below the methods being investigated to modulate mitophagy.",
"Increasing mitophagy in transgenic mouse models of neurodegeneration have shown mostly beneficial effects. In AD models (iPSC derived, transgenic mice, and C. elegans), increasing mitophagy using nicotinamide mononucleotide (NMN), UA, or actinonin (AC) reduced Aβ and tau aggregation. In AD transgenic mice, mitophagy induction benefited cognition [260,261]. These compounds are NAD + precursors, which may drive mitophagy through alterations in redox balance (NAD + /NADH). UA likely drives mitophagy through a PINK1/Parkin/Nix axis.",
"Broad autophagy induction with Rilmenidine in the G93ASOD1 mouse model of ALS did not change disease progression [262]. The mechanism(s) of Rilmenidine autophagy/mitophagy induction are currently unknown. Rapamaycin (an mTOR inhibitor) treatment of this same mouse model was detrimental unless mature lymphocytes were depleted [263]. These studies highlight the importance of understanding the non-cell autonomous effects of autophagy and mitophagy pathways.",
"In PD rodent models (MPTP injection), a drug, Salidroside, increased Parkin and PINK1 expression and preserved dopaminergic neurons in the substantia nigra [264]. A cell permeable form of Parkin rescued cells from aggregating α-synuclein, partially restored motor function, and protected dopaminergic neurons in the 6-OHDA PD (6-hydroxydopamine) mouse model [265].",
"In an MS-related mouse model of EAE administration of rapamycin, an mTOR inhibitor improves outcomes [211]. Further studies of the EAE mouse model show that excessive activation of Drp1 through nitration leads to an overaction of mitophagy [266]. Blocking this pathway alleviated the disease burden in the EAE mouse mode [267]. Genetic ablation of Beclin 1 was also protective in the EAE mouse model [268]. Overall, in MS inhibition of mitophagy specifically in T cells could be beneficial.",
"UA has been shown to be safe and well-tolerated in elderly adults, with plasma concentrations detectable at a range of doses. Furthermore, UA affected mitochondrial gene expression in muscle [269]. A separate study in healthy adults is registered for UA (NCT04160312), but no results have been posted. Clinical trials for NMN (NCT04228640 safety trial) are recruiting or ongoing (NCT03151239 effects on cardiometabolic health). In Japan, the first human clinical trial of NMN showed no deleterious effects, suggesting NMN is tolerable and safe [270,271]. No clinical trials for these NAD + precursor mitophagy modulators are currently registered for neurodegenerative diseases.",
"Lifestyle interventions could be useful tools to boost mitophagy. Exercise and diet have been shown to induce mitophagy [272][273][274][275][276]. In both AD animal models and human clinical trials, exercise has shown cognitive benefit [275,[277][278][279][280][281]. The exercise effects in ALS and PD are more controversial, but overall, exercise seems to improve physical and cognitive outcomes [282][283][284][285][286][287]. Intermittent fasting and ketogenic diets have also been shown to induce mitophagy and improve cognition/motor performance [288][289][290][291][292][293][294].",
"Current clinical trials aimed at increasing autophagy, mitophagy, or mitochondrial function are ongoing or recently completed. For AD, these include treatment with nicotinamide riboside (NR; NCT04430517; NAD + precursor), Dimebon (NCT00675623, NCT00829374; stimulates mTOR-dependent mitophagy), resveratrol (NCT00678431; mTOR inhibitory), ketogenic diets (NCT03860792), and caloric restriction diets (NCT02460783). In PD, these include nicotinamide supplementation (NCT03568968; NAD + precursor), ubiquinol/Coenzyme Q10 (NCT03061513; autophagy mechanism unknown), ketogenic diets, and ketone esters (NCT01364545, NCT04477161). In MS, clinical trials include ketogenic diet, dimethyl fumarate, and MitoQ (NCT03740295, NCT04267926, NCT02461069). In ALS, one clinical trial for ubiquinol/Coenzyme Q10 (NCT00243932; autophagy mechanism unknown) was completed. Overall, the clinical trials directly modulating mitophagy are lacking and require more attention. The majority of mitophagy inducers in clinical trials have unknown mechanisms and pleotropic affects.",
"Targeting specific pathways and tissues could be advantageous in avoiding deleterious or off-target effects. Designing new therapeutic strategies should focus on modulating specific mitophagy targets while also enhancing mitochondrial function and biogenesis.",
"Mitophagy and mitochondrial quality control are important mechanisms which should be further studied in the context of brain aging and neurodegeneration. Novel mechanisms of mitochondrial quality control in neurons and glia have illuminated the knowledge gaps in this field of study. Mitochondria are dynamic and multifaceted organelles at the forefront of pathways associated with aging and neurodegeneration (proteostasis, metabolism, inflammation, and synapse loss). Thus, targeting mitochondrial health and mitochondrial quality control will target the most common pathological mechanisms in neurodegeneration. 19 "
] | [] | [
"Introduction",
"Non-Receptor Mediated Mitophagy",
"Non-Receptor Mediated Mitophagy",
"Receptor Mediated Mitophagy",
"Mitoptosis",
"Transcellular Mitophagy",
"Mitophagy in Aging and Neurodegeneration",
"Aging",
"Alzheimer's Disease",
"Parkinson's Disease",
"Multiple Sclerosis",
"Amyotrophic Lateral Sclerosis",
"Modulating Mitophagy in Neurodegeneration",
"Concluding Remarks",
"Figure 1 .",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 3 ."
] | [] | [] | [
"Molecular Sciences Mitophagy and the Brain",
"Molecular Sciences Mitophagy and the Brain"
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5,696,267 | 2022-03-24T20:39:29Z | CCBY | http://www.jbc.org/content/281/21/14882.full.pdf | HYBRID | 6a1c2b462b06a77810c1df9478b0416fab954a94 | null | null | null | null | 10.1074/jbc.m511747200 | 1997683520 | 16556595 | null |
p204 Is Required for the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes ITS EXPRESSION IS ACTIVATED BY THE CARDIAC GATA4, NKX2.5, AND TBX5 PROTEINS * □ S
JBC Papers in PressCopyright JBC Papers in PressMarch 22, 2006. 2006
B Published ; Ding
C Liu
Y Huang
J Yu
W Kong
P Lengyel
p204 Is Required for the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes ITS EXPRESSION IS ACTIVATED BY THE CARDIAC GATA4, NKX2.5, AND TBX5 PROTEINS * □ S
J. Biol. Chem
JBC Papers in Press281March 22, 2006. 200610.1074/jbc.M511747200Received for publication, October 31, 2005, and in revised form, February 15, 2006
Among 10 adult mouse tissues tested, the p204 protein levels were highest in heart and skeletal muscle.We described previously that the MyoD-inducible p204 protein is required for the differentiation of cultured murine C2C12 skeletal muscle myoblasts to myotubes. Here we report that p204 was also required for the differentiation of cultured P19 murine embryonal carcinoma stem cells to beating cardiac myocytes. As shown by others, this process can be triggered by dimethyl sulfoxide (DMSO). We established that DMSO induced the formation of 204RNA and p204. Ectopic p204 could partially substitute for DMSO in inducing differentiation, whereas ectopic 204 antisense RNA inhibited the differentiation. Experiments with reporter constructs, including regulatory regions from the Ifi204 gene (encoding p204) in P19 cells and in cultured newborn rat cardiac myocytes, as well as chromatin coimmunoprecipitations with transcription factors, revealed that p204 expression was synergistically transactivated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. Furthermore, ectopic p204 triggered the expression of Gata4 and Nkx2.5 in P19 cells. p204 contains a nuclear export signal and was partially translocated to the cytoplasm during the differentiation. p204 from which the nuclear export signal was deleted was not translocated, and it did not induce differentiation. The various mechanisms by which p204 promoted the differentiation are reported in the accompanying article (2 The abbreviations used are: Id, inhibitor of differentiation; NES, nuclear export signal; 204AS, 204 antisense; ␣protein (e.g. ␣Gata4), antiserum to the protein in question (e.g. to Gata4); DMSO, dimethyl sulfoxide; MHC, myosin heavy chain; p204⌬NES, p204 lacking the NES; p204MNES, p204 with a mutated NES; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; RT, reverse transcription.
The interferons are vertebrate cytokines with antimicrobial, cell growth regulatory, and immunomodulatory activities that also affect differentiation (1)(2)(3). They function by modulating the expression of many genes, including those of the gene 200 cluster (4 -10). In the mouse this cluster consists of at least 10 genes, which encode the p200 family proteins (11). The human counterpart of the cluster is smaller; it consists apparently of only four genes (MNDA, IFI16, AIM2, and IFIX) that encode the Hin200 family proteins (12)(13)(14)(15).
Among the two best characterized members of the murine p200 family proteins is p202a (which was designated earlier as p202) (16 -26). p202a modulates transcription, cell proliferation, and apoptosis. It functions primarily by binding various sequence-specific transcription fac-tors and inhibiting their activity generally, but not exclusively, by binding them and blocking their sequence-specific binding to DNA (24). p202a overexpression was implicated in the susceptibility of mice to the autoimmune disease lupus (27).
The other much studied p200 family protein p204 is encoded by the Ifi204 gene (5). p204 is structurally related to p202a and is inducible by interferons, as is p202a. Depending on the cell type and the state of differentiation, p204 can be located in the nucleolus, the nucleoplasm, or the cytoplasm (28 -30). Its overexpression in cultured mammalian cells is growth-inhibitory (28,31). It can delay the progression of cells from the G 0 /G 1 to the S phase of the cell cycle (31). p204 can inhibit the transcription of ribosomal RNA by binding to the ribosomal DNAspecific UBF transcription factor and by inhibiting its binding to DNA (30). p204, similarly to p202a, contains the pRb-, p107-, and p130-binding motif LXCXE and can bind these pocket proteins (19,30,32).
Among 10 adult mouse tissues tested, the level of p204 is highest in the heart and second highest in skeletal muscle (29). During the differentiation of cultured C2C12 mouse skeletal muscle myoblasts to myotubes, the p204 (and p202a) levels increased severalfold as a consequence of the transactivation of the Ifi204 gene by the muscle-specific MyoD, myogenin, and E12/E47 transcription factors (29). This increase did not depend on interferon action (33). p204 is required for the differentiation of C2C12 myoblasts to myotubes. It enables the differentiation, at least in part, by overcoming the inhibition of the activities of MyoD, E12/E47, and other myogenic basic region-helix-loop-helix transcription factors by the inhibitor of differentiation (Id) 2 proteins Id1, Id2, and Id3 (33).
p204 is also involved in osteogenesis. It is expressed in osteoblasts of various tissues in embryonic and neonatal mice. It is induced in the course of the BMP-2-triggered differentiation of C2C12 cells to osteoblasts. p204 binds and acts as a cofactor of Cbfa-1 (Core binding factor ␣-1), which is an essential transcription factor in osteoblasts differentiation and bone formation (26).
The aim of this study has been to explore whether p204 is involved in and is required for cardiac myocyte differentiation and the basis of the high level of expression of p204 in mouse heart. As a model system for cardiac myocyte differentiation, we used cultured murine P19 embryonal carcinoma cells (34 -37). The P19 line of pluripotent embryonal stem cells was derived from a teratocarcinoma induced in C3H/HC mice (38). The cells can be maintained in culture in an undifferentiated state. When injected into early mouse embryos, * This work was supported by NIAID Research Grant AI-12320 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. P19 cells are capable of contributing to a variety of apparently normal tissues suggesting that the mechanisms of their differentiation are similar to those of normal embryonic cells (39). P19 cells can also be induced to differentiate in vitro into multiple cell types, including beating cardiac myocytes, skeletal myocytes, and myotubes, as well as neurons (34,36). The differentiation of P19 cells to cardiac and skeletal myocytes in vitro is initiated by plating P19 cells grown in tissue culture dishes and dissociated with trypsin into bacteriological culture dishes to which the cells do not attach and in which they form aggregates. The cultures are supplemented with DMSO. The aggregates are transferred to tissue culture dishes and further incubated in the presence of DMSO. Proliferating cells migrate out from the aggregates, including many that are differentiated to cardiac myocyte-type beating cells (and later also skeletal myocyte-type cells). In the absence of DMSO (whose mode of action remains to be clarified), there occurs little or no differentiation of P19 cells in vitro (34,36). DMSO causes an immediate, transient increase in Ca 2ϩ ions in the cells, and it was proposed that permeabilization and membrane penetration by DMSO may alter molecular interactions in the cells (40,41).
We report here that in P19 cells (i) DMSO induced p204 formation, (ii) ectopic 204AS RNA blocked the DMSO-induced differentiation (as well as p204 formation), and (iii) ectopic p204 could partially substitute for DMSO in inducing the differentiation of P19 cells to beating myocytes. p204 contains an NES, and in the course of differentiation to myocytes a portion of p204 was translocated from the nucleus to the cytoplasm of P19 cells (29). p204 lacking an NES or carrying a mutated NES was not translocated in P19 cultures treated with DMSO and could not substitute for p204 in triggering differentiation.
The 5Ј-flanking region of the gene encoding p204 (i.e. Ifi204) (11) contains several binding sites for the Gata4, Nkx2.5, and Tbx5 transcription factors. All these factors are important transcriptional regulators of cardiac gene expression and are members of protein families conserved in evolution from Drosophila to vertebrates (42)(43)(44)(45)(46)(47). Nkx2.5 is an NK-2 class homeodomain protein (48). Mouse Nkx2.5 is expressed throughout the development of the heart and is required for this process. In DNA it binds to the 5Ј-AAGTG-3Ј sequence (NKE element), among others. Gata4 is a zinc finger protein (49). It is expressed in the developing mouse heart almost simultaneously and spatially overlapping with Nkx2.5 (46, 50 -52). Gata4 binds in DNA to a sequence element containing a core 5Ј-GATA-3Ј motif. This motif occurs in the promoter region of many genes encoding cardiac proteins, including the atrial natriuretic factor gene (53). Tbx5 is a member of the family of transcription factors containing T box-type DNA binding domains (42). It is also expressed throughout heart development. Its consensus DNAbinding sequence is 5Ј-(A/G/T)GGTG(T/C)(C/T/G)(A/G) (43). This also occurs in the promoter regions of various cardiac-specific genes, including that encoding atrial natriuretic factor (43). Nkx2.5, Gata4, and Tbx5 can pairwise bind one another and can cooperatively or synergistically activate gene expression (52, 54 -56).
We report results showing that ectopic Gata4, Nkx2.5, and Tbx5 synergistically transactivated the expression of reporter constructs, including segments from the 5Ј-flanking region of the Ifi204 gene in murine 10T1/2 fibroblasts and in cardiac myocytes from rats, and also cooperatively transactivated the expression of endogenous p204 in 10T1/2 cells. Ectopic p204 (also DMSO) triggered P19 cells (to differentiate and) to increase their expression of the Gata4 and Nkx2.5 transcription factors. An examination of P19 cells differentiated to myocytes by chromatin coimmunoprecipitation assays with ␣Gata4 or ␣Nkx2.5 antibodies and PCR revealed the binding of endogenous Gata4 and Nkx2.5 to a segment of the 5Ј-flanking region of the Ifi204 gene, which contains a cluster of Gata4-and Nkx2.5-specific sequences. These findings indicate the following: (i) p204 is required for cardiac type myocyte differentiation from P19 embryonal stem cells, and (ii) the level of p204 increases during myocyte differentiation in consequence of the transactivation of the Ifi204 gene by cardiac transcription factors. The various mechanisms by which p204 enabled the differentiation of P19 cells to cardiac-type myocytes are reported in the accompanying article (76).
EXPERIMENTAL PROCEDURES
Plasmid Constructs-The following plasmids were constructed as described in detail in supplement M: p204 (sense) expression plasmid pCMV204, 204 antisense RNA expression plasmid pCMV204AS (5); GFP-p204, GFP-204⌬NES (29); pCMV204⌬NES, pCMV204MNES; pCMVFLAG-p204, pCMVFLAG-p204⌬NES, and FLAG-p204MNES; pCGNGata4 and pCGNNkx2.5 (52); and pCMVTbx5, pCMVHATbx5, pACCMVTbx5 (57). The various Ifi204-based reporters (based on the pGL3 expression vector (Promega)) included wild type Ifi204 gene sequences or sequences with deletions or mutations. For further details see supplement M.
Cell Culture-P19 cells (a gift from M. W. McBurney) were derived from mouse teratocarcinoma induced in C3H/HC mice. The cells were cultured and induced to differentiate to cardiac-type myocytes as described previously (34) and in supplement M. Neonatal rat cardiac myocytes were isolated from hearts of 3-day-old rats as described previously (58,59). 10T1/2 cells, cloned murine embryonic fibroblasts (ATCC 226 CCL) (60), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For further details see supplement M.
Transfection-P19 cells were transfected as specified with expression plasmids encoding p204, p204ASRNA, p204MNES, p204⌬NES, Id3, p204 plus Id3, or p204⌬NES plus FLAGId3 using Lipofectamine 2000 in minimum essential medium-␣ (Invitrogen), supplemented with 2.5% fetal bovine serum (Invitrogen) and 7.5% donor calf serum (TerraCell). Stable cell lines were established by selection with G418. Clones were assayed to ascertain high level expression of the ectopic RNA or protein of interest by Northern or Western blotting, respectively. For further details see supplement M.
Assay of p204, Gata4, Nkx2.5, and MHC by Immunofluorescence Microscopy-P19 cells growing on 35-mm culture dishes were treated as described previously (34) and incubated as specified with mouse MF20, the Developmental Studies Hybridoma Bank, and M2 monoclonal ␣FLAG (Sigma), rabbit ␣p204 and rabbit ␣Id3 (Santa Cruz Biotechnology), and goat ␣Gata4 or ␣Nkx2.5 antibodies (Santa Cruz Biotechnology). As secondary antibodies, fluorescein isothiocyanate or Texas Red-linked goat anti-rabbit and anti-mouse IgG and donkey antigoat IgG (Santa Cruz Biotechnology) were used. Nuclei were stained with DAPI.
RT-PCR-P19 cells in 35-mm tissue culture dishes were lysed in 1 ml of RNA STAT-60 TM (Tel-Test Inc.), and total RNA was isolated following the instructions of the manufacturer and subjected to RT-PCR using the Omniscript RT kit (Qiagen). Sequences of primers are shown in supplement M.
Western Blotting-P19 cells were lysed as described previously (61), and 100 g of total protein was subjected to 4 -20% gradient PAGE and transferred to a polyvinylidene difluoride membrane and blotted with rabbit anti-p204 antibody (30). The blots were visualized using ECL (Pierce, SuperSignal Western Blotting kit). Rat anti-HA antibodies (Santa Cruz Biotechnology) and mouse anti--tubulin antibody (the Developmental Studies Hybridoma Bank) were used to detect HA-tagged Tbx5 and -tubulin, respectively. ␣Gata4 and ␣Nkx2.5 antibodies were used for detection of Gata4 and Nkx2.5, respectively.
Reporter Gene Assay-Synergistic transactivation of p204 reporter constructs was assayed as indicated by cotransfection with pCGNN-kx2.5 (3 g) and/or pCGNGata4 (3 g) and/or pCMVTbx5 (3 g) with pSVGal control plasmid (1 g) in 10T1/2 cells (62) by Lipofectamine 2000 (Invitrogen) (2 l/g DNA) in 35-mm dishes as described previously (62). pCGN and/or pCMV vector was used to bring the total amount of DNA transfected to 8 g. After a 36-h incubation, the cultures were harvested. Luciferase and -galactosidase were assayed using the luciferase reporter gene assay (Roche Applied Science) and the -galactosidase assay system (Promega), respectively. The experiments were repeated 3-4 times.
Sequence Analysis-To search for Gata4, Nkx2.5, and Tbx5 recognition sequences in the Ifi204 gene 5Ј-flanking region (11) (GenBank TM accession number AC006944), the MatInspector, version 2.1, data base was used, and the parameter selected for both core similarity and matrix was 0.8.
Chromatin Immunoprecipitation-The chromatin immunoprecipitation assay kit (Upstate) was used following the instructions of the manufacturer. Published procedures (63) were followed in the treatment of the cells with formaldehyde to cross-link the DNA protein interactions, the preparation of the nuclear extracts, the shearing of the chromatin by sonication, and the immunoprecipitation of DNA-protein complexes using immobilized antibodies (i.e. ␣Gata4 and ␣Nkx2.5). The nucleotide sequence of the 5Ј-flanking region of the Ifi204 gene was reported (Gen-Bank TM accession number AC006944). The sequences of the primers used in the PCR analysis of the DNA segments immunoprecipitated are available upon request. The % of beating P19 cell aggregates on day 12 of differentiation was as follows: curve 1, ectopic p204 and treatment with 0.8% DMSO (39%); curve 2, treatment with 0.8% DMSO (38%); curve 3, 0.8% DMSO and transfection with the pCMV vector decreased this to 29%; curve 4, ectopic p204 and treatment with 0.1% DMSO (16%); curve 5, ectopic p204 and no DMSO (10%). No beating cultures arose in the absence of DMSO (curve 6) or in the presence of the pCMV vector (curve 7), and even in the presence of 0.8% DMSO if ectopic 204ASRNA was present (curve 8). No beating cultures arose (in the absence of DMSO) when p204 lacking a nuclear export signal (p204⌬NES) (curve 9) or p204 with a mutated nuclear export signal (p204MNES; see Fig. 3) was substituted for p204 (curve 10). No beating cultures arose in the presence of 0.1% DMSO and pCMV vector (curve 11). The standard deviations are indicated. For further details, see "Experimental Procedures." p204 in Cardiac Myocyte Differentiation clusters of beating cardiac myocytes by DMSO, we grew the cells in tissue culture dishes, shifted them (on day 0) to bacteriological Petri dishes in the presence of DMSO where they form aggregates, and shifted the aggregates to tissue culture dishes (on day 5). Within two days, many clusters, composed mainly of cells with the shape of cardiac myocytes, started beating (day 8; data not shown).
RESULTS AND DISCUSSION
As shown in the Western blots in Fig. 1A in P19 cells proliferating in tissue culture dishes (in the absence or presence of DMSO), no p204 was detected (day 0). Cells aggregated in Petri dishes without DMSO contained very low levels, if any, of p204, which increased slightly after their shift to tissue culture dishes (between day 6 and day 8). Cells aggregated in the presence of DMSO contained very low levels of p204 (on days 2-4) but much higher levels after their shift to tissue culture dishes (between day 6 and day 8). Thus, DMSO induced p204 in differentiating P19 cultures.
The immunofluorescence microscopic pictures (day 8; Fig. 1C represent the time course of the increase in the percent of cell aggregates with beating myocytes in P19 cultures exposed to various conditions. The cultures that were transfected with expression plasmids were selected in the presence of G418 to eliminate untransfected cells. The conditions (Fig. 1C, curves [1][2][3][4][5][6][7][8][9][10][11] are specified in the figure. Curve 2, by day 12 of the differentiation process, 38% of the P19 cell aggregates exposed to 0.8% DMSO included beating myocytes. Transfection of pCMV204 did not increase significantly the yield of beating aggregates (39%) in a culture exposed to 0.8% DMSO (curve 1). Curve 6, no beating cultures arose in the absence of DMSO or (curve 8) in the presence of 0.8% DMSO if pCMV204AS (a plasmid encoding 204AS RNA) was transfected. Curve 5, transfection of pCMV204 resulted in the formation of aggregates, including beating myocytes (even) in the absence of DMSO. However, the percent of such aggregates (10%) was lower than in the corresponding culture (29%) (curve 3), which was transfected with pCMV, the control vector, and was incubated in the presence of DMSO. Curve 4, DMSO at a concentration as low as 0.1% increased the yield of aggregates with beating cells in a culture expressing ectopic p204 (16%), whereas as already noted (curve 5) ectopic p204 alone resulted only in a lower yield (10%). Curve 11, 0.1% DMSO treatment of cells expressing pCMV vector produced no beating culture.
The data in Fig. 1, A-C, are in line with the following conclusions: (i) DMSO induced p204 expression in aggregated P19 cells; (ii) p204 was required for the induction of differentiation by DMSO; and (iii) p204 could partially substitute for DMSO in inducing the differentiation.
The RT-PCR assays in Fig. 2A reveal that DMSO did not trigger the transcription of the Ifi204 gene to 204 RNA in proliferating P19 cells (day 0). DMSO did, however, trigger the transcription to 204 RNA in differentiating P19 cells, and the 204 RNA was present from day 6 at least until day 12, the last day when it was tested. Transfection of pCMV204 (but not of pCMV) into P19 cells (without or after elimination of the untransfected cells) resulted in 204 RNA expression even in proliferating cells (Fig. 2B). This expression persisted during the differentiation of the cells at least until day 12 (data not shown). The ectopic p204 level was determined by Western blotting to confirm that 204 RNA in P19 cells was translated into p204 (Fig. 2C). Transfection of a pCMV204AS plasmid resulted in the expression of a high level of 204AS RNA in the cells (Fig. 2D). This ectopic 204AS RNA (but not ectopic pCMV vector) decreased the amount of p204 induced by DMSO to a barely detectable level (Fig. 2E). These results suggest that the inhibition of the DMSO-induced differentiation of P19 cells to beating myocytes by 204AS RNA was a consequence of the inhibition by 204AS RNA of the accumulation of p204 as induced by DMSO.
The (64) amino acid sequence in p204, and the sequence of nucleotides encoding it in Ifi204. Fig. 3A-(2) is a schematic representation of p204 lacking an NES (p204⌬NES). The generation of p204⌬NES involved the deletion of a 12-amino acid segment from p204. It is possible that this might have altered the structure of p204 resulting in the loss of some of its activities. Therefore, as a control we also generated p204 with a mutated NES (p204MNES), in which only three amino acid residues were replaced ( Fig. 3A-(3)). This shows the altered amino acid sequence of p204MNES in which three Leu residues from the NES were replaced by Ala residues. The mutated nucleotide sequence encoding the altered amino acid sequence is also shown. The Western blots in Fig. 3, A-(4), A- (5), and A- (6), established that the pCMV plasmids encoding p204, p204⌬NES, and p204MNES transfected into P19 cells resulted in the expression of similar amounts of protein.
The fluorescence microscopic analysis in Fig. 3B involved the use of two types of fusion proteins as follows: (i) green fluorescent pro- MAY 26, 2006 • VOLUME 281 • NUMBER 21 tein-204 (GFP-p204) and (ii) GFP linked to a p204 derivative from which the NES oligopeptide segment was lacking (GFP-p204⌬NES). GFP serving as a control was shown to be distributed between the nuclei and the cytoplasm in both proliferating (Fig. 3B, PROLIF.) and differentiated (DIFF.) P19 cells (Fig. 3B, bottom panels). GFP-p204 was nuclear in proliferating cells, but a portion of GFP-p204 was A-(3), in p204MNES, the sequence of NES is altered; three Leu residues are replaced by Ala residues. These Ala residues and the nucleotides encoding Leu residues or Ala residues replacing Leu residues are in boldface letters. A-(4), A- (5), and A- (6), the levels of expression of ectopic p204, p204⌬NES, and p204MNES were very similar in differentiating P19 cells transfected with pCMV204, pCMV204⌬NES, or pCMV204MNES (as determined by Western blotting on day 8 of the differentiation). Con, transfected by vector; UC, untransfected control. B, distribution of ectopic green fluorescent protein, GFP-p204, and GFP-p204⌬NES between the nucleus and the cytoplasm in proliferating and differentiating P19 cells. P19 cells were transfected, as indicated, with plasmids encoding green fluorescent protein or the fusion proteins GFP-p204 or GFP-p204⌬NES. Two cultures from each of the three types of transfectants were incubated in tissue culture dishes (without DMSO). After a 36-h incubation, one of each two of the proliferating cultures was examined by fluorescence microscopy (in panel PROLIF). Second cultures from each of the three types of transfectants were shifted after 36 h to Petri dishes and supplemented with 0.8% DMSO (in panel DIFF). Four days later, these were transferred in the same medium to tissue culture dishes to allow differentiation during the next 48 -72 h. This was followed by fluorescence microscopy. Bar is 30 m. C, subcellular location of endogenous p204 and of ectopic FLAG-p204, FLAG-p204⌬NES, and FLAG-p204MNES in proliferating P19 cells and in differentiating P19 cells (tested on D6-D7 of the differentiation). Fluorescence microscopy, using ␣FLAG (top and bottom panels) and, as secondary antibodies, anti-mouse IgG tagged with fluorescein (top panel) or with rhodamine (bottom panel) is shown. In the middle panel, to reveal endogenous p204, ␣p204, and as secondary antibody, anti-rabbit IgG, tagged with fluorescein were used. Nuclei were stained with DAPI. The bar corresponds to 30 m. For further details see "Experimental Procedures." p204 in Cardiac Myocyte Differentiation translocated from the nucleus to the cytoplasm in differentiated cells (Fig. 3B, top panels). GFP-p204⌬NES, however, was nuclear in proliferating P19 cells and remained nuclear also in differentiated P19 cells (Fig. 3B, middle panels).
To ascertain that the presence of GFP in the fusion proteins did not affect the translocation (in Fig. 3B), the tests were also performed using ectopic FLAG-p204, FLAG-p204⌬NES, and FLAG-p204MNES. Confirming the observations in Fig. 3B, the immunofluorescence microscopic analysis (in Fig. 3C, bottom panel) revealed that in proliferating P19 cells FLAG-p204 was nuclear (and so were FLAG-p204⌬NES and FLAG-p204MNES (not shown)). In P19 cells whose differentiation to cardiac myocytes cells was triggered by DMSO, much of the FLAG-p204 was translocated to the cytoplasm; however, FLAG-p204⌬NES and FLAG-p204MNES, which lacked an intact NES, remained in the nuclei (Fig. 3C, upper panels). The level of endogenous p204 in proliferating P19 cells is too low for immunofluorescent detection. However, we have reported earlier that in proliferating C2C12 myoblasts, in which the endogenous p204 level is higher and thus detectable, p204 is nuclear (29). In differentiating P19 cells the endogenous 204 was distributed between the nuclei and the cytoplasm (Fig. 3C, middle panel).
The findings in Fig. 1C indicate that (in addition to controlling the subcellular location of p204, as shown in Fig. 3, B and C) the NES was also required for the differentiation of P19 cells to beating cardiac-type myocytes. Thus, although the transfection of pCMV204 triggered the differentiation of P19 cells (even in the absence of DMSO) (Fig. 1C (curve 5)), the transfection of pCMV204⌬NES or pCMV204MNES did not trigger the formation of beating myocytes from P19 cells in the absence of DMSO (Fig. 1C (curves 9 and 10)). Furthermore, ectopic p204⌬NES or p204MNES did not substitute for ectopic p204 in triggering MHC protein formation in aggregated P19 cells in the absence of DMSO (data not shown).
p204, but Not p204 Lacking the NES, Could Substitute for DMSO in the Triggering of the Expression of the Gata4 and Nkx2.5 Transcription Factors in Aggregated P19 Cultures-As reported earlier (49,65), two of the transcription factors expressed early in the course of differentiation of aggregated P19 cultures to cardiac-type myocytes in the presence of DMSO (and also in the course of murine heart development) are Gata4 and Nkx2.5. Moreover, in the absence of DMSO, the P19 aggregates (even if transfected with an empty vector) contained primarily undifferentiated carcinoma cells, and neither Gata4 nor Nkx2.5 expression could be detected (65,66).
As shown in the Western blots (Fig. 4A), aggregated P19 cells, incubated with DMSO or incubated without DMSO but transfected with pCMV204 and thus expressing p204 (p204), expressed both Gata4 and Nkx2.5 (left panel). Aggregated cells incubated without DMSO and transfected with empty vector (Fig. 4A, Con), however, did not express either Gata4 or Nkx2.5 (left panel). Moreover, the expression of ectopic 204ASRNA strongly inhibited the expression of Nkx2.5 and Gata4 as induced by DMSO (Fig. 4A, middle and 4A, left panel). This was the case although the level of p204⌬NES expressed was similar to the level of p204 in cells transfected with pCMV204 (see Fig. 3, A-(4) and A-(5)). These results are in line with the observations that p204⌬NES or p204MNES could not substitute for wild type p204 in inducing MHC either (data not shown). All these findings are in accord with the results in Fig. 1C in demonstrating that p204 lacking an active NES was unable to trigger the differentiation of P19 cells to beating cardiac myocytes.
The immunofluorescence microscopic pictures in Fig. 4B are in accord with the Western blots in Fig. 4A in showing the pronounced induction of Gata4 and Nkx2.5 in differentiating P19 cells by ectopic p204, but little if any, if it was lacking the NES. In the pictures of cultures with ectopic p204 (but not in those with ectopic p204⌬NES) (see Fig. 4B), some of the p204 has spread into the cytoplasm. This (as noted earlier, see Fig. 3C) is in consequence of the differentiation of the P19 cells induced by p204 (but not by p204⌬NES).
The RT-PCR patterns in Fig. 4C revealed that the induction of MHC protein by DMSO or ectopic p204 in differentiating P19 cells (Fig. 1B) was (i) the consequence of the induction of ␣and MHCRNA, and (ii) the ectopic 204ASRNA inhibited the induction of the transcription of the MHC genes by DMSO.
The Expression of the Ifi204 Gene Could Be Synergistically Transactivated in 10T1/2 Cells, Rat Cardiac Myocytes, and P19 Cells Differentiating to Myocytes by the Gata4, Nkx2.5, and Tbx5 Cardiac Transcription Factors-Searching for cardiac transcription factor recognition sequences in the 5Ј-flanking region of the murine Ifi204 gene (11), we noted the occurrence of numerous recognition sequences for Gata4, Nkx2.5, and Tbx5 (Fig. 5A).
These transcription factors that function in cardiac differentiation can bind each other and are capable of synergistically transactivating gene expression (51, 52, 54 -56, 67). We assayed the activity of the three factors to transactivate Ifi204 gene expression in transfected 10T1/2 murine embryonic fibroblasts (Figs. 5B and 6), cultured myocytes from newborn rats (Fig. 7), and proliferating and differentiating P19 cultures (Fig. 8). In the bulk of the assays we used reporter constructs, including various segments from the 5Ј-flanking region of the Ifi204 gene driving luciferase expression.
One of the short Ifi204 segments (of 160 nucleotides) used in a reporter construct (Fig. 5A) included parts of exon 1 and intron 1 with three recognition sites for Gata4 (designated as GATA), one for Nkx2.5 (designated as NKE), and two for Tbx5 (designated as TBX). It should be noted that a cluster of several short (about 6 -8 bp) transcription factor
p204 in Cardiac Myocyte Differentiation
recognition sites, such as in this region, is rare in the genome, although single recognition sites are common. Multiple sequence-specific factors in such a cluster typically function synergistically (68).
The expression of the reporter construct, including the Ifi204 segment in Fig. 5A in transfected 10T1/2 cells, is shown in Fig. 5B (wild type panel). The low level of expression in control cells (lane 1) (taken as 1 to serve as the standard for comparison) was only slightly increased by ectopic Gata4, Nkx2.5, or Tbx5 (lanes 2-4). Any two of these transcription factors activated the expression synergistically ( lanes 5-7 ), and all three factors activated it even further (25-fold) (lane 8). Mutation in the reporter of any one of the two TBX sequences and especially of both strongly decreased the synergistic boost of the expression by TBX5 but affected the boosts by Gata4 and Nkx2.5 less, if at all (Fig. 5B, panels Mutated TBX (1), Mutated TBX (2), and Doubly mutated TBX ). These results are in accord with the finding that synergistic transactivation by Tbx5 (with Nkx2.5 or Gata4) requires the binding of Tbx5 to DNA (54 -56).
The importance of the GATA and NKE sequences in the control of the transactivation by ectopic Gata4 and Nkx2.5 in 10T1/2 cells was demonstrated (see supplemental Fig. S1). The results (i) confirmed that Gata4 and Nkx2.5 can synergize by protein-protein interaction even if only one of the factors was bound to DNA (50,51,69), and (ii) they demonstrated that the deletion or mutation of a GATA or NKE sequence may decrease the synergistic transactivation even in reporters with two or more binding sites for the above transcription factors.
The effects of ectopic Nkx2.5, Gata4, and Tbx5 on the expression of endogenous p204 in 10T1/2 cells, together with the levels of expression of the three ectopic transfection factors, are shown in the Western blots in Fig. 6. The numbers below the p204 panel in Fig. 6 reveal the cooperativity or in some cases the slight synergy between any two of the three ectopic transcription factors. (The lack of a further increase in the level of endogenous p204 upon addition of the third transcription factor may only be apparent because it may be due, at least in part, to the fact that in this case in order to keep the total DNA concentration constant less DNA was transfected for each factor (2 g) than in all other cases (3 g).) The lesser increase in the level of p204 protein (Fig. 6) than of p204 RNA in 10T1/2 cells (Fig. 5B) by ectopic Gata4, Nkx2.5, and Tbx5 is likely to be the consequence of, at least in part, a post-transcriptional control of p204 expression. Such control was reported in the case of the expression of p202b, another protein of the p200 family (17,23).
To establish whether the increase in the expression of p204 by ectopic Nkx2.5, Gata4, and Tbx5 in 10T1/2 cells may be relevant for cardiac myocyte differentiation, we also tested the effect of these transcription factors on p204 expression in cultures of primary cardiac myocytes from newborn rats (Fig. 7). Both the endogenous level of Nkx2.5 protein and the endogenous Gata4 activity were reported to be very low in cultured newborn rat myocytes (70,71). We transfected newborn rat myocytes in culture with a reporter construct, including a sequence from the Ifi204 gene 5Ј-flanking segment (shown in Fig. 5A). The Gata4, Nkx2.5, and Tbx5 transcription factor binding sites in the construct are shown in Fig. 7. Transfection of an expression plasmid encoding any one of the above listed transcription factors transactivated p204 expression weakly (less than 2-fold). Transfection of any two of them had a synergistic effect (an over 8-fold increase). Transfection of all three had an even stronger synergistic effect (an over 14-fold increase). These results establish that ectopic Gata4, Nkx2.5, and Tbx5 can synergistically transactivate FIGURE 6. Ectopic Gata4, Nkx2.5, and Tbx5 (actually HA-tagged Tbx5 was used) cooperatively promoted the expression of endogenous p204 in cultures of 10T1/2 cells. The fold increase is indicated below the Western blot of endogenous p204. The protein levels of each of the ectopic transcription factors in cultures transfected with the appropriate expression plasmids are shown, together with the level of endogenous tubulin serving as an internal protein control. Con, culture transfected with empty vector. 36 -40 h after transfection the cultures were lysed, and p204 was detected using p204 antibodies and the ECL system. Thereafter the membrane was stripped and blotted using antibodies against Nkx2.5, Gata4, and Tbx5 (in the case of Tbx5 antibodies to HA were used) and tubulin (each after stripping). The number 3.05 is in parentheses to indicate that less DNA was transfected of each transcription factor when using Gata4/Nxk2.5 and Tbx5 than in all other cases in which only 1 or 2 transcription factors were used. See also the text. The standard deviations are indicated. For further details see "Experimental Procedures." FIGURE 7. Synergistic transactivation of the expression of an Ifi204 reporter construct by ectopic Gata4, Nkx2. 5, and Tbx5 transcription factors in rat cardiac myocytes. Cardiac myocytes isolated from 3-day-old rats were seeded on collagen-coated dishes. The Ifi204 reporter construct inserted into a pGL3 vector and driving luciferase expression included a segment from the 5Ј-flanking region of Ifi204 extending, as indicated, from nucleotide Ϫ98 to ϩ47. The binding sites for Gata4, Nkx2.5, or Tbx5 are indicated in the figure. The Ifi204 reporter constructs were cotransfected, as indicated, with the pCGN vector (Con), and pCGNGata4, pCGNNkx2.5, pCMVTbx5, as well as with pSVGal. The activities of -galactosidase and luciferase were assayed in the extracts after 36 h. The luciferase activities were normalized to -galactosidase activities, and the normalized activities of the wild type and mutated reporters in extracts from cells transfected with the plasmids encoding transcription factors were related to the activity of the extract from cells transfected with vector only. The standard deviations are indicated. For further details see "Experimental Procedures." p204 expression not only in 10T1/2 cells but also in cardiac myocytes.
Proliferating P19 cultures are lacking endogenous Nkx2.5 as well as Gata4 and Tbx5 transcription factor activity (51,65,66,72), whereas P19 cultures in the course of differentiation to myocytes express Nkx2.5 and Gata4. Thus, we expected that Ifi204 reporter constructs with binding sites only for Gata4, Nkx2.5, and/or Tbx5 would be expressed only in differentiating but not in proliferating P19 cultures. The data in Fig. 8, (a and b) demonstrate high level expression of 204 reporter constructs in differentiating and not, however, in proliferating P19 cells, thus proving the validity of this expectation. The data also indicate that the expression of the p204 reporter constructs decreases if the sequence of a Gata4-binding site (Fig. 8a) or an Nkx2.5-binding site (Fig. 8b) is mutated.
Coimmunoprecipitation of Chromatin Segments from the 5Ј-Flanking Region of the Ifi204 Gene Containing Gata4-and Nkx2.5-specific Sequences by Antibodies to Gata4 and Nkx2.5-As indicated earlier, undifferentiated proliferating P19 cells do not contain detectable levels of Gata4 and Nkx2.5 transcription factors (65,66,72) and express the Ifi204 gene very weakly, if at all ( Fig. 2A). P19 cells differentiating to myocytes, however, contain pronounced levels of Gata4 and Nkx2.5 (73) and strongly express the Ifi204 gene (Figs. 1A and 2B). These findings prompted us to test if antibodies to Gata4 and Nkx2.5 (but not control IgG) can coimmunoprecipitate chromatin segments (generated by sonication (Fig. 9A, panel b)) containing Gata4-and Nkx2.5-specific sequences from the 5Ј-flanking region of the Ifi204 gene in nuclear extracts from differentiating P19 cells but not from proliferating P19 cells. The data in Fig. 9A, panel a, indicate that this was the case.
As determined by PCR, of the five chromatin segments tested (i.e. four from the 5Ј-flanking region and one from the coding region, including the DNA with the last exon of p204 RNA), two segments, segment 1 (extending from nucleotide Ϫ5124 to Ϫ4705) and segment 4 (extending from nucleotide Ϫ184 to ϩ93), were coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5 (but not by IgG), but only in a nuclear extract from differentiating P19 cells and not in that from proliferating P19 cells (Fig. 9A, panel a). (As noted earlier, the first nucleotide of intron 1 of the gene Ifi204 was taken as ϩ1.) Segments 2 and 3, located between segments 1 and 4, were not coimmunoprecipitated despite the occurrence in them of potential Gata4-and Nkx2.5-specific sequences. This indicates that the pres-ence of such a sequence does not ensure the binding of the appropriate transcription factor.
The coimmunoprecipitable segment 4 included a sequence (Fig. 5A) that was used (i) in a reporter construct whose expression in transfected cells was synergistically transactivated by Gata4 and Nkx2.5) (Fig. 5B), and (ii) to which Gata4 and Nkx2.5 bound in an electrophoretic mobility shift assay in vitro (not shown).
The reporter construct (in Fig. 9B) contained an ϳ5.5-kb sequence from the Ifi204 5Ј-flanking region. This included all the chromatin segments tested in the chromatin immunoprecipitation in Fig. 9A. The diagram in Fig. 9B reveals that the expression of this construct was cooperatively transactivated by Gata4 and Nkx2.5.
The results of the chromatin coimmunoprecipitation assays indicated that Gata4 and Nkx2.5 were bound in vivo to the chromatin from the 5Ј-flanking region of the Ifi204 gene in P19 cells differentiating to myocytes but not in proliferating P19 cells. These findings are in accord with other findings in this study by suggesting that the expression of p204 in P19 cells differentiating to myocytes is the consequence, at least in part, of the transactivation of the gene by Gata4 and Nkx2.5. Chromatin segment 1, which was also coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5, also contained Gata4-and Nkx2.5-specific sequences. This segment was part of the 3Ј-terminal transcribed but not translated region of the Ifi203a gene, the 5Ј-terminal neighbor of the Ifi204 gene (11). According to a recent report (74), about a third of the transcription factor binding sites (as examined in human chromosomes 21 and 22) are located within or immediately 3Ј to well characterized genes and are significantly correlated with noncoding RNAs. Furthermore, overlapping pairs of protein coding and noncoding RNAs are often coregulated. Consequently, it will be interesting to see whether this segment 1 is a site in which a noncoding RNA transcript is initiated, and if so whether the transcript affects the expression of the Ifi203a and/or Ifi204 genes.
Various Inducers and Transcription Factors Can Promote the Tissuespecific Expression of p204-p204 was discovered as an interferoninducible protein (5). Studies on the differentiation of skeletal muscle revealed that in the course of this process p204 expression is activated by the skeletal muscle-specific MyoD and myogenin transcription factors (29). The results presented in this study established that in cardiac myocyte differentiation, p204 expression is synergis- The wild type or mutated binding sites for the Gata4, Nkx2.5, and Tbx5 transcription factors in the various Ifi204 reporters are indicated. In the mutated binding site in (a), the GATA sequence (in the 3Ј to 5Ј direction) TATC was mutated to CGTC, and in (b), the NKE sequence (in the 3Ј to 5Ј direction) CAC T T was mutated to CAAGG. A pSVGal internal control plasmid was cotransfected. Proliferating cells were transfected in the absence of DMSO and differentiating cells in the presence of DMSO. -Galactosidase and luciferase activities were determined 36 h after transfection. Relative luciferase activity (R.L.A.) was calculated by comparing the normalized luciferase activities of extracts from cells transfected with the wild type or mutated reporter constructs to that of an extract from cells transfected with vector. The standard deviations are indicated. For further details see "Experimental Procedures." p204 in Cardiac Myocyte Differentiation tically activated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. In all the above cases, distinct transcription factors and cis-acting sequences in the Ifi204 gene mediated p204 expression. Furthermore, the expression of p204 in differentiating T cells was reported to be activated by Notch 1 (75); however, the mediators of the activation remain to be identified. Thus the expression of p204 in different tissues can be activated by a variety of tissue-specific mechanisms, i.e. by distinct inducers, transcription factors, and cis-acting sequences.
Indications for the Existence of a Positive Feedback Loop Including the p204, Gata4, and Nkx2.5 Proteins-The results presented reveal that in P19 cells differentiating to cardiac myocytes, (i) p204 (but not if lacking its NES) induced the expression of the Gata4 and Nkx2.5 transcription factors, and (ii) Gata4 and Nkx2.5 synergistically activated the expression of p204. These findings indicate the existence of a positive feedback loop, including the p204, Gata4, and Nkx2.5 proteins. The mechanisms of action of p204 in this feedback loop, together with the other mechanisms by which p204 and its NES FIGURE 9. A, antibodies to Nkx2. 5 and Gata4 (but not IgG controls) coimmunoprecipitate some of the chromatin segments from the 5Ј-flanking region of the Ifi204 gene containing Nkx2.5-and Gata4-specific binding sites. This is the case in nuclear extracts from P19 cells differentiating to myocytes but not from undifferentiated, proliferating P19 cells. (a), cultures of P19 cells proliferating in the absence of DMSO (PROLIF ) and cultures of P19 cells undergoing differentiation to cardiac myocytes in the presence of DMSO (DIFF ) on day 6 to day 7 of the process were incubated with formaldehyde to cross-link the transcription factors to DNA. Nuclear extracts were prepared, and the chromatin was sheared by sonication to 200 -1000-bp segments (see the gel electrophoretic pattern of the DNA segments in (b). The suspension was incubated with protein A beads without or after precoating with control IgG or antibodies to Gata4 (␣G) or Nkx2.5 (␣N). (The specificities of the antibodies (Ab) used were verified.) The DNA-protein complexes were eluted from the beads. The cross-linking was reversed, and the DNA was recovered by phenol/chloroform extraction and was assayed by PCR using pairs of primers for various segments of the 5Ј-flanking region of Ifi204 containing GATA or NKE sequences. The amounts of PCR products from the input DNA (Input) and from the DNA coimmunoprecipitated with ␣G or ␣N from the indicated segments of the Ifi204 gene 5Ј-flanking region (specified by the numbering of their 5Ј-and 3Ј-terminal nucleotides, taking as nucleotide ϩ1 the first nucleotide of intron 1 of the Ifi204 gene) were visualized by ethidium bromide-agarose gel electrophoresis. (c) is a schematic drawing of the Ifi204 gene showing the exons (numbered 1-9) as well as the segments S1 to S5 whose presence in the coimmunoprecipitates obtained using ␣G or ␣N was examined by PCR. Of the three negative control segments that were not coimmunoprecipitated by antibodies ␣G or ␣N, two, i.e. S2 and S3, were from the 5Ј-flanking region of the Ifi204 gene and are located between the S1 and S4 segments that were coimmunoprecipitated with ␣G and ␣N. The third negative control segment, S5, corresponds to the translated 3Ј-terminal exon of the Ifi204 gene. Control IgG did not coimmunoprecipitate any of the DNA segments tested. B, the reporter construct driving luciferase expression was generated by inserting into the pGL3 vector a segment from the 5Ј-flanking region of the Ifi204 gene extending from nucleotide Ϫ5482 to ϩ78. Ectopic Nxk2.5 and Gata4 cooperatively boosted the expression of the construct in 10T1/2 cells. For further details see "Experimental Procedures." enable the differentiation of P19 embryonal carcinoma stem cells to cardiac myocytes, are the topics of the accompanying article (76).
□ S The on-line version of this article (available at http://www.jbc.org) contains supplement M, Fig. S1, and Videos 1 and 2.
p204 Is a Mediator of the Induction of the Differentiation of P19 Embryonal Carcinoma Cells to Beating Cardiac Type Myocytes by DMSO; DMSO Induced p204 Expression, and p204 Could Induce the Differentiation in the Absence of DMSO-Following the published procedure (34, 38) for the induction of the differentiation of P19 cells to
FIGURE 1 .
1p204 was required for the differentiation of cultured P19 cells to beating cardiac myocytes. A, time course of p204 accumulation during the differentiation as triggered by DMSO. P19 cultures were incubated in the absence or presence of 0.8% DMSO, and the level of p204 was determined daily until day 8 by Western blotting. As internal control, tubulin levels were followed. B, ectopic p204 (generated by transfection of pCMV204) could partially substitute for DMSO in the induction of MHC protein. Ectopic 204ASRNA (generated by transfection of pCMV204AS) inhibited the induction of MHC by DMSO. Top panel, on day 8 of the differentiation process, regions from the tissue culture dishes containing beating cell clusters were assayed by fluorescence microscopy using monoclonal MF20 antibodies against MHC. Bottom panel, in the absence of DMSO, or in the presence of 204ASRNA (even if DMSO was added), neither beating clusters nor MHC was detected. Nuclei were stained with DAPI. The bar corresponds to 30 m. C, effect of different agents on the differentiation of P19 cells to beating myocytes. The time courses of the increase in the % of beating P19 cell aggregates in different culture conditions are shown (curves 1-11).
Fig. 1B) illustrate the following: (i) the need for DMSO in the triggering of the formation of beating cardiac myocyte-like cells expressing MHC proteins(Fig. 1B, compare panels a and e); (ii) the ability of ectopic p204 to trigger the formation of beating myocyte-like cells expressing MHC (in the absence of DMSO(Fig. 1B, panel b)) (see supplemental Videos 1 and 2); and (iii) the inhibition of the induction of beating and MHC-expressing cells by DMSO in P19 cells that expressed 204ASRNA(Fig. 1B, panel f ). (As will be demonstrated inFig. 2E, the ectopic 204ASRNA inhibited the induction of p204 by DMSO.) The curves in
FIGURE 2 .
2DMSO triggered the transcription of the Ifi204 gene in P19 cells; ectopic 204ASRNA inhibited the expression of p204 as triggered by DMSO. A, time course of 204RNA expression, as triggered by DMSO. As loading control 18 S rRNA was assayed (RT-PCR). B, expression of 204RNA in proliferating P19 cells transfected with pCMV204 but not with pCMV only (RT-PCR). C, expression of p204 in cells transfected with pCMV204 (Western blot). D, expression of 204ASRNA in cells transfected with a pCMV204AS expression plasmid. As loading controls the levels of 18 S rRNA were assayed (RT-PCR). E, expression of 204ASRNA in cells transfected with a pCMV204AS construct strongly decreased the induction of p204 expression by DMSO to a level below that in a control culture transfected with pCMV. As loading controls, the levels of tubulin were assayed (Western blots). For further details, see "Experimental Procedures." p204 in Cardiac Myocyte Differentiation
FIGURE 3 .
3NES was required for the translocation of p204 from the nucleus to the cytoplasm during the differentiation of P19 cells to myocytes, as induced by DMSO. Schematic structures and levels of p204 (wild type), p204⌬NES, and p204MNES are shown. A-(1), sequence and location of the NES in p204. The nucleotides encoding Leu residues are in boldface letters. A-(2), p204⌬NES is lacking the NES.
right panels). P19 cells transfected with pCMV204⌬NES and incubated in the absence of DMSO expressed little if any Gata4 and Nkx2.5 (Fig.
FIGURE 4 .
4Effects of DMSO, ectopic p204, p204⌬NES, and 204ASRNA on the expression of Gata4 and Nkx2.5 proteins and ␣and MHCRNA in P19 cells. A, left panel, induction of Nkx2.5 and Gata4 proteins in differentiating P19 cultures incubated with DMSO or transfected with pCMV204 (p204), but not in cultures incubated without DMSO and transfected with pCMV vector (Con) or pCMV204⌬NES (p204⌬NES) (Western blotting on day 6). Middle and right panels, transfection with pCMV204AS (204ASRNA) decreased the levels of Nkx2.5 and Gata4 induced by DMSO in P19 cultures in conditions of differentiation. As internal control, the levels of tubulin were assayed. B, transfection with pCMV204 (p204), but not with pCMV204⌬NES (p204⌬NES), induced the formation of Gata4 and Nkx2.5 in P19 cultures in differentiation conditions (immunofluorescence assays). Nuclei were stained with DAPI. C, incubation with DMSO (0.8% DMSO) or transfection with pCMV204 (p204) induced the expression of ␣and MHCRNA. Transfection with pCMV204AS inhibited the induction by DMSO (204ASRNA ϩ 0.8% DMSO) in P19 cultures in conditions of differentiation (RT-PCR assays). For further details see "Experimental Procedures."
FIGURE 5 .
5Synergistic transactivation of wild type and of mutated Ifi204 reporter constructs by ectopic Gata4, Nkx2. 5, and Tbx5 transcription factors in 10T1/2 cells. A, nucleotide sequence of an Ifi204 reporter construct. This extends from nucleotide Ϫ108 to nucleotide ϩ52. As nucleotide ϩ1 (indicated in the figure), the first nucleotide of intron 1 was chosen. This is the case because the transcription of the gene is initiated at several sites. The numbers (to the left of the sequences) indicate distances in nucleotides from the first nucleotide of intron 1. GATA sequences (recognized by Gata4), an NKE sequence (recognized by Nkx2.5), and TBX sequences (recognized by Tbx5) are printed in boldface and are also indicated in parentheses above the sequence.B, the Gata4, Nkx2.5, and Tbx5 transcription factors synergistically transactivated four types of p204 reporter constructs (in pGL3 vectors) driving the expression of luciferase. The different constructs included either the wild type Ifi204 gene segment as shown in A, or the same segment but with mutations in the TBX site 1, TBX site 2, or both TBX sites. The sequences of the wild type and mutated TBX sites (mutated TBX1) and (mutated TBX2) are indicated. The reporter constructs were transfected into 10T1/2 cells as indicated together with the following: lane 1, the pCGN vector (Con); lane 2, pCGNGata4 (Gata4); lane 3, pCGNNkx2.5 (Nkx2.5); lane 4, pCMVTbx5 (Tbx5); lane 5, pCGNGata4 and pCGNNkx2.5 (Gata4/Nkx2.5); lane 6, pCGNGata4 and pCMVTbx5 (Gata4/Tbx5); lane 7, pCGNNkx2.5 and pCMVTbx5 (Nkx2.5/Tbx5); or lane 8, pCGNGata4 and pCGNNkx2.5 and pCMV-Tbx5 expression plasmids (Gata4/Nkx2.5/Tbx5), together with the pSVGal internal control plasmid. 36 h after transfection the cultures were harvested and lysed, and the -galactosidase and luciferase activities were determined. The luciferase activities were normalized to the -galactosidase activities. The normalized activity of the control lysates (lane 1) was taken as 1 and was compared with the normalized luciferase activities of the other lysates (lanes 2-8). The relative luciferase activities (R.L.A.) for the experiments with the wild type reporters and three mutated reporters and the standard deviations are shown. The standard deviations are indicated. For further details see "Experimental Procedures."
FIGURE 8 .
8Transactivation of wild type and mutated Ifi204 reporter constructs by endogenous transcription factors in differentiating but not in undifferentiated, proliferating P19 cultures. Cultures of P19 cells proliferating in the absence of DMSO (PROLIF P19) or differentiating (on day 6 of the process) in the presence of DMSO (DIFF P19) were transfected with the wild type or the mutated version of the Ifi204 reporter (Wild type R and Mutated R) specified (a) and (b) or with the Vector.
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| [
"Among 10 adult mouse tissues tested, the p204 protein levels were highest in heart and skeletal muscle.We described previously that the MyoD-inducible p204 protein is required for the differentiation of cultured murine C2C12 skeletal muscle myoblasts to myotubes. Here we report that p204 was also required for the differentiation of cultured P19 murine embryonal carcinoma stem cells to beating cardiac myocytes. As shown by others, this process can be triggered by dimethyl sulfoxide (DMSO). We established that DMSO induced the formation of 204RNA and p204. Ectopic p204 could partially substitute for DMSO in inducing differentiation, whereas ectopic 204 antisense RNA inhibited the differentiation. Experiments with reporter constructs, including regulatory regions from the Ifi204 gene (encoding p204) in P19 cells and in cultured newborn rat cardiac myocytes, as well as chromatin coimmunoprecipitations with transcription factors, revealed that p204 expression was synergistically transactivated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. Furthermore, ectopic p204 triggered the expression of Gata4 and Nkx2.5 in P19 cells. p204 contains a nuclear export signal and was partially translocated to the cytoplasm during the differentiation. p204 from which the nuclear export signal was deleted was not translocated, and it did not induce differentiation. The various mechanisms by which p204 promoted the differentiation are reported in the accompanying article (2 The abbreviations used are: Id, inhibitor of differentiation; NES, nuclear export signal; 204AS, 204 antisense; ␣protein (e.g. ␣Gata4), antiserum to the protein in question (e.g. to Gata4); DMSO, dimethyl sulfoxide; MHC, myosin heavy chain; p204⌬NES, p204 lacking the NES; p204MNES, p204 with a mutated NES; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; RT, reverse transcription."
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"B E Teunissen, ",
"A T Jansen, ",
"S C Van Amersfoorth, ",
"T X O'brien, ",
"H J Jongsma, ",
"M F Bierhuizen, ",
"C Ventura, ",
"M Maioli, ",
"C Grepin, ",
"G Nemer, ",
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"H H Ng, ",
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"R Wheeler, ",
"B Wong, ",
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"H Tammana, ",
"G Helt, ",
"K Struhl, ",
"T R Gingeras, ",
"M L Deftos, ",
"E Huang, ",
"E W Ojala, ",
"K A Forbush, ",
"M J Bevan, ",
"B Ding, ",
"C Liu, ",
"Y Huang, ",
"J Yu, ",
"W Kong, ",
"P Lengyel, "
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". D Choubey, P Lengyel, J. Cell Biol. 116Choubey, D., and Lengyel, P. (1992) J. Cell Biol. 116, 1333-1341",
". C Liu, H Wang, Z Zhao, S Yu, Y B Lu, J Meyer, G Chatterjee, S Deschamps, B A Roe, P Lengyel, Mol. Cell. Biol. 20Liu, C., Wang, H., Zhao, Z., Yu, S., Lu, Y. B., Meyer, J., Chatterjee, G., Deschamps, S., Roe, B. A., and Lengyel, P. (2000) Mol. Cell. Biol. 20, 7024 -7036",
". C J Liu, H Wang, P Lengyel, EMBO J. 18Liu, C. J., Wang, H., and Lengyel, P. (1999) EMBO J. 18, 2845-2854",
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"[1]",
"[2]",
"[3]",
"[4]",
"[5]",
"[6]",
"[7]",
"[8]",
"[9]",
"[10]",
"[11]",
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"MAY 26, 2006",
"(5)",
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] | [
"\n\n□ S The on-line version of this article (available at http://www.jbc.org) contains supplement M, Fig. S1, and Videos 1 and 2.",
"\n\np204 Is a Mediator of the Induction of the Differentiation of P19 Embryonal Carcinoma Cells to Beating Cardiac Type Myocytes by DMSO; DMSO Induced p204 Expression, and p204 Could Induce the Differentiation in the Absence of DMSO-Following the published procedure (34, 38) for the induction of the differentiation of P19 cells to",
"\nFIGURE 1 .\n1p204 was required for the differentiation of cultured P19 cells to beating cardiac myocytes. A, time course of p204 accumulation during the differentiation as triggered by DMSO. P19 cultures were incubated in the absence or presence of 0.8% DMSO, and the level of p204 was determined daily until day 8 by Western blotting. As internal control, tubulin levels were followed. B, ectopic p204 (generated by transfection of pCMV204) could partially substitute for DMSO in the induction of MHC protein. Ectopic 204ASRNA (generated by transfection of pCMV204AS) inhibited the induction of MHC by DMSO. Top panel, on day 8 of the differentiation process, regions from the tissue culture dishes containing beating cell clusters were assayed by fluorescence microscopy using monoclonal MF20 antibodies against MHC. Bottom panel, in the absence of DMSO, or in the presence of 204ASRNA (even if DMSO was added), neither beating clusters nor MHC was detected. Nuclei were stained with DAPI. The bar corresponds to 30 m. C, effect of different agents on the differentiation of P19 cells to beating myocytes. The time courses of the increase in the % of beating P19 cell aggregates in different culture conditions are shown (curves 1-11).",
"\n\nFig. 1B) illustrate the following: (i) the need for DMSO in the triggering of the formation of beating cardiac myocyte-like cells expressing MHC proteins(Fig. 1B, compare panels a and e); (ii) the ability of ectopic p204 to trigger the formation of beating myocyte-like cells expressing MHC (in the absence of DMSO(Fig. 1B, panel b)) (see supplemental Videos 1 and 2); and (iii) the inhibition of the induction of beating and MHC-expressing cells by DMSO in P19 cells that expressed 204ASRNA(Fig. 1B, panel f ). (As will be demonstrated inFig. 2E, the ectopic 204ASRNA inhibited the induction of p204 by DMSO.) The curves in",
"\nFIGURE 2 .\n2DMSO triggered the transcription of the Ifi204 gene in P19 cells; ectopic 204ASRNA inhibited the expression of p204 as triggered by DMSO. A, time course of 204RNA expression, as triggered by DMSO. As loading control 18 S rRNA was assayed (RT-PCR). B, expression of 204RNA in proliferating P19 cells transfected with pCMV204 but not with pCMV only (RT-PCR). C, expression of p204 in cells transfected with pCMV204 (Western blot). D, expression of 204ASRNA in cells transfected with a pCMV204AS expression plasmid. As loading controls the levels of 18 S rRNA were assayed (RT-PCR). E, expression of 204ASRNA in cells transfected with a pCMV204AS construct strongly decreased the induction of p204 expression by DMSO to a level below that in a control culture transfected with pCMV. As loading controls, the levels of tubulin were assayed (Western blots). For further details, see \"Experimental Procedures.\" p204 in Cardiac Myocyte Differentiation",
"\nFIGURE 3 .\n3NES was required for the translocation of p204 from the nucleus to the cytoplasm during the differentiation of P19 cells to myocytes, as induced by DMSO. Schematic structures and levels of p204 (wild type), p204⌬NES, and p204MNES are shown. A-(1), sequence and location of the NES in p204. The nucleotides encoding Leu residues are in boldface letters. A-(2), p204⌬NES is lacking the NES.",
"\n\nright panels). P19 cells transfected with pCMV204⌬NES and incubated in the absence of DMSO expressed little if any Gata4 and Nkx2.5 (Fig.",
"\nFIGURE 4 .\n4Effects of DMSO, ectopic p204, p204⌬NES, and 204ASRNA on the expression of Gata4 and Nkx2.5 proteins and ␣and MHCRNA in P19 cells. A, left panel, induction of Nkx2.5 and Gata4 proteins in differentiating P19 cultures incubated with DMSO or transfected with pCMV204 (p204), but not in cultures incubated without DMSO and transfected with pCMV vector (Con) or pCMV204⌬NES (p204⌬NES) (Western blotting on day 6). Middle and right panels, transfection with pCMV204AS (204ASRNA) decreased the levels of Nkx2.5 and Gata4 induced by DMSO in P19 cultures in conditions of differentiation. As internal control, the levels of tubulin were assayed. B, transfection with pCMV204 (p204), but not with pCMV204⌬NES (p204⌬NES), induced the formation of Gata4 and Nkx2.5 in P19 cultures in differentiation conditions (immunofluorescence assays). Nuclei were stained with DAPI. C, incubation with DMSO (0.8% DMSO) or transfection with pCMV204 (p204) induced the expression of ␣and MHCRNA. Transfection with pCMV204AS inhibited the induction by DMSO (204ASRNA ϩ 0.8% DMSO) in P19 cultures in conditions of differentiation (RT-PCR assays). For further details see \"Experimental Procedures.\"",
"\nFIGURE 5 .\n5Synergistic transactivation of wild type and of mutated Ifi204 reporter constructs by ectopic Gata4, Nkx2. 5, and Tbx5 transcription factors in 10T1/2 cells. A, nucleotide sequence of an Ifi204 reporter construct. This extends from nucleotide Ϫ108 to nucleotide ϩ52. As nucleotide ϩ1 (indicated in the figure), the first nucleotide of intron 1 was chosen. This is the case because the transcription of the gene is initiated at several sites. The numbers (to the left of the sequences) indicate distances in nucleotides from the first nucleotide of intron 1. GATA sequences (recognized by Gata4), an NKE sequence (recognized by Nkx2.5), and TBX sequences (recognized by Tbx5) are printed in boldface and are also indicated in parentheses above the sequence.B, the Gata4, Nkx2.5, and Tbx5 transcription factors synergistically transactivated four types of p204 reporter constructs (in pGL3 vectors) driving the expression of luciferase. The different constructs included either the wild type Ifi204 gene segment as shown in A, or the same segment but with mutations in the TBX site 1, TBX site 2, or both TBX sites. The sequences of the wild type and mutated TBX sites (mutated TBX1) and (mutated TBX2) are indicated. The reporter constructs were transfected into 10T1/2 cells as indicated together with the following: lane 1, the pCGN vector (Con); lane 2, pCGNGata4 (Gata4); lane 3, pCGNNkx2.5 (Nkx2.5); lane 4, pCMVTbx5 (Tbx5); lane 5, pCGNGata4 and pCGNNkx2.5 (Gata4/Nkx2.5); lane 6, pCGNGata4 and pCMVTbx5 (Gata4/Tbx5); lane 7, pCGNNkx2.5 and pCMVTbx5 (Nkx2.5/Tbx5); or lane 8, pCGNGata4 and pCGNNkx2.5 and pCMV-Tbx5 expression plasmids (Gata4/Nkx2.5/Tbx5), together with the pSVGal internal control plasmid. 36 h after transfection the cultures were harvested and lysed, and the -galactosidase and luciferase activities were determined. The luciferase activities were normalized to the -galactosidase activities. The normalized activity of the control lysates (lane 1) was taken as 1 and was compared with the normalized luciferase activities of the other lysates (lanes 2-8). The relative luciferase activities (R.L.A.) for the experiments with the wild type reporters and three mutated reporters and the standard deviations are shown. The standard deviations are indicated. For further details see \"Experimental Procedures.\"",
"\nFIGURE 8 .\n8Transactivation of wild type and mutated Ifi204 reporter constructs by endogenous transcription factors in differentiating but not in undifferentiated, proliferating P19 cultures. Cultures of P19 cells proliferating in the absence of DMSO (PROLIF P19) or differentiating (on day 6 of the process) in the presence of DMSO (DIFF P19) were transfected with the wild type or the mutated version of the Ifi204 reporter (Wild type R and Mutated R) specified (a) and (b) or with the Vector.",
"\n\nNES in p204 Is Required for the Translocation of a Portion of p204 from the Nucleus to the Cytoplasm during the Differentiation of P19 Cells to Beating Myocytes; p204 Lacking the NES or Carrying a Mutated NES Did Not Trigger P19 Differentiation-Fig. 3A-(1)shows the NES"
] | [
"□ S The on-line version of this article (available at http://www.jbc.org) contains supplement M, Fig. S1, and Videos 1 and 2.",
"p204 Is a Mediator of the Induction of the Differentiation of P19 Embryonal Carcinoma Cells to Beating Cardiac Type Myocytes by DMSO; DMSO Induced p204 Expression, and p204 Could Induce the Differentiation in the Absence of DMSO-Following the published procedure (34, 38) for the induction of the differentiation of P19 cells to",
"p204 was required for the differentiation of cultured P19 cells to beating cardiac myocytes. A, time course of p204 accumulation during the differentiation as triggered by DMSO. P19 cultures were incubated in the absence or presence of 0.8% DMSO, and the level of p204 was determined daily until day 8 by Western blotting. As internal control, tubulin levels were followed. B, ectopic p204 (generated by transfection of pCMV204) could partially substitute for DMSO in the induction of MHC protein. Ectopic 204ASRNA (generated by transfection of pCMV204AS) inhibited the induction of MHC by DMSO. Top panel, on day 8 of the differentiation process, regions from the tissue culture dishes containing beating cell clusters were assayed by fluorescence microscopy using monoclonal MF20 antibodies against MHC. Bottom panel, in the absence of DMSO, or in the presence of 204ASRNA (even if DMSO was added), neither beating clusters nor MHC was detected. Nuclei were stained with DAPI. The bar corresponds to 30 m. C, effect of different agents on the differentiation of P19 cells to beating myocytes. The time courses of the increase in the % of beating P19 cell aggregates in different culture conditions are shown (curves 1-11).",
"Fig. 1B) illustrate the following: (i) the need for DMSO in the triggering of the formation of beating cardiac myocyte-like cells expressing MHC proteins(Fig. 1B, compare panels a and e); (ii) the ability of ectopic p204 to trigger the formation of beating myocyte-like cells expressing MHC (in the absence of DMSO(Fig. 1B, panel b)) (see supplemental Videos 1 and 2); and (iii) the inhibition of the induction of beating and MHC-expressing cells by DMSO in P19 cells that expressed 204ASRNA(Fig. 1B, panel f ). (As will be demonstrated inFig. 2E, the ectopic 204ASRNA inhibited the induction of p204 by DMSO.) The curves in",
"DMSO triggered the transcription of the Ifi204 gene in P19 cells; ectopic 204ASRNA inhibited the expression of p204 as triggered by DMSO. A, time course of 204RNA expression, as triggered by DMSO. As loading control 18 S rRNA was assayed (RT-PCR). B, expression of 204RNA in proliferating P19 cells transfected with pCMV204 but not with pCMV only (RT-PCR). C, expression of p204 in cells transfected with pCMV204 (Western blot). D, expression of 204ASRNA in cells transfected with a pCMV204AS expression plasmid. As loading controls the levels of 18 S rRNA were assayed (RT-PCR). E, expression of 204ASRNA in cells transfected with a pCMV204AS construct strongly decreased the induction of p204 expression by DMSO to a level below that in a control culture transfected with pCMV. As loading controls, the levels of tubulin were assayed (Western blots). For further details, see \"Experimental Procedures.\" p204 in Cardiac Myocyte Differentiation",
"NES was required for the translocation of p204 from the nucleus to the cytoplasm during the differentiation of P19 cells to myocytes, as induced by DMSO. Schematic structures and levels of p204 (wild type), p204⌬NES, and p204MNES are shown. A-(1), sequence and location of the NES in p204. The nucleotides encoding Leu residues are in boldface letters. A-(2), p204⌬NES is lacking the NES.",
"right panels). P19 cells transfected with pCMV204⌬NES and incubated in the absence of DMSO expressed little if any Gata4 and Nkx2.5 (Fig.",
"Effects of DMSO, ectopic p204, p204⌬NES, and 204ASRNA on the expression of Gata4 and Nkx2.5 proteins and ␣and MHCRNA in P19 cells. A, left panel, induction of Nkx2.5 and Gata4 proteins in differentiating P19 cultures incubated with DMSO or transfected with pCMV204 (p204), but not in cultures incubated without DMSO and transfected with pCMV vector (Con) or pCMV204⌬NES (p204⌬NES) (Western blotting on day 6). Middle and right panels, transfection with pCMV204AS (204ASRNA) decreased the levels of Nkx2.5 and Gata4 induced by DMSO in P19 cultures in conditions of differentiation. As internal control, the levels of tubulin were assayed. B, transfection with pCMV204 (p204), but not with pCMV204⌬NES (p204⌬NES), induced the formation of Gata4 and Nkx2.5 in P19 cultures in differentiation conditions (immunofluorescence assays). Nuclei were stained with DAPI. C, incubation with DMSO (0.8% DMSO) or transfection with pCMV204 (p204) induced the expression of ␣and MHCRNA. Transfection with pCMV204AS inhibited the induction by DMSO (204ASRNA ϩ 0.8% DMSO) in P19 cultures in conditions of differentiation (RT-PCR assays). For further details see \"Experimental Procedures.\"",
"Synergistic transactivation of wild type and of mutated Ifi204 reporter constructs by ectopic Gata4, Nkx2. 5, and Tbx5 transcription factors in 10T1/2 cells. A, nucleotide sequence of an Ifi204 reporter construct. This extends from nucleotide Ϫ108 to nucleotide ϩ52. As nucleotide ϩ1 (indicated in the figure), the first nucleotide of intron 1 was chosen. This is the case because the transcription of the gene is initiated at several sites. The numbers (to the left of the sequences) indicate distances in nucleotides from the first nucleotide of intron 1. GATA sequences (recognized by Gata4), an NKE sequence (recognized by Nkx2.5), and TBX sequences (recognized by Tbx5) are printed in boldface and are also indicated in parentheses above the sequence.B, the Gata4, Nkx2.5, and Tbx5 transcription factors synergistically transactivated four types of p204 reporter constructs (in pGL3 vectors) driving the expression of luciferase. The different constructs included either the wild type Ifi204 gene segment as shown in A, or the same segment but with mutations in the TBX site 1, TBX site 2, or both TBX sites. The sequences of the wild type and mutated TBX sites (mutated TBX1) and (mutated TBX2) are indicated. The reporter constructs were transfected into 10T1/2 cells as indicated together with the following: lane 1, the pCGN vector (Con); lane 2, pCGNGata4 (Gata4); lane 3, pCGNNkx2.5 (Nkx2.5); lane 4, pCMVTbx5 (Tbx5); lane 5, pCGNGata4 and pCGNNkx2.5 (Gata4/Nkx2.5); lane 6, pCGNGata4 and pCMVTbx5 (Gata4/Tbx5); lane 7, pCGNNkx2.5 and pCMVTbx5 (Nkx2.5/Tbx5); or lane 8, pCGNGata4 and pCGNNkx2.5 and pCMV-Tbx5 expression plasmids (Gata4/Nkx2.5/Tbx5), together with the pSVGal internal control plasmid. 36 h after transfection the cultures were harvested and lysed, and the -galactosidase and luciferase activities were determined. The luciferase activities were normalized to the -galactosidase activities. The normalized activity of the control lysates (lane 1) was taken as 1 and was compared with the normalized luciferase activities of the other lysates (lanes 2-8). The relative luciferase activities (R.L.A.) for the experiments with the wild type reporters and three mutated reporters and the standard deviations are shown. The standard deviations are indicated. For further details see \"Experimental Procedures.\"",
"Transactivation of wild type and mutated Ifi204 reporter constructs by endogenous transcription factors in differentiating but not in undifferentiated, proliferating P19 cultures. Cultures of P19 cells proliferating in the absence of DMSO (PROLIF P19) or differentiating (on day 6 of the process) in the presence of DMSO (DIFF P19) were transfected with the wild type or the mutated version of the Ifi204 reporter (Wild type R and Mutated R) specified (a) and (b) or with the Vector.",
"NES in p204 Is Required for the Translocation of a Portion of p204 from the Nucleus to the Cytoplasm during the Differentiation of P19 Cells to Beating Myocytes; p204 Lacking the NES or Carrying a Mutated NES Did Not Trigger P19 Differentiation-Fig. 3A-(1)shows the NES"
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"The interferons are vertebrate cytokines with antimicrobial, cell growth regulatory, and immunomodulatory activities that also affect differentiation (1)(2)(3). They function by modulating the expression of many genes, including those of the gene 200 cluster (4 -10). In the mouse this cluster consists of at least 10 genes, which encode the p200 family proteins (11). The human counterpart of the cluster is smaller; it consists apparently of only four genes (MNDA, IFI16, AIM2, and IFIX) that encode the Hin200 family proteins (12)(13)(14)(15).",
"Among the two best characterized members of the murine p200 family proteins is p202a (which was designated earlier as p202) (16 -26). p202a modulates transcription, cell proliferation, and apoptosis. It functions primarily by binding various sequence-specific transcription fac-tors and inhibiting their activity generally, but not exclusively, by binding them and blocking their sequence-specific binding to DNA (24). p202a overexpression was implicated in the susceptibility of mice to the autoimmune disease lupus (27).",
"The other much studied p200 family protein p204 is encoded by the Ifi204 gene (5). p204 is structurally related to p202a and is inducible by interferons, as is p202a. Depending on the cell type and the state of differentiation, p204 can be located in the nucleolus, the nucleoplasm, or the cytoplasm (28 -30). Its overexpression in cultured mammalian cells is growth-inhibitory (28,31). It can delay the progression of cells from the G 0 /G 1 to the S phase of the cell cycle (31). p204 can inhibit the transcription of ribosomal RNA by binding to the ribosomal DNAspecific UBF transcription factor and by inhibiting its binding to DNA (30). p204, similarly to p202a, contains the pRb-, p107-, and p130-binding motif LXCXE and can bind these pocket proteins (19,30,32).",
"Among 10 adult mouse tissues tested, the level of p204 is highest in the heart and second highest in skeletal muscle (29). During the differentiation of cultured C2C12 mouse skeletal muscle myoblasts to myotubes, the p204 (and p202a) levels increased severalfold as a consequence of the transactivation of the Ifi204 gene by the muscle-specific MyoD, myogenin, and E12/E47 transcription factors (29). This increase did not depend on interferon action (33). p204 is required for the differentiation of C2C12 myoblasts to myotubes. It enables the differentiation, at least in part, by overcoming the inhibition of the activities of MyoD, E12/E47, and other myogenic basic region-helix-loop-helix transcription factors by the inhibitor of differentiation (Id) 2 proteins Id1, Id2, and Id3 (33).",
"p204 is also involved in osteogenesis. It is expressed in osteoblasts of various tissues in embryonic and neonatal mice. It is induced in the course of the BMP-2-triggered differentiation of C2C12 cells to osteoblasts. p204 binds and acts as a cofactor of Cbfa-1 (Core binding factor ␣-1), which is an essential transcription factor in osteoblasts differentiation and bone formation (26).",
"The aim of this study has been to explore whether p204 is involved in and is required for cardiac myocyte differentiation and the basis of the high level of expression of p204 in mouse heart. As a model system for cardiac myocyte differentiation, we used cultured murine P19 embryonal carcinoma cells (34 -37). The P19 line of pluripotent embryonal stem cells was derived from a teratocarcinoma induced in C3H/HC mice (38). The cells can be maintained in culture in an undifferentiated state. When injected into early mouse embryos, * This work was supported by NIAID Research Grant AI-12320 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked \"advertisement\" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. P19 cells are capable of contributing to a variety of apparently normal tissues suggesting that the mechanisms of their differentiation are similar to those of normal embryonic cells (39). P19 cells can also be induced to differentiate in vitro into multiple cell types, including beating cardiac myocytes, skeletal myocytes, and myotubes, as well as neurons (34,36). The differentiation of P19 cells to cardiac and skeletal myocytes in vitro is initiated by plating P19 cells grown in tissue culture dishes and dissociated with trypsin into bacteriological culture dishes to which the cells do not attach and in which they form aggregates. The cultures are supplemented with DMSO. The aggregates are transferred to tissue culture dishes and further incubated in the presence of DMSO. Proliferating cells migrate out from the aggregates, including many that are differentiated to cardiac myocyte-type beating cells (and later also skeletal myocyte-type cells). In the absence of DMSO (whose mode of action remains to be clarified), there occurs little or no differentiation of P19 cells in vitro (34,36). DMSO causes an immediate, transient increase in Ca 2ϩ ions in the cells, and it was proposed that permeabilization and membrane penetration by DMSO may alter molecular interactions in the cells (40,41).",
"We report here that in P19 cells (i) DMSO induced p204 formation, (ii) ectopic 204AS RNA blocked the DMSO-induced differentiation (as well as p204 formation), and (iii) ectopic p204 could partially substitute for DMSO in inducing the differentiation of P19 cells to beating myocytes. p204 contains an NES, and in the course of differentiation to myocytes a portion of p204 was translocated from the nucleus to the cytoplasm of P19 cells (29). p204 lacking an NES or carrying a mutated NES was not translocated in P19 cultures treated with DMSO and could not substitute for p204 in triggering differentiation.",
"The 5Ј-flanking region of the gene encoding p204 (i.e. Ifi204) (11) contains several binding sites for the Gata4, Nkx2.5, and Tbx5 transcription factors. All these factors are important transcriptional regulators of cardiac gene expression and are members of protein families conserved in evolution from Drosophila to vertebrates (42)(43)(44)(45)(46)(47). Nkx2.5 is an NK-2 class homeodomain protein (48). Mouse Nkx2.5 is expressed throughout the development of the heart and is required for this process. In DNA it binds to the 5Ј-AAGTG-3Ј sequence (NKE element), among others. Gata4 is a zinc finger protein (49). It is expressed in the developing mouse heart almost simultaneously and spatially overlapping with Nkx2.5 (46, 50 -52). Gata4 binds in DNA to a sequence element containing a core 5Ј-GATA-3Ј motif. This motif occurs in the promoter region of many genes encoding cardiac proteins, including the atrial natriuretic factor gene (53). Tbx5 is a member of the family of transcription factors containing T box-type DNA binding domains (42). It is also expressed throughout heart development. Its consensus DNAbinding sequence is 5Ј-(A/G/T)GGTG(T/C)(C/T/G)(A/G) (43). This also occurs in the promoter regions of various cardiac-specific genes, including that encoding atrial natriuretic factor (43). Nkx2.5, Gata4, and Tbx5 can pairwise bind one another and can cooperatively or synergistically activate gene expression (52, 54 -56).",
"We report results showing that ectopic Gata4, Nkx2.5, and Tbx5 synergistically transactivated the expression of reporter constructs, including segments from the 5Ј-flanking region of the Ifi204 gene in murine 10T1/2 fibroblasts and in cardiac myocytes from rats, and also cooperatively transactivated the expression of endogenous p204 in 10T1/2 cells. Ectopic p204 (also DMSO) triggered P19 cells (to differentiate and) to increase their expression of the Gata4 and Nkx2.5 transcription factors. An examination of P19 cells differentiated to myocytes by chromatin coimmunoprecipitation assays with ␣Gata4 or ␣Nkx2.5 antibodies and PCR revealed the binding of endogenous Gata4 and Nkx2.5 to a segment of the 5Ј-flanking region of the Ifi204 gene, which contains a cluster of Gata4-and Nkx2.5-specific sequences. These findings indicate the following: (i) p204 is required for cardiac type myocyte differentiation from P19 embryonal stem cells, and (ii) the level of p204 increases during myocyte differentiation in consequence of the transactivation of the Ifi204 gene by cardiac transcription factors. The various mechanisms by which p204 enabled the differentiation of P19 cells to cardiac-type myocytes are reported in the accompanying article (76).",
"Plasmid Constructs-The following plasmids were constructed as described in detail in supplement M: p204 (sense) expression plasmid pCMV204, 204 antisense RNA expression plasmid pCMV204AS (5); GFP-p204, GFP-204⌬NES (29); pCMV204⌬NES, pCMV204MNES; pCMVFLAG-p204, pCMVFLAG-p204⌬NES, and FLAG-p204MNES; pCGNGata4 and pCGNNkx2.5 (52); and pCMVTbx5, pCMVHATbx5, pACCMVTbx5 (57). The various Ifi204-based reporters (based on the pGL3 expression vector (Promega)) included wild type Ifi204 gene sequences or sequences with deletions or mutations. For further details see supplement M.",
"Cell Culture-P19 cells (a gift from M. W. McBurney) were derived from mouse teratocarcinoma induced in C3H/HC mice. The cells were cultured and induced to differentiate to cardiac-type myocytes as described previously (34) and in supplement M. Neonatal rat cardiac myocytes were isolated from hearts of 3-day-old rats as described previously (58,59). 10T1/2 cells, cloned murine embryonic fibroblasts (ATCC 226 CCL) (60), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For further details see supplement M.",
"Transfection-P19 cells were transfected as specified with expression plasmids encoding p204, p204ASRNA, p204MNES, p204⌬NES, Id3, p204 plus Id3, or p204⌬NES plus FLAGId3 using Lipofectamine 2000 in minimum essential medium-␣ (Invitrogen), supplemented with 2.5% fetal bovine serum (Invitrogen) and 7.5% donor calf serum (TerraCell). Stable cell lines were established by selection with G418. Clones were assayed to ascertain high level expression of the ectopic RNA or protein of interest by Northern or Western blotting, respectively. For further details see supplement M.",
"Assay of p204, Gata4, Nkx2.5, and MHC by Immunofluorescence Microscopy-P19 cells growing on 35-mm culture dishes were treated as described previously (34) and incubated as specified with mouse MF20, the Developmental Studies Hybridoma Bank, and M2 monoclonal ␣FLAG (Sigma), rabbit ␣p204 and rabbit ␣Id3 (Santa Cruz Biotechnology), and goat ␣Gata4 or ␣Nkx2.5 antibodies (Santa Cruz Biotechnology). As secondary antibodies, fluorescein isothiocyanate or Texas Red-linked goat anti-rabbit and anti-mouse IgG and donkey antigoat IgG (Santa Cruz Biotechnology) were used. Nuclei were stained with DAPI.",
"RT-PCR-P19 cells in 35-mm tissue culture dishes were lysed in 1 ml of RNA STAT-60 TM (Tel-Test Inc.), and total RNA was isolated following the instructions of the manufacturer and subjected to RT-PCR using the Omniscript RT kit (Qiagen). Sequences of primers are shown in supplement M.",
"Western Blotting-P19 cells were lysed as described previously (61), and 100 g of total protein was subjected to 4 -20% gradient PAGE and transferred to a polyvinylidene difluoride membrane and blotted with rabbit anti-p204 antibody (30). The blots were visualized using ECL (Pierce, SuperSignal Western Blotting kit). Rat anti-HA antibodies (Santa Cruz Biotechnology) and mouse anti--tubulin antibody (the Developmental Studies Hybridoma Bank) were used to detect HA-tagged Tbx5 and -tubulin, respectively. ␣Gata4 and ␣Nkx2.5 antibodies were used for detection of Gata4 and Nkx2.5, respectively.",
"Reporter Gene Assay-Synergistic transactivation of p204 reporter constructs was assayed as indicated by cotransfection with pCGNN-kx2.5 (3 g) and/or pCGNGata4 (3 g) and/or pCMVTbx5 (3 g) with pSVGal control plasmid (1 g) in 10T1/2 cells (62) by Lipofectamine 2000 (Invitrogen) (2 l/g DNA) in 35-mm dishes as described previously (62). pCGN and/or pCMV vector was used to bring the total amount of DNA transfected to 8 g. After a 36-h incubation, the cultures were harvested. Luciferase and -galactosidase were assayed using the luciferase reporter gene assay (Roche Applied Science) and the -galactosidase assay system (Promega), respectively. The experiments were repeated 3-4 times.",
"Sequence Analysis-To search for Gata4, Nkx2.5, and Tbx5 recognition sequences in the Ifi204 gene 5Ј-flanking region (11) (GenBank TM accession number AC006944), the MatInspector, version 2.1, data base was used, and the parameter selected for both core similarity and matrix was 0.8.",
"Chromatin Immunoprecipitation-The chromatin immunoprecipitation assay kit (Upstate) was used following the instructions of the manufacturer. Published procedures (63) were followed in the treatment of the cells with formaldehyde to cross-link the DNA protein interactions, the preparation of the nuclear extracts, the shearing of the chromatin by sonication, and the immunoprecipitation of DNA-protein complexes using immobilized antibodies (i.e. ␣Gata4 and ␣Nkx2.5). The nucleotide sequence of the 5Ј-flanking region of the Ifi204 gene was reported (Gen-Bank TM accession number AC006944). The sequences of the primers used in the PCR analysis of the DNA segments immunoprecipitated are available upon request. The % of beating P19 cell aggregates on day 12 of differentiation was as follows: curve 1, ectopic p204 and treatment with 0.8% DMSO (39%); curve 2, treatment with 0.8% DMSO (38%); curve 3, 0.8% DMSO and transfection with the pCMV vector decreased this to 29%; curve 4, ectopic p204 and treatment with 0.1% DMSO (16%); curve 5, ectopic p204 and no DMSO (10%). No beating cultures arose in the absence of DMSO (curve 6) or in the presence of the pCMV vector (curve 7), and even in the presence of 0.8% DMSO if ectopic 204ASRNA was present (curve 8). No beating cultures arose (in the absence of DMSO) when p204 lacking a nuclear export signal (p204⌬NES) (curve 9) or p204 with a mutated nuclear export signal (p204MNES; see Fig. 3) was substituted for p204 (curve 10). No beating cultures arose in the presence of 0.1% DMSO and pCMV vector (curve 11). The standard deviations are indicated. For further details, see \"Experimental Procedures.\" p204 in Cardiac Myocyte Differentiation clusters of beating cardiac myocytes by DMSO, we grew the cells in tissue culture dishes, shifted them (on day 0) to bacteriological Petri dishes in the presence of DMSO where they form aggregates, and shifted the aggregates to tissue culture dishes (on day 5). Within two days, many clusters, composed mainly of cells with the shape of cardiac myocytes, started beating (day 8; data not shown).",
"As shown in the Western blots in Fig. 1A in P19 cells proliferating in tissue culture dishes (in the absence or presence of DMSO), no p204 was detected (day 0). Cells aggregated in Petri dishes without DMSO contained very low levels, if any, of p204, which increased slightly after their shift to tissue culture dishes (between day 6 and day 8). Cells aggregated in the presence of DMSO contained very low levels of p204 (on days 2-4) but much higher levels after their shift to tissue culture dishes (between day 6 and day 8). Thus, DMSO induced p204 in differentiating P19 cultures.",
"The immunofluorescence microscopic pictures (day 8; Fig. 1C represent the time course of the increase in the percent of cell aggregates with beating myocytes in P19 cultures exposed to various conditions. The cultures that were transfected with expression plasmids were selected in the presence of G418 to eliminate untransfected cells. The conditions (Fig. 1C, curves [1][2][3][4][5][6][7][8][9][10][11] are specified in the figure. Curve 2, by day 12 of the differentiation process, 38% of the P19 cell aggregates exposed to 0.8% DMSO included beating myocytes. Transfection of pCMV204 did not increase significantly the yield of beating aggregates (39%) in a culture exposed to 0.8% DMSO (curve 1). Curve 6, no beating cultures arose in the absence of DMSO or (curve 8) in the presence of 0.8% DMSO if pCMV204AS (a plasmid encoding 204AS RNA) was transfected. Curve 5, transfection of pCMV204 resulted in the formation of aggregates, including beating myocytes (even) in the absence of DMSO. However, the percent of such aggregates (10%) was lower than in the corresponding culture (29%) (curve 3), which was transfected with pCMV, the control vector, and was incubated in the presence of DMSO. Curve 4, DMSO at a concentration as low as 0.1% increased the yield of aggregates with beating cells in a culture expressing ectopic p204 (16%), whereas as already noted (curve 5) ectopic p204 alone resulted only in a lower yield (10%). Curve 11, 0.1% DMSO treatment of cells expressing pCMV vector produced no beating culture.",
"The data in Fig. 1, A-C, are in line with the following conclusions: (i) DMSO induced p204 expression in aggregated P19 cells; (ii) p204 was required for the induction of differentiation by DMSO; and (iii) p204 could partially substitute for DMSO in inducing the differentiation.",
"The RT-PCR assays in Fig. 2A reveal that DMSO did not trigger the transcription of the Ifi204 gene to 204 RNA in proliferating P19 cells (day 0). DMSO did, however, trigger the transcription to 204 RNA in differentiating P19 cells, and the 204 RNA was present from day 6 at least until day 12, the last day when it was tested. Transfection of pCMV204 (but not of pCMV) into P19 cells (without or after elimination of the untransfected cells) resulted in 204 RNA expression even in proliferating cells (Fig. 2B). This expression persisted during the differentiation of the cells at least until day 12 (data not shown). The ectopic p204 level was determined by Western blotting to confirm that 204 RNA in P19 cells was translated into p204 (Fig. 2C). Transfection of a pCMV204AS plasmid resulted in the expression of a high level of 204AS RNA in the cells (Fig. 2D). This ectopic 204AS RNA (but not ectopic pCMV vector) decreased the amount of p204 induced by DMSO to a barely detectable level (Fig. 2E). These results suggest that the inhibition of the DMSO-induced differentiation of P19 cells to beating myocytes by 204AS RNA was a consequence of the inhibition by 204AS RNA of the accumulation of p204 as induced by DMSO.",
"The (64) amino acid sequence in p204, and the sequence of nucleotides encoding it in Ifi204. Fig. 3A-(2) is a schematic representation of p204 lacking an NES (p204⌬NES). The generation of p204⌬NES involved the deletion of a 12-amino acid segment from p204. It is possible that this might have altered the structure of p204 resulting in the loss of some of its activities. Therefore, as a control we also generated p204 with a mutated NES (p204MNES), in which only three amino acid residues were replaced ( Fig. 3A-(3)). This shows the altered amino acid sequence of p204MNES in which three Leu residues from the NES were replaced by Ala residues. The mutated nucleotide sequence encoding the altered amino acid sequence is also shown. The Western blots in Fig. 3, A-(4), A- (5), and A- (6), established that the pCMV plasmids encoding p204, p204⌬NES, and p204MNES transfected into P19 cells resulted in the expression of similar amounts of protein.",
"The fluorescence microscopic analysis in Fig. 3B involved the use of two types of fusion proteins as follows: (i) green fluorescent pro- MAY 26, 2006 • VOLUME 281 • NUMBER 21 tein-204 (GFP-p204) and (ii) GFP linked to a p204 derivative from which the NES oligopeptide segment was lacking (GFP-p204⌬NES). GFP serving as a control was shown to be distributed between the nuclei and the cytoplasm in both proliferating (Fig. 3B, PROLIF.) and differentiated (DIFF.) P19 cells (Fig. 3B, bottom panels). GFP-p204 was nuclear in proliferating cells, but a portion of GFP-p204 was A-(3), in p204MNES, the sequence of NES is altered; three Leu residues are replaced by Ala residues. These Ala residues and the nucleotides encoding Leu residues or Ala residues replacing Leu residues are in boldface letters. A-(4), A- (5), and A- (6), the levels of expression of ectopic p204, p204⌬NES, and p204MNES were very similar in differentiating P19 cells transfected with pCMV204, pCMV204⌬NES, or pCMV204MNES (as determined by Western blotting on day 8 of the differentiation). Con, transfected by vector; UC, untransfected control. B, distribution of ectopic green fluorescent protein, GFP-p204, and GFP-p204⌬NES between the nucleus and the cytoplasm in proliferating and differentiating P19 cells. P19 cells were transfected, as indicated, with plasmids encoding green fluorescent protein or the fusion proteins GFP-p204 or GFP-p204⌬NES. Two cultures from each of the three types of transfectants were incubated in tissue culture dishes (without DMSO). After a 36-h incubation, one of each two of the proliferating cultures was examined by fluorescence microscopy (in panel PROLIF). Second cultures from each of the three types of transfectants were shifted after 36 h to Petri dishes and supplemented with 0.8% DMSO (in panel DIFF). Four days later, these were transferred in the same medium to tissue culture dishes to allow differentiation during the next 48 -72 h. This was followed by fluorescence microscopy. Bar is 30 m. C, subcellular location of endogenous p204 and of ectopic FLAG-p204, FLAG-p204⌬NES, and FLAG-p204MNES in proliferating P19 cells and in differentiating P19 cells (tested on D6-D7 of the differentiation). Fluorescence microscopy, using ␣FLAG (top and bottom panels) and, as secondary antibodies, anti-mouse IgG tagged with fluorescein (top panel) or with rhodamine (bottom panel) is shown. In the middle panel, to reveal endogenous p204, ␣p204, and as secondary antibody, anti-rabbit IgG, tagged with fluorescein were used. Nuclei were stained with DAPI. The bar corresponds to 30 m. For further details see \"Experimental Procedures.\" p204 in Cardiac Myocyte Differentiation translocated from the nucleus to the cytoplasm in differentiated cells (Fig. 3B, top panels). GFP-p204⌬NES, however, was nuclear in proliferating P19 cells and remained nuclear also in differentiated P19 cells (Fig. 3B, middle panels).",
"To ascertain that the presence of GFP in the fusion proteins did not affect the translocation (in Fig. 3B), the tests were also performed using ectopic FLAG-p204, FLAG-p204⌬NES, and FLAG-p204MNES. Confirming the observations in Fig. 3B, the immunofluorescence microscopic analysis (in Fig. 3C, bottom panel) revealed that in proliferating P19 cells FLAG-p204 was nuclear (and so were FLAG-p204⌬NES and FLAG-p204MNES (not shown)). In P19 cells whose differentiation to cardiac myocytes cells was triggered by DMSO, much of the FLAG-p204 was translocated to the cytoplasm; however, FLAG-p204⌬NES and FLAG-p204MNES, which lacked an intact NES, remained in the nuclei (Fig. 3C, upper panels). The level of endogenous p204 in proliferating P19 cells is too low for immunofluorescent detection. However, we have reported earlier that in proliferating C2C12 myoblasts, in which the endogenous p204 level is higher and thus detectable, p204 is nuclear (29). In differentiating P19 cells the endogenous 204 was distributed between the nuclei and the cytoplasm (Fig. 3C, middle panel).",
"The findings in Fig. 1C indicate that (in addition to controlling the subcellular location of p204, as shown in Fig. 3, B and C) the NES was also required for the differentiation of P19 cells to beating cardiac-type myocytes. Thus, although the transfection of pCMV204 triggered the differentiation of P19 cells (even in the absence of DMSO) (Fig. 1C (curve 5)), the transfection of pCMV204⌬NES or pCMV204MNES did not trigger the formation of beating myocytes from P19 cells in the absence of DMSO (Fig. 1C (curves 9 and 10)). Furthermore, ectopic p204⌬NES or p204MNES did not substitute for ectopic p204 in triggering MHC protein formation in aggregated P19 cells in the absence of DMSO (data not shown).",
"p204, but Not p204 Lacking the NES, Could Substitute for DMSO in the Triggering of the Expression of the Gata4 and Nkx2.5 Transcription Factors in Aggregated P19 Cultures-As reported earlier (49,65), two of the transcription factors expressed early in the course of differentiation of aggregated P19 cultures to cardiac-type myocytes in the presence of DMSO (and also in the course of murine heart development) are Gata4 and Nkx2.5. Moreover, in the absence of DMSO, the P19 aggregates (even if transfected with an empty vector) contained primarily undifferentiated carcinoma cells, and neither Gata4 nor Nkx2.5 expression could be detected (65,66).",
"As shown in the Western blots (Fig. 4A), aggregated P19 cells, incubated with DMSO or incubated without DMSO but transfected with pCMV204 and thus expressing p204 (p204), expressed both Gata4 and Nkx2.5 (left panel). Aggregated cells incubated without DMSO and transfected with empty vector (Fig. 4A, Con), however, did not express either Gata4 or Nkx2.5 (left panel). Moreover, the expression of ectopic 204ASRNA strongly inhibited the expression of Nkx2.5 and Gata4 as induced by DMSO (Fig. 4A, middle and 4A, left panel). This was the case although the level of p204⌬NES expressed was similar to the level of p204 in cells transfected with pCMV204 (see Fig. 3, A-(4) and A-(5)). These results are in line with the observations that p204⌬NES or p204MNES could not substitute for wild type p204 in inducing MHC either (data not shown). All these findings are in accord with the results in Fig. 1C in demonstrating that p204 lacking an active NES was unable to trigger the differentiation of P19 cells to beating cardiac myocytes.",
"The immunofluorescence microscopic pictures in Fig. 4B are in accord with the Western blots in Fig. 4A in showing the pronounced induction of Gata4 and Nkx2.5 in differentiating P19 cells by ectopic p204, but little if any, if it was lacking the NES. In the pictures of cultures with ectopic p204 (but not in those with ectopic p204⌬NES) (see Fig. 4B), some of the p204 has spread into the cytoplasm. This (as noted earlier, see Fig. 3C) is in consequence of the differentiation of the P19 cells induced by p204 (but not by p204⌬NES).",
"The RT-PCR patterns in Fig. 4C revealed that the induction of MHC protein by DMSO or ectopic p204 in differentiating P19 cells (Fig. 1B) was (i) the consequence of the induction of ␣and MHCRNA, and (ii) the ectopic 204ASRNA inhibited the induction of the transcription of the MHC genes by DMSO.",
"The Expression of the Ifi204 Gene Could Be Synergistically Transactivated in 10T1/2 Cells, Rat Cardiac Myocytes, and P19 Cells Differentiating to Myocytes by the Gata4, Nkx2.5, and Tbx5 Cardiac Transcription Factors-Searching for cardiac transcription factor recognition sequences in the 5Ј-flanking region of the murine Ifi204 gene (11), we noted the occurrence of numerous recognition sequences for Gata4, Nkx2.5, and Tbx5 (Fig. 5A).",
"These transcription factors that function in cardiac differentiation can bind each other and are capable of synergistically transactivating gene expression (51, 52, 54 -56, 67). We assayed the activity of the three factors to transactivate Ifi204 gene expression in transfected 10T1/2 murine embryonic fibroblasts (Figs. 5B and 6), cultured myocytes from newborn rats (Fig. 7), and proliferating and differentiating P19 cultures (Fig. 8). In the bulk of the assays we used reporter constructs, including various segments from the 5Ј-flanking region of the Ifi204 gene driving luciferase expression.",
"One of the short Ifi204 segments (of 160 nucleotides) used in a reporter construct (Fig. 5A) included parts of exon 1 and intron 1 with three recognition sites for Gata4 (designated as GATA), one for Nkx2.5 (designated as NKE), and two for Tbx5 (designated as TBX). It should be noted that a cluster of several short (about 6 -8 bp) transcription factor ",
"recognition sites, such as in this region, is rare in the genome, although single recognition sites are common. Multiple sequence-specific factors in such a cluster typically function synergistically (68).",
"The expression of the reporter construct, including the Ifi204 segment in Fig. 5A in transfected 10T1/2 cells, is shown in Fig. 5B (wild type panel). The low level of expression in control cells (lane 1) (taken as 1 to serve as the standard for comparison) was only slightly increased by ectopic Gata4, Nkx2.5, or Tbx5 (lanes 2-4). Any two of these transcription factors activated the expression synergistically ( lanes 5-7 ), and all three factors activated it even further (25-fold) (lane 8). Mutation in the reporter of any one of the two TBX sequences and especially of both strongly decreased the synergistic boost of the expression by TBX5 but affected the boosts by Gata4 and Nkx2.5 less, if at all (Fig. 5B, panels Mutated TBX (1), Mutated TBX (2), and Doubly mutated TBX ). These results are in accord with the finding that synergistic transactivation by Tbx5 (with Nkx2.5 or Gata4) requires the binding of Tbx5 to DNA (54 -56).",
"The importance of the GATA and NKE sequences in the control of the transactivation by ectopic Gata4 and Nkx2.5 in 10T1/2 cells was demonstrated (see supplemental Fig. S1). The results (i) confirmed that Gata4 and Nkx2.5 can synergize by protein-protein interaction even if only one of the factors was bound to DNA (50,51,69), and (ii) they demonstrated that the deletion or mutation of a GATA or NKE sequence may decrease the synergistic transactivation even in reporters with two or more binding sites for the above transcription factors.",
"The effects of ectopic Nkx2.5, Gata4, and Tbx5 on the expression of endogenous p204 in 10T1/2 cells, together with the levels of expression of the three ectopic transfection factors, are shown in the Western blots in Fig. 6. The numbers below the p204 panel in Fig. 6 reveal the cooperativity or in some cases the slight synergy between any two of the three ectopic transcription factors. (The lack of a further increase in the level of endogenous p204 upon addition of the third transcription factor may only be apparent because it may be due, at least in part, to the fact that in this case in order to keep the total DNA concentration constant less DNA was transfected for each factor (2 g) than in all other cases (3 g).) The lesser increase in the level of p204 protein (Fig. 6) than of p204 RNA in 10T1/2 cells (Fig. 5B) by ectopic Gata4, Nkx2.5, and Tbx5 is likely to be the consequence of, at least in part, a post-transcriptional control of p204 expression. Such control was reported in the case of the expression of p202b, another protein of the p200 family (17,23).",
"To establish whether the increase in the expression of p204 by ectopic Nkx2.5, Gata4, and Tbx5 in 10T1/2 cells may be relevant for cardiac myocyte differentiation, we also tested the effect of these transcription factors on p204 expression in cultures of primary cardiac myocytes from newborn rats (Fig. 7). Both the endogenous level of Nkx2.5 protein and the endogenous Gata4 activity were reported to be very low in cultured newborn rat myocytes (70,71). We transfected newborn rat myocytes in culture with a reporter construct, including a sequence from the Ifi204 gene 5Ј-flanking segment (shown in Fig. 5A). The Gata4, Nkx2.5, and Tbx5 transcription factor binding sites in the construct are shown in Fig. 7. Transfection of an expression plasmid encoding any one of the above listed transcription factors transactivated p204 expression weakly (less than 2-fold). Transfection of any two of them had a synergistic effect (an over 8-fold increase). Transfection of all three had an even stronger synergistic effect (an over 14-fold increase). These results establish that ectopic Gata4, Nkx2.5, and Tbx5 can synergistically transactivate FIGURE 6. Ectopic Gata4, Nkx2.5, and Tbx5 (actually HA-tagged Tbx5 was used) cooperatively promoted the expression of endogenous p204 in cultures of 10T1/2 cells. The fold increase is indicated below the Western blot of endogenous p204. The protein levels of each of the ectopic transcription factors in cultures transfected with the appropriate expression plasmids are shown, together with the level of endogenous tubulin serving as an internal protein control. Con, culture transfected with empty vector. 36 -40 h after transfection the cultures were lysed, and p204 was detected using p204 antibodies and the ECL system. Thereafter the membrane was stripped and blotted using antibodies against Nkx2.5, Gata4, and Tbx5 (in the case of Tbx5 antibodies to HA were used) and tubulin (each after stripping). The number 3.05 is in parentheses to indicate that less DNA was transfected of each transcription factor when using Gata4/Nxk2.5 and Tbx5 than in all other cases in which only 1 or 2 transcription factors were used. See also the text. The standard deviations are indicated. For further details see \"Experimental Procedures.\" FIGURE 7. Synergistic transactivation of the expression of an Ifi204 reporter construct by ectopic Gata4, Nkx2. 5, and Tbx5 transcription factors in rat cardiac myocytes. Cardiac myocytes isolated from 3-day-old rats were seeded on collagen-coated dishes. The Ifi204 reporter construct inserted into a pGL3 vector and driving luciferase expression included a segment from the 5Ј-flanking region of Ifi204 extending, as indicated, from nucleotide Ϫ98 to ϩ47. The binding sites for Gata4, Nkx2.5, or Tbx5 are indicated in the figure. The Ifi204 reporter constructs were cotransfected, as indicated, with the pCGN vector (Con), and pCGNGata4, pCGNNkx2.5, pCMVTbx5, as well as with pSVGal. The activities of -galactosidase and luciferase were assayed in the extracts after 36 h. The luciferase activities were normalized to -galactosidase activities, and the normalized activities of the wild type and mutated reporters in extracts from cells transfected with the plasmids encoding transcription factors were related to the activity of the extract from cells transfected with vector only. The standard deviations are indicated. For further details see \"Experimental Procedures.\" p204 expression not only in 10T1/2 cells but also in cardiac myocytes.",
"Proliferating P19 cultures are lacking endogenous Nkx2.5 as well as Gata4 and Tbx5 transcription factor activity (51,65,66,72), whereas P19 cultures in the course of differentiation to myocytes express Nkx2.5 and Gata4. Thus, we expected that Ifi204 reporter constructs with binding sites only for Gata4, Nkx2.5, and/or Tbx5 would be expressed only in differentiating but not in proliferating P19 cultures. The data in Fig. 8, (a and b) demonstrate high level expression of 204 reporter constructs in differentiating and not, however, in proliferating P19 cells, thus proving the validity of this expectation. The data also indicate that the expression of the p204 reporter constructs decreases if the sequence of a Gata4-binding site (Fig. 8a) or an Nkx2.5-binding site (Fig. 8b) is mutated.",
"Coimmunoprecipitation of Chromatin Segments from the 5Ј-Flanking Region of the Ifi204 Gene Containing Gata4-and Nkx2.5-specific Sequences by Antibodies to Gata4 and Nkx2.5-As indicated earlier, undifferentiated proliferating P19 cells do not contain detectable levels of Gata4 and Nkx2.5 transcription factors (65,66,72) and express the Ifi204 gene very weakly, if at all ( Fig. 2A). P19 cells differentiating to myocytes, however, contain pronounced levels of Gata4 and Nkx2.5 (73) and strongly express the Ifi204 gene (Figs. 1A and 2B). These findings prompted us to test if antibodies to Gata4 and Nkx2.5 (but not control IgG) can coimmunoprecipitate chromatin segments (generated by sonication (Fig. 9A, panel b)) containing Gata4-and Nkx2.5-specific sequences from the 5Ј-flanking region of the Ifi204 gene in nuclear extracts from differentiating P19 cells but not from proliferating P19 cells. The data in Fig. 9A, panel a, indicate that this was the case.",
"As determined by PCR, of the five chromatin segments tested (i.e. four from the 5Ј-flanking region and one from the coding region, including the DNA with the last exon of p204 RNA), two segments, segment 1 (extending from nucleotide Ϫ5124 to Ϫ4705) and segment 4 (extending from nucleotide Ϫ184 to ϩ93), were coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5 (but not by IgG), but only in a nuclear extract from differentiating P19 cells and not in that from proliferating P19 cells (Fig. 9A, panel a). (As noted earlier, the first nucleotide of intron 1 of the gene Ifi204 was taken as ϩ1.) Segments 2 and 3, located between segments 1 and 4, were not coimmunoprecipitated despite the occurrence in them of potential Gata4-and Nkx2.5-specific sequences. This indicates that the pres-ence of such a sequence does not ensure the binding of the appropriate transcription factor.",
"The coimmunoprecipitable segment 4 included a sequence (Fig. 5A) that was used (i) in a reporter construct whose expression in transfected cells was synergistically transactivated by Gata4 and Nkx2.5) (Fig. 5B), and (ii) to which Gata4 and Nkx2.5 bound in an electrophoretic mobility shift assay in vitro (not shown).",
"The reporter construct (in Fig. 9B) contained an ϳ5.5-kb sequence from the Ifi204 5Ј-flanking region. This included all the chromatin segments tested in the chromatin immunoprecipitation in Fig. 9A. The diagram in Fig. 9B reveals that the expression of this construct was cooperatively transactivated by Gata4 and Nkx2.5.",
"The results of the chromatin coimmunoprecipitation assays indicated that Gata4 and Nkx2.5 were bound in vivo to the chromatin from the 5Ј-flanking region of the Ifi204 gene in P19 cells differentiating to myocytes but not in proliferating P19 cells. These findings are in accord with other findings in this study by suggesting that the expression of p204 in P19 cells differentiating to myocytes is the consequence, at least in part, of the transactivation of the gene by Gata4 and Nkx2.5. Chromatin segment 1, which was also coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5, also contained Gata4-and Nkx2.5-specific sequences. This segment was part of the 3Ј-terminal transcribed but not translated region of the Ifi203a gene, the 5Ј-terminal neighbor of the Ifi204 gene (11). According to a recent report (74), about a third of the transcription factor binding sites (as examined in human chromosomes 21 and 22) are located within or immediately 3Ј to well characterized genes and are significantly correlated with noncoding RNAs. Furthermore, overlapping pairs of protein coding and noncoding RNAs are often coregulated. Consequently, it will be interesting to see whether this segment 1 is a site in which a noncoding RNA transcript is initiated, and if so whether the transcript affects the expression of the Ifi203a and/or Ifi204 genes.",
"Various Inducers and Transcription Factors Can Promote the Tissuespecific Expression of p204-p204 was discovered as an interferoninducible protein (5). Studies on the differentiation of skeletal muscle revealed that in the course of this process p204 expression is activated by the skeletal muscle-specific MyoD and myogenin transcription factors (29). The results presented in this study established that in cardiac myocyte differentiation, p204 expression is synergis- The wild type or mutated binding sites for the Gata4, Nkx2.5, and Tbx5 transcription factors in the various Ifi204 reporters are indicated. In the mutated binding site in (a), the GATA sequence (in the 3Ј to 5Ј direction) TATC was mutated to CGTC, and in (b), the NKE sequence (in the 3Ј to 5Ј direction) CAC T T was mutated to CAAGG. A pSVGal internal control plasmid was cotransfected. Proliferating cells were transfected in the absence of DMSO and differentiating cells in the presence of DMSO. -Galactosidase and luciferase activities were determined 36 h after transfection. Relative luciferase activity (R.L.A.) was calculated by comparing the normalized luciferase activities of extracts from cells transfected with the wild type or mutated reporter constructs to that of an extract from cells transfected with vector. The standard deviations are indicated. For further details see \"Experimental Procedures.\" p204 in Cardiac Myocyte Differentiation tically activated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. In all the above cases, distinct transcription factors and cis-acting sequences in the Ifi204 gene mediated p204 expression. Furthermore, the expression of p204 in differentiating T cells was reported to be activated by Notch 1 (75); however, the mediators of the activation remain to be identified. Thus the expression of p204 in different tissues can be activated by a variety of tissue-specific mechanisms, i.e. by distinct inducers, transcription factors, and cis-acting sequences.",
"Indications for the Existence of a Positive Feedback Loop Including the p204, Gata4, and Nkx2.5 Proteins-The results presented reveal that in P19 cells differentiating to cardiac myocytes, (i) p204 (but not if lacking its NES) induced the expression of the Gata4 and Nkx2.5 transcription factors, and (ii) Gata4 and Nkx2.5 synergistically activated the expression of p204. These findings indicate the existence of a positive feedback loop, including the p204, Gata4, and Nkx2.5 proteins. The mechanisms of action of p204 in this feedback loop, together with the other mechanisms by which p204 and its NES FIGURE 9. A, antibodies to Nkx2. 5 and Gata4 (but not IgG controls) coimmunoprecipitate some of the chromatin segments from the 5Ј-flanking region of the Ifi204 gene containing Nkx2.5-and Gata4-specific binding sites. This is the case in nuclear extracts from P19 cells differentiating to myocytes but not from undifferentiated, proliferating P19 cells. (a), cultures of P19 cells proliferating in the absence of DMSO (PROLIF ) and cultures of P19 cells undergoing differentiation to cardiac myocytes in the presence of DMSO (DIFF ) on day 6 to day 7 of the process were incubated with formaldehyde to cross-link the transcription factors to DNA. Nuclear extracts were prepared, and the chromatin was sheared by sonication to 200 -1000-bp segments (see the gel electrophoretic pattern of the DNA segments in (b). The suspension was incubated with protein A beads without or after precoating with control IgG or antibodies to Gata4 (␣G) or Nkx2.5 (␣N). (The specificities of the antibodies (Ab) used were verified.) The DNA-protein complexes were eluted from the beads. The cross-linking was reversed, and the DNA was recovered by phenol/chloroform extraction and was assayed by PCR using pairs of primers for various segments of the 5Ј-flanking region of Ifi204 containing GATA or NKE sequences. The amounts of PCR products from the input DNA (Input) and from the DNA coimmunoprecipitated with ␣G or ␣N from the indicated segments of the Ifi204 gene 5Ј-flanking region (specified by the numbering of their 5Ј-and 3Ј-terminal nucleotides, taking as nucleotide ϩ1 the first nucleotide of intron 1 of the Ifi204 gene) were visualized by ethidium bromide-agarose gel electrophoresis. (c) is a schematic drawing of the Ifi204 gene showing the exons (numbered 1-9) as well as the segments S1 to S5 whose presence in the coimmunoprecipitates obtained using ␣G or ␣N was examined by PCR. Of the three negative control segments that were not coimmunoprecipitated by antibodies ␣G or ␣N, two, i.e. S2 and S3, were from the 5Ј-flanking region of the Ifi204 gene and are located between the S1 and S4 segments that were coimmunoprecipitated with ␣G and ␣N. The third negative control segment, S5, corresponds to the translated 3Ј-terminal exon of the Ifi204 gene. Control IgG did not coimmunoprecipitate any of the DNA segments tested. B, the reporter construct driving luciferase expression was generated by inserting into the pGL3 vector a segment from the 5Ј-flanking region of the Ifi204 gene extending from nucleotide Ϫ5482 to ϩ78. Ectopic Nxk2.5 and Gata4 cooperatively boosted the expression of the construct in 10T1/2 cells. For further details see \"Experimental Procedures.\" enable the differentiation of P19 embryonal carcinoma stem cells to cardiac myocytes, are the topics of the accompanying article (76)."
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"JBC Papers in Press"
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"EXPERIMENTAL PROCEDURES",
"RESULTS AND DISCUSSION",
"p204 in Cardiac Myocyte Differentiation",
"FIGURE 1 .",
"FIGURE 2 .",
"FIGURE 3 .",
"FIGURE 4 .",
"FIGURE 5 .",
"FIGURE 8 ."
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"p204 Is Required for the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes ITS EXPRESSION IS ACTIVATED BY THE CARDIAC GATA4, NKX2.5, AND TBX5 PROTEINS * □ S",
"p204 Is Required for the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes ITS EXPRESSION IS ACTIVATED BY THE CARDIAC GATA4, NKX2.5, AND TBX5 PROTEINS * □ S"
] | [
"J. Biol. Chem"
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15,866,905 | 2022-03-17T01:07:39Z | CCBY | http://downloads.hindawi.com/journals/jo/2009/342391.pdf | GOLD | bf3f4f34be90857392a4fb61ebc5e8a6fa031ab7 | null | null | null | null | 10.1155/2009/342391 | 2025669733 | 19365582 | 2667932 |
TGF-β1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy
Hindawi Publishing CorporationCopyright Hindawi Publishing Corporation2009
Rohan Samarakoon
Center for Cell Biology and Cancer Research
Albany Medical College
47 New Scotland Avenue12208AlbanyNYUSA
Craig E Higgins [email protected]
Center for Cell Biology and Cancer Research
Albany Medical College
47 New Scotland Avenue12208AlbanyNYUSA
Stephen P Higgins
Center for Cell Biology and Cancer Research
Albany Medical College
47 New Scotland Avenue12208AlbanyNYUSA
Paul J Higgins
Center for Cell Biology and Cancer Research
Albany Medical College
47 New Scotland Avenue12208AlbanyNYUSA
TGF-β1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy
Journal of Oncology
Hindawi Publishing Corporation6200910.1155/2009/342391Received 29 November 2008; Accepted 30 January 2009 Recommended by Daniel ChuaReview Article Correspondence should be addressed to Paul J. Higgins,
Increased transforming growth factor-β (TGF-β) expression and epidermal growth factor receptor (EGFR) amplification accompany the emergence of highly aggressive human carcinomas. Cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors (i.e., proteases, protease inhibitors) that, in turn, regulate tumor invasion, neo-angiogenesis and inflammation. ranscript profiling of transformed human cells revealed that genes encoding wound healing, matrix remodeling and cell cycle proteins (i.e., the "tissue repair" transcriptome) are significantly upregulated early after growth factor stimulation. The major inhibitor of plasmin generation, plasminogen activator inhibitor-1 (PAI-1), is among the most highly induced transcripts during the phenotypic transition initiated by TGF-β maximal expression requires EGFR signaling. PAI-1 induction occurs early in the progression of incipient epidermal squamous cell carcinoma (SCC) and is a significant indicator of poor prognosis in epithelial malignancies. Mouse modeling and molecular genetic analysis of complex systems indicates that PAI-1 regulates the temporal/spatial control of pericellular proteolysis, promotes epithelial plasticity, inhibits capillary regression and facilitates stromal invasion. Defining TGF-β1-initiated signaling events that cooperate with an activated EGFR to impact the protease-protease inhibitor balance in the tumor microenvironment is critical to the development of novel therapies for the clinical management of human cancers.
Introduction
Transition of a normal epithelial cell to an early malignant phenotype often involves mutation of the p53 and p21 ras genes and progressive increases in autocrine TGF-β1 expression [1][2][3][4][5][6][7][8][9][10]. Elevated TGF-β1 production, in fact, typifies advanced pathologies in both mouse and human SCC [8,10,11]. Despite relatively high concentrations of TGF-β in the immediate tumor microenvironment, some malignant epithelial cells become refractory to TGF-β1initiated proliferative arrest likely due to reductions in either TGF-βRII and/or SMAD4 levels as well as the now recognized p21 ras -dependent antagonism of TGF-β1mediated growth inhibition/apoptosis [10][11][12][13]. In certain epithelial malignancies, moreover, resistance to TGF-β1mediated growth suppression is often coupled with EGFR amplification or dysregulated EGFR signaling, particularly during the later stages of tumor development [14][15][16][17][18][19]. The associated reprogramming of gene expression initiates and perpetuates TGF-β1-induced cellular "plasticity" (usually referred to as epithelial-to-mesenchymal transition or EMT) which facilites tumor invasion and metastasis [8,[20][21][22][23][24][25].
Microarray of the EMT transcriptome in several clinically relevant model systems has provided insights into the specific repertoire of "plasticity" genes. Plasminogen activator inhibitor type-1 (PAI-1; SERPINE1), the major physiologic regulator of the pericellular plasmin-generating cascade, is a prominent member of the subset of TGF-β1-induced, EMT-associated genes in human malignant keratinocytes [21,26,27]. In epithelial cells undergoing a mesenchymallike conversion in response to the E-cadherin transcriptional repressors Snail, Slug or E47, PAI-1 upregulation appears to be an essential characteristic of the plastic phenotype [28]. The association between PAI-1 expression and tumor 2 Journal of Oncology "progression" has significant clinical implications. Current data suggest that this serine protease inhibitor maintains an angiogenic "scaffold," stabilizes nascent capillary vessel structure, and facilitates tumor cell invasion through precise control of the peritumor proteolytic microenvironment [29][30][31]. Increased PAI-1 expression is, in fact, an early event in the progression of epidermal SCC, often localizing to tumor cells and myofibroblasts at the invasive front [24,[32][33][34][35][36] and, most importantly, is a biomarker with significant prognostic value [37]. Indeed, two of the best-validated prognostic indicators (level of evidence [LOE] = 1) in breast carcinoma are the serine protease urokinase plasminogen activator (uPA) and its endogenous inhibitor PAI-1 [38]. Certain PAI-1 tumor thresholds predict both poor prognosis and reduced disease-free survival in patients with breast, lung, ovarian, and oral SCC [29,38] with the expression amplitude frequently associated with the 4G polymorphism at the PE1 E box motif in the PAI-1 promoter [37]. Identification of PAI-1 in tumor-proximal stromal myofibroblasts, furthermore, implies a more global involvement in modulating cellular invasive potential [34][35][36], perhaps as a matricellular effector of epithelial motility [39], invasion and the associated angiogenic response [24,30,31,40,41].
Recent findings clearly implicate EGFR/MEK/rho-ROCK signaling as required for PAI-1 expression in TGF-β1stimulated cells. E box motifs (CACGTG) in the PAI-1 PE1/PE2 promoter regions, moreover, are platforms for a MAP kinase-directed upstream stimulatory factor (USF) subtype switch (USF-1 → USF-2) in response to growth factor addition [42][43][44] suggesting that the EGFR/MEK/rho-ROCK axis impacts PAI-1 expression through USFdependent transcriptional controls. The continued definition of TGF-β1-activated pathways that influence expression of this important target gene may lead to therapeutically useful approaches to manage human cancer. This paper, therefore, reviews data regarding the rapid transactivation of the EGFR in TGF-β1-stimulated cells suggesting cooperativity between TGF-β1 and EGFR → MAP kinase pathways in PAI-1 gene expression.
EGFR Signaling Is Required for TGF-β1-Induced PAI-1 Expression
TGF-β1 mobilizes both SMAD-dependent and -independent signaling [45] although the individual roles of specific crosspathway events on PAI-1 expression are not well understood. Several recent studies demonstrated that TGF-β1induced rapid EGFR transactivation highlighting cooperativity between TGF-β1 and EGFR signaling events in vascular, epithelial, and endothelial cells. Indeed, PAI-1 induction in response to TGF-β1 is significantly attenuated by an EGFR pharmacologic inhibitor (AG1478), by molecular targeting of EGFR activity (i.e., by adenoviral delivery of EGFR Y721A kinase-dead constructs) and, more importantly, by genetic ablation of the EGFR in mouse fibroblasts [43,46,47] with PAI-1 "rescue" evident in EGFR −/− cells engineered to express an EGFR construct. TGF-β1 treatment, moreover, specifically increased EGFR phosphorylation at the Y845 src-target residue; either mutation of this residue (EGFR Y845F ) or transfection of a DN pp60 c-src construct completely blocked TGF-β1-dependent PAI-1 induction. Similarly, TGF-β1 failed to stimulate PAI-1 expression in cultured mouse embryonic fibroblasts (MEFs) genetically deficient in three src family kinases (i.e., c-src, c-yes-, cfyn-null fibroblasts; SYF −/−/− ) compared to identically stimulated wild-type SYF +/+/+ cells. PAI-1 synthesis was restored in SYF −/−/− MEFs engineered to re-express a wild-type pp60 c-src [47] providing proof-of-principle for involvement of this particular src kinase in the inductive response. The highly specific src family kinase inhibitor SU6656, morevover, effectively blocked TGF-β1-initiated increases in both pp60 c-src and EGFR phosphorylation as well as pp60 c-src and EGFR activation (at the Y416 and Y845 residues, resp.). pEGFR Y845 phosphorylation in response to TGF-β1 was evident, furthermore, in wild type but not SYF −/−/− fibroblasts. The TGF-β1-dependent formation of EGFR/pp60 c-src complexes [46] and EGFR Y845 phosphorylation and the inhibition of TGF-β1-(but not PDGF-) induced PAI-1 expression by the EGFR Y845F mutant as well as a DN-Src construct [47] collectively implicate EGFR/pp60 c-src interactions and, in particular, the EGFR Y845 pp60 c-src site in the kinase domain activation loop in signal propagation [48]. The time course of TGF-β1-initiated SMAD2/3 activation, in contrast, was similar in both wild type and SYF −/−/− MEFs confirming that, in the context of either EGFR or src family kinase deficiency, SMAD2/3 activation occurs but is not sufficient for PAI-1 induction. TGF-β1 stimulated ERK1/2 phosphorylation in EGFR +/+ but not in EGFR −/− cells consistent with prior observations that TGF-β1-dependent ERK1/2 activation is downstream of EGFR signaling [43,46]. EGFR −/− MEFs, however, are fully capable of responding to exogenous TGF-β1 as SMAD2 was effectively activated (i.e., phosphorylated) in both wild type and EGFR −/− fibroblasts [47].
The PAI-1 Gene Is a Model of TGF-β1-Initiated Cooperative EGFR Signaling
While TGF-β1 receptors phosphorylate SMADs downstream of growth factor engagement, it appears that the Rho/ROCK pathway modulates the duration of SMAD2/3 phosphorylation [47]. How Rho/ROCK impact TGF-β1-initiated SMAD2/3 activation and subcellular localization [49,50] is not known but this pathway may function to provide efficient SMAD2/3 activation for extended periods. Alternatively, Rho/ROCK signaling may be required to inhibit negative regulation of SMAD2/3 function by inactivation of SMAD phosphatases sustaining, thereby, SMAD2/3 transcriptional actions (e.g., [51,52]). TGF-β1-induced SMAD2 phosphorylation is not altered by EGFR blockade either pharmacologically (with AG1478), molecularly (by expression of EGFR Y721A or EGFR Y845F ), or by the genetic absence of EGFR [47]. Clearly, while SMAD2/3 activation may be necessary it is not sufficient for TGF-β1-stimulated PAI-1 expression in the absence of EGFR signaling. Figure 1: Model for TGF-β1-induced PAI-1 expression. TGF-β1 activates two distinct signaling pathways to initiate PAI-1 transcription. Rho/ROCK are required to maintain SMAD phosphorylation and ERK activation (through to be defined mechanisms) while the pp60 c-srcactivated EGFR (at the Y845 site) signals to MEK-ERK initiating ERK/USF interactions resulting in USF phosphorylation and a subtype (USF-1 → USF-2) switch (e.g., [44]) at the PAI-1 PE1/PE2 E box sites. Collectively, these promoter-level events stimulate high level PAI-1 expression in response to TGF-βR occupancy. The actual mechanism underlying EGFR activation in response to TGF-β1 may involve direct recruitment of src kinases to the EGFR or the processing/release of a membrane-anchored EGFR ligand (e.g., HB-EGF). Events associated with TGF-β1 stimulation of the RhoA/ROCK pathway are similarly unclear. Rho/ROCK may regulate the activity and/or function of the SMAD phosphatase PPM1A impacting, thereby, the duration of SMAD-dependent transcription of target genes such as PAI-1. (modified from [47]).
It is apparent, therefore, that TGF-β1 stimulates PAI-1 expression through two distinct but cooperating pathways that involve EGFR/pp60 c-src → MEK/ERK signaling and EGFR-independent, but Rho/ROCK-modulated, TGF-βRdirected SMAD and ERK activation [47]. Interference with any of the specific individual elements in this dual cascade (EGFR/pp60 c-src /MEK or Rho/p160ROCK) markedly reduced, and in some cases, completely inhibited PAI-1 expression. One model consistent with the available data [24,40,43,44,47,53] suggests that SMADs and specific MAP kinase-targeted bHLH-LZ factors (such as USF) occupy their separate binding motifs at the critical TGF-β1-responsive PE2 region E box in the PAI-1 promoter (Figure 1). Dominant-negative interference with USF DNAbinding ability significantly reduced TGF-β1-mediated PAI-1 transcription [43,44,53]. Since MAP kinases regulate the DNA-binding and transcriptional activites of USF [40,43], TGF-βR signaling through SMAD2/3 may actually cooperate with EGFR/MEK-ERK-activated USF to attain high level PAI-1 expression [40,47]. SMADs are known to interact with E box-binding HLH-LZ factors such as TFE3 at the PE2 site in the PAI-1 geneat least in one cell type [54]. There is evidence, in fact, to suggest that such interacting complexes impact PAI-1 gene control since USF occupancy of the PAI-1 PE2 region E box site, which is juxtaposed to three SMADrecognition elements, modulates transcription in response to TGF-β1 or serum [40,43,44,53]. Current data indicate that recruitment of this multicomponent complex likely requires participation of the TGF-β1-stimulated EGFR → MEK/ERK and Rho/ROCK pathways for the optimal response of the PAI-1 gene to TGF-β1.
The mechanism of MAP kinase activation in TGF-β1-stimulated cells is just becoming clear. Upon ligand binding, the TGF-βRII undergoes autophosphorylation on three tyrosines (Y259, Y336, Y424), while Y284 is a target site for src kinases [55]. TGF-βRI is also subject to tyrosine phosphorylation postreceptor accupancy [56]. Such phosphorylated tyrosine residues provide docking sites for recruitment of Grb2/Shc/SOS complexes with subsequent mobilization of the ras-raf -MEK-ERK cascade [46,47,55]. Although ERKs are prominently activated in response to TGF-β1 [40,43], perhaps the JNK and p38 MAP kinase pathways are better characterized targets of TGF-β1-initiated signaling. TGF-β1 rapidly activates JNK through MKK4 and p38 via MKK3/6 perhaps even in a cell type-specific fashion contributing to the mechanistic complexity of pathway crosstalk. Each of these kinase systems, moreover, has been implicated in a cell type-dependency of PAI-1 gene control [40,43,55]. Should such pathways prove uniquely or, at least, preferentially utilized in specific cellular lineages, they may provide tumor type-specific targets for intervention therapy.
EGFR as a Potential Therapeutic Target for Regulating PAI-1 Expression
Modulation of EGFR/HER1 signaling by specific receptor function (kinase domain) inhibitors or neutralizing antibodies against specific EGFR1 ligands (e.g., HB-EGF antibodies) can be an attractive therapeutic modality (particularly in the context of neoplastic diseases associated with elevated TGF-β1 levels). This strategy would likely impact not only PAI-1 suppression but has the potential to regulate other proinvasive target genes. There is, in fact, increasing evidence that TGF-β1-induced connective tissue growth factor and fibronectin expression similarly involve EGFR/HER1 cooperative pathways (Samarakoon and Higgins, unpublished data). Moreover, PAI-1 repression by EGFR signaling blockade may also suppress tumor angiogenesis consistent with the well-established role of PAI-1 as an inhibitor of endothelial apoptosis and neovessel regression [40]. Combinatorial targeting of PAI-1 function using established small molecule PAI-1 inhibitors and genetic-based PAI-1 expression attenuation [40] coupled with disruption of EGFR signaling (e.g., with cetuximab or erlotinib) may impact, therefore, both cancer invasion and the associated angiogenic response, particularly in the context of a TGF-β1rich tumor microenvironment.
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AcknowledgmentThis research is supported by NIH Grant GM57242 to PJH.
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"Increased transforming growth factor-β (TGF-β) expression and epidermal growth factor receptor (EGFR) amplification accompany the emergence of highly aggressive human carcinomas. Cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors (i.e., proteases, protease inhibitors) that, in turn, regulate tumor invasion, neo-angiogenesis and inflammation. ranscript profiling of transformed human cells revealed that genes encoding wound healing, matrix remodeling and cell cycle proteins (i.e., the \"tissue repair\" transcriptome) are significantly upregulated early after growth factor stimulation. The major inhibitor of plasmin generation, plasminogen activator inhibitor-1 (PAI-1), is among the most highly induced transcripts during the phenotypic transition initiated by TGF-β maximal expression requires EGFR signaling. PAI-1 induction occurs early in the progression of incipient epidermal squamous cell carcinoma (SCC) and is a significant indicator of poor prognosis in epithelial malignancies. Mouse modeling and molecular genetic analysis of complex systems indicates that PAI-1 regulates the temporal/spatial control of pericellular proteolysis, promotes epithelial plasticity, inhibits capillary regression and facilitates stromal invasion. Defining TGF-β1-initiated signaling events that cooperate with an activated EGFR to impact the protease-protease inhibitor balance in the tumor microenvironment is critical to the development of novel therapies for the clinical management of human cancers."
] | [
"Rohan Samarakoon \nCenter for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA\n",
"Craig E Higgins [email protected] \nCenter for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA\n",
"Stephen P Higgins \nCenter for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA\n",
"Paul J Higgins \nCenter for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA\n"
] | [
"Center for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA",
"Center for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA",
"Center for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA",
"Center for Cell Biology and Cancer Research\nAlbany Medical College\n47 New Scotland Avenue12208AlbanyNYUSA"
] | [
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"D Breitkreutz, ",
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"H.-J Stark, ",
"D R Roop, ",
"N E Fusenig, ",
"A Dlugosz, ",
"G Merlino, ",
"S H Yuspa, ",
"P Boukamp, ",
"P Boukamp, ",
"W Peter, ",
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"H Tsao, ",
"R J Akhurst, ",
"A Balmain, ",
"W Cui, ",
"D J Fowlis, ",
"S Bryson, ",
"G Portella, ",
"S A Cumming, ",
"J Liddell, ",
"R Derynck, ",
"R J Akhurst, ",
"A Balmain, ",
"M Oft, ",
"R J Akhurst, ",
"A Balmain, ",
"G Han, ",
"S.-L Lu, ",
"A G Li, ",
"M Kretzschmar, ",
"J Doody, ",
"I Timokhina, ",
"J Massagué, ",
"O Rho, ",
"L M Beltrán, ",
"I B Gimenez-Conti, ",
"J Digiovanni, ",
"S H Yuspa, ",
"P O-Charoenrat, ",
"P Rhys-Evans, ",
"H Modjtahedi, ",
"W Court, ",
"G Box, ",
"S Eccles, ",
"P O-Charoenrat, ",
"P H Rhys-Evans, ",
"D J Archer, ",
"S A Eccles, ",
"N Moghal, ",
"P W Sternberg, ",
"T Shimizu, ",
"H Izumi, ",
"A Oga, ",
"J Zavadil, ",
"E P Böttinger, ",
"J Zavadil, ",
"M Bitzer, ",
"D Liang, ",
"J P Thiery, ",
"J P Sleeman, ",
"H Peinado, ",
"M Quintanilla, ",
"A Cano, ",
"C E Wilkins-Port, ",
"C E Higgins, ",
"J Freytag, ",
"S P Higgins, ",
"J A Carlson, ",
"P J Higgins, ",
"C E Wilkins-Port, ",
"P J Higgins, ",
"L Qi, ",
"S P Higgins, ",
"Q Lu, ",
"S Akiyoshi, ",
"M Ishii, ",
"N Nemoto, ",
"M Kawabata, ",
"H Aburatani, ",
"K Miyazono, ",
"G Moreno-Bueno, ",
"E Cubillo, ",
"D Sarrió, ",
"P A Andreasen, ",
"L Kjøller, ",
"L Christensen, ",
"M J Duffy, ",
"K Bajou, ",
"V Masson, ",
"R D Gerard, ",
"K Bajou, ",
"A Noël, ",
"R D Gerard, ",
"Y.-J Chen, ",
"S.-C Lin, ",
"T Kao, ",
"P Lindberg, ",
"Å Larsson, ",
"B S Nielsen, ",
"M Illemann, ",
"U Hansen, ",
"H J Nielsen, ",
"B V Offersen, ",
"B S Nielsen, ",
"G Høyer-Hansen, ",
"C Robert, ",
"I Bolon, ",
"S Gazzeri, ",
"S Veyrenc, ",
"C Brambilla, ",
"E Brambilla, ",
"E Vairaktaris, ",
"C Yapijakis, ",
"Z Serefoglou, ",
"B Hundsdorfer, ",
"H.-F Zeilhofer, ",
"K P Bock, ",
"F Maquerlot, ",
"S Galiacy, ",
"M Malo, ",
"P J Higgins, ",
"C Maillard, ",
"M Jost, ",
"M U Rømer, ",
"M.-D Galibert, ",
"S Carreira, ",
"C R Goding, ",
"S M Kutz, ",
"C E Higgins, ",
"R Samarakoon, ",
"L Qi, ",
"R R Allen, ",
"Q Lu, ",
"C E Higgins, ",
"R Garone, ",
"L Staiano-Coico, ",
"P J Higgins, ",
"R Derynck, ",
"Y E Zhang, ",
"R Samarakoon, ",
"C E Higgins, ",
"S P Higgins, ",
"S M Kutz, ",
"P J Higgins, ",
"R Samarakoon, ",
"S P Higgins, ",
"C E Higgins, ",
"P J Higgins, ",
"R Ishizawar, ",
"S J Parsons, ",
"S Chen, ",
"M Crawford, ",
"R M Day, ",
"T Kita, ",
"Y Hata, ",
"K Kano, ",
"S Itoh, ",
"P Ten Dijke, ",
"X Lin, ",
"X Duan, ",
"Y.-Y Liang, ",
"R R Allen, ",
"L Qi, ",
"P J Higgins, ",
"X Hua, ",
"Z A Miller, ",
"G Wu, ",
"Y Shi, ",
"H F Lodish, ",
"Y E Zhang, ",
"M K Lee, ",
"C Pardoux, ",
"M C Hall, "
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] | [
"Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line. D Breitkreutz, P Boukamp, C M Ryle, H.-J Stark, D R Roop, N E Fusenig, Cancer Research. 5116D. Breitkreutz, P. Boukamp, C. M. Ryle, H.-J. Stark, D. R. Roop, and N. E. Fusenig, \"Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line,\" Cancer Research, vol. 51, no. 16, pp. 4402-4409, 1991.",
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"Metastasis is driven by sequental elevation of H-ras and Smad2 levels. M Oft, R J Akhurst, A Balmain, Nature Cell Biology. 47M. Oft, R. J. Akhurst, and A. Balmain, \"Metastasis is driven by sequental elevation of H-ras and Smad2 levels,\" Nature Cell Biology, vol. 4, no. 7, pp. 487-494, 2002.",
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"Epidermal growth factor receptor overexpression and genetic aberrations in metastatic squamous-cell carcinoma of the skin. T Shimizu, H Izumi, A Oga, Dermatology. 2023T. Shimizu, H. Izumi, A. Oga, et al., \"Epidermal growth factor receptor overexpression and genetic aberrations in metastatic squamous-cell carcinoma of the skin,\" Dermatology, vol. 202, no. 3, pp. 203-206, 2001.",
"TGF-β and epithelial-tomesenchymal transitions. J Zavadil, E P Böttinger, Oncogene. 2437J. Zavadil and E. P. Böttinger, \"TGF-β and epithelial-to- mesenchymal transitions,\" Oncogene, vol. 24, no. 37, pp. 5764- 5774, 2005.",
"Genetic programs of epithelial cell plasticity directed by transforming growth factor-β. J Zavadil, M Bitzer, D Liang, Proceedings of the National Academy of Sciences of the United States of America. the National Academy of Sciences of the United States of America98J. Zavadil, M. Bitzer, D. Liang, et al., \"Genetic programs of epithelial cell plasticity directed by transforming growth factor-β,\" Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 12, pp. 6686-6691, 2001.",
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"PAI-1 is a critical upstream regulator of the TGF-β1/EGF-induced invasive phenotype in mutant p53 human cutaneous squamous cell carcinoma. C E Wilkins-Port, C E Higgins, J Freytag, S P Higgins, J A Carlson, P J Higgins, Journal of Biomedicine and Biotechnology. Article ID 85208, 8 pagesC. E. Wilkins-Port, C. E. Higgins, J. Freytag, S. P. Higgins, J. A. Carlson, and P. J. Higgins, \"PAI-1 is a critical upstream regulator of the TGF-β1/EGF-induced invasive phenotype in mutant p53 human cutaneous squamous cell carcinoma,\" Journal of Biomedicine and Biotechnology, vol. 2007, Article ID 85208, 8 pages, 2007.",
"Regulation of extracellular matrix remodeling following transforming growth factor-β1/epidermal growth factor-stimulated epithelial-mesenchymal transition in human premalignant keratinocytes. C E Wilkins-Port, P J Higgins, Cells Tissues Organs. 1851-3C. E. Wilkins-Port and P. J. Higgins, \"Regulation of extracellular matrix remodeling following transforming growth factor-β1/epidermal growth factor-stimulated epithelial-mesenchymal transition in human premalignant keratinocytes,\" Cells Tissues Organs, vol. 185, no. 1-3, pp. 116-122, 2007.",
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"Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for snail, slug, and E47 Factors in epithelial-mesenchymal transition. G Moreno-Bueno, E Cubillo, D Sarrió, Cancer Research. 6619G. Moreno-Bueno, E. Cubillo, D. Sarrió, et al., \"Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for snail, slug, and E47 Factors in epithelial-mesenchymal transition,\" Cancer Research, vol. 66, no. 19, pp. 9543-9556, 2006.",
"The urokinase-type plasminogen activator system in cancer metastasis: a review. P A Andreasen, L Kjøller, L Christensen, M J Duffy, International Journal of Cancer. 721P. A. Andreasen, L. Kjøller, L. Christensen, and M. J. Duffy, \"The urokinase-type plasminogen activator system in cancer metastasis: a review,\" International Journal of Cancer, vol. 72, no. 1, pp. 1-22, 1997.",
"The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin: implications for antiangiogenic strategies. K Bajou, V Masson, R D Gerard, Journal of Cell Biology. 1524K. Bajou, V. Masson, R. D. Gerard, et al., \"The plas- minogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin: implications for antiangiogenic strategies,\" Journal of Cell Biology, vol. 152, no. 4, pp. 777-784, 2001.",
"Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. K Bajou, A Noël, R D Gerard, Nature Medicine. 48K. Bajou, A. Noël, R. D. Gerard, et al., \"Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization,\" Nature Medicine, vol. 4, no. 8, pp. 923- 928, 1998.",
"Genome-wide profiling of oral squamous cell carcinoma. Y.-J Chen, S.-C Lin, T Kao, The Journal of Pathology. 2043Y.-J. Chen, S.-C. Lin, T. Kao, et al., \"Genome-wide profiling of oral squamous cell carcinoma,\" The Journal of Pathology, vol. 204, no. 3, pp. 326-332, 2004.",
"Expression of plasminogen activator inhibitor-1, urokinase receptor and laminin γ-2 chain is an early coordinated event in incipient oral squamous cell carcinoma. P Lindberg, Å Larsson, B S Nielsen, International Journal of Cancer. 11812P. Lindberg,Å. Larsson, and B. S. Nielsen, \"Expression of plas- minogen activator inhibitor-1, urokinase receptor and laminin γ-2 chain is an early coordinated event in incipient oral squamous cell carcinoma,\" International Journal of Cancer, vol. 118, no. 12, pp. 2948-2956, 2006.",
"Leading-edge myofibroblasts in human colon cancer express plasminogen activator inhibitor-1. M Illemann, U Hansen, H J Nielsen, American Journal of Clinical Pathology. 1222M. Illemann, U. Hansen, H. J. Nielsen, et al., \"Leading-edge myofibroblasts in human colon cancer express plasminogen activator inhibitor-1,\" American Journal of Clinical Pathology, vol. 122, no. 2, pp. 256-265, 2004.",
"The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas. B V Offersen, B S Nielsen, G Høyer-Hansen, American Journal of Pathology. 1635B. V. Offersen, B. S. Nielsen, G. Høyer-Hansen, et al., \"The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas,\" American Journal of Pathology, vol. 163, no. 5, pp. 1887-1899, 2003.",
"Expression of plasminogen activator inhibitors 1 and 2 in lung cancer and their role in tumor progression. C Robert, I Bolon, S Gazzeri, S Veyrenc, C Brambilla, E Brambilla, Clinical Cancer Research. 58C. Robert, I. Bolon, S. Gazzeri, S. Veyrenc, C. Brambilla, and E. Brambilla, \"Expression of plasminogen activator inhibitors 1 and 2 in lung cancer and their role in tumor progression,\" Clinical Cancer Research, vol. 5, no. 8, pp. 2094-2102, 1999.",
"Plasminogen activator inhibitor-1 polymorphism is associated with increased risk for oral cancer. E Vairaktaris, C Yapijakis, Z Serefoglou, Oral Oncology. 429E. Vairaktaris, C. Yapijakis, Z. Serefoglou, et al., \"Plasmino- gen activator inhibitor-1 polymorphism is associated with increased risk for oral cancer,\" Oral Oncology, vol. 42, no. 9, pp. 888-892, 2006.",
"Tumourassociated urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity-clinical relevance and prognostic value. B Hundsdorfer, H.-F Zeilhofer, K P Bock, Journal of Cranio-Maxillofacial Surgery. 333B. Hundsdorfer, H.-F. Zeilhofer, K. P. Bock, et al., \"Tumour- associated urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity-clinical relevance and prognostic value,\" Journal of Cranio-Maxillofacial Surgery, vol. 33, no. 3, pp. 191-196, 2005.",
"Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing. F Maquerlot, S Galiacy, M Malo, American Journal of Pathology. 1695F. Maquerlot, S. Galiacy, M. Malo, et al., \"Dual role for plasminogen activator inhibitor type 1 as soluble and as matri- cellular regulator of epithelial alveolar cell wound healing,\" American Journal of Pathology, vol. 169, no. 5, pp. 1624-1632, 2006.",
"TGF-β1-stimulated p21 ras -ERK signaling regulates expression of the angiogenic SERPIN PAI-1. P J Higgins, Recent Research Developments in Biochemistry. 7P. J. Higgins, \"TGF-β1-stimulated p21 ras -ERK signaling reg- ulates expression of the angiogenic SERPIN PAI-1,\" Recent Research Developments in Biochemistry, vol. 7, pp. 31-45, 2006.",
"Host plasminogen activator inhibitor-1 promotes human skin carcinoma progression in a stage-dependent manner. C Maillard, M Jost, M U Rømer, Neoplasia. 71C. Maillard, M. Jost, M. U. Rømer, et al., \"Host plasminogen activator inhibitor-1 promotes human skin carcinoma pro- gression in a stage-dependent manner,\" Neoplasia, vol. 7, no. 1, pp. 57-66, 2005.",
"The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression. M.-D Galibert, S Carreira, C R Goding, The EMBO Journal. 2017M.-D. Galibert, S. Carreira, and C. R. Goding, \"The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression,\" The EMBO Journal, vol. 20, no. 17, pp. 5022-5031, 2001.",
"TGF-β1-induced PAI-1 expression is e box/USF-dependent and requires EGFR signaling. S M Kutz, C E Higgins, R Samarakoon, Experimental Cell Research. 3127S. M. Kutz, C. E. Higgins, R. Samarakoon, et al., \"TGF- β1-induced PAI-1 expression is e box/USF-dependent and requires EGFR signaling,\" Experimental Cell Research, vol. 312, no. 7, pp. 1093-1105, 2006.",
"PAI-1 transcriptional regulation during the G 0 → G 1 transition in human epidermal keratinocytes. L Qi, R R Allen, Q Lu, C E Higgins, R Garone, L Staiano-Coico, P J Higgins, Journal of Cellular Biochemistry. 992L. Qi, R. R. Allen, Q. Lu, C. E. Higgins, R. Garone, L. Staiano- Coico, and P. J. Higgins, \"PAI-1 transcriptional regulation during the G 0 → G 1 transition in human epidermal keratinocytes,\" Journal of Cellular Biochemistry, vol. 99, no. 2, pp. 495-507, 2006.",
"Smad-dependent and Smadindependent pathways in TGF-β family signalling. R Derynck, Y E Zhang, Nature. 4256958R. Derynck and Y. E. Zhang, \"Smad-dependent and Smad- independent pathways in TGF-β family signalling,\" Nature, vol. 425, no. 6958, pp. 577-584, 2003.",
"Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-β1-stimulated smooth muscle cells is pp60 c−src /MEK-dependent. R Samarakoon, C E Higgins, S P Higgins, S M Kutz, P J Higgins, Journal of Cellular Physiology. 2041R. Samarakoon, C. E. Higgins, S. P. Higgins, S. M. Kutz, and P. J. Higgins, \"Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-β1-stimulated smooth muscle cells is pp60 c−src /MEK-dependent,\" Journal of Cellular Physiology, vol. 204, no. 1, pp. 236-246, 2005.",
"TGF-β1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60 c−src /EGFR Y 845 and Rho/ROCK signaling. R Samarakoon, S P Higgins, C E Higgins, P J Higgins, Journal of Molecular and Cellular Cardiology. 443R. Samarakoon, S. P. Higgins, C. E. Higgins, and P. J. Higgins, \"TGF-β1-induced plasminogen activator inhibitor- 1 expression in vascular smooth muscle cells requires pp60 c−src /EGFR Y 845 and Rho/ROCK signaling,\" Journal of Molecular and Cellular Cardiology, vol. 44, no. 3, pp. 527-538, 2008.",
"C-Src and cooperating partners in human cancer. R Ishizawar, S J Parsons, Cancer Cell. 63R. Ishizawar and S. J. Parsons, \"C-Src and cooperating partners in human cancer,\" Cancer Cell, vol. 6, no. 3, pp. 209-214, 2004.",
"RhoA modulates Smad signaling during transforming growth factor-β-induced smooth muscle differentiation. S Chen, M Crawford, R M Day, The Journal of Biological Chemistry. 2813S. Chen, M. Crawford, R. M. Day, et al., \"RhoA modulates Smad signaling during transforming growth factor-β-induced smooth muscle differentiation,\" The Journal of Biological Chemistry, vol. 281, no. 3, pp. 1765-1770, 2006.",
"Transforming growth factor-β2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor. T Kita, Y Hata, K Kano, Diabetes. 561T. Kita, Y. Hata, K. Kano, et al., \"Transforming growth factor-β2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor,\" Diabetes, vol. 56, no. 1, pp. 231-238, 2007.",
"Negative regulation of TGF-β receptor/Smad signal transduction. S Itoh, P Ten Dijke, Current Opinion in Cell Biology. 192S. Itoh and P. ten Dijke, \"Negative regulation of TGF-β receptor/Smad signal transduction,\" Current Opinion in Cell Biology, vol. 19, no. 2, pp. 176-184, 2007.",
"PPM1A functions as a Smad phosphatase to terminate TGFβ signaling. X Lin, X Duan, Y.-Y Liang, Cell. 1255X. Lin, X. Duan, Y.-Y. Liang, et al., \" PPM1A functions as a Smad phosphatase to terminate TGFβ signaling,\" Cell, vol. 125, no. 5, pp. 915-928, 2006.",
"Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes. R R Allen, L Qi, P J Higgins, Journal of Cellular Physiology. 2031Journal of OncologyR. R. Allen, L. Qi, and P. J. Higgins, \"Upstream stimulatory fac- tor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes,\" Journal of Cellular Physiology, vol. 203, no. 1, pp. 156-165, 2005. 6 Journal of Oncology",
"Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor μE3, and Smad proteins. X Hua, Z A Miller, G Wu, Y Shi, H F Lodish, Proceedings of the National Academy of Sciences of the United States of America. 9623X. Hua, Z. A. Miller, G. Wu, Y. Shi, and H. F. Lodish, \"Speci- ficity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor μE3, and Smad proteins,\" Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 23, pp. 13130-13135, 1999.",
"Non-Smad pathways in TGF-β signaling. Y E Zhang, Cell Research. 191Y. E. Zhang, \"Non-Smad pathways in TGF-β signaling,\" Cell Research, vol. 19, no. 1, pp. 128-139, 2009.",
"TGF-β activates Erk MAP kinase signalling through direct phsophorylation of ShcA. M K Lee, C Pardoux, M C Hall, The EMBO Journal. 26M. K. Lee, C. Pardoux, M. C. Hall, et al., \"TGF-β activates Erk MAP kinase signalling through direct phsophorylation of ShcA,\" The EMBO Journal, vol. 26, pp. 3957-3967, 2007."
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"Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line",
"Progress in cutaneous cancer research",
"UV-induced skin cancer: similarities-variations",
"Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, rasH oncogene activation and additional chromosome loss",
"Squamous cell carcinoma: from precursor lesions to high-risk variants",
"The genetics of skin cancer",
"Genetic events and the role of TGFβ in epithelial tumour progression",
"TGFβ1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice",
"Transforming growth factor β is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion",
"TGF-β signaling in tumor suppression and cancer progression",
"Metastasis is driven by sequental elevation of H-ras and Smad2 levels",
"Distinct mechanisms of TGF-β1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis",
"A mechanism of repression of TGFfβ/Smad signaling by oncogenic Ras",
"Altered expression of the epidermal growth factor receptor and transforming growth factor-α during multistage skin carcinogenesis in SENCAR mice",
"The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis",
"Overexpression of epidermal growth factor receptor in human head and neck squamous carcinoma cell lines correlates with matrix metalloproteinase-9 expression and in vitro invasion",
"C-erbB receptors in squamous cell carcinomas of the head and neck: clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors",
"Multiple positive and negative regulators of signaling by the EGF-receptor",
"Epidermal growth factor receptor overexpression and genetic aberrations in metastatic squamous-cell carcinoma of the skin",
"TGF-β and epithelial-tomesenchymal transitions",
"Genetic programs of epithelial cell plasticity directed by transforming growth factor-β",
"Complex networks orchestrate epithelial-mesenchymal transitions",
"Transforming growth factor β-1 induces snail transcription factor in epithelial cell lines. Mechanisms for epithelial mesenchymal transitions",
"PAI-1 is a critical upstream regulator of the TGF-β1/EGF-induced invasive phenotype in mutant p53 human cutaneous squamous cell carcinoma",
"Regulation of extracellular matrix remodeling following transforming growth factor-β1/epidermal growth factor-stimulated epithelial-mesenchymal transition in human premalignant keratinocytes",
"SERPINE1 (PAI-1) is a prominent member of the early G 0 → G 1 transition \"wound repair\" transcriptome in p53 mutant human keratinocytes",
"Targets of transcriptional regulation by transforming growth factor-β: expression profile analysis using oligonucleotide arrays",
"Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for snail, slug, and E47 Factors in epithelial-mesenchymal transition",
"The urokinase-type plasminogen activator system in cancer metastasis: a review",
"The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin: implications for antiangiogenic strategies",
"Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization",
"Genome-wide profiling of oral squamous cell carcinoma",
"Expression of plasminogen activator inhibitor-1, urokinase receptor and laminin γ-2 chain is an early coordinated event in incipient oral squamous cell carcinoma",
"Leading-edge myofibroblasts in human colon cancer express plasminogen activator inhibitor-1",
"The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas",
"Expression of plasminogen activator inhibitors 1 and 2 in lung cancer and their role in tumor progression",
"Plasminogen activator inhibitor-1 polymorphism is associated with increased risk for oral cancer",
"Tumourassociated urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity-clinical relevance and prognostic value",
"Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing",
"TGF-β1-stimulated p21 ras -ERK signaling regulates expression of the angiogenic SERPIN PAI-1",
"Host plasminogen activator inhibitor-1 promotes human skin carcinoma progression in a stage-dependent manner",
"The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression",
"TGF-β1-induced PAI-1 expression is e box/USF-dependent and requires EGFR signaling",
"PAI-1 transcriptional regulation during the G 0 → G 1 transition in human epidermal keratinocytes",
"Smad-dependent and Smadindependent pathways in TGF-β family signalling",
"Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-β1-stimulated smooth muscle cells is pp60 c−src /MEK-dependent",
"TGF-β1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60 c−src /EGFR Y 845 and Rho/ROCK signaling",
"C-Src and cooperating partners in human cancer",
"RhoA modulates Smad signaling during transforming growth factor-β-induced smooth muscle differentiation",
"Transforming growth factor-β2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor",
"Negative regulation of TGF-β receptor/Smad signal transduction",
"PPM1A functions as a Smad phosphatase to terminate TGFβ signaling",
"Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes",
"Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor μE3, and Smad proteins",
"Non-Smad pathways in TGF-β signaling",
"TGF-β activates Erk MAP kinase signalling through direct phsophorylation of ShcA"
] | [
"the National Academy of Sciences of the United States of America",
"Cancer Research",
"Journal of Investigative Dermatology Symposium Proceedings",
"Journal der Deutschen Dermatologischen Gesellschaft",
"Oncogene",
"Modern Pathology",
"American Journal of Medical Genetics Part C",
"The Journal of Pathology",
"Cell",
"Cell Growth & Differentiation",
"Nature Genetics",
"Nature Cell Biology",
"The Journal of Clinical Investigation",
"Genes & Development",
"Molecular Carcinogenesis",
"Journal of Dermatological Science",
"International Journal of Cancer",
"Oral Oncology",
"Current Opinion in Cell Biology",
"Dermatology",
"Oncogene",
"Proceedings of the National Academy of Sciences of the United States of America",
"Nature Reviews Molecular Cell Biology",
"The Journal of Biological Chemistry",
"Journal of Biomedicine and Biotechnology",
"Cells Tissues Organs",
"Journal of Investigative Dermatology",
"Japanese Journal of Cancer Research",
"Cancer Research",
"International Journal of Cancer",
"Journal of Cell Biology",
"Nature Medicine",
"The Journal of Pathology",
"International Journal of Cancer",
"American Journal of Clinical Pathology",
"American Journal of Pathology",
"Clinical Cancer Research",
"Oral Oncology",
"Journal of Cranio-Maxillofacial Surgery",
"American Journal of Pathology",
"Recent Research Developments in Biochemistry",
"Neoplasia",
"The EMBO Journal",
"Experimental Cell Research",
"Journal of Cellular Biochemistry",
"Nature",
"Journal of Cellular Physiology",
"Journal of Molecular and Cellular Cardiology",
"Cancer Cell",
"The Journal of Biological Chemistry",
"Diabetes",
"Current Opinion in Cell Biology",
"Cell",
"Journal of Cellular Physiology",
"Proceedings of the National Academy of Sciences of the United States of America",
"Cell Research",
"The EMBO Journal"
] | [
"\n?\n"
] | [] | [
"Figure 1",
"(Figure 1"
] | [] | [
"Transition of a normal epithelial cell to an early malignant phenotype often involves mutation of the p53 and p21 ras genes and progressive increases in autocrine TGF-β1 expression [1][2][3][4][5][6][7][8][9][10]. Elevated TGF-β1 production, in fact, typifies advanced pathologies in both mouse and human SCC [8,10,11]. Despite relatively high concentrations of TGF-β in the immediate tumor microenvironment, some malignant epithelial cells become refractory to TGF-β1initiated proliferative arrest likely due to reductions in either TGF-βRII and/or SMAD4 levels as well as the now recognized p21 ras -dependent antagonism of TGF-β1mediated growth inhibition/apoptosis [10][11][12][13]. In certain epithelial malignancies, moreover, resistance to TGF-β1mediated growth suppression is often coupled with EGFR amplification or dysregulated EGFR signaling, particularly during the later stages of tumor development [14][15][16][17][18][19]. The associated reprogramming of gene expression initiates and perpetuates TGF-β1-induced cellular \"plasticity\" (usually referred to as epithelial-to-mesenchymal transition or EMT) which facilites tumor invasion and metastasis [8,[20][21][22][23][24][25].",
"Microarray of the EMT transcriptome in several clinically relevant model systems has provided insights into the specific repertoire of \"plasticity\" genes. Plasminogen activator inhibitor type-1 (PAI-1; SERPINE1), the major physiologic regulator of the pericellular plasmin-generating cascade, is a prominent member of the subset of TGF-β1-induced, EMT-associated genes in human malignant keratinocytes [21,26,27]. In epithelial cells undergoing a mesenchymallike conversion in response to the E-cadherin transcriptional repressors Snail, Slug or E47, PAI-1 upregulation appears to be an essential characteristic of the plastic phenotype [28]. The association between PAI-1 expression and tumor 2 Journal of Oncology \"progression\" has significant clinical implications. Current data suggest that this serine protease inhibitor maintains an angiogenic \"scaffold,\" stabilizes nascent capillary vessel structure, and facilitates tumor cell invasion through precise control of the peritumor proteolytic microenvironment [29][30][31]. Increased PAI-1 expression is, in fact, an early event in the progression of epidermal SCC, often localizing to tumor cells and myofibroblasts at the invasive front [24,[32][33][34][35][36] and, most importantly, is a biomarker with significant prognostic value [37]. Indeed, two of the best-validated prognostic indicators (level of evidence [LOE] = 1) in breast carcinoma are the serine protease urokinase plasminogen activator (uPA) and its endogenous inhibitor PAI-1 [38]. Certain PAI-1 tumor thresholds predict both poor prognosis and reduced disease-free survival in patients with breast, lung, ovarian, and oral SCC [29,38] with the expression amplitude frequently associated with the 4G polymorphism at the PE1 E box motif in the PAI-1 promoter [37]. Identification of PAI-1 in tumor-proximal stromal myofibroblasts, furthermore, implies a more global involvement in modulating cellular invasive potential [34][35][36], perhaps as a matricellular effector of epithelial motility [39], invasion and the associated angiogenic response [24,30,31,40,41].",
"Recent findings clearly implicate EGFR/MEK/rho-ROCK signaling as required for PAI-1 expression in TGF-β1stimulated cells. E box motifs (CACGTG) in the PAI-1 PE1/PE2 promoter regions, moreover, are platforms for a MAP kinase-directed upstream stimulatory factor (USF) subtype switch (USF-1 → USF-2) in response to growth factor addition [42][43][44] suggesting that the EGFR/MEK/rho-ROCK axis impacts PAI-1 expression through USFdependent transcriptional controls. The continued definition of TGF-β1-activated pathways that influence expression of this important target gene may lead to therapeutically useful approaches to manage human cancer. This paper, therefore, reviews data regarding the rapid transactivation of the EGFR in TGF-β1-stimulated cells suggesting cooperativity between TGF-β1 and EGFR → MAP kinase pathways in PAI-1 gene expression.",
"TGF-β1 mobilizes both SMAD-dependent and -independent signaling [45] although the individual roles of specific crosspathway events on PAI-1 expression are not well understood. Several recent studies demonstrated that TGF-β1induced rapid EGFR transactivation highlighting cooperativity between TGF-β1 and EGFR signaling events in vascular, epithelial, and endothelial cells. Indeed, PAI-1 induction in response to TGF-β1 is significantly attenuated by an EGFR pharmacologic inhibitor (AG1478), by molecular targeting of EGFR activity (i.e., by adenoviral delivery of EGFR Y721A kinase-dead constructs) and, more importantly, by genetic ablation of the EGFR in mouse fibroblasts [43,46,47] with PAI-1 \"rescue\" evident in EGFR −/− cells engineered to express an EGFR construct. TGF-β1 treatment, moreover, specifically increased EGFR phosphorylation at the Y845 src-target residue; either mutation of this residue (EGFR Y845F ) or transfection of a DN pp60 c-src construct completely blocked TGF-β1-dependent PAI-1 induction. Similarly, TGF-β1 failed to stimulate PAI-1 expression in cultured mouse embryonic fibroblasts (MEFs) genetically deficient in three src family kinases (i.e., c-src, c-yes-, cfyn-null fibroblasts; SYF −/−/− ) compared to identically stimulated wild-type SYF +/+/+ cells. PAI-1 synthesis was restored in SYF −/−/− MEFs engineered to re-express a wild-type pp60 c-src [47] providing proof-of-principle for involvement of this particular src kinase in the inductive response. The highly specific src family kinase inhibitor SU6656, morevover, effectively blocked TGF-β1-initiated increases in both pp60 c-src and EGFR phosphorylation as well as pp60 c-src and EGFR activation (at the Y416 and Y845 residues, resp.). pEGFR Y845 phosphorylation in response to TGF-β1 was evident, furthermore, in wild type but not SYF −/−/− fibroblasts. The TGF-β1-dependent formation of EGFR/pp60 c-src complexes [46] and EGFR Y845 phosphorylation and the inhibition of TGF-β1-(but not PDGF-) induced PAI-1 expression by the EGFR Y845F mutant as well as a DN-Src construct [47] collectively implicate EGFR/pp60 c-src interactions and, in particular, the EGFR Y845 pp60 c-src site in the kinase domain activation loop in signal propagation [48]. The time course of TGF-β1-initiated SMAD2/3 activation, in contrast, was similar in both wild type and SYF −/−/− MEFs confirming that, in the context of either EGFR or src family kinase deficiency, SMAD2/3 activation occurs but is not sufficient for PAI-1 induction. TGF-β1 stimulated ERK1/2 phosphorylation in EGFR +/+ but not in EGFR −/− cells consistent with prior observations that TGF-β1-dependent ERK1/2 activation is downstream of EGFR signaling [43,46]. EGFR −/− MEFs, however, are fully capable of responding to exogenous TGF-β1 as SMAD2 was effectively activated (i.e., phosphorylated) in both wild type and EGFR −/− fibroblasts [47].",
"While TGF-β1 receptors phosphorylate SMADs downstream of growth factor engagement, it appears that the Rho/ROCK pathway modulates the duration of SMAD2/3 phosphorylation [47]. How Rho/ROCK impact TGF-β1-initiated SMAD2/3 activation and subcellular localization [49,50] is not known but this pathway may function to provide efficient SMAD2/3 activation for extended periods. Alternatively, Rho/ROCK signaling may be required to inhibit negative regulation of SMAD2/3 function by inactivation of SMAD phosphatases sustaining, thereby, SMAD2/3 transcriptional actions (e.g., [51,52]). TGF-β1-induced SMAD2 phosphorylation is not altered by EGFR blockade either pharmacologically (with AG1478), molecularly (by expression of EGFR Y721A or EGFR Y845F ), or by the genetic absence of EGFR [47]. Clearly, while SMAD2/3 activation may be necessary it is not sufficient for TGF-β1-stimulated PAI-1 expression in the absence of EGFR signaling. Figure 1: Model for TGF-β1-induced PAI-1 expression. TGF-β1 activates two distinct signaling pathways to initiate PAI-1 transcription. Rho/ROCK are required to maintain SMAD phosphorylation and ERK activation (through to be defined mechanisms) while the pp60 c-srcactivated EGFR (at the Y845 site) signals to MEK-ERK initiating ERK/USF interactions resulting in USF phosphorylation and a subtype (USF-1 → USF-2) switch (e.g., [44]) at the PAI-1 PE1/PE2 E box sites. Collectively, these promoter-level events stimulate high level PAI-1 expression in response to TGF-βR occupancy. The actual mechanism underlying EGFR activation in response to TGF-β1 may involve direct recruitment of src kinases to the EGFR or the processing/release of a membrane-anchored EGFR ligand (e.g., HB-EGF). Events associated with TGF-β1 stimulation of the RhoA/ROCK pathway are similarly unclear. Rho/ROCK may regulate the activity and/or function of the SMAD phosphatase PPM1A impacting, thereby, the duration of SMAD-dependent transcription of target genes such as PAI-1. (modified from [47]).",
"It is apparent, therefore, that TGF-β1 stimulates PAI-1 expression through two distinct but cooperating pathways that involve EGFR/pp60 c-src → MEK/ERK signaling and EGFR-independent, but Rho/ROCK-modulated, TGF-βRdirected SMAD and ERK activation [47]. Interference with any of the specific individual elements in this dual cascade (EGFR/pp60 c-src /MEK or Rho/p160ROCK) markedly reduced, and in some cases, completely inhibited PAI-1 expression. One model consistent with the available data [24,40,43,44,47,53] suggests that SMADs and specific MAP kinase-targeted bHLH-LZ factors (such as USF) occupy their separate binding motifs at the critical TGF-β1-responsive PE2 region E box in the PAI-1 promoter (Figure 1). Dominant-negative interference with USF DNAbinding ability significantly reduced TGF-β1-mediated PAI-1 transcription [43,44,53]. Since MAP kinases regulate the DNA-binding and transcriptional activites of USF [40,43], TGF-βR signaling through SMAD2/3 may actually cooperate with EGFR/MEK-ERK-activated USF to attain high level PAI-1 expression [40,47]. SMADs are known to interact with E box-binding HLH-LZ factors such as TFE3 at the PE2 site in the PAI-1 geneat least in one cell type [54]. There is evidence, in fact, to suggest that such interacting complexes impact PAI-1 gene control since USF occupancy of the PAI-1 PE2 region E box site, which is juxtaposed to three SMADrecognition elements, modulates transcription in response to TGF-β1 or serum [40,43,44,53]. Current data indicate that recruitment of this multicomponent complex likely requires participation of the TGF-β1-stimulated EGFR → MEK/ERK and Rho/ROCK pathways for the optimal response of the PAI-1 gene to TGF-β1.",
"The mechanism of MAP kinase activation in TGF-β1-stimulated cells is just becoming clear. Upon ligand binding, the TGF-βRII undergoes autophosphorylation on three tyrosines (Y259, Y336, Y424), while Y284 is a target site for src kinases [55]. TGF-βRI is also subject to tyrosine phosphorylation postreceptor accupancy [56]. Such phosphorylated tyrosine residues provide docking sites for recruitment of Grb2/Shc/SOS complexes with subsequent mobilization of the ras-raf -MEK-ERK cascade [46,47,55]. Although ERKs are prominently activated in response to TGF-β1 [40,43], perhaps the JNK and p38 MAP kinase pathways are better characterized targets of TGF-β1-initiated signaling. TGF-β1 rapidly activates JNK through MKK4 and p38 via MKK3/6 perhaps even in a cell type-specific fashion contributing to the mechanistic complexity of pathway crosstalk. Each of these kinase systems, moreover, has been implicated in a cell type-dependency of PAI-1 gene control [40,43,55]. Should such pathways prove uniquely or, at least, preferentially utilized in specific cellular lineages, they may provide tumor type-specific targets for intervention therapy.",
"Modulation of EGFR/HER1 signaling by specific receptor function (kinase domain) inhibitors or neutralizing antibodies against specific EGFR1 ligands (e.g., HB-EGF antibodies) can be an attractive therapeutic modality (particularly in the context of neoplastic diseases associated with elevated TGF-β1 levels). This strategy would likely impact not only PAI-1 suppression but has the potential to regulate other proinvasive target genes. There is, in fact, increasing evidence that TGF-β1-induced connective tissue growth factor and fibronectin expression similarly involve EGFR/HER1 cooperative pathways (Samarakoon and Higgins, unpublished data). Moreover, PAI-1 repression by EGFR signaling blockade may also suppress tumor angiogenesis consistent with the well-established role of PAI-1 as an inhibitor of endothelial apoptosis and neovessel regression [40]. Combinatorial targeting of PAI-1 function using established small molecule PAI-1 inhibitors and genetic-based PAI-1 expression attenuation [40] coupled with disruption of EGFR signaling (e.g., with cetuximab or erlotinib) may impact, therefore, both cancer invasion and the associated angiogenic response, particularly in the context of a TGF-β1rich tumor microenvironment."
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"EGFR Signaling Is Required for TGF-β1-Induced PAI-1 Expression",
"The PAI-1 Gene Is a Model of TGF-β1-Initiated Cooperative EGFR Signaling",
"EGFR as a Potential Therapeutic Target for Regulating PAI-1 Expression",
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"TGF-β1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy",
"TGF-β1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy"
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232,342,143 | 2022-01-14T14:03:03Z | CCBY | https://www.frontiersin.org/articles/10.3389/fonc.2021.675442/pdf | GOLD | 8efdb98879b79f5bbae97f8b245e4fa78b29b2fd | null | null | null | null | 10.3389/fonc.2021.675442 | null | 33842379 | 8027261 |
Article 675442 1 (2021) CircRNA circ_0092314 Induces Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Elevating the Expression of S100P by Sponging miR-671
March 2021
Qian Shen
Department of Oncology
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Gang Zheng†
†
Yi Zhou
Department of General Surgery
The Fifth Hospital of Wuhan
WuhanChina
Department of Gastrointestinal Surgery
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Jin Tong
Department of PICC
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
†
Sanpeng Xu
Department of Pathology
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Hui Gao
Department of Clinical Nutrition
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Xiaofan Zhang
Department of Neurology
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Hokkaido University
Japan
Qiang Fu
Department of Oncology
Tongji Hospital
Tongji Medical College
Huazhong University of Science and Technology
WuhanChina
Peixin Dong
Qiang Fu
Shen Q
Zheng G
Zhou Y
Tong J
Xu S
Gao H
Zhang X
Fu Q
Article 675442 1 (2021) CircRNA circ_0092314 Induces Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Elevating the Expression of S100P by Sponging miR-671
Front. Oncol
11675442March 202110.3389/fonc.2021.675442Received: 03 March 2021 Accepted: 11 March 2021 Published: 25 March 2021 Citation:Edited by: Reviewed by: Daozhi Xu, Shenyang Medical College, China Rui Chen, Sun Yat-Sen University, China *Correspondence: These authors have contributed equally to this work and share first authorship Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncologycircular RNAcirc_0092314microRNAemtmiR-671s100ppancreatic cancer
Background: Circular RNAs (circRNAs) is a novel class of non-coding RNAs that regulate gene expression during cancer progression. Circ_0092314 is a newly discovered circRNA that was upregulated in pancreatic cancer (PAAD) tissues. However, the detailed functions and underlying mechanisms of circ_0092314 in PAAD cells remain unclear.Methods: We first determined the expression of circ_0092314 in PAAD and normal tissues and further investigated the functional roles of circ_0092314 in regulating epithelial-mesenchymal transition (EMT) of PAAD cells. We also assessed the regulatory action of circ_0092314 on the microRNA-671 (miR-671) and its target S100P.Results: Circ_0092314 was markedly upregulated in PAAD tissues and cells, and its overexpression was closely correlated with worse prognosis of PAAD patients. Functionally, circ_0092314 promotes proliferation, invasion and EMT in vitro and tumor growth in vivo. Mechanistically, we demonstrated that circ_0092314 directly binds to miR-671 and relieve its suppression of the downstream target S100P, which induces EMT and activates the AKT signaling pathway. The tumor-promoting effects caused by overexpression of circ_0092314 could be revered by re-expression of miR-671 in PAAD cells.Conclusions: Overall, our study demonstrates that circ_0092314 exerts critical roles in promoting the EMT features of PAAD cells, and provides insight into how elevated expression of circ_0092314 might influence PAAD progression.
BACKGROUND
Pancreatic ductal adenocarcinoma (PAAD) is a highly aggressive cancer type, being characterized by high rates of metastasis and resistance to chemotherapy (1). Epithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells lose contact between neighboring cells and acquire migratory mesenchymal phenotypes (2). Hallmarks of EMT include loss of epithelial markers, such as E-cadherin, and gain of mesenchymal markers, such as Vimentin (2). Cancer stem cells (CSCs) are subpopulations of cancer cells that show stem cell characteristics and influence tumorigenesis, metastasis and chemoresistance (3). The pancreatic cancer stem cells express cell surface markers, such as CD44 and CD133 (3). It is considered that cancer cells can enter the CSC state via activation of the EMT program (4). S100P protein, a small isoform of the S100 protein family, is frequently overexpressed in human tumors including PAAD (5). S100P was shown to increase the migratory and invasive capabilities of PAAD cells (6). Although a previous study in pancreatic cancer has shown that S100P contributes to the aggressive nature and EMT features of colon cancer cells (7), the association of S100P expression with EMT induction in PAAD needs further exploration.
MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that mediate gene expression through interaction with 3′untranslated regions (3′-UTRs) of mRNAs (8). Circular RNAs (circRNAs) are an emerging subgroup of non-coding RNAs that are more stable than other RNAs and can act as competing endogenous RNAs (ceRNAs) to sponge miRNAs, thus indirectly regulating the expression of target genes of miRNAs (9,10). Growing evidence has shown that the dysregulation of circRNAs is associated with the initiation and development of many cancers, including PAAD (9)(10)(11). For example, circ_0092314 is a newly discovered circRNA that was upregulated in PAAD tissues (12). However, the detailed functions and the molecular mechanism underlying the role of circ_0092314 in PAAD cells are not fully understood.
Here, we identified a novel role of circ_0092314 in regulating EMT and invasion of PAAD cells. Circ_0092314 induces EMT of PAAD cells by competing for miR-671 to subsequently increase the expression of its downstream target of S100P. Our results suggest that circ_0092314 would be a promising target for PAAD therapy.
MATERIALS AND METHODS
Clinical Tissue Samples
This research has been approved by the Helsinki declaration and approved by the Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. Each patient signed the informed consent. Forty PAAD tissues and adjacent normal tissues were collected during surgical excision. Tissues were immediately frozen in liquid nitrogen and stored at -80°C.
Cell Lines, Cell Culture, and Transfection Four PAAD cell lines (AsPC-1, BxPC-3, SW-1990 and PaCa-2) and a human pancreatic duct epithelial cell line HPDE6-C7 were provided by the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) in a 37°C, 5% CO2 humidified atmosphere.
Transient transfection was done using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. The overexpression vectors for circ_0092314 and S100P were synthesized by Geneseed (Guangzhou, China). SiRNAs targeting circ_0092314 and S100P, as well as miR-671 mimics and miR-671 inhibitors, were purchased from Genechem (Shanghai, China).
Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted from tissues or cells by TRIzol reagent (Invitrogen). Total RNA from PAAD cells was divided into two groups and one group was treated with 3 U/µg RNase R (Geneseed, Guangzhou, China) at 37°C, for 30 min according to the manufacturer's instructions. The nuclear and cytoplasmic RNA fractionation was isolated with PARIS Kit (Invitrogen). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara, Dalian, China). Quantitation of circ_0092314 and S100P mRNA was performed using a SYBR PCR kit (Takara), as previously described (13). The expression level of miR-671 was evaluated using the NCode miRNA qRT-PCR analysis (Invitrogen). The levels of circ_0092314 and S100P were normalized to the control GAPDH. The expression of miR-671 was normalized to the control U6. All primers used in this study were obtained from Genechem (Shanghai, China).
Western Blotting
Total protein was extracted using a RIPA buffer (Beyotime, Beijing, China). SDS-polyacrylamide gel electrophoresis was performed using an equal amount of total protein. Then, the protein was transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Next, membranes were incubated with the primary antibodies: E-cadherin (Cell Signaling, MA, USA; 1:1000), Vimentin (Cell Signaling; 1:1000), S100P (Cell Signaling; 1:1000), AKT (Cell Signaling; 1:1000), p-AKT (Cell Signaling; 1:1000) and b-actin (Cell Signaling; 1:5000). Next day, the secondary antibodies were added and each protein band was detected by the ECL detection system (Amersham Biosciences, Buckinghamshire, UK).
Cell Counting Kit-8 Assay
The proliferation rate of PAAD cells were assessed by Cell Counting Kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. 5 × 10 3 cells were seeded into 96-well plates. At 24, 48, 72 and 96 h after transfection, 10 ml of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using an automatic microplate reader (BioTek, VT, USA).
Transwell Invasion Assay
Cell invasion assay was performed as previously reported (14). We placed 750 ml medium containing 10% FBS in the bottom chamber, and then seeded PAAD cells into the upper chamber for invasion assays (8-mm pore size, Corning, CA, USA) in 500 ml serum-free media. After 24 h, the invaded cells were stained with Giemsa (Sigma, St. Louis, MO, USA) for 15 minutes. Finally, the number of cells was counted using an Olympus microscope.
Tumor Sphere Formation Assay
Single cells were seeded in 24-well ultra-low attachment plates (Corning, Acton, MA) containing serum-free medium supplemented with B27 (1:50; Invitrogen, Carlsbad, CA, USA), 20 ng/ml basic FGF (BD Biosciences, CA, USA) and 20 ng/ml EGF (Sigma, St. Louis, MO, USA). The cultures were fed with fresh serum-free medium and growth factors every other day. After 14 days of incubation, images of cells were captured and tumor spheres with a diameter more than 50 mm were counted using Image J software.
In Vivo Tumor Formation Assay
All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. Four-week-old Nude mice (n = 6 per group) were purchased from Beijing HFK Bioscience (Beijing, China). PAAD cells transfected as indicated were transplanted subcutaneously into the right flank of nude mice. The tumor volumes were measured every week and calculated as length (mm) × width 2 (mm 2 ) × 0.5. After 3 weeks after injection, the mice were sacrificed. The tumor were carefully removed, photographed and weighed.
Luciferase Reporter Assay
A circ_0092314 fragment or a 3′-UTR sequence of S100P was inserted into a luciferase reporter plasmid (Ribobio, Guangzhou, China). Mutagenesis was performed using a QuickChange Site-Directed Mutagenesis kit (Stratagene, CA, USA). PAAD cells were transiently co-transfected with luciferase reporter plasmid with miR-671 mimic (or miR-671 inhibitor) using Lipofectamine 2000 Reagent (Invitrogen), along with the Renilla luciferase plasmid pRL-CMV (Promega, WI, USA). After 48 h of transfection, the cells were harvested and subjected to a Dual-Luciferase Reporter Assay system (Promega). The relative luciferase activities were normalized with the Renilla luciferase activities.
RNA Immunoprecipitation (RIP) Assay
RIP assay was conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). PAAD cells were lysed with a RIP-lysis buffer and cell lysates were incubated with magnetic beads conjugated with anti-Ago2 antibody (Millipore) or control anti-IgG antibody (Millipore). After incubating with proteinase K, the immunoprecipitated RNAs were extracted and subjected to the qRT-PCR analysis.
Statistical Analysis
All results were representative of at least three independent experiments. All data were shown as the mean ± standard deviation, and experimental data were evaluated using the Student's t-tests, one-way ANOVA tests and Wilcoxon signedrank tests. The statistical analysis was performed using SPSS 25.0 statistical software package. * P < 0.05 was considered statistically significant.
RESULTS
Circ_0092314 Is Highly Expressed in PAAD Tissues and Cells
Using the circBase database (http://www.circbase.org/), we found that circ_0092314 was produced from human RANBP1 gene located at chr22: 20113099-20113439, and finally formed a circular transcript of 340 nt ( Figure 1A). To investigate the biological role of circ_0092314 in PAAD, we first analyzed its expression in 40 pairs of PAAD specimens and adjacent normal tissues using qRT-PCR assay. The expression level of circ_0092314 was significantly upregulated in PAAD tissues compared with adjacent normal tissues ( Figure 1B). Subsequently, we divided PAAD tissues as well/moderately and poorly differentiated groups, low TNM stages (I-II) and high TNM stages (III-IV) groups, as well as lymph node metastasisnegative and -positive groups. As shown in Figures 1C, D, E, the levels of circ_0092314 in the poorly differentiated group, high TNM stage group, or lymph node metastasis-positive group were significantly higher than those in the well/moderately group, low TNM stage or lymph node metastasis-negative group. Furthermore, the expression of circ_0092314 in PAAD tissues was divided into the low expression group and the high expression group based on the median values. Our Kaplan-Meier analysis showed that PAAD patients with high circ_0092314 expression had a significantly poorer overall survival and disease-free survival compared with patients with low circ_0092314 expression ( Figures 1F, G).
Then, the expression of circ_0092314 in four PAAD cell lines (AsPC-1, BxPC-3, SW-1990 and PaCa-2) and human pancreatic duct epithelial cell line (HPDE6-C7) was assessed by qRT-PCR experiments. Increased expression of circ_0092314 was observed in all PAAD cell lines compared with HPDE6-C7 cells ( Figure 1H). Among these cell lines, PaCa-2 cells expressed the highest level of circ_0092314, while AsPC-1 cells had the lowest level of circ_0092314 ( Figure 1G). RNase R was administrated to the extracted RNA from PaCa-2 cells. We found that the circular form (circ_0092314) can be resistant to RNase R, whereas the linear form (RANBP1 mRNA) was significantly decayed ( Figure 1I). In addition, qRT-PCR analysis of nuclear and cytoplasmic fractions of RNAs demonstrated that circ_0092314 was predominantly localized in the cytoplasm ( Figure 1J). Taken together, our results suggest that circ_0092314 is an abundant and stable circRNA overexpressed in PAAD cells.
Circ_0092314 Enhances the Proliferation of PAAD Cells In Vitro and In Vivo
We determined the biological roles of circ_0092314 in PAAD cells. To this end, we transfected PaCa-2 cells with specific siRNAs targeting circ_0092314, and transfected AsPC-1 cells with overexpression plasmid of circ_0092314, respectively.
Circ_0092314 expression was significantly silenced in PaCa-2 cells, whereas its expression was clearly increased in AsPC-1 cells following overexpression of circ_0092314 (Figures 2A, B). The proliferation of PaCa-2 cells was decreased by downregulation of circ_0092314, and overexpression of circ_0092314 promoted the proliferation of AsPC-1 cells (Figures 2C, D). To investigate the function of circ_0092314 in vivo, we established a nude mice xenograft model of PAAD by subcutaneous inoculation of PaCa-2 cells transfected with specific siRNA against circ_0092314, and of AsPC-1 cells transfected with circ_0092314 overexpression plasmid ( Figures 2E, F). Compared with tumors derived from The nuclearcytoplasmic fractionation assay showed that circ_0092314 was mainly located in the cytoplasm. *P < 0.05, **P < 0.01, ***P < 0.001.
the control cells, those derived from circ_0092314 siRNAtransfected PaCa-2 cells grew slowly ( Figure 2E). Moreover, overexpression of circ_0092314 significantly increased tumor growth ( Figure 2F). The results showed that the tumor volume of xenograft in circ_0092314 knockdown group was obviously smaller than the control group ( Figure 2G). The weight of tumors was lighter in circ_0092314 knockdown group than the control group, consistently ( Figure 2H). Conversely, the tumor volume and weight were significantly increased in the circ_0092314 overexpressed group (Figures 2G, H). These results confirmed the promoting effects of circ_0092314 on PAAD proliferation.
Circ_0092314 Induces EMT in PAAD Cells
EMT is a critical process that enhances the aggressive properties of cancer cells (2). Therefore, we investigated whether circ_0092314 could promote the EMT features of PAAD cells. Western blotting analysis showed that the expression of E-cadherin was increased, whereas the expression of Vimentin was decreased in PaCa-2 cells after the knockdown of circ_0092314 ( Figure 3A). In contrast, overexpression of circ_0092314 led to downregulation of Ecadherin and upregulation of Vimentin ( Figure 3A). Furthermore, we found that knockdown of circ_0092314 could significantly inhibit the invasive abilities and sphere-formation of PaCa-2 cells (Figures 3B-D). However, overexpression of circ_0092314 in AsPC-1 cells increased invasive abilities and sphere-forming abilities ( Figures 3B-D). Our qRT-PCR assay confirmed the downregulated expression of CD133 and CD44 in circ_0092314-silenced PaCa-2 cells, and the upregulated expression of CD133 and CD44 in circ_0092314-overexpressing AsPC-1 cells ( Figure 3E). These findings indicated that in circ_0092314 promotes EMT and CSC properties of PAAD cells.
Circ_0092314 Binds to miR-671 and Suppresses Its Expression in PAAD Cells
According to the CircInteractome prediction database, a complementary sequence in circ_0092314 for miR-671 was found ( Figure 4A). The qRT-PCR assay was performed to study the expression of miR-671 in PAAD tissues and adjacent normal tissues. MiR-671 was expressed at lower levels in PAAD tissues compared with normal tissues ( Figure 4B). The expression of miR-671 in four PAAD cell lines was significantly decreased when compared with HPDE6-C7 cells ( Figure 4C). The survival curves from Kaplan-Meier Plotter database demonstrated that low level of miR-671 predicted poor outcome in PAAD patients ( Figure 4D). Then, a dual-luciferase reporter assay was conducted to confirm the interaction between circ_0092314 and miR-671. The results suggested that transfection with miR-671 mimic significantly attenuated the activity of luciferase reporter containing wild-type (WT) circ_0092314 fragment compared with control mimic, while transfection of miR-671 mimic failed to affect the activity of luciferase reporter containing mutant (MUT) circ_0092314 fragment (Figures 5A, B). Transfection with miR-671 inhibitor significantly increased the luciferase activity of the WT circ_0092314 fragment, but not the MUT circ_0092314 fragment (Figures 5A, B). RIP experiments suggested that circ_0092314 and miR-671 were preferentially enriched in anti-AGO2-containing complexes compared with anti-IgG ( Figure 5C). In addition, silencing of circ_0092314 can upregulate the expression of miR-671 in PaCa-2 cells, and overexpression of circ_0092314 in AsPC-1 cells can reduce the levels of miR-671 ( Figure 5D). Our rescue experiments suggested that knockdown of circ_0092314 suppressed the invasion and sphere formation in PaCa-2 cells, however coexpression of miR-671 inhibitor could abolish these effects of circ_0092314 knockdown ( Figure 5E). Also, we observed that overexpression of circ_0092314 induced cell invasion and sphere formation, and co-transfection with miR-671 mimic significantly reversed these effects ( Figure 5F). Collectively, these data demonstrated that circ_0092314 acts as a sponge of miR-761 and suppresses its expression in PAAD cells.
MiR-671 Suppresses EMT and PAAD Cell Invasion by Inhibiting S100P Expression
We used the TargetScan database to predict the potential targets of miR-671 and found that S100P was one of the targets of miR-671 ( Figure 4A). Then, our qRT-PCR results showed the upregulation of S100P in PAAD tissues compared with normal tissues ( Figure 6A). The protein expression of S100P was further validated in the Human Protein Atlas database (https://www. proteinatlas.org/). The results indicated that the expression levels of S100P were strongly expressed in PAAD tissues, however S100P protein expression was undetectable in adjacent normal tissues ( Figure 6B). Consistent with these results, the mRNA expression of S100P was highly expressed in PAAD cells than that in HPDE6-C7 cells ( Figure 6C). Kaplan-Meier survival curves suggested that high levels of S100P expression were associated with a significantly unfavorable overall survival ( Figure 6D). In order to explore whether miR-671 could directly bind to the 3′-UTR of S100P mRNA, we performed the luciferase reporter assay. Our results showed that overexpression of miR-671 remarkably reduced the luciferase activity of WT S100P 3′-UTR, but had no significant effect on the mutant one ( Figures 7A, B). Alternatively, inhibition of miR-671 increased the luciferase activity of WT S100P 3′-UTR, without affecting the MUT S100P 3′-UTR ( Figures 7A, B). S100P was known to promote EMT in colorectal cancer cells by activating the AKT pathway (7). In line with this finding, our western blotting analysis indicated that miR-671 overexpression led to the downregulation of S100P, Vimentin, and inactivation of AKT signaling, but the upregulation of E-cadherin in PaCa-2 cells ( Figure 7C). By contrast, inhibition of miR-671 in AsPC-1 cells led to suppression of E-cadherin, induction of Vimentin, and activation of the AKT pathway ( Figure 7C). We further examined the contribution of S100P in miR-671dependent EMT by performing rescue experiments. Remarkably, the ability of miR-671 in suppressing EMT as presented by decreased cell invasion/sphere formation, upregulation of E-cadherin, downregulation of Vimentin, as well as inactivation of the AKT pathway, was largely reversed by overexpression of S100P ( Figure 7C-E). Conversely, miR-671 inhibitor-induced EMT properties were partially abrogated by knockdown of S100P ( Figure 7C-E). These data implicated that miR-671 behave as an EMT suppressor in PAAD by targeting S100P and probably by repressing the AKT pathway. Overexpression of circ_0092314 induced S100P expression and activated the AKT pathway in AsPC-1 cells ( Figure 3A). Consistently, S100P was decreased, and the activity of the AKT pathway was suppressed following circ_0092314 knockdown in PaCa-2 cells ( Figure 3A).
Furthermore, Pearson correlation analysis indicated that the expression of circ_0092314 was negatively correlated with miR-671 expression, and positively correlated with S100P mRNA expression in PAAD cancer tissues ( Figure 8A). We also observed that there was a negative correlation between miR-671 expression and S100P mRNA expression in PAAD cancer tissues ( Figure 8A). Overall, in this study, we identified circ_0092314, as a novel oncogenic circRNA induces EMT and invasion of PAAD cells via elevating the abundance of S100P by sponging a tumor suppressor miR-671 ( Figure 8B).
DISCUSSION
The traditional prognostic markers for PAAD include histological subtype, vascular and perineural invasion, the presence of desmoplastic reaction, tumor budding and EMT (15). In addition, new and emerging prognostic biomarkers, including miRNA, long non-coding RNA and recently circRNA, have been reported (15,16). For instance, circ-LDLRAD3 was significantly upregulated in PAAD tissues and plasma, and a high level of circ-LDLRAD3 was positively associated with tumor venous invasion and lymphatic metastasis (17). Interestingly, circ-LDLRAD3 combined with CA19-9 was confirmed to have higher sensitivity and specificity for the diagnosis of PAAD (17). In addition, high expression of circ_0030235 was an independent prognostic indicator of unfavorable overall survival for PAAD patients according to a multivariate Cox analysis (18).
Here, we provided new evidence to show that the expression of circ_0092314 was significantly higher in clinical PAAD tissues than in adjacent normal tissues, and its expression was closely associated with aggressive behavior of PAAD. Importantly, we have shown that those PAAD patients with high circ_0092314 expression had worse clinical outcomes, indicating the potential of circ_0092314 as a promising prognostic biomarker for PAAD patients. Whether its expression was changed in the plasma of patients with PAAD, whether circ_0092314 expression is correlated with CA19-9 levels, deserve further investigation. Growing studies have supported the existence of complex interactions between circRNAs and miRNAs in PAAD cells (19,20), in which circRNAs regulates the expression and activity of miRNAs by competitively binding their target sites on protein-coding mRNAs. Here, we showed for the first time that circ_0092314 promotes EMT features by binding to miR-671 to induce the expression of S100P. Our bioinformatic analysis using the CircInteractome database has shown that circ_0092314 hosts multiple binding sites for diverse miRNAs and RNA-binding proteins (data not shown), suggesting that circ_0092314 probably act as sponges for different cytoplasmic miRNAs, or interact with RNA-binding proteins to generate RNA-protein complexes that can also control the EMT process and PAAD metastasis.
Over the last several years, although we should acknowledge that numerous efforts to improve the efficacy of surgery and chemotherapy in PAAD patients have been made, there are still few reliable biomarkers or effective therapeutic strategies for daily clinical practice in PAAD. Increasing evidence indicated that circRNAs have great potential to regulate cancer cell proliferation, apoptosis, invasion, EMT, metastasis and response to chemotherapy, implying that circRNAs may be used as novel potential therapeutic targets for treating various tumors including PAAD (9,10,21,22). Here, we confirmed that the knockdown of circ_0092314 suppressed the growth and invasion of PAAD cells in vitro, and resulted in reduced tumor A B D C FIGURE 6 | High S100P expression predicts poor prognosis in PAAD. (A) qRT-PCR analysis of S100P mRNA expression in PAAD tissues and adjacent normal tissues. (B) Immunohistochemical data was downloaded from the Human Protein Atlas database. The staining pattern for S100P protein in PAAD tissues and adjacent normal tissues were shown. (C) S100P mRNA expression in PAAD cells and HPDE6-C7 cells. (D) Kaplan-Meier curves for the overall survival of PAAD patients with high or low S100P expression (KM Plotter database). ***P < 0.001. size and tumor weight in vivo, suggesting that targeting circ_0092314 is a potential therapeutic strategy for PAAD.
Downregulation of miR-671 has been observed in a panel of human tumors, such as osteosarcoma (23), prostate cancer (24), lung cancer (25), breast cancer (26), and gastric cancer (27). In these tumors, miR-671 was demonstrated to show inhibit the malignant phenotypes of tumor cells by targeting SOX6, CCND2, and FOXM1 (23)(24)(25)(26)(27). However, miR-671 may function as an oncogenic miRNA in other types of cancer (28,29). In our study, miR-671 was identified as a downstream effector of circ_0092314 in PAAD cells, in which the tumor-promoting effects of c i r c _ 0 0 9 2 3 1 4 c o u l d b e bl o c k e d t h r o u g h mi R -6 7 1 overexpression. This ability of miR-671 to suppress the EMT properties of PAAD cells provides insights into the molecular mechanisms of PAAD metastasis and a path for inhibiting the metastatic spread of PAAD or other cancers. The detailed mechanisms by which miR-671 mediates EMT and PAAD cell invasion deserve future studies. S100P, a calcium-binding protein, can advance tumor progression and metastasis in pancreatic and several other cancers (6,30,31). S100P has previously been demonstrated to regulate the proliferation, migratory and invasive capabilities of PAAD cells (6). In addition, decreased PAAD growth was observed following S100P silencing in an orthotropic mouse model (6). S100P promotes EMT, migration and invasion of colon cancer cells by up-regulating S100A4 through AKT activation (7). Consistent with these reports, we found that S100P acts as an important EMT activator in PAAD cells, and its oncogenic functions might be involved in the activation of the AKT pathway. Together, our results suggest that dysregulation of the circ_0092314/miR-671/S100P axis is responsible for PAAD progression, and these molecules are potential therapeutic targets for suppressing the EMT in metastatic PAAD. The levels of S100P mRNA in PAAD cells transfected with miR-671 mimic or miR-671 inhibitor, respectively. (B) The activities of S100P 3′-UTR reporter containing WT or MUT miR-671 binding sites were determined using luciferase assay following co-transfection with miR-671 mimic or miR-671 inhibitor. (C) Western blotting analysis of the indicated proteins in PAAD cells transfected as indicated. (D, E) Cell invasion assay (left) and tumor sphere formation assay (right) in PaCa-2 cells transfected with (or without) miR-671 mimic, with (or without) S100P expression vector (D), and in AsPC-1 cells transfected with (or without) miR-671 inhibitor, with (or without) S100P siRNA (E). ***P < 0.001.
In summary, our findings support the idea that the circ_0092314/miR-671/S100P signaling plays crucial roles in regulating EMT phenotypes of PAAD cells and suggest that this signaling pathway might be an effective target for PAAD therapy.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China.
AUTHOR CONTRIBUTIONS
QF designed the experiments. QS, GZ, YZ, and JT performed the experiments. SX, HG, and XZ made significant revisions to the manuscript. All authors contributed to the article and approved the submitted version.
FUNDING
This study was supported by the National Natural Science Foundation of China (No. 81974381). A B FIGURE 8 | The correlation between circ_0092314 and miR-671/S100P expression in PAAD tissues. (A) The correlation between circ_0092314 expression and miR-671/S100P expression in PAAD tissues was examined using qRT-PCR assay. (B) A proposed mechanistic model in which circ_0092314 can sponge miR-671 to increase S100P expression, thereby promoting EMT and PAAD cell invasion.
FIGURE 1 |
1Circ_0092314 is highly expressed in PAAD tissues and cells. (A) Human RANBP1 gene was identified as the host gene of circ_0092314 by browsing circBase database. (B) The expression of circ_0092314 in PAAD tissues (n = 40) and their adjacent normal pancreatic tissues (n = 40) was determined using qRT-PCR assay. (C) The expression of circ_0092314 in well/moderate and poor differentiated PAAD tissues from patients. (D) The expression of circ_0092314 in PAAD tissues from patients with stages I and II, or stage III and IV disease. (E) The expression of circ_0092314 in PAAD tissues from patients with (or without) lymph node metastasis. (F, G) Kaplan-Meier survival analysis showed that PAAD patients with high circ_0092314 expression have a lower overall survival (F) and disease-free survival (G) than those with low circ_0092314 expression. (H) qRT-PCR analysis of circ_0092314 expression in PAAD cell lines and HPDE6-C7 cells. (I) The expression of circ_0092314 and RANBP1 mRNA in PaCa-2 cells was detected using qRT-PCR assay in the presence or absence of RNase R. (J)
FIGURE 2 |
2Circ_0092314 enhances the proliferation of PAAD cells in vitro and in vivo. (A) The expression of circ_0092314 in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (B) The expression of circ_0092314 in AsPC-1 cells transfected with circ_0092314 overexpression vector or control vector. (C) CCK-8 assays in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (D) CCK-8 assays in AsPC-1 cells transfected with circ_0092314 overexpression vector or the control vector. (E, F) The expression of circ_0092314 in PaCa-2 (E) and AsPC-1 (F) cells was examined using qRT-PCR assay. The lower panels showed the representative images of xenograft tumors in nude mice. (G, H) Tumor volume (G) and tumor weight (H) were measured from each group after 21 days. **P < 0.01, ***P < 0.001.
FIGURE 3 |
3Circ_0092314 induces EMT in PAAD cells. (A) Western blotting analysis of the indicated proteins in circ_0092314-silenced or circ_0092314overexpressing PAAD cells. (B, C) Images of representative invasive PAAD cells are shown (B), and quantification analysis is presented (C). (D) Tumor sphere formation assay for circ_0092314-silenced or circ_0092314-overexpressing PAAD cells, shown by representative images (left) and by quantification (right). (E) qRT-PCR assay of CD133 and CD44 expression in circ_0092314-silenced or circ_0092314-overexpressing PAAD cells. ***P < 0.001.
FIGURE 4 |
4MiR-671 is downregulated in PAAD tissues and positively correlates with patient prognosis. (A) The predicted binding site of miR-671 within circ_0092314 and the S100P 3′-UTR sequence were shown. (B) The expression of miR-671 in PAAD tissues and adjacent normal tissues. (C) qRT-PCR analysis of miR-671 expression in four PAAD cell lines and normal pancreatic cells. (D) Kaplan-Meier curves for the overall survival of PAAD patients with high or low levels of miR-671 (KM Plotter database). **P < 0.01, ***P < 0.001.
FIGURE 5 |
5Circ_0092314 binds to miR-671 and suppresses its expression in PAAD cells. (A) qRT-PCR analysis of miR-671 in PAAD cells following overexpression or knockdown of miR-671. (B) The activities of circ_0092314 reporter containing wild-type (WT) or mutant (MUT) binding sites were determined using luciferase assay following co-transfection with miR-671 mimic or miR-671 inhibitor. (C) RIP assay was performed in PaCa-2 or AsPC-1 cells. The levels of circ_0092314 and miR-671 were detected by qRT-PCR assay. (D) The expression of miR-671 in PAAD cells following knockdown or overexpression of circ_0092314. (E) Cell invasion (left) and sphere formation (right) assay in PaCa-2 cells transfected with (or without) circ_0092314 siRNA, along with (or without) miR-671 inhibitor. (F) Cell invasion (left) and sphere formation (right) assay in AsPC-1 cells transfected with (or without) circ_0092314 vector, along with (or without) miR-671 mimic. ***P < 0.001.
FIGURE 7 |
7MiR-671 suppresses EMT via targeting S100P. (A)
Frontiers in Oncology | www.frontiersin.org March 2021 | Volume 11 | Article 675442
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| [
"Background: Circular RNAs (circRNAs) is a novel class of non-coding RNAs that regulate gene expression during cancer progression. Circ_0092314 is a newly discovered circRNA that was upregulated in pancreatic cancer (PAAD) tissues. However, the detailed functions and underlying mechanisms of circ_0092314 in PAAD cells remain unclear.Methods: We first determined the expression of circ_0092314 in PAAD and normal tissues and further investigated the functional roles of circ_0092314 in regulating epithelial-mesenchymal transition (EMT) of PAAD cells. We also assessed the regulatory action of circ_0092314 on the microRNA-671 (miR-671) and its target S100P.Results: Circ_0092314 was markedly upregulated in PAAD tissues and cells, and its overexpression was closely correlated with worse prognosis of PAAD patients. Functionally, circ_0092314 promotes proliferation, invasion and EMT in vitro and tumor growth in vivo. Mechanistically, we demonstrated that circ_0092314 directly binds to miR-671 and relieve its suppression of the downstream target S100P, which induces EMT and activates the AKT signaling pathway. The tumor-promoting effects caused by overexpression of circ_0092314 could be revered by re-expression of miR-671 in PAAD cells.Conclusions: Overall, our study demonstrates that circ_0092314 exerts critical roles in promoting the EMT features of PAAD cells, and provides insight into how elevated expression of circ_0092314 might influence PAAD progression."
] | [
"Qian Shen \nDepartment of Oncology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"Gang Zheng† ",
"† ",
"Yi Zhou \nDepartment of General Surgery\nThe Fifth Hospital of Wuhan\nWuhanChina\n\nDepartment of Gastrointestinal Surgery\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"Jin Tong \nDepartment of PICC\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"† ",
"Sanpeng Xu \nDepartment of Pathology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"Hui Gao \nDepartment of Clinical Nutrition\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"Xiaofan Zhang \nDepartment of Neurology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n\nHokkaido University\nJapan\n",
"Qiang Fu \nDepartment of Oncology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina\n",
"Peixin Dong ",
"Qiang Fu ",
"Shen Q ",
"Zheng G ",
"Zhou Y ",
"Tong J ",
"Xu S ",
"Gao H ",
"Zhang X ",
"Fu Q "
] | [
"Department of Oncology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Department of General Surgery\nThe Fifth Hospital of Wuhan\nWuhanChina",
"Department of Gastrointestinal Surgery\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Department of PICC\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Department of Pathology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Department of Clinical Nutrition\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Department of Neurology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina",
"Hokkaido University\nJapan",
"Department of Oncology\nTongji Hospital\nTongji Medical College\nHuazhong University of Science and Technology\nWuhanChina"
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"I Garrido-Laguna, ",
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"G Ercan, ",
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"K Ohuchida, ",
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"T Egami, ",
"H Yamaguchi, ",
"K Fujii, ",
"H Konomi, ",
"T Arumugam, ",
"D M Simeone, ",
"K Van Golen, ",
"C D Logsdon, ",
"Z Zuo, ",
"P Zhang, ",
"F Lin, ",
"W Shang, ",
"R Bi, ",
"F Lu, ",
"D Xu, ",
"P Dong, ",
"Y Xiong, ",
"J Yue, ",
"K Ihira, ",
"Y Konno, ",
"S Bai, ",
"Y Wu, ",
"Y Yan, ",
"S Shao, ",
"J Zhang, ",
"J Liu, ",
"P Dong, ",
"D Xu, ",
"Y Xiong, ",
"J Yue, ",
"K Ihira, ",
"Y Konno, ",
"Z Rong, ",
"J Xu, ",
"S Shi, ",
"Z Tan, ",
"Q Meng, ",
"J Hua, ",
"W Song, ",
"W J Wang, ",
"T Fu, ",
"L Chen, ",
"D L Miao, ",
"W Xia, ",
"M Qiu, ",
"R Chen, ",
"S Wang, ",
"X Leng, ",
"J Wang, ",
"D Xu, ",
"P Dong, ",
"Y Xiong, ",
"J Yue, ",
"Y Konno, ",
"K Ihira, ",
"E Dell'aquila, ",
"Cam Fulgenzi, ",
"A Minelli, ",
"F Citarella, ",
"M Stellato, ",
"F Pantano, ",
"Y Lv, ",
"S Huang, ",
"F Yang, ",
"D Y Liu, ",
"J T Guo, ",
"N Ge, ",
"P Zhu, ",
"X Liu, ",
"Y Xu, ",
"Y Yao, ",
"P Gao, ",
"Y Cui, ",
"L Liu, ",
"F B Liu, ",
"M Huang, ",
"K Xie, ",
"Q S Xie, ",
"C H Liu, ",
"L Hao, ",
"W Rong, ",
"L Bai, ",
"H Cui, ",
"S Zhang, ",
"Y Li, ",
"B Wan, ",
"H Hu, ",
"R Wang, ",
"W Liu, ",
"D Chen, ",
"C Limb, ",
"Dsk Liu, ",
"M T Veno, ",
"E Rees, ",
"J Krell, ",
"I N Bagwan, ",
"C Xin, ",
"S Lu, ",
"Y Li, ",
"Y Zhang, ",
"J Tian, ",
"S Zhang, ",
"Y Yu, ",
"Z Wang, ",
"D Sun, ",
"X Zhou, ",
"X Wei, ",
"W Hou, ",
"Y Yao, ",
"Y Zhou, ",
"X Fu, ",
"X Tan, ",
"Y Fu, ",
"L Chen, ",
"W Lee, ",
"Y Lai, ",
"K Rezaei, ",
"H Song, ",
"Y Xu, ",
"T Xu, ",
"R Fan, ",
"T Jiang, ",
"M Cao, ",
"Z Zhu, ",
"L Luo, ",
"Q Xiang, ",
"J Wang, ",
"Y Liu, ",
"Y Deng, ",
"X G Chi, ",
"X X Meng, ",
"D L Ding, ",
"X H Xuan, ",
"Y Z Chen, ",
"Q Cai, ",
"R Camara, ",
"D Ogbeni, ",
"L Gerstmann, ",
"M Ostovar, ",
"E Hurer, ",
"M Scott, ",
"H Nakayama, ",
"K Ohuchida, ",
"A Yonenaga, ",
"A Sagara, ",
"Y Ando, ",
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] | [
"Pancreatic cancer: from state-of-the-art treatments to promising novel therapies. I Garrido-Laguna, M Hidalgo, 10.1038/nrclinonc.2015.53Nat Rev Clin Oncol. 126Garrido-Laguna I, Hidalgo M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies. Nat Rev Clin Oncol (2015) 12 (6):319-34. doi: 10.1038/nrclinonc.2015.53",
"Complex networks orchestrate epithelialmesenchymal transitions. J P Thiery, J P Sleeman, 10.1038/nrm1835Nat Rev Mol Cell Biol. 72Thiery JP, Sleeman JP. Complex networks orchestrate epithelial- mesenchymal transitions. Nat Rev Mol Cell Biol (2006) 7(2):131-42. doi: 10.1038/nrm1835",
"Pancreatic Cancer Stem Cells and Therapeutic Approaches. G Ercan, A Karlitepe, B Ozpolat, 10.21873/anticanres.11628doi: 10. 21873/anticanres.11628Anticancer Res. 376Ercan G, Karlitepe A, Ozpolat B. Pancreatic Cancer Stem Cells and Therapeutic Approaches. Anticancer Res (2017) 37(6):2761-75. doi: 10. 21873/anticanres.11628",
"EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. T Shibue, R A Weinberg, 10.1038/nrclinonc.2017.44Nat Rev Clin Oncol. 1410Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol (2017) 14(10):611-29. doi: 10.1038/nrclinonc.2017.44",
"S100P is an early developmental marker of pancreatic carcinogenesis. K Ohuchida, K Mizumoto, T Egami, H Yamaguchi, K Fujii, H Konomi, 10.1158/1078-0432.CCR-06-0298Clin Cancer Res. 1218Ohuchida K, Mizumoto K, Egami T, Yamaguchi H, Fujii K, Konomi H, et al. S100P is an early developmental marker of pancreatic carcinogenesis. Clin Cancer Res (2006) 12(18):5411-6. doi: 10.1158/1078-0432.CCR-06-0298",
"S100P promotes pancreatic cancer growth, survival, and invasion. T Arumugam, D M Simeone, K Van Golen, C D Logsdon, 10.1158/1078-0432.CCR-05-0092Clin Cancer Res. 1115Arumugam T, Simeone DM, Van Golen K, Logsdon CD. S100P promotes pancreatic cancer growth, survival, and invasion. Clin Cancer Res (2005) 11 (15):5356-64. doi: 10.1158/1078-0432.CCR-05-0092",
"Interplay between Trx-1 and S100P promotes colorectal cancer cell epithelial-mesenchymal transition by up-regulating S100A4 through AKT activation. Z Zuo, P Zhang, F Lin, W Shang, R Bi, F Lu, 10.1111/jcmm.13541J Cell Mol Med. 224Zuo Z, Zhang P, Lin F, Shang W, Bi R, Lu F, et al. Interplay between Trx-1 and S100P promotes colorectal cancer cell epithelial-mesenchymal transition by up-regulating S100A4 through AKT activation. J Cell Mol Med (2018) 22 (4):2430-41. doi: 10.1111/jcmm.13541",
"MicroRNA-361: A Multifaceted Player Regulating Tumor Aggressiveness and Tumor Microenvironment Formation. D Xu, P Dong, Y Xiong, J Yue, K Ihira, Y Konno, 10.3390/cancers11081130Cancers (Basel). 81130Xu D, Dong P, Xiong Y, Yue J, Ihira K, Konno Y, et al. MicroRNA-361: A Multifaceted Player Regulating Tumor Aggressiveness and Tumor Microenvironment Formation. Cancers (Basel) (2019) 11(8):1130. doi: 10.3390/cancers11081130",
"Construct a circRNA/ miRNA/mRNA regulatory network to explore potential pathogenesis and therapy options of clear cell renal cell carcinoma. S Bai, Y Wu, Y Yan, S Shao, J Zhang, J Liu, 10.1038/s41598-020-70484-2Sci Rep. 10113659Bai S, Wu Y, Yan Y, Shao S, Zhang J, Liu J, et al. Construct a circRNA/ miRNA/mRNA regulatory network to explore potential pathogenesis and therapy options of clear cell renal cell carcinoma. Sci Rep (2020) 10(1):13659. doi: 10.1038/s41598-020-70484-2",
"The Expression, Functions and Mechanisms of Circular RNAs in Gynecological Cancers. P Dong, D Xu, Y Xiong, J Yue, K Ihira, Y Konno, 10.3390/cancers12061472Cancers (Basel). 61472Dong P, Xu D, Xiong Y, Yue J, Ihira K, Konno Y, et al. The Expression, Functions and Mechanisms of Circular RNAs in Gynecological Cancers. Cancers (Basel) (2020) 12(6):1472. doi: 10.3390/cancers12061472",
"Circular RNA in pancreatic cancer: a novel avenue for the roles of diagnosis and treatment. Z Rong, J Xu, S Shi, Z Tan, Q Meng, J Hua, 10.7150/thno.56174Theranostics. 116Rong Z, Xu J, Shi S, Tan Z, Meng Q, Hua J, et al. Circular RNA in pancreatic cancer: a novel avenue for the roles of diagnosis and treatment. Theranostics (2021) 11(6):2755-69. doi: 10.7150/thno.56174",
"Integrated analysis of circular RNA-associated ceRNA network in pancreatic ductal adenocarcinoma. W Song, W J Wang, T Fu, L Chen, D L Miao, 10.3892/ol.2020.11306Oncol Lett. 193Song W, Wang WJ, Fu T, Chen L, Miao DL. Integrated analysis of circular RNA-associated ceRNA network in pancreatic ductal adenocarcinoma. Oncol Lett (2020) 19(3):2175-84. doi: 10.3892/ol.2020.11306",
"Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation. W Xia, M Qiu, R Chen, S Wang, X Leng, J Wang, 10.1038/srep35576Sci Rep. 635576Xia W, Qiu M, Chen R, Wang S, Leng X, Wang J, et al. Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation. Sci Rep (2016) 6:35576. doi: 10.1038/srep35576",
"MicroRNA-361-Mediated Inhibition of HSP90 Expression and EMT in Cervical Cancer Is Counteracted by Oncogenic lncRNA NEAT1. D Xu, P Dong, Y Xiong, J Yue, Y Konno, K Ihira, 10.3390/cells9030632Cells. 93632Xu D, Dong P, Xiong Y, Yue J, Konno Y, Ihira K, et al. MicroRNA-361- Mediated Inhibition of HSP90 Expression and EMT in Cervical Cancer Is Counteracted by Oncogenic lncRNA NEAT1. Cells (2020) 9(3):632. doi: 10.3390/cells9030632",
"Prognostic and predictive factors in pancreatic cancer. E Dell'aquila, Cam Fulgenzi, A Minelli, F Citarella, M Stellato, F Pantano, 10.18632/oncotarget.27518Oncotarget. 1110Dell'Aquila E, Fulgenzi CAM, Minelli A, Citarella F, Stellato M, Pantano F, et al. Prognostic and predictive factors in pancreatic cancer. Oncotarget (2020) 11(10):924-41. doi: 10.18632/oncotarget.27518",
"Role of non-coding RNA in pancreatic cancer. Y Lv, S Huang, 10.3892/ol.2019.10758Oncol Lett. 184Lv Y, Huang S. Role of non-coding RNA in pancreatic cancer. Oncol Lett (2019) 18(4):3963-73. doi: 10.3892/ol.2019.10758",
"Circular RNA circ-LDLRAD3 as a biomarker in diagnosis of pancreatic cancer. F Yang, D Y Liu, J T Guo, N Ge, P Zhu, X Liu, 10.3748/wjg.v23.i47.8345World J Gastroenterol. 2347Yang F, Liu DY, Guo JT, Ge N, Zhu P, Liu X, et al. Circular RNA circ- LDLRAD3 as a biomarker in diagnosis of pancreatic cancer. World J Gastroenterol (2017) 23(47):8345-54. doi: 10.3748/wjg.v23.i47.8345",
"Upregulated circular RNA circ_0030235 predicts unfavorable prognosis in pancreatic ductal adenocarcinoma and facilitates cell progression by sponging miR-1253 and miR-1294. Y Xu, Y Yao, P Gao, Y Cui, 10.1016/j.bbrc.2018.12.088Biochem Biophys Res Commun. 5091Xu Y, Yao Y, Gao P, Cui Y. Upregulated circular RNA circ_0030235 predicts unfavorable prognosis in pancreatic ductal adenocarcinoma and facilitates cell progression by sponging miR-1253 and miR-1294. Biochem Biophys Res Commun (2019) 509(1):138-42. doi: 10.1016/j.bbrc.2018.12.088",
"Circular RNA ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miR-7-mediated EGFR/STAT3 signaling pathway. L Liu, F B Liu, M Huang, K Xie, Q S Xie, C H Liu, 10.1016/j.hbpd.2019.03.003Hepatobiliary Pancreat Dis Int. 186Liu L, Liu FB, Huang M, Xie K, Xie QS, Liu CH, et al. Circular RNA ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miR-7-mediated EGFR/STAT3 signaling pathway. Hepatobiliary Pancreat Dis Int (2019) 18(6):580-6. doi: 10.1016/j.hbpd.2019.03.003",
"Upregulated circular RNA circ_0007534 indicates an unfavorable prognosis in pancreatic ductal adenocarcinoma and regulates cell proliferation, apoptosis, and invasion by sponging miR-625 and miR-892b. L Hao, W Rong, L Bai, H Cui, S Zhang, Y Li, 10.1002/jcb.27658J Cell Biochem. 1203Hao L, Rong W, Bai L, Cui H, Zhang S, Li Y, et al. Upregulated circular RNA circ_0007534 indicates an unfavorable prognosis in pancreatic ductal adenocarcinoma and regulates cell proliferation, apoptosis, and invasion by sponging miR-625 and miR-892b. J Cell Biochem (2019) 120(3):3780-9. doi: 10.1002/jcb.27658",
"Therapeutic Potential of Circular RNAs in Osteosarcoma. B Wan, H Hu, R Wang, W Liu, D Chen, 10.3389/fonc.2020.00370doi: 10.3389/ fonc.2020.00370Front Oncol. 10370Wan B, Hu H, Wang R, Liu W, Chen D. Therapeutic Potential of Circular RNAs in Osteosarcoma. Front Oncol (2020) 10:370. doi: 10.3389/ fonc.2020.00370",
"The Role of Circular RNAs in Pancreatic Ductal Adenocarcinoma and Biliary-Tract Cancers. C Limb, Dsk Liu, M T Veno, E Rees, J Krell, I N Bagwan, 10.3390/cancers12113250Cancers (Basel). 12113250Limb C, Liu DSK, Veno MT, Rees E, Krell J, Bagwan IN, et al. The Role of Circular RNAs in Pancreatic Ductal Adenocarcinoma and Biliary-Tract Cancers. Cancers (Basel) (2020) 12(11):3250. doi: 10.3390/cancers12113250",
"miR-671-5p Inhibits Tumor Proliferation by Blocking Cell Cycle in Osteosarcoma. C Xin, S Lu, Y Li, Y Zhang, J Tian, S Zhang, 10.1089/dna.2019.4870DNA Cell Biol. 389Xin C, Lu S, Li Y, Zhang Y, Tian J, Zhang S, et al. miR-671-5p Inhibits Tumor Proliferation by Blocking Cell Cycle in Osteosarcoma. DNA Cell Biol (2019) 38 (9):996-1004. doi: 10.1089/dna.2019.4870",
"miR-671 promotes prostate cancer cell proliferation by targeting tumor suppressor SOX6. Y Yu, Z Wang, D Sun, X Zhou, X Wei, W Hou, 10.1016/j.ejphar.2018.01.016Eur J Pharmacol. 823Yu Y, Wang Z, Sun D, Zhou X, Wei X, Hou W, et al. miR-671 promotes prostate cancer cell proliferation by targeting tumor suppressor SOX6. Eur J Pharmacol (2018) 823:65-71. doi: 10.1016/j.ejphar.2018.01.016",
"miR−671−3p is downregulated in non−small cell lung cancer and inhibits cancer progression by directly targeting CCND2. Y Yao, Y Zhou, X Fu, 10.3892/mmr.2019.9858Mol Med Rep. 193Yao Y, Zhou Y, Fu X. miR−671−3p is downregulated in non−small cell lung cancer and inhibits cancer progression by directly targeting CCND2. Mol Med Rep (2019) 19(3):2407-12. doi: 10.3892/mmr.2019.9858",
"miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer. X Tan, Y Fu, L Chen, W Lee, Y Lai, K Rezaei, 10.18632/oncotarget.6344doi: 10.18632/ oncotarget.6344Oncotarget. 71Tan X, Fu Y, Chen L, Lee W, Lai Y, Rezaei K, et al. miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer. Oncotarget (2016) 7(1):293-307. doi: 10.18632/ oncotarget.6344",
"CircPIP5K1A activates KRT80 and PI3K/AKT pathway to promote gastric cancer development through sponging miR-671-5p. H Song, Y Xu, T Xu, R Fan, T Jiang, M Cao, 10.1016/j.biopha.2020.109941BioMed Pharmacother. 126109941Song H, Xu Y, Xu T, Fan R, Jiang T, Cao M, et al. CircPIP5K1A activates KRT80 and PI3K/AKT pathway to promote gastric cancer development through sponging miR-671-5p. BioMed Pharmacother (2020) 126:109941. doi: 10.1016/j.biopha.2020.109941",
"MiRNA-671-5p Promotes prostate cancer development and metastasis by targeting NFIA/ CRYAB axis. Z Zhu, L Luo, Q Xiang, J Wang, Y Liu, Y Deng, 10.1038/s41419-020-03138-wCell Death Dis. 1111949Zhu Z, Luo L, Xiang Q, Wang J, Liu Y, Deng Y, et al. MiRNA-671-5p Promotes prostate cancer development and metastasis by targeting NFIA/ CRYAB axis. Cell Death Dis (2020) 11(11):949. doi: 10.1038/s41419-020- 03138-w",
"HMGA1-mediated miR-671-5p targets APC to promote metastasis of clear cell renal cell carcinoma through Wnt signaling. X G Chi, X X Meng, D L Ding, X H Xuan, Y Z Chen, Q Cai, 10.4149/neo_2019_190217N135Neoplasma. 671Chi XG, Meng XX, Ding DL, Xuan XH, Chen YZ, Cai Q, et al. HMGA1- mediated miR-671-5p targets APC to promote metastasis of clear cell renal cell carcinoma through Wnt signaling. Neoplasma (2020) 67(1):46-53. doi: 10.4149/neo_2019_190217N135",
"Discovery of novel small molecule inhibitors of S100P with in vitro antimetastatic effects on pancreatic cancer cells. R Camara, D Ogbeni, L Gerstmann, M Ostovar, E Hurer, M Scott, 10.1016/j.ejmech.2020.112621Eur J Med Chem. 203112621Camara R, Ogbeni D, Gerstmann L, Ostovar M, Hurer E, Scott M, et al. Discovery of novel small molecule inhibitors of S100P with in vitro anti- metastatic effects on pancreatic cancer cells. Eur J Med Chem (2020) 203:112621. doi: 10.1016/j.ejmech.2020.112621",
"S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer. H Nakayama, K Ohuchida, A Yonenaga, A Sagara, Y Ando, S Kibe, 10.3892/ijo.2019.4812Int J Oncol. 551Nakayama H, Ohuchida K, Yonenaga A, Sagara A, Ando Y, Kibe S, et al. S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer. Int J Oncol (2019)55(1):211-22. doi: 10.3892/ijo.2019.4812"
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"Pancreatic cancer: from state-of-the-art treatments to promising novel therapies",
"Complex networks orchestrate epithelialmesenchymal transitions",
"Pancreatic Cancer Stem Cells and Therapeutic Approaches",
"EMT, CSCs, and drug resistance: the mechanistic link and clinical implications",
"S100P is an early developmental marker of pancreatic carcinogenesis",
"S100P promotes pancreatic cancer growth, survival, and invasion",
"Interplay between Trx-1 and S100P promotes colorectal cancer cell epithelial-mesenchymal transition by up-regulating S100A4 through AKT activation",
"MicroRNA-361: A Multifaceted Player Regulating Tumor Aggressiveness and Tumor Microenvironment Formation",
"Construct a circRNA/ miRNA/mRNA regulatory network to explore potential pathogenesis and therapy options of clear cell renal cell carcinoma",
"The Expression, Functions and Mechanisms of Circular RNAs in Gynecological Cancers",
"Circular RNA in pancreatic cancer: a novel avenue for the roles of diagnosis and treatment",
"Integrated analysis of circular RNA-associated ceRNA network in pancreatic ductal adenocarcinoma",
"Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation",
"MicroRNA-361-Mediated Inhibition of HSP90 Expression and EMT in Cervical Cancer Is Counteracted by Oncogenic lncRNA NEAT1",
"Prognostic and predictive factors in pancreatic cancer",
"Role of non-coding RNA in pancreatic cancer",
"Circular RNA circ-LDLRAD3 as a biomarker in diagnosis of pancreatic cancer",
"Upregulated circular RNA circ_0030235 predicts unfavorable prognosis in pancreatic ductal adenocarcinoma and facilitates cell progression by sponging miR-1253 and miR-1294",
"Circular RNA ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miR-7-mediated EGFR/STAT3 signaling pathway",
"Upregulated circular RNA circ_0007534 indicates an unfavorable prognosis in pancreatic ductal adenocarcinoma and regulates cell proliferation, apoptosis, and invasion by sponging miR-625 and miR-892b",
"Therapeutic Potential of Circular RNAs in Osteosarcoma",
"The Role of Circular RNAs in Pancreatic Ductal Adenocarcinoma and Biliary-Tract Cancers",
"miR-671-5p Inhibits Tumor Proliferation by Blocking Cell Cycle in Osteosarcoma",
"miR-671 promotes prostate cancer cell proliferation by targeting tumor suppressor SOX6",
"miR−671−3p is downregulated in non−small cell lung cancer and inhibits cancer progression by directly targeting CCND2",
"miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer",
"CircPIP5K1A activates KRT80 and PI3K/AKT pathway to promote gastric cancer development through sponging miR-671-5p",
"MiRNA-671-5p Promotes prostate cancer development and metastasis by targeting NFIA/ CRYAB axis",
"HMGA1-mediated miR-671-5p targets APC to promote metastasis of clear cell renal cell carcinoma through Wnt signaling",
"Discovery of novel small molecule inhibitors of S100P with in vitro antimetastatic effects on pancreatic cancer cells",
"S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer"
] | [
"Nat Rev Clin Oncol",
"Nat Rev Mol Cell Biol",
"Anticancer Res",
"Nat Rev Clin Oncol",
"Clin Cancer Res",
"Clin Cancer Res",
"J Cell Mol Med",
"Cancers (Basel)",
"Sci Rep",
"Cancers (Basel)",
"Theranostics",
"Oncol Lett",
"Sci Rep",
"Cells",
"Oncotarget",
"Oncol Lett",
"World J Gastroenterol",
"Biochem Biophys Res Commun",
"Hepatobiliary Pancreat Dis Int",
"J Cell Biochem",
"Front Oncol",
"Cancers (Basel)",
"DNA Cell Biol",
"Eur J Pharmacol",
"Mol Med Rep",
"Oncotarget",
"BioMed Pharmacother",
"Cell Death Dis",
"Neoplasma",
"Eur J Med Chem",
"Int J Oncol"
] | [
"\nFIGURE 1 |\n1Circ_0092314 is highly expressed in PAAD tissues and cells. (A) Human RANBP1 gene was identified as the host gene of circ_0092314 by browsing circBase database. (B) The expression of circ_0092314 in PAAD tissues (n = 40) and their adjacent normal pancreatic tissues (n = 40) was determined using qRT-PCR assay. (C) The expression of circ_0092314 in well/moderate and poor differentiated PAAD tissues from patients. (D) The expression of circ_0092314 in PAAD tissues from patients with stages I and II, or stage III and IV disease. (E) The expression of circ_0092314 in PAAD tissues from patients with (or without) lymph node metastasis. (F, G) Kaplan-Meier survival analysis showed that PAAD patients with high circ_0092314 expression have a lower overall survival (F) and disease-free survival (G) than those with low circ_0092314 expression. (H) qRT-PCR analysis of circ_0092314 expression in PAAD cell lines and HPDE6-C7 cells. (I) The expression of circ_0092314 and RANBP1 mRNA in PaCa-2 cells was detected using qRT-PCR assay in the presence or absence of RNase R. (J)",
"\nFIGURE 2 |\n2Circ_0092314 enhances the proliferation of PAAD cells in vitro and in vivo. (A) The expression of circ_0092314 in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (B) The expression of circ_0092314 in AsPC-1 cells transfected with circ_0092314 overexpression vector or control vector. (C) CCK-8 assays in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (D) CCK-8 assays in AsPC-1 cells transfected with circ_0092314 overexpression vector or the control vector. (E, F) The expression of circ_0092314 in PaCa-2 (E) and AsPC-1 (F) cells was examined using qRT-PCR assay. The lower panels showed the representative images of xenograft tumors in nude mice. (G, H) Tumor volume (G) and tumor weight (H) were measured from each group after 21 days. **P < 0.01, ***P < 0.001.",
"\nFIGURE 3 |\n3Circ_0092314 induces EMT in PAAD cells. (A) Western blotting analysis of the indicated proteins in circ_0092314-silenced or circ_0092314overexpressing PAAD cells. (B, C) Images of representative invasive PAAD cells are shown (B), and quantification analysis is presented (C). (D) Tumor sphere formation assay for circ_0092314-silenced or circ_0092314-overexpressing PAAD cells, shown by representative images (left) and by quantification (right). (E) qRT-PCR assay of CD133 and CD44 expression in circ_0092314-silenced or circ_0092314-overexpressing PAAD cells. ***P < 0.001.",
"\nFIGURE 4 |\n4MiR-671 is downregulated in PAAD tissues and positively correlates with patient prognosis. (A) The predicted binding site of miR-671 within circ_0092314 and the S100P 3′-UTR sequence were shown. (B) The expression of miR-671 in PAAD tissues and adjacent normal tissues. (C) qRT-PCR analysis of miR-671 expression in four PAAD cell lines and normal pancreatic cells. (D) Kaplan-Meier curves for the overall survival of PAAD patients with high or low levels of miR-671 (KM Plotter database). **P < 0.01, ***P < 0.001.",
"\nFIGURE 5 |\n5Circ_0092314 binds to miR-671 and suppresses its expression in PAAD cells. (A) qRT-PCR analysis of miR-671 in PAAD cells following overexpression or knockdown of miR-671. (B) The activities of circ_0092314 reporter containing wild-type (WT) or mutant (MUT) binding sites were determined using luciferase assay following co-transfection with miR-671 mimic or miR-671 inhibitor. (C) RIP assay was performed in PaCa-2 or AsPC-1 cells. The levels of circ_0092314 and miR-671 were detected by qRT-PCR assay. (D) The expression of miR-671 in PAAD cells following knockdown or overexpression of circ_0092314. (E) Cell invasion (left) and sphere formation (right) assay in PaCa-2 cells transfected with (or without) circ_0092314 siRNA, along with (or without) miR-671 inhibitor. (F) Cell invasion (left) and sphere formation (right) assay in AsPC-1 cells transfected with (or without) circ_0092314 vector, along with (or without) miR-671 mimic. ***P < 0.001.",
"\nFIGURE 7 |\n7MiR-671 suppresses EMT via targeting S100P. (A)"
] | [
"Circ_0092314 is highly expressed in PAAD tissues and cells. (A) Human RANBP1 gene was identified as the host gene of circ_0092314 by browsing circBase database. (B) The expression of circ_0092314 in PAAD tissues (n = 40) and their adjacent normal pancreatic tissues (n = 40) was determined using qRT-PCR assay. (C) The expression of circ_0092314 in well/moderate and poor differentiated PAAD tissues from patients. (D) The expression of circ_0092314 in PAAD tissues from patients with stages I and II, or stage III and IV disease. (E) The expression of circ_0092314 in PAAD tissues from patients with (or without) lymph node metastasis. (F, G) Kaplan-Meier survival analysis showed that PAAD patients with high circ_0092314 expression have a lower overall survival (F) and disease-free survival (G) than those with low circ_0092314 expression. (H) qRT-PCR analysis of circ_0092314 expression in PAAD cell lines and HPDE6-C7 cells. (I) The expression of circ_0092314 and RANBP1 mRNA in PaCa-2 cells was detected using qRT-PCR assay in the presence or absence of RNase R. (J)",
"Circ_0092314 enhances the proliferation of PAAD cells in vitro and in vivo. (A) The expression of circ_0092314 in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (B) The expression of circ_0092314 in AsPC-1 cells transfected with circ_0092314 overexpression vector or control vector. (C) CCK-8 assays in PaCa-2 cells transfected with siRNAs targeting circ_0092314 or control siRNA. (D) CCK-8 assays in AsPC-1 cells transfected with circ_0092314 overexpression vector or the control vector. (E, F) The expression of circ_0092314 in PaCa-2 (E) and AsPC-1 (F) cells was examined using qRT-PCR assay. The lower panels showed the representative images of xenograft tumors in nude mice. (G, H) Tumor volume (G) and tumor weight (H) were measured from each group after 21 days. **P < 0.01, ***P < 0.001.",
"Circ_0092314 induces EMT in PAAD cells. (A) Western blotting analysis of the indicated proteins in circ_0092314-silenced or circ_0092314overexpressing PAAD cells. (B, C) Images of representative invasive PAAD cells are shown (B), and quantification analysis is presented (C). (D) Tumor sphere formation assay for circ_0092314-silenced or circ_0092314-overexpressing PAAD cells, shown by representative images (left) and by quantification (right). (E) qRT-PCR assay of CD133 and CD44 expression in circ_0092314-silenced or circ_0092314-overexpressing PAAD cells. ***P < 0.001.",
"MiR-671 is downregulated in PAAD tissues and positively correlates with patient prognosis. (A) The predicted binding site of miR-671 within circ_0092314 and the S100P 3′-UTR sequence were shown. (B) The expression of miR-671 in PAAD tissues and adjacent normal tissues. (C) qRT-PCR analysis of miR-671 expression in four PAAD cell lines and normal pancreatic cells. (D) Kaplan-Meier curves for the overall survival of PAAD patients with high or low levels of miR-671 (KM Plotter database). **P < 0.01, ***P < 0.001.",
"Circ_0092314 binds to miR-671 and suppresses its expression in PAAD cells. (A) qRT-PCR analysis of miR-671 in PAAD cells following overexpression or knockdown of miR-671. (B) The activities of circ_0092314 reporter containing wild-type (WT) or mutant (MUT) binding sites were determined using luciferase assay following co-transfection with miR-671 mimic or miR-671 inhibitor. (C) RIP assay was performed in PaCa-2 or AsPC-1 cells. The levels of circ_0092314 and miR-671 were detected by qRT-PCR assay. (D) The expression of miR-671 in PAAD cells following knockdown or overexpression of circ_0092314. (E) Cell invasion (left) and sphere formation (right) assay in PaCa-2 cells transfected with (or without) circ_0092314 siRNA, along with (or without) miR-671 inhibitor. (F) Cell invasion (left) and sphere formation (right) assay in AsPC-1 cells transfected with (or without) circ_0092314 vector, along with (or without) miR-671 mimic. ***P < 0.001.",
"MiR-671 suppresses EMT via targeting S100P. (A)"
] | [
"Figure 1A)",
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] | [] | [
"Pancreatic ductal adenocarcinoma (PAAD) is a highly aggressive cancer type, being characterized by high rates of metastasis and resistance to chemotherapy (1). Epithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells lose contact between neighboring cells and acquire migratory mesenchymal phenotypes (2). Hallmarks of EMT include loss of epithelial markers, such as E-cadherin, and gain of mesenchymal markers, such as Vimentin (2). Cancer stem cells (CSCs) are subpopulations of cancer cells that show stem cell characteristics and influence tumorigenesis, metastasis and chemoresistance (3). The pancreatic cancer stem cells express cell surface markers, such as CD44 and CD133 (3). It is considered that cancer cells can enter the CSC state via activation of the EMT program (4). S100P protein, a small isoform of the S100 protein family, is frequently overexpressed in human tumors including PAAD (5). S100P was shown to increase the migratory and invasive capabilities of PAAD cells (6). Although a previous study in pancreatic cancer has shown that S100P contributes to the aggressive nature and EMT features of colon cancer cells (7), the association of S100P expression with EMT induction in PAAD needs further exploration.",
"MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that mediate gene expression through interaction with 3′untranslated regions (3′-UTRs) of mRNAs (8). Circular RNAs (circRNAs) are an emerging subgroup of non-coding RNAs that are more stable than other RNAs and can act as competing endogenous RNAs (ceRNAs) to sponge miRNAs, thus indirectly regulating the expression of target genes of miRNAs (9,10). Growing evidence has shown that the dysregulation of circRNAs is associated with the initiation and development of many cancers, including PAAD (9)(10)(11). For example, circ_0092314 is a newly discovered circRNA that was upregulated in PAAD tissues (12). However, the detailed functions and the molecular mechanism underlying the role of circ_0092314 in PAAD cells are not fully understood.",
"Here, we identified a novel role of circ_0092314 in regulating EMT and invasion of PAAD cells. Circ_0092314 induces EMT of PAAD cells by competing for miR-671 to subsequently increase the expression of its downstream target of S100P. Our results suggest that circ_0092314 would be a promising target for PAAD therapy.",
"This research has been approved by the Helsinki declaration and approved by the Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. Each patient signed the informed consent. Forty PAAD tissues and adjacent normal tissues were collected during surgical excision. Tissues were immediately frozen in liquid nitrogen and stored at -80°C.",
"Cell Lines, Cell Culture, and Transfection Four PAAD cell lines (AsPC-1, BxPC-3, SW-1990 and PaCa-2) and a human pancreatic duct epithelial cell line HPDE6-C7 were provided by the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) in a 37°C, 5% CO2 humidified atmosphere.",
"Transient transfection was done using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. The overexpression vectors for circ_0092314 and S100P were synthesized by Geneseed (Guangzhou, China). SiRNAs targeting circ_0092314 and S100P, as well as miR-671 mimics and miR-671 inhibitors, were purchased from Genechem (Shanghai, China).",
"Total RNA was extracted from tissues or cells by TRIzol reagent (Invitrogen). Total RNA from PAAD cells was divided into two groups and one group was treated with 3 U/µg RNase R (Geneseed, Guangzhou, China) at 37°C, for 30 min according to the manufacturer's instructions. The nuclear and cytoplasmic RNA fractionation was isolated with PARIS Kit (Invitrogen). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara, Dalian, China). Quantitation of circ_0092314 and S100P mRNA was performed using a SYBR PCR kit (Takara), as previously described (13). The expression level of miR-671 was evaluated using the NCode miRNA qRT-PCR analysis (Invitrogen). The levels of circ_0092314 and S100P were normalized to the control GAPDH. The expression of miR-671 was normalized to the control U6. All primers used in this study were obtained from Genechem (Shanghai, China).",
"Total protein was extracted using a RIPA buffer (Beyotime, Beijing, China). SDS-polyacrylamide gel electrophoresis was performed using an equal amount of total protein. Then, the protein was transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Next, membranes were incubated with the primary antibodies: E-cadherin (Cell Signaling, MA, USA; 1:1000), Vimentin (Cell Signaling; 1:1000), S100P (Cell Signaling; 1:1000), AKT (Cell Signaling; 1:1000), p-AKT (Cell Signaling; 1:1000) and b-actin (Cell Signaling; 1:5000). Next day, the secondary antibodies were added and each protein band was detected by the ECL detection system (Amersham Biosciences, Buckinghamshire, UK).",
"The proliferation rate of PAAD cells were assessed by Cell Counting Kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. 5 × 10 3 cells were seeded into 96-well plates. At 24, 48, 72 and 96 h after transfection, 10 ml of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using an automatic microplate reader (BioTek, VT, USA).",
"Cell invasion assay was performed as previously reported (14). We placed 750 ml medium containing 10% FBS in the bottom chamber, and then seeded PAAD cells into the upper chamber for invasion assays (8-mm pore size, Corning, CA, USA) in 500 ml serum-free media. After 24 h, the invaded cells were stained with Giemsa (Sigma, St. Louis, MO, USA) for 15 minutes. Finally, the number of cells was counted using an Olympus microscope.",
"Single cells were seeded in 24-well ultra-low attachment plates (Corning, Acton, MA) containing serum-free medium supplemented with B27 (1:50; Invitrogen, Carlsbad, CA, USA), 20 ng/ml basic FGF (BD Biosciences, CA, USA) and 20 ng/ml EGF (Sigma, St. Louis, MO, USA). The cultures were fed with fresh serum-free medium and growth factors every other day. After 14 days of incubation, images of cells were captured and tumor spheres with a diameter more than 50 mm were counted using Image J software.",
"All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. Four-week-old Nude mice (n = 6 per group) were purchased from Beijing HFK Bioscience (Beijing, China). PAAD cells transfected as indicated were transplanted subcutaneously into the right flank of nude mice. The tumor volumes were measured every week and calculated as length (mm) × width 2 (mm 2 ) × 0.5. After 3 weeks after injection, the mice were sacrificed. The tumor were carefully removed, photographed and weighed.",
"A circ_0092314 fragment or a 3′-UTR sequence of S100P was inserted into a luciferase reporter plasmid (Ribobio, Guangzhou, China). Mutagenesis was performed using a QuickChange Site-Directed Mutagenesis kit (Stratagene, CA, USA). PAAD cells were transiently co-transfected with luciferase reporter plasmid with miR-671 mimic (or miR-671 inhibitor) using Lipofectamine 2000 Reagent (Invitrogen), along with the Renilla luciferase plasmid pRL-CMV (Promega, WI, USA). After 48 h of transfection, the cells were harvested and subjected to a Dual-Luciferase Reporter Assay system (Promega). The relative luciferase activities were normalized with the Renilla luciferase activities.",
"RIP assay was conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). PAAD cells were lysed with a RIP-lysis buffer and cell lysates were incubated with magnetic beads conjugated with anti-Ago2 antibody (Millipore) or control anti-IgG antibody (Millipore). After incubating with proteinase K, the immunoprecipitated RNAs were extracted and subjected to the qRT-PCR analysis.",
"All results were representative of at least three independent experiments. All data were shown as the mean ± standard deviation, and experimental data were evaluated using the Student's t-tests, one-way ANOVA tests and Wilcoxon signedrank tests. The statistical analysis was performed using SPSS 25.0 statistical software package. * P < 0.05 was considered statistically significant.",
"Using the circBase database (http://www.circbase.org/), we found that circ_0092314 was produced from human RANBP1 gene located at chr22: 20113099-20113439, and finally formed a circular transcript of 340 nt ( Figure 1A). To investigate the biological role of circ_0092314 in PAAD, we first analyzed its expression in 40 pairs of PAAD specimens and adjacent normal tissues using qRT-PCR assay. The expression level of circ_0092314 was significantly upregulated in PAAD tissues compared with adjacent normal tissues ( Figure 1B). Subsequently, we divided PAAD tissues as well/moderately and poorly differentiated groups, low TNM stages (I-II) and high TNM stages (III-IV) groups, as well as lymph node metastasisnegative and -positive groups. As shown in Figures 1C, D, E, the levels of circ_0092314 in the poorly differentiated group, high TNM stage group, or lymph node metastasis-positive group were significantly higher than those in the well/moderately group, low TNM stage or lymph node metastasis-negative group. Furthermore, the expression of circ_0092314 in PAAD tissues was divided into the low expression group and the high expression group based on the median values. Our Kaplan-Meier analysis showed that PAAD patients with high circ_0092314 expression had a significantly poorer overall survival and disease-free survival compared with patients with low circ_0092314 expression ( Figures 1F, G).",
"Then, the expression of circ_0092314 in four PAAD cell lines (AsPC-1, BxPC-3, SW-1990 and PaCa-2) and human pancreatic duct epithelial cell line (HPDE6-C7) was assessed by qRT-PCR experiments. Increased expression of circ_0092314 was observed in all PAAD cell lines compared with HPDE6-C7 cells ( Figure 1H). Among these cell lines, PaCa-2 cells expressed the highest level of circ_0092314, while AsPC-1 cells had the lowest level of circ_0092314 ( Figure 1G). RNase R was administrated to the extracted RNA from PaCa-2 cells. We found that the circular form (circ_0092314) can be resistant to RNase R, whereas the linear form (RANBP1 mRNA) was significantly decayed ( Figure 1I). In addition, qRT-PCR analysis of nuclear and cytoplasmic fractions of RNAs demonstrated that circ_0092314 was predominantly localized in the cytoplasm ( Figure 1J). Taken together, our results suggest that circ_0092314 is an abundant and stable circRNA overexpressed in PAAD cells.",
"We determined the biological roles of circ_0092314 in PAAD cells. To this end, we transfected PaCa-2 cells with specific siRNAs targeting circ_0092314, and transfected AsPC-1 cells with overexpression plasmid of circ_0092314, respectively.",
"Circ_0092314 expression was significantly silenced in PaCa-2 cells, whereas its expression was clearly increased in AsPC-1 cells following overexpression of circ_0092314 (Figures 2A, B). The proliferation of PaCa-2 cells was decreased by downregulation of circ_0092314, and overexpression of circ_0092314 promoted the proliferation of AsPC-1 cells (Figures 2C, D). To investigate the function of circ_0092314 in vivo, we established a nude mice xenograft model of PAAD by subcutaneous inoculation of PaCa-2 cells transfected with specific siRNA against circ_0092314, and of AsPC-1 cells transfected with circ_0092314 overexpression plasmid ( Figures 2E, F). Compared with tumors derived from The nuclearcytoplasmic fractionation assay showed that circ_0092314 was mainly located in the cytoplasm. *P < 0.05, **P < 0.01, ***P < 0.001.",
"the control cells, those derived from circ_0092314 siRNAtransfected PaCa-2 cells grew slowly ( Figure 2E). Moreover, overexpression of circ_0092314 significantly increased tumor growth ( Figure 2F). The results showed that the tumor volume of xenograft in circ_0092314 knockdown group was obviously smaller than the control group ( Figure 2G). The weight of tumors was lighter in circ_0092314 knockdown group than the control group, consistently ( Figure 2H). Conversely, the tumor volume and weight were significantly increased in the circ_0092314 overexpressed group (Figures 2G, H). These results confirmed the promoting effects of circ_0092314 on PAAD proliferation. ",
"EMT is a critical process that enhances the aggressive properties of cancer cells (2). Therefore, we investigated whether circ_0092314 could promote the EMT features of PAAD cells. Western blotting analysis showed that the expression of E-cadherin was increased, whereas the expression of Vimentin was decreased in PaCa-2 cells after the knockdown of circ_0092314 ( Figure 3A). In contrast, overexpression of circ_0092314 led to downregulation of Ecadherin and upregulation of Vimentin ( Figure 3A). Furthermore, we found that knockdown of circ_0092314 could significantly inhibit the invasive abilities and sphere-formation of PaCa-2 cells (Figures 3B-D). However, overexpression of circ_0092314 in AsPC-1 cells increased invasive abilities and sphere-forming abilities ( Figures 3B-D). Our qRT-PCR assay confirmed the downregulated expression of CD133 and CD44 in circ_0092314-silenced PaCa-2 cells, and the upregulated expression of CD133 and CD44 in circ_0092314-overexpressing AsPC-1 cells ( Figure 3E). These findings indicated that in circ_0092314 promotes EMT and CSC properties of PAAD cells.",
"According to the CircInteractome prediction database, a complementary sequence in circ_0092314 for miR-671 was found ( Figure 4A). The qRT-PCR assay was performed to study the expression of miR-671 in PAAD tissues and adjacent normal tissues. MiR-671 was expressed at lower levels in PAAD tissues compared with normal tissues ( Figure 4B). The expression of miR-671 in four PAAD cell lines was significantly decreased when compared with HPDE6-C7 cells ( Figure 4C). The survival curves from Kaplan-Meier Plotter database demonstrated that low level of miR-671 predicted poor outcome in PAAD patients ( Figure 4D). Then, a dual-luciferase reporter assay was conducted to confirm the interaction between circ_0092314 and miR-671. The results suggested that transfection with miR-671 mimic significantly attenuated the activity of luciferase reporter containing wild-type (WT) circ_0092314 fragment compared with control mimic, while transfection of miR-671 mimic failed to affect the activity of luciferase reporter containing mutant (MUT) circ_0092314 fragment (Figures 5A, B). Transfection with miR-671 inhibitor significantly increased the luciferase activity of the WT circ_0092314 fragment, but not the MUT circ_0092314 fragment (Figures 5A, B). RIP experiments suggested that circ_0092314 and miR-671 were preferentially enriched in anti-AGO2-containing complexes compared with anti-IgG ( Figure 5C). In addition, silencing of circ_0092314 can upregulate the expression of miR-671 in PaCa-2 cells, and overexpression of circ_0092314 in AsPC-1 cells can reduce the levels of miR-671 ( Figure 5D). Our rescue experiments suggested that knockdown of circ_0092314 suppressed the invasion and sphere formation in PaCa-2 cells, however coexpression of miR-671 inhibitor could abolish these effects of circ_0092314 knockdown ( Figure 5E). Also, we observed that overexpression of circ_0092314 induced cell invasion and sphere formation, and co-transfection with miR-671 mimic significantly reversed these effects ( Figure 5F). Collectively, these data demonstrated that circ_0092314 acts as a sponge of miR-761 and suppresses its expression in PAAD cells.",
"We used the TargetScan database to predict the potential targets of miR-671 and found that S100P was one of the targets of miR-671 ( Figure 4A). Then, our qRT-PCR results showed the upregulation of S100P in PAAD tissues compared with normal tissues ( Figure 6A). The protein expression of S100P was further validated in the Human Protein Atlas database (https://www. proteinatlas.org/). The results indicated that the expression levels of S100P were strongly expressed in PAAD tissues, however S100P protein expression was undetectable in adjacent normal tissues ( Figure 6B). Consistent with these results, the mRNA expression of S100P was highly expressed in PAAD cells than that in HPDE6-C7 cells ( Figure 6C). Kaplan-Meier survival curves suggested that high levels of S100P expression were associated with a significantly unfavorable overall survival ( Figure 6D). In order to explore whether miR-671 could directly bind to the 3′-UTR of S100P mRNA, we performed the luciferase reporter assay. Our results showed that overexpression of miR-671 remarkably reduced the luciferase activity of WT S100P 3′-UTR, but had no significant effect on the mutant one ( Figures 7A, B). Alternatively, inhibition of miR-671 increased the luciferase activity of WT S100P 3′-UTR, without affecting the MUT S100P 3′-UTR ( Figures 7A, B). S100P was known to promote EMT in colorectal cancer cells by activating the AKT pathway (7). In line with this finding, our western blotting analysis indicated that miR-671 overexpression led to the downregulation of S100P, Vimentin, and inactivation of AKT signaling, but the upregulation of E-cadherin in PaCa-2 cells ( Figure 7C). By contrast, inhibition of miR-671 in AsPC-1 cells led to suppression of E-cadherin, induction of Vimentin, and activation of the AKT pathway ( Figure 7C). We further examined the contribution of S100P in miR-671dependent EMT by performing rescue experiments. Remarkably, the ability of miR-671 in suppressing EMT as presented by decreased cell invasion/sphere formation, upregulation of E-cadherin, downregulation of Vimentin, as well as inactivation of the AKT pathway, was largely reversed by overexpression of S100P ( Figure 7C-E). Conversely, miR-671 inhibitor-induced EMT properties were partially abrogated by knockdown of S100P ( Figure 7C-E). These data implicated that miR-671 behave as an EMT suppressor in PAAD by targeting S100P and probably by repressing the AKT pathway. Overexpression of circ_0092314 induced S100P expression and activated the AKT pathway in AsPC-1 cells ( Figure 3A). Consistently, S100P was decreased, and the activity of the AKT pathway was suppressed following circ_0092314 knockdown in PaCa-2 cells ( Figure 3A).",
"Furthermore, Pearson correlation analysis indicated that the expression of circ_0092314 was negatively correlated with miR-671 expression, and positively correlated with S100P mRNA expression in PAAD cancer tissues ( Figure 8A). We also observed that there was a negative correlation between miR-671 expression and S100P mRNA expression in PAAD cancer tissues ( Figure 8A). Overall, in this study, we identified circ_0092314, as a novel oncogenic circRNA induces EMT and invasion of PAAD cells via elevating the abundance of S100P by sponging a tumor suppressor miR-671 ( Figure 8B).",
"The traditional prognostic markers for PAAD include histological subtype, vascular and perineural invasion, the presence of desmoplastic reaction, tumor budding and EMT (15). In addition, new and emerging prognostic biomarkers, including miRNA, long non-coding RNA and recently circRNA, have been reported (15,16). For instance, circ-LDLRAD3 was significantly upregulated in PAAD tissues and plasma, and a high level of circ-LDLRAD3 was positively associated with tumor venous invasion and lymphatic metastasis (17). Interestingly, circ-LDLRAD3 combined with CA19-9 was confirmed to have higher sensitivity and specificity for the diagnosis of PAAD (17). In addition, high expression of circ_0030235 was an independent prognostic indicator of unfavorable overall survival for PAAD patients according to a multivariate Cox analysis (18).",
"Here, we provided new evidence to show that the expression of circ_0092314 was significantly higher in clinical PAAD tissues than in adjacent normal tissues, and its expression was closely associated with aggressive behavior of PAAD. Importantly, we have shown that those PAAD patients with high circ_0092314 expression had worse clinical outcomes, indicating the potential of circ_0092314 as a promising prognostic biomarker for PAAD patients. Whether its expression was changed in the plasma of patients with PAAD, whether circ_0092314 expression is correlated with CA19-9 levels, deserve further investigation. Growing studies have supported the existence of complex interactions between circRNAs and miRNAs in PAAD cells (19,20), in which circRNAs regulates the expression and activity of miRNAs by competitively binding their target sites on protein-coding mRNAs. Here, we showed for the first time that circ_0092314 promotes EMT features by binding to miR-671 to induce the expression of S100P. Our bioinformatic analysis using the CircInteractome database has shown that circ_0092314 hosts multiple binding sites for diverse miRNAs and RNA-binding proteins (data not shown), suggesting that circ_0092314 probably act as sponges for different cytoplasmic miRNAs, or interact with RNA-binding proteins to generate RNA-protein complexes that can also control the EMT process and PAAD metastasis.",
"Over the last several years, although we should acknowledge that numerous efforts to improve the efficacy of surgery and chemotherapy in PAAD patients have been made, there are still few reliable biomarkers or effective therapeutic strategies for daily clinical practice in PAAD. Increasing evidence indicated that circRNAs have great potential to regulate cancer cell proliferation, apoptosis, invasion, EMT, metastasis and response to chemotherapy, implying that circRNAs may be used as novel potential therapeutic targets for treating various tumors including PAAD (9,10,21,22). Here, we confirmed that the knockdown of circ_0092314 suppressed the growth and invasion of PAAD cells in vitro, and resulted in reduced tumor A B D C FIGURE 6 | High S100P expression predicts poor prognosis in PAAD. (A) qRT-PCR analysis of S100P mRNA expression in PAAD tissues and adjacent normal tissues. (B) Immunohistochemical data was downloaded from the Human Protein Atlas database. The staining pattern for S100P protein in PAAD tissues and adjacent normal tissues were shown. (C) S100P mRNA expression in PAAD cells and HPDE6-C7 cells. (D) Kaplan-Meier curves for the overall survival of PAAD patients with high or low S100P expression (KM Plotter database). ***P < 0.001. size and tumor weight in vivo, suggesting that targeting circ_0092314 is a potential therapeutic strategy for PAAD.",
"Downregulation of miR-671 has been observed in a panel of human tumors, such as osteosarcoma (23), prostate cancer (24), lung cancer (25), breast cancer (26), and gastric cancer (27). In these tumors, miR-671 was demonstrated to show inhibit the malignant phenotypes of tumor cells by targeting SOX6, CCND2, and FOXM1 (23)(24)(25)(26)(27). However, miR-671 may function as an oncogenic miRNA in other types of cancer (28,29). In our study, miR-671 was identified as a downstream effector of circ_0092314 in PAAD cells, in which the tumor-promoting effects of c i r c _ 0 0 9 2 3 1 4 c o u l d b e bl o c k e d t h r o u g h mi R -6 7 1 overexpression. This ability of miR-671 to suppress the EMT properties of PAAD cells provides insights into the molecular mechanisms of PAAD metastasis and a path for inhibiting the metastatic spread of PAAD or other cancers. The detailed mechanisms by which miR-671 mediates EMT and PAAD cell invasion deserve future studies. S100P, a calcium-binding protein, can advance tumor progression and metastasis in pancreatic and several other cancers (6,30,31). S100P has previously been demonstrated to regulate the proliferation, migratory and invasive capabilities of PAAD cells (6). In addition, decreased PAAD growth was observed following S100P silencing in an orthotropic mouse model (6). S100P promotes EMT, migration and invasion of colon cancer cells by up-regulating S100A4 through AKT activation (7). Consistent with these reports, we found that S100P acts as an important EMT activator in PAAD cells, and its oncogenic functions might be involved in the activation of the AKT pathway. Together, our results suggest that dysregulation of the circ_0092314/miR-671/S100P axis is responsible for PAAD progression, and these molecules are potential therapeutic targets for suppressing the EMT in metastatic PAAD. The levels of S100P mRNA in PAAD cells transfected with miR-671 mimic or miR-671 inhibitor, respectively. (B) The activities of S100P 3′-UTR reporter containing WT or MUT miR-671 binding sites were determined using luciferase assay following co-transfection with miR-671 mimic or miR-671 inhibitor. (C) Western blotting analysis of the indicated proteins in PAAD cells transfected as indicated. (D, E) Cell invasion assay (left) and tumor sphere formation assay (right) in PaCa-2 cells transfected with (or without) miR-671 mimic, with (or without) S100P expression vector (D), and in AsPC-1 cells transfected with (or without) miR-671 inhibitor, with (or without) S100P siRNA (E). ***P < 0.001.",
"In summary, our findings support the idea that the circ_0092314/miR-671/S100P signaling plays crucial roles in regulating EMT phenotypes of PAAD cells and suggest that this signaling pathway might be an effective target for PAAD therapy.",
"The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.",
"The studies involving human participants were reviewed and approved by The Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China.",
"QF designed the experiments. QS, GZ, YZ, and JT performed the experiments. SX, HG, and XZ made significant revisions to the manuscript. All authors contributed to the article and approved the submitted version.",
"This study was supported by the National Natural Science Foundation of China (No. 81974381). A B FIGURE 8 | The correlation between circ_0092314 and miR-671/S100P expression in PAAD tissues. (A) The correlation between circ_0092314 expression and miR-671/S100P expression in PAAD tissues was examined using qRT-PCR assay. (B) A proposed mechanistic model in which circ_0092314 can sponge miR-671 to increase S100P expression, thereby promoting EMT and PAAD cell invasion."
] | [] | [
"BACKGROUND",
"MATERIALS AND METHODS",
"Clinical Tissue Samples",
"Quantitative Real-Time PCR (qRT-PCR)",
"Western Blotting",
"Cell Counting Kit-8 Assay",
"Transwell Invasion Assay",
"Tumor Sphere Formation Assay",
"In Vivo Tumor Formation Assay",
"Luciferase Reporter Assay",
"RNA Immunoprecipitation (RIP) Assay",
"Statistical Analysis",
"RESULTS",
"Circ_0092314 Is Highly Expressed in PAAD Tissues and Cells",
"Circ_0092314 Enhances the Proliferation of PAAD Cells In Vitro and In Vivo",
"Circ_0092314 Induces EMT in PAAD Cells",
"Circ_0092314 Binds to miR-671 and Suppresses Its Expression in PAAD Cells",
"MiR-671 Suppresses EMT and PAAD Cell Invasion by Inhibiting S100P Expression",
"DISCUSSION",
"DATA AVAILABILITY STATEMENT",
"ETHICS STATEMENT",
"AUTHOR CONTRIBUTIONS",
"FUNDING",
"FIGURE 1 |",
"FIGURE 2 |",
"FIGURE 3 |",
"FIGURE 4 |",
"FIGURE 5 |",
"FIGURE 7 |"
] | [] | [] | [
"Article 675442 1 (2021) CircRNA circ_0092314 Induces Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Elevating the Expression of S100P by Sponging miR-671",
"Article 675442 1 (2021) CircRNA circ_0092314 Induces Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Elevating the Expression of S100P by Sponging miR-671"
] | [
"Front. Oncol"
] |
210,933,014 | 2022-01-31T17:21:18Z | CCBY | https://doi.org/10.3389/fbioe.2020.00023 | GOLD | b949512a069c191518e9b1621acf392815896434 | null | null | null | null | 10.3389/fbioe.2020.00023 | 3004027425 | 32117912 | 7025592 |
A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs
published: 29 January 2020 Published: 29 January 2020
Roberto Narcisi
Jane Ru Choi
Jérôme Guicheux [email protected]
Cécile Boyer
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Gildas Réthoré
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
CHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale
PHU4 OTONNNantesFrance
Pierre Weiss
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
CHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale
PHU4 OTONNNantesFrance
Cyril D'arros
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Julie Lesoeur
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
SC3M -"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes
Université de Nantes
NantesFrance
Claire Vinatier
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
SC3M -"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes
Université de Nantes
NantesFrance
Boris Halgand
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
CHU Nantes
PHU4 OTONNNantesFrance
Olivier Geffroy
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS
NantesFrance
Marion Fusellier
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS
NantesFrance
Gildas Vaillant
CHU Nantes
PHU4 OTONNNantesFrance
Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS
NantesFrance
Patrice Roy
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Olivier Gauthier
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS
NantesFrance
Jérôme Guicheux
Inserm
UMR 1229, RMeS, Regenerative Medicine and Skeleton
Université de Nantes
ONIRIS
NantesFrance
Université de Nantes
UFR Odontologie
NantesFrance
SC3M -"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes
Université de Nantes
NantesFrance
CHU Nantes
PHU4 OTONNNantesFrance
Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS
NantesFrance
Erasmus University Rotterdam
Netherlands
University of British Columbia
Canada
A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs
Front. Bioeng. Biotechnol
823published: 29 January 2020 Published: 29 January 202010.3389/fbioe.2020.00023Received: 25 September 2019 Accepted: 10 January 2020ORIGINAL RESEARCH Edited by: Martijn van Griensven, Maastricht University, Netherlands Reviewed by: *Correspondence: Specialty section: This article was submitted to Tissue Engineering and Regenerative Medicine, a section of the journal Frontiers in Bioengineering and Biotechnology Citation: Boyer C, Réthoré G, Weiss P, d'Arros C, Lesoeur J, Vinatier C, Halgand B, Geffroy O, Fusellier M, Vaillant G, Roy P, Gauthier O and Guicheux J (2020) A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs.hydrogelcell therapycartilageregenerative medicineosteoarthritis
Articular cartilage (AC) may be affected by many injuries including traumatic lesions that predispose to osteoarthritis. Currently there is no efficient cure for cartilage lesions. In that respect, new strategies for regenerating AC are contemplated with interest. In this context, we aim to develop and characterize an injectable, self-hardening, mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropymethyl cellulose (Si-HPMC) mixed with silanised chitosan. The in vitro cytocompatibility of Si-HPCH was tested using human adipose stromal cells (hASC). In vivo, we first mixed Si-HPCH with hASC to observe cell viability after implantation in nude mice subcutis. Si-HPCH associated or not with canine ASC (cASC), was then tested for the repair of osteochondral defects in canine femoral condyles. Our data demonstrated that Si-HPCH supports hASC viability in culture. Moreover, Si-HPCH allows the transplantation of hASC in the subcutis of nude mice while maintaining their viability and secretory activity. In the canine osteochondral defect model, while the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without cASC, revealed a significant osteochondral regeneration. To conclude, Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC as well as the regeneration of osteochondral defects in dogs when implanted alone or with ASC.
INTRODUCTION
Articular cartilage (AC) is a complex porous permeable extracellular matrix that surrounds specific cell type called chondrocytes. Due primarily to its avascular and aneural structural organization (Clouet et al., 2009), AC does not heal spontaneously when injured. AC defects affect approximately two million patients every year in Europe and in the United States (Mumme et al., 2016), and they remain a clinical challenge due to the paucity of routinely translatable therapeutics to treat this condition. Degenerative changes affected the cartilage and the bone surrounding the lesion (Schinhan et al., 2012). Therapeutic options to repair AC defects include microfracture, autologous osteochondral grafting (Brittberg et al., 2016), and autologous chondrocyte implantation. These therapeutic options are, however, subject to major logistical and clinical issues, such as their short-lived clinical benefits in particular .
Articular cartilage defects, when left untreated or partially healed, dramatically increase the predisposition to osteoarthritis (OA). OA is a painful, debilitating, and slowly progressing degenerative and inflammatory joint disease that is initiated by cartilage damage prior to its culmination in alteration of all of the joint tissues (i.e., synovium, bone, muscle, tendon, and ligament) (Hunter and Bierma-Zeinstra, 2019). As there is no curative treatment, prosthetic joint replacement remains the last-recourse for alleviation of OA-associated pain symptoms . In light of this, a number of innovative strategies to repair or regenerate cartilage and subchondral bone very early in the degenerative joint cascade have been proposed. Among these strategies, tissue engineering (TE) is thought to be one of the most promising approaches (Makris et al., 2015). It consists primarily of combining reparative cells with a biomaterial capable of supporting cell transplantation as well as their engraftment, viability, growth, differentiation, and secretory activity.
Cartilage tissue engineering was initially focused on the use of chondrocytes of articular origin (Brittberg et al., 1994). However, the difficulties encountered, particularly with loss of the chondrocyte phenotype during their in vitro expansion phase and the morbidity of the donor site (Benya and Shaffer, 1982;Vinatier et al., 2009a), has led researchers to explore new cell sources. Nasal chondrocytes have been proposed as an alternative to articular chondrocytes (Vinatier et al., 2007), and their successful clinical use in association with a collagen membrane was recently reported (Mumme et al., 2016). Another alternative to articular chondrocytes that has been widely investigated is adult mesenchymal stromal cells (MSC). MSC can readily be isolated from various tissues such as bone marrow (BMMSC) (Friedenstein et al., 1968), adipose tissue (ASC) (Fraser et al., 2006), and synovial fluid (SFAC) (Jones et al., 2004). They have the capacity for self-renewal and differentiation into a range of cell types encompassing chondrocytes in particular, as well as other cell types of the musculoskeletal system (Pittenger, 1999). In addition, recent studies have shown that MSC are able to secrete a myriad of biological factors, either directly or through the release of extracellular vesicles (Heldring et al., 2015). These cytokines, chemokines, and growth factors can exert immunomodulatory effects (Le Blanc and Davies, 2015), reduce tissue damage, and promote repair or healing processes. Finally, as MSC generally fail to express the class two major histocompatibility complex molecules, they may be candidates for allogeneic transplantation without host alloreactivity (Ankrum et al., 2014) and hence of particular clinical interest for cartilage TE. This immune privilege of MSC, although it is still a matter of considerable debate (Ankrum et al., 2014), paves the way of the development of "off-theshelf " MSC therapies.
A number of biomaterials have been proposed in recent years as cell carriers for cartilage and subchondral bone TE (Morris et al., 2010). Among these scaffolding biomaterials, hydrogels have been widely investigated in light of their favorable physicochemical and biological properties (e.g., high water content, biocompatibility, tunable mechanical properties, and permeability) (Hoffman, 2001;Guan et al., 2017). With the aim of developing a self-crosslinking hydrogel, we have devised and patented injectable silanized hydroxypropylmethyl cellulose (Si-HPMC). Si-HPMC was initially used for the 3D culture of MSC and chondrocytes (Vinatier et al., 2005(Vinatier et al., , 2009bMerceron et al., 2011). More recently, Si-HPMC has also been successfully used for MSC based regeneration of soft tissues such as myocardium (Mathieu et al., 2012) and colon (Moussa et al., 2017). It has also been preclinically tested, albeit with less encouraging results, for the repair of stiffer tissues such as cartilage (Vinatier et al., 2007;Portron et al., 2013). Interestingly, a converging body of proof has recently indicated that MSC are "touchy-feely" cells that are particularly able to sense the biomechanical properties (e.g., stiffness, elasticity, and relaxability) of their micro-environment (Discher et al., 2009). When cultured on stiff materials, they preferentially engage in osteochondral differentiation, while on soft materials they commit to brain or cardiac differentiation pathways (Engler et al., 2006;Murphy et al., 2014). In light of these data, there appears to be ample merit in developing hydrogels for cartilage TE that have mechanical properties that mimic those of the target tissue. To address this issue, we sought to determine whether adding mechanical reinforcement to Si-HPMC may be a viable strategy to develop materials that not only have improved mechanical properties but also increased cartilage regenerative capacity. In light of the silanol-dependent reticulation process of Si-HPMC and our ability to silanize a large panel of biomolecules including polysaccharides (patent WO2011089267), we have synthesized a hybrid hydrogel of Si-HPMC and Si-chitosan. Chitosan is a natural chitin-derived polymer extracted from the exoskeleton of crustaceans. It is composed of D-glucosamine and N-acetyl-D-glucosamine (Oprenyeszk et al., 2015), which can mimic the glycosaminoglycan content of cartilaginous extracellular matrix (Lahiji et al., 2000;Hoemann et al., 2007). Several studies have also shown that chitosan enhances the viability of bovine chondrocytes (Sechriest et al., 2000) and the chondrogenic differentiation of MSC (Cho et al., 2004;Dang et al., 2006) in culture. In addition, chitosan has mucoadhesive properties that are useful for scaffold anchorage to native cartilage (de Campos et al., 2005). Chitosan is also positively charged (Morris et al., 2010), sterilizable, biocompatible (Debnath et al., 2015), and biodegradable (Lee and Mooney, 2001). All of these characteristics make chitosan an appropriate candidate for silanization and further combination with Si-HPMC for use in cartilage TE.
The aim of this study was to develop and characterize a new mechanically reinforced hybrid hydrogel made of Si-HPMC and Si-chitosan (referred to here as Si-HPCH) and to evaluate its ability to repair osteochondral defect in a dog model. We first characterized the mechanical and rheological properties of Si-HPCH. The viability of human MSC was then assayed after in vitro culture either in contact with or embedded in Si-HPCH. The viability of human MSC was also investigated after transplantation with Si-HPCH in the subcutis of nude mice. Lastly, we assessed the preclinical potential of Si-HPCH in cartilage repair using a canine model of osteochondral knee defects.
MATERIALS AND METHODS
Si-HPMC and Si-Chitosan Synthesis and Hydrogel Preparation
Silanized hydroxypropyl methylcellulose and an acidic buffer solution (ABS, pH 3.6) were prepared as previously described (Bourges et al., 2002a,b). Silanized chitosan (Si-chitosan) was synthesized as follows. One hundred milliliters of chitosan solution (1% wt/v, HCl, pH 3.0) was poured into a round bottom flask and 1 eq (1.55 mL) of (3-Isocyanatopropyl)triethoxysilane was added. The mixture was then stirred for 3 h at room temperature and then initially dialyzed (molecular weight cutoff of 6-8 kDa) for 2 days against NaOH (0.1 M) with frequent replacement of the dialysis solution. The sample was then dialyzed against water with frequent replacement of the dialysis solution until the conductimetry monitoring indicated no further release. The dialyzes eliminated the non-grafted silane derivatives used for siloxane grafting onto the HPMC and chitosan. As a final step, the purified gels were lyophilized.
Two types of hydrogel were prepared. The first one was made of Si-HPMC alone and prepared by dissolving Si-HPMC polymer (3 wt%) in 0.1 M NaOH aqueous solution. The second one, referred to as Si-HPCH, was composed of Si-HMPC and Sichitosan. It was prepared as follows: Si-HPMC polymer (3 wt%) was dissolved in 0.1 M NaOH aqueous solution; Si-chitosan (3 wt%) was then dissolved in the 3 wt% Si-HPMC aqueous solution (0.1 M NaOH).
The hydrogel precursor solution was then obtained by mixing 1 volume of the above Si-HPMC or Si-HPCH basic solution contained in a Luer lock syringe, with a 0.5 volume of an ABS prepared in our laboratory (Bourges et al., 2002a,b) at pH 3.6 in another Luer lock syringe, by interconnection of both syringes; the final pH of the mixture was 7.4. This precursor mixture was injectable for 30-40 min, at which time the gel point was reached.
Mechanical and Rheological Characterization
The viscoelastic modulus (storage modulus G ) and the breaking strength (σ) were obtained using a HAAKE MARS rheometer (Thermo Fisher Scientific, United States) using a plate geometry (PP20Ti, titanium plateau with a 20 mm diameter). Immediately after mixing, the liquid hydrogel precursor solutions were injected into a mold (20 mm in diameter and 5 mm in height) and the measurements started 5 days later (full cross-linking of the network). The storage modulus (G ) and the breaking strength (σ) were monitored as a function of strain (from 0.1 to 3,000 Pa) at a constant frequency and temperature (1 Hz and 23 • C). Each condition was measured in quintuplicate from 2 different batches of both hydrogels.
Rheological measurements were performed using a Haake MARS rheometer (ThermoHaake R , Germany) with a titanium cone-plate geometry (60 mm in diameter, 1 • cone angle, 52 µm gap). Steady shear tests were carried out at 23 • C on Si-HPMC (2 wt%) and Si-HPCH (4 wt%) solutions. The operating shear rate ranged from 0.1 to 100 s −1 . Different flow curves were fitted and extrapolated to lower shear rates by the Cross equation (Cross, 1969). The injectability properties were investigated using a compression testing device (TAHDplus) with a 5 kg load cell for the measurements at an injectability rate of 2 mm per second through a 3 mL syringe equipped with an 18G needle. A syringe containing 1 mL Si-HPMC and Si-HPCH was set and the injection force was measured.
Cell Viability
Adipose Stromal Cell (ASC) Culture Adipose Stromal Cell were obtained from human patients (hASC) undergoing liposuction and who had provided written consent (ethics committees: Agence de BioMedecine no PFS08-018, the legislation code L.1211-3 to L.1211-9: residues obtained during a surgical procedure, performed in the interest of the person operated, can be used for scientific research), or from autologous canine adipose tissue (cASC) harvested from the gluteal area (APAFIS #4446). Briefly, and as previously described , human lipoaspirate and canine adipose tissue were shredded into small pieces and washed extensively with Hanks' balanced salt solution (HBSS) to remove debris. The washed adipose tissue was treated with collagenase (0.025%) in HBSS for 1 h at 37 • C under gentle agitation. The collagenase (Sigma-Aldrich C2674) was inactivated by the addition of an equal volume of Dulbecco's modified Eagle's medium (DMEM-Glutamax; Gibco) supplemented with 1% penicillin/streptomycin (P/S) containing 10% fetal calf serum (FCS; control medium). The digested product was then centrifuged at 250 × g for 5 min to separate the adipose fraction from the stromal fraction. The supernatant was removed, and the stromal cells were resuspended in the control medium and filtered through a 70 µm nylon mesh filter. The filtrate was centrifuged and the cells were resuspended in red blood cell lysis buffer. The lysis reaction was stopped by the addition of control medium. The suspension was centrifuged, and the cells were finally resuspended in control medium and plated at a density of 5 × 10 4 cells/cm 2 .
Adipose Stromal Cell isolated using the protocol described above have been extensively characterized in our laboratory (for details see Merceron et al., 2009Merceron et al., , 2011. The medium was replaced 24 h after seeding to remove non-adherent cells. To prevent spontaneous differentiation, primary cultures (P0) of ASC were grown to approximately 80% confluency and then detached from the cell culture flask using trypsin-EDTA. All of the culture incubations were performed at 37 • C in a humidified atmosphere containing 5% CO 2 , and the medium was replaced every 2 days. The different experiments with hASC and cASC were carried out with cells at passage 2.
Cell Viability in 2D
Human adipose stromal cells (n = 3) and cASC (n = 1) viability was evaluated by the methyl tetrazolium salt (MTS) assay (Promega, United States). The cells were cultured in physical contact with Si-HPMC or Si-HPCH. hASC and cASC were seeded onto culture plates and were allowed to attach to 48well plates at a final density of 10,000 cells per cm 2 . After 24 h, the culture medium was removed and either Si-HPMC or Si-HPCH hydrogel (200 µL/well) was added on top of the cell layer. After 1 h of gelation at 37 • C, 200 µL of culture medium was added to each well and replaced every 2 days. As a negative control, cells were cultured in the presence of actinomycin-D (5 µg/mL), a well-known inducer of cell death. Finally, the MTS assay was performed on days 0, 1, and 7 for the hASC and on days 0 and 7 for the cASC. The MTS assay is based on the reduction of MTS tetrazolium compound by viable cells, thereby generating a colored and soluble formazan product in the culture medium. The amount of the colored product was determined by the optical density reading at 490 nm (Victor3V 1420 Multilabel Counter). Each condition was tested in triplicate from three different donors of hASC and one donor of cASC.
Cell Viability in 3D
3D cell viability was evaluated by the Live/Dead Cell Viability assay (Thermo Fisher Scientific, MA, United States) using hASC (n = 3) or cASC (n = 1) cultured three-dimensionally in hydrogels at a final density of 1 × 10 6 cells per mL of hydrogel. The ASC were collected and gently mixed with Si-HPMC or Si-HPCH. Cellularized hydrogels were molded and allowed to gelate in the wells of a 48-well plate at 37 • C for 1 h. After gelation, 200 µL of culture medium were added to each well and replaced every 2 days (Vinatier et al., 2007). As a negative control, cells were cultured in 3D in Si-HPMC hydrogel in the presence of actinomycin-D (5 µg/mL). A Live/Dead Cell Viability assay was performed according to the manufacturer's instructions on days 0, 1, and 7 for hASC and on days 0 and 7 for cASC. Green fluorescence can be observed in the living cells due to the conversion of non-fluorescent calcein AM into green-fluorescent calcein by the intracellular esterase activity in live cells. Dead cells become red-fluorescent due to the uptake of ethidium homodimer-1 as a result of the loss of plasma membrane integrity. The red and green fluorescence were observed with a confocal microscope [Nikon D-eclipse C1 (Ar/Kr)]. The numbers of green and red cells were quantified with Volocity software and the results shown as the percentage of living cells. Each condition was tested in triplicate from three different donors of hASC and one donor of cASC.
In vivo Experiments
Nude Mice Subcutis
To investigate whether our biomaterial was able to support the viability of cells in vivo, a single-cell suspension of 1 × 10 6 hASC was gently mixed with Si-HPMC or Si-HPCH hydrogel prior to subcutaneous implantation in 7-week-old female Swiss nude mice (Charles River, L'Arbresle, France), as described previously (Vinatier et al., 2009b;Merceron et al., 2011). All of the animals were treated in accordance with the Medical Animal Care Guidelines of Nantes University (APAFIS#4213). General anesthesia was achieved in an induction chamber with isoflurane (2%) delivered in O 2 and maintained through an individual mask. The Si-HPMC and Si-HPCH cellularized hydrogels were injected subcutaneously in the back along each side of the dorsum of nude mice. The implantations were performed under aseptic conditions. The two different conditions ("Si-HPMC" and "Si-HPCH") were tested (n = 4), four animals received implants (two implants per animal). The animals were sacrificed 6 weeks after implantation. The mice were euthanized by an overdose of isoflurane in an induction chamber. The hydrogels were individually explanted and the samples were processed histologically as described below.
Canine Osteochondral Defects
To investigate whether or not Si-HPCH biomaterial associated with autologous ASC was able to support the repair of osteochondral defects, we undertook a preclinical experiment in dogs. All animal handling and surgical procedures were conducted according to European Community guidelines for the care and use of laboratory animals (EU Directive 2010/63), after approval by the Pays de la Loire Animal Ethical Committee (APAFIS #4446) and the Oniris College of Veterinary Medicine Animal Welfare Committee. Surgical procedures were performed with the animals under general anesthesia and under surgical aseptic conditions. Self-hardening, mechanically reinforced hydrogel was implanted into calibrated 6 mm-diameter osteochondral defects. Twelve clinically healthy adult Beagle female dogs with body weights ranging from 10 to 15 kg, split into two groups, were used in this study. Osteochondral defects were generated using a 6-mm in diameter orthopedic bur to create a calibrated 6 mm × 5 mm deep osteochondral defect.
A total of 14 defects were created on the medial femoral condyles, one defect per dog (except for two dogs that had two defects, one on each femur). The defects of six dogs were filled with Si-HPCH alone (the Si-HPCH group), while the defects of the six other dogs were filled with Si-HPCH associated with autologous ASC (2 × 10 6 cells/mL; the Si-HPCH/cASC group).
In an attempt to reduce the number of animal lives, a second defect was generated on the contralateral limb in two dogs of the Si-HPCH group and left empty (the Empty Defect group). These two empty defects were used as critical-sized controls. The dogs were sacrificed 4 months after the implantation using intravenous overdose of pentobarbital. Femoral extremities were immediately dissected and prepared for further histological analysis.
Histological Analyses
Subcutaneous samples were fixed in 10% formalin and embedded in paraffin. Embedded samples were stained as described previously (Vinatier et al., 2009b;Merceron et al., 2011). Briefly, the paraffin-embedded explants were cut into 5 µmthick sections passing through the middle of the sample. The sections were deparaffinized by immersion in methylcyclohexane, rehydrated by a graded series of ethanol, and then rinsed in distilled water. The tissue sections were stained with Alcian blue (AB). The sections were then visualized and scanned using a NanoZoomer device (Hamamatsu Photonics, Japan).
Canine femoral extremities were fixed in 10% formalin and embedded in resin (Tecknovit R 9100, Heraeus Kulzer, Japan). The embedded samples provided 7 µm thick sections according to the osteochondral defect axis. The resin sections were then deplastified using acetone, rehydrated by a graded series of ethanol, and rinsed in distilled water. The tissue sections were stained with hematoxylin-eosin (HE), Safranin-O (SO), or Movat pentachrome (Movat). For IHC, the sections were incubated with primary antibodies against type II (MP Biomedicals 08631711, United States) or type I (Abcam ab6308, United Kingdom) collagen. The primary antibodies were detected using a kit (DAKO Agilent, Agilent Technologies, United States) with DAB substrate as per the manufacturer's instructions. The sections were then visualized and scanned using a NanoZoomer device (Hamamatsu Photonics, Japan).
To quantify the histological repair of osteochondral defects, we used a modified version of the grading scale described by O'Driscoll et al. (1986). This histological scoring was based on 10 histological parameters ( Table 1). We assigned a score ranging from 0 to 4 points to each criterion for a total of 40 points. Three investigators with extensive experience in cartilage histological analyses performed a blind rating of the stained sections.
Statistical Analyses
The results are expressed as means ± SEM of replicate determinations. Statistical analyses were performed using GraphPad Prism software (San Diego, United States). Comparative studies of means were performed by using the Mann-Whitney test when two conditions had to be compared and the Kruskal-Wallis test (post hoc: Dunn's Multiple comparison test) when more than two conditions had to be compared.
RESULTS
Rheological and Mechanical Properties
The objective was to develop a new hybrid hydrogel (Si-HPCH) by adding Si-Chitosan to an Si-HPMC hydrogel that was already being used for tissue repair. The formulation was characterized to determine its potential usefulness as a hydrogel for tissue engineering. Throughout the physicochemical characterizations, we evaluated the selected formulation for biological investigation. A score ranging from 0 to 4 points was assigned to each criterion.
First, we measured the viscosity of the solutions. As reported in Figure 1A, the viscosity increased significantly when Si-chitosan was added compared to Si-HPMC alone. The viscosity profile of a solution is usually indicative of the injectability behavior. Nevertheless, the syringeability of the Si-HPCH solutions was monitored. As expected, the force required to inject increased when Si-chitosan was added, but the mixed solution remained readily injectable, with a value of 2.50 N ( Figure 1B). Having evaluated the rheological properties of our hydrogels, we sought to investigate the mechanical properties of Si-HPCH. Its storage modulus (G ) and breaking strength (σ) were hence measured.
The results, plotted in Figure 1C, show a 9-fold increase in G when Si-HPMC was combined with Si-Chitosan. In parallel, Figure 1D indicates that Si-HPCH exhibits a breaking strength 5.5-fold lower than that of Si-HPMC. Moreover, at the same concentration (4 wt%) of total polymer, Si-HPCH exhibited a 30% higher stiffness (G ) and a 30% decrease in the breaking strength.
Cell Viability
To investigate the viability of ASC cultured in contact or embedded in Si-HPCH, we performed several in vitro and in vivo tests, (i) in 2D culture with an MTS-based metabolic assay, (ii) in 3D culture with a viability assay, and (iii) after subcutis implantation in nude mice. The cytotoxicity of Si-HPCH hydrogel was first evaluated using hASC. As shown in Figure 2A, the MTS activity of hASC cultured in contact with Si-HPCH or Si-HPMC gradually increased from day 0 to day 7 (a 2-fold increase). After 7 days of culture, no difference in metabolic activity was observed between the three conditions (i.e., CTRL, Si-HPMC, and Si-HPCH). As expected, in the presence of actinomycin-D, the MTS activity decreased drastically from day 0 to day 7. Taken together, these data indicate that neither of the hydrogels resulted in a major alteration of the metabolic activity of 2D-cultured hASC. To further analyze whether Si-HPCH can influence the behavior of clinically relevant MSC, we also cultured hASC embedded three-dimensionally in Si-HPCH. Figure 2B illustrates the viability of hASC after 7 days of culture in 3D into Si-HPMC hydrogel in the presence of actinomycin-D (Panel Actino) or not (Panel Si-HPMC) and into Si-HPCH hydrogel (Panel Si-HPCH). Figure 2C shows the number of viable hASC when cultured three-dimensionally in Si-HPCH or Si-HPMC compared to cells cultured in Si-HPMC in the presence of actinomycin D.
Interestingly, a slight but significant increase was observed at day 7 for Si-HPCH, while a barely discernible decrease was observed for cells cultured in Si-HPMC. As expected, actinomycin-D induced a progressive decrease in hASC viability. Collectively, these data indicate that the addition of Si-chitosan to Si-HPMC did not significantly alter the viability of 3Dcultured hASC.
Finally, to determine whether Si-HPCH can support the viability of hASC in vivo, we mixed hASC (1 × 10 6 cells/mL of hydrogel) with Si-HPMC or Si-HPCH prior to subcutis injection (250 µL) in nude mice. Both hydrogels were implanted alone as negative control. Six weeks after implantation, samples were retrieved and histologically analyzed. Macroscopically, no inflammation was observed in any condition. Interestingly, no cellular infiltration was observed in the Si-HPMC and Si-HPCH conditions as shown in Figure 2D (panels a and c).
To assess whether the embedded hASC could produce extracellular matrix components, we performed Alcian Blue staining to detect glycosaminoglycans (GAG). As indicated in Figure 2D (panels b and d), when the cells were implanted with Si-HPMC enriched with Si-chitosan (panel d), the cells produced a high amount of GAG as evidenced by the intense blue staining compared to cells implanted with Si-HPMC alone (panel b). Altogether, these in vivo results confirm the data generated by 2D and 3D culture and they highlight that Si-HPCH allows transplantation of viable hASC in the subcutis of nude mice while maintaining their viability and GAG-production activity for 6 weeks.
Prior to embarking on a preclinical experiment of canine osteochondral defect repair with Si-HPCH and autologous ASC, we sought to check whether Si-HPCH can also support the viability of canine ASC. As shown in Figure 3A, we observed that the metabolic activity of cASC cultured in 2D while in contact with Si-HPMC or Si-HPCH increased from day 0 to day 7.
Whereas this increase was found to be statistically significant for cells cultured in contact with Si-HPMC, we failed to detect a significant increase in the metabolic activity with Si-HPCH. As expected, the MTS activity of cells cultured on a plastic substrate (CTRL) increased significantly, while that of cells cultured in the presence of actinomycin-D (Actino) dropped significantly from day 0 to day 7. To further document the effects of hydrogels on cell viability, we also analyzed the viability of cASC cultured 3D in Si-HPMC or Si-HPCH. Figure 3B illustrates the viability of cASC after 7 days of culture in 3D into Si-HPMC hydrogel in the presence of actinomycin-D (Panel Actino) or not (Panel Si-HPMC) and into Si-HPCH hydrogel (Panel Si-HPCH). As shown in Figure 3C, when cASC were 3D-cultured in Si-HPMC, there was a significant decrease in viability. Notably, cells cultured in Si-HPCH did not exhibit a decrease in cell viability. Viewed together, these data strongly suggest that Si-HPCH is a 3D-supportive scaffold for cASC viability.
Repair of Osteochondral Defects in Dog
Having demonstrated that enrichment of Si-HPMC with Sichitosan yields a mechanically reinforced hydrogel that is able to support the viability of canine and human ASC, we wanted to test the ability of this newly developed hydrogel to regenerate FIGURE 1 | Rheological and mechanical.1pc properties of Si-HPMC and Si-HPCH hydrogels. As described in the section "Materials and Methods," the viscosity (A) was measured with a MARS rheometer with a cone/plate geometry; the injectability (B) was investigated using a TAXT2 Texture Analyzer at an injectability rate of 17 sec per mL; the storage modulus (C) and the breaking strength (D) were measured with a MARS rheometer using a plate geometry. * * p < 0.01 compared to the Si-HPMC condition (Mann-Whitney). articular cartilage in a canine model of osteochondral defects. Osteochondral defects (6 mm in diameter and 5 mm in depth) were created in the knees of adult dogs and filled with Si-HPCH in the absence or presence of autologous ASC. All of the animals survived and fully recovered from the surgery. At 4 months after the surgery, the dogs were euthanized and condyles isolated for histological analyses of tissue repair. Macroscopic observation of the condyles revealed that one sample from the Si-HPCH/cASC group exhibited a fracture at the implanted site. This sample was, therefore, excluded from the study.
As illustrated in Figure 4, macroscopic observation ( Figure 4A) of the samples did not reveal any abnormal findings indicative of inflammation. To further compare the histological features of the repair tissues, we then performed HE staining ( Figure 4B). Figure 4A illustrates the worst (panels b, d, and f) and the best (panels c, e, and g) cartilage defect repair outcomes for each group compared to healthy cartilage (panel a). Notably, while there was barely any detectable repaired tissue in the empty defects, a newly formed tissue, partially covering the entire surface of the defects, was found in all the filled defects. Figure 4B illustrates the worst (panels b, d, and f) and the best (panels c, e, and g) cartilage defect repair outcomes for each group compared to healthy cartilage (panel a). These analyses of the repair tissue in the empty defect group (Figure 4B, panel b and c) revealed the presence of a fibrous tissue that was unable to induce an ad integrum covering of the defects.
To more specifically characterize the repaired tissue, histological (Safranin O and Movat pentachrome staining) and immunohistological analyses (type I and II collagen) were then performed (Figure 5). The repair tissue in the empty defect group (panel Empty Defect) was Safranin O-negative, thus indicating the absence of GAG, and did not exhibit any positivity for the immunodetection of type II collagen. A faint but detectable positive immunostaining for type I collagen was also observed, thereby confirming the fibrous nature of the repaired tissue. The defects treated with Si-HPCH or Si-HPCH/ASC were almost completely covered (Figure 5, panel Si-HPCH and Si-HPCH/ASC). Si-HPCH alone was found to induce an almost complete covering of the defects, while Si-HPCH associated with cASC, induced an ad integrum filling of the defects. In both conditions, the repaired tissue stained positively for the presence of GAG (Figure 5, SO) and collagen (Figure 5, Movat). Movat staining also revealed substantial bone remodeling in the subchondral area. Interestingly, in both conditions, the repaired tissue exhibited strong and diffuse immunostaining for type II collagen (Figure 5, Coll2) while remaining negative for type I collagen (Figure 5, Coll1). Altogether, these data very much suggest the production of a repaired tissue with cartilaginous FIGURE 2 | In vitro and in vivo viability of hASC. (A) MTS activity of hASC cultured in 2D. Cell viability was evaluated in 2D after molding Si-HPMC or Si-HPCH hydrogels on top of the cell layer (10,000 cells/cm 2 ). As described in the section "Materials and Methods," an MTS assay was performed on days 0, 1, and 7. The positive control (CTRL) was obtained by growing hASC alone, while the negative control was obtained by growing hASC in the presence of actinomycin D (Actino; 5 µg/mL). The results are expressed as the percentage of day 0 for each respective condition. * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (B,C) 3D viability of hASC cultured in Si-HPMC or Si-HPCH hydrogels. Cell viability was evaluated in 3D after molding hydrogels mixed with 1 × 10 6 hASC on days 0, 1, and 7 by the Live/Dead Cell Viability assay. The negative control was obtained by adding actinomycin-D (Actino; 5 µg/mL) to the culture medium. Pictures of representative samples of hASC into Si-HPMC cultured in the presence of actinomycin-D (Actino), into Si-HPMC (Si-HPMC) or into Si-HPMC (Si-HPCH) at day 7 are shown (B). The scale bar represents 100 µm. Live and dead cells were then counted (C). The results are expressed as the percentage of day 0 for each respective condition. * p < 0.05; * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (D) In vivo hASC viability after subcutaneous implantation. 250 µL of Si-HPMC (panels a and b) or Si-HPCH (panels c and d) were implanted alone (panels a and c) or mixed with human ASC (panels b and d; 1 × 10 6 cells/mL of hydrogel) in the subcutis of nude mice. After 6 weeks, explanted samples were histologically prepared for Alcian Blue staining. The back arrows indicate hASC while the white arrows indicate chitosan. The scale bar represents 100 µm.
features including the presence of GAG and type II collagen and the absence of type I collagen. Finally, to quantitatively assess the formation of a repaired tissue in the different conditions, HE staining (Figure 4) in association with the macroscopic appearance as well as SO, Movat, and anti-type I/II collagen staining ( Figure 5) were used to provide a modified O'Driscoll scoring (Figure 6). Three independent assessors blindly scored the various samples. For the filled conditions, the O'Driscoll-based repair scoring (Figure 6) indicated that the Si-HPCH samples were more consistently repaired than the Si-HPCH/ASC-treated defects. Indeed, the mean score of the Si-HPCH samples was 27.6 ± 2.82 while for the Si-HPCH/cASC samples the mean score was 22.31 ± 8.14. Unfortunately, and probably because of the reduced number of samples, no statistical difference was recorded between the different groups, even though a clear tendency was noted.
DISCUSSION
In this study, we showed in a dog model of osteochondral defects that it is possible to repair articular cartilage with a hybrid hydrogel made of cellulose-and chitosan-derived polymers. After having shown that associating Si-HPMC with Si-chitosan led to the formation of a mechanically reinforced hydrogel that can support MSC viability in vitro and in vivo, we were able to clearly demonstrate the ability of this novel self-setting hydrogel to contribute to the repair of osteochondral defects in a canine model in the absence or presence of MSCs.
Due to the limited ability of cartilage to heal, untreated joint cartilage damage leads to progressive tissue degeneration that drastically increases the risk of the development of osteoarthritis (Clouet et al., 2009). It has been estimated that patients with cartilage lesions have a 5-fold increased risk of osteoarthritis (Gelber, 2000). The development of new therapies in cartilage regenerative medicine is, therefore, an issue of considerable importance. In order to devise treatments for cartilage damage, strategies combining cells and biomaterials have been developed. As a result of their physicochemical and biological properties, hydrogels are presently considered to be ideal candidates for cartilage tissue engineering. Over the past decade, our team has developed a hydrogel made of cellulose, referred to as Si-HPMC (Bourges et al., 2002a,b). Previous studies have shown that this Si-HPMC hydrogel is an injectable, self-setting, and biocompatible hydrogel that allows MSCs to regenerate soft tissues such as heart muscle (Mathieu et al., 2012) and colon (Moussa et al., 2017). As bone and joint tissues are chronically exposed to high mechanical stress, hydrogels need to have exceptional mechanically properties if they are to be suitable for repair of these tissues. In addition, in light of the role of FIGURE 3 | In vitro viability of cASC. (A) MTS activity of cASC cultured in 2D. Cell viability was evaluated in 2D after molding Si-HPMC or Si-HPCH hydrogels on top of the cell layer (10,000 cells/cm 2 ). As described in the section "Materials and Methods" an MTS assay was performed on days 0 and 7. The positive control (CTRL) was obtained by growing cASC alone, while the negative control was obtained by growing cASC in the presence of actinomycin D (Actino; 5 µg/mL). The results are expressed as the percentage of day 0 for each respective condition. * * p < 0.01; * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (B,C) 3D viability of cASC cultured in Si-HPMC or Si-HPCH hydrogels. Cell viability was evaluated in 3D after molding hydrogels mixed with 1 × 10 6 cASC on days 0, 1, and 7 by the Live/Dead Cell Viability assay. The negative control was obtained by adding actinomycin-D (Actino; 5 µg/mL) to the culture medium. Pictures of representative samples of cASC into Si-HPMC cultured in the presence of actinomycin-D (Actino), into Si-HPMC (Si-HPMC) or into Si-HPCH (Si-HPCH) at day 7 (B). The scale bar represents 100 µm. Live and dead cells were then counted (C). The results are expressed as the percentage of day 0 for each respective condition. * * * p < 0.001 compared to day 0 (Kruskal-Wallis).
FIGURE 4 | Macroscopic observation and histological characterization of tissue repair. Canine osteochondral defects (6 mm × 5 mm) were performed as described in the section "Materials and Methods." The defects were either left empty ("empty" defects) of filled with Si-HPCH alone ("Si-HPCH") or Si-HPCH associated with cASC ("Si-HPCH/cASC"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and the samples were macroscopically observed (A) or histologically characterized after hematoxylin-eosin-safran staining (B). For each tested condition, the worst (b,d,f) and the best (c,e,g) repair outcomes are presented. Arrowheads indicate the edges of the defect. Healthy cartilage (a) of intact canine condyle is shown as an internal comparator. The scale bar represents 2.5 mm.
FIGURE 5 | Extracellular matrix-specific histological and immunohistological analyses of tissue repair. Canine osteochondral defects (6 mm × 5 mm) were performed as described in the section "Materials and Methods." The defects were left empty ("empty" defects) or filled with Si-HPCH alone ("Si-HPCH") or Si-HPCH associated with cASC ("Si-PHCH/cASC"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and samples were histologically characterized by Safranin O (SO) and Movat pentachrome (Movat) staining. The samples were also immunostained for type II collagen ("Coll2") and type I collagen ("Coll1"). Healthy cartilage of intact canine condyle is shown as an internal comparator. Representative histological slides are presented. The scale bar represents 2.5 mm.
ECM stiffness and rigidity on stem cell fate (Engler et al., 2008;Discher et al., 2009), it has become clear that synthetic ECM, such as hydrogels used to guide tissue regeneration, have to exhibit biomechanical properties that largely mimic those of FIGURE 6 | O'Driscoll-based quantitative scoring of cartilage repair. Canine osteochondral defects (6 mm × 5 mm) were performed and the defects left empty ("empty" defects) or filled with Si-HPCH alone ("Si-HPCH") or Si-HPCH associated with cASC ("Si-HPCH/cASC"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and samples were histologically characterized (see Figure 5). An O'Driscoll-based histological scoring was then performed according to 10 histological parameters as described ( Table 1). Each parameter was scored 0 to 4, with 4 being the best score. The results are expressed as individual plots for each condition. the native tissue. In this context and considering the limited mechanical properties of Si-HPMC, we hypothesized that the addition of silanized chitosan to Si-HPMC could be a way to improve its mechanical properties. Interestingly, it has also been widely demonstrated that mechanical loading also influences the chondrogenic activities of MSC (Panadero et al., 2016;Choi et al., 2018). It could have been interesting to address whether combining mechanical loading and our Si-HPCH hydrogel may be a relevant strategy to improve MSC chondrogenesis.
In addition, chitosan was also considered due to its structural similarity to GAGs, which are a major component of the extracellular matrix of cartilage. Chitosan has been used for many years as a dressing and has been shown to improve wound healing with a high level of collagen deposition (Hamilton et al., 2006). Furthermore, like some GAGs such as hyaluronic acid, chitosan contains N-acetylglucosamine chains. As a result of this characteristic, chitosan may exhibit some of the biological activities of GAGs such as their ability to bind growth factors or their mucoadhesive properties (Martins et al., 2014). Therefore, this study aimed to improve the properties of an already developed Si-HPMC-based hydrogel by addition of Si-Chitosan as an additive within its network.
Moreover, previous studies have suggested that the mechanical properties of Si-HPMC remain far from those of native cartilage tissue. The stiffness of hydrogels is often reported has being two orders of magnitude lower than cartilage's (100-1000 kPa) (Levental et al., 2007;Boyer et al., 2018). These low mechanical properties have drastically limited their clinical translatability and hydrogels are mainly used as space-filling scaffolds used for the delivery of bioactive molecules and cells (Drury and Mooney, 2003;Kopeček, 2007). To address this issue, we also focused our efforts on the development of a mechanically reinforced hydrogel.
In light of this, the first objective of this study was to characterize the mechanical properties of the hybrid Si-HPCH hydrogel that we had developed. We found that the addition of silanized chitosan to Si-HPMC increased the viscoelastic storage modulus G (nearly 10-fold). Interestingly, while the mechanical properties of Si-HPCH were determined to be higher than those of Si-HPMC, the Si-HPCH remained manually injectable, as indicated by its injectability force of less than 80 N (Vo et al., 2016). The increase in the viscosity and the storage modulus can be explained by the higher polymer concentration within the hybrid hydrogel (4 wt% for Si-HPCH compared to 2 wt% for Si-HPMC hydrogel). Shear mechanical results, storage modulus, and breaking strength, with the same Si-HPMC and Si-HPCH concentration of 4%, revealed an effect of the chitosan backbone on the mechanical properties of the hydrogel, with an increase of the G (8 vs. 14 KPa p < 0.05, data not shown) and a decrease in the breaking stress (1,300 vs. 800 Pa, p < 0.05, data not shown). However, the influence of the chitosan backbone on the rheological and mechanical properties of the hydrogel was not investigated throughout this study. These data clearly demonstrate that Si-HPCH, while exhibiting improved mechanical properties, remains manually injectable and can hence be used for mini-invasive surgical purposes.
In addition to their mechanical properties, the cytocompatibility of newly developed hydrogels is also a basic requirement for their use in tissue engineering. Since it is well known that improvement of the mechanical properties of a hydrogel often results in a decrease in its cytocompatibility, we performed an in-depth analysis of the cytocompatibility of Si-HPCH with respect to MSC. This observation can be explained by an increase in shear forces, as suggested by several studies (Gefen et al., 2008;Hong et al., 2016).
We first addressed whether MSC from human adipose tissue retain their viability when cultured in 2D or 3D while in contact with or embedded in Si-HPCH. The choice of MSCs was guided by the increasing use of these cells in regenerative medicine and tissue engineering studies (Ruiz et al., 2016). Indeed, although chondrocytes were the first cell type used for cartilage regenerative medicine, these cells exhibit many disadvantages, such as morbidity of the donor site and their in vitro dedifferentiation (Benya and Shaffer, 1982). These shortcomings led researchers to find a new source of cells for tissue engineering. In this context, MSCs are an attractive option because they can readily be harvested from different tissues [e.g., bone marrow (Friedenstein et al., 1968), adipose tissue (Fraser et al., 2006), etc.,]. In addition, MSCs have the capacity to differentiate into the chondrogenic lineage and to exhibiting immunomodulatory properties (Tyndall, 2015) that are clinically relevant for cartilage tissue engineering. Surprisingly, and while cell-cell contact have been largely proposed as a necessary prerequisite for MSC chondrogenesis (DeLise et al., 2000), our data strongly suggest that MSC embedded into a polysaccharidic hydrogel without any external pro-chondrogenic factors, may conserve their ability to commit into the chondrogenic lineage. Whether these data may be exploited to propose a cell-free approach for the repair of osteochondral defects now deserve to be further investigated.
The results obtained in our various viability tests indicate good cytocompatibility of Si-HPCH with respect to ASC (of human or canine origin) cultured either in 2D or 3D. To complement these in vitro viability assays, we also performed implantation of Si-HPCH associated with hASC in subcutaneous sites of nude mice. This in vivo experiment confirmed that Si-HPCH was able to support the viability of hASC even after 6 weeks of in vivo implantation. Interestingly, our histological analyses of samples retrieved from the injection site also indicate that hASC, when implanted with Si-HPCH, exhibited a notable ability to produce GAG. Whether this ability of cells to produce ECM component when embedded in chitosan-containing cellulose hydrogel is related to the biological properties of chitosan, including its ability to interact with growth factors (Raftery et al., 2013), warrants further exploration.
Finally, to assess whether Si-HPCH may be a good candidate for cartilage repair, we undertook animal testing in dog, as they remain a clinically relevant veterinary target for cartilage repair strategies. Dogs are commonly affected by joint dysplasia (Ytrehus et al., 2007;Pascual-Garrido et al., 2018;Soo et al., 2018) and there is still not an effective therapeutic treatment for the resulting osteochondral defects. As is the case in humans, these untreated osteochondral defects often lead to an increased risk of osteoarthritis and severe disability.
The results of our preclinical testing in dogs showed that, 4 months after the implantation of hydrogels in osteochondral defects, Si-HPCH allowed for an almost complete filling of the osteochondral defects irrespective of the presence of autologous ASC.
Histological and immunohistological analyses of the newly formed tissue indicated the presence of a hyaline cartilage-like tissue with a GAG-and type II collagen-rich ECM. While these analyses are evidence for the presence of a tissue resembling healthy articular cartilage, several in situ biomechanical analyses can now be performed to further document the cartilaginous nature of this repaired tissue.
Unexpectedly, our data also revealed that defects filled with Si-HPCH alone exhibited a degree of repair that was quite comparable to what was observed for defects filled with Si-HPCH/ASC. Of particular interest for the clinical translation of our data, O'Driscoll scoring revealed that defects treated with Si-HPCH alone had a highly consistent and reproducible repair process while those treated with Si-HPCH/ASC exhibited a greater degree of inter individual variability. These data, which are presumably related to the great variability of ASC properties between individuals, are likely to raise some important issues regarding the ability to exploit the regenerative properties of ASC. The ability of Si-HPCH to support cartilage repair also raises the possibility that this hydrogel, due to the growth factor interacting properties of its chitosan moieties, may promote chemoattraction of regenerative cells emanating from subchondral bone marrow. This hypothesis, although appealing due to the clinical potential, should, however, be tested further before definitive conclusions are drawn.
To conclude, we have developed a new hybrid hydrogel made of silylated chitosan and HPMC that has better mechanical properties, hand-compatible injectability, and in vitro and in vivo cytocompatibility. This Si-HPCH hydrogel supports the repair of load-bearing osteochondral defects in a canine model in the presence or absence of adipose stromal cells.
The possibility of using Si-HPCH for the development of a cell-free repair strategy of osteochondral defects may be of great value for cartilage clinicians, notably by reducing cell dependent inter-individual variability, cost, logistical complexity, and regulatory concerns. Testing of its efficacy in canine or equine patients with naturally occurring and OA-related osteochondral defects should now be undertaken to further address the clinical value of Si-HPCH in cartilage repair.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be available by the authors, without undue reservation, to any qualified researcher.
AUTHOR CONTRIBUTIONS
CB, PW, CV, OGe, OGa, and JG contributed to conception and design of the study. CB, GR, Cd'A, JL, CV, BH, OGe, MF, GV, PR, and OGa carried out the experiments. CB performed the statistical analysis. CB, GR, PW, OGa, and JG wrote sections of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.
FUNDING
This research was supported by the SATT Ouest Valorisation in the framework of the "MAN & ANIMAL: ONE HEALTH" call for proposals, the Agence Nationale de la Recherche in the framework of ANR-11-BSV5-0022 (HYCAR). This work was also funded by the French Research on Osteo Arthritis Diseases (ROAD) Network (Arthritis Foundation).
TABLE 1 |
1Histological parameters used for O'Driscoll-based quantitative scoring of cartilage repair.Histological parameters
Degree of filling
-80 to 100%
4
-60 to 80%
3
-40 to 60%
2
-20 to 40%
1
-0 to 20%
0
Surface regularity
-Flush
4
-Rough
3
-Slight depressed
2
-Depressed
1
-Overgrown
0
Glycosaminoglycan content
-Normal
4
-Nearly normal
3
-Moderate
2
-Weak
1
-None
0
Cartilage thickness
-Similar to the surrounding cartilage
4
-Greater than the surrounding cartilage
3
-Less than the surrounding cartilage
2
-Very thin layer of cartilage
1
-No cartilage
0
Cellular morphology
-Hyaline cartilage
4
-Mostly hyaline cartilage
3
-Mostly fibrocartilage
2
-Fibrocartilage
1
-No cartilage
0
Type 2 collagen staining in the defect
-Normal
4
-Nearly normal
3
-Moderate
2
-Slight
1
-None
0
Type 1 collagen staining in the defect
-None
4
-Slight
3
-Moderate
2
-Important
1
-Complete
0
Adjacent cartilage quality
-Normal
4
-Nearly normal
3
-Moderate
2
-Poor
1
-Degraded
0
Subchondral bone integrity
-Normal
4
-Nearly normal
3
-Moderate
2
-Poor
1
-Degraded
0
(Continued)
TABLE 1 |
1ContinuedHistological parameters
Osteochondral junction
-Normal
4
-Nearly normal
3
-Slight disruption
2
-Severe disintegration
1
-Disruption
0
January 2020 | Volume 8 | Article 23
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org
ACKNOWLEDGMENTSWe would like to thank Dr. F. Lejeune (Brétéché Clinic, Nantes, France) and the Centre of Research and Preclinical Investigation (C.R.I.P., ONIRIS, Nantes).Conflict of Interest:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Copyright © 2020Boyer, Réthoré, Weiss, d'Arros, Lesoeur, Vinatier, Halgand, Geffroy, Fusellier, Vaillant, Roy, Gauthier and Guicheux.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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Cartilage tissue engineering: towards a biomaterialassisted mesenchymal stem cell therapy. C Vinatier, C Bouffi, C Merceron, J Gordeladze, J.-M Brondello, C Jorgensen, 10.2174/157488809789649205doi: 10.2174/ 1574888097896492054Vinatier, C., Bouffi, C., Merceron, C., Gordeladze, J., Brondello, J.-M., Jorgensen, C., et al. (2009a). Cartilage tissue engineering: towards a biomaterial- assisted mesenchymal stem cell therapy. CSCR 4, 318-329. doi: 10.2174/ 157488809789649205
An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects. C Vinatier, O Gauthier, A Fatimi, C Merceron, M Masson, A Moreau, 10.1002/bit.22137Biotechnol. Bioeng. 102Vinatier, C., Gauthier, O., Fatimi, A., Merceron, C., Masson, M., Moreau, A., et al. (2009b). An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects. Biotechnol. Bioeng. 102, 1259- 1267. doi: 10.1002/bit.22137
Cartilage tissue engineering: from biomaterials and stem cells to osteoarthritis treatments. C Vinatier, J Guicheux, 10.1016/j.rehab.2016.03.002Ann. Phys. Rehabil. Med. 59Vinatier, C., and Guicheux, J. (2016). Cartilage tissue engineering: from biomaterials and stem cells to osteoarthritis treatments. Ann. Phys. Rehabil. Med. 59, 139-144. doi: 10.1016/j.rehab.2016.03.002
Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel. C Vinatier, D Magne, A Moreau, O Gauthier, O Malard, C Vignes-Colombeix, 10.1002/jbm.a.30867J. Biomed. Mater. Res. 80Vinatier, C., Magne, D., Moreau, A., Gauthier, O., Malard, O., Vignes-Colombeix, C., et al. (2007). Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel. J. Biomed. Mater. Res. 80A, 66-74. doi: 10.1002/jbm.a.30867
A silanized hydroxypropyl methylcellulose hydrogel for the threedimensional culture of chondrocytes. C Vinatier, D Magne, P Weiss, C Trojani, N Rochet, G F Carle, 10.1016/j.biomaterials.2005.04.057doi: 10.1016/ j.biomaterials.2005.04.057Biomaterials. 26Vinatier, C., Magne, D., Weiss, P., Trojani, C., Rochet, N., Carle, G. F., et al. (2005). A silanized hydroxypropyl methylcellulose hydrogel for the three- dimensional culture of chondrocytes. Biomaterials 26, 6643-6651. doi: 10.1016/ j.biomaterials.2005.04.057
Osteoarthritis: from pathogenic mechanisms and recent clinical developments to novel prospective therapeutic options. C Vinatier, C Merceron, J Guicheux, 10.1016/j.drudis.2016.08.011Drug Discov. Today. 21Vinatier, C., Merceron, C., and Guicheux, J. (2016). Osteoarthritis: from pathogenic mechanisms and recent clinical developments to novel prospective therapeutic options. Drug Discov. Today 21, 1932-1937. doi: 10.1016/j.drudis.2016. 08.011
The Biomechanics and optimization of the needle-syringe system for injecting triamcinolone acetonide into keloids. A Vo, M Doumit, Rockwell , G , 10.1155/2016/5162394J. Med. Eng. Vo, A., Doumit, M., and Rockwell, G. (2016). The Biomechanics and optimization of the needle-syringe system for injecting triamcinolone acetonide into keloids. J. Med. Eng. 2016, 1-8. doi: 10.1155/2016/5162394
Etiology and Pathogenesis of Osteochondrosis. B Ytrehus, C S Carlson, S Ekman, 10.1354/vp.44-4-429Vet. Pathol. 44Ytrehus, B., Carlson, C. S., and Ekman, S. (2007). Etiology and Pathogenesis of Osteochondrosis. Vet. Pathol. 44, 429-448. doi: 10.1354/vp.44-4-429
| [
"Articular cartilage (AC) may be affected by many injuries including traumatic lesions that predispose to osteoarthritis. Currently there is no efficient cure for cartilage lesions. In that respect, new strategies for regenerating AC are contemplated with interest. In this context, we aim to develop and characterize an injectable, self-hardening, mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropymethyl cellulose (Si-HPMC) mixed with silanised chitosan. The in vitro cytocompatibility of Si-HPCH was tested using human adipose stromal cells (hASC). In vivo, we first mixed Si-HPCH with hASC to observe cell viability after implantation in nude mice subcutis. Si-HPCH associated or not with canine ASC (cASC), was then tested for the repair of osteochondral defects in canine femoral condyles. Our data demonstrated that Si-HPCH supports hASC viability in culture. Moreover, Si-HPCH allows the transplantation of hASC in the subcutis of nude mice while maintaining their viability and secretory activity. In the canine osteochondral defect model, while the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without cASC, revealed a significant osteochondral regeneration. To conclude, Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC as well as the regeneration of osteochondral defects in dogs when implanted alone or with ASC."
] | [
"Roberto Narcisi ",
"Jane Ru Choi ",
"Jérôme Guicheux [email protected] ",
"Cécile Boyer \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n",
"Gildas Réthoré \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale\nPHU4 OTONNNantesFrance\n",
"Pierre Weiss \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale\nPHU4 OTONNNantesFrance\n",
"Cyril D'arros \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n",
"Julie Lesoeur \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nSC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance\n",
"Claire Vinatier \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nSC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance\n",
"Boris Halgand \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCHU Nantes\nPHU4 OTONNNantesFrance\n",
"Olivier Geffroy \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCentre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance\n",
"Marion Fusellier \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCentre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance\n",
"Gildas Vaillant \nCHU Nantes\nPHU4 OTONNNantesFrance\n\nCentre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance\n",
"Patrice Roy \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n",
"Olivier Gauthier \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nCentre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance\n",
"Jérôme Guicheux \nInserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance\n\nUniversité de Nantes\nUFR Odontologie\nNantesFrance\n\nSC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance\n\nCHU Nantes\nPHU4 OTONNNantesFrance\n\nCentre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance\n",
"\nErasmus University Rotterdam\nNetherlands\n",
"\nUniversity of British Columbia\nCanada\n"
] | [
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"CHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale\nPHU4 OTONNNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"CHU Nantes, Service d'Odontologie Restauratrice et Chirurgicale\nPHU4 OTONNNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"SC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"SC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"CHU Nantes\nPHU4 OTONNNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance",
"CHU Nantes\nPHU4 OTONNNantesFrance",
"Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance",
"Inserm\nUMR 1229, RMeS, Regenerative Medicine and Skeleton\nUniversité de Nantes\nONIRIS\nNantesFrance",
"Université de Nantes\nUFR Odontologie\nNantesFrance",
"SC3M -\"Electron Microscopy, Microcharacterization and Functional Morphohistology Imaging\" Core Facility, Structure Fédérative de Recherche Franc˛ois Bonamy, INSERM -UMS016, CNRS 3556, CHU Nantes\nUniversité de Nantes\nNantesFrance",
"CHU Nantes\nPHU4 OTONNNantesFrance",
"Centre of Research and Preclinical Investigation (C.R.I.P.), ONIRIS\nNantesFrance",
"Erasmus University Rotterdam\nNetherlands",
"University of British Columbia\nCanada"
] | [
"Roberto",
"Jane",
"Ru",
"Jérôme",
"Cécile",
"Gildas",
"Pierre",
"Cyril",
"Julie",
"Claire",
"Boris",
"Olivier",
"Marion",
"Gildas",
"Patrice",
"Olivier",
"Jérôme"
] | [
"Narcisi",
"Choi",
"Guicheux",
"Boyer",
"Réthoré",
"Weiss",
"D'arros",
"Lesoeur",
"Vinatier",
"Halgand",
"Geffroy",
"Fusellier",
"Vaillant",
"Roy",
"Gauthier",
"Guicheux"
] | [
"J A Ankrum, ",
"J F Ong, ",
"J M Karp, ",
"P D Benya, ",
"J D Shaffer, ",
"X Bourges, ",
"P Weiss, ",
"A Coudreuse, ",
"G Daculsi, ",
"G Legeay, ",
"X Bourges, ",
"P Weiss, ",
"G Daculsi, ",
"G Legeay, ",
"I Levental, ",
"P C Georges, ",
"P A Janmey, ",
"C Boyer, ",
"L Figueiredo, ",
"R Pace, ",
"J Lesoeur, ",
"T Rouillon, ",
"C L Visage, ",
"M Brittberg, ",
"A H Gomoll, ",
"J A Canseco, ",
"J Far, ",
"M Lind, ",
"J Hui, ",
"M Brittberg, ",
"A Lindahl, ",
"A Nilsson, ",
"C Ohlsson, ",
"O Isaksson, ",
"L Peterson, ",
"J H Cho, ",
"S.-H Kim, ",
"K D Park, ",
"M C Jung, ",
"W I Yang, ",
"S W Han, ",
"J R Choi, ",
"K W Yong, ",
"J Y Choi, ",
"J Clouet, ",
"C Vinatier, ",
"C Merceron, ",
"M Pot-Vaucel, ",
"Y Maugars, ",
"P Weiss, ",
"M M Cross, ",
"J M Dang, ",
"D D N Sun, ",
"Y Shin-Ya, ",
"A N Sieber, ",
"J P Kostuik, ",
"K W Leong, ",
"A M De Campos, ",
"Y Diebold, ",
"E L S Carbalho, ",
"A Sánchez, ",
"José Alonso, ",
"M , ",
"T Debnath, ",
"S Ghosh, ",
"U S Potlapuvu, ",
"L Kona, ",
"S R Kamaraju, ",
"S Sarkar, ",
"A M Delise, ",
"L Fischer, ",
"R S Tuan, ",
"D E Discher, ",
"D J Mooney, ",
"P W Zandstra, ",
"J L Drury, ",
"D J Mooney, ",
"A J Engler, ",
"C Carag-Krieger, ",
"C P Johnson, ",
"M Raab, ",
"H.-Y Tang, ",
"D W Speicher, ",
"A J Engler, ",
"S Sen, ",
"H L Sweeney, ",
"D E Discher, ",
"J K Fraser, ",
"I Wulur, ",
"Z Alfonso, ",
"M H Hedrick, ",
"A J Friedenstein, ",
"K V Petrakova, ",
"A I Kurolesova, ",
"G P Frolova, ",
"A Gefen, ",
"B Van Nierop, ",
"D L Bader, ",
"C W Oomens, ",
"A C Gelber, ",
"X Guan, ",
"M Avci-Adali, ",
"E Alarçin, ",
"H Cheng, ",
"S S Kashaf, ",
"Y Li, ",
"V Hamilton, ",
"Y Yuan, ",
"D A Rigney, ",
"A D Puckett, ",
"J L Ong, ",
"Y Yang, ",
"N Heldring, ",
"I Mäger, ",
"M J A Wood, ",
"K Le Blanc, ",
"S E L Andaloussi, ",
"C D Hoemann, ",
"J Sun, ",
"M D Mckee, ",
"A Chevrier, ",
"E Rossomacha, ",
"G.-E Rivard, ",
"A S Hoffman, ",
"Y Hong, ",
"Y Yao, ",
"S Wong, ",
"L Bian, ",
"A F T Mak, ",
"D J Hunter, ",
"S Bierma-Zeinstra, ",
"E A Jones, ",
"A English, ",
"K Henshaw, ",
"S E Kinsey, ",
"A F Markham, ",
"P Emery, ",
"J Kopeček, ",
"A Lahiji, ",
"A Sohrabi, ",
"D S Hungerford, ",
"C G Frondoza, ",
"Le Blanc, ",
"K Davies, ",
"L C , ",
"K Y Lee, ",
"D J Mooney, ",
"E A Makris, ",
"A H Gomoll, ",
"K N Malizos, ",
"J C Hu, ",
"K A Athanasiou, ",
"E A Martins, ",
"Y M Michelacci, ",
"R Y Baccarin, ",
"B Cogliati, ",
"L C Silva, ",
"E Mathieu, ",
"G Lamirault, ",
"C Toquet, ",
"P Lhommet, ",
"E Rederstorff, ",
"S Sourice, ",
"C Merceron, ",
"S Portron, ",
"M Masson, ",
"J Lesoeur, ",
"B H Fellah, ",
"O Gauthier, ",
"C Merceron, ",
"C Vinatier, ",
"S Portron, ",
"M Masson, ",
"J Amiaud, ",
"L Guigand, ",
"G A Morris, ",
"S M Kök, ",
"S E Harding, ",
"Adams , ",
"G G , ",
"L Moussa, ",
"G Pattappa, ",
"B Doix, ",
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"C Demarquay, ",
"M Benderitter, ",
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"S Miot, ",
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"S Feliciano, ",
"F Wolf, ",
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"T C Mcdevitt, ",
"A J Engler, ",
"S W O'driscoll, ",
"F W Keeley, ",
"R B Salter, ",
"F Oprenyeszk, ",
"C Sanchez, ",
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"P Compère, ",
"J A Panadero, ",
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"C Pascual-Garrido, ",
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"M F Rai, ",
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"M J Lopez, ",
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"S Portron, ",
"C Merceron, ",
"O Gauthier, ",
"J Lesoeur, ",
"S Sourice, ",
"M Masson, ",
"R Raftery, ",
"F O'brien, ",
"S.-A Cryan, ",
"M Ruiz, ",
"S Cosenza, ",
"M Maumus, ",
"C Jorgensen, ",
"D Noël, ",
"M Schinhan, ",
"M Gruber, ",
"P Vavken, ",
"R Dorotka, ",
"L Samouh, ",
"C Chiari, ",
"V F Sechriest, ",
"Y J Miao, ",
"C Niyibizi, ",
"A Westerhausen-Larson, ",
"H W Matthew, ",
"C H Evans, ",
"M Soo, ",
"N Lopez-Villalobos, ",
"A Worth, ",
"A Tyndall, ",
"C Vinatier, ",
"C Bouffi, ",
"C Merceron, ",
"J Gordeladze, ",
"J.-M Brondello, ",
"C Jorgensen, ",
"C Vinatier, ",
"O Gauthier, ",
"A Fatimi, ",
"C Merceron, ",
"M Masson, ",
"A Moreau, ",
"C Vinatier, ",
"J Guicheux, ",
"C Vinatier, ",
"D Magne, ",
"A Moreau, ",
"O Gauthier, ",
"O Malard, ",
"C Vignes-Colombeix, ",
"C Vinatier, ",
"D Magne, ",
"P Weiss, ",
"C Trojani, ",
"N Rochet, ",
"G F Carle, ",
"C Vinatier, ",
"C Merceron, ",
"J Guicheux, ",
"A Vo, ",
"M Doumit, ",
"Rockwell , ",
"G , ",
"B Ytrehus, ",
"C S Carlson, ",
"S Ekman, "
] | [
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"\nTABLE 1 |\n1Histological parameters used for O'Driscoll-based quantitative scoring of cartilage repair.Histological parameters \n\nDegree of filling \n\n-80 to 100% \n4 \n\n-60 to 80% \n3 \n\n-40 to 60% \n2 \n\n-20 to 40% \n1 \n\n-0 to 20% \n0 \n\nSurface regularity \n\n-Flush \n4 \n\n-Rough \n3 \n\n-Slight depressed \n2 \n\n-Depressed \n1 \n\n-Overgrown \n0 \n\nGlycosaminoglycan content \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Weak \n1 \n\n-None \n0 \n\nCartilage thickness \n\n-Similar to the surrounding cartilage \n4 \n\n-Greater than the surrounding cartilage \n3 \n\n-Less than the surrounding cartilage \n2 \n\n-Very thin layer of cartilage \n1 \n\n-No cartilage \n0 \n\nCellular morphology \n\n-Hyaline cartilage \n4 \n\n-Mostly hyaline cartilage \n3 \n\n-Mostly fibrocartilage \n2 \n\n-Fibrocartilage \n1 \n\n-No cartilage \n0 \n\nType 2 collagen staining in the defect \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Slight \n1 \n\n-None \n0 \n\nType 1 collagen staining in the defect \n\n-None \n4 \n\n-Slight \n3 \n\n-Moderate \n2 \n\n-Important \n1 \n\n-Complete \n0 \n\nAdjacent cartilage quality \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Poor \n1 \n\n-Degraded \n0 \n\nSubchondral bone integrity \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Poor \n1 \n\n-Degraded \n0 \n\n(Continued) \n\n",
"\nTABLE 1 |\n1ContinuedHistological parameters \n\nOsteochondral junction \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Slight disruption \n2 \n\n-Severe disintegration \n1 \n\n-Disruption \n0 \n\n"
] | [
"Histological parameters used for O'Driscoll-based quantitative scoring of cartilage repair.",
"Continued"
] | [
"Figure 1A",
"Figure 1B)",
"Figure 1C",
"Figure 1D",
"Figure 2A",
"Figure 2B",
"Figure 2C",
"Figure 2D",
"Figure 2D",
"Figure 3A",
"Figure 3B",
"Figure 3C",
"Figure 4",
"Figure 4A",
"Figure 4B",
"Figure 4A",
"Figure 4B",
"(Figure 4B, panel b",
"(Figure 5)",
"(Figure 5",
"(Figure 5",
"(Figure 5, Movat)",
"(Figure 5",
"(Figure 5, Coll1)",
"(Figure 4)",
"Figure 5)",
"(Figure 6)",
"(Figure 6",
"Figure 5"
] | [] | [
"Articular cartilage (AC) is a complex porous permeable extracellular matrix that surrounds specific cell type called chondrocytes. Due primarily to its avascular and aneural structural organization (Clouet et al., 2009), AC does not heal spontaneously when injured. AC defects affect approximately two million patients every year in Europe and in the United States (Mumme et al., 2016), and they remain a clinical challenge due to the paucity of routinely translatable therapeutics to treat this condition. Degenerative changes affected the cartilage and the bone surrounding the lesion (Schinhan et al., 2012). Therapeutic options to repair AC defects include microfracture, autologous osteochondral grafting (Brittberg et al., 2016), and autologous chondrocyte implantation. These therapeutic options are, however, subject to major logistical and clinical issues, such as their short-lived clinical benefits in particular .",
"Articular cartilage defects, when left untreated or partially healed, dramatically increase the predisposition to osteoarthritis (OA). OA is a painful, debilitating, and slowly progressing degenerative and inflammatory joint disease that is initiated by cartilage damage prior to its culmination in alteration of all of the joint tissues (i.e., synovium, bone, muscle, tendon, and ligament) (Hunter and Bierma-Zeinstra, 2019). As there is no curative treatment, prosthetic joint replacement remains the last-recourse for alleviation of OA-associated pain symptoms . In light of this, a number of innovative strategies to repair or regenerate cartilage and subchondral bone very early in the degenerative joint cascade have been proposed. Among these strategies, tissue engineering (TE) is thought to be one of the most promising approaches (Makris et al., 2015). It consists primarily of combining reparative cells with a biomaterial capable of supporting cell transplantation as well as their engraftment, viability, growth, differentiation, and secretory activity.",
"Cartilage tissue engineering was initially focused on the use of chondrocytes of articular origin (Brittberg et al., 1994). However, the difficulties encountered, particularly with loss of the chondrocyte phenotype during their in vitro expansion phase and the morbidity of the donor site (Benya and Shaffer, 1982;Vinatier et al., 2009a), has led researchers to explore new cell sources. Nasal chondrocytes have been proposed as an alternative to articular chondrocytes (Vinatier et al., 2007), and their successful clinical use in association with a collagen membrane was recently reported (Mumme et al., 2016). Another alternative to articular chondrocytes that has been widely investigated is adult mesenchymal stromal cells (MSC). MSC can readily be isolated from various tissues such as bone marrow (BMMSC) (Friedenstein et al., 1968), adipose tissue (ASC) (Fraser et al., 2006), and synovial fluid (SFAC) (Jones et al., 2004). They have the capacity for self-renewal and differentiation into a range of cell types encompassing chondrocytes in particular, as well as other cell types of the musculoskeletal system (Pittenger, 1999). In addition, recent studies have shown that MSC are able to secrete a myriad of biological factors, either directly or through the release of extracellular vesicles (Heldring et al., 2015). These cytokines, chemokines, and growth factors can exert immunomodulatory effects (Le Blanc and Davies, 2015), reduce tissue damage, and promote repair or healing processes. Finally, as MSC generally fail to express the class two major histocompatibility complex molecules, they may be candidates for allogeneic transplantation without host alloreactivity (Ankrum et al., 2014) and hence of particular clinical interest for cartilage TE. This immune privilege of MSC, although it is still a matter of considerable debate (Ankrum et al., 2014), paves the way of the development of \"off-theshelf \" MSC therapies.",
"A number of biomaterials have been proposed in recent years as cell carriers for cartilage and subchondral bone TE (Morris et al., 2010). Among these scaffolding biomaterials, hydrogels have been widely investigated in light of their favorable physicochemical and biological properties (e.g., high water content, biocompatibility, tunable mechanical properties, and permeability) (Hoffman, 2001;Guan et al., 2017). With the aim of developing a self-crosslinking hydrogel, we have devised and patented injectable silanized hydroxypropylmethyl cellulose (Si-HPMC). Si-HPMC was initially used for the 3D culture of MSC and chondrocytes (Vinatier et al., 2005(Vinatier et al., , 2009bMerceron et al., 2011). More recently, Si-HPMC has also been successfully used for MSC based regeneration of soft tissues such as myocardium (Mathieu et al., 2012) and colon (Moussa et al., 2017). It has also been preclinically tested, albeit with less encouraging results, for the repair of stiffer tissues such as cartilage (Vinatier et al., 2007;Portron et al., 2013). Interestingly, a converging body of proof has recently indicated that MSC are \"touchy-feely\" cells that are particularly able to sense the biomechanical properties (e.g., stiffness, elasticity, and relaxability) of their micro-environment (Discher et al., 2009). When cultured on stiff materials, they preferentially engage in osteochondral differentiation, while on soft materials they commit to brain or cardiac differentiation pathways (Engler et al., 2006;Murphy et al., 2014). In light of these data, there appears to be ample merit in developing hydrogels for cartilage TE that have mechanical properties that mimic those of the target tissue. To address this issue, we sought to determine whether adding mechanical reinforcement to Si-HPMC may be a viable strategy to develop materials that not only have improved mechanical properties but also increased cartilage regenerative capacity. In light of the silanol-dependent reticulation process of Si-HPMC and our ability to silanize a large panel of biomolecules including polysaccharides (patent WO2011089267), we have synthesized a hybrid hydrogel of Si-HPMC and Si-chitosan. Chitosan is a natural chitin-derived polymer extracted from the exoskeleton of crustaceans. It is composed of D-glucosamine and N-acetyl-D-glucosamine (Oprenyeszk et al., 2015), which can mimic the glycosaminoglycan content of cartilaginous extracellular matrix (Lahiji et al., 2000;Hoemann et al., 2007). Several studies have also shown that chitosan enhances the viability of bovine chondrocytes (Sechriest et al., 2000) and the chondrogenic differentiation of MSC (Cho et al., 2004;Dang et al., 2006) in culture. In addition, chitosan has mucoadhesive properties that are useful for scaffold anchorage to native cartilage (de Campos et al., 2005). Chitosan is also positively charged (Morris et al., 2010), sterilizable, biocompatible (Debnath et al., 2015), and biodegradable (Lee and Mooney, 2001). All of these characteristics make chitosan an appropriate candidate for silanization and further combination with Si-HPMC for use in cartilage TE.",
"The aim of this study was to develop and characterize a new mechanically reinforced hybrid hydrogel made of Si-HPMC and Si-chitosan (referred to here as Si-HPCH) and to evaluate its ability to repair osteochondral defect in a dog model. We first characterized the mechanical and rheological properties of Si-HPCH. The viability of human MSC was then assayed after in vitro culture either in contact with or embedded in Si-HPCH. The viability of human MSC was also investigated after transplantation with Si-HPCH in the subcutis of nude mice. Lastly, we assessed the preclinical potential of Si-HPCH in cartilage repair using a canine model of osteochondral knee defects.",
"Silanized hydroxypropyl methylcellulose and an acidic buffer solution (ABS, pH 3.6) were prepared as previously described (Bourges et al., 2002a,b). Silanized chitosan (Si-chitosan) was synthesized as follows. One hundred milliliters of chitosan solution (1% wt/v, HCl, pH 3.0) was poured into a round bottom flask and 1 eq (1.55 mL) of (3-Isocyanatopropyl)triethoxysilane was added. The mixture was then stirred for 3 h at room temperature and then initially dialyzed (molecular weight cutoff of 6-8 kDa) for 2 days against NaOH (0.1 M) with frequent replacement of the dialysis solution. The sample was then dialyzed against water with frequent replacement of the dialysis solution until the conductimetry monitoring indicated no further release. The dialyzes eliminated the non-grafted silane derivatives used for siloxane grafting onto the HPMC and chitosan. As a final step, the purified gels were lyophilized.",
"Two types of hydrogel were prepared. The first one was made of Si-HPMC alone and prepared by dissolving Si-HPMC polymer (3 wt%) in 0.1 M NaOH aqueous solution. The second one, referred to as Si-HPCH, was composed of Si-HMPC and Sichitosan. It was prepared as follows: Si-HPMC polymer (3 wt%) was dissolved in 0.1 M NaOH aqueous solution; Si-chitosan (3 wt%) was then dissolved in the 3 wt% Si-HPMC aqueous solution (0.1 M NaOH).",
"The hydrogel precursor solution was then obtained by mixing 1 volume of the above Si-HPMC or Si-HPCH basic solution contained in a Luer lock syringe, with a 0.5 volume of an ABS prepared in our laboratory (Bourges et al., 2002a,b) at pH 3.6 in another Luer lock syringe, by interconnection of both syringes; the final pH of the mixture was 7.4. This precursor mixture was injectable for 30-40 min, at which time the gel point was reached.",
"The viscoelastic modulus (storage modulus G ) and the breaking strength (σ) were obtained using a HAAKE MARS rheometer (Thermo Fisher Scientific, United States) using a plate geometry (PP20Ti, titanium plateau with a 20 mm diameter). Immediately after mixing, the liquid hydrogel precursor solutions were injected into a mold (20 mm in diameter and 5 mm in height) and the measurements started 5 days later (full cross-linking of the network). The storage modulus (G ) and the breaking strength (σ) were monitored as a function of strain (from 0.1 to 3,000 Pa) at a constant frequency and temperature (1 Hz and 23 • C). Each condition was measured in quintuplicate from 2 different batches of both hydrogels.",
"Rheological measurements were performed using a Haake MARS rheometer (ThermoHaake R , Germany) with a titanium cone-plate geometry (60 mm in diameter, 1 • cone angle, 52 µm gap). Steady shear tests were carried out at 23 • C on Si-HPMC (2 wt%) and Si-HPCH (4 wt%) solutions. The operating shear rate ranged from 0.1 to 100 s −1 . Different flow curves were fitted and extrapolated to lower shear rates by the Cross equation (Cross, 1969). The injectability properties were investigated using a compression testing device (TAHDplus) with a 5 kg load cell for the measurements at an injectability rate of 2 mm per second through a 3 mL syringe equipped with an 18G needle. A syringe containing 1 mL Si-HPMC and Si-HPCH was set and the injection force was measured.",
"Adipose Stromal Cell (ASC) Culture Adipose Stromal Cell were obtained from human patients (hASC) undergoing liposuction and who had provided written consent (ethics committees: Agence de BioMedecine no PFS08-018, the legislation code L.1211-3 to L.1211-9: residues obtained during a surgical procedure, performed in the interest of the person operated, can be used for scientific research), or from autologous canine adipose tissue (cASC) harvested from the gluteal area (APAFIS #4446). Briefly, and as previously described , human lipoaspirate and canine adipose tissue were shredded into small pieces and washed extensively with Hanks' balanced salt solution (HBSS) to remove debris. The washed adipose tissue was treated with collagenase (0.025%) in HBSS for 1 h at 37 • C under gentle agitation. The collagenase (Sigma-Aldrich C2674) was inactivated by the addition of an equal volume of Dulbecco's modified Eagle's medium (DMEM-Glutamax; Gibco) supplemented with 1% penicillin/streptomycin (P/S) containing 10% fetal calf serum (FCS; control medium). The digested product was then centrifuged at 250 × g for 5 min to separate the adipose fraction from the stromal fraction. The supernatant was removed, and the stromal cells were resuspended in the control medium and filtered through a 70 µm nylon mesh filter. The filtrate was centrifuged and the cells were resuspended in red blood cell lysis buffer. The lysis reaction was stopped by the addition of control medium. The suspension was centrifuged, and the cells were finally resuspended in control medium and plated at a density of 5 × 10 4 cells/cm 2 .",
"Adipose Stromal Cell isolated using the protocol described above have been extensively characterized in our laboratory (for details see Merceron et al., 2009Merceron et al., , 2011. The medium was replaced 24 h after seeding to remove non-adherent cells. To prevent spontaneous differentiation, primary cultures (P0) of ASC were grown to approximately 80% confluency and then detached from the cell culture flask using trypsin-EDTA. All of the culture incubations were performed at 37 • C in a humidified atmosphere containing 5% CO 2 , and the medium was replaced every 2 days. The different experiments with hASC and cASC were carried out with cells at passage 2.",
"Human adipose stromal cells (n = 3) and cASC (n = 1) viability was evaluated by the methyl tetrazolium salt (MTS) assay (Promega, United States). The cells were cultured in physical contact with Si-HPMC or Si-HPCH. hASC and cASC were seeded onto culture plates and were allowed to attach to 48well plates at a final density of 10,000 cells per cm 2 . After 24 h, the culture medium was removed and either Si-HPMC or Si-HPCH hydrogel (200 µL/well) was added on top of the cell layer. After 1 h of gelation at 37 • C, 200 µL of culture medium was added to each well and replaced every 2 days. As a negative control, cells were cultured in the presence of actinomycin-D (5 µg/mL), a well-known inducer of cell death. Finally, the MTS assay was performed on days 0, 1, and 7 for the hASC and on days 0 and 7 for the cASC. The MTS assay is based on the reduction of MTS tetrazolium compound by viable cells, thereby generating a colored and soluble formazan product in the culture medium. The amount of the colored product was determined by the optical density reading at 490 nm (Victor3V 1420 Multilabel Counter). Each condition was tested in triplicate from three different donors of hASC and one donor of cASC.",
"3D cell viability was evaluated by the Live/Dead Cell Viability assay (Thermo Fisher Scientific, MA, United States) using hASC (n = 3) or cASC (n = 1) cultured three-dimensionally in hydrogels at a final density of 1 × 10 6 cells per mL of hydrogel. The ASC were collected and gently mixed with Si-HPMC or Si-HPCH. Cellularized hydrogels were molded and allowed to gelate in the wells of a 48-well plate at 37 • C for 1 h. After gelation, 200 µL of culture medium were added to each well and replaced every 2 days (Vinatier et al., 2007). As a negative control, cells were cultured in 3D in Si-HPMC hydrogel in the presence of actinomycin-D (5 µg/mL). A Live/Dead Cell Viability assay was performed according to the manufacturer's instructions on days 0, 1, and 7 for hASC and on days 0 and 7 for cASC. Green fluorescence can be observed in the living cells due to the conversion of non-fluorescent calcein AM into green-fluorescent calcein by the intracellular esterase activity in live cells. Dead cells become red-fluorescent due to the uptake of ethidium homodimer-1 as a result of the loss of plasma membrane integrity. The red and green fluorescence were observed with a confocal microscope [Nikon D-eclipse C1 (Ar/Kr)]. The numbers of green and red cells were quantified with Volocity software and the results shown as the percentage of living cells. Each condition was tested in triplicate from three different donors of hASC and one donor of cASC.",
"To investigate whether our biomaterial was able to support the viability of cells in vivo, a single-cell suspension of 1 × 10 6 hASC was gently mixed with Si-HPMC or Si-HPCH hydrogel prior to subcutaneous implantation in 7-week-old female Swiss nude mice (Charles River, L'Arbresle, France), as described previously (Vinatier et al., 2009b;Merceron et al., 2011). All of the animals were treated in accordance with the Medical Animal Care Guidelines of Nantes University (APAFIS#4213). General anesthesia was achieved in an induction chamber with isoflurane (2%) delivered in O 2 and maintained through an individual mask. The Si-HPMC and Si-HPCH cellularized hydrogels were injected subcutaneously in the back along each side of the dorsum of nude mice. The implantations were performed under aseptic conditions. The two different conditions (\"Si-HPMC\" and \"Si-HPCH\") were tested (n = 4), four animals received implants (two implants per animal). The animals were sacrificed 6 weeks after implantation. The mice were euthanized by an overdose of isoflurane in an induction chamber. The hydrogels were individually explanted and the samples were processed histologically as described below.",
"To investigate whether or not Si-HPCH biomaterial associated with autologous ASC was able to support the repair of osteochondral defects, we undertook a preclinical experiment in dogs. All animal handling and surgical procedures were conducted according to European Community guidelines for the care and use of laboratory animals (EU Directive 2010/63), after approval by the Pays de la Loire Animal Ethical Committee (APAFIS #4446) and the Oniris College of Veterinary Medicine Animal Welfare Committee. Surgical procedures were performed with the animals under general anesthesia and under surgical aseptic conditions. Self-hardening, mechanically reinforced hydrogel was implanted into calibrated 6 mm-diameter osteochondral defects. Twelve clinically healthy adult Beagle female dogs with body weights ranging from 10 to 15 kg, split into two groups, were used in this study. Osteochondral defects were generated using a 6-mm in diameter orthopedic bur to create a calibrated 6 mm × 5 mm deep osteochondral defect.",
"A total of 14 defects were created on the medial femoral condyles, one defect per dog (except for two dogs that had two defects, one on each femur). The defects of six dogs were filled with Si-HPCH alone (the Si-HPCH group), while the defects of the six other dogs were filled with Si-HPCH associated with autologous ASC (2 × 10 6 cells/mL; the Si-HPCH/cASC group).",
"In an attempt to reduce the number of animal lives, a second defect was generated on the contralateral limb in two dogs of the Si-HPCH group and left empty (the Empty Defect group). These two empty defects were used as critical-sized controls. The dogs were sacrificed 4 months after the implantation using intravenous overdose of pentobarbital. Femoral extremities were immediately dissected and prepared for further histological analysis.",
"Subcutaneous samples were fixed in 10% formalin and embedded in paraffin. Embedded samples were stained as described previously (Vinatier et al., 2009b;Merceron et al., 2011). Briefly, the paraffin-embedded explants were cut into 5 µmthick sections passing through the middle of the sample. The sections were deparaffinized by immersion in methylcyclohexane, rehydrated by a graded series of ethanol, and then rinsed in distilled water. The tissue sections were stained with Alcian blue (AB). The sections were then visualized and scanned using a NanoZoomer device (Hamamatsu Photonics, Japan).",
"Canine femoral extremities were fixed in 10% formalin and embedded in resin (Tecknovit R 9100, Heraeus Kulzer, Japan). The embedded samples provided 7 µm thick sections according to the osteochondral defect axis. The resin sections were then deplastified using acetone, rehydrated by a graded series of ethanol, and rinsed in distilled water. The tissue sections were stained with hematoxylin-eosin (HE), Safranin-O (SO), or Movat pentachrome (Movat). For IHC, the sections were incubated with primary antibodies against type II (MP Biomedicals 08631711, United States) or type I (Abcam ab6308, United Kingdom) collagen. The primary antibodies were detected using a kit (DAKO Agilent, Agilent Technologies, United States) with DAB substrate as per the manufacturer's instructions. The sections were then visualized and scanned using a NanoZoomer device (Hamamatsu Photonics, Japan).",
"To quantify the histological repair of osteochondral defects, we used a modified version of the grading scale described by O'Driscoll et al. (1986). This histological scoring was based on 10 histological parameters ( Table 1). We assigned a score ranging from 0 to 4 points to each criterion for a total of 40 points. Three investigators with extensive experience in cartilage histological analyses performed a blind rating of the stained sections.",
"The results are expressed as means ± SEM of replicate determinations. Statistical analyses were performed using GraphPad Prism software (San Diego, United States). Comparative studies of means were performed by using the Mann-Whitney test when two conditions had to be compared and the Kruskal-Wallis test (post hoc: Dunn's Multiple comparison test) when more than two conditions had to be compared.",
"The objective was to develop a new hybrid hydrogel (Si-HPCH) by adding Si-Chitosan to an Si-HPMC hydrogel that was already being used for tissue repair. The formulation was characterized to determine its potential usefulness as a hydrogel for tissue engineering. Throughout the physicochemical characterizations, we evaluated the selected formulation for biological investigation. A score ranging from 0 to 4 points was assigned to each criterion.",
"First, we measured the viscosity of the solutions. As reported in Figure 1A, the viscosity increased significantly when Si-chitosan was added compared to Si-HPMC alone. The viscosity profile of a solution is usually indicative of the injectability behavior. Nevertheless, the syringeability of the Si-HPCH solutions was monitored. As expected, the force required to inject increased when Si-chitosan was added, but the mixed solution remained readily injectable, with a value of 2.50 N ( Figure 1B). Having evaluated the rheological properties of our hydrogels, we sought to investigate the mechanical properties of Si-HPCH. Its storage modulus (G ) and breaking strength (σ) were hence measured.",
"The results, plotted in Figure 1C, show a 9-fold increase in G when Si-HPMC was combined with Si-Chitosan. In parallel, Figure 1D indicates that Si-HPCH exhibits a breaking strength 5.5-fold lower than that of Si-HPMC. Moreover, at the same concentration (4 wt%) of total polymer, Si-HPCH exhibited a 30% higher stiffness (G ) and a 30% decrease in the breaking strength.",
"To investigate the viability of ASC cultured in contact or embedded in Si-HPCH, we performed several in vitro and in vivo tests, (i) in 2D culture with an MTS-based metabolic assay, (ii) in 3D culture with a viability assay, and (iii) after subcutis implantation in nude mice. The cytotoxicity of Si-HPCH hydrogel was first evaluated using hASC. As shown in Figure 2A, the MTS activity of hASC cultured in contact with Si-HPCH or Si-HPMC gradually increased from day 0 to day 7 (a 2-fold increase). After 7 days of culture, no difference in metabolic activity was observed between the three conditions (i.e., CTRL, Si-HPMC, and Si-HPCH). As expected, in the presence of actinomycin-D, the MTS activity decreased drastically from day 0 to day 7. Taken together, these data indicate that neither of the hydrogels resulted in a major alteration of the metabolic activity of 2D-cultured hASC. To further analyze whether Si-HPCH can influence the behavior of clinically relevant MSC, we also cultured hASC embedded three-dimensionally in Si-HPCH. Figure 2B illustrates the viability of hASC after 7 days of culture in 3D into Si-HPMC hydrogel in the presence of actinomycin-D (Panel Actino) or not (Panel Si-HPMC) and into Si-HPCH hydrogel (Panel Si-HPCH). Figure 2C shows the number of viable hASC when cultured three-dimensionally in Si-HPCH or Si-HPMC compared to cells cultured in Si-HPMC in the presence of actinomycin D.",
"Interestingly, a slight but significant increase was observed at day 7 for Si-HPCH, while a barely discernible decrease was observed for cells cultured in Si-HPMC. As expected, actinomycin-D induced a progressive decrease in hASC viability. Collectively, these data indicate that the addition of Si-chitosan to Si-HPMC did not significantly alter the viability of 3Dcultured hASC.",
"Finally, to determine whether Si-HPCH can support the viability of hASC in vivo, we mixed hASC (1 × 10 6 cells/mL of hydrogel) with Si-HPMC or Si-HPCH prior to subcutis injection (250 µL) in nude mice. Both hydrogels were implanted alone as negative control. Six weeks after implantation, samples were retrieved and histologically analyzed. Macroscopically, no inflammation was observed in any condition. Interestingly, no cellular infiltration was observed in the Si-HPMC and Si-HPCH conditions as shown in Figure 2D (panels a and c).",
"To assess whether the embedded hASC could produce extracellular matrix components, we performed Alcian Blue staining to detect glycosaminoglycans (GAG). As indicated in Figure 2D (panels b and d), when the cells were implanted with Si-HPMC enriched with Si-chitosan (panel d), the cells produced a high amount of GAG as evidenced by the intense blue staining compared to cells implanted with Si-HPMC alone (panel b). Altogether, these in vivo results confirm the data generated by 2D and 3D culture and they highlight that Si-HPCH allows transplantation of viable hASC in the subcutis of nude mice while maintaining their viability and GAG-production activity for 6 weeks.",
"Prior to embarking on a preclinical experiment of canine osteochondral defect repair with Si-HPCH and autologous ASC, we sought to check whether Si-HPCH can also support the viability of canine ASC. As shown in Figure 3A, we observed that the metabolic activity of cASC cultured in 2D while in contact with Si-HPMC or Si-HPCH increased from day 0 to day 7.",
"Whereas this increase was found to be statistically significant for cells cultured in contact with Si-HPMC, we failed to detect a significant increase in the metabolic activity with Si-HPCH. As expected, the MTS activity of cells cultured on a plastic substrate (CTRL) increased significantly, while that of cells cultured in the presence of actinomycin-D (Actino) dropped significantly from day 0 to day 7. To further document the effects of hydrogels on cell viability, we also analyzed the viability of cASC cultured 3D in Si-HPMC or Si-HPCH. Figure 3B illustrates the viability of cASC after 7 days of culture in 3D into Si-HPMC hydrogel in the presence of actinomycin-D (Panel Actino) or not (Panel Si-HPMC) and into Si-HPCH hydrogel (Panel Si-HPCH). As shown in Figure 3C, when cASC were 3D-cultured in Si-HPMC, there was a significant decrease in viability. Notably, cells cultured in Si-HPCH did not exhibit a decrease in cell viability. Viewed together, these data strongly suggest that Si-HPCH is a 3D-supportive scaffold for cASC viability.",
"Having demonstrated that enrichment of Si-HPMC with Sichitosan yields a mechanically reinforced hydrogel that is able to support the viability of canine and human ASC, we wanted to test the ability of this newly developed hydrogel to regenerate FIGURE 1 | Rheological and mechanical.1pc properties of Si-HPMC and Si-HPCH hydrogels. As described in the section \"Materials and Methods,\" the viscosity (A) was measured with a MARS rheometer with a cone/plate geometry; the injectability (B) was investigated using a TAXT2 Texture Analyzer at an injectability rate of 17 sec per mL; the storage modulus (C) and the breaking strength (D) were measured with a MARS rheometer using a plate geometry. * * p < 0.01 compared to the Si-HPMC condition (Mann-Whitney). articular cartilage in a canine model of osteochondral defects. Osteochondral defects (6 mm in diameter and 5 mm in depth) were created in the knees of adult dogs and filled with Si-HPCH in the absence or presence of autologous ASC. All of the animals survived and fully recovered from the surgery. At 4 months after the surgery, the dogs were euthanized and condyles isolated for histological analyses of tissue repair. Macroscopic observation of the condyles revealed that one sample from the Si-HPCH/cASC group exhibited a fracture at the implanted site. This sample was, therefore, excluded from the study.",
"As illustrated in Figure 4, macroscopic observation ( Figure 4A) of the samples did not reveal any abnormal findings indicative of inflammation. To further compare the histological features of the repair tissues, we then performed HE staining ( Figure 4B). Figure 4A illustrates the worst (panels b, d, and f) and the best (panels c, e, and g) cartilage defect repair outcomes for each group compared to healthy cartilage (panel a). Notably, while there was barely any detectable repaired tissue in the empty defects, a newly formed tissue, partially covering the entire surface of the defects, was found in all the filled defects. Figure 4B illustrates the worst (panels b, d, and f) and the best (panels c, e, and g) cartilage defect repair outcomes for each group compared to healthy cartilage (panel a). These analyses of the repair tissue in the empty defect group (Figure 4B, panel b and c) revealed the presence of a fibrous tissue that was unable to induce an ad integrum covering of the defects.",
"To more specifically characterize the repaired tissue, histological (Safranin O and Movat pentachrome staining) and immunohistological analyses (type I and II collagen) were then performed (Figure 5). The repair tissue in the empty defect group (panel Empty Defect) was Safranin O-negative, thus indicating the absence of GAG, and did not exhibit any positivity for the immunodetection of type II collagen. A faint but detectable positive immunostaining for type I collagen was also observed, thereby confirming the fibrous nature of the repaired tissue. The defects treated with Si-HPCH or Si-HPCH/ASC were almost completely covered (Figure 5, panel Si-HPCH and Si-HPCH/ASC). Si-HPCH alone was found to induce an almost complete covering of the defects, while Si-HPCH associated with cASC, induced an ad integrum filling of the defects. In both conditions, the repaired tissue stained positively for the presence of GAG (Figure 5, SO) and collagen (Figure 5, Movat). Movat staining also revealed substantial bone remodeling in the subchondral area. Interestingly, in both conditions, the repaired tissue exhibited strong and diffuse immunostaining for type II collagen (Figure 5, Coll2) while remaining negative for type I collagen (Figure 5, Coll1). Altogether, these data very much suggest the production of a repaired tissue with cartilaginous FIGURE 2 | In vitro and in vivo viability of hASC. (A) MTS activity of hASC cultured in 2D. Cell viability was evaluated in 2D after molding Si-HPMC or Si-HPCH hydrogels on top of the cell layer (10,000 cells/cm 2 ). As described in the section \"Materials and Methods,\" an MTS assay was performed on days 0, 1, and 7. The positive control (CTRL) was obtained by growing hASC alone, while the negative control was obtained by growing hASC in the presence of actinomycin D (Actino; 5 µg/mL). The results are expressed as the percentage of day 0 for each respective condition. * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (B,C) 3D viability of hASC cultured in Si-HPMC or Si-HPCH hydrogels. Cell viability was evaluated in 3D after molding hydrogels mixed with 1 × 10 6 hASC on days 0, 1, and 7 by the Live/Dead Cell Viability assay. The negative control was obtained by adding actinomycin-D (Actino; 5 µg/mL) to the culture medium. Pictures of representative samples of hASC into Si-HPMC cultured in the presence of actinomycin-D (Actino), into Si-HPMC (Si-HPMC) or into Si-HPMC (Si-HPCH) at day 7 are shown (B). The scale bar represents 100 µm. Live and dead cells were then counted (C). The results are expressed as the percentage of day 0 for each respective condition. * p < 0.05; * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (D) In vivo hASC viability after subcutaneous implantation. 250 µL of Si-HPMC (panels a and b) or Si-HPCH (panels c and d) were implanted alone (panels a and c) or mixed with human ASC (panels b and d; 1 × 10 6 cells/mL of hydrogel) in the subcutis of nude mice. After 6 weeks, explanted samples were histologically prepared for Alcian Blue staining. The back arrows indicate hASC while the white arrows indicate chitosan. The scale bar represents 100 µm.",
"features including the presence of GAG and type II collagen and the absence of type I collagen. Finally, to quantitatively assess the formation of a repaired tissue in the different conditions, HE staining (Figure 4) in association with the macroscopic appearance as well as SO, Movat, and anti-type I/II collagen staining ( Figure 5) were used to provide a modified O'Driscoll scoring (Figure 6). Three independent assessors blindly scored the various samples. For the filled conditions, the O'Driscoll-based repair scoring (Figure 6) indicated that the Si-HPCH samples were more consistently repaired than the Si-HPCH/ASC-treated defects. Indeed, the mean score of the Si-HPCH samples was 27.6 ± 2.82 while for the Si-HPCH/cASC samples the mean score was 22.31 ± 8.14. Unfortunately, and probably because of the reduced number of samples, no statistical difference was recorded between the different groups, even though a clear tendency was noted.",
"In this study, we showed in a dog model of osteochondral defects that it is possible to repair articular cartilage with a hybrid hydrogel made of cellulose-and chitosan-derived polymers. After having shown that associating Si-HPMC with Si-chitosan led to the formation of a mechanically reinforced hydrogel that can support MSC viability in vitro and in vivo, we were able to clearly demonstrate the ability of this novel self-setting hydrogel to contribute to the repair of osteochondral defects in a canine model in the absence or presence of MSCs.",
"Due to the limited ability of cartilage to heal, untreated joint cartilage damage leads to progressive tissue degeneration that drastically increases the risk of the development of osteoarthritis (Clouet et al., 2009). It has been estimated that patients with cartilage lesions have a 5-fold increased risk of osteoarthritis (Gelber, 2000). The development of new therapies in cartilage regenerative medicine is, therefore, an issue of considerable importance. In order to devise treatments for cartilage damage, strategies combining cells and biomaterials have been developed. As a result of their physicochemical and biological properties, hydrogels are presently considered to be ideal candidates for cartilage tissue engineering. Over the past decade, our team has developed a hydrogel made of cellulose, referred to as Si-HPMC (Bourges et al., 2002a,b). Previous studies have shown that this Si-HPMC hydrogel is an injectable, self-setting, and biocompatible hydrogel that allows MSCs to regenerate soft tissues such as heart muscle (Mathieu et al., 2012) and colon (Moussa et al., 2017). As bone and joint tissues are chronically exposed to high mechanical stress, hydrogels need to have exceptional mechanically properties if they are to be suitable for repair of these tissues. In addition, in light of the role of FIGURE 3 | In vitro viability of cASC. (A) MTS activity of cASC cultured in 2D. Cell viability was evaluated in 2D after molding Si-HPMC or Si-HPCH hydrogels on top of the cell layer (10,000 cells/cm 2 ). As described in the section \"Materials and Methods\" an MTS assay was performed on days 0 and 7. The positive control (CTRL) was obtained by growing cASC alone, while the negative control was obtained by growing cASC in the presence of actinomycin D (Actino; 5 µg/mL). The results are expressed as the percentage of day 0 for each respective condition. * * p < 0.01; * * * p < 0.001 compared to day 0 (Kruskal-Wallis). (B,C) 3D viability of cASC cultured in Si-HPMC or Si-HPCH hydrogels. Cell viability was evaluated in 3D after molding hydrogels mixed with 1 × 10 6 cASC on days 0, 1, and 7 by the Live/Dead Cell Viability assay. The negative control was obtained by adding actinomycin-D (Actino; 5 µg/mL) to the culture medium. Pictures of representative samples of cASC into Si-HPMC cultured in the presence of actinomycin-D (Actino), into Si-HPMC (Si-HPMC) or into Si-HPCH (Si-HPCH) at day 7 (B). The scale bar represents 100 µm. Live and dead cells were then counted (C). The results are expressed as the percentage of day 0 for each respective condition. * * * p < 0.001 compared to day 0 (Kruskal-Wallis).",
"FIGURE 4 | Macroscopic observation and histological characterization of tissue repair. Canine osteochondral defects (6 mm × 5 mm) were performed as described in the section \"Materials and Methods.\" The defects were either left empty (\"empty\" defects) of filled with Si-HPCH alone (\"Si-HPCH\") or Si-HPCH associated with cASC (\"Si-HPCH/cASC\"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and the samples were macroscopically observed (A) or histologically characterized after hematoxylin-eosin-safran staining (B). For each tested condition, the worst (b,d,f) and the best (c,e,g) repair outcomes are presented. Arrowheads indicate the edges of the defect. Healthy cartilage (a) of intact canine condyle is shown as an internal comparator. The scale bar represents 2.5 mm.",
"FIGURE 5 | Extracellular matrix-specific histological and immunohistological analyses of tissue repair. Canine osteochondral defects (6 mm × 5 mm) were performed as described in the section \"Materials and Methods.\" The defects were left empty (\"empty\" defects) or filled with Si-HPCH alone (\"Si-HPCH\") or Si-HPCH associated with cASC (\"Si-PHCH/cASC\"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and samples were histologically characterized by Safranin O (SO) and Movat pentachrome (Movat) staining. The samples were also immunostained for type II collagen (\"Coll2\") and type I collagen (\"Coll1\"). Healthy cartilage of intact canine condyle is shown as an internal comparator. Representative histological slides are presented. The scale bar represents 2.5 mm.",
"ECM stiffness and rigidity on stem cell fate (Engler et al., 2008;Discher et al., 2009), it has become clear that synthetic ECM, such as hydrogels used to guide tissue regeneration, have to exhibit biomechanical properties that largely mimic those of FIGURE 6 | O'Driscoll-based quantitative scoring of cartilage repair. Canine osteochondral defects (6 mm × 5 mm) were performed and the defects left empty (\"empty\" defects) or filled with Si-HPCH alone (\"Si-HPCH\") or Si-HPCH associated with cASC (\"Si-HPCH/cASC\"). After 4 months of implantation, the dogs were sacrificed, the knees were retrieved, and samples were histologically characterized (see Figure 5). An O'Driscoll-based histological scoring was then performed according to 10 histological parameters as described ( Table 1). Each parameter was scored 0 to 4, with 4 being the best score. The results are expressed as individual plots for each condition. the native tissue. In this context and considering the limited mechanical properties of Si-HPMC, we hypothesized that the addition of silanized chitosan to Si-HPMC could be a way to improve its mechanical properties. Interestingly, it has also been widely demonstrated that mechanical loading also influences the chondrogenic activities of MSC (Panadero et al., 2016;Choi et al., 2018). It could have been interesting to address whether combining mechanical loading and our Si-HPCH hydrogel may be a relevant strategy to improve MSC chondrogenesis.",
"In addition, chitosan was also considered due to its structural similarity to GAGs, which are a major component of the extracellular matrix of cartilage. Chitosan has been used for many years as a dressing and has been shown to improve wound healing with a high level of collagen deposition (Hamilton et al., 2006). Furthermore, like some GAGs such as hyaluronic acid, chitosan contains N-acetylglucosamine chains. As a result of this characteristic, chitosan may exhibit some of the biological activities of GAGs such as their ability to bind growth factors or their mucoadhesive properties (Martins et al., 2014). Therefore, this study aimed to improve the properties of an already developed Si-HPMC-based hydrogel by addition of Si-Chitosan as an additive within its network.",
"Moreover, previous studies have suggested that the mechanical properties of Si-HPMC remain far from those of native cartilage tissue. The stiffness of hydrogels is often reported has being two orders of magnitude lower than cartilage's (100-1000 kPa) (Levental et al., 2007;Boyer et al., 2018). These low mechanical properties have drastically limited their clinical translatability and hydrogels are mainly used as space-filling scaffolds used for the delivery of bioactive molecules and cells (Drury and Mooney, 2003;Kopeček, 2007). To address this issue, we also focused our efforts on the development of a mechanically reinforced hydrogel.",
"In light of this, the first objective of this study was to characterize the mechanical properties of the hybrid Si-HPCH hydrogel that we had developed. We found that the addition of silanized chitosan to Si-HPMC increased the viscoelastic storage modulus G (nearly 10-fold). Interestingly, while the mechanical properties of Si-HPCH were determined to be higher than those of Si-HPMC, the Si-HPCH remained manually injectable, as indicated by its injectability force of less than 80 N (Vo et al., 2016). The increase in the viscosity and the storage modulus can be explained by the higher polymer concentration within the hybrid hydrogel (4 wt% for Si-HPCH compared to 2 wt% for Si-HPMC hydrogel). Shear mechanical results, storage modulus, and breaking strength, with the same Si-HPMC and Si-HPCH concentration of 4%, revealed an effect of the chitosan backbone on the mechanical properties of the hydrogel, with an increase of the G (8 vs. 14 KPa p < 0.05, data not shown) and a decrease in the breaking stress (1,300 vs. 800 Pa, p < 0.05, data not shown). However, the influence of the chitosan backbone on the rheological and mechanical properties of the hydrogel was not investigated throughout this study. These data clearly demonstrate that Si-HPCH, while exhibiting improved mechanical properties, remains manually injectable and can hence be used for mini-invasive surgical purposes.",
"In addition to their mechanical properties, the cytocompatibility of newly developed hydrogels is also a basic requirement for their use in tissue engineering. Since it is well known that improvement of the mechanical properties of a hydrogel often results in a decrease in its cytocompatibility, we performed an in-depth analysis of the cytocompatibility of Si-HPCH with respect to MSC. This observation can be explained by an increase in shear forces, as suggested by several studies (Gefen et al., 2008;Hong et al., 2016).",
"We first addressed whether MSC from human adipose tissue retain their viability when cultured in 2D or 3D while in contact with or embedded in Si-HPCH. The choice of MSCs was guided by the increasing use of these cells in regenerative medicine and tissue engineering studies (Ruiz et al., 2016). Indeed, although chondrocytes were the first cell type used for cartilage regenerative medicine, these cells exhibit many disadvantages, such as morbidity of the donor site and their in vitro dedifferentiation (Benya and Shaffer, 1982). These shortcomings led researchers to find a new source of cells for tissue engineering. In this context, MSCs are an attractive option because they can readily be harvested from different tissues [e.g., bone marrow (Friedenstein et al., 1968), adipose tissue (Fraser et al., 2006), etc.,]. In addition, MSCs have the capacity to differentiate into the chondrogenic lineage and to exhibiting immunomodulatory properties (Tyndall, 2015) that are clinically relevant for cartilage tissue engineering. Surprisingly, and while cell-cell contact have been largely proposed as a necessary prerequisite for MSC chondrogenesis (DeLise et al., 2000), our data strongly suggest that MSC embedded into a polysaccharidic hydrogel without any external pro-chondrogenic factors, may conserve their ability to commit into the chondrogenic lineage. Whether these data may be exploited to propose a cell-free approach for the repair of osteochondral defects now deserve to be further investigated.",
"The results obtained in our various viability tests indicate good cytocompatibility of Si-HPCH with respect to ASC (of human or canine origin) cultured either in 2D or 3D. To complement these in vitro viability assays, we also performed implantation of Si-HPCH associated with hASC in subcutaneous sites of nude mice. This in vivo experiment confirmed that Si-HPCH was able to support the viability of hASC even after 6 weeks of in vivo implantation. Interestingly, our histological analyses of samples retrieved from the injection site also indicate that hASC, when implanted with Si-HPCH, exhibited a notable ability to produce GAG. Whether this ability of cells to produce ECM component when embedded in chitosan-containing cellulose hydrogel is related to the biological properties of chitosan, including its ability to interact with growth factors (Raftery et al., 2013), warrants further exploration.",
"Finally, to assess whether Si-HPCH may be a good candidate for cartilage repair, we undertook animal testing in dog, as they remain a clinically relevant veterinary target for cartilage repair strategies. Dogs are commonly affected by joint dysplasia (Ytrehus et al., 2007;Pascual-Garrido et al., 2018;Soo et al., 2018) and there is still not an effective therapeutic treatment for the resulting osteochondral defects. As is the case in humans, these untreated osteochondral defects often lead to an increased risk of osteoarthritis and severe disability.",
"The results of our preclinical testing in dogs showed that, 4 months after the implantation of hydrogels in osteochondral defects, Si-HPCH allowed for an almost complete filling of the osteochondral defects irrespective of the presence of autologous ASC.",
"Histological and immunohistological analyses of the newly formed tissue indicated the presence of a hyaline cartilage-like tissue with a GAG-and type II collagen-rich ECM. While these analyses are evidence for the presence of a tissue resembling healthy articular cartilage, several in situ biomechanical analyses can now be performed to further document the cartilaginous nature of this repaired tissue.",
"Unexpectedly, our data also revealed that defects filled with Si-HPCH alone exhibited a degree of repair that was quite comparable to what was observed for defects filled with Si-HPCH/ASC. Of particular interest for the clinical translation of our data, O'Driscoll scoring revealed that defects treated with Si-HPCH alone had a highly consistent and reproducible repair process while those treated with Si-HPCH/ASC exhibited a greater degree of inter individual variability. These data, which are presumably related to the great variability of ASC properties between individuals, are likely to raise some important issues regarding the ability to exploit the regenerative properties of ASC. The ability of Si-HPCH to support cartilage repair also raises the possibility that this hydrogel, due to the growth factor interacting properties of its chitosan moieties, may promote chemoattraction of regenerative cells emanating from subchondral bone marrow. This hypothesis, although appealing due to the clinical potential, should, however, be tested further before definitive conclusions are drawn.",
"To conclude, we have developed a new hybrid hydrogel made of silylated chitosan and HPMC that has better mechanical properties, hand-compatible injectability, and in vitro and in vivo cytocompatibility. This Si-HPCH hydrogel supports the repair of load-bearing osteochondral defects in a canine model in the presence or absence of adipose stromal cells.",
"The possibility of using Si-HPCH for the development of a cell-free repair strategy of osteochondral defects may be of great value for cartilage clinicians, notably by reducing cell dependent inter-individual variability, cost, logistical complexity, and regulatory concerns. Testing of its efficacy in canine or equine patients with naturally occurring and OA-related osteochondral defects should now be undertaken to further address the clinical value of Si-HPCH in cartilage repair.",
"The raw data supporting the conclusions of this article will be available by the authors, without undue reservation, to any qualified researcher.",
"CB, PW, CV, OGe, OGa, and JG contributed to conception and design of the study. CB, GR, Cd'A, JL, CV, BH, OGe, MF, GV, PR, and OGa carried out the experiments. CB performed the statistical analysis. CB, GR, PW, OGa, and JG wrote sections of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.",
"This research was supported by the SATT Ouest Valorisation in the framework of the \"MAN & ANIMAL: ONE HEALTH\" call for proposals, the Agence Nationale de la Recherche in the framework of ANR-11-BSV5-0022 (HYCAR). This work was also funded by the French Research on Osteo Arthritis Diseases (ROAD) Network (Arthritis Foundation)."
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Si-HPMC and Si-Chitosan Synthesis and Hydrogel Preparation",
"Mechanical and Rheological Characterization",
"Cell Viability",
"Cell Viability in 2D",
"Cell Viability in 3D",
"In vivo Experiments",
"Nude Mice Subcutis",
"Canine Osteochondral Defects",
"Histological Analyses",
"Statistical Analyses",
"RESULTS",
"Rheological and Mechanical Properties",
"Cell Viability",
"Repair of Osteochondral Defects in Dog",
"DISCUSSION",
"DATA AVAILABILITY STATEMENT",
"AUTHOR CONTRIBUTIONS",
"FUNDING",
"TABLE 1 |",
"TABLE 1 |"
] | [
"Histological parameters \n\nDegree of filling \n\n-80 to 100% \n4 \n\n-60 to 80% \n3 \n\n-40 to 60% \n2 \n\n-20 to 40% \n1 \n\n-0 to 20% \n0 \n\nSurface regularity \n\n-Flush \n4 \n\n-Rough \n3 \n\n-Slight depressed \n2 \n\n-Depressed \n1 \n\n-Overgrown \n0 \n\nGlycosaminoglycan content \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Weak \n1 \n\n-None \n0 \n\nCartilage thickness \n\n-Similar to the surrounding cartilage \n4 \n\n-Greater than the surrounding cartilage \n3 \n\n-Less than the surrounding cartilage \n2 \n\n-Very thin layer of cartilage \n1 \n\n-No cartilage \n0 \n\nCellular morphology \n\n-Hyaline cartilage \n4 \n\n-Mostly hyaline cartilage \n3 \n\n-Mostly fibrocartilage \n2 \n\n-Fibrocartilage \n1 \n\n-No cartilage \n0 \n\nType 2 collagen staining in the defect \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Slight \n1 \n\n-None \n0 \n\nType 1 collagen staining in the defect \n\n-None \n4 \n\n-Slight \n3 \n\n-Moderate \n2 \n\n-Important \n1 \n\n-Complete \n0 \n\nAdjacent cartilage quality \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Poor \n1 \n\n-Degraded \n0 \n\nSubchondral bone integrity \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Moderate \n2 \n\n-Poor \n1 \n\n-Degraded \n0 \n\n(Continued) \n\n",
"Histological parameters \n\nOsteochondral junction \n\n-Normal \n4 \n\n-Nearly normal \n3 \n\n-Slight disruption \n2 \n\n-Severe disintegration \n1 \n\n-Disruption \n0 \n\n"
] | [
"Table 1)",
"Table 1)"
] | [
"A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs",
"A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs"
] | [
"Front. Bioeng. Biotechnol"
] |
238,357,290 | 2021-12-25T19:32:40Z | CCBYNC | https://journals.sagepub.com/doi/pdf/10.1177/09636897211048786 | GOLD | 14e73bb65083e90b5ba244adf53e04d178a1b572 | null | null | null | null | 10.1177/09636897211048786 | null | 34606735 | 8493326 |
Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat
Samaneh Khazaei
Masoud Soleimani
Seyed Hossein
Ahmadi Tafti
Rouhollah Mehdinavaz Aghdam
Zohreh Hojati
Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat
10.1177/09636897211048786Special Issue Cell Medicinecardiovascular diseasemiR-13D cultureencapsulationdifferentiationmesenchymal stem cellstransplantation
Cardiovascular disease is one of the most common causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources in cell-based therapies in heart regeneration. There are several methods to differentiate MSCs into cardiac-like cells, such as gene induction. Moreover, using a three-dimensional (3D) culture, such as hydrogels increases efficiency of differentiation. In the current study, mouse adipose-derived MSCs were co-transduced with lentiviruses containing microRNA-1 (miR-1) and Myocardin (Myocd). Then, expression of cardiac markers, such as NK2 homeobox 5(Nkx2-5), GATA binding protein 4 (Gata4), and troponin T type 2 (Tnnt2) was investigated, at both gene and protein levels in two-dimensional (2D) culture and chitosan/collagen hydrogel (CS/CO) as a 3D culture. Additionally, after induction of myocardial infarction (MI) in rats, a patch containing the encapsulated induced cardiomyocytes (iCM/P) was implanted to MI zone. Subsequently, 30 days after MI induction, echocardiography, immunohistochemistry staining, and histological examination were performed to evaluate cardiac function. The results of quantitative real -time polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by 3D culture of transduced cells increased expression of cardiac markers. Besides, results of in vivo study implicated that heart function was improved in MI model of rats in iCM/P-treated group. The results suggested that miR-1/Myocd induction combined with encapsulation of transduced cells in CS/CO hydrogel increased efficiency of MSCs differentiation into iCMs and could improve heart function in MI model of rats after implantation.
Introduction
Cardiovascular disease (CVD) is main reason of death globally 1 . In the recent decades, there have been significant advances in surgical techniques and pharmacological therapies. Nevertheless, CVDs have remained a leading cause of heart failure worldwide 2 . For this reason, it seems necessary to adopt new methods, such as cardiac regenerative approaches and cell-based therapies 3 . Among cellular sources, mesenchymal stem cells (MSCs) are widely used due to their unique properties including paracrine effects, strong immunomodulation, low immunogenicity 4 , and differentiation ability into several cell lines like cardiomyocyte 5 . MSCs are found in various sources, such as bone marrow, placenta, cord blood, and adipose tissue 6 . Among these, adipose-derived MSCs have several advantages such as their accessibility, easy harvesting, low morbidity, and high expansion capacity 7 . Recently, combined strategies, such as genetic modification 8 and using biomaterials 9 have been developed to increase therapeutic efficacy of MSCs.
In several studies, using genetic modification, MSCs have been differentiated into induced cardiomyocyte-like cells (iCMs), for which both different genes and microRNAs (miRNAs) are used [10][11][12] . The miRNAs bind to the 3 0 untranslated region (3 0 UTR) of target genes and downregulate the expression of them 13 . Various studies implicate that miRNAs are involved in cell proliferation, differentiation, and development 14 . Moreover, different miRNAs are involved in cardiogenesis, which are known as cardiac miR-NAs, such as miR-208a/b, miR-499, miR-133a, and miR-1. The miR-1 and miR-133 are involved in early stages of cardiogenesis, while miR-208a/b and miR-499 regulate late stages of cardiac development 15 . The miR-1 is one of miR-NAs differentiating MSCs into cardiomyocyte-like cells by induction of NK2 homeobox 5(Nkx2.5) and GATA binding protein 4(Gata4) (earliest cardiac markers) expression and activating Wnt/b-catenin signaling pathway 16 .
In muscle differentiation, serum response factor (SRF) acts as an activator of muscle genes including miR-1 17 . However, it has been shown that SRF is a weak activator, requiring a co-activator called as Myocardin (Myocd) to function properly. SRF and Myocd synergistically increase miR-1 expression by binding to miR-1 enhancer sequences 18 . In this regard, herein, the effect of simultaneous overexpression of miR-1 and Myocd on differentiation of MSCs into iCMs was investigated.
Despite many advances in genetic modification of MSCs and their differentiation into iCMs, these cells are not sufficiently effective in cell-based therapy because MSC-derived cardiomyocytes in a two-dimensional (2D) cell culture are often immature and have a phenotype similar to that of fetal cardiomyocyte instead of adult cardiomyocyte 19 . Therefore, many studies have investigated various approaches of 3D cell culture, such as using polymer scaffold, decellularized extracellular matrix, and different hydrogels for further differentiation and maturation of MSCs [20][21][22] . Furthermore, in myocardial infarction (MI) models, most of the cells injected straightly in infarction zone would undergo apoptosis 23 . Therefore, for increasing retention of the injected cells as well as protecting them from apoptosis in the infarction site, appropriate scaffolds can be utilized to improve healing process 23 .
Hydrogels as the first water-swollen biomaterials used in tissue engineering can create a 3D structure 24 . Chitosan is one of the widely used materials in hydrogels because of its properties such as biodegradability, biocompatibility, hydrophilicity, non-toxicity, and compressive strength due to its positive charge 25 . In cardiac regeneration, chitosan has been used in various studies as a cell carrier, increasing cell retention and viability, improving heart function by reducing infarct size and neovascularization in MI model [26][27][28] .
One of the strategies in tissue engineering is the use of composite hydrogels. Chitosan-collagen (CS/CO) hydrogel is one of the most widely used composite hydrogels to improve cardiac cell differentiation and regeneration 29 . Collagen is one of the main components of extracellular matrix promoting tensile strength in heart wall. Collagen promotes survival, proliferation, and cell attachment 30 . Nevertheless, it has high biodegradability rate with weak mechanical strength 31 . But due to mechanical stability and positive charge of chitosan, combination of chitosan and collagen improves mechanical strength and reduces degradation rate of collagen 32 . Thus, in the current study, the effect of simultaneous induction of miR-1 and Myocd on differentiation of MSCs into iCMs in 2D and 3D cultures (CS/CO hydrogel) was investigated. In addition, iCMs encapsulated in CS/CO hydrogel were transplanted in MI model of rats and various tests were performed to evaluate improvement of heart function.
Materials and Methods
Fabrication of Composite Hydrogel
Composite hydrogel consisting of chitosan (CS), human collagen type I (CO), and b-glycerophosphate (b-GP), as a crosslinker, was fabricated according to the previous studies 33 . Briefly, CS and CO (Sigma Aldrich, UK) were dissolved in 0.1 N and 0.02 N acetic acid and were stirred for 6 h to make 2.0% w/w and 4 mg/ml of stock solution, respectively. Then, CS and CO solutions were mixed in a ratio of 3:1 and b-GP (7.5%) was added dropwise as a cross-linker on ice bath. Next, the solution was pipetted into a 48-well plate and gel formation was performed by addition of sterile saturated NaHCO3 at 37 C for 15 min. In order to investigate the porous structure, the hydrogel was freeze-dried at -80 C for 48 h. Subsequently, the freeze-dried samples were covered with gold particles and were studied by a scanning electron microscope (SEM) (Hitachi, SU3500) with 15 kV of accelerating voltage.
Physiochemical Properties of CS/CO Hydrogel
Compressive strength of CS and CS/CO hydrogels was measured on a mechanical test machine (Model: INSTRON 5566). All the samples were compressed to 50% of their initial height and compressive strength of each sample was calculated with respect to compressive strain ratio. For testing swelling ratio, the freeze-dried samples were allowed to hydrate in phosphatebuffered saline (PBS) at room temperature (RT). The swollen samples were weighed at 50 minute-intervals, after removal of excess PBS by gentle blotting. Swelling ratio was calculated as follows: Q ¼ (Ms-Md) / Md; where Q is swelling ratio, Ms is mass at the swollen state, and Md is mass at the dried state. Finally, Fourier-transform infrared (FTIR) spectroscopy was performed for evaluation of chemical structure of CS and CS/CO. FTIR spectrum was documented at the range of 400 -4000 cm -1 , using KBr pellet by FTIR spectrophotometer (FT-IR 8400 S, Shimadzu, Japan).
Isolation and Characterization of MSCs
Isolation and characterization of MSCs was mentioned in supplementary file.
Lentiviral Production and Transduction of MSCs
For lentiviral generation of miR-1 and Myocd, HEK-293 T cell line (Pasteur institute, Iran) was cultured at a concentration of 3 Â 10 6 cells in 10-cm plates. The mmu-mir-1 vector (containing the cytomegalovirus (CMV) and Simian virus 40(SV40) promoters) (Applied Biological Materials, Canada, mm10023), as well as Myocd (abm, 3130606) and pLenti-III-miR-GFP-Blank (abm, m001) as control were separately co-transfected with psPAX2 (containing gag/pol element) and pMD2. G (vesicular stomatitis virus (VSV) element) into HEK-293 T cell line using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's protocols. Then, 48 and 72 h after transfection, the supernatant containing lentiviral particles were collected. Collected viruses were concentrated by ultracentrifuge at 40000 g for 2 h at 4 C.
MSCs at passage 3 were used for transduction. The cells were seeded at a concentration of 4 Â 10 5 in 6-cm petri dishes and the next day were transduced in three groups; first a group of cells that were transduced with pLenti-GFP-blank lentiviruses containing green fluorescent protein (GFP) (MSC null ), another group of cells that were infected with miR-1 lentiviruses containing GFP (MSC miR-1 ), and the third group of cells that were transduced with both of miR-1 and Myocd lentiviruses containing GFP (MSC miR-1/Myocd ). The fourth group consisting of non-transduced MSCs was considered as a control (MSC). The composition of the medium used during transduction is the same as the composition of the medium during cell culture, that composed high glucose DMEM, FBS10%, penicillin (100U/ml), and streptomycin (100mg/ml). For transduction, a mixture of the concentrated viruses and polybrene (8 mg/mL) was added into the MSCs at a multiplicity of infection (MOI) of 30. Infection efficiency was evaluated 3 days after transduction by examination of GFP expression using a fluorescence microscope (Olympus, Tokyo, Japan). After puromycin selection (2mg/ml), the transduced cells were cultivated in fresh medium containing DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) for 21 days.
Viability Assay of Transduced Cells
For evaluating their viability, transduced cells were pipetted in equal volumes at a concentration of 1Â10 4 in 96-well plate and were kept at 37 C for 7, 14, and 21 days. Cell viability rate was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide (MTT; Sigma Aldrich) assay. At given time points, 100 ml/well of MTT was added into the cells, then was kept at 37 C for 4 h, followed by adding 100 ml/well of dimethyl sulfoxide (DMSO). The samples were monitored at 580 nm of wavelength using enzyme-linked immunosorbent assay (ELISA) reader. The non-transduced MSCs were used as a control.
Evaluation of Differentiation of Transduced Cells
Measuring expression levels of miR-1 and Myocd, and target genes in transduced cells by qRT-PCR. For verifying transduction efficiency, expression levels of miR-1 and Myocd in treated and control groups was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Total RNA was extracted from the transduced cells on day 4 using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Moreover, total RNA was isolated from different groups of transduced cells and MSCs control group on 7, 14, and 21 days after transduction for evaluation of Tnnt2, Nkx2-5 and Gata4 expression. cDNA was obtained through reverse transcription of total RNA by cDNA synthesis kit (Takara, Korea). Then, qRT-PCR was performed by SYBR Green PCR master mix (Ampliqon) in an Applied Biosystems 7500 real -time PCR system, according to the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the house keeping gene for target gene expression and U6 small nuclear RNA (U6) was selected as the house keeping gene for miRNA expression. The 2 -DDCt approach was used for data analysis. The sequences of primers used for target amplification are listed in Table 1.
Immunocytochemistry (ICC) analysis.
Twenty-one day after transduction in the 48-well plate, fixation of MSCs miR-1 and MSCs miR-1/Myocd was performed by 4% paraformaldehyde (PFA) for 20 min at RT and permeabilization was performed with 0.2% Triton X-100 (Sigma, USA) for 5 min at RT. Then, cells were incubated overnight with primary antibodies, at 4 C. cTnT (ab8295, Abcam, MA, UK), NKX2-5 (ab272914) and GATA4 (ab227512) were used as primary antibodies. Subsequently, cells were incubated with fluorescence-labeled secondary antibodies for 1 h at RT. The cells nuclei were stained with 0.1mg/ml of blue-fluorescent 4', 6-Diamidino-2-phenylindole (DAPI) DNA stain (Sigma, USA) at RT for 5 min. Finally, cells were observed by a fluorescence microscope (Olympus, Tokyo, Japan). ICC analysis for MSCs group was mentioned in supplementary file.
3D Cell Culture
Cell viability and proliferation assay for encapsulated cells into CS/CO hydrogel was performed and non-transduced MSCs were used because of eliminate transduction interference on viability test results. For this regard, MSCs were encapsulated to CS/CO hydrogel and were homogenized by stirrer for 1 min. After transferring the mixture to the culture plate, it was neutralized using sterile saturated NaHCO3 solution to pH level of 7, and then it was incubated at 37 C for 15 min. Finally, cell culture medium was added to the encapsulated cells.
Viability and proliferation assays in CS/CO hydrogel. For evaluating their viability, MSCs were pipetted in equal volumes with a density of 1Â10 5 in 96-well plate and were kept at 37 C for 1, 3,7, and 14 days. Cell viability rate was examined by MTT assay, as previously described. Cell free hydrogel and 2D culture of MSCs were used as control. Moreover, for showing cell proliferation potential in the hydrogel, the cells were labeled with 1,1'-dioctadecyl-3,3,3',3' -tetramethylindocarbocyanine perchlorate (DiI) (Thermofisher), a red cell membrane dye, according to manufacturer's instructions. Finally, on days 1, 3, and 7, fluorescence microscopy was performed.
SEM analysis. Twenty-one days after transduction, the MSCs embedded in hydrogel was prepared for SEM analysis. The medium was aspirated, and glutaraldehyde (2.5%) was added to the cells and they were kept for 1 h at 4 C and after washing with PBS, dehydration was performed by ascending series of ethanol and SEM was performed as previously described 34 .
Encapsulation of Transduced Cells in CS/CO Hydrogel and Evaluation of their Differentiation into iCMs
3 days after transduction, MSCs miR-1/Myocd were trypsinized and encapsulated in CS/CO hydrogel. Then, like the 2D culture, qRT-PCR and ICC assay for differentiation assessment of encapsulated transduced cells were performed. Total RNA was obtained from the cells on 7, 14, and 21 days after infection. Subsequently, after cDNA synthesis, qRT-PCR was performed for evaluation of Tnnt2, Nkx2-5, and Gata4 expression. MSCs miR-1/Myocd in 2D culture was used as a control group. Moreover, ICC assay was implemented on MSCs miR-1/Myocd for cTnT, NKX2-5 and GATA4 markers.
In vivo Studies
Induction of myocardial infarction (MI) in rats and patch transplantation. In the current study, 20 male Sprague Dawley (SD) rats (6-8 weeks old, and 180-250 g) were used. All the animal experiments were conducted according to the guidelines approved by Care and Use of Laboratory Animals and the Ethics Committee of the Tarbiat Modares University, Iran (IR.MODARES.REC.1398.125). The rats were anesthetized by intramuscular injection of 50 ml/kg of ketamine hydrochloride and 5 mg/kg of xylazine. Next, the thoracotomy was performed, the pericardium was opened and a 5-0 silk suture was narrowed close the left anterior descending (LAD) coronary artery. After 10 min, color of the area under the suture was changed and patches were transplanted on MI zone. The transplanted patches (P), comprising of CS/CO hydrogel placed on Polydimethylsiloxane (PDMS) were used in three groups; P (cell-free patch), MSC/P, and iCM/P. In MSC/P and iCM/P groups, MSCs and iCMs were encapsulated in CS/CO hydrogel and then were placed on PDMS. For tracking the transplanted cells in rat heart tissue, DiI staining was used to label cells before transplantation according to manufacturer's structure. In MI group, the LAD was sutured but no patches were transplanted on MI zone. Finally, rats were sacrificed by an intravenous injection of sodium thiopental (100 ml/kg) and the hearts were harvested for qRT-PCR, SEM, and histological analyses.
Echocardiography. Echocardiographic images of the hearts from 20 rats were obtained using a GE Vivid 7 echocardiography machine before MI, 3 days after MI (baseline), and 30 days after MI from different groups. For measuring left ventricular (LV) function, fractional shortening (FS), ejection fraction (EF), End-systolic volume (ESV), and Enddiastolic volume (EDV) were assessed by echocardiography. The percentage of fractional shortening (FS) was calculated as follows:
FS (%) ¼[(LVEDdÀLVESd)/LVEDd] Â 100
where LVEDd is LV end-diastolic dimension and LVESd is LV end-systolic dimension. All the measurements were performed based on averaged three sequential cycles of cardiac and were assessed by two independent operators who were blinded to treat and control groups of the animals.
RNA isolation and qRT-PCR analysis. Total RNA isolation, cDNA synthesis, and qRT-PCR were performed for Tnnt2, Nkx2-5, and Gata4 genes for intervention and control groups, as previously described. MI group was used as negative control. Histological analysis and immunohistochemistry (IHC). Samples were prepared for histological analysis according to previous study 35 . Briefly, samples underwent 10% formalin fixation for 24 h and were embedded into paraffin wax. Paraffinembedded tissues were cut at a thickness of 3mm and were prepared for histological assay. For the routine histological assessment was performed on tissue sections by Hematoxylin and Eosin (H & E) staining 36 . Moreover, IHC analysis was performed according to previous protocols 37 and paraffinembedded sections were immunostained with cTnT, NKX2-5, and GATA4 antibodies. The images were observed by inverted fluorescence microscope (Olympus, Japan).
Statistical analysis
All data were represented as mean + standard deviation (SD) of at least three experiments. The differences between test and control groups were analyzed with the unpaired t-test, one-way or two-way ANOVA using GraphPad Prism v9.0 (USA). P-values < 0.05 were regarded to be statistically significant.
Results
Morphology and characterization of the CS/CO hydrogel
SEM analysis was performed for determining morphological structure of CS and CS/CO hydrogels. The composite hydrogel exhibited a structure with CO fibrils dispersed within CS matrix. Moreover, interconnecting pores and creasing patterns were formed inside the hydrogels (Fig. 1A). CS and CS/CO hydrogel showed porous structure with 51.57% and 56.68% porosity, 17-89 mm and 21-109 mm pore size, respectively. This means that the pore size in both hydrogels allows the exchange of nutrients and biological molecules between cells. Stress-strain curve showed that pure CS and CS/CO hydrogels indicated a gradually enhancement in stiffness. Moreover, the CS/CO hydrogel showed a significant increase (**P < 0.01) in compression strength in comparison with the pure CS hydrogel because CO-containing hydrogels are stiffer than pure CS due to higher density (Fig. 1B). As shown in Fig. 1C, the swelling ratio in CS/CO hydrogel was significantly lower than pure CS hydrogel (*P < 0.05), because of higher hydrophilicity of CS compared to CO. FTIR spectrum of CS recorded absorption at 3150 cm -1 and 2870 cm -1 , which are attributed to groups of OH-and CH3-, respectively. In addition, the absorptions observed at 1570 cm -1 and 1410 cm -1 are related to stretching vibration of N-H group and vibration of OH-group (primary alcohol), respectively. The bond observed at 1318 cm -1 and 1070 cm -1 corresponds to tensile vibrations of C-O-N and C-O. Additionally, the adsorption intensities observed in 1152 cm -1 and 837 cm -1 are attributed to glucoside bonds, respectively. The peak observed at 1686 cm -1 indicates tensile vibration of C¼O and the band seen at 1705 cm -1 is related to free acetic acid. FTIR spectrum of the CS / CO hydrogel included all CS or CO adsorption peaks separately. No extra peaks were observed. It should be noted that the interaction between CO and CS occurs through formation of hydrogen bonds. When the amount of CO in the hydrogel is low, intensity of Amide I peak is decreased, whereas intensity corresponding to Amide II is increased. Amide I peak at 1623 cm -1 for pure CO was almost not found in the hydrogel sample, but it can be seen in the case of Amide II (the corresponding peak at 1560 cm -1 ) with a slight change at 1554 cm -1 in the hydrogel, proving well the proper interaction between CO and CS. In addition, triple helix continuity in CO can be evaluated at 1235 cm -1 and 1450 cm -1 peaks. Intensity ratio of these two peaks in pure CO was equal to 1; however, it was increased to 1.06 through addition of CS in the hydrogel, confirming the proper interaction between CO and CS in the hydrogel. On the other hand, this implies that structural properties of CO have been well preserved (Fig. 1D).
Up-Regulation of miR-1 and Myocd Expression in Transduced Cells
Transduction efficiency was performed by fluorescent microscopy and flow cytometry assessment, which about 80% of cells were transduced ( Fig. 2A). Expression levels of miR-1 and Myocd were determined on day 4 after transduction in intervention and control groups using qRT-PCR assay. The results indicated that expression of miR-1 in MSC miR-1 and MSC miR-1/Myocd groups was increased by 68-and 88-folds, respectively, compared to MSCs control group. Moreover, Myocd expression was up-regulated by 43-and 57-folds in MSC Myocd and MSC miR-1/Myocd groups, respectively, compared to MSCs control group (Fig. 2B). Expectedly, when MSCs were transduced with empty lentiviruses, miR-1 and Myocd expression showed no significant changes in comparison with MSCs group. Viability of the transduced cells in intervention groups evaluated by MTT assay was lower than control groups (Fig. 2C).
Differentiation of Transduced Cells into iCM
Seven days after transduction, morphological changes were observed in different groups of transduced cells. On day 7, morphology of MSC miR-1 and MSC miR-1/Myocd groups was found to be short spindle-or star-shaped (Fig. 3A). As expected, morphology of control groups (MSC and MSC null ) did not change (Data were not shown). Twenty-one days after transduction, further differentiation and maturation was observed. The cells became polygonal or spindle-shaped and were connected to adjacent cells (Fig. 3A).
As shown in Fig. 3B, mRNA expression of the genes involved in cardiogenesis, such as Nkx2-5 and Gata4 (key transcription factors), Tnnt2 (cardiac specific marker) was analyzed by qRT-PCR on days 7, 14, and 21 posttransduction. The results indicated that Nkx2-5 and Gata4 genes were expressed at maximum levels 7 days after transduction and then, their expression was gradually decreased, while the maximum level of Tnnt2 expression was observed on day 14 in MSC miR-1 and MSC miR-1/Myocd groups. The results also indicated that expression levels of target genes were significantly higher in the MSC miR-1/Myocd group than the MSC miR-1 . For ICC analysis, MSC miR-1 and MSC miR-1/Myocd were immunostained with cTnT, NKX2.5, and GATA4 antibodies on day 21 after transduction. The images indicated that the markers in the MSC miR-1/Myocd group were more upregulated than the MSC miR-1 group (Fig. 3C). The results confirmed that Myocd gene had a synergistic effect on miR-1 expression resulting in increased expression of cardiac markers. Images of MSCs ICC analysis was mentioned in supplementary file (Fig. S2).
Viability and Proliferation Analyses of Cells in CS/CO Hydrogel
For viability and proliferation analysis of composite hydrogel, MSCs were encapsulated in CS/CO hydrogel. Dil staining of the encapsulated cells in the CS/CO hydrogel showed that the cells were proliferated over time (Fig. 4A). In addition, quantified results regarding assessment of proliferation showed that the number of DiI-positive encapsulated cells were significantly increased on days 3 and 7 compared to day 1 (*P < 0.05, **P < 0.01, respectively) (Fig. 4B). In addition, viability and proliferation assays were performed for 1, 3, 7, and 14 days after 3D culture. The MTT assay results showed percentage of surviving cells grown in the CS/CO hydrogel. Fig. 4C illustrates that the cells had the ability to survive in the hydrogel at different time points. Cell free hydrogel and 2D culture of MSCs were used as control. Besides, 21 days after encapsulation, MSCs were prepared for SEM analysis. As shown in Fig. 4D, the cells were well attached and encapsulated inside the hydrogel with round morphologies.
Differentiation of Encapsulated Transduced Cells in CS/CO Hydrogel
3 days after transduction, transduced cells (MSC miR-1/Myocd ) were trypsinized and cultured in CS/CO hydrogel. Subsequently, qRT-PCR and ICC analysis were performed to evaluate the differentiation of encapsulated cells. As in the 2D culture, mRNA gene expression of Tnnt2, Nkx2-5 and Gata4, were measured 7, 14, and 21 days after transduction. According to Fig. 5A, expression level of target genes in encapsulated transduced cells were significantly increased compared to the transduced cells in 2D cell culture (*P < 0.05). Additionally, ICC images showed that the expression of cardiac markers in 3D culture were upregulated in comparison with 2D culture (Fig. 5B). Taken together, these results implicated that encapsulation of transduced cells into CS/CO hydrogel promotes the differentiation of MSCs into iCMs, due to increased cell-cell and cell-environment interactions.
Effect of iCM/P on Cardiac Function
After MI induction (Fig. 6A), transplantation of patch was performed in different groups (Fig. 6B). Echocardiograph analysis was performed 30 days after patch transplantation in different groups (Fig. 6C). The increased EF and FS in both iCM/P and MSC/P groups indicated functional recovery after cell/patch transplantation in comparison with MI control group. Additionally, the increase in EF and FS was significantly higher in the iCM/P group compared to MSC/P group by *P < 0.05 and **P < 0.01, respectively. A significant decrease in EDV and ESV was found 30 days after MI compared to 3 days after MI in the iCM/P group (Fig. 6D), meaning that 30 days after MI induction in the intervention group, volume of blood remaining in the heart ventricle was reduced after ejection, similar to normal heart.
Effect of iCM/P on Cardiac gene Expression in MI Zone
The expression levels of Tnnt2, Nkx2-5, and Gata4 was significantly increased in iCM/P group with mean fold changes of 10.4, 7.2, and 5, respectively compared to MI control group. Moreover, expression of the genes was significantly increased in iCM/P group much more than MSC/P group (**P < 0.01) (Fig. 7A). Besides, in Patch group, expression of target genes did not show a significant difference in comparison with MI group.
Histological and IHC Staining in MI Zones
For histological examination, H&E staining was performed for MI zone of patch transplantation-treated and MI groups. As shown in Fig. 7B, myocytes in iCM/P and MSC/P groups were arranged regularly and vascular structures and red blood cells were detectable. In contrast, no regularly and vascular structures were detected in MI zone of MI group. On day 30 post-transplantation, the patch-treated groups showed the grafted cells with DiI labeling in MI zone. In heart sections with DiI labeling in iCM/P and MSC/P groups, co-localization of DiI labeling and cTnT, NKX2.5, and GATA4 markers was observed. Expression of markers in Patch and MI groups was observed only in border of MI zones (Fig. 7C).
Discussion
The miRNAs are considered as regulators in bioprocesses, such as differentiation, proliferation, and development and they have important roles in cardiogenesis 38 . Among cardiac-specific miRNAs, miR-1 is involved in early stage of cardiomyocyte development by targeting different signaling pathways including Wnt/b-catenin pathway. Various studies have shown that miR-1 activates Wnt/b-catenin signaling pathway by downregulation of genes that are inhibitor of Wnt pathway. Activation of the Wnt/b-catenin pathway has been found to enhance expression of genes, such as wnt11, b-catenin, c-Jun N-terminal kinase (JNK), and ternary complex factor(TCF), which in turn enhanced expression of cardiac-specific genes including Nkx2-5, Gata4, and Tnnt2 16 . Also, level of cardiac miRNAs, in particular miR-1 has been indicated to be increased in differentiation of MSCs into iCMs. Therefore, miR-1 overexpression causes further differentiation of MSCs into iCMs via targeting Wnt pathway and regulation of target genes expression 39 . Myocyte enhancer factor-2 (Mef2), SRF, and Myocd are miR-1 transcriptional activators, binding to its enhancer sequences. Myocd is a co-activator for SRF because it is a weak activator. Thus, Myocd is an important factor for miR-1 expression in early stages of heart development 17 the previous studies 40,42 , our results showed that expression of cardiac markers including Tnnt2, Nkx2-5, and Gata4 at gene and protein levels was higher in MSCs transduced with a combination of miR-1 and Myocd than MSCs transduced with miR-1 alone.
Most studies on cells function have been performed in 2D microenvironment, but recently 3D culture has been developed because of its advantages over 2D culture. In 3D cell culture, different cells interact with each other and with extracellular matrix. Whereas, in 2D cell culture, cells interact only with their surrounding cells 43,44 . In addition, mechanical forces between cell-cell and cell-environment in 3D culture provide a set of signals, regulating different functions of cells, such as proliferation, differentiation, and migration 45 . Moreover, structure and organization of cells in 3D culture change compared to a 2D culture, influencing function and cell signaling 46 . Therefore, it can be concluded that cell behaviors in 3D culture are different from 2D culture.
3D culture promotes an environment for better signaling between cells and their neighboring, and causes more realistic physiological, biomechanical, and biochemical properties, and less substrate stiffness than 2D environment, collectively increasing differentiation capability of MSCs into different cell lines. A combination of these conditions alters gene expression of MSCs and thus, activates different signaling pathways ultimately improving efficacy of MSCs differentiation 47 . Pineda et al., investigated myocardial differentiation of embedded mouse embryonic stem cells (M-ESCs) in collagen type I gels (GELs) 48 . They indicated that expression of Gata4, FMS-like tyrosine kinase 1(Flt1), and endothelin type B receptor (Endbr) was upregulated in GELs compared to 2D culture. As a result, 3D culture is a supportive environment for cardiovascular differentiation. Histological analysis showed that myocytes in iCM/P group had regularly structure and red blood cells were observed, while in MI group, inflammatory cells infiltrated in MI zone and large area of fibrosis was detectable. C) Thirty days after MI induction, IHC staining was performed for NKX2-5, GATA4, and cTnT markers of MI zone tissue. The images showed that the immunostained cells in the iCM/P group were more than the other groups. Encapsulated cells of iCM/P and MSC/P groups were labeled with DiI stain for tracking in rats heart tissues.
Hydrogels are more common than other biomaterials due to their properties, such as similarity to natural extracellular matrices (ECM), biocompatibility, encapsulation of cells, integrating with host cells, and enhancement of cell proliferation, survival, and differentiation 49 .
CS as a biocompatible polysaccharide is frequently used in tissue engineering due to its antioxidant activity, lack of immunogenicity, and bioactive properties 26 . Studies have shown that CS as a scaffold in combination with MSCs can improve heart function in MI models of rats 27,28 . There are several mechanisms for improving function of ischemic heart and reduction of infarct size for example, (1) CS, as a carrier of MSCs increases their survival and engraftment, which is related to antioxidant activity of CS and eliminating of reactive oxygen species (ROS), diminishing cell adhesion, and eventually improving MI environment 50 . (2) CS is involved in formation of neovasculature. In addition, MSCs in combination with CS play a synergistic role in ischemic myocardial angiogenesis because they have paracrine effect and release angiogenic factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and hepatocyte growth factor (HGF) 51 . (3) CS increases differentiation of MSCs into cardiac-like cells due to collagen synthesis 28 . CO is one of the main components of ECM playing an important role in process of cardiac maturation and differentiation of MSCs 30 . Therefore, heart function is improved by replacing cardiac differentiated cells in MI zone.
CO facilitates cell adhesion by integrin receptors, activating signaling pathways, which in turn regulate cell survival, proliferation, and differentiation 30 . Therefore, in combination with CS, efficiency of differentiation of MSCs into cardiac-like cells is improved. Moreover, due to similarity of CO to the pericardium and native ECM, and enhancement of tensile strength, composite hydrogel of CS and CO improves heart function in a MI model of rat 33 . The prepared integrated porous CS/CO hydrogel network provided a microenvironment for cell proliferation and differentiation by permeability of nutrients and biological molecules and development of signaling pathway among embedded cells in hydrogel 33 . Moreover, the presence of CO in the hydrogel increases compaction. As a result, CS/CO hydrogel is more compact and stiffer than pure CS 33 . Additionally, results of viability test and Dil staining showed that the composite CS/CO hydrogel promoted growth and proliferation of the encapsulated cells and had no cytotoxicity effect. In the current study, first mouse MSCs were co-transduced with miR-1 and Myocd lentiviruses, and after 3 days, the transduced cells were encapsulated in CS/CO hydrogel to improve efficiency of differentiation and maturation. In the encapsulated cells, expression of cardiac markers of Tnnt2, Nkx2-5, and Gata4, and, at levels of gene and protein was significantly upregulated in comparison with the transduced cells in 2D culture.
Previous studies have shown that transplantation of MSCs in MI zone does not effectively improve heart function due to high apoptosis rate of the transplanted cells in the infarct environment and low efficiency of their differentiating into cardiomyocytes and endothelial cells 48,52 . Therefore, recently, using different hydrogels has been considered for injection or transplantation of MSCs in the MI zone 53,54 . In the present report, for in vivo studies, after co-transduction of MSCs with miR-1 and Myocd lentiviruses, iCMs were encapsulated into CS/CO hydrogel followed by placed on PDMS for making patch. PDMS is a biocompatible and biodegradable substrate that has been shown in several studies to use as a substrate to promote the self-renewal and differentiation of stem cells into cardiomyocyte 58 . PDMS have also been used in several studies as a suitable substrate for cultivation 59 and implantation 60 .
After MI induction, patches were implanted to the MI zone in different groups including iCM/P, MSC/P, and cell-free/P groups. Echocardiography of rat heart tissue was performed for assessing heart function. The results confirmed that EF and FS were increased in iCM/P and MSC/P groups, indicating an improvement in the impaired heart function after transplantation of the encapsulated cells 55 . Moreover, cardiac markers, at the level of gene and protein, in iCM/P group significantly increased in comparison with control group.
As mentioned in previous studies 56,57 , MSCs transplantation in MI zone can partially improve heart function due to induction of angiogenesis, paracrine effects, and stimulating differentiation of the implanted MSCs into cardiomyocyte. In the iCM/P group, not all MSCs are differentiated into iCMs, and this group includes a heterogeneous population of differentiated iCMs and undifferentiated MSCs. Therefore, in the iCM/P group, both iCMs and MSCs are effective in improving heart function after MI induction. As a result, in the MSC/P group, a relative improvement in heart function is observed.
Conclusion
Our results revealed that co-induction of miR-1 and Myocd followed by culture in CS/CO hydrogel led to greater differentiation and maturation of MSCs. Additionally, heart function was improved after transplantation of encapsulated iCMs in MI model of rat. Therefore, our findings indicated importance of 3D culture in stem cell differentiation. So, in the future studies, progress in cardiac regeneration based on cell therapy can be achieved by improving differentiation conditions of MSCs and subsequently, obtaining mature cardiac cells.
Figure 1 .
1Fabrication and characterization of CS/CO hydrogel. (A)Results of SEM analysis of CS/CO and pure CS hydrogels showed a porous structure. (B) Stress-strain curve of CS/CO hydrogel and pure CS showed that CS/CO hydrogel had significantly higher compressive strength than CS. **P < 0.01. (C) Swelling ratio was significantly lower in CS/CO hydrogel than CS because pure CS is more hydrophilic than CS/CO hydrogel. *P < 0.05. (D) FTIR spectra of CS and CS/CO hydrogel showed a successful cross-link between CS and CO. CS: Chitosan, CS/CO: Chitosan/collagen, FTIR: Fourier-transform infrared.
Figure 2 .
2Transduction efficiency of MSCs with lentiviruses containing miR-1 and Myocd. (A) Transduced cells were observed by fluorescence microscopy regarding expression of GFP reporter. Moreover, percentage of transduced cells was measured by flow cytometry. About 80% of MSCs were transduced compared to the control cells. (B) Expression of miR-1 and Myocd in different groups was evaluated by qRT-PCR. **P < 0.01, ***P < 0.001; data were expressed as mean + SD, n ¼ 3. (C) Cell viability of transduced cells was studied by MTT analysis. In intervention groups, cell viability was significantly reduced on days 14 and 21. *P < 0.05, **P < 0.01, ***P < 0.001. GFP: Green fluorescent protein.
Figure 3 .
3Differentiation of transduced cells into iCMs. (A)Seven days after transduction, morphology of MSC miR-1 and MSC miR-1/Myocd groups became spindle-shaped or star-shaped. Twenty-one days after transduction, the cells became more differentiated and matured and short spindle-shaped or polygonal cells were observed, similar to cardiomyocytes. (B) qRT-PCR analysis was performed for cardiac specific markers of Tnnt2, Nkx2-5, and Gata4. Expression of markers was compared in MSC miR-1 and MSC miR-1/Myocd groups with MSC null as a negative control. The results showed that expression of Tnnt2, Nkx2-5, and Gata4 was increased significantly in MSC miR-1/Myocd group in comparison with other groups, on days 7, 14, and 21 after transduction. *P < 0.05, **P < 0.01, ***P < 0.001; Transcript value was shown as mean+SD. (C) ICC analysis detected cTnT, Nkx2-5, and GATA4 markers in MSC miR-1 and MSC miR-1/Myocd groups on day 21. Nucleus staining was conducted by DAPI stain. An Olympus fluorescence microscope was used to record images.
Figure 4 .
43D culture of MSCs into the CS/CO hydrogel and viability assay. (A) Dil staining of the encapsulated cells in the CS/CO hydrogel implicated that the cells were proliferated over time. (B) The number of the stained cells was increased significantly on day 7 compared to day 1 after cultivation in the CS/CO hydrogel **P < 0.01. (C) Results of MTT assay implicated that cell viability percentage was increased significantly on 3, 7, and 14 days after encapsulation in the CS/CO hydrogel. *P < 0.05, ****P < 0.0001. (D) SEM image of the encapsulated transduced cells into the CS/CO hydrogel with two magnifications. 3D culture: Three dimensional culture, CS/CO: Chitosan/ collagen.
. Moreover, in various studies, the effect of Myocd on cardiac cell differentiation and enhancement of cardiac genes expression has been investigated 40,41 . Zhang et al. studied transdifferentiation of fibroblasts into iCMs by transduction of miR-1 alone or in combination with five cardiac transcription factors including GATA4, T-box transcription factor 5 (TBX5), Mef2, MYOCD, and NKX2-5 (GTMMN). TNNT2 þ cells were significantly enhanced when miR-1 was used in combination with GTMMN 42 . In the current study, cotransduction effect of miR-1 and Myocd was evaluated on efficacy of MSCs differentiation into iCMs. Consistent with
Figure 5 .
5Differentiation of transduced cells into iCM after encapsulation in the CS/CO hydrogel. Three days after co-transduction of MSCs with miR-1 and Myocd lentiviruses, the infected cells were trypsinized and embedded in the CS/CO hydrogel. (A) Expression of Tnnt2, Nkx2-5, and Gata4, and was evaluated by qRT-PCR in 2D and 3D cell cultures (CS/CO hydrogel). Expression of markers was increased significantly in the 3D cell culture in comparison with the 2D cell culture. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Transcript value was shown as mean+SD. (B) Immunofluorescence staining of transduced cells 21 days after transduction showed that expression of the markers was enhanced in 3D culture. iCMs: Induced cardiomyocytes, CS/CO: Chitosan/collagen, 3D culture: Three dimensional culture.
Figure 6 .
6Evaluation of heart function after patch transplantation. (A)Schematic representation of the heart before and after MI induction and patch transplantation. (B) MI induction was performed by tightening the silk suture around the LAD. After changing color of the area, patch containing CS/CO, placed on PDMS, was transplanted on the MI zone. (C) Representative M-mode echocardiogram of the MI, P, MSC/P, and iCM/P groups is shown. D) FS, EF, ESV, and EDV values of different groups were quantified. FS and EF values were increased significantly in MSC/P and iCM/P groups versus MI group. Moreover, ESV and EDV values were significantly reduced in MSC/P and iCM/P groups compared to MI group. In addition, FS and EF values were enhanced significantly in iCM/P group versus MSC/P group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MI: Myocardial infarction, LAD: Left anterior descending, PDMS: Polydimethylsiloxane, FS: Fractional shortening, EF: Ejection fraction, ESV: End-systolic volume, EDV: End-diastolic volume, iCMs: Induced cardiomyocytes.
Figure 7 .
7qRT PCR, histological studies and immunohistochemical staining of heart tissues after patch implantation. (A) qRT-PCR assessment of MI zone tissue in the intervention and control groups. (B)
Table 1 .
1Sequences of Primers Used in qRT-PCR.Gene
symbol
Primer
Product
size (bp)
miR-1
F: GTAGGCACCTGAAATGGAA
85
R: TTGATGGTGCCTACAGTACAT
U6
F: CTCGCTTCGGCAGCACA
94
R: AACGCTTCACGAATTTGCGT
Myocd
F: CAAGCCAAAGGTGAAGAAGC
177
R: TAGCTGAATCGGTGTTGCTG
Gata4
F: TCATCTCACTACGGGCACAG
233
R: GGGAAGAGGGAAGATTACGC
Nkx2-5
F: CCTCAACAGCTCCCTGACTC
201
R: GGGGACAGCTAAGACACCAG
Tnnt2
F: GGCAGCTGCTGTTCTGAGGGAG
191
R: TGCCCTGGTCTCCTCGGTCT
Gapdh
F: CCTGGAGAAACCTGCCAAGTA
148
R: GGCATCGAAGGTGGAAGAGT
AcknowledgmentThe authors would like to thank the Tehran Heart Center for helping us in this study.Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.Ethics approvalEthical approval to report this case was obtained from the Ethics Committee of the Tarbiat Modares University, Iran (IR.MOD-ARES.REC.1398.125).Statement of human and animal rightsAll procedures in this study were conducted in accordance with the Ethics Committee of the Tarbiat Modares University, Iran (IR.MO-DARES.REC.1398.125) approved protocols.Statement of informed consentThere are no human subjects in this article and informed consent is not applicable.FundingThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the Tarbiat Modares University and in part by the Isfahan University.ORCID iDsRouhollah Mehdinavaz Aghdam https://orcid.org/0000-0003-2325-5121 Zohreh Hojati https://orcid.org/0000-0003-4831-0123Supplemental MaterialSupplemental material for this article is available online.
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| [
"Cardiovascular disease is one of the most common causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources in cell-based therapies in heart regeneration. There are several methods to differentiate MSCs into cardiac-like cells, such as gene induction. Moreover, using a three-dimensional (3D) culture, such as hydrogels increases efficiency of differentiation. In the current study, mouse adipose-derived MSCs were co-transduced with lentiviruses containing microRNA-1 (miR-1) and Myocardin (Myocd). Then, expression of cardiac markers, such as NK2 homeobox 5(Nkx2-5), GATA binding protein 4 (Gata4), and troponin T type 2 (Tnnt2) was investigated, at both gene and protein levels in two-dimensional (2D) culture and chitosan/collagen hydrogel (CS/CO) as a 3D culture. Additionally, after induction of myocardial infarction (MI) in rats, a patch containing the encapsulated induced cardiomyocytes (iCM/P) was implanted to MI zone. Subsequently, 30 days after MI induction, echocardiography, immunohistochemistry staining, and histological examination were performed to evaluate cardiac function. The results of quantitative real -time polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by 3D culture of transduced cells increased expression of cardiac markers. Besides, results of in vivo study implicated that heart function was improved in MI model of rats in iCM/P-treated group. The results suggested that miR-1/Myocd induction combined with encapsulation of transduced cells in CS/CO hydrogel increased efficiency of MSCs differentiation into iCMs and could improve heart function in MI model of rats after implantation."
] | [
"Samaneh Khazaei ",
"Masoud Soleimani ",
"Seyed Hossein ",
"Ahmadi Tafti ",
"Rouhollah Mehdinavaz Aghdam ",
"Zohreh Hojati "
] | [] | [
"Samaneh",
"Masoud",
"Seyed",
"Ahmadi",
"Rouhollah",
"Mehdinavaz",
"Zohreh"
] | [
"Khazaei",
"Soleimani",
"Hossein",
"Tafti",
"Aghdam",
"Hojati"
] | [
"Y Zhao, ",
"P Londono, ",
"Y Cao, ",
"E J Sharpe, ",
"C Proenza, ",
"R O'rourke, ",
"K L Jones, ",
"M Y Jeong, ",
"L A Walker, ",
"P M Buttrick, ",
"T A Mckinsey, ",
"M L Muiesan, ",
"A Paini, ",
"C A Rosei, ",
"F Bertacchini, ",
"D Stassaldi, ",
"M Salvetti, ",
"P Hénon, ",
"V Karantalis, ",
"J M Hare, ",
"S Ohnishi, ",
"H Ohgushi, ",
"S Kitamura, ",
"N Nagaya, ",
"P Musialek, ",
"A Mazurek, ",
"D Jarocha, ",
"L Tekieli, ",
"W Szot, ",
"M Kostkiewicz, ",
"R P Banys, ",
"M Urbanczyk, ",
"A Kadzielski, ",
"M Trystula, ",
"J Kijowski, ",
"E C Perin, ",
"R Sanz-Ruiz, ",
"P L Sánchez, ",
"J Lasso, ",
"R Pérez-Cano, ",
"Alonso-Farto , ",
"J C Pérez-David, ",
"E Fernández-Santos, ",
"M E Serruys, ",
"P W Duckers, ",
"H J Kastrup, ",
"J , ",
"J S Park, ",
"S Suryaprakash, ",
"Y H Lao, ",
"K W Leong, ",
"S Martino, ",
"F Angelo, ",
"I Armentano, ",
"J M Kenny, ",
"A Orlacchio, ",
"L Mcginley, ",
"J Mcmahon, ",
"P Strappe, ",
"F Barry, ",
"M Murphy, ",
"D O'toole, ",
"O 'brien, ",
"T , ",
"N K Satija, ",
"V K Singh, ",
"Y K Verma, ",
"P Gupta, ",
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"R P Tripathi, ",
"G U Gurudutta, ",
"V Neshati, ",
"S Mollazadeh, ",
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"De Vries, ",
"A A Mojarrad, ",
"M Naderi-Meshkin, ",
"H Neshati, ",
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"M Kerachian, ",
"M A , ",
"T Treiber, ",
"N Treiber, ",
"G Meister, ",
"C Torrini, ",
"R J Cubero, ",
"E Dirkx, ",
"L Braga, ",
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"M I Gutierrez, ",
"C Collesi, ",
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"Y Colombo, ",
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"A , ",
"X Shen, ",
"B Pan, ",
"H Zhou, ",
"L Liu, ",
"T Lv, ",
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"X Huang, ",
"J Tian, ",
"Y Zhao, ",
"E Samal, ",
"D Srivastava, ",
"D Z Wang, ",
"P S Chang, ",
"Z Wang, ",
"L Sutherland, ",
"J A Richardson, ",
"E Small, ",
"P A Krieg, ",
"E N Olson, ",
"A H Fong, ",
"M Romero-López, ",
"C M Heylman, ",
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"J Blumling, ",
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"N C Ni, ",
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"R K Li, ",
"J Chen, ",
"Y Zhan, ",
"Y Wang, ",
"D Han, ",
"B Tao, ",
"Z Luo, ",
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"Q Wang, ",
"X Li, ",
"L Fan, ",
"C Li, ",
"H Wang, ",
"J Shi, ",
"Y Wang, ",
"Y Yin, ",
"L Wang, ",
"J Liu, ",
"Z Liu, ",
"C Duan, ",
"P Zhu, ",
"C Wang, ",
"H Wang, ",
"J Shi, ",
"Y Wang, ",
"Y Yin, ",
"L Wang, ",
"J Liu, ",
"Z Liu, ",
"C Duan, ",
"P Zhu, ",
"C Wang, ",
"J P Orgel, ",
"A V Persikov, ",
"O Antipova, ",
"J P Orgel, ",
"A V Persikov, ",
"O Antipova, ",
"C Deng, ",
"P Zhang, ",
"B Vulesevic, ",
"D Kuraitis, ",
"F Li, ",
"A F Yang, ",
"M Griffith, ",
"M Ruel, ",
"E J Suuronen, ",
"L Wang, ",
"J P Stegemann, ",
"A Koroleva, ",
"A Deiwick, ",
"A Nguyen, ",
"S Schlie-Wolter, ",
"R Narayan, ",
"P Timashev, ",
"V Popov, ",
"V Bagratashvili, ",
"B Chichkov, ",
"D Baudouy, ",
"J F Michiels, ",
"A Vukolic, ",
"K D Wagner, ",
"N Wagner, ",
"J Valentin, ",
"A Frobert, ",
"G Ajalbert, ",
"S Cook, ",
"M N Giraud, ",
"G Yang, ",
"J Tian, ",
"C Feng, ",
"L L Zhao, ",
"Z Liu, ",
"J Zhu, ",
"J Synnergren, ",
"C Améen, ",
"A Lindahl, ",
"B Olsson, ",
"P Sartipy, ",
"X L Zhao, ",
"B Yang, ",
"L N Ma, ",
"Y H Dong, ",
"Y J Nam, ",
"K Song, ",
"X Luo, ",
"E Daniel, ",
"K Lambeth, ",
"K West, ",
"J A Hill, ",
"J M Dimaio, ",
"L A Baker, ",
"R Bassel-Duby, ",
"E N Olson, ",
"N Christoforou, ",
"M Chellappan, ",
"A F Adler, ",
"R D Kirkton, ",
"T Wu, ",
"R C Addis, ",
"N Bursac, ",
"K W Leong, ",
"N Christoforou, ",
"S Chakraborty, ",
"R D Kirkton, ",
"A F Adler, ",
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"K W Leong, ",
"D Zhang, ",
"I Y Shadrin, ",
"J Lam, ",
"H Q Xian, ",
"H R Snodgrass, ",
"N Bursac, ",
"K Ronaldson-Bouchard, ",
"S P Ma, ",
"K Yeager, ",
"T Chen, ",
"L Song, ",
"D Sirabella, ",
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"M Yazawa, ",
"G Vunjak-Novakovic, ",
"I Y Shadrin, ",
"B W Allen, ",
"Y Qian, ",
"C P Jackman, ",
"A L Carlson, ",
"M E Juhas, ",
"N Bursac, ",
"N Chaicharoenaudomrung, ",
"P Kunhorm, ",
"P Noisa, ",
"P Sokolowska, ",
"K Zukowski, ",
"I Lasocka, ",
"L Szulc-Dabrowska, ",
"E Jastrzebska, ",
"E T Pineda, ",
"R M Nerem, ",
"T Ahsan, ",
"M Domenech, ",
"L Polo-Corrales, ",
"J E Ramirez-Vick, ",
"D O Freytes, ",
"Z Liu, ",
"H Wang, ",
"Y Wang, ",
"Q Lin, ",
"A Yao, ",
"F Cao, ",
"D Li, ",
"J Zhou, ",
"C Duan, ",
"Z Du, ",
"Y Wang, ",
"Y Yamada, ",
"S I Yokoyama, ",
"X D Wang, ",
"N Fukuda, ",
"N Takakura, ",
"Y C Yeh, ",
"W Y Lee, ",
"C L Yu, ",
"S M Hwang, ",
"M F Chung, ",
"L W Hsu, ",
"Y Chang, ",
"W W Lin, ",
"M S Tsai, ",
"H J Wei, ",
"H W Sung, ",
"K L Fujimoto, ",
"Z Ma, ",
"D M Nelson, ",
"R Hashizume, ",
"J Guan, ",
"K Tobita, ",
"W R Wagner, ",
"E Ruvinov, ",
"J Leor, ",
"S Cohen, ",
"N H Chi, ",
"M C Yang, ",
"T W Chung, ",
"J Y Chen, ",
"N K Chou, ",
"S S Wang, ",
"H Tao, ",
"Z Han, ",
"Z C Han, ",
"Z Li, ",
"X R Gao, ",
"H J Xu, ",
"L F Wang, ",
"C B Liu, ",
"F Yu, ",
"J Fu, ",
"Y Chuah, ",
"J Liu, ",
"S Tan, ",
"D Wang, ",
"N E Oyunbaatar, ",
"D H Lee, ",
"S J Patil, ",
"E S Kim, ",
"D W Lee, ",
"C P Jackman, ",
"A M Ganapathi, ",
"H Asfour, ",
"Y Qian, ",
"B W Allen, ",
"Y Li, ",
"N Bursac, "
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] | [
"High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling. Y Zhao, P Londono, Y Cao, E J Sharpe, C Proenza, R O'rourke, K L Jones, M Y Jeong, L A Walker, P M Buttrick, T A Mckinsey, Nat Commun. 6Zhao Y, Londono P, Cao Y, Sharpe EJ, Proenza C, O'Rourke R, Jones KL, Jeong MY, Walker LA, Buttrick PM, McKinsey TA, et al. High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling. Nat Commun. 2015;6:1-15.",
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"High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling",
"Key success factors for regenerative medicine in acquired heart diseases",
"Use of mesenchymal stem cells for therapy of cardiac disease",
"Mesenchymal stem cells for the treatment of heart failure",
"Myocardial regeneration strategy using Wharton's jelly mesenchymal stem cells as an off-the-shelf 'unlimited'therapeutic agent: results from the Acute Myocardial Infarction First-in-Man study",
"Adipose-derived regenerative cells in patients with ischemic cardiomyopathy: the PRECISE trial",
"Engineering mesenchymal stem cells for regenerative medicine and drug delivery",
"Stem cell-biomaterial interactions for regenerative medicine",
"Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia",
"Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine",
"MicroRNA-499a-5p promotes differentiation of human bone marrow-derived mesenchymal stem cells to cardiomyocytes",
"Regulation of microRNA biogenesis and its crosstalk with other cellular pathways",
"Common regulatory pathways mediate activity of microRNAs inducing cardiomyocyte proliferation",
"microRNAs and cardiac cell fate",
"Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway",
"Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis",
"Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor",
"Three-dimensional adult cardiac extracellular matrix promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes",
"Tissue engineering of vascularized cardiac muscle from human embryonic stem cells",
"Decellularized extracellular matrix materials for cardiac repair and regeneration",
"Hydrogel based approaches for cardiac tissue engineering",
"Retention and biodistribution of microspheres injected into ischemic myocardium",
"Hydrogel scaffolds for tissue engineering: the importance of polymer choice",
"Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures",
"Polypyrrole-chitosan conductive biomaterial synchronizes cardiomyocyte contraction and improves myocardial electrical impulse propagation",
"Chitosan/silk fibroin modified nanofibrous patches with mesenchymal stem cells prevent heart remodeling post-myocardial infarction in rats",
"Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction",
"Synthesis and characterization of injectable hydrogels with varying collagen-chitosan-thymosin b4 composition for myocardial infarction therapy",
"Variation in the helical structure of native collagen",
"Collagen-chitosan polymer as a scaffold for the proliferation of human adipose tissuederived stem cells",
"A collagen-chitosan hydrogel for endothelial differentiation and angiogenesis",
"Thermogelling chitosan and collagen composite hydrogels initiated with b-glycerophosphate for bone tissue engineering",
"Osteogenic differentiation of human mesenchymal stem cells in 3-D Zr-Si organic-inorganic scaffolds produced by two-photon polymerization technique",
"Echocardiographic and histological examination of cardiac morphology in the mouse",
"Histological quantification of chronic myocardial infarct in rats",
"Trichostatin a promotes cardiomyocyte differentiation of rat mesenchymal stem cells after 5-azacytidine induction or during coculture with neonatal cardiomyocytes via a mechanism independent of histone deacetylase inhibition",
"Expression of microRNAs and their target mRNAs in human stem cell-derived cardiomyocyte clusters and in heart tissue",
"MicroRNA-1 effectively induces differentiation of myocardial cells from mouse bone marrow mesenchymal stem cells",
"Reprogramming of human fibroblasts toward a cardiac fate",
"Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardioinducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming",
"Core transcription factors, microRNAs, and small molecules drive transdifferentiation of human fibroblasts towards the cardiac cell lineage",
"Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes",
"Advanced maturation of human cardiac tissue grown from pluripotent stem cells",
"Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues",
"Threedimensional cell culture systems as an in vitro platform for cancer and stem cell modeling",
"Human mesenchymal stem cell (hMSC) differentiation towards cardiac cells using a new microbioanalytical method",
"Differentiation patterns of embryonic stem cells in two-versus three-dimensional culture",
"Tissue engineering strategies for myocardial regeneration: acellular versus cellular scaffolds?",
"The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment",
"Cardiac stem cells in brown adipose tissue express CD133 and induce bone marrow nonhematopoietic cells to differentiate into cardiomyocytes",
"Cardiac repair with injectable cell sheet fragments of human amniotic fluid stem cells in an immune-suppressed rat model",
"Synthesis, characterization and therapeutic efficacy of a biodegradable, thermoresponsive hydrogel designed for application in chronic infarcted myocardium",
"The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction",
"Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model",
"S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells",
"Mesenchymal stem cell transplantation carried in SVVYGLR modified selfassembling peptide promoted cardiac repair and angiogenesis after myocardial infarction",
"Respective effects of gelatin-coated polydimethylsiloxane (PDMS) substrates on self-renewal and cardiac differentiation of induced pluripotent stem cells (iPSCs)",
"Biomechanical characterization of cardiomyocyte using PDMS pillar with microgrooves",
"Engineered cardiac tissue patch maintains structural and electrical properties after epicardial implantation"
] | [
"Nat Commun",
"Current pharmacological therapies in heart failure patients",
"Stem Cell Rev Rep",
"Circ Res",
"Int J Hematol",
"Postepy Kardiol Interwencyjne",
"Am Heart J",
"Methods",
"Biotechnol Adv",
"Stem Cell Res Ther",
"J Cell Mol Med",
"Appl Biochem Biotechnol",
"Nat Rev Mol Cell Biol",
"Cell Rep",
"Cells",
"J Biomed Sci",
"Nature",
"Cell",
"Tissue Eng Part A",
"Circ Res",
"Adv Healthc Mater",
"Int J Pharm",
"J Biomed Mater Res A",
"Polymer Chemistry",
"Biomaterials",
"Theranostics",
"Acta Biomater",
"Biomaterials",
"J Funct Biomater",
"PLoS One",
"J Mater Sci Mater Med",
"Tissue Eng Part A",
"Biomaterials",
"PloS One",
"J Vis Exp",
"J Vis Exp",
"Cell Transplant",
"Physiol Genomics",
"Artif Cells Nanomed Biotechnol",
"Proc Natl Acad Sci U S A",
"PloS one",
"Sci Rep",
"Biomaterials",
"Nature",
"Nat Commun",
"World J Stem Cells",
"Analyst",
"Cells Tissues Organs",
"Tissue Eng Part B Rev",
"Biomaterials",
"Stem Cells",
"Biomaterials",
"Biomaterials",
"Biomaterials",
"Biomaterials",
"Proceed Nat Academy Sci",
"Biochem Biophys Res Commun",
"ACS Biomater Sci Eng",
"Sensors",
"Biomaterials"
] | [
"\nFigure 1 .\n1Fabrication and characterization of CS/CO hydrogel. (A)Results of SEM analysis of CS/CO and pure CS hydrogels showed a porous structure. (B) Stress-strain curve of CS/CO hydrogel and pure CS showed that CS/CO hydrogel had significantly higher compressive strength than CS. **P < 0.01. (C) Swelling ratio was significantly lower in CS/CO hydrogel than CS because pure CS is more hydrophilic than CS/CO hydrogel. *P < 0.05. (D) FTIR spectra of CS and CS/CO hydrogel showed a successful cross-link between CS and CO. CS: Chitosan, CS/CO: Chitosan/collagen, FTIR: Fourier-transform infrared.",
"\nFigure 2 .\n2Transduction efficiency of MSCs with lentiviruses containing miR-1 and Myocd. (A) Transduced cells were observed by fluorescence microscopy regarding expression of GFP reporter. Moreover, percentage of transduced cells was measured by flow cytometry. About 80% of MSCs were transduced compared to the control cells. (B) Expression of miR-1 and Myocd in different groups was evaluated by qRT-PCR. **P < 0.01, ***P < 0.001; data were expressed as mean + SD, n ¼ 3. (C) Cell viability of transduced cells was studied by MTT analysis. In intervention groups, cell viability was significantly reduced on days 14 and 21. *P < 0.05, **P < 0.01, ***P < 0.001. GFP: Green fluorescent protein.",
"\nFigure 3 .\n3Differentiation of transduced cells into iCMs. (A)Seven days after transduction, morphology of MSC miR-1 and MSC miR-1/Myocd groups became spindle-shaped or star-shaped. Twenty-one days after transduction, the cells became more differentiated and matured and short spindle-shaped or polygonal cells were observed, similar to cardiomyocytes. (B) qRT-PCR analysis was performed for cardiac specific markers of Tnnt2, Nkx2-5, and Gata4. Expression of markers was compared in MSC miR-1 and MSC miR-1/Myocd groups with MSC null as a negative control. The results showed that expression of Tnnt2, Nkx2-5, and Gata4 was increased significantly in MSC miR-1/Myocd group in comparison with other groups, on days 7, 14, and 21 after transduction. *P < 0.05, **P < 0.01, ***P < 0.001; Transcript value was shown as mean+SD. (C) ICC analysis detected cTnT, Nkx2-5, and GATA4 markers in MSC miR-1 and MSC miR-1/Myocd groups on day 21. Nucleus staining was conducted by DAPI stain. An Olympus fluorescence microscope was used to record images.",
"\nFigure 4 .\n43D culture of MSCs into the CS/CO hydrogel and viability assay. (A) Dil staining of the encapsulated cells in the CS/CO hydrogel implicated that the cells were proliferated over time. (B) The number of the stained cells was increased significantly on day 7 compared to day 1 after cultivation in the CS/CO hydrogel **P < 0.01. (C) Results of MTT assay implicated that cell viability percentage was increased significantly on 3, 7, and 14 days after encapsulation in the CS/CO hydrogel. *P < 0.05, ****P < 0.0001. (D) SEM image of the encapsulated transduced cells into the CS/CO hydrogel with two magnifications. 3D culture: Three dimensional culture, CS/CO: Chitosan/ collagen.",
"\n\n. Moreover, in various studies, the effect of Myocd on cardiac cell differentiation and enhancement of cardiac genes expression has been investigated 40,41 . Zhang et al. studied transdifferentiation of fibroblasts into iCMs by transduction of miR-1 alone or in combination with five cardiac transcription factors including GATA4, T-box transcription factor 5 (TBX5), Mef2, MYOCD, and NKX2-5 (GTMMN). TNNT2 þ cells were significantly enhanced when miR-1 was used in combination with GTMMN 42 . In the current study, cotransduction effect of miR-1 and Myocd was evaluated on efficacy of MSCs differentiation into iCMs. Consistent with",
"\nFigure 5 .\n5Differentiation of transduced cells into iCM after encapsulation in the CS/CO hydrogel. Three days after co-transduction of MSCs with miR-1 and Myocd lentiviruses, the infected cells were trypsinized and embedded in the CS/CO hydrogel. (A) Expression of Tnnt2, Nkx2-5, and Gata4, and was evaluated by qRT-PCR in 2D and 3D cell cultures (CS/CO hydrogel). Expression of markers was increased significantly in the 3D cell culture in comparison with the 2D cell culture. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Transcript value was shown as mean+SD. (B) Immunofluorescence staining of transduced cells 21 days after transduction showed that expression of the markers was enhanced in 3D culture. iCMs: Induced cardiomyocytes, CS/CO: Chitosan/collagen, 3D culture: Three dimensional culture.",
"\nFigure 6 .\n6Evaluation of heart function after patch transplantation. (A)Schematic representation of the heart before and after MI induction and patch transplantation. (B) MI induction was performed by tightening the silk suture around the LAD. After changing color of the area, patch containing CS/CO, placed on PDMS, was transplanted on the MI zone. (C) Representative M-mode echocardiogram of the MI, P, MSC/P, and iCM/P groups is shown. D) FS, EF, ESV, and EDV values of different groups were quantified. FS and EF values were increased significantly in MSC/P and iCM/P groups versus MI group. Moreover, ESV and EDV values were significantly reduced in MSC/P and iCM/P groups compared to MI group. In addition, FS and EF values were enhanced significantly in iCM/P group versus MSC/P group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MI: Myocardial infarction, LAD: Left anterior descending, PDMS: Polydimethylsiloxane, FS: Fractional shortening, EF: Ejection fraction, ESV: End-systolic volume, EDV: End-diastolic volume, iCMs: Induced cardiomyocytes.",
"\nFigure 7 .\n7qRT PCR, histological studies and immunohistochemical staining of heart tissues after patch implantation. (A) qRT-PCR assessment of MI zone tissue in the intervention and control groups. (B)",
"\nTable 1 .\n1Sequences of Primers Used in qRT-PCR.Gene \nsymbol \nPrimer \n\nProduct \nsize (bp) \n\nmiR-1 \nF: GTAGGCACCTGAAATGGAA \n85 \nR: TTGATGGTGCCTACAGTACAT \nU6 \nF: CTCGCTTCGGCAGCACA \n94 \nR: AACGCTTCACGAATTTGCGT \nMyocd \nF: CAAGCCAAAGGTGAAGAAGC \n177 \nR: TAGCTGAATCGGTGTTGCTG \nGata4 \nF: TCATCTCACTACGGGCACAG \n233 \nR: GGGAAGAGGGAAGATTACGC \nNkx2-5 \nF: CCTCAACAGCTCCCTGACTC \n201 \nR: GGGGACAGCTAAGACACCAG \nTnnt2 \nF: GGCAGCTGCTGTTCTGAGGGAG \n191 \nR: TGCCCTGGTCTCCTCGGTCT \nGapdh \nF: CCTGGAGAAACCTGCCAAGTA \n148 \nR: GGCATCGAAGGTGGAAGAGT \n"
] | [
"Fabrication and characterization of CS/CO hydrogel. (A)Results of SEM analysis of CS/CO and pure CS hydrogels showed a porous structure. (B) Stress-strain curve of CS/CO hydrogel and pure CS showed that CS/CO hydrogel had significantly higher compressive strength than CS. **P < 0.01. (C) Swelling ratio was significantly lower in CS/CO hydrogel than CS because pure CS is more hydrophilic than CS/CO hydrogel. *P < 0.05. (D) FTIR spectra of CS and CS/CO hydrogel showed a successful cross-link between CS and CO. CS: Chitosan, CS/CO: Chitosan/collagen, FTIR: Fourier-transform infrared.",
"Transduction efficiency of MSCs with lentiviruses containing miR-1 and Myocd. (A) Transduced cells were observed by fluorescence microscopy regarding expression of GFP reporter. Moreover, percentage of transduced cells was measured by flow cytometry. About 80% of MSCs were transduced compared to the control cells. (B) Expression of miR-1 and Myocd in different groups was evaluated by qRT-PCR. **P < 0.01, ***P < 0.001; data were expressed as mean + SD, n ¼ 3. (C) Cell viability of transduced cells was studied by MTT analysis. In intervention groups, cell viability was significantly reduced on days 14 and 21. *P < 0.05, **P < 0.01, ***P < 0.001. GFP: Green fluorescent protein.",
"Differentiation of transduced cells into iCMs. (A)Seven days after transduction, morphology of MSC miR-1 and MSC miR-1/Myocd groups became spindle-shaped or star-shaped. Twenty-one days after transduction, the cells became more differentiated and matured and short spindle-shaped or polygonal cells were observed, similar to cardiomyocytes. (B) qRT-PCR analysis was performed for cardiac specific markers of Tnnt2, Nkx2-5, and Gata4. Expression of markers was compared in MSC miR-1 and MSC miR-1/Myocd groups with MSC null as a negative control. The results showed that expression of Tnnt2, Nkx2-5, and Gata4 was increased significantly in MSC miR-1/Myocd group in comparison with other groups, on days 7, 14, and 21 after transduction. *P < 0.05, **P < 0.01, ***P < 0.001; Transcript value was shown as mean+SD. (C) ICC analysis detected cTnT, Nkx2-5, and GATA4 markers in MSC miR-1 and MSC miR-1/Myocd groups on day 21. Nucleus staining was conducted by DAPI stain. An Olympus fluorescence microscope was used to record images.",
"3D culture of MSCs into the CS/CO hydrogel and viability assay. (A) Dil staining of the encapsulated cells in the CS/CO hydrogel implicated that the cells were proliferated over time. (B) The number of the stained cells was increased significantly on day 7 compared to day 1 after cultivation in the CS/CO hydrogel **P < 0.01. (C) Results of MTT assay implicated that cell viability percentage was increased significantly on 3, 7, and 14 days after encapsulation in the CS/CO hydrogel. *P < 0.05, ****P < 0.0001. (D) SEM image of the encapsulated transduced cells into the CS/CO hydrogel with two magnifications. 3D culture: Three dimensional culture, CS/CO: Chitosan/ collagen.",
". Moreover, in various studies, the effect of Myocd on cardiac cell differentiation and enhancement of cardiac genes expression has been investigated 40,41 . Zhang et al. studied transdifferentiation of fibroblasts into iCMs by transduction of miR-1 alone or in combination with five cardiac transcription factors including GATA4, T-box transcription factor 5 (TBX5), Mef2, MYOCD, and NKX2-5 (GTMMN). TNNT2 þ cells were significantly enhanced when miR-1 was used in combination with GTMMN 42 . In the current study, cotransduction effect of miR-1 and Myocd was evaluated on efficacy of MSCs differentiation into iCMs. Consistent with",
"Differentiation of transduced cells into iCM after encapsulation in the CS/CO hydrogel. Three days after co-transduction of MSCs with miR-1 and Myocd lentiviruses, the infected cells were trypsinized and embedded in the CS/CO hydrogel. (A) Expression of Tnnt2, Nkx2-5, and Gata4, and was evaluated by qRT-PCR in 2D and 3D cell cultures (CS/CO hydrogel). Expression of markers was increased significantly in the 3D cell culture in comparison with the 2D cell culture. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Transcript value was shown as mean+SD. (B) Immunofluorescence staining of transduced cells 21 days after transduction showed that expression of the markers was enhanced in 3D culture. iCMs: Induced cardiomyocytes, CS/CO: Chitosan/collagen, 3D culture: Three dimensional culture.",
"Evaluation of heart function after patch transplantation. (A)Schematic representation of the heart before and after MI induction and patch transplantation. (B) MI induction was performed by tightening the silk suture around the LAD. After changing color of the area, patch containing CS/CO, placed on PDMS, was transplanted on the MI zone. (C) Representative M-mode echocardiogram of the MI, P, MSC/P, and iCM/P groups is shown. D) FS, EF, ESV, and EDV values of different groups were quantified. FS and EF values were increased significantly in MSC/P and iCM/P groups versus MI group. Moreover, ESV and EDV values were significantly reduced in MSC/P and iCM/P groups compared to MI group. In addition, FS and EF values were enhanced significantly in iCM/P group versus MSC/P group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MI: Myocardial infarction, LAD: Left anterior descending, PDMS: Polydimethylsiloxane, FS: Fractional shortening, EF: Ejection fraction, ESV: End-systolic volume, EDV: End-diastolic volume, iCMs: Induced cardiomyocytes.",
"qRT PCR, histological studies and immunohistochemical staining of heart tissues after patch implantation. (A) qRT-PCR assessment of MI zone tissue in the intervention and control groups. (B)",
"Sequences of Primers Used in qRT-PCR."
] | [
"(Fig. 1A)",
"(Fig. 1B)",
"Fig. 1C",
"(Fig. 1D",
"Fig. 2A)",
"(Fig. 2B)",
"(Fig. 2C)",
"(Fig. 3A)",
"(Fig. 3A)",
"Fig. 3B",
"(Fig. 3C)",
"(Fig. S2",
"(Fig. 4A)",
"(Fig. 4B",
"Fig. 4C",
"Fig. 4D",
"Fig. 5A",
"(Fig. 5B)",
"(Fig. 6A)",
"(Fig. 6B)",
"(Fig. 6C)",
"(Fig. 6D)",
"(Fig. 7A)",
"Fig. 7B",
"(Fig. 7C"
] | [
"FS (%) ¼[(LVEDdÀLVESd)/LVEDd] Â 100"
] | [
"Cardiovascular disease (CVD) is main reason of death globally 1 . In the recent decades, there have been significant advances in surgical techniques and pharmacological therapies. Nevertheless, CVDs have remained a leading cause of heart failure worldwide 2 . For this reason, it seems necessary to adopt new methods, such as cardiac regenerative approaches and cell-based therapies 3 . Among cellular sources, mesenchymal stem cells (MSCs) are widely used due to their unique properties including paracrine effects, strong immunomodulation, low immunogenicity 4 , and differentiation ability into several cell lines like cardiomyocyte 5 . MSCs are found in various sources, such as bone marrow, placenta, cord blood, and adipose tissue 6 . Among these, adipose-derived MSCs have several advantages such as their accessibility, easy harvesting, low morbidity, and high expansion capacity 7 . Recently, combined strategies, such as genetic modification 8 and using biomaterials 9 have been developed to increase therapeutic efficacy of MSCs.",
"In several studies, using genetic modification, MSCs have been differentiated into induced cardiomyocyte-like cells (iCMs), for which both different genes and microRNAs (miRNAs) are used [10][11][12] . The miRNAs bind to the 3 0 untranslated region (3 0 UTR) of target genes and downregulate the expression of them 13 . Various studies implicate that miRNAs are involved in cell proliferation, differentiation, and development 14 . Moreover, different miRNAs are involved in cardiogenesis, which are known as cardiac miR-NAs, such as miR-208a/b, miR-499, miR-133a, and miR-1. The miR-1 and miR-133 are involved in early stages of cardiogenesis, while miR-208a/b and miR-499 regulate late stages of cardiac development 15 . The miR-1 is one of miR-NAs differentiating MSCs into cardiomyocyte-like cells by induction of NK2 homeobox 5(Nkx2.5) and GATA binding protein 4(Gata4) (earliest cardiac markers) expression and activating Wnt/b-catenin signaling pathway 16 .",
"In muscle differentiation, serum response factor (SRF) acts as an activator of muscle genes including miR-1 17 . However, it has been shown that SRF is a weak activator, requiring a co-activator called as Myocardin (Myocd) to function properly. SRF and Myocd synergistically increase miR-1 expression by binding to miR-1 enhancer sequences 18 . In this regard, herein, the effect of simultaneous overexpression of miR-1 and Myocd on differentiation of MSCs into iCMs was investigated.",
"Despite many advances in genetic modification of MSCs and their differentiation into iCMs, these cells are not sufficiently effective in cell-based therapy because MSC-derived cardiomyocytes in a two-dimensional (2D) cell culture are often immature and have a phenotype similar to that of fetal cardiomyocyte instead of adult cardiomyocyte 19 . Therefore, many studies have investigated various approaches of 3D cell culture, such as using polymer scaffold, decellularized extracellular matrix, and different hydrogels for further differentiation and maturation of MSCs [20][21][22] . Furthermore, in myocardial infarction (MI) models, most of the cells injected straightly in infarction zone would undergo apoptosis 23 . Therefore, for increasing retention of the injected cells as well as protecting them from apoptosis in the infarction site, appropriate scaffolds can be utilized to improve healing process 23 .",
"Hydrogels as the first water-swollen biomaterials used in tissue engineering can create a 3D structure 24 . Chitosan is one of the widely used materials in hydrogels because of its properties such as biodegradability, biocompatibility, hydrophilicity, non-toxicity, and compressive strength due to its positive charge 25 . In cardiac regeneration, chitosan has been used in various studies as a cell carrier, increasing cell retention and viability, improving heart function by reducing infarct size and neovascularization in MI model [26][27][28] .",
"One of the strategies in tissue engineering is the use of composite hydrogels. Chitosan-collagen (CS/CO) hydrogel is one of the most widely used composite hydrogels to improve cardiac cell differentiation and regeneration 29 . Collagen is one of the main components of extracellular matrix promoting tensile strength in heart wall. Collagen promotes survival, proliferation, and cell attachment 30 . Nevertheless, it has high biodegradability rate with weak mechanical strength 31 . But due to mechanical stability and positive charge of chitosan, combination of chitosan and collagen improves mechanical strength and reduces degradation rate of collagen 32 . Thus, in the current study, the effect of simultaneous induction of miR-1 and Myocd on differentiation of MSCs into iCMs in 2D and 3D cultures (CS/CO hydrogel) was investigated. In addition, iCMs encapsulated in CS/CO hydrogel were transplanted in MI model of rats and various tests were performed to evaluate improvement of heart function.",
"Composite hydrogel consisting of chitosan (CS), human collagen type I (CO), and b-glycerophosphate (b-GP), as a crosslinker, was fabricated according to the previous studies 33 . Briefly, CS and CO (Sigma Aldrich, UK) were dissolved in 0.1 N and 0.02 N acetic acid and were stirred for 6 h to make 2.0% w/w and 4 mg/ml of stock solution, respectively. Then, CS and CO solutions were mixed in a ratio of 3:1 and b-GP (7.5%) was added dropwise as a cross-linker on ice bath. Next, the solution was pipetted into a 48-well plate and gel formation was performed by addition of sterile saturated NaHCO3 at 37 C for 15 min. In order to investigate the porous structure, the hydrogel was freeze-dried at -80 C for 48 h. Subsequently, the freeze-dried samples were covered with gold particles and were studied by a scanning electron microscope (SEM) (Hitachi, SU3500) with 15 kV of accelerating voltage.",
"Compressive strength of CS and CS/CO hydrogels was measured on a mechanical test machine (Model: INSTRON 5566). All the samples were compressed to 50% of their initial height and compressive strength of each sample was calculated with respect to compressive strain ratio. For testing swelling ratio, the freeze-dried samples were allowed to hydrate in phosphatebuffered saline (PBS) at room temperature (RT). The swollen samples were weighed at 50 minute-intervals, after removal of excess PBS by gentle blotting. Swelling ratio was calculated as follows: Q ¼ (Ms-Md) / Md; where Q is swelling ratio, Ms is mass at the swollen state, and Md is mass at the dried state. Finally, Fourier-transform infrared (FTIR) spectroscopy was performed for evaluation of chemical structure of CS and CS/CO. FTIR spectrum was documented at the range of 400 -4000 cm -1 , using KBr pellet by FTIR spectrophotometer (FT-IR 8400 S, Shimadzu, Japan).",
"Isolation and characterization of MSCs was mentioned in supplementary file.",
"For lentiviral generation of miR-1 and Myocd, HEK-293 T cell line (Pasteur institute, Iran) was cultured at a concentration of 3 Â 10 6 cells in 10-cm plates. The mmu-mir-1 vector (containing the cytomegalovirus (CMV) and Simian virus 40(SV40) promoters) (Applied Biological Materials, Canada, mm10023), as well as Myocd (abm, 3130606) and pLenti-III-miR-GFP-Blank (abm, m001) as control were separately co-transfected with psPAX2 (containing gag/pol element) and pMD2. G (vesicular stomatitis virus (VSV) element) into HEK-293 T cell line using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's protocols. Then, 48 and 72 h after transfection, the supernatant containing lentiviral particles were collected. Collected viruses were concentrated by ultracentrifuge at 40000 g for 2 h at 4 C.",
"MSCs at passage 3 were used for transduction. The cells were seeded at a concentration of 4 Â 10 5 in 6-cm petri dishes and the next day were transduced in three groups; first a group of cells that were transduced with pLenti-GFP-blank lentiviruses containing green fluorescent protein (GFP) (MSC null ), another group of cells that were infected with miR-1 lentiviruses containing GFP (MSC miR-1 ), and the third group of cells that were transduced with both of miR-1 and Myocd lentiviruses containing GFP (MSC miR-1/Myocd ). The fourth group consisting of non-transduced MSCs was considered as a control (MSC). The composition of the medium used during transduction is the same as the composition of the medium during cell culture, that composed high glucose DMEM, FBS10%, penicillin (100U/ml), and streptomycin (100mg/ml). For transduction, a mixture of the concentrated viruses and polybrene (8 mg/mL) was added into the MSCs at a multiplicity of infection (MOI) of 30. Infection efficiency was evaluated 3 days after transduction by examination of GFP expression using a fluorescence microscope (Olympus, Tokyo, Japan). After puromycin selection (2mg/ml), the transduced cells were cultivated in fresh medium containing DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) for 21 days.",
"For evaluating their viability, transduced cells were pipetted in equal volumes at a concentration of 1Â10 4 in 96-well plate and were kept at 37 C for 7, 14, and 21 days. Cell viability rate was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide (MTT; Sigma Aldrich) assay. At given time points, 100 ml/well of MTT was added into the cells, then was kept at 37 C for 4 h, followed by adding 100 ml/well of dimethyl sulfoxide (DMSO). The samples were monitored at 580 nm of wavelength using enzyme-linked immunosorbent assay (ELISA) reader. The non-transduced MSCs were used as a control.",
"Measuring expression levels of miR-1 and Myocd, and target genes in transduced cells by qRT-PCR. For verifying transduction efficiency, expression levels of miR-1 and Myocd in treated and control groups was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Total RNA was extracted from the transduced cells on day 4 using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Moreover, total RNA was isolated from different groups of transduced cells and MSCs control group on 7, 14, and 21 days after transduction for evaluation of Tnnt2, Nkx2-5 and Gata4 expression. cDNA was obtained through reverse transcription of total RNA by cDNA synthesis kit (Takara, Korea). Then, qRT-PCR was performed by SYBR Green PCR master mix (Ampliqon) in an Applied Biosystems 7500 real -time PCR system, according to the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the house keeping gene for target gene expression and U6 small nuclear RNA (U6) was selected as the house keeping gene for miRNA expression. The 2 -DDCt approach was used for data analysis. The sequences of primers used for target amplification are listed in Table 1.",
"Twenty-one day after transduction in the 48-well plate, fixation of MSCs miR-1 and MSCs miR-1/Myocd was performed by 4% paraformaldehyde (PFA) for 20 min at RT and permeabilization was performed with 0.2% Triton X-100 (Sigma, USA) for 5 min at RT. Then, cells were incubated overnight with primary antibodies, at 4 C. cTnT (ab8295, Abcam, MA, UK), NKX2-5 (ab272914) and GATA4 (ab227512) were used as primary antibodies. Subsequently, cells were incubated with fluorescence-labeled secondary antibodies for 1 h at RT. The cells nuclei were stained with 0.1mg/ml of blue-fluorescent 4', 6-Diamidino-2-phenylindole (DAPI) DNA stain (Sigma, USA) at RT for 5 min. Finally, cells were observed by a fluorescence microscope (Olympus, Tokyo, Japan). ICC analysis for MSCs group was mentioned in supplementary file.",
"Cell viability and proliferation assay for encapsulated cells into CS/CO hydrogel was performed and non-transduced MSCs were used because of eliminate transduction interference on viability test results. For this regard, MSCs were encapsulated to CS/CO hydrogel and were homogenized by stirrer for 1 min. After transferring the mixture to the culture plate, it was neutralized using sterile saturated NaHCO3 solution to pH level of 7, and then it was incubated at 37 C for 15 min. Finally, cell culture medium was added to the encapsulated cells.",
"Viability and proliferation assays in CS/CO hydrogel. For evaluating their viability, MSCs were pipetted in equal volumes with a density of 1Â10 5 in 96-well plate and were kept at 37 C for 1, 3,7, and 14 days. Cell viability rate was examined by MTT assay, as previously described. Cell free hydrogel and 2D culture of MSCs were used as control. Moreover, for showing cell proliferation potential in the hydrogel, the cells were labeled with 1,1'-dioctadecyl-3,3,3',3' -tetramethylindocarbocyanine perchlorate (DiI) (Thermofisher), a red cell membrane dye, according to manufacturer's instructions. Finally, on days 1, 3, and 7, fluorescence microscopy was performed.",
"SEM analysis. Twenty-one days after transduction, the MSCs embedded in hydrogel was prepared for SEM analysis. The medium was aspirated, and glutaraldehyde (2.5%) was added to the cells and they were kept for 1 h at 4 C and after washing with PBS, dehydration was performed by ascending series of ethanol and SEM was performed as previously described 34 .",
"3 days after transduction, MSCs miR-1/Myocd were trypsinized and encapsulated in CS/CO hydrogel. Then, like the 2D culture, qRT-PCR and ICC assay for differentiation assessment of encapsulated transduced cells were performed. Total RNA was obtained from the cells on 7, 14, and 21 days after infection. Subsequently, after cDNA synthesis, qRT-PCR was performed for evaluation of Tnnt2, Nkx2-5, and Gata4 expression. MSCs miR-1/Myocd in 2D culture was used as a control group. Moreover, ICC assay was implemented on MSCs miR-1/Myocd for cTnT, NKX2-5 and GATA4 markers.",
"Induction of myocardial infarction (MI) in rats and patch transplantation. In the current study, 20 male Sprague Dawley (SD) rats (6-8 weeks old, and 180-250 g) were used. All the animal experiments were conducted according to the guidelines approved by Care and Use of Laboratory Animals and the Ethics Committee of the Tarbiat Modares University, Iran (IR.MODARES.REC.1398.125). The rats were anesthetized by intramuscular injection of 50 ml/kg of ketamine hydrochloride and 5 mg/kg of xylazine. Next, the thoracotomy was performed, the pericardium was opened and a 5-0 silk suture was narrowed close the left anterior descending (LAD) coronary artery. After 10 min, color of the area under the suture was changed and patches were transplanted on MI zone. The transplanted patches (P), comprising of CS/CO hydrogel placed on Polydimethylsiloxane (PDMS) were used in three groups; P (cell-free patch), MSC/P, and iCM/P. In MSC/P and iCM/P groups, MSCs and iCMs were encapsulated in CS/CO hydrogel and then were placed on PDMS. For tracking the transplanted cells in rat heart tissue, DiI staining was used to label cells before transplantation according to manufacturer's structure. In MI group, the LAD was sutured but no patches were transplanted on MI zone. Finally, rats were sacrificed by an intravenous injection of sodium thiopental (100 ml/kg) and the hearts were harvested for qRT-PCR, SEM, and histological analyses.",
"Echocardiography. Echocardiographic images of the hearts from 20 rats were obtained using a GE Vivid 7 echocardiography machine before MI, 3 days after MI (baseline), and 30 days after MI from different groups. For measuring left ventricular (LV) function, fractional shortening (FS), ejection fraction (EF), End-systolic volume (ESV), and Enddiastolic volume (EDV) were assessed by echocardiography. The percentage of fractional shortening (FS) was calculated as follows:",
"where LVEDd is LV end-diastolic dimension and LVESd is LV end-systolic dimension. All the measurements were performed based on averaged three sequential cycles of cardiac and were assessed by two independent operators who were blinded to treat and control groups of the animals.",
"RNA isolation and qRT-PCR analysis. Total RNA isolation, cDNA synthesis, and qRT-PCR were performed for Tnnt2, Nkx2-5, and Gata4 genes for intervention and control groups, as previously described. MI group was used as negative control. Histological analysis and immunohistochemistry (IHC). Samples were prepared for histological analysis according to previous study 35 . Briefly, samples underwent 10% formalin fixation for 24 h and were embedded into paraffin wax. Paraffinembedded tissues were cut at a thickness of 3mm and were prepared for histological assay. For the routine histological assessment was performed on tissue sections by Hematoxylin and Eosin (H & E) staining 36 . Moreover, IHC analysis was performed according to previous protocols 37 and paraffinembedded sections were immunostained with cTnT, NKX2-5, and GATA4 antibodies. The images were observed by inverted fluorescence microscope (Olympus, Japan).",
"All data were represented as mean + standard deviation (SD) of at least three experiments. The differences between test and control groups were analyzed with the unpaired t-test, one-way or two-way ANOVA using GraphPad Prism v9.0 (USA). P-values < 0.05 were regarded to be statistically significant.",
"SEM analysis was performed for determining morphological structure of CS and CS/CO hydrogels. The composite hydrogel exhibited a structure with CO fibrils dispersed within CS matrix. Moreover, interconnecting pores and creasing patterns were formed inside the hydrogels (Fig. 1A). CS and CS/CO hydrogel showed porous structure with 51.57% and 56.68% porosity, 17-89 mm and 21-109 mm pore size, respectively. This means that the pore size in both hydrogels allows the exchange of nutrients and biological molecules between cells. Stress-strain curve showed that pure CS and CS/CO hydrogels indicated a gradually enhancement in stiffness. Moreover, the CS/CO hydrogel showed a significant increase (**P < 0.01) in compression strength in comparison with the pure CS hydrogel because CO-containing hydrogels are stiffer than pure CS due to higher density (Fig. 1B). As shown in Fig. 1C, the swelling ratio in CS/CO hydrogel was significantly lower than pure CS hydrogel (*P < 0.05), because of higher hydrophilicity of CS compared to CO. FTIR spectrum of CS recorded absorption at 3150 cm -1 and 2870 cm -1 , which are attributed to groups of OH-and CH3-, respectively. In addition, the absorptions observed at 1570 cm -1 and 1410 cm -1 are related to stretching vibration of N-H group and vibration of OH-group (primary alcohol), respectively. The bond observed at 1318 cm -1 and 1070 cm -1 corresponds to tensile vibrations of C-O-N and C-O. Additionally, the adsorption intensities observed in 1152 cm -1 and 837 cm -1 are attributed to glucoside bonds, respectively. The peak observed at 1686 cm -1 indicates tensile vibration of C¼O and the band seen at 1705 cm -1 is related to free acetic acid. FTIR spectrum of the CS / CO hydrogel included all CS or CO adsorption peaks separately. No extra peaks were observed. It should be noted that the interaction between CO and CS occurs through formation of hydrogen bonds. When the amount of CO in the hydrogel is low, intensity of Amide I peak is decreased, whereas intensity corresponding to Amide II is increased. Amide I peak at 1623 cm -1 for pure CO was almost not found in the hydrogel sample, but it can be seen in the case of Amide II (the corresponding peak at 1560 cm -1 ) with a slight change at 1554 cm -1 in the hydrogel, proving well the proper interaction between CO and CS. In addition, triple helix continuity in CO can be evaluated at 1235 cm -1 and 1450 cm -1 peaks. Intensity ratio of these two peaks in pure CO was equal to 1; however, it was increased to 1.06 through addition of CS in the hydrogel, confirming the proper interaction between CO and CS in the hydrogel. On the other hand, this implies that structural properties of CO have been well preserved (Fig. 1D).",
"Transduction efficiency was performed by fluorescent microscopy and flow cytometry assessment, which about 80% of cells were transduced ( Fig. 2A). Expression levels of miR-1 and Myocd were determined on day 4 after transduction in intervention and control groups using qRT-PCR assay. The results indicated that expression of miR-1 in MSC miR-1 and MSC miR-1/Myocd groups was increased by 68-and 88-folds, respectively, compared to MSCs control group. Moreover, Myocd expression was up-regulated by 43-and 57-folds in MSC Myocd and MSC miR-1/Myocd groups, respectively, compared to MSCs control group (Fig. 2B). Expectedly, when MSCs were transduced with empty lentiviruses, miR-1 and Myocd expression showed no significant changes in comparison with MSCs group. Viability of the transduced cells in intervention groups evaluated by MTT assay was lower than control groups (Fig. 2C).",
"Seven days after transduction, morphological changes were observed in different groups of transduced cells. On day 7, morphology of MSC miR-1 and MSC miR-1/Myocd groups was found to be short spindle-or star-shaped (Fig. 3A). As expected, morphology of control groups (MSC and MSC null ) did not change (Data were not shown). Twenty-one days after transduction, further differentiation and maturation was observed. The cells became polygonal or spindle-shaped and were connected to adjacent cells (Fig. 3A).",
"As shown in Fig. 3B, mRNA expression of the genes involved in cardiogenesis, such as Nkx2-5 and Gata4 (key transcription factors), Tnnt2 (cardiac specific marker) was analyzed by qRT-PCR on days 7, 14, and 21 posttransduction. The results indicated that Nkx2-5 and Gata4 genes were expressed at maximum levels 7 days after transduction and then, their expression was gradually decreased, while the maximum level of Tnnt2 expression was observed on day 14 in MSC miR-1 and MSC miR-1/Myocd groups. The results also indicated that expression levels of target genes were significantly higher in the MSC miR-1/Myocd group than the MSC miR-1 . For ICC analysis, MSC miR-1 and MSC miR-1/Myocd were immunostained with cTnT, NKX2.5, and GATA4 antibodies on day 21 after transduction. The images indicated that the markers in the MSC miR-1/Myocd group were more upregulated than the MSC miR-1 group (Fig. 3C). The results confirmed that Myocd gene had a synergistic effect on miR-1 expression resulting in increased expression of cardiac markers. Images of MSCs ICC analysis was mentioned in supplementary file (Fig. S2).",
"For viability and proliferation analysis of composite hydrogel, MSCs were encapsulated in CS/CO hydrogel. Dil staining of the encapsulated cells in the CS/CO hydrogel showed that the cells were proliferated over time (Fig. 4A). In addition, quantified results regarding assessment of proliferation showed that the number of DiI-positive encapsulated cells were significantly increased on days 3 and 7 compared to day 1 (*P < 0.05, **P < 0.01, respectively) (Fig. 4B). In addition, viability and proliferation assays were performed for 1, 3, 7, and 14 days after 3D culture. The MTT assay results showed percentage of surviving cells grown in the CS/CO hydrogel. Fig. 4C illustrates that the cells had the ability to survive in the hydrogel at different time points. Cell free hydrogel and 2D culture of MSCs were used as control. Besides, 21 days after encapsulation, MSCs were prepared for SEM analysis. As shown in Fig. 4D, the cells were well attached and encapsulated inside the hydrogel with round morphologies.",
"3 days after transduction, transduced cells (MSC miR-1/Myocd ) were trypsinized and cultured in CS/CO hydrogel. Subsequently, qRT-PCR and ICC analysis were performed to evaluate the differentiation of encapsulated cells. As in the 2D culture, mRNA gene expression of Tnnt2, Nkx2-5 and Gata4, were measured 7, 14, and 21 days after transduction. According to Fig. 5A, expression level of target genes in encapsulated transduced cells were significantly increased compared to the transduced cells in 2D cell culture (*P < 0.05). Additionally, ICC images showed that the expression of cardiac markers in 3D culture were upregulated in comparison with 2D culture (Fig. 5B). Taken together, these results implicated that encapsulation of transduced cells into CS/CO hydrogel promotes the differentiation of MSCs into iCMs, due to increased cell-cell and cell-environment interactions.",
"After MI induction (Fig. 6A), transplantation of patch was performed in different groups (Fig. 6B). Echocardiograph analysis was performed 30 days after patch transplantation in different groups (Fig. 6C). The increased EF and FS in both iCM/P and MSC/P groups indicated functional recovery after cell/patch transplantation in comparison with MI control group. Additionally, the increase in EF and FS was significantly higher in the iCM/P group compared to MSC/P group by *P < 0.05 and **P < 0.01, respectively. A significant decrease in EDV and ESV was found 30 days after MI compared to 3 days after MI in the iCM/P group (Fig. 6D), meaning that 30 days after MI induction in the intervention group, volume of blood remaining in the heart ventricle was reduced after ejection, similar to normal heart.",
"The expression levels of Tnnt2, Nkx2-5, and Gata4 was significantly increased in iCM/P group with mean fold changes of 10.4, 7.2, and 5, respectively compared to MI control group. Moreover, expression of the genes was significantly increased in iCM/P group much more than MSC/P group (**P < 0.01) (Fig. 7A). Besides, in Patch group, expression of target genes did not show a significant difference in comparison with MI group.",
"For histological examination, H&E staining was performed for MI zone of patch transplantation-treated and MI groups. As shown in Fig. 7B, myocytes in iCM/P and MSC/P groups were arranged regularly and vascular structures and red blood cells were detectable. In contrast, no regularly and vascular structures were detected in MI zone of MI group. On day 30 post-transplantation, the patch-treated groups showed the grafted cells with DiI labeling in MI zone. In heart sections with DiI labeling in iCM/P and MSC/P groups, co-localization of DiI labeling and cTnT, NKX2.5, and GATA4 markers was observed. Expression of markers in Patch and MI groups was observed only in border of MI zones (Fig. 7C).",
"The miRNAs are considered as regulators in bioprocesses, such as differentiation, proliferation, and development and they have important roles in cardiogenesis 38 . Among cardiac-specific miRNAs, miR-1 is involved in early stage of cardiomyocyte development by targeting different signaling pathways including Wnt/b-catenin pathway. Various studies have shown that miR-1 activates Wnt/b-catenin signaling pathway by downregulation of genes that are inhibitor of Wnt pathway. Activation of the Wnt/b-catenin pathway has been found to enhance expression of genes, such as wnt11, b-catenin, c-Jun N-terminal kinase (JNK), and ternary complex factor(TCF), which in turn enhanced expression of cardiac-specific genes including Nkx2-5, Gata4, and Tnnt2 16 . Also, level of cardiac miRNAs, in particular miR-1 has been indicated to be increased in differentiation of MSCs into iCMs. Therefore, miR-1 overexpression causes further differentiation of MSCs into iCMs via targeting Wnt pathway and regulation of target genes expression 39 . Myocyte enhancer factor-2 (Mef2), SRF, and Myocd are miR-1 transcriptional activators, binding to its enhancer sequences. Myocd is a co-activator for SRF because it is a weak activator. Thus, Myocd is an important factor for miR-1 expression in early stages of heart development 17 the previous studies 40,42 , our results showed that expression of cardiac markers including Tnnt2, Nkx2-5, and Gata4 at gene and protein levels was higher in MSCs transduced with a combination of miR-1 and Myocd than MSCs transduced with miR-1 alone.",
"Most studies on cells function have been performed in 2D microenvironment, but recently 3D culture has been developed because of its advantages over 2D culture. In 3D cell culture, different cells interact with each other and with extracellular matrix. Whereas, in 2D cell culture, cells interact only with their surrounding cells 43,44 . In addition, mechanical forces between cell-cell and cell-environment in 3D culture provide a set of signals, regulating different functions of cells, such as proliferation, differentiation, and migration 45 . Moreover, structure and organization of cells in 3D culture change compared to a 2D culture, influencing function and cell signaling 46 . Therefore, it can be concluded that cell behaviors in 3D culture are different from 2D culture.",
"3D culture promotes an environment for better signaling between cells and their neighboring, and causes more realistic physiological, biomechanical, and biochemical properties, and less substrate stiffness than 2D environment, collectively increasing differentiation capability of MSCs into different cell lines. A combination of these conditions alters gene expression of MSCs and thus, activates different signaling pathways ultimately improving efficacy of MSCs differentiation 47 . Pineda et al., investigated myocardial differentiation of embedded mouse embryonic stem cells (M-ESCs) in collagen type I gels (GELs) 48 . They indicated that expression of Gata4, FMS-like tyrosine kinase 1(Flt1), and endothelin type B receptor (Endbr) was upregulated in GELs compared to 2D culture. As a result, 3D culture is a supportive environment for cardiovascular differentiation. Histological analysis showed that myocytes in iCM/P group had regularly structure and red blood cells were observed, while in MI group, inflammatory cells infiltrated in MI zone and large area of fibrosis was detectable. C) Thirty days after MI induction, IHC staining was performed for NKX2-5, GATA4, and cTnT markers of MI zone tissue. The images showed that the immunostained cells in the iCM/P group were more than the other groups. Encapsulated cells of iCM/P and MSC/P groups were labeled with DiI stain for tracking in rats heart tissues.",
"Hydrogels are more common than other biomaterials due to their properties, such as similarity to natural extracellular matrices (ECM), biocompatibility, encapsulation of cells, integrating with host cells, and enhancement of cell proliferation, survival, and differentiation 49 .",
"CS as a biocompatible polysaccharide is frequently used in tissue engineering due to its antioxidant activity, lack of immunogenicity, and bioactive properties 26 . Studies have shown that CS as a scaffold in combination with MSCs can improve heart function in MI models of rats 27,28 . There are several mechanisms for improving function of ischemic heart and reduction of infarct size for example, (1) CS, as a carrier of MSCs increases their survival and engraftment, which is related to antioxidant activity of CS and eliminating of reactive oxygen species (ROS), diminishing cell adhesion, and eventually improving MI environment 50 . (2) CS is involved in formation of neovasculature. In addition, MSCs in combination with CS play a synergistic role in ischemic myocardial angiogenesis because they have paracrine effect and release angiogenic factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and hepatocyte growth factor (HGF) 51 . (3) CS increases differentiation of MSCs into cardiac-like cells due to collagen synthesis 28 . CO is one of the main components of ECM playing an important role in process of cardiac maturation and differentiation of MSCs 30 . Therefore, heart function is improved by replacing cardiac differentiated cells in MI zone.",
"CO facilitates cell adhesion by integrin receptors, activating signaling pathways, which in turn regulate cell survival, proliferation, and differentiation 30 . Therefore, in combination with CS, efficiency of differentiation of MSCs into cardiac-like cells is improved. Moreover, due to similarity of CO to the pericardium and native ECM, and enhancement of tensile strength, composite hydrogel of CS and CO improves heart function in a MI model of rat 33 . The prepared integrated porous CS/CO hydrogel network provided a microenvironment for cell proliferation and differentiation by permeability of nutrients and biological molecules and development of signaling pathway among embedded cells in hydrogel 33 . Moreover, the presence of CO in the hydrogel increases compaction. As a result, CS/CO hydrogel is more compact and stiffer than pure CS 33 . Additionally, results of viability test and Dil staining showed that the composite CS/CO hydrogel promoted growth and proliferation of the encapsulated cells and had no cytotoxicity effect. In the current study, first mouse MSCs were co-transduced with miR-1 and Myocd lentiviruses, and after 3 days, the transduced cells were encapsulated in CS/CO hydrogel to improve efficiency of differentiation and maturation. In the encapsulated cells, expression of cardiac markers of Tnnt2, Nkx2-5, and Gata4, and, at levels of gene and protein was significantly upregulated in comparison with the transduced cells in 2D culture.",
"Previous studies have shown that transplantation of MSCs in MI zone does not effectively improve heart function due to high apoptosis rate of the transplanted cells in the infarct environment and low efficiency of their differentiating into cardiomyocytes and endothelial cells 48,52 . Therefore, recently, using different hydrogels has been considered for injection or transplantation of MSCs in the MI zone 53,54 . In the present report, for in vivo studies, after co-transduction of MSCs with miR-1 and Myocd lentiviruses, iCMs were encapsulated into CS/CO hydrogel followed by placed on PDMS for making patch. PDMS is a biocompatible and biodegradable substrate that has been shown in several studies to use as a substrate to promote the self-renewal and differentiation of stem cells into cardiomyocyte 58 . PDMS have also been used in several studies as a suitable substrate for cultivation 59 and implantation 60 .",
"After MI induction, patches were implanted to the MI zone in different groups including iCM/P, MSC/P, and cell-free/P groups. Echocardiography of rat heart tissue was performed for assessing heart function. The results confirmed that EF and FS were increased in iCM/P and MSC/P groups, indicating an improvement in the impaired heart function after transplantation of the encapsulated cells 55 . Moreover, cardiac markers, at the level of gene and protein, in iCM/P group significantly increased in comparison with control group.",
"As mentioned in previous studies 56,57 , MSCs transplantation in MI zone can partially improve heart function due to induction of angiogenesis, paracrine effects, and stimulating differentiation of the implanted MSCs into cardiomyocyte. In the iCM/P group, not all MSCs are differentiated into iCMs, and this group includes a heterogeneous population of differentiated iCMs and undifferentiated MSCs. Therefore, in the iCM/P group, both iCMs and MSCs are effective in improving heart function after MI induction. As a result, in the MSC/P group, a relative improvement in heart function is observed.",
"Our results revealed that co-induction of miR-1 and Myocd followed by culture in CS/CO hydrogel led to greater differentiation and maturation of MSCs. Additionally, heart function was improved after transplantation of encapsulated iCMs in MI model of rat. Therefore, our findings indicated importance of 3D culture in stem cell differentiation. So, in the future studies, progress in cardiac regeneration based on cell therapy can be achieved by improving differentiation conditions of MSCs and subsequently, obtaining mature cardiac cells."
] | [] | [
"Introduction",
"Materials and Methods",
"Fabrication of Composite Hydrogel",
"Physiochemical Properties of CS/CO Hydrogel",
"Isolation and Characterization of MSCs",
"Lentiviral Production and Transduction of MSCs",
"Viability Assay of Transduced Cells",
"Evaluation of Differentiation of Transduced Cells",
"Immunocytochemistry (ICC) analysis.",
"3D Cell Culture",
"Encapsulation of Transduced Cells in CS/CO Hydrogel and Evaluation of their Differentiation into iCMs",
"In vivo Studies",
"Statistical analysis",
"Results",
"Morphology and characterization of the CS/CO hydrogel",
"Up-Regulation of miR-1 and Myocd Expression in Transduced Cells",
"Differentiation of Transduced Cells into iCM",
"Viability and Proliferation Analyses of Cells in CS/CO Hydrogel",
"Differentiation of Encapsulated Transduced Cells in CS/CO Hydrogel",
"Effect of iCM/P on Cardiac Function",
"Effect of iCM/P on Cardiac gene Expression in MI Zone",
"Histological and IHC Staining in MI Zones",
"Discussion",
"Conclusion",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 .",
"Figure 6 .",
"Figure 7 .",
"Table 1 ."
] | [
"Gene \nsymbol \nPrimer \n\nProduct \nsize (bp) \n\nmiR-1 \nF: GTAGGCACCTGAAATGGAA \n85 \nR: TTGATGGTGCCTACAGTACAT \nU6 \nF: CTCGCTTCGGCAGCACA \n94 \nR: AACGCTTCACGAATTTGCGT \nMyocd \nF: CAAGCCAAAGGTGAAGAAGC \n177 \nR: TAGCTGAATCGGTGTTGCTG \nGata4 \nF: TCATCTCACTACGGGCACAG \n233 \nR: GGGAAGAGGGAAGATTACGC \nNkx2-5 \nF: CCTCAACAGCTCCCTGACTC \n201 \nR: GGGGACAGCTAAGACACCAG \nTnnt2 \nF: GGCAGCTGCTGTTCTGAGGGAG \n191 \nR: TGCCCTGGTCTCCTCGGTCT \nGapdh \nF: CCTGGAGAAACCTGCCAAGTA \n148 \nR: GGCATCGAAGGTGGAAGAGT \n"
] | [
"Table 1"
] | [
"Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat",
"Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat"
] | [] |
14,532,393 | 2022-03-18T07:43:45Z | CCBY | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043137&type=printable | GOLD | c0b8323a8ee9be4f83c58092b0b86fa8651c289e | null | null | null | null | 10.1371/journal.pone.0043137 | 2042177858 | 22905217 | 3419196 |
SUMO Modification of Stra13 Is Required for Repression of Cyclin D1 Expression and Cellular Growth Arrest
2012
Y Wang
V K Rao
W K Kok
D N Roy
S Sethi
SUMO Modification of Stra13 Is Required for Repression of Cyclin D1 Expression and Cellular Growth Arrest
PLoS ONE
78431372012Received March 19, 2012; Accepted July 16, 2012;Editor: Swati Palit Deb, Virginia Commonwealth University, United States of America Funding: This work was supported by funds from the National Medical Research Foundation (R.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G 1 cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G 1 /S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation.
Introduction
Stra13, a member of the bHLH-O repressor subfamily is widely expressed both during embryonic development as well as in a number of adult tissues [1,2]. In addition to being constitutively expressed in several cell types, its expression is up regulated in response to multiple stimuli including retinoic acid, TGFb, serum deprivation, genotoxic agents and trichostatin A (TSA). Several gain of function and loss of function studies have shown its involvement in cellular differentiation programs, cell cycle progression, senescence, apoptosis, immune responses, tissue regeneration and circadian rhythms [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. However, the molecular mechanisms through which Stra13 regulates these diverse biological responses are largely unclear. Previous studies have shown that Stra13 overexpression results in growth suppression, cell cycle arrest, and cellular senescence, which are important tumor suppression mechanisms [4,6,10,12,17]. Consistent with these observations, Stra13 expression is indeed down regulated in some tumors. Intriguingly however, it is also overexpressed in many cancers [2,[18][19][20][21][22]. Similarly, while Stra13 inhibits differentiation of some cell types, it promotes others [3,8,9]. The seemingly paradoxical functions of Stra13 could potentially occur by altered sub-cellular localization, or association with distinct co-factors in different cell types. Alternatively, posttranslational modifications may allow it to rapidly and reversibly alter functions in diverse cellular contexts.
We and others have previously demonstrated that Stra13 associates with the co-repressor HDAC1 through its C-terminal repression domain that contains three a-helices [4] and regulates transcriptional repression of specific target genes [4,23,24]. However the mechanism by which HDAC1 regulates Stra13dependent biological functions is unclear.
SUMO (Small Ubiquitin-related Modifier) modification, or sumoylation, is an important post-translational modification that modulates the biological functions of proteins [25,26]. Sumoylation is a highly dynamic process, whereby SUMO is covalently conjugated to an obligatory lysine in canonical yKXE SUMO motifs (where y is a hydrophobic amino acid, K is the acceptor lysine for covalent attachment of SUMO, and X is any residue, and E is glutamic acid) in the substrate. Sumoylation is a three-step reaction consisting of SUMO activation, transfer, and ligation that are catalyzed by E1 heterodimeric enzyme (SAE1/SAE1), E2 enzyme (Ubc9) and E3 SUMO ligases, of which the Protein Inhibitor of Activated Stats (PIAS) proteins have been wellcharacterized [27,28]. Protein sumoylation is readily reversed by cellular isopeptidases or Sentrin/SUMO-specific proteases (SENP, SUSP), which cleave SUMO from its substrate. Unlike ubiquitination, which usually facilitates protein degradation, sumoylation results in pleiotropic functional consequences that include changes in subcellular localization, protein stability, alterations in DNAbinding and transcriptional activity. Transcription factors, coactivators and co-repressors are predominant targets of sumoylation, which alters their activity resulting in changes in gene expression and function [29,30].
In this study, we demonstrate that Stra13 can be SUMO modified at conserved residues Lys 159 and Lys 279 that is enhanced by the SUMO E3 ligases PIAS3 and PIAS1. Mutation of these target residues, or co-expression of the SUMO protease SENP1 with wild type Stra13, impairs its ability to repress cyclin D1 expression and attenuates its function as a growth suppressor. In addition, mutation of sumoylation sites reduces association of Stra13 with HDAC1, which plays an essential role in cell cycle progression. HDAC1 inhibits Stra13 sumoylation in a deacetylaseactivity dependent manner and blocks its anti-proliferative effects. Together these studies identify sumoylation as a key posttranslational modification that modulates Stra13 transcriptional repression activity and function in cell cycle arrest.
Results
Stra13 Sumoylation is Enhanced by PIAS3 and PIAS1
We have previously demonstrated that the co-repressor HDAC1 interacts with the C-terminal region of Stra13 spanning amino acid residues 111-343 ( Fig. 1A; and 4). Alignment of this region from several species revealed two potential sumoylation motifs AKHE and IKQE. K279 within the IKQE motif was phylogenetically conserved, whereas K159 within AKHE was less conserved through various species (Fig. 1A). To examine whether Stra13 undergoes sumoylation, we transfected cells with constructs encoding Myc-Stra13 in the absence or presence of SUMO1. Lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. A putative sumoylated band was detected in the presence of SUMO1 (Fig. 1B). To examine whether the slower migrating band corresponds to sumoylated Stra13, we co-transfected the Sentrin-specific protease (SENP), which is able to remove SUMO conjugates from substrates. In the presence of SENP1, sumoylation was abrogated, confirming that Stra13 is indeed sumoylated in cells. To examine whether K159 and K279 serve as acceptor sites for sumoylation, we generated lysine (K) to arginine (R) point mutants at each site individually (Stra13 K159R and Stra13 K279R respectively) and together (2KR) by site-directed mutagenesis. Cells were transfected with Stra13, Stra13 K159R, Stra13 K279R as well as the double mutant Stra13 2KR (Fig. 1C). Immunoprecipitation and western blotting analysis revealed that in presence of SUMO1, both Stra13 and Stra13 K159R mutant were sumoylated. In contrast, neither Stra13 K279R, nor Stra13 2KR were sumoylated, suggesting SUMO conjugation occurs predominantly at K279. The PIAS protein family act as E3 SUMO ligases and enhance sumoylation of target proteins [28]. To examine whether PIAS proteins modulate Stra13 sumoylation, we co-transfected cells with Myc-Stra13, SUMO1 and Flag-PIAS1, PIAS3, PIASxa, and PIASy. Cell lysates were immunoprecipitated with Myc-agarose beads, and analyzed by western blotting with anti-SUMO1 antibody. Both PIAS3 and PIAS1 enhanced Stra13 sumoylation, whereas expression of PIASxa and PIASy had a minimal impact (Fig. 1D).
Stra13 Sumoylation is Required for its Anti-proliferative Effects
Stra13 mediates growth suppression in a number of cell types and has also been implicated in cellular senescence [4,6,10,12]. We therefore examined whether sumoylation impacts Stra13mediated growth arrest. NIH3T3 cells were co-transfected with Stra13, or Stra13 2KR, along with pD503, which confers resistance to puromycin. Western blot analysis showed equivalent expression of Stra13 and Stra13 2KR ( Fig. 2A). After selection, cells were seeded at a low density and analyzed for colony formation two weeks later. Consistent with our previous studies [4], overexpression of Stra13 resulted in significant reduction in colony numbers compared with vector-transfected cells (Fig. 2B, C). Interestingly, in contrast to wild type Stra13, Stra13 2KR was unable to inhibit colony formation (Fig. 2B, C). To examine the underlying mechanisms, we measured proliferation of cells expressing Stra13 and Stra13 2KR relative to vector controls. Stra13 overexpressing cells resulted in reduced cell numbers over a five-day period, whereas Stra13 2KR expressing cells proliferated similar to control cells (Fig. 2D). Consistently, re-expression of wild type Stra13 but not Stra13 2KR in mouse embryonic fibroblast (MEFs) derived from Stra13 2/2 mice [5] led to growth suppression (Fig. 2E). We then examined the cell cycle profile of Stra13 and Stra13 2KR overexpressing cells by flow cytometry (Fig. 2F). Compared to controls, Stra13 expressing cells exhibited delayed progression in the G 1 /S phase of the cell cycle, resulting in G 1 arrest. In contrast, the cell cycle profile of Stra13 2KR cells was similar to controls. However, expression of either protein had no impact on the sub-G 1 phase. Together these results suggest that reduced colony formation upon Stra13 overexpression is due to reduced proliferation rates and G 1 arrest rather than increased apoptosis.
The inability of Stra13 2KR mutant to mediate growth suppression suggested that sumoylation may be involved in the anti-proliferative effects of Stra13. To determine the impact of sumoylation on Stra13-mediated growth inhibition, we asked whether inhibition of Stra13 sumoylation recapitulates the phenotype of Stra13 2KR expressing cells. To examine this possibility, we performed colony assays in cells expressing the SUMO protease SENP1 along with equivalent levels of Stra13 and Stra13 2KR (Fig. 3A). Co-expression of SENP1 reversed the anti-proliferative effect of Stra13 whereas, as expected, Stra13 2KR was insensitive to SENP1 ( Fig. 3B-C) confirming that sumoylation of Stra13 is indeed critical in mediating growth arrest in fibroblast cells.
SUMO Modification of Stra13 does not Affect its Subcellular Localization but Enhances its Ability to Repress Cyclin D1
To examine the molecular basis underlying Stra13-mediated G 1 arrest, the expression of endogenous cyclin D1 and p21 that regulate G 1 /S transition was analyzed by Q-PCR. Stra13 significantly inhibited cyclin D1 expression, and up-regulated the levels of p21 Cip/WAF (Fig. 4A). In contrast, Stra13 2KR expressing cells did not show a significant change in the expression of either gene relative to control cells. Cyclin B1 and cyclin E1 expression was similarly regulated in cells expressing Stra13 and Stra13 2KR. Since sumoylation typically enhances transcriptional repression, we examined whether it is required for Stra13-mediated repression of cyclin D1 expression. A cyclin D1 promoter reporter [31] was co-expressed with Stra13, SUMO1 and SENP1. Consistent with repression of endogenous cyclin D1 expression, Stra13 repressed the cyclin D1 reporter, which was further augmented in presence of SUMO1, and attenuated in the presence of SENP1 (Fig. 4B). Stra13 2KR was unable to repress cyclin D1 promoter to the levels achieved with Stra13, confirming that sumoylation is relevant in Stra13-mediated repression of cyclin D1 expression. Since sumoylation can regulate the subcellular localization of target proteins [25][26][27], we examined whether the inability of Stra13 2KR to repress cyclin D1 was due to altered cellular localization. Stra13 and Stra13 2KR transfected cells were immunostained with anti-Myc antibody, and visualized by confocal microscopy. Both proteins showed nearly identical patterns of nuclear localization that was independent of sumoylation sites (Fig. 4C). Similarly, no differences were apparent between wild type and Stra13 2KR in the presence of SUMO1. Thus, the inability of Stra13 2KR to transcriptionally repress cyclin D1 is not due to altered subcellular localization. To determine whether cyclin D1 is directly regulated by Stra13, we performed chromatin immunoprecipitation (ChIP) assays in NIH3T3 cells that were left untreated or treated with trichostatin A (TSA) which causes growth arrest [4]. Binding of endogenous Stra13 was evident on the cyclin D1 promoter both in the absence and presence of TSA treatment (Fig. 4D). These findings are consistent with a recent study [17] demonstrating that cyclin D1 is a Stra13 target gene.
HDAC1 Regulates Stra13 Sumoylation, Cyclin D1 Repression and Growth Arrest
As Stra13 sumoylation sites are located within the HDAC1 interaction region, we tested whether the association between HDAC1 and Stra13 is SUMO-dependent. Cells were transfected with Stra13 or Stra13 2KR mutant together with Flag-HDAC1. Lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-Flag antibody (Fig. 5A). As previously reported, Stra13 interacted with HDAC1 [4], and consistent with a recent report [32], sumoylation-defective Stra13 2KR interacted less efficiently with HDAC1 compared to wild type Stra13. Several studies have demonstrated that HDACs modulate sumoylation of proteins. To investigate whether HDAC1 modulates Stra13 sumoylation, we co-expressed the two proteins. Interestingly, Stra13 sumoylation was almost completely abolished by HDAC1. Moreover, TSA (a histone deacetylase inhibitor) reversed HDAC1-mediated inhibition of Stra13 sumoylation (Fig. 5B). To validate these findings, endogenous Stra13 was immunoprecipitated from cells in the absence and presence of TSA treatment. Indeed, Stra13 sumoylation (Fig. 5C), and its association with HDAC1 (Fig. 5D), was enhanced in TSA treated cells. To further examine the role of endogenous HDAC1 in Stra13 sumoylation, its expression was down-regulated with HDAC1 specific siRNA (siHDAC1). Control cells were transfected with scrambled siRNA (siRNA). The down-regulation of HDAC1 expression in siHDAC1 cells (Fig. 5E) led to enhanced Stra13 sumoylation compared to controls (Fig. 5F) demonstrating that endogenous HDAC1 regulates Stra13 sumoylation.
In contrast to the anti-proliferative effect of Stra13, HDAC1 is known to promote proliferation via regulation of G 1 /S progression. Given its impact on sumoylation, we examined whether HDAC1 antagonizes Stra13-dependent growth suppression that is sumoylation-dependent. NIH3T3 cells were co-transfected with equivalent amounts of Stra13 and Stra13 2KR along with HDAC1 (Fig. 5G). In its presence, Stra13-mediated growth suppression was abrogated and phenotypically resembled Stra13 2KR cells (Fig. 5H, I). To further investigate whether the loss of growth suppression occurs due to an impact on cyclin D1, we performed reporter assays. In presence of HDAC1, repression of the cyclin D1 promoter by Stra13 and SUMO1 was attenuated in a dose-dependent manner (Fig. 5J). Conversely, in siHDAC1 cells, Stra13-mediated repression of cyclin D1 was augmented compared to controls (Fig. 5K) Together, these results demonstrate that HDAC1 inhibits Stra13 sumoylation, and consequently its ability to repress cyclin D1 that is essential for growth suppression in fibroblast cells.
Discussion
In this study we have identified sumoylation as a key modification that impacts Stra13-mediated transcriptional repres- sion with an overt impact on its function in cell cycle arrest. Mutation of sumoylation sites attenuates the anti-proliferative effect of Stra13, at least in part by abrogating its ability to inhibit cyclin D1 expression.
Post-translational modifications play a significant role in the regulation of transcription factors. Co-regulator proteins can either promote or inhibit these modifications. SUMO modification of many transcription factors appears to correlate with transcriptional repression, which may reflect altered proteinprotein interactions. For instance, association with co-repressors such as HDACs is generally enhanced by sumoylation, and conversely interaction with co-activators is reduced [30,33]. Consistent with altered recruitment of co-regulators, sumoylated Stra13 efficiently interacted with HDAC1, whereas the sumoylation defective mutant Stra13 2KR exhibited reduced association. HDACs enhance sumoylation of target proteins such as MEF2 and HIC1 that may occur via deacetylation of lysine residues, allowing them to be subsequently modified by sumoylation [34,35]. Intriguingly however, HDAC1 inhibits Stra13 sumoylation and its ability to repress cyclin D1 thereby countering its anti-proliferative impact in fibroblast cells. HDAC1-mediated inhibition of Stra13 sumoylation and cyclin D1 repression is consistent with the opposing functions of the two proteins in cellular proliferation. Stra13 has been reported to repress cyclin D1 levels that correlate with its ability to mediate G 1 arrest and cause growth suppression. In contrast, HDAC1 and HDAC2 promote cellular proliferation and cell cycle progression by inhibiting the cyclin dependent kinases (CDK) p21 WAF1/CIP1 and p57 Kip2 through direct regulation of their promoters resulting in transcriptional repression [36,37]. Correspondingly, mouse embryonic fibroblasts lacking HDAC1 and HDAC2 are arrested in G 1 , and express elevated levels p21 WAF1/CIP1 and p57 Kip2 . Moreover, increased expression of HDACs has been found in several cancers confirming their roles in cellular proliferation [38,39]. Our studies suggest that in addition to direct regulation of CDK levels, HDAC1 may indirectly enhance proliferation by blocking growth suppressive signals via desumoylation of Stra13 relieving repression of cyclin D1. Given the impact of HDAC1 activity on Stra13 sumoylation, it is conceivable that in cells that overexpress both proteins, Stra13 may exist in a desumoylated state, and unable to block cellular proliferation allowing cells to bypass its growth suppressive function. The mechanism by which HDAC1 inhibits Stra13 sumoylation remains to be investigated. Since TSA can antagonize the effect of HDAC1 on Stra13 sumoylation, endogenous deacetylase activity is involved and may reflect a requirement for acetylation-dependent sumoylation similar to PML [40]. Alternatively, histone deacetylation by HDAC1 may release promoter bound sumoylated Stra13, which could then become accessible for desumoylation. Nonetheless, our studies demonstrate sumoylation is an important mechanism by which Stra13 transcriptional activity and function is modulated. Such post-translational modifications may underlie the seemingly paradoxical functions of Stra13 to either promote or inhibit cellular differentiation and growth in different contexts.
Materials and Methods
Plasmids, Mutagenesis, and HDAC1 siRNA
Flag-mPIAS1, Flag-mPIAS3, Flag-mPIASxa, Flag-mPIASy, SUMO1, SENP1, Flag-HDAC1, and pD1luc harboring the cyclin D1 promoter have been described [4,31,41]. pCS2-Myc-Stra13 was derived from pCS2-Flag-Stra13 by PCR using primers 59-GAA TTC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG GAA CGG ATC CCC AGC GCG-39 and 59-GAA TTC TTA GTC TTT GGT TTC TAA GTT-39. The PCR product was TA-cloned into pCRII (Invitrogen), and then subcloned into the EcoR1 site of pCS2. K279R, K159R, and 2KR mutants were generated from pCS2-Myc-Stra13 using the QuickChange TM site-directed mutagenesis kit (Stratagene). Primers used for generating Stra13K279R are: 59-GTC AGC ACA ATT AGG CAA GAA TCC GAA-39 and 59-TTC GGA TTC TTG CCT AAT TGT GCT GAC-39; and for generating Stra13 K159R are: 59-CAG TAC CTG GCG AGG CAT GAG AAC ACT-39 and 59-AGT GTT CTC ATG CCT CGC CAG GTA CTG-39. The entire cDNA was sequenced to confirm the presence of directed mutations. For HDAC1 knockdown, NIH3T3 cells were transfected with 100 nM siRNA specific for mouse HDAC1 (Ambion); or with control scrambled siRNA using Lipofectamine RNAiMAX (Invitrogen). The efficiency of knockdown was determined using anti-HDAC1 antibody.
Cell Culture and Transient Transfections
HEK293 cells, COS-7 cells NIH3T3 and cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HEK293), 10% calf serum (COS-7) and 10% bovine serum (NIH3T3) respectively. MEFs were cultured as described [5]. Transfections were performed using Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen).
Proliferation, Cell Cycle Analysis, and Colony Suppression Assays
NIH3T3 cells or MEFs were transfected with Stra13 or Stra13 2KR with pD503 that confers resistance to puromycin. Control cells were transfected with empty vector and pD503. 24 hours (hr) after transfection, cells were selected with 1.2 mg/ml puromycin for three days. Selected cells were used for the following assays:
Proliferation/Viability assays. Cells were seeded at 1610 4 per well in 6-well plates in triplicates. Attached cells were trypsinized and proliferation was measured by counting cells daily over a period of five days. Alternatively, 3000 cells were seeded in 96-well plates and 72 hr later, MTT assays were performed using MTT cell proliferation assay kit (Invitrogen).
Cell cycle. Selected cells were seeded at a density of 5610 5 / 10-cm plates. 24 hr later, cells were fixed with cold 70% ethanol. Twenty thousand cells were acquired and analyzed for DNA content by propidium iodide staining (50 mg/ml) as described previously [4,42]. Cell cycle distribution was analyzed by flow cytometer (Becton Dickinson) using WINMDI software. Colony suppression assays. Colony assays were done as described [4,42]. Briefly cells were seeded at 1610 3 /10-cm plate. Two weeks later, colonies were fixed with 70% ethanol stained with 0.02% crystal violet and photographed. For quantification, the dye was extracted in 1% SDS and the absorbance read at 570 nm.
Chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed as described [43] using 3 mg of anti-Stra13 (Bethyl Laboratories) antibody. The following primers were used for amplification of the cyclin D1 promoter: 59-GAGAGCT-TAGGGCTCGTCTG-39 and 59-TGGGTGCGTTTCCGAG-TAC-39;
and for b-actin promoter: 59-GCTTCTTTGCAGCTCCTTCGTTG-39 and 59-TTTGCA-CATGCCGGAGCCGTTGT-39.
Quantitative RT-PCR (Q-PCR). Selected cells were synchronized in mitosis by adding nocodazole at a final concentration of 500 ng/ml. After 16 hr, nocodazole was removed by washing with media. Total RNA was extracted from cells using TRIZOL according to the manufacturer's instructions (Invitrogen). Genomic DNA was eliminated by treatment with TURBO DNase (Ambion) and cDNA synthesis was carried out using AMV Reverse Transcriptase (Promega) according to manufacturer's instructions. Q-PCR was performed using Light cycler 480 SYBR green I master (Roche) as described [43].
Luciferase Assays
Cells were transfected with the cyclin D1 promoter reporter pD1luc, Stra13, Stra13 2KR, SUMO1, SENP1 as indicated in the figures along with 5 ng of Renilla luciferase. Empty expression vector was added to normalize the amount of total DNA. 48 hr after transfection, luciferase activity was measured by the Dual-Luciferase Reporter Assay system (Promega). All transfections were performed in triplicates, and repeated at least twice.
Immunofluorescence COS-7 cells were used to examine subcellular localization of Stra13 and Stra13 2KR. 48 hr after transfection, cells were fixed in 4% formaldehyde, incubated with anti-Myc antibody and detected with secondary antibody coupled with Texas-red (Invitrogen). Slides were mounted in Vectashield (Vector Laboratories) supplemented with DAPI (49, 69-diamidino-2-phenylindole) to identify nuclei. Cells were visualized on a Zeiss LSM 510 META confocal laser-scanning microscope.
Statistical Analysis
Error bars indicate mean 6 standard deviation (S.D.). Statistical analysis was performed with by Student's t-test and P values ,0.05 were considered statistically significant [*p,0.05; **p,0.01].
Figure 1 .
1Stra13 is sumoylated. (A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three a-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. b-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxa, or PIASy as indicated. Lysates were immunoprecipitated with Mycagarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS. doi:10.1371/journal.pone.0043137.g001
Figure 2 .
2Mutation of sumoylation sites abrogates Stra13-mediated growth suppression. (A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B-C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean 6SD. (E) Stra13 2/2 MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments. doi:10.1371/journal.pone.0043137.g002
Figure 3 .
3Sumoylation is essential for Stra13-dependent growth inhibition. (A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B-C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean 6SD. doi:10.1371/journal.pone.0043137.g003Immunoprecipitation and Western Blot AnalysisCells were washed twice in cold PBS, lysed in 50 mM Tris-HCl pH 8.0, 50 mM NaC1, 1 mM EDTA, 0.1% Triton X-100, 0.5 mM PMSF and protease inhibitors (Roche). To detect sumoylation, 20 mM N-ethylmaleimide (Sigma) was added to lysis buffer. Protein concentrations were determined by the Bradford method (BioRad). Equal amounts of total protein were loaded for western blotting. Lysates were incubated with Mycagarose beads (Sigma) in pull down buffer (as described above), and precipitates analyzed by western blotting using the following antibodies: anti-SUMO1 (1:200 Zymed), anti-Flag (1:5000 Sigma), anti-Myc (1:2000 Roche) and anti-b-actin (1:10,000 Sigma). For endogenous IP, 1.5 mg lysate was immunoprecipitated with 2 mg anti-Stra13 antibody (Bethyl Laboratories and Novus Laboratories), and immunoblotted with anti-SUMO1 antibody and anti-HDAC1 antibody respectively (Upstate).
Figure 4 .
4Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization. (A) mRNA levels of cyclin D1, p21 Cip/ WAF , cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean 6SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter. doi:10.1371/journal.pone.0043137.g004
PLOS ONE | www.plosone.org
August 2012 | Volume 7 | Issue 8 | e43137
AcknowledgmentsWe thank M. Hinz for pD1luc, and S. Schreiber for Flag-HDAC1.Author Contributions
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bHLH-O transcription factors in development and disease. H Sun, S Ghaffari, R Taneja, Translational Oncogenomics. 2Sun H, Ghaffari S, Taneja R (2007) bHLH-O transcription factors in development and disease. Translational Oncogenomics 2: 107-120.
Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helixloop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. M Boudjelal, R Taneja, M Shyuichiro, P Bouillet, P Dolle, Genes Dev. 11Boudjelal M, Taneja R, Shyuichiro M, Bouillet P, Dolle P, et al. (1997) Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix- loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev 11: 2052-2065.
Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. H Sun, R Taneja, Proc Natl Acad Sci U S A. 97Sun H, Taneja R (2000) Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Proc Natl Acad Sci U S A 97: 4058-4063.
Defective T Cell activation and autoimmune disorder in Stra13-deficient mice. H Sun, B Lu, R-Q Li, R A Flavell, R Taneja, Nat Immunology. 2Sun H, Lu B, Li R-Q, Flavell RA, Taneja R (2001) Defective T Cell activation and autoimmune disorder in Stra13-deficient mice Nat Immunology 2: 1040- 1047.
Cytokine response gene 8 (CR8) regulates the cell cycle G1-S phase transition and promotes cellular survival. C Beadling, A Cereseto, W Fan, M Naramura, K A Smith, Oncogene. 20Beadling C, Cereseto A, Fan W, Naramura M, Smith KA (2001) Cytokine response gene 8 (CR8) regulates the cell cycle G1-S phase transition and promotes cellular survival. Oncogene 20: 1771-1783.
Abundant expression of Dec1/ stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivationinduced apoptosis and selective inhibition of procaspase activation. Y Li, H Zhang, M Xie, M Hu, S Ge, Biochem J. 367Li Y, Zhang H, Xie M, Hu M, Ge S, et al. (2002) Abundant expression of Dec1/ stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivation- induced apoptosis and selective inhibition of procaspase activation. Biochem J 367: 413-422.
Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Z Yun, H L Maecker, R S Johnson, A J Giaccia, Dev Cell. 2Yun Z, Maecker HL, Johnson RS, Giaccia AJ (2002) Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Dev Cell 2: 331-341.
Basic helixloop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. M Shen, E Yoshida, W Yan, T Kawamoto, K Suardita, J Biol Chem. 277Shen M, Yoshida E, Yan W, Kawamoto T, Suardita K, et al. (2002) Basic helix- loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. J Biol Chem 277: 50112-50120.
Clast5/ Stra13 is a negative regulator of B lymphocyte activation. M Seimiya, R Bahar, Y Wang, K Kawamura, Y Tada, Biochem Biophys Res Commun. 292Seimiya M, Bahar R, Wang Y, Kawamura K, Tada Y, et al. (2002) Clast5/ Stra13 is a negative regulator of B lymphocyte activation. Biochem Biophys Res Commun 292: 121-127.
The transcriptional repressor STRA13 regulates a subset of peripheral circadian outputs. A Grechez-Cassiau, S Panda, S Lacoche, M Teboul, S Azmi, J Biol Chem. 279Grechez-Cassiau A, Panda S, Lacoche S, Teboul M, Azmi S, et al. (2004) The transcriptional repressor STRA13 regulates a subset of peripheral circadian outputs. J Biol Chem 279: 1141-1150.
Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G-I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng). 10.1371/journal.pone.0043137.g005Figure 5. HDAC1 regulates Stra13 sumoylation. (A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti-Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. (C-D) NIH3T3 cells were left untreated (2) or treated (+) with TSA. Endogenous Stra13 was immunoprecipitated and analyzed for sumoylation (C), as well as for association with HDAC1 (D). (E-F) Down-regulation of endogenous HDAC1 expression in siHDAC1 cells compared to control cells was examined by western blotting (E). 48 hr later, luciferase activity was assayed. (K) siHDAC1 cells and controls were transfected with pD1luc in the absence and presence of Stra13. Luciferase activity was measured 48 hr later. Error bars indicate mean 6SDFigure 5. HDAC1 regulates Stra13 sumoylation. (A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti- Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1. TSA was added at a concentration of 300 nM. Cell lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. (C-D) NIH3T3 cells were left untreated (2) or treated (+) with TSA. Endogenous Stra13 was immunoprecipitated and analyzed for sumoylation (C), as well as for association with HDAC1 (D). (E-F) Down-regulation of endogenous HDAC1 expression in siHDAC1 cells compared to control cells was examined by western blotting (E). Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G-I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng). 48 hr later, luciferase activity was assayed. (K) siHDAC1 cells and controls were transfected with pD1luc in the absence and presence of Stra13. Luciferase activity was measured 48 hr later. Error bars indicate mean 6SD. doi:10.1371/journal.pone.0043137.g005
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The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells. K Miyazaki, M Miyazaki, Y Guo, N Yamasaki, M Kanno, J Immunol. 185Miyazaki K, Miyazaki M, Guo Y, Yamasaki N, Kanno M, et al. (2010) The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells. J Immunol 185: 7330-7339.
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DEC1 (STRA13) protein expression relates to hypoxia-inducible factor1-alpha and carbonic anhydrase-9 overexpression in non small cell lung cancer. A Giatromanolaki, M I Koukourakis, E Sivridis, H Turley, C C Wykoff, J Pathol. 200Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Wykoff CC, et al. (2003) DEC1 (STRA13) protein expression relates to hypoxia-inducible factor1- alpha and carbonic anhydrase-9 overexpression in non small cell lung cancer. J Pathol 200: 222-228.
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The transcription factor DEC1 (Stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers. J Chakrabarti, H Turley, L Campo, C Han, A L Harris, Br J Cancer. 91Chakrabarti J, Turley H, Campo L, Han C, Harris AL, et al. (2004) The transcription factor DEC1 (Stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers. Br J Cancer 91: 954- 958.
STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker. A Ivanova, S Y Liao, M I Lerman, S Ivanov, E J Stanbridge, J Med Genet. 42Ivanova A, Liao SY, Lerman MI, Ivanov S, Stanbridge EJ (2005) STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker. J Med Genet 42: 565-576.
The hypoxia-regulated transcription factor DEC1(Stra13, SHARP-2) and its expression in gastric cancer. Y Zheng, Y Jia, Y Wang, M Wang, B Li, Omics. 13Zheng Y, Jia Y, Wang Y, Wang M, Li B, et al. (2009) The hypoxia-regulated transcription factor DEC1(Stra13, SHARP-2) and its expression in gastric cancer. Omics 13: 301-306.
Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. K Fujimoto, H Hamaguchi, T Hashiba, T Nakamura, T Kawamoto, Int J Mol Med. 19Fujimoto K, Hamaguchi H, Hashiba T, Nakamura T, Kawamoto T, et al. (2007) Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. Int J Mol Med 19: 925-932.
Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13. S V Ivanov, K Salnikow, A V Ivanova, L Bai, M I Lerman, Oncogene. 26Ivanov SV, Salnikow K, Ivanova AV, Bai L, Lerman MI (2007) Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13. Oncogene 26: 802-812.
SUMO: a regulator of gene expression and genome integrity. S Muller, A Ledl, D Schmidt, Oncogene. 23Muller S, Ledl A, Schmidt D (2004) SUMO: a regulator of gene expression and genome integrity. Oncogene 23: 1998-2008.
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Something about SUMO inhibits transcription. G Gill, Curr Opin Genet Dev. 15Gill G (2005) Something about SUMO inhibits transcription. Curr Opin Genet Dev 15: 536-551.
NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/ G1-to-S-phase transition. M Hinz, D Krappmann, A Eichten, A Heder, C Scheidereit, Mol Cell Biol. 19Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, et al. (1999) NF- kappaB function in growth control: regulation of cyclin D1 expression and G0/ G1-to-S-phase transition. Mol Cell Biol 19: 2690-2698.
SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability. Y Hong, X Xing, S Li, H Bi, C Yang, PLoS One. 623046Hong Y, Xing X, Li S, Bi H, Yang C, et al. (2011) SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability. PLoS One 6: e23046.
Modification with SUMO: A role in transcriptional regulation. A Verger, J Perdomo, M Crossley, EMBO Reports. 4Verger A, Perdomo J, Crossley M (2003) Modification with SUMO: A role in transcriptional regulation. EMBO Reports 4: 137-143.
Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors. S Grégoire, X J Yang, Mol Cell Biol. 25Grégoire S, Yang XJ (2005) Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors. Mol Cell Biol 25: 2273-2287.
An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity. N Stankovic-Valentin, S Deltour, J Seeler, S Pinte, G Vergoten, Mol Cell Biol. 27Stankovic-Valentin N, Deltour S, Seeler J, Pinte S, Vergoten G, et al. (2007) An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcrip- tional repression activity. Mol Cell Biol 27: 2661-2675.
Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. G Lagger, D O'carroll, O' Carroll, D , EMBO Journal. 21Lagger G, O'Carroll D, O'Carroll D (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO Journal 21: 2672-2681.
Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression. T Yamaguchi, F Cubizolles, Y Zhang, N Reichert, H Kohler, Genes Dev. 24Yamaguchi T, Cubizolles F, Zhang Y, Reichert N, Kohler H, et al. (2010) Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression. Genes Dev 24: 455-469.
Histone deacetylases and cancer. M A Glozak, E Seto, Oncogene. 26Glozak MA, Seto E (2007) Histone deacetylases and cancer. Oncogene 26: 5420-5432.
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Acetylation of PML is involved in histone deacetylase inhibitor-mediated apoptosis. F Hayakawa, A Abe, I Kitabayashi, P P Pandolfi, T Naoe, J Biol Chem. 283Hayakawa F, Abe A, Kitabayashi I, Pandolfi PP, Naoe T (2008) Acetylation of PML is involved in histone deacetylase inhibitor-mediated apoptosis. J Biol Chem 283: 24420-24425.
The DEAD-Box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification. M B Lee, L A Lebedeva, M Suzawa, S A Wadekar, M Desclozeaus, Mol Cell Biol. 25Lee MB, Lebedeva LA, Suzawa M, Wadekar SA, Desclozeaus M, et al. (2005) The DEAD-Box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification. Mol Cell Biol 25: 1879-1889.
Sharp-1 modulates the cellular response to DNA damage. J J Liu, T K Chung, J Li, R Taneja, FEBS Lett. 584Liu JJ, Chung TK, Li J, Taneja R (2010) Sharp-1 modulates the cellular response to DNA damage. FEBS Lett 584: 619-624.
Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation. B M Ling, N Bharathy, T K Chung, W K Kok, S Li, Proc Natl Acad Sci U S A. 109Ling BM, Bharathy N, Chung TK, Kok WK, Li S, et al. (2012) Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation. Proc Natl Acad Sci U S A 109: 841-846.
| [
"Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G 1 cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G 1 /S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation."
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"Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helixloop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. M Boudjelal, R Taneja, M Shyuichiro, P Bouillet, P Dolle, Genes Dev. 11Boudjelal M, Taneja R, Shyuichiro M, Bouillet P, Dolle P, et al. (1997) Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix- loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev 11: 2052-2065.",
"Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. H Sun, R Taneja, Proc Natl Acad Sci U S A. 97Sun H, Taneja R (2000) Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Proc Natl Acad Sci U S A 97: 4058-4063.",
"Defective T Cell activation and autoimmune disorder in Stra13-deficient mice. H Sun, B Lu, R-Q Li, R A Flavell, R Taneja, Nat Immunology. 2Sun H, Lu B, Li R-Q, Flavell RA, Taneja R (2001) Defective T Cell activation and autoimmune disorder in Stra13-deficient mice Nat Immunology 2: 1040- 1047.",
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"Clast5/ Stra13 is a negative regulator of B lymphocyte activation. M Seimiya, R Bahar, Y Wang, K Kawamura, Y Tada, Biochem Biophys Res Commun. 292Seimiya M, Bahar R, Wang Y, Kawamura K, Tada Y, et al. (2002) Clast5/ Stra13 is a negative regulator of B lymphocyte activation. Biochem Biophys Res Commun 292: 121-127.",
"The transcriptional repressor STRA13 regulates a subset of peripheral circadian outputs. A Grechez-Cassiau, S Panda, S Lacoche, M Teboul, S Azmi, J Biol Chem. 279Grechez-Cassiau A, Panda S, Lacoche S, Teboul M, Azmi S, et al. (2004) The transcriptional repressor STRA13 regulates a subset of peripheral circadian outputs. J Biol Chem 279: 1141-1150.",
"Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G-I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng). 10.1371/journal.pone.0043137.g005Figure 5. HDAC1 regulates Stra13 sumoylation. (A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti-Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. (C-D) NIH3T3 cells were left untreated (2) or treated (+) with TSA. Endogenous Stra13 was immunoprecipitated and analyzed for sumoylation (C), as well as for association with HDAC1 (D). (E-F) Down-regulation of endogenous HDAC1 expression in siHDAC1 cells compared to control cells was examined by western blotting (E). 48 hr later, luciferase activity was assayed. (K) siHDAC1 cells and controls were transfected with pD1luc in the absence and presence of Stra13. Luciferase activity was measured 48 hr later. Error bars indicate mean 6SDFigure 5. HDAC1 regulates Stra13 sumoylation. (A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti- Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1. TSA was added at a concentration of 300 nM. Cell lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. (C-D) NIH3T3 cells were left untreated (2) or treated (+) with TSA. Endogenous Stra13 was immunoprecipitated and analyzed for sumoylation (C), as well as for association with HDAC1 (D). (E-F) Down-regulation of endogenous HDAC1 expression in siHDAC1 cells compared to control cells was examined by western blotting (E). Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G-I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng). 48 hr later, luciferase activity was assayed. (K) siHDAC1 cells and controls were transfected with pD1luc in the absence and presence of Stra13. Luciferase activity was measured 48 hr later. Error bars indicate mean 6SD. doi:10.1371/journal.pone.0043137.g005",
"DEC1, a basic helix-loop-helix transcription factor and a novel target gene of the p53 family, mediates p53-dependent premature senescence. Y Qian, J Zhang, B Yan, X Chen, 283Qian Y, Zhang J, Yan B, Chen X (2008) DEC1, a basic helix-loop-helix transcription factor and a novel target gene of the p53 family, mediates p53- dependent premature senescence 283: 2896-2905.",
"Stra13 is induced by genotoxic stress and regulates ionizing-radiation-induced apoptosis. T H Thin, L Li, T K Chung, H Sun, R Taneja, EMBO Rep. 8Thin TH, Li L, Chung TK, Sun H, Taneja R (2007) Stra13 is induced by genotoxic stress and regulates ionizing-radiation-induced apoptosis. EMBO Rep 8: 401-407.",
"Stra13 regulates satellite cell activation by antagonizing Notch signaling. H Sun, L Li, C Vercherat, N T Gulbagci, S Acharjee, J Cell Biol. 177Sun H, Li L, Vercherat C, Gulbagci NT, Acharjee S, et al. (2007) Stra13 regulates satellite cell activation by antagonizing Notch signaling. J Cell Biol 177: 647-657.",
"Stra13 regulates oxidative stress mediated skeletal muscle degeneration. C Vercherat, T K Chung, S Yalcin, N Gulbagci, S Gopinadhan, Hum Mol Genet. 18Vercherat C, Chung TK, Yalcin S, Gulbagci N, Gopinadhan S, et al. (2009) Stra13 regulates oxidative stress mediated skeletal muscle degeneration. Hum Mol Genet 18: 4304-4316.",
"The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells. K Miyazaki, M Miyazaki, Y Guo, N Yamasaki, M Kanno, J Immunol. 185Miyazaki K, Miyazaki M, Guo Y, Yamasaki N, Kanno M, et al. (2010) The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells. J Immunol 185: 7330-7339.",
"Basic helix-loop-helix transcription factor DEC1 negatively regulates cyclin D1. U K Bhawal, F Sato, Y Arakawa, K Fujimoto, T Kawamoto, J Pathol. 24Bhawal UK, Sato F, Arakawa Y, Fujimoto K, Kawamoto T, et al. (2011) Basic helix-loop-helix transcription factor DEC1 negatively regulates cyclin D1. J Pathol 24: 420-429.",
"DEC1 (STRA13) protein expression relates to hypoxia-inducible factor1-alpha and carbonic anhydrase-9 overexpression in non small cell lung cancer. A Giatromanolaki, M I Koukourakis, E Sivridis, H Turley, C C Wykoff, J Pathol. 200Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Wykoff CC, et al. (2003) DEC1 (STRA13) protein expression relates to hypoxia-inducible factor1- alpha and carbonic anhydrase-9 overexpression in non small cell lung cancer. J Pathol 200: 222-228.",
"The hypoxia-regulated transcription factor DEC1 (Stra13, SHARP-2) and its expression in human tissues and tumors. H Turley, C C Wykoff, S Troup, P H Watson, K C Gatter, J Pathol. 203Turley H, Wykoff CC, Troup S, Watson PH, Gatter KC, et al. (2004) The hypoxia-regulated transcription factor DEC1 (Stra13, SHARP-2) and its expression in human tissues and tumors. J Pathol 203: 808-813.",
"The transcription factor DEC1 (Stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers. J Chakrabarti, H Turley, L Campo, C Han, A L Harris, Br J Cancer. 91Chakrabarti J, Turley H, Campo L, Han C, Harris AL, et al. (2004) The transcription factor DEC1 (Stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers. Br J Cancer 91: 954- 958.",
"STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker. A Ivanova, S Y Liao, M I Lerman, S Ivanov, E J Stanbridge, J Med Genet. 42Ivanova A, Liao SY, Lerman MI, Ivanov S, Stanbridge EJ (2005) STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker. J Med Genet 42: 565-576.",
"The hypoxia-regulated transcription factor DEC1(Stra13, SHARP-2) and its expression in gastric cancer. Y Zheng, Y Jia, Y Wang, M Wang, B Li, Omics. 13Zheng Y, Jia Y, Wang Y, Wang M, Li B, et al. (2009) The hypoxia-regulated transcription factor DEC1(Stra13, SHARP-2) and its expression in gastric cancer. Omics 13: 301-306.",
"Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. K Fujimoto, H Hamaguchi, T Hashiba, T Nakamura, T Kawamoto, Int J Mol Med. 19Fujimoto K, Hamaguchi H, Hashiba T, Nakamura T, Kawamoto T, et al. (2007) Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. Int J Mol Med 19: 925-932.",
"Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13. S V Ivanov, K Salnikow, A V Ivanova, L Bai, M I Lerman, Oncogene. 26Ivanov SV, Salnikow K, Ivanova AV, Bai L, Lerman MI (2007) Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13. Oncogene 26: 802-812.",
"SUMO: a regulator of gene expression and genome integrity. S Muller, A Ledl, D Schmidt, Oncogene. 23Muller S, Ledl A, Schmidt D (2004) SUMO: a regulator of gene expression and genome integrity. Oncogene 23: 1998-2008.",
"Concepts in sumoylation: a decade on. R Geiss-Friedlander, F Melchior, Nat Rev Mol Cell Biol. 8Geiss-Friedlander R, Melchior F (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 8: 947-956.",
"SUMO-nonclassical ubiquitin. F Melchior, Annu Rev Cell Dev Bio. 16Melchior F (2000) SUMO-nonclassical ubiquitin. Annu Rev Cell Dev Bio 16: 591-626.",
"PIAS proteins modulate transcription factors by functioning as SUMO-1 ligase. N Kotaja, U Karvonen, O A Janne, J J Pavimo, Mol Cell Biol. 22Kotaja N, Karvonen U, Janne OA, Pavimo JJ (2002) PIAS proteins modulate transcription factors by functioning as SUMO-1 ligase. Mol Cell Biol 22: 5222- 5234.",
"Protein modification by SUMO. E S Johnson, Annu Rev Biochem. 73Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73: 355-382.",
"Something about SUMO inhibits transcription. G Gill, Curr Opin Genet Dev. 15Gill G (2005) Something about SUMO inhibits transcription. Curr Opin Genet Dev 15: 536-551.",
"NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/ G1-to-S-phase transition. M Hinz, D Krappmann, A Eichten, A Heder, C Scheidereit, Mol Cell Biol. 19Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, et al. (1999) NF- kappaB function in growth control: regulation of cyclin D1 expression and G0/ G1-to-S-phase transition. Mol Cell Biol 19: 2690-2698.",
"SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability. Y Hong, X Xing, S Li, H Bi, C Yang, PLoS One. 623046Hong Y, Xing X, Li S, Bi H, Yang C, et al. (2011) SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability. PLoS One 6: e23046.",
"Modification with SUMO: A role in transcriptional regulation. A Verger, J Perdomo, M Crossley, EMBO Reports. 4Verger A, Perdomo J, Crossley M (2003) Modification with SUMO: A role in transcriptional regulation. EMBO Reports 4: 137-143.",
"Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors. S Grégoire, X J Yang, Mol Cell Biol. 25Grégoire S, Yang XJ (2005) Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors. Mol Cell Biol 25: 2273-2287.",
"An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity. N Stankovic-Valentin, S Deltour, J Seeler, S Pinte, G Vergoten, Mol Cell Biol. 27Stankovic-Valentin N, Deltour S, Seeler J, Pinte S, Vergoten G, et al. (2007) An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcrip- tional repression activity. Mol Cell Biol 27: 2661-2675.",
"Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. G Lagger, D O'carroll, O' Carroll, D , EMBO Journal. 21Lagger G, O'Carroll D, O'Carroll D (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO Journal 21: 2672-2681.",
"Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression. T Yamaguchi, F Cubizolles, Y Zhang, N Reichert, H Kohler, Genes Dev. 24Yamaguchi T, Cubizolles F, Zhang Y, Reichert N, Kohler H, et al. (2010) Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression. Genes Dev 24: 455-469.",
"Histone deacetylases and cancer. M A Glozak, E Seto, Oncogene. 26Glozak MA, Seto E (2007) Histone deacetylases and cancer. Oncogene 26: 5420-5432.",
"Histone deacetylase inhibitors: Potential in cancer therapy. P A Marks, W S Xu, J Cell Biochem. 107Marks PA, Xu WS (2009) Histone deacetylase inhibitors: Potential in cancer therapy. J Cell Biochem 107: 600-608.",
"Acetylation of PML is involved in histone deacetylase inhibitor-mediated apoptosis. F Hayakawa, A Abe, I Kitabayashi, P P Pandolfi, T Naoe, J Biol Chem. 283Hayakawa F, Abe A, Kitabayashi I, Pandolfi PP, Naoe T (2008) Acetylation of PML is involved in histone deacetylase inhibitor-mediated apoptosis. J Biol Chem 283: 24420-24425.",
"The DEAD-Box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification. M B Lee, L A Lebedeva, M Suzawa, S A Wadekar, M Desclozeaus, Mol Cell Biol. 25Lee MB, Lebedeva LA, Suzawa M, Wadekar SA, Desclozeaus M, et al. (2005) The DEAD-Box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification. Mol Cell Biol 25: 1879-1889.",
"Sharp-1 modulates the cellular response to DNA damage. J J Liu, T K Chung, J Li, R Taneja, FEBS Lett. 584Liu JJ, Chung TK, Li J, Taneja R (2010) Sharp-1 modulates the cellular response to DNA damage. FEBS Lett 584: 619-624.",
"Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation. B M Ling, N Bharathy, T K Chung, W K Kok, S Li, Proc Natl Acad Sci U S A. 109Ling BM, Bharathy N, Chung TK, Kok WK, Li S, et al. (2012) Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation. Proc Natl Acad Sci U S A 109: 841-846."
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"Basic helix-loop-helix transcription factors, BHLHB2 and BHLHB3; their gene expressions are regulated by multiple extracellular stimuli",
"bHLH-O transcription factors in development and disease",
"Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helixloop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells",
"Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms",
"Defective T Cell activation and autoimmune disorder in Stra13-deficient mice",
"Cytokine response gene 8 (CR8) regulates the cell cycle G1-S phase transition and promotes cellular survival",
"Abundant expression of Dec1/ stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivationinduced apoptosis and selective inhibition of procaspase activation",
"Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia",
"Basic helixloop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages",
"Clast5/ Stra13 is a negative regulator of B lymphocyte activation",
"The transcriptional repressor STRA13 regulates a subset of peripheral circadian outputs",
"Control and siHDAC1 cells were transfected with Stra13 and SUMO1. Lysates were immunoprecipitated as indicated and analyzed with anti-SUMO1 antibody (F). (G-I) Cells were co-transfected with Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. Lysates were subject to western blotting with anti-Myc and anti-Flag antibodies to detect expression of Stra13 and HDAC1 (G). Colony assays were performed, and representative plates stained with crystal violet are shown (H). Colony assays were quantified by measuring the absorbance of extracted crystal violet dye at 570 nm (I). (J) Cells were transfected with the pD1luc reporter (100 ng) with Myc-Stra13 (25 ng) and SUMO1 (25 ng) in the presence of increasing amounts of HDAC1 (25, 50 and 100 ng)",
"Stra13 is induced by genotoxic stress and regulates ionizing-radiation-induced apoptosis",
"Stra13 regulates satellite cell activation by antagonizing Notch signaling",
"Stra13 regulates oxidative stress mediated skeletal muscle degeneration",
"The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells",
"Basic helix-loop-helix transcription factor DEC1 negatively regulates cyclin D1",
"DEC1 (STRA13) protein expression relates to hypoxia-inducible factor1-alpha and carbonic anhydrase-9 overexpression in non small cell lung cancer",
"The hypoxia-regulated transcription factor DEC1 (Stra13, SHARP-2) and its expression in human tissues and tumors",
"The transcription factor DEC1 (Stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers",
"STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker",
"The hypoxia-regulated transcription factor DEC1(Stra13, SHARP-2) and its expression in gastric cancer",
"Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements",
"Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13",
"SUMO: a regulator of gene expression and genome integrity",
"Concepts in sumoylation: a decade on",
"SUMO-nonclassical ubiquitin",
"PIAS proteins modulate transcription factors by functioning as SUMO-1 ligase",
"Protein modification by SUMO",
"Something about SUMO inhibits transcription",
"NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/ G1-to-S-phase transition",
"SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability",
"Modification with SUMO: A role in transcriptional regulation",
"Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors",
"An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity",
"Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression",
"Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression",
"Histone deacetylases and cancer",
"Histone deacetylase inhibitors: Potential in cancer therapy",
"Acetylation of PML is involved in histone deacetylase inhibitor-mediated apoptosis",
"The DEAD-Box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification",
"Sharp-1 modulates the cellular response to DNA damage",
"Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation"
] | [
"Front Biosci",
"Translational Oncogenomics",
"Genes Dev",
"Proc Natl Acad Sci U S A",
"Nat Immunology",
"Oncogene",
"Biochem J",
"Dev Cell",
"J Biol Chem",
"Biochem Biophys Res Commun",
"J Biol Chem",
"Figure 5. HDAC1 regulates Stra13 sumoylation. (A) Cells were co-transfected with plasmids expressing Flag-HDAC1 and Myc-Stra13 or Stra13 2KR. 48 hr after transfection, lysates were immunoprecipitated with Myc-agarose beads and analyzed for interaction by western blotting with anti-Flag antibody. (B) Cells were co-transfected with constructs encoding Myc-Stra13, Flag-HDAC1 and SUMO1",
"DEC1, a basic helix-loop-helix transcription factor and a novel target gene of the p53 family, mediates p53-dependent premature senescence",
"EMBO Rep",
"J Cell Biol",
"Hum Mol Genet",
"J Immunol",
"J Pathol",
"J Pathol",
"J Pathol",
"Br J Cancer",
"J Med Genet",
"Omics",
"Int J Mol Med",
"Oncogene",
"Oncogene",
"Nat Rev Mol Cell Biol",
"Annu Rev Cell Dev Bio",
"Mol Cell Biol",
"Annu Rev Biochem",
"Curr Opin Genet Dev",
"Mol Cell Biol",
"PLoS One",
"EMBO Reports",
"Mol Cell Biol",
"Mol Cell Biol",
"EMBO Journal",
"Genes Dev",
"Oncogene",
"J Cell Biochem",
"J Biol Chem",
"Mol Cell Biol",
"FEBS Lett",
"Proc Natl Acad Sci U S A"
] | [
"\nFigure 1 .\n1Stra13 is sumoylated. (A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three a-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. b-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxa, or PIASy as indicated. Lysates were immunoprecipitated with Mycagarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS. doi:10.1371/journal.pone.0043137.g001",
"\nFigure 2 .\n2Mutation of sumoylation sites abrogates Stra13-mediated growth suppression. (A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B-C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean 6SD. (E) Stra13 2/2 MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments. doi:10.1371/journal.pone.0043137.g002",
"\nFigure 3 .\n3Sumoylation is essential for Stra13-dependent growth inhibition. (A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B-C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean 6SD. doi:10.1371/journal.pone.0043137.g003Immunoprecipitation and Western Blot AnalysisCells were washed twice in cold PBS, lysed in 50 mM Tris-HCl pH 8.0, 50 mM NaC1, 1 mM EDTA, 0.1% Triton X-100, 0.5 mM PMSF and protease inhibitors (Roche). To detect sumoylation, 20 mM N-ethylmaleimide (Sigma) was added to lysis buffer. Protein concentrations were determined by the Bradford method (BioRad). Equal amounts of total protein were loaded for western blotting. Lysates were incubated with Mycagarose beads (Sigma) in pull down buffer (as described above), and precipitates analyzed by western blotting using the following antibodies: anti-SUMO1 (1:200 Zymed), anti-Flag (1:5000 Sigma), anti-Myc (1:2000 Roche) and anti-b-actin (1:10,000 Sigma). For endogenous IP, 1.5 mg lysate was immunoprecipitated with 2 mg anti-Stra13 antibody (Bethyl Laboratories and Novus Laboratories), and immunoblotted with anti-SUMO1 antibody and anti-HDAC1 antibody respectively (Upstate).",
"\nFigure 4 .\n4Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization. (A) mRNA levels of cyclin D1, p21 Cip/ WAF , cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean 6SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter. doi:10.1371/journal.pone.0043137.g004"
] | [
"Stra13 is sumoylated. (A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three a-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. b-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxa, or PIASy as indicated. Lysates were immunoprecipitated with Mycagarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS. doi:10.1371/journal.pone.0043137.g001",
"Mutation of sumoylation sites abrogates Stra13-mediated growth suppression. (A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B-C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean 6SD. (E) Stra13 2/2 MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments. doi:10.1371/journal.pone.0043137.g002",
"Sumoylation is essential for Stra13-dependent growth inhibition. (A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B-C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean 6SD. doi:10.1371/journal.pone.0043137.g003Immunoprecipitation and Western Blot AnalysisCells were washed twice in cold PBS, lysed in 50 mM Tris-HCl pH 8.0, 50 mM NaC1, 1 mM EDTA, 0.1% Triton X-100, 0.5 mM PMSF and protease inhibitors (Roche). To detect sumoylation, 20 mM N-ethylmaleimide (Sigma) was added to lysis buffer. Protein concentrations were determined by the Bradford method (BioRad). Equal amounts of total protein were loaded for western blotting. Lysates were incubated with Mycagarose beads (Sigma) in pull down buffer (as described above), and precipitates analyzed by western blotting using the following antibodies: anti-SUMO1 (1:200 Zymed), anti-Flag (1:5000 Sigma), anti-Myc (1:2000 Roche) and anti-b-actin (1:10,000 Sigma). For endogenous IP, 1.5 mg lysate was immunoprecipitated with 2 mg anti-Stra13 antibody (Bethyl Laboratories and Novus Laboratories), and immunoblotted with anti-SUMO1 antibody and anti-HDAC1 antibody respectively (Upstate).",
"Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization. (A) mRNA levels of cyclin D1, p21 Cip/ WAF , cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean 6SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter. doi:10.1371/journal.pone.0043137.g004"
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"Stra13, a member of the bHLH-O repressor subfamily is widely expressed both during embryonic development as well as in a number of adult tissues [1,2]. In addition to being constitutively expressed in several cell types, its expression is up regulated in response to multiple stimuli including retinoic acid, TGFb, serum deprivation, genotoxic agents and trichostatin A (TSA). Several gain of function and loss of function studies have shown its involvement in cellular differentiation programs, cell cycle progression, senescence, apoptosis, immune responses, tissue regeneration and circadian rhythms [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. However, the molecular mechanisms through which Stra13 regulates these diverse biological responses are largely unclear. Previous studies have shown that Stra13 overexpression results in growth suppression, cell cycle arrest, and cellular senescence, which are important tumor suppression mechanisms [4,6,10,12,17]. Consistent with these observations, Stra13 expression is indeed down regulated in some tumors. Intriguingly however, it is also overexpressed in many cancers [2,[18][19][20][21][22]. Similarly, while Stra13 inhibits differentiation of some cell types, it promotes others [3,8,9]. The seemingly paradoxical functions of Stra13 could potentially occur by altered sub-cellular localization, or association with distinct co-factors in different cell types. Alternatively, posttranslational modifications may allow it to rapidly and reversibly alter functions in diverse cellular contexts.",
"We and others have previously demonstrated that Stra13 associates with the co-repressor HDAC1 through its C-terminal repression domain that contains three a-helices [4] and regulates transcriptional repression of specific target genes [4,23,24]. However the mechanism by which HDAC1 regulates Stra13dependent biological functions is unclear.",
"SUMO (Small Ubiquitin-related Modifier) modification, or sumoylation, is an important post-translational modification that modulates the biological functions of proteins [25,26]. Sumoylation is a highly dynamic process, whereby SUMO is covalently conjugated to an obligatory lysine in canonical yKXE SUMO motifs (where y is a hydrophobic amino acid, K is the acceptor lysine for covalent attachment of SUMO, and X is any residue, and E is glutamic acid) in the substrate. Sumoylation is a three-step reaction consisting of SUMO activation, transfer, and ligation that are catalyzed by E1 heterodimeric enzyme (SAE1/SAE1), E2 enzyme (Ubc9) and E3 SUMO ligases, of which the Protein Inhibitor of Activated Stats (PIAS) proteins have been wellcharacterized [27,28]. Protein sumoylation is readily reversed by cellular isopeptidases or Sentrin/SUMO-specific proteases (SENP, SUSP), which cleave SUMO from its substrate. Unlike ubiquitination, which usually facilitates protein degradation, sumoylation results in pleiotropic functional consequences that include changes in subcellular localization, protein stability, alterations in DNAbinding and transcriptional activity. Transcription factors, coactivators and co-repressors are predominant targets of sumoylation, which alters their activity resulting in changes in gene expression and function [29,30].",
"In this study, we demonstrate that Stra13 can be SUMO modified at conserved residues Lys 159 and Lys 279 that is enhanced by the SUMO E3 ligases PIAS3 and PIAS1. Mutation of these target residues, or co-expression of the SUMO protease SENP1 with wild type Stra13, impairs its ability to repress cyclin D1 expression and attenuates its function as a growth suppressor. In addition, mutation of sumoylation sites reduces association of Stra13 with HDAC1, which plays an essential role in cell cycle progression. HDAC1 inhibits Stra13 sumoylation in a deacetylaseactivity dependent manner and blocks its anti-proliferative effects. Together these studies identify sumoylation as a key posttranslational modification that modulates Stra13 transcriptional repression activity and function in cell cycle arrest.",
"We have previously demonstrated that the co-repressor HDAC1 interacts with the C-terminal region of Stra13 spanning amino acid residues 111-343 ( Fig. 1A; and 4). Alignment of this region from several species revealed two potential sumoylation motifs AKHE and IKQE. K279 within the IKQE motif was phylogenetically conserved, whereas K159 within AKHE was less conserved through various species (Fig. 1A). To examine whether Stra13 undergoes sumoylation, we transfected cells with constructs encoding Myc-Stra13 in the absence or presence of SUMO1. Lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-SUMO1 antibody. A putative sumoylated band was detected in the presence of SUMO1 (Fig. 1B). To examine whether the slower migrating band corresponds to sumoylated Stra13, we co-transfected the Sentrin-specific protease (SENP), which is able to remove SUMO conjugates from substrates. In the presence of SENP1, sumoylation was abrogated, confirming that Stra13 is indeed sumoylated in cells. To examine whether K159 and K279 serve as acceptor sites for sumoylation, we generated lysine (K) to arginine (R) point mutants at each site individually (Stra13 K159R and Stra13 K279R respectively) and together (2KR) by site-directed mutagenesis. Cells were transfected with Stra13, Stra13 K159R, Stra13 K279R as well as the double mutant Stra13 2KR (Fig. 1C). Immunoprecipitation and western blotting analysis revealed that in presence of SUMO1, both Stra13 and Stra13 K159R mutant were sumoylated. In contrast, neither Stra13 K279R, nor Stra13 2KR were sumoylated, suggesting SUMO conjugation occurs predominantly at K279. The PIAS protein family act as E3 SUMO ligases and enhance sumoylation of target proteins [28]. To examine whether PIAS proteins modulate Stra13 sumoylation, we co-transfected cells with Myc-Stra13, SUMO1 and Flag-PIAS1, PIAS3, PIASxa, and PIASy. Cell lysates were immunoprecipitated with Myc-agarose beads, and analyzed by western blotting with anti-SUMO1 antibody. Both PIAS3 and PIAS1 enhanced Stra13 sumoylation, whereas expression of PIASxa and PIASy had a minimal impact (Fig. 1D).",
"Stra13 mediates growth suppression in a number of cell types and has also been implicated in cellular senescence [4,6,10,12]. We therefore examined whether sumoylation impacts Stra13mediated growth arrest. NIH3T3 cells were co-transfected with Stra13, or Stra13 2KR, along with pD503, which confers resistance to puromycin. Western blot analysis showed equivalent expression of Stra13 and Stra13 2KR ( Fig. 2A). After selection, cells were seeded at a low density and analyzed for colony formation two weeks later. Consistent with our previous studies [4], overexpression of Stra13 resulted in significant reduction in colony numbers compared with vector-transfected cells (Fig. 2B, C). Interestingly, in contrast to wild type Stra13, Stra13 2KR was unable to inhibit colony formation (Fig. 2B, C). To examine the underlying mechanisms, we measured proliferation of cells expressing Stra13 and Stra13 2KR relative to vector controls. Stra13 overexpressing cells resulted in reduced cell numbers over a five-day period, whereas Stra13 2KR expressing cells proliferated similar to control cells (Fig. 2D). Consistently, re-expression of wild type Stra13 but not Stra13 2KR in mouse embryonic fibroblast (MEFs) derived from Stra13 2/2 mice [5] led to growth suppression (Fig. 2E). We then examined the cell cycle profile of Stra13 and Stra13 2KR overexpressing cells by flow cytometry (Fig. 2F). Compared to controls, Stra13 expressing cells exhibited delayed progression in the G 1 /S phase of the cell cycle, resulting in G 1 arrest. In contrast, the cell cycle profile of Stra13 2KR cells was similar to controls. However, expression of either protein had no impact on the sub-G 1 phase. Together these results suggest that reduced colony formation upon Stra13 overexpression is due to reduced proliferation rates and G 1 arrest rather than increased apoptosis.",
"The inability of Stra13 2KR mutant to mediate growth suppression suggested that sumoylation may be involved in the anti-proliferative effects of Stra13. To determine the impact of sumoylation on Stra13-mediated growth inhibition, we asked whether inhibition of Stra13 sumoylation recapitulates the phenotype of Stra13 2KR expressing cells. To examine this possibility, we performed colony assays in cells expressing the SUMO protease SENP1 along with equivalent levels of Stra13 and Stra13 2KR (Fig. 3A). Co-expression of SENP1 reversed the anti-proliferative effect of Stra13 whereas, as expected, Stra13 2KR was insensitive to SENP1 ( Fig. 3B-C) confirming that sumoylation of Stra13 is indeed critical in mediating growth arrest in fibroblast cells.",
"To examine the molecular basis underlying Stra13-mediated G 1 arrest, the expression of endogenous cyclin D1 and p21 that regulate G 1 /S transition was analyzed by Q-PCR. Stra13 significantly inhibited cyclin D1 expression, and up-regulated the levels of p21 Cip/WAF (Fig. 4A). In contrast, Stra13 2KR expressing cells did not show a significant change in the expression of either gene relative to control cells. Cyclin B1 and cyclin E1 expression was similarly regulated in cells expressing Stra13 and Stra13 2KR. Since sumoylation typically enhances transcriptional repression, we examined whether it is required for Stra13-mediated repression of cyclin D1 expression. A cyclin D1 promoter reporter [31] was co-expressed with Stra13, SUMO1 and SENP1. Consistent with repression of endogenous cyclin D1 expression, Stra13 repressed the cyclin D1 reporter, which was further augmented in presence of SUMO1, and attenuated in the presence of SENP1 (Fig. 4B). Stra13 2KR was unable to repress cyclin D1 promoter to the levels achieved with Stra13, confirming that sumoylation is relevant in Stra13-mediated repression of cyclin D1 expression. Since sumoylation can regulate the subcellular localization of target proteins [25][26][27], we examined whether the inability of Stra13 2KR to repress cyclin D1 was due to altered cellular localization. Stra13 and Stra13 2KR transfected cells were immunostained with anti-Myc antibody, and visualized by confocal microscopy. Both proteins showed nearly identical patterns of nuclear localization that was independent of sumoylation sites (Fig. 4C). Similarly, no differences were apparent between wild type and Stra13 2KR in the presence of SUMO1. Thus, the inability of Stra13 2KR to transcriptionally repress cyclin D1 is not due to altered subcellular localization. To determine whether cyclin D1 is directly regulated by Stra13, we performed chromatin immunoprecipitation (ChIP) assays in NIH3T3 cells that were left untreated or treated with trichostatin A (TSA) which causes growth arrest [4]. Binding of endogenous Stra13 was evident on the cyclin D1 promoter both in the absence and presence of TSA treatment (Fig. 4D). These findings are consistent with a recent study [17] demonstrating that cyclin D1 is a Stra13 target gene.",
"As Stra13 sumoylation sites are located within the HDAC1 interaction region, we tested whether the association between HDAC1 and Stra13 is SUMO-dependent. Cells were transfected with Stra13 or Stra13 2KR mutant together with Flag-HDAC1. Lysates were immunoprecipitated with Myc-agarose beads followed by western blotting with anti-Flag antibody (Fig. 5A). As previously reported, Stra13 interacted with HDAC1 [4], and consistent with a recent report [32], sumoylation-defective Stra13 2KR interacted less efficiently with HDAC1 compared to wild type Stra13. Several studies have demonstrated that HDACs modulate sumoylation of proteins. To investigate whether HDAC1 modulates Stra13 sumoylation, we co-expressed the two proteins. Interestingly, Stra13 sumoylation was almost completely abolished by HDAC1. Moreover, TSA (a histone deacetylase inhibitor) reversed HDAC1-mediated inhibition of Stra13 sumoylation (Fig. 5B). To validate these findings, endogenous Stra13 was immunoprecipitated from cells in the absence and presence of TSA treatment. Indeed, Stra13 sumoylation (Fig. 5C), and its association with HDAC1 (Fig. 5D), was enhanced in TSA treated cells. To further examine the role of endogenous HDAC1 in Stra13 sumoylation, its expression was down-regulated with HDAC1 specific siRNA (siHDAC1). Control cells were transfected with scrambled siRNA (siRNA). The down-regulation of HDAC1 expression in siHDAC1 cells (Fig. 5E) led to enhanced Stra13 sumoylation compared to controls (Fig. 5F) demonstrating that endogenous HDAC1 regulates Stra13 sumoylation.",
"In contrast to the anti-proliferative effect of Stra13, HDAC1 is known to promote proliferation via regulation of G 1 /S progression. Given its impact on sumoylation, we examined whether HDAC1 antagonizes Stra13-dependent growth suppression that is sumoylation-dependent. NIH3T3 cells were co-transfected with equivalent amounts of Stra13 and Stra13 2KR along with HDAC1 (Fig. 5G). In its presence, Stra13-mediated growth suppression was abrogated and phenotypically resembled Stra13 2KR cells (Fig. 5H, I). To further investigate whether the loss of growth suppression occurs due to an impact on cyclin D1, we performed reporter assays. In presence of HDAC1, repression of the cyclin D1 promoter by Stra13 and SUMO1 was attenuated in a dose-dependent manner (Fig. 5J). Conversely, in siHDAC1 cells, Stra13-mediated repression of cyclin D1 was augmented compared to controls (Fig. 5K) Together, these results demonstrate that HDAC1 inhibits Stra13 sumoylation, and consequently its ability to repress cyclin D1 that is essential for growth suppression in fibroblast cells.",
"In this study we have identified sumoylation as a key modification that impacts Stra13-mediated transcriptional repres- sion with an overt impact on its function in cell cycle arrest. Mutation of sumoylation sites attenuates the anti-proliferative effect of Stra13, at least in part by abrogating its ability to inhibit cyclin D1 expression.",
"Post-translational modifications play a significant role in the regulation of transcription factors. Co-regulator proteins can either promote or inhibit these modifications. SUMO modification of many transcription factors appears to correlate with transcriptional repression, which may reflect altered proteinprotein interactions. For instance, association with co-repressors such as HDACs is generally enhanced by sumoylation, and conversely interaction with co-activators is reduced [30,33]. Consistent with altered recruitment of co-regulators, sumoylated Stra13 efficiently interacted with HDAC1, whereas the sumoylation defective mutant Stra13 2KR exhibited reduced association. HDACs enhance sumoylation of target proteins such as MEF2 and HIC1 that may occur via deacetylation of lysine residues, allowing them to be subsequently modified by sumoylation [34,35]. Intriguingly however, HDAC1 inhibits Stra13 sumoylation and its ability to repress cyclin D1 thereby countering its anti-proliferative impact in fibroblast cells. HDAC1-mediated inhibition of Stra13 sumoylation and cyclin D1 repression is consistent with the opposing functions of the two proteins in cellular proliferation. Stra13 has been reported to repress cyclin D1 levels that correlate with its ability to mediate G 1 arrest and cause growth suppression. In contrast, HDAC1 and HDAC2 promote cellular proliferation and cell cycle progression by inhibiting the cyclin dependent kinases (CDK) p21 WAF1/CIP1 and p57 Kip2 through direct regulation of their promoters resulting in transcriptional repression [36,37]. Correspondingly, mouse embryonic fibroblasts lacking HDAC1 and HDAC2 are arrested in G 1 , and express elevated levels p21 WAF1/CIP1 and p57 Kip2 . Moreover, increased expression of HDACs has been found in several cancers confirming their roles in cellular proliferation [38,39]. Our studies suggest that in addition to direct regulation of CDK levels, HDAC1 may indirectly enhance proliferation by blocking growth suppressive signals via desumoylation of Stra13 relieving repression of cyclin D1. Given the impact of HDAC1 activity on Stra13 sumoylation, it is conceivable that in cells that overexpress both proteins, Stra13 may exist in a desumoylated state, and unable to block cellular proliferation allowing cells to bypass its growth suppressive function. The mechanism by which HDAC1 inhibits Stra13 sumoylation remains to be investigated. Since TSA can antagonize the effect of HDAC1 on Stra13 sumoylation, endogenous deacetylase activity is involved and may reflect a requirement for acetylation-dependent sumoylation similar to PML [40]. Alternatively, histone deacetylation by HDAC1 may release promoter bound sumoylated Stra13, which could then become accessible for desumoylation. Nonetheless, our studies demonstrate sumoylation is an important mechanism by which Stra13 transcriptional activity and function is modulated. Such post-translational modifications may underlie the seemingly paradoxical functions of Stra13 to either promote or inhibit cellular differentiation and growth in different contexts.",
"Flag-mPIAS1, Flag-mPIAS3, Flag-mPIASxa, Flag-mPIASy, SUMO1, SENP1, Flag-HDAC1, and pD1luc harboring the cyclin D1 promoter have been described [4,31,41]. pCS2-Myc-Stra13 was derived from pCS2-Flag-Stra13 by PCR using primers 59-GAA TTC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG GAA CGG ATC CCC AGC GCG-39 and 59-GAA TTC TTA GTC TTT GGT TTC TAA GTT-39. The PCR product was TA-cloned into pCRII (Invitrogen), and then subcloned into the EcoR1 site of pCS2. K279R, K159R, and 2KR mutants were generated from pCS2-Myc-Stra13 using the QuickChange TM site-directed mutagenesis kit (Stratagene). Primers used for generating Stra13K279R are: 59-GTC AGC ACA ATT AGG CAA GAA TCC GAA-39 and 59-TTC GGA TTC TTG CCT AAT TGT GCT GAC-39; and for generating Stra13 K159R are: 59-CAG TAC CTG GCG AGG CAT GAG AAC ACT-39 and 59-AGT GTT CTC ATG CCT CGC CAG GTA CTG-39. The entire cDNA was sequenced to confirm the presence of directed mutations. For HDAC1 knockdown, NIH3T3 cells were transfected with 100 nM siRNA specific for mouse HDAC1 (Ambion); or with control scrambled siRNA using Lipofectamine RNAiMAX (Invitrogen). The efficiency of knockdown was determined using anti-HDAC1 antibody.",
"HEK293 cells, COS-7 cells NIH3T3 and cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HEK293), 10% calf serum (COS-7) and 10% bovine serum (NIH3T3) respectively. MEFs were cultured as described [5]. Transfections were performed using Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen). ",
"NIH3T3 cells or MEFs were transfected with Stra13 or Stra13 2KR with pD503 that confers resistance to puromycin. Control cells were transfected with empty vector and pD503. 24 hours (hr) after transfection, cells were selected with 1.2 mg/ml puromycin for three days. Selected cells were used for the following assays:",
"Proliferation/Viability assays. Cells were seeded at 1610 4 per well in 6-well plates in triplicates. Attached cells were trypsinized and proliferation was measured by counting cells daily over a period of five days. Alternatively, 3000 cells were seeded in 96-well plates and 72 hr later, MTT assays were performed using MTT cell proliferation assay kit (Invitrogen).",
"Cell cycle. Selected cells were seeded at a density of 5610 5 / 10-cm plates. 24 hr later, cells were fixed with cold 70% ethanol. Twenty thousand cells were acquired and analyzed for DNA content by propidium iodide staining (50 mg/ml) as described previously [4,42]. Cell cycle distribution was analyzed by flow cytometer (Becton Dickinson) using WINMDI software. Colony suppression assays. Colony assays were done as described [4,42]. Briefly cells were seeded at 1610 3 /10-cm plate. Two weeks later, colonies were fixed with 70% ethanol stained with 0.02% crystal violet and photographed. For quantification, the dye was extracted in 1% SDS and the absorbance read at 570 nm.",
"Chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed as described [43] using 3 mg of anti-Stra13 (Bethyl Laboratories) antibody. The following primers were used for amplification of the cyclin D1 promoter: 59-GAGAGCT-TAGGGCTCGTCTG-39 and 59-TGGGTGCGTTTCCGAG-TAC-39;",
"and for b-actin promoter: 59-GCTTCTTTGCAGCTCCTTCGTTG-39 and 59-TTTGCA-CATGCCGGAGCCGTTGT-39.",
"Quantitative RT-PCR (Q-PCR). Selected cells were synchronized in mitosis by adding nocodazole at a final concentration of 500 ng/ml. After 16 hr, nocodazole was removed by washing with media. Total RNA was extracted from cells using TRIZOL according to the manufacturer's instructions (Invitrogen). Genomic DNA was eliminated by treatment with TURBO DNase (Ambion) and cDNA synthesis was carried out using AMV Reverse Transcriptase (Promega) according to manufacturer's instructions. Q-PCR was performed using Light cycler 480 SYBR green I master (Roche) as described [43]. ",
"Cells were transfected with the cyclin D1 promoter reporter pD1luc, Stra13, Stra13 2KR, SUMO1, SENP1 as indicated in the figures along with 5 ng of Renilla luciferase. Empty expression vector was added to normalize the amount of total DNA. 48 hr after transfection, luciferase activity was measured by the Dual-Luciferase Reporter Assay system (Promega). All transfections were performed in triplicates, and repeated at least twice.",
"Immunofluorescence COS-7 cells were used to examine subcellular localization of Stra13 and Stra13 2KR. 48 hr after transfection, cells were fixed in 4% formaldehyde, incubated with anti-Myc antibody and detected with secondary antibody coupled with Texas-red (Invitrogen). Slides were mounted in Vectashield (Vector Laboratories) supplemented with DAPI (49, 69-diamidino-2-phenylindole) to identify nuclei. Cells were visualized on a Zeiss LSM 510 META confocal laser-scanning microscope.",
"Error bars indicate mean 6 standard deviation (S.D.). Statistical analysis was performed with by Student's t-test and P values ,0.05 were considered statistically significant [*p,0.05; **p,0.01]."
] | [] | [
"Introduction",
"Results",
"Stra13 Sumoylation is Enhanced by PIAS3 and PIAS1",
"Stra13 Sumoylation is Required for its Anti-proliferative Effects",
"SUMO Modification of Stra13 does not Affect its Subcellular Localization but Enhances its Ability to Repress Cyclin D1",
"HDAC1 Regulates Stra13 Sumoylation, Cyclin D1 Repression and Growth Arrest",
"Discussion",
"Materials and Methods",
"Plasmids, Mutagenesis, and HDAC1 siRNA",
"Cell Culture and Transient Transfections",
"Proliferation, Cell Cycle Analysis, and Colony Suppression Assays",
"Luciferase Assays",
"Statistical Analysis",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 ."
] | [] | [] | [
"SUMO Modification of Stra13 Is Required for Repression of Cyclin D1 Expression and Cellular Growth Arrest",
"SUMO Modification of Stra13 Is Required for Repression of Cyclin D1 Expression and Cellular Growth Arrest"
] | [
"PLoS ONE"
] |
231,603,159 | 2022-01-15T15:55:28Z | CCBYNCND | https://doi.org/10.1016/j.neo.2021.06.009 | GOLD | c97b94130c2c9961b158501bbf7ad8fc0c30e423 | [
"https://arxiv.org/pdf/2101.05746v2.pdf"
] | null | 2101.05746 | null | 10.1016/j.neo.2021.06.009 | null | 34237504 | 8267495 |
A mutual information-based in vivo monitoring of adaptive response to targeted therapies in melanoma
Aurore Bugi-Marteyn
Dermato-Oncology Unit
Division of Dermatology
University Hospital of Geneva
Switzerland
Department of Pathology and Immunology
Faculty of Medicine
University of Geneva
Switzerland
Fanny Noulet
Dermato-Oncology Unit
Division of Dermatology
University Hospital of Geneva
Switzerland
Department of Pathology and Immunology
Faculty of Medicine
University of Geneva
Switzerland
Nicolas Liaudet
Bioimaging core Facility
Faculty of Medicine
University of Geneva
Switzerland
†
Rastine Merat
Dermato-Oncology Unit
Division of Dermatology
University Hospital of Geneva
Switzerland
Department of Pathology and Immunology
Faculty of Medicine
University of Geneva
Switzerland
A mutual information-based in vivo monitoring of adaptive response to targeted therapies in melanoma
(Dated: December 7, 2020)Adaptive resistanceBRAF inhibitorELAVL1/HuRinformation theorymelanomamutual information
The mechanisms of adaptive resistance to genetic-based targeted therapies of solid malignancies have been the subject of intense research. These studies hold great promise for finding co-targetable hub/pathways which in turn would control the downstream non-genetic mechanisms of adaptive resistance. Many such mechanisms have been described in the paradigmatic BRAF-mutated melanoma model of adaptive response to BRAF inhibition. Currently, a major challenge for these mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existence of dependencies between the co-targeted hub/pathways and their downstream effectors. Moreover, the drug-induced in vivo modulation of these dependencies needs to be demonstrated. Here, we implement such single-cell-based in vivo expression dependency quantification using immunohistochemistry (IHC)-based analyses of sequential biopsies in two xenograft models. These mimic phase 2 and 3 trials in our own therapeutic strategy to prevent the adaptive response to BRAF inhibition. In this mechanistic model, the dependencies between the targeted Li2CO3-inducible hub HuR and the resistance effectors are more likely time-shifted and transient since the minority of HuR Low cells, which act as a reservoir of adaptive plasticity, switch to a HuR High state as they paradoxically proliferate under BRAF inhibition. Nevertheless, we show that a copula/kernel density estimator (KDE)-based quantification of mutual information (MI) efficiently captures, at the individual level, the dependencies between HuR and two relevant resistance markers pERK and EGFR, and outperforms classic expression correlation coefficients. Ultimately, the validation of MI as a predictive IHC-based metric of response to our therapeutic strategy will be carried in clinical trials.
I. INTRODUCTION
Based on a large amount of experimental research, our comprehension of the mechanisms of adaptive resistance to targeted therapies of solid mutated malignancies has significantly improved during the last decade [1]. Regardless of the current controversies on the scale-free properties of the cell/cancer signaling network [2], one of the most prevalent intuitions behind this experimental research has been to overcome the redundancy and robustness of such network by targeting its most essential connected nodes. Within the paradigmatic model of the adaptive response of BRAFmutated melanoma to BRAF inhibition on which we focus here, the convergence on MYC activation of various upstream signaling pathways and downstream targets [3] or the targetable WNT5A-induced activation of the AKT pathway associated with transcriptional reprograming [4], stand as good examples of "hub-directed" strategies in the recent literature. Currently, one of the major challenges of such mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existing dependencies between these hubs and their connected nodes. Similarly, the drug-targeted in vivo modulation of these dependencies needs to be demonstrated. These in vivo analyses are often not performed because of the lack of sensitivity to change of the available quantification techniques and the non-linear and time-shifted mutual dependencies of the involved mechanistic factors.
Such quantification becomes even more challenging if the proportion of cells in which such dependency operates is small within the tumor tissue, particularly at the initial timepoints of the adaptive response. This would be the case for rare, highly plastic cells that reprogram under therapeutic selection [5]. It would also occur in any embryonic signaling network that operates in the minority of senescent stem celllike cells that give rise to adaptive resistance [6]. Nevertheless, these quantifications are necessary for any further clinical implementation of these hub-directed strategies, not only to confirm their involvement in the observed clinical outcome but also as to obtain predictive markers of response to these strategies.
Practically, these analyses would need to be performed using techniques that can be routinely performed on patient biopsies as immunohistochemistry (IHC). Here, to implement such single-cell based in vivo expression dependency quantification, we use mutual information and compare it to more commonly used approaches in our own mechanistic strategy to reduce the adaptive response to BRAF inhibition in melanoma. In our model, the targeted hub HuR/ ELAVL1 (HuR) operates on the adaptive response in a minority of cells intermittently only if its expression becomes insufficient. Any dependency is therefore more likely time-shifted and transient. This model is therefore ideal to challenge the sensitivity of our approach.
II. MATERIALS AND METHODS
A. Descriptive statistics and mutual information estimation
All analyses, including tumor mean values Pearson's correlation, single-cell-based Spearman's correlation and mutual information estimation as well as the statistical tests, were conducted in MATLAB2020b (code for mutual information estimation and example dataset available upon request).
Mutual information (MI) is invariant to reparameterization, consequently, as a first step to decrease the impact of variability inherent to use of IHC, we use a copula-transform of values obtained for both markers (i.e., rank order them between 0 and 1) at the single-cell level. This initial step leads to a uniform distribution of their marginal distributions. We then estimate MI using a "smoothing" nonparametric Gaussian kernel density estimator (KDE), which is a local weighted average of the relative frequency of observations in the neighborhood of each estimate [7] given as
̂( ) = 1 ∑ ( ), =1(1)
where is the two-dimensional signal-measured intensities for any single cell and is the number of samples (cells) and
= ( − ) −1 ( − ) ℎ 2 ,(2)
where ℎ is the bandwidth of the kernel smoothing window and is the covariance matrix of . ( ), the kernel function, is given as
( ) = 1 (2 ) /2 ℎ det( ) 1/2 exp(− /2).(3)
Since ( ) is a multivariate normal density function of dimension , ℎ is calculated using the "optimal" Silverman's bandwidth that minimizes the mean integrated square error (MISE),
ℎ = ( 4 ( + 2) ) 1 +4 .(4)
Compared to classical histogram binning, this method is insensitive to the choice of origin, and most importantly provides a continuous better estimate of the underlying probability density, which avoids biases related to binning [7] or assumptions about the underlying distributions. The one-and two-dimensional estimates obtained with this pipeline are then used to calculate the mutual information between the two variables , (markers intensity) defined as
̂( , ) = 1 ∑ 2 , (̂( , ) ( ) ̂( ) ).(5)
To ensure the robustness of the MI estimates in each tumor and for each pair of markers, we randomly select the subpopulation of cells used to estimate the MI (one hundred assays per estimate and surrogate). The fixed number of cells used in this subpopulation across all biopsies being compared is defined as the smallest number of cells detected within all biopsies.
B. Mouse xenografts
Animal experiments were approved by the Animal Welfare Commission of the Canton of Geneva (approval n° GE/108/18) and followed the Swiss guidelines for animal experimentation. For model 1, two million shCtrl or shHuR SK-MEL28 cells (FACS-determined 70% apparent shift), generated as previously described [8], were resuspended in 100 µl of PBS and mixed with an equal volume of Matrigel (Corning ® Matrigel ® Matrix High Concentration, Phenol-Red free) and injected subcutaneously into the posterior left flanks of six-week-old female immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NSG, Charles River). Tumor formation was monitored twice a week using calliper measurements and calculated by the ellipsoidal formula: tumor volume ꞊ (length x width 2 ) x 0.5. The vemurafenib was prepared using 240 mg vemurafenib tablets (for human use) that were manually ground and suspended in a water containing solution of carboxymethylcellulose (CMC, 1%) and DMSO (5%). The final concentration of vemurafenib was 16.5 mg/mL as confirmed using an in-house liquid chromatography-electrospray ionization-tandem mass spectrometry method. Once the tumors reached a volume of approximately 0.2 cm 3 , all mice were treated by oral gavage once a day with 150 μL of this suspension (fixed vemurafenib dosage of 100 mg/kg/day). All mice also received a lithium carbonate (Li2CO3) containing chow dosed at 0.25% (2.5g/kg). However, considering that in the initially conducted fast growing A375 cells experiment (model 2) [8], tumor growth was affected in the Li2CO3 arm and that in this experiment tumor growth was initially extremely slow in both shCtrl and shHuR arms, the Li2CO3 therapy was initiated at the regrowth time-point. This was mandatory to obtain largeenough tumors that were, upon mice sacrifice, immediately collected and formalin-fixed and subsequently paraffinembedded (FFPE) (time-point 2, TP2, no time-point 1, TP1). The methodology for the A375 xenografts (model 2), comprising two groups receiving or not Li2CO3, has been previously reported [8]. In model 2, punch biopsies were collected under local anesthesia and similarly prepared (FFPE) in a separate-cohort of mice before treatment initiation (TP1J0) and 10 days later (average volume-doubling time) in a subgroup of this cohort that did not receive any therapy during that time (TP1J10). Samples were similarly prepared (FFPE) upon mice sacrifice at the end of the experiment (TP2).
C. Immunohistochemistry (IHC)-based single-cell automated quantification
Standard fluorescence-based immunohistochemical staining on deparaffinized tumor sections was performed as previously described [8]. In short, 5-µm thick sections were deparaffinized and cleared using UltraClear TM reagent and rehydrated in ethanol. Following antigen retrieval in citrate buffer, tissues were permeabilized with 0,1% tween 20 and blocked in 5% normal goat serum (NGS). The mouse monoclonal anti-human HuR antibody 3A2 (1:100) was used for co-staining (2h exposure at room temperature in PBS tween 0,1% NGS 5%) with one of the following rabbit primary antibodies: anti-S100 A1 antibody (SAB502708, Sigma 1:100), anti-EGFR antibody (D38B1, Cell signaling, 1:50), anti-phospho-p44/42 MAPK (pERK1/2) antibody (520G11, Cell signaling, 1:200). Following washing, Alexa 488-conjugated anti-mouse, or Alexa 555-conjugated antirabbit antibodies (1:500) were used as secondary antibodies (1 h exposure at room temperature). Following additional washing, slides were mounted with Dapi FluoromountG (Southern Biotech). Images were acquired using an automated Zeiss Axioscan.Z1 with a 20x 0.8NA Plan Apochromat objective (lateral resolution 0.325 µm/pixel). Nuclei were segmented using QuPath v0.2.3 based on the Dapi fluorescent channel. All areas of homogeneous S100 staining available within the tumor section were initially defined as region of interest (ROI) and subsequently manually transposed for all other stainings. Each individual nucleus area was expanded of 5 m to simulate a cytoplasmic area. Both areas were used to measure the nuclear and the wholecell mean signal fluorescence intensity in each cell. HuR being mainly a nuclear protein, its signal was measured in the nuclear area, whereas EGFR, pERK, and S100 signals were measured in the whole-cell surface area.
Human biopsies obtained from patients with metastatic melanoma disease were similarly analyzed. Their use was approved by the Geneva ethics committee (study n°2017-01346). According to the Swiss Federal Law for research, a positive vote of an ethical committee in a retrospective study is sufficient to use patient data and materials for research purposes without further need of individual informed consents. All patient-related data were identified as previously described [8] at the University Hospital of Geneva and selected on the availability of the samples.
III. RESULTS
The RNA-binding protein HuR has been extensively characterized as a ubiquitously expressed post-transcriptional orchestrator of differentiation [9], cell death [10] and an expression synchronizer of cell-cycle regulatory genes (11).
HuR has the ability to regulate many of the previously identified hubs within the adaptive network to BRAF inhibition and could potentially represent a "super-hub" within this network [3,4,[12][13][14]. We have recently shown that a heterogeneous and intermittently lower expression of HuR, within a subpopulation of BRAF-mutated melanoma cells (HuR Low state), is induced upon their exposure to a BRAF inhibitor (BRAFi). This heterogeneous state is increasingly detected during the adaptive response and is not dependent on the proliferation status of the cell population. The HuR Low heterogeneous state in turn induces a heterogeneous and adaptive plastic expression of resistance markers and an adaptive response of the whole cell population to chronic BRAF inhibition. Experimentally, in order to increase the adaptive response, the insufficient expression of HuR needs to be reversible between two attractor sets (reversible knockdown). Indeed, a stable knockdown of HuR has no effect on the adaptive response [8]. This observation indirectly indicates that although the heterogeneous HuR Low cells are a reservoir of adaptive plasticity, they need to switch to a HuR High state in order to proliferate. At the single-cell and at steady state, although the adapted highly proliferating cells are in a HuR High state and have the highest expression level of resistance markers, their emergence occurs within the cell subpopulation carrying the heterogeneous HuR Low cell component. From a therapeutic standpoint, a slight lithium salt-induced suppression of the heterogeneous HuR Low cell component attenuates the adaptive paradoxical expression and proliferative response to BRAF inhibition [8].
To develop in vivo predictive expression dependency markers of response to the resulting therapeutic strategy of combining lithium salts with small-molecule inhibitors in BRAF-mutated (i) shHuR (HuR lithium-non inducible) carrying an unstable proportion of HuR Low cells oscillating between two attractor sets [8] (blue) (n=7), (ii) shCtrl (HuR lithium-inducible) (red) (n=8). Mice were treated as in a single-arm phase 2 trial and received the BRAFi vemurafenib (100mg/kg/day) with subsequent addition of lithium carbonate (Li2CO3) containing chow dosed at 0.25% (2.5g/kg) at the indicated time-points. Considering that before exposure to the vemurafenib, HuR heterogeneous expression was already operating at a much higher extent in the shHuR panel than the one expected to be induced by the treatment, excision-biopsies were performed only at the end of the experiment (TP2, no TP1). (B) IHC-based HuR expression distribution in shHuR (blue) and shCtrl (red) xenografts shown as violin plots. (C) Model 2: A375 BRAF-mutated xenografts. Mice were treated as in a double-arm phase 3 trial and assigned to control (blue) or Li2CO3 chow (red) dosed as in (A) and subsequently all received the BRAFi vemurafenib dosed as in (A) at the indicated time-points. Biopsies were performed before initiating the Li2CO3 therapy (TP1j 0 , n=15) and following an average volume-doubling time period of 10 days in a subcohort of mice that did not receive any therapy during that time (TP1j 10 , n=7, purple). Fig. 1 legend). In each panel, the Pearson's correlation r and p-value for testing the hypothesis of no correlation is indicated.
metastatic BRAF-mutated melanoma, we conduct here a series of analyses on biopsies obtained from two mouse xenograft models established according to the design of a phase 2 (model 1) and phase 3 (model 2) clinical trials in which biopsy samples would be obtained for translational studies. We focus our IHC-based monitoring on the expression of HuR and its dependent resistance markers, EGFR and pERK. Ex vivo, the increased expression of EGFR or pERK under reversible knockdown of HuR or conversely, their decreased expression under suppression of the HuR Low cells by lithium salts, are clearly detected at the whole cell population level in synchronized cells adapted to, and treated with, a BRAF inhibitor [8]. However, at steady state and under physiologic variation of HuR expression, these changes are difficult to detect, considering that they occur at any moment in a subpopulation of adapting cells. Nevertheless, at the single-cell level, the dependencies between HuR and these resistance markers are expected to change in adapting cells and be detectable in vivo. Conversely, our results predict that these changes will be modulated in tumors concomitantly exposed to lithium salts.
In model 1 (Fig. 1A), designed as a single-arm phase 2 trial, all individuals received the BRAFi vemurafenib with subsequent addition of lithium carbonate (Li2CO3) to their regimen. The SK-MEL28 HuR reversible knockdown cells (shHuR) carrying an unstable proportion of HuR Low cells [8], were used to generate a panel of xenografts (n=7) in which the average expression of HuR is variable yet distinguishable from, and inferior to, the average expression of HuR observed in the control (shCtrl) panel (n=8) (Fig. 1B). Based on our previous ex vivo-made sensitive immunocytochemistry analyses, HuR expression is not inducible in these cells as opposed to their shCtrl counterparts in which a positive shift in HuR nuclear and cytoplasmic content is detected upon exposure to therapeutic concentrations of Li2CO3 [8]. This in vivo experimental model is therefore perfectly suited to measure to what extent the dependencies between HuR and both EGFR and pERK are detectable and distinguishable between non-responders' tumors carrying a more heterogeneous expression of HuR and the responders' carrying a more homogeneous expression of HuR. In this model, before treatment initiation, a HuR heterogeneous expression is already operating to a much higher extent in the shHuR panel than the one expected to be induced by the BRAFi; the comparative analyses were therefore performed only on biopsies obtained at the end of the experiment, from tumors exposed to sustained BRAF inhibition (time-point 2, TP2, no time-point 1, TP1).
In model 2 (Fig. 1C), designed as a double-arm phase 3 trial, individuals (n=16) were randomly assigned into two arms to receive or not Li2CO3 in addition to the vemurafenib treatment. The A375 BRAF-mutated melanoma cells were used to generate the xenografts. These cells have a high deterministic behavior for simulating tumor relapse following an initial response to BRAF inhibition and are therefore often used as a model of adaptive response to BRAF inhibition [15]. Our ex vivo observations indicate that HuR expression is more heterogeneous in A375 cells having a higher propensity for adaptive response than in cell lines having a lower propensity for adaptive response, e.g., the SK-MEL28 cells used in model 1. Moreover, this expression heterogeneity is highly increasing in A375 cells following exposure to a BRAFi [8]. Therefore, contrary to model 1, a change in the baseline expression dependencies between HuR and both EGFR and pERK is expected to occur in BRAFi-treated tumors. For this reason, the comparative analyses were performed on biopsies obtained both before treatment initiation (n=15, separate cohort, TP1j 0 , see materials & methods) and upon tumor regrowth (TP2). Moreover, the tumor volume is rapidly increasing in this model and might affect the expression dependencies of the markers. Consequently, additional biopsies were performed following an average volumedoubling time period of 10 days in a sub-cohort of individuals (n=7) that did not receive any treatment during that time (TP1j 10 ). The overall outcome of this experiment was previously reported and showed that following the initial response to vemurafenib, relative tumor regrowth was clearly attenuated in the Li2CO3 receiving arm (Fig. 1C) [8].
A. Average expression correlations
HuR/pERK and HuR/EGFR dependencies were first examined based on the strength of Pearson's correlation between the tumor average expression values of markers (Fig. 2). In model 1 ( Fig. 2A), an overall positive correlation for HuR/pERK and HuR/EGFR was apparent and consistent with FIG. 3. HuR/pERK, HuR/EGFR and HuR/S100 dependencies examined based on the Spearman's rank correlation value of expression of the markers at the single-cell level in tumor biopsies performed as in Fig. 2. For the two models, Fisher transformations were applied on each correlation. In model 1, Wilcoxon rank sum tests were used to compare medians. In model 2, Kruskal-Wallis tests were used to compare the distributions (upper left p-value), when the null hypothesis was rejected post-hoc Tukey-Kramer tests were performed.
our ex vivo single-cell observations, in which the adapted HuR High proliferating cells showed the highest expression level of resistance markers [8]. Nevertheless, for HuR/pERK, the strength of correlation was higher in the shHuR panel, and for HuR/EGFR, a positive correlation was only observed in the shHuR panel. In model 2 (Fig. 2B), HuR/EGFR already positively correlated at baseline in TP1j 0 biopsies and a HuR/pERK correlation was induced following exposure to BRAFi in TP2 tumors, compared with baseline. However, the suppression of these correlations/dependencies in TP2Li 2 CO 3 tumors was only apparent for HuR/EGFR. Overall, these results were consistent with the mechanistic model deduced from our ex vivo experimental results in which the dependency of resistance markers toward HuR increases when HuR expression is rendered insufficient: whether experimentally as in model 1 reversible knockdown, or therapeutically following exposure to a BRAFi, as for HuR/pERK in model 2. Yet, the Li2CO3-induced suppression of these dependencies in model 2 was only captured for HuR/EGFR. Moreover, the overall sensitivity of this commonly used approach was undoubtedly insufficient to predict the phenotypic outcome of individual tumors i.e., in terms of predicting in model 2 the therapeutic arm to which they belong.
B. Single-cell Spearman's correlations
Next, in order to improve our sensitivity to detect nonlinear dependencies, we calculated for each sample the Spearman's (rank) correlation for HuR/pERK and HuR/EGFR at the single-cell level (Fig. 3). Henceforward, the expression of the differentiation marker S100 was also included as a "negative" marker of dependency. Indeed, the dependency between HuR and S100 is expected to either be low in the undifferentiated cell lines used in both models or at least not to be positively selected in the adaptive response to BRAF inhibition. In model 1 (Fig. 3A), strikingly, no significant differences in the values were observed between the shHuR and the control panels. In model 2 (Fig. 3B), consistent with the average expression correlations analyses, the coefficient was significantly higher for HuR/pERK upon exposure to the BRAFi in TP2 tumors compared with the baseline TP1j 0 biopsies. Compared with TP2 tumors, here, a significant reduction of was observed in TP2Li 2 CO 3 tumors for both HuR/pERK and HuR/EGFR but was more apparent for the latter. For HuR/S100, as expected no significant differences were observed for values among the samples. These analyses were therefore confirmatory and more sensitive for detecting the Li2CO3-induced suppression of both HuR/pERK and HuR/EGFR dependencies in model 2. However again, they were insufficient in predicting the phenotypic outcome of individual tumors and even in detecting the differences in dependencies in model 1. In this model, the lack of sensitivity was presumably due to an averaging effect between HuR Low and HuR High cells, both types being, according to our ex vivo experimental results, timely involved in these dependencies.
C. Mutual information
MI is a universal metric for quantifying any type rather than just linear dependencies between two variables. Information theory and entropy-based description of MI provide the formalism to define MI between two variables and as
( ; ) = ( ) + ( ) − ( , ),(6)
where is the entropy associated with each of the variables, i.e. the amount of information gained (or reduced uncertainty) on each of them when measuring them separately, and ( , ) is the joint entropy symmetrically associated with both variables, i.e. the amount of information gained on one of the variables when measuring the other variable. Most importantly, MI is scale-free and its quantification using rank-ordered data (see materials and methods) allows to To calculate surrogates, one hundred assays were conducted by randomly shuffling the intensity of the second marker intensity in the subpopulation of cells used for MI estimate (grey histogram). (B) Tumor growth effect on HuR/pERK and HuR/EGFR MI estimates. Biopsies from baseline (TP1j 0 , green) are compared with identical tumors following a volume-doubling time of 10 days in a subcohort of mice that did not receive any therapy during that time (TP1j 10 , purple). Testing the hypothesis of equal MI under tumor growth was done by using Wilcoxon rank sum tests. (C, D) Sample size effect on HuR/pERK and HuR/EGFR MI estimates in human metastatic melanoma. (C) Example of HuR/pERK co-staining performed on a small intestine metastatic melanoma disease. Note that the region of interest (ROI) was chosen as to be similar to the additional tumor sections that were used for other stainings. (D) The fixed number of cells (percentage of the smallest sample) being used for HuR/pERK and HuR/EGFR MI estimates is changed across eight biopsies of metastatic disease. Biopsies were performed before small-molecule inhibitor therapies were initiated but only half of the patients had a complete response (cream, red and brown color range, n=4), the remaining patients had either partial response or progressive disease (blue color range, n=4) on the biopsied tumor two to three months after treatment initiation (radiologically-assessed response according to RECIST1.1). overcome some of the limitations of semi-quantitative techniques as IHC [16,17]. These include variability in sample preparation and imaging. However, as extensively discussed in the physics literature, MI is highly dependent on the size of the probability distribution and is sample-sizedependent [18]. This is even true for discrete data for which, in order to get a reliable estimate of MI, the number of samples needs to be significantly larger than the cardinality of the underlying distribution. To overcome these limitations, we used a computationally efficient Gaussian kernel estimator (KDE see materials and methods) and applied it throughout this study on a fixed smallest number of cells detected within the samples being compared. With this fixed number, cells were randomly chosen in the one hundred assays which were performed to ensure the stability of each MI estimate.
We first demonstrated that in each tumor sample and for each pair of markers, the MI estimates were distinguishable from a "null" MI obtained by shuffling one of the two signals across cells (Fig. 4A). An above null MI was quantifiable even for samples having a very low HuR/pERK or HuR/EGFR MI estimate e.g., the TP2Li 2 CO 3 tumors in model 2. Although using a KDE, we then asked if the size of the probability distribution had an impact on the MI estimates. We considered that a natural increase in the probability distribution might occur during tumor growth even if the fixed-size sample is randomly chosen in the tumor section, and estimated the HuR/pERK and HuR/EGFR MI of biopsies obtained in a subcohort of mice that did not receive any treatment during an average volume-doubling time period of 10 days (TP1j 10 ). These MI estimates were not significantly different from the one obtained in biopsies performed on smaller TP1j 0 identical tumors ( Fig. 1C and 4B). Next, we checked if the sample size would affect the MI estimate by varying the number of cells analyzed within tumors. Considering that the xenografts in our mouse models were likely homogeneous in their cell composition, these analyses were carried on biopsies (n=8) obtained from patients with BRAF-mutated metastatic melanoma disease before targeted therapy was initiated. Once treated, these patients had various types of response in their biopsied tumors which were therefore more likely heterogeneous in their cell composition. Yet the change in sample size across all metastatic tumors had no effect in their MI estimates and ranking ( Fig. 4C and D). Overall, we concluded that our copula-transformed/KDEbased estimate of MI was robust enough to overcome both the probability distribution size and the sample size biases.
In model 1, MI estimates for both HuR/pERK and HuR/EGFR were significantly higher in the shHuR tumors than in the control panel (Fig. 5A). In contrast, HuR/S100 MI, used here as a control, was slightly but significantly higher in the control panel. Remarkably, HuR/EGFR MI estimates were almost sufficient to individually distinguish the shHuR from the shCtrl tumors. In addition, contrary to the HuR/pERK MI estimates for the 20 th percentile of the highest HuR-expressing cells, these estimates for the 20 th lowest percentile were almost as high as the ones obtained for the total cell population in most shHuR tumors, suggesting that most of the HuR/pERK dependency was operating in the HuR Low cells in these tumors. Overall, the increased HuR/pERK and HuR/EGFR MI-based dependencies observed in shHuR tumors were consistent with the average expression Pearson's coefficient-based analyses discussed in section 3.1, however, MI performed better, particularly in capturing HuR/EGFR dependency in individual tumors. ..nIn model 2 (Fig. 5B), in accordance with 3.1 results, HuR/EGFR MI-determined dependency was already detected at baseline in TP1j 0 biopsies and became significant compared to baseline for HuR/pERK in adapted TP2 tumors. As expected, HuR/S100 MI estimate was low and not affected in BRAFi-treated tumors. Importantly, the Li2CO3 suppression of HuR/EGFR and BRAFi-induced HuR/pERK dependencies, under physiologic expression levels of HuR, was clearly captured here. With the exception of a few mice, MI performed well to distinguish the mice co-treated with Li2CO3 from the ones treated only with the BRAFi.
Taken together, these results support the use of MI, rather than the classical metrics used in sections 3.1 and 3.2, for quantifying HuR/pERK and HuR/EGFR dependencies in our therapeutic strategy. The increase in these dependencies under an experimentally induced insufficient expression of HuR occurred similarly, at least for one pair of markers, under BRAFi therapy and were reduced under concomitant Li2CO3 therapy. More importantly, the predictive value of MI at the individual level was far more effective. Ultimately, the validation of MI as a biologic, predictive IHC-based metric of response to combining small-molecule inhibitors with Li2CO3 in BRAF-mutated metastatic melanoma will be carried in trials equivalent to either one of the models developed here.
IV. DISCUSSION
Although extensively used in the literature to elucidate gene regulatory networks [17,19] and quantify cellular signal processing [20,21], MI-based quantification of expression dependencies has not been used, to our knowledge, as an in vivo confirmatory approach for mechanistic studies, nor as a quantitative metric for clinical use. As shown in this work, MI is far more sensitive to change and performs better to predict drug effect than average co-expression or even single-cell-based co-expression correlation coefficients. MI should be particularly used when suspecting complex patterns of dependencies and when dealing with dynamic, reversible dependencies as observed during the adaptive response to targeted therapies in solid malignancies. This approach may help clarify contradictory observations made in this field. As an example, the melanocytic lineage transcription factor MITF has been reported to be either upregulated [22] or downregulated [23,24] in association with an increased level of pERK in melanoma cells adapted to and treated with a MAPK inhibitor. These contradictory observations could either be attributed to the use of different experimental settings including cell types or be related to partial independencies between these markers (in which case the presumed mechanistic effect is at least partially refuted). However, they could as well be related to a heterogeneous, complex, pattern of dependency [25] that could be captured by MI as the one described in this study between HuR and EGFR or pERK.
Generally, the in vivo dynamic change in MI between the main regulatory hub and its targets should be first assessed in an experimentally-induced, presumed pathologic expression condition of the targeted hub and subsequently, under its physiologic expression where the incriminated pathologic change of MI is expected to be therapeutically reversed or at least modulated according to the observed phenotypic outcome. Importantly, the integration of the methodology developed here into the translational analytic pipeline used to develop predictive markers of response to therapy in clinical trials is highly feasible. Its potential future use in routine pathology as an automated procedure is therefore predicted.
FIG. 1 .
1Mice xenografts melanoma models of adaptive response to BRAF inhibition used in this study. (A) Model 1: SK-MEL28
Final excision-biopsies were performed in both arms at the end of the experiment (TP2 and TP2Li 2 CO 3 ). For (A) and (C) data shown are mean tumor volume (indicated as fold change) ± SEM. FIG. 2. HuR/pERK and HuR/EGFR linear dependencies examined based on the strength of Pearson's correlation between the average expression value of markers in tumor biopsies performed, (A) in model 1 in SK-MEL28 shHuR (blue) and shCtrl (red) xenografts, (B) in model 2 in A375 cells xenografts before initiating any therapy (TP1j 0 , green) and at the end of the experiment in the vemurafenib + control chow arm (TP2, blue) or the vemurafenib + Li2CO3 chow arm (TP2 Li 2 CO 3 , red) (see
FIG. 4 .
4Robustness of the MI estimates including in respect to the probability distribution size and the sample size biases. (A) Examples of HuR/pERK and HuR/EGFR MI estimates in the three types of biopsies obtained in model 2.
FIG. 5 .
5HuR/pERK, HuR/EGFR and HuR/S100 dependencies examined based on MI estimate as inFig. 2. For model 1, additional estimates of MI were calculated for the 20 th percentile of the highest HuR-expressing cells (80-100) and for the 20 th percentile of the lowest HuR-expressing cells (0-20). In model 1, Wilcoxon rank sum tests were used to compare medians. In model 2, Kruskal-Wallis tests were used to compare the distributions (upper left p-value), when the null hypothesis was rejected post-hoc Tukey-Kramer tests were performed.
ACKNOWLEDGMENTSWe thank Wolf-Henning Boehncke for continuous support, Ludovic Wrobel for his assistance for performing the xenografts and animal biopsies, Youssef Daali for conducting the vemurafenib dosage in the gavage solution, Pierre Bonnaventure and his team for performing the mice gavage.This work was supported by the Ligue Genevoise contre le Cancer and the Fondation pour la lutte contre le cancer (Zurich).AUTHOR CONTRIBUTIONSAurore Bugi-Marteyn performed the animal experiments and the immunohistochemistry procedures. Fanny Noulet performed all the confirmatory immunohistochemistry procedures. Nicolas Liaudet designed the code, analyzed the data and designed the figures. Rastine Merat designed the theoretical framework, supervised the project, analyzed the data, designed the figures and wrote the manuscript.DECLARATION OF INTERESTRastine Merat is inventor on a patent on the use of agents enhancing HuR/ELAV protein levels in the treatment of BRAF-mutated cancers. Declarations of interest for Aurore Bugi-Marteyn, Fanny Noulet and Nicolas Liaudet: none.
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. Biochem. Biophys. Res. Commun. 517Biochem. Biophys. Res. Commun. 517, 181-187 (2019).
Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes. A Figueroa, A Cuadrado, J Fan, U Atasoy, G E Muscat, P Muñoz-Canoves, Mol. Cell. Biol. 23A. Figueroa, A. Cuadrado, J. Fan , U. Atasoy, G.E. Muscat, P. Muñoz-Canoves, et al. Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes. Mol. Cell. Biol. 23, 4991-5004 (2003).
Posttranscriptional orchestration of an anti-apoptotic program by HuR. K Abdelmohsen, A Lal, H H Kim, M Gorospe, Cell Cycle. 6K. Abdelmohsen, A. Lal, H.H. Kim, M . Gorospe. Posttranscriptional orchestration of an anti-apoptotic program by HuR. Cell Cycle 6, 1288-1292 (2007).
Phosphorylated HuR shuttles in cycles. H H Kim, M Gorospe, Cell Cycle. 7H.H. Kim, M. Gorospe. Phosphorylated HuR shuttles in cycles. Cell Cycle 7, 3124-3126 (2008).
HuR recruits let-7/RISC to repress c-Myc expression. H H Kim, Y Kuwano, S Srikantan, E K Lee, J L Martindale, M Gorospe, Genes Dev. 23H.H. Kim, Y. Kuwano, S. Srikantan, E.K. Lee, J.L. Martindale, M. Gorospe. HuR recruits let-7/RISC to repress c-Myc expression. Genes Dev. 23, 1743-1748 (2009).
Wnt-5a mRNA Translation is suppressed by the Elav-like protein HuR in human breast epithelial cells. K Leandersson, K Riesbeck, T Andersson, Nucleic Acids Res. 34K. Leandersson, K. Riesbeck, T. Andersson .Wnt-5a mRNA Translation is suppressed by the Elav-like protein HuR in human breast epithelial cells. Nucleic Acids Res. 34, 3988-3999 (2006).
The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation. Z Meng, P H King, L B Nabors, N L Jackson, C Y Chen, P D Emanuel, Nucleic Acids Res. 33Z. Meng, P.H. King, L.B. Nabors, N.L. Jackson, C.Y. Chen, P.D. Emanuel, et al. The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF- IR transcript and differentially represses cap-dependent and IRES-mediated translation. Nucleic Acids Res. 33, 2962-2979 (2005).
An acquired vulnerability of drug-resistant melanoma with therapeutic potential. L Wang, R Leite De Oliveira, S Huijberts, E Bosdriesz, N Pencheva, D Brunen, Cell. 173L. Wang, R. Leite de Oliveira, S. Huijberts, E. Bosdriesz, N. Pencheva, D. Brunen , et al. An acquired vulnerability of drug-resistant melanoma with therapeutic potential. Cell 173, 1413-1425 (2018).
Equitability, mutual information, And the maximal information coefficient. J B Kinney, G S , Proc. Natl. Acad. Sci. 111J.B. Kinney, G.S. Atwal. Equitability, mutual information, And the maximal information coefficient. Proc. Natl. Acad. Sci. 111, 3354-3359 (2014).
ARACNE: An Algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. A A Margolin, I Nemenman, K Basso, C Wiggins, G Stolovitzky, R Dalla Favera, BMC Bioinformatics. 717SupplA.A. Margolin, I. Nemenman, K. Basso, C. Wiggins, G. Stolovitzky, R. Dalla Favera, et al. ARACNE: An Algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. BMC Bioinformatics. 7 Suppl 1, S7 (2006).
Estimation of mutual information for real-valued data with error bars and controlled bias. C M Holmes, I Nemenman, Phys. Rev. E. 10022404C. M. Holmes, I. Nemenman. Estimation of mutual information for real-valued data with error bars and controlled bias Phys. Rev. E 100, 022404 (2019).
Estimation of the proteomic cancer co-expression sub networks by usin g association estimators. C Erdoğan, Z Kurt, B Diri, PLOS ONE. 12188016C. Erdoğan, Z. Kurt, B. Diri. Estimation of the proteomic cancer co-expression sub networks by usin g association estimators PLOS ONE 12, e0188016 (2017).
Robustness and compensation of information transmission of signaling pathways. S Uda, T H Saito, T Kudo, T Kokaji, T Tsuchiya, H Kubota, Science. 341S. Uda, T.H. Saito, T. Kudo, T. Kokaji, T. Tsuchiya, H. Kubota, et al. Robustness and compensation of information transmission of signaling pathways. Science 341, 558-561 (2013).
Information processing by simple molecular motifs and susceptibility to noise. S S Mc Mahon, O Lenive, S Filippi, M P Stumpf, J. R. Soc. Interface. 1220150597S.S. Mc Mahon., O. Lenive, S. Filippi, M.P. Stumpf. Information processing by simple molecular motifs and susceptibility to noise. J. R. Soc. Interface 12, 20150597 (2015).
Inhibiting drivers of nonmutational drug tolerance is a salvage strategy for targeted melanoma therapy. M P Smith, H Brunton, E J Rowling, J Ferguson, I Arozarena, Z Miskolczi, Cancer Cell. 29M.P. Smith, H. Brunton, E.J . Rowling, J. Ferguson, I. Arozarena, Z. Miskolczi, et al. Inhibiting drivers of non- mutational drug tolerance is a salvage strategy for targeted melanoma therapy. Cancer Cell 29, 270-284 (2016).
A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. D J Konieczkowski, C M Johannessen, O Abudayyeh, J W Kim, Z A Cooper, A Piris, Cancer Discov. 4D.J. Konieczkowski, C.M. Johannessen, O. Abudayyeh, J.W. Kim, Z.A. Cooper, A. Piris, et al. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov. 4, 816-827 (2014).
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors. G Richard, S Dalle, M A Monet, M Ligier, A Boespflug, R M Pommier, EMBO Mol. Med. 8G. Richard, S. Dalle, M.A. Monet, M. Ligier, A. Boespflug, R.M. Pommier, et al. ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors. EMBO Mol. Med. 8, 1143-1161 (2016).
Intratumor and intertumor heterogeneity in melanoma. T M Grzywa, W Paskal, P K Włodarski, Transl. Oncol. 10T.M. Grzywa, W. Paskal, P.K. Włodarski. Intratumor and intertumor heterogeneity in melanoma. Transl. Oncol. 10, 956-975 (2017).
| [
"The mechanisms of adaptive resistance to genetic-based targeted therapies of solid malignancies have been the subject of intense research. These studies hold great promise for finding co-targetable hub/pathways which in turn would control the downstream non-genetic mechanisms of adaptive resistance. Many such mechanisms have been described in the paradigmatic BRAF-mutated melanoma model of adaptive response to BRAF inhibition. Currently, a major challenge for these mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existence of dependencies between the co-targeted hub/pathways and their downstream effectors. Moreover, the drug-induced in vivo modulation of these dependencies needs to be demonstrated. Here, we implement such single-cell-based in vivo expression dependency quantification using immunohistochemistry (IHC)-based analyses of sequential biopsies in two xenograft models. These mimic phase 2 and 3 trials in our own therapeutic strategy to prevent the adaptive response to BRAF inhibition. In this mechanistic model, the dependencies between the targeted Li2CO3-inducible hub HuR and the resistance effectors are more likely time-shifted and transient since the minority of HuR Low cells, which act as a reservoir of adaptive plasticity, switch to a HuR High state as they paradoxically proliferate under BRAF inhibition. Nevertheless, we show that a copula/kernel density estimator (KDE)-based quantification of mutual information (MI) efficiently captures, at the individual level, the dependencies between HuR and two relevant resistance markers pERK and EGFR, and outperforms classic expression correlation coefficients. Ultimately, the validation of MI as a predictive IHC-based metric of response to our therapeutic strategy will be carried in clinical trials."
] | [
"Aurore Bugi-Marteyn \nDermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland\n\nDepartment of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland\n",
"Fanny Noulet \nDermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland\n\nDepartment of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland\n",
"Nicolas Liaudet \nBioimaging core Facility\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland\n",
"† ",
"Rastine Merat \nDermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland\n\nDepartment of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland\n"
] | [
"Dermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland",
"Department of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland",
"Dermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland",
"Department of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland",
"Bioimaging core Facility\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland",
"Dermato-Oncology Unit\nDivision of Dermatology\nUniversity Hospital of Geneva\nSwitzerland",
"Department of Pathology and Immunology\nFaculty of Medicine\nUniversity of Geneva\nSwitzerland"
] | [
"Aurore",
"Fanny",
"Nicolas",
"†",
"Rastine"
] | [
"Bugi-Marteyn",
"Noulet",
"Liaudet",
"Merat"
] | [
"D J Konieczkowski, ",
"C M Johannessen, ",
"L A Garraway, ",
"A D Broido, ",
"A Clauset, ",
"K R Singleton, ",
"L Crawford, ",
"E Tsui, ",
"H E Manchester, ",
"O Maertens, ",
"X Liu, ",
"J N Anastas, ",
"R M Kulikauskas, ",
"T Tamir, ",
"H Rizos, ",
"G V Long, ",
"E M Von Euw, ",
"S M Shaffer, ",
"M C Dunagin, ",
"S R Torborg, ",
"E A Torre, ",
"B Emert, ",
"C Krepler, ",
"M Milanovic, ",
"D N Y Fan, ",
"D Belenki, ",
"J H M Däbritz, ",
"Z Zhao, ",
"Y Yu, ",
"Y I Moon, ",
"B Rajagopalan, ",
"U Lail, ",
"R Merat, ",
"A Bugi-Marteyn, ",
"L J Wrobel, ",
"C Py, ",
"Y Daali, ",
"C Schwärzler, ",
"A Figueroa, ",
"A Cuadrado, ",
"J Fan, ",
"U Atasoy, ",
"G E Muscat, ",
"P Muñoz-Canoves, ",
"K Abdelmohsen, ",
"A Lal, ",
"H H Kim, ",
"M Gorospe, ",
"H H Kim, ",
"M Gorospe, ",
"H H Kim, ",
"Y Kuwano, ",
"S Srikantan, ",
"E K Lee, ",
"J L Martindale, ",
"M Gorospe, ",
"K Leandersson, ",
"K Riesbeck, ",
"T Andersson, ",
"Z Meng, ",
"P H King, ",
"L B Nabors, ",
"N L Jackson, ",
"C Y Chen, ",
"P D Emanuel, ",
"L Wang, ",
"R Leite De Oliveira, ",
"S Huijberts, ",
"E Bosdriesz, ",
"N Pencheva, ",
"D Brunen, ",
"J B Kinney, ",
"G S , ",
"A A Margolin, ",
"I Nemenman, ",
"K Basso, ",
"C Wiggins, ",
"G Stolovitzky, ",
"R Dalla Favera, ",
"C M Holmes, ",
"I Nemenman, ",
"C Erdoğan, ",
"Z Kurt, ",
"B Diri, ",
"S Uda, ",
"T H Saito, ",
"T Kudo, ",
"T Kokaji, ",
"T Tsuchiya, ",
"H Kubota, ",
"S S Mc Mahon, ",
"O Lenive, ",
"S Filippi, ",
"M P Stumpf, ",
"M P Smith, ",
"H Brunton, ",
"E J Rowling, ",
"J Ferguson, ",
"I Arozarena, ",
"Z Miskolczi, ",
"D J Konieczkowski, ",
"C M Johannessen, ",
"O Abudayyeh, ",
"J W Kim, ",
"Z A Cooper, ",
"A Piris, ",
"G Richard, ",
"S Dalle, ",
"M A Monet, ",
"M Ligier, ",
"A Boespflug, ",
"R M Pommier, ",
"T M Grzywa, ",
"W Paskal, ",
"P K Włodarski, "
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] | [
"Konieczkowski",
"Johannessen",
"Garraway",
"Broido",
"Clauset",
"Singleton",
"Crawford",
"Tsui",
"Manchester",
"Maertens",
"Liu",
"Anastas",
"Kulikauskas",
"Tamir",
"Rizos",
"Long",
"Von Euw",
"Shaffer",
"Dunagin",
"Torborg",
"Torre",
"Emert",
"Krepler",
"Milanovic",
"Fan",
"Belenki",
"Däbritz",
"Zhao",
"Yu",
"Moon",
"Rajagopalan",
"Lail",
"Merat",
"Bugi-Marteyn",
"Wrobel",
"Py",
"Daali",
"Schwärzler",
"Figueroa",
"Cuadrado",
"Fan",
"Atasoy",
"Muscat",
"Muñoz-Canoves",
"Abdelmohsen",
"Lal",
"Kim",
"Gorospe",
"Kim",
"Gorospe",
"Kim",
"Kuwano",
"Srikantan",
"Lee",
"Martindale",
"Gorospe",
"Leandersson",
"Riesbeck",
"Andersson",
"Meng",
"King",
"Nabors",
"Jackson",
"Chen",
"Emanuel",
"Wang",
"Leite De Oliveira",
"Huijberts",
"Bosdriesz",
"Pencheva",
"Brunen",
"Kinney",
"Margolin",
"Nemenman",
"Basso",
"Wiggins",
"Stolovitzky",
"Favera",
"Holmes",
"Nemenman",
"Erdoğan",
"Kurt",
"Diri",
"Uda",
"Saito",
"Kudo",
"Kokaji",
"Tsuchiya",
"Kubota",
"Mc Mahon",
"Lenive",
"Filippi",
"Stumpf",
"Smith",
"Brunton",
"Rowling",
"Ferguson",
"Arozarena",
"Miskolczi",
"Konieczkowski",
"Johannessen",
"Abudayyeh",
"Kim",
"Cooper",
"Piris",
"Richard",
"Dalle",
"Monet",
"Ligier",
"Boespflug",
"Pommier",
"Grzywa",
"Paskal",
"Włodarski"
] | [
"A Convergence-Based Framework for Cancer Drug Resistance. D J Konieczkowski, C M Johannessen, L A Garraway, Cancer Cell. 33D. J. Konieczkowski, C.M. Johannessen, L.A. Garraway. A Convergence-Based Framework for Cancer Drug Resistance. Cancer Cell 33, 801-815 (2018).",
"Scale-free networks are rare. A D Broido, A Clauset, Nat. Commun. 101017A.D. Broido, A. Clauset. Scale-free networks are rare. Nat. Commun. 10, 1017 (2019).",
"Melanoma therapeutic strategies that select against resistance by exploiting MYC-driven evolutionary convergence. K R Singleton, L Crawford, E Tsui, H E Manchester, O Maertens, X Liu, Cell Rep. 21K. R. Singleton, L. Crawford, E. Tsui H.E. Manchester, O. Maertens, X. Liu, et al. Melanoma therapeutic strategies that select against resistance by exploiting MYC-driven evolutionary convergence. Cell Rep. 21, 2796-2812 (2017).",
"WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors. J N Anastas, R M Kulikauskas, T Tamir, H Rizos, G V Long, E M Von Euw, J. Clin. Invest. 124J.N. Anastas, R.M. Kulikauskas, T. Tamir, H. Rizos, G.V. Long, E.M. von Euw, et al. WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors. J. Clin. Invest. 124, 2877-2890 (2014).",
"Rare cell variability and druginduced reprogramming as a mode of cancer drug resistance. S M Shaffer, M C Dunagin, S R Torborg, E A Torre, B Emert, C Krepler, Nature. 546S.M. Shaffer, M.C. Dunagin, S.R. Torborg, E.A. Torre, B. Emert, C. Krepler, et al. Rare cell variability and drug- induced reprogramming as a mode of cancer drug resistance. Nature 546, 431-435 (2017).",
"Senescence-associated reprogramming promotes cancer stemness. M Milanovic, D N Y Fan, D Belenki, J H M Däbritz, Z Zhao, Y Yu, Nature. 553M. Milanovic, D.N.Y. Fan, D. Belenki, J.H.M. Däbritz, Z. Zhao, Y. Yu, et al. Senescence-associated reprogramming promotes cancer stemness. Nature 553, 96-100 (2018).",
"Estimation of mutual information using kernel density estimators. Y I Moon, B Rajagopalan, U Lail, Phys. Rev. E. 52Y.I. Moon, B. Rajagopalan, U. Lail. Estimation of mutual information using kernel density estimators. Phys. Rev. E 52, 2318-2321 (1995).",
"Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma. R Merat, A Bugi-Marteyn, L J Wrobel, C Py, Y Daali, C Schwärzler, R. Merat, A. Bugi-Marteyn, L.J. Wrobel, C. Py, Y. Daali, C. Schwärzler, et al. Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma.",
". Biochem. Biophys. Res. Commun. 517Biochem. Biophys. Res. Commun. 517, 181-187 (2019).",
"Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes. A Figueroa, A Cuadrado, J Fan, U Atasoy, G E Muscat, P Muñoz-Canoves, Mol. Cell. Biol. 23A. Figueroa, A. Cuadrado, J. Fan , U. Atasoy, G.E. Muscat, P. Muñoz-Canoves, et al. Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes. Mol. Cell. Biol. 23, 4991-5004 (2003).",
"Posttranscriptional orchestration of an anti-apoptotic program by HuR. K Abdelmohsen, A Lal, H H Kim, M Gorospe, Cell Cycle. 6K. Abdelmohsen, A. Lal, H.H. Kim, M . Gorospe. Posttranscriptional orchestration of an anti-apoptotic program by HuR. Cell Cycle 6, 1288-1292 (2007).",
"Phosphorylated HuR shuttles in cycles. H H Kim, M Gorospe, Cell Cycle. 7H.H. Kim, M. Gorospe. Phosphorylated HuR shuttles in cycles. Cell Cycle 7, 3124-3126 (2008).",
"HuR recruits let-7/RISC to repress c-Myc expression. H H Kim, Y Kuwano, S Srikantan, E K Lee, J L Martindale, M Gorospe, Genes Dev. 23H.H. Kim, Y. Kuwano, S. Srikantan, E.K. Lee, J.L. Martindale, M. Gorospe. HuR recruits let-7/RISC to repress c-Myc expression. Genes Dev. 23, 1743-1748 (2009).",
"Wnt-5a mRNA Translation is suppressed by the Elav-like protein HuR in human breast epithelial cells. K Leandersson, K Riesbeck, T Andersson, Nucleic Acids Res. 34K. Leandersson, K. Riesbeck, T. Andersson .Wnt-5a mRNA Translation is suppressed by the Elav-like protein HuR in human breast epithelial cells. Nucleic Acids Res. 34, 3988-3999 (2006).",
"The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation. Z Meng, P H King, L B Nabors, N L Jackson, C Y Chen, P D Emanuel, Nucleic Acids Res. 33Z. Meng, P.H. King, L.B. Nabors, N.L. Jackson, C.Y. Chen, P.D. Emanuel, et al. The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF- IR transcript and differentially represses cap-dependent and IRES-mediated translation. Nucleic Acids Res. 33, 2962-2979 (2005).",
"An acquired vulnerability of drug-resistant melanoma with therapeutic potential. L Wang, R Leite De Oliveira, S Huijberts, E Bosdriesz, N Pencheva, D Brunen, Cell. 173L. Wang, R. Leite de Oliveira, S. Huijberts, E. Bosdriesz, N. Pencheva, D. Brunen , et al. An acquired vulnerability of drug-resistant melanoma with therapeutic potential. Cell 173, 1413-1425 (2018).",
"Equitability, mutual information, And the maximal information coefficient. J B Kinney, G S , Proc. Natl. Acad. Sci. 111J.B. Kinney, G.S. Atwal. Equitability, mutual information, And the maximal information coefficient. Proc. Natl. Acad. Sci. 111, 3354-3359 (2014).",
"ARACNE: An Algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. A A Margolin, I Nemenman, K Basso, C Wiggins, G Stolovitzky, R Dalla Favera, BMC Bioinformatics. 717SupplA.A. Margolin, I. Nemenman, K. Basso, C. Wiggins, G. Stolovitzky, R. Dalla Favera, et al. ARACNE: An Algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. BMC Bioinformatics. 7 Suppl 1, S7 (2006).",
"Estimation of mutual information for real-valued data with error bars and controlled bias. C M Holmes, I Nemenman, Phys. Rev. E. 10022404C. M. Holmes, I. Nemenman. Estimation of mutual information for real-valued data with error bars and controlled bias Phys. Rev. E 100, 022404 (2019).",
"Estimation of the proteomic cancer co-expression sub networks by usin g association estimators. C Erdoğan, Z Kurt, B Diri, PLOS ONE. 12188016C. Erdoğan, Z. Kurt, B. Diri. Estimation of the proteomic cancer co-expression sub networks by usin g association estimators PLOS ONE 12, e0188016 (2017).",
"Robustness and compensation of information transmission of signaling pathways. S Uda, T H Saito, T Kudo, T Kokaji, T Tsuchiya, H Kubota, Science. 341S. Uda, T.H. Saito, T. Kudo, T. Kokaji, T. Tsuchiya, H. Kubota, et al. Robustness and compensation of information transmission of signaling pathways. Science 341, 558-561 (2013).",
"Information processing by simple molecular motifs and susceptibility to noise. S S Mc Mahon, O Lenive, S Filippi, M P Stumpf, J. R. Soc. Interface. 1220150597S.S. Mc Mahon., O. Lenive, S. Filippi, M.P. Stumpf. Information processing by simple molecular motifs and susceptibility to noise. J. R. Soc. Interface 12, 20150597 (2015).",
"Inhibiting drivers of nonmutational drug tolerance is a salvage strategy for targeted melanoma therapy. M P Smith, H Brunton, E J Rowling, J Ferguson, I Arozarena, Z Miskolczi, Cancer Cell. 29M.P. Smith, H. Brunton, E.J . Rowling, J. Ferguson, I. Arozarena, Z. Miskolczi, et al. Inhibiting drivers of non- mutational drug tolerance is a salvage strategy for targeted melanoma therapy. Cancer Cell 29, 270-284 (2016).",
"A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. D J Konieczkowski, C M Johannessen, O Abudayyeh, J W Kim, Z A Cooper, A Piris, Cancer Discov. 4D.J. Konieczkowski, C.M. Johannessen, O. Abudayyeh, J.W. Kim, Z.A. Cooper, A. Piris, et al. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov. 4, 816-827 (2014).",
"ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors. G Richard, S Dalle, M A Monet, M Ligier, A Boespflug, R M Pommier, EMBO Mol. Med. 8G. Richard, S. Dalle, M.A. Monet, M. Ligier, A. Boespflug, R.M. Pommier, et al. ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors. EMBO Mol. Med. 8, 1143-1161 (2016).",
"Intratumor and intertumor heterogeneity in melanoma. T M Grzywa, W Paskal, P K Włodarski, Transl. Oncol. 10T.M. Grzywa, W. Paskal, P.K. Włodarski. Intratumor and intertumor heterogeneity in melanoma. Transl. Oncol. 10, 956-975 (2017)."
] | [
"[1]",
"[2]",
"[3]",
"[4]",
"[5]",
"[6]",
"[7]",
"[7]",
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"[8]",
"[8]",
"[8]",
"[9]",
"[10]",
"(11)",
"[3,",
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] | [
"A Convergence-Based Framework for Cancer Drug Resistance",
"Scale-free networks are rare",
"Melanoma therapeutic strategies that select against resistance by exploiting MYC-driven evolutionary convergence",
"WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors",
"Rare cell variability and druginduced reprogramming as a mode of cancer drug resistance",
"Senescence-associated reprogramming promotes cancer stemness",
"Estimation of mutual information using kernel density estimators",
"Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes",
"Posttranscriptional orchestration of an anti-apoptotic program by HuR",
"Phosphorylated HuR shuttles in cycles",
"HuR recruits let-7/RISC to repress c-Myc expression",
"Wnt-5a mRNA Translation is suppressed by the Elav-like protein HuR in human breast epithelial cells",
"The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation",
"An acquired vulnerability of drug-resistant melanoma with therapeutic potential",
"Equitability, mutual information, And the maximal information coefficient",
"ARACNE: An Algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context",
"Estimation of mutual information for real-valued data with error bars and controlled bias",
"Estimation of the proteomic cancer co-expression sub networks by usin g association estimators",
"Robustness and compensation of information transmission of signaling pathways",
"Information processing by simple molecular motifs and susceptibility to noise",
"Inhibiting drivers of nonmutational drug tolerance is a salvage strategy for targeted melanoma therapy",
"A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors",
"ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors",
"Intratumor and intertumor heterogeneity in melanoma"
] | [
"Cancer Cell",
"Nat. Commun",
"Cell Rep",
"J. Clin. Invest",
"Nature",
"Nature",
"Phys. Rev. E",
"Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma",
"Biochem. Biophys. Res. Commun",
"Mol. Cell. Biol",
"Cell Cycle",
"Cell Cycle",
"Genes Dev",
"Nucleic Acids Res",
"Nucleic Acids Res",
"Cell",
"Proc. Natl. Acad. Sci",
"BMC Bioinformatics",
"Phys. Rev. E",
"PLOS ONE",
"Science",
"J. R. Soc. Interface",
"Cancer Cell",
"Cancer Discov",
"EMBO Mol. Med",
"Transl. Oncol"
] | [
"\nFIG. 1 .\n1Mice xenografts melanoma models of adaptive response to BRAF inhibition used in this study. (A) Model 1: SK-MEL28",
"\n\nFinal excision-biopsies were performed in both arms at the end of the experiment (TP2 and TP2Li 2 CO 3 ). For (A) and (C) data shown are mean tumor volume (indicated as fold change) ± SEM. FIG. 2. HuR/pERK and HuR/EGFR linear dependencies examined based on the strength of Pearson's correlation between the average expression value of markers in tumor biopsies performed, (A) in model 1 in SK-MEL28 shHuR (blue) and shCtrl (red) xenografts, (B) in model 2 in A375 cells xenografts before initiating any therapy (TP1j 0 , green) and at the end of the experiment in the vemurafenib + control chow arm (TP2, blue) or the vemurafenib + Li2CO3 chow arm (TP2 Li 2 CO 3 , red) (see",
"\nFIG. 4 .\n4Robustness of the MI estimates including in respect to the probability distribution size and the sample size biases. (A) Examples of HuR/pERK and HuR/EGFR MI estimates in the three types of biopsies obtained in model 2.",
"\nFIG. 5 .\n5HuR/pERK, HuR/EGFR and HuR/S100 dependencies examined based on MI estimate as inFig. 2. For model 1, additional estimates of MI were calculated for the 20 th percentile of the highest HuR-expressing cells (80-100) and for the 20 th percentile of the lowest HuR-expressing cells (0-20). In model 1, Wilcoxon rank sum tests were used to compare medians. In model 2, Kruskal-Wallis tests were used to compare the distributions (upper left p-value), when the null hypothesis was rejected post-hoc Tukey-Kramer tests were performed."
] | [
"Mice xenografts melanoma models of adaptive response to BRAF inhibition used in this study. (A) Model 1: SK-MEL28",
"Final excision-biopsies were performed in both arms at the end of the experiment (TP2 and TP2Li 2 CO 3 ). For (A) and (C) data shown are mean tumor volume (indicated as fold change) ± SEM. FIG. 2. HuR/pERK and HuR/EGFR linear dependencies examined based on the strength of Pearson's correlation between the average expression value of markers in tumor biopsies performed, (A) in model 1 in SK-MEL28 shHuR (blue) and shCtrl (red) xenografts, (B) in model 2 in A375 cells xenografts before initiating any therapy (TP1j 0 , green) and at the end of the experiment in the vemurafenib + control chow arm (TP2, blue) or the vemurafenib + Li2CO3 chow arm (TP2 Li 2 CO 3 , red) (see",
"Robustness of the MI estimates including in respect to the probability distribution size and the sample size biases. (A) Examples of HuR/pERK and HuR/EGFR MI estimates in the three types of biopsies obtained in model 2.",
"HuR/pERK, HuR/EGFR and HuR/S100 dependencies examined based on MI estimate as inFig. 2. For model 1, additional estimates of MI were calculated for the 20 th percentile of the highest HuR-expressing cells (80-100) and for the 20 th percentile of the lowest HuR-expressing cells (0-20). In model 1, Wilcoxon rank sum tests were used to compare medians. In model 2, Kruskal-Wallis tests were used to compare the distributions (upper left p-value), when the null hypothesis was rejected post-hoc Tukey-Kramer tests were performed."
] | [
"Fig. 1 legend)",
"(Fig. 1A)",
"(Fig. 1B)",
"(Fig. 1C)",
"(Fig. 1C)",
"(Fig. 2)",
"Fig. 2A)",
"Fig. 2",
"(Fig. 2B)",
"(Fig. 3)",
"(Fig. 3A)",
"(Fig. 3B)",
"(Fig. 4A",
"Fig. 1C and 4B",
"Fig. 4C and D)",
"(Fig. 5A)",
"(Fig. 5B"
] | [
"̂( ) = 1 ∑ ( ), =1(1)",
"= ( − ) −1 ( − ) ℎ 2 ,(2)",
"( ) = 1 (2 ) /2 ℎ det( ) 1/2 exp(− /2).(3)",
"ℎ = ( 4 ( + 2) ) 1 +4 .(4)",
"̂( , ) = 1 ∑ 2 , (̂( , ) ( ) ̂( ) ).(5)",
"( ; ) = ( ) + ( ) − ( , ),(6)"
] | [
"Based on a large amount of experimental research, our comprehension of the mechanisms of adaptive resistance to targeted therapies of solid mutated malignancies has significantly improved during the last decade [1]. Regardless of the current controversies on the scale-free properties of the cell/cancer signaling network [2], one of the most prevalent intuitions behind this experimental research has been to overcome the redundancy and robustness of such network by targeting its most essential connected nodes. Within the paradigmatic model of the adaptive response of BRAFmutated melanoma to BRAF inhibition on which we focus here, the convergence on MYC activation of various upstream signaling pathways and downstream targets [3] or the targetable WNT5A-induced activation of the AKT pathway associated with transcriptional reprograming [4], stand as good examples of \"hub-directed\" strategies in the recent literature. Currently, one of the major challenges of such mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existing dependencies between these hubs and their connected nodes. Similarly, the drug-targeted in vivo modulation of these dependencies needs to be demonstrated. These in vivo analyses are often not performed because of the lack of sensitivity to change of the available quantification techniques and the non-linear and time-shifted mutual dependencies of the involved mechanistic factors.",
"Such quantification becomes even more challenging if the proportion of cells in which such dependency operates is small within the tumor tissue, particularly at the initial timepoints of the adaptive response. This would be the case for rare, highly plastic cells that reprogram under therapeutic selection [5]. It would also occur in any embryonic signaling network that operates in the minority of senescent stem celllike cells that give rise to adaptive resistance [6]. Nevertheless, these quantifications are necessary for any further clinical implementation of these hub-directed strategies, not only to confirm their involvement in the observed clinical outcome but also as to obtain predictive markers of response to these strategies.",
"Practically, these analyses would need to be performed using techniques that can be routinely performed on patient biopsies as immunohistochemistry (IHC). Here, to implement such single-cell based in vivo expression dependency quantification, we use mutual information and compare it to more commonly used approaches in our own mechanistic strategy to reduce the adaptive response to BRAF inhibition in melanoma. In our model, the targeted hub HuR/ ELAVL1 (HuR) operates on the adaptive response in a minority of cells intermittently only if its expression becomes insufficient. Any dependency is therefore more likely time-shifted and transient. This model is therefore ideal to challenge the sensitivity of our approach.",
"All analyses, including tumor mean values Pearson's correlation, single-cell-based Spearman's correlation and mutual information estimation as well as the statistical tests, were conducted in MATLAB2020b (code for mutual information estimation and example dataset available upon request).",
"Mutual information (MI) is invariant to reparameterization, consequently, as a first step to decrease the impact of variability inherent to use of IHC, we use a copula-transform of values obtained for both markers (i.e., rank order them between 0 and 1) at the single-cell level. This initial step leads to a uniform distribution of their marginal distributions. We then estimate MI using a \"smoothing\" nonparametric Gaussian kernel density estimator (KDE), which is a local weighted average of the relative frequency of observations in the neighborhood of each estimate [7] given as",
"where is the two-dimensional signal-measured intensities for any single cell and is the number of samples (cells) and",
"where ℎ is the bandwidth of the kernel smoothing window and is the covariance matrix of . ( ), the kernel function, is given as",
"Since ( ) is a multivariate normal density function of dimension , ℎ is calculated using the \"optimal\" Silverman's bandwidth that minimizes the mean integrated square error (MISE),",
"Compared to classical histogram binning, this method is insensitive to the choice of origin, and most importantly provides a continuous better estimate of the underlying probability density, which avoids biases related to binning [7] or assumptions about the underlying distributions. The one-and two-dimensional estimates obtained with this pipeline are then used to calculate the mutual information between the two variables , (markers intensity) defined as",
"To ensure the robustness of the MI estimates in each tumor and for each pair of markers, we randomly select the subpopulation of cells used to estimate the MI (one hundred assays per estimate and surrogate). The fixed number of cells used in this subpopulation across all biopsies being compared is defined as the smallest number of cells detected within all biopsies.",
"Animal experiments were approved by the Animal Welfare Commission of the Canton of Geneva (approval n° GE/108/18) and followed the Swiss guidelines for animal experimentation. For model 1, two million shCtrl or shHuR SK-MEL28 cells (FACS-determined 70% apparent shift), generated as previously described [8], were resuspended in 100 µl of PBS and mixed with an equal volume of Matrigel (Corning ® Matrigel ® Matrix High Concentration, Phenol-Red free) and injected subcutaneously into the posterior left flanks of six-week-old female immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NSG, Charles River). Tumor formation was monitored twice a week using calliper measurements and calculated by the ellipsoidal formula: tumor volume ꞊ (length x width 2 ) x 0.5. The vemurafenib was prepared using 240 mg vemurafenib tablets (for human use) that were manually ground and suspended in a water containing solution of carboxymethylcellulose (CMC, 1%) and DMSO (5%). The final concentration of vemurafenib was 16.5 mg/mL as confirmed using an in-house liquid chromatography-electrospray ionization-tandem mass spectrometry method. Once the tumors reached a volume of approximately 0.2 cm 3 , all mice were treated by oral gavage once a day with 150 μL of this suspension (fixed vemurafenib dosage of 100 mg/kg/day). All mice also received a lithium carbonate (Li2CO3) containing chow dosed at 0.25% (2.5g/kg). However, considering that in the initially conducted fast growing A375 cells experiment (model 2) [8], tumor growth was affected in the Li2CO3 arm and that in this experiment tumor growth was initially extremely slow in both shCtrl and shHuR arms, the Li2CO3 therapy was initiated at the regrowth time-point. This was mandatory to obtain largeenough tumors that were, upon mice sacrifice, immediately collected and formalin-fixed and subsequently paraffinembedded (FFPE) (time-point 2, TP2, no time-point 1, TP1). The methodology for the A375 xenografts (model 2), comprising two groups receiving or not Li2CO3, has been previously reported [8]. In model 2, punch biopsies were collected under local anesthesia and similarly prepared (FFPE) in a separate-cohort of mice before treatment initiation (TP1J0) and 10 days later (average volume-doubling time) in a subgroup of this cohort that did not receive any therapy during that time (TP1J10). Samples were similarly prepared (FFPE) upon mice sacrifice at the end of the experiment (TP2).",
"Standard fluorescence-based immunohistochemical staining on deparaffinized tumor sections was performed as previously described [8]. In short, 5-µm thick sections were deparaffinized and cleared using UltraClear TM reagent and rehydrated in ethanol. Following antigen retrieval in citrate buffer, tissues were permeabilized with 0,1% tween 20 and blocked in 5% normal goat serum (NGS). The mouse monoclonal anti-human HuR antibody 3A2 (1:100) was used for co-staining (2h exposure at room temperature in PBS tween 0,1% NGS 5%) with one of the following rabbit primary antibodies: anti-S100 A1 antibody (SAB502708, Sigma 1:100), anti-EGFR antibody (D38B1, Cell signaling, 1:50), anti-phospho-p44/42 MAPK (pERK1/2) antibody (520G11, Cell signaling, 1:200). Following washing, Alexa 488-conjugated anti-mouse, or Alexa 555-conjugated antirabbit antibodies (1:500) were used as secondary antibodies (1 h exposure at room temperature). Following additional washing, slides were mounted with Dapi FluoromountG (Southern Biotech). Images were acquired using an automated Zeiss Axioscan.Z1 with a 20x 0.8NA Plan Apochromat objective (lateral resolution 0.325 µm/pixel). Nuclei were segmented using QuPath v0.2.3 based on the Dapi fluorescent channel. All areas of homogeneous S100 staining available within the tumor section were initially defined as region of interest (ROI) and subsequently manually transposed for all other stainings. Each individual nucleus area was expanded of 5 m to simulate a cytoplasmic area. Both areas were used to measure the nuclear and the wholecell mean signal fluorescence intensity in each cell. HuR being mainly a nuclear protein, its signal was measured in the nuclear area, whereas EGFR, pERK, and S100 signals were measured in the whole-cell surface area.",
"Human biopsies obtained from patients with metastatic melanoma disease were similarly analyzed. Their use was approved by the Geneva ethics committee (study n°2017-01346). According to the Swiss Federal Law for research, a positive vote of an ethical committee in a retrospective study is sufficient to use patient data and materials for research purposes without further need of individual informed consents. All patient-related data were identified as previously described [8] at the University Hospital of Geneva and selected on the availability of the samples.",
"The RNA-binding protein HuR has been extensively characterized as a ubiquitously expressed post-transcriptional orchestrator of differentiation [9], cell death [10] and an expression synchronizer of cell-cycle regulatory genes (11).",
"HuR has the ability to regulate many of the previously identified hubs within the adaptive network to BRAF inhibition and could potentially represent a \"super-hub\" within this network [3,4,[12][13][14]. We have recently shown that a heterogeneous and intermittently lower expression of HuR, within a subpopulation of BRAF-mutated melanoma cells (HuR Low state), is induced upon their exposure to a BRAF inhibitor (BRAFi). This heterogeneous state is increasingly detected during the adaptive response and is not dependent on the proliferation status of the cell population. The HuR Low heterogeneous state in turn induces a heterogeneous and adaptive plastic expression of resistance markers and an adaptive response of the whole cell population to chronic BRAF inhibition. Experimentally, in order to increase the adaptive response, the insufficient expression of HuR needs to be reversible between two attractor sets (reversible knockdown). Indeed, a stable knockdown of HuR has no effect on the adaptive response [8]. This observation indirectly indicates that although the heterogeneous HuR Low cells are a reservoir of adaptive plasticity, they need to switch to a HuR High state in order to proliferate. At the single-cell and at steady state, although the adapted highly proliferating cells are in a HuR High state and have the highest expression level of resistance markers, their emergence occurs within the cell subpopulation carrying the heterogeneous HuR Low cell component. From a therapeutic standpoint, a slight lithium salt-induced suppression of the heterogeneous HuR Low cell component attenuates the adaptive paradoxical expression and proliferative response to BRAF inhibition [8].",
"To develop in vivo predictive expression dependency markers of response to the resulting therapeutic strategy of combining lithium salts with small-molecule inhibitors in BRAF-mutated (i) shHuR (HuR lithium-non inducible) carrying an unstable proportion of HuR Low cells oscillating between two attractor sets [8] (blue) (n=7), (ii) shCtrl (HuR lithium-inducible) (red) (n=8). Mice were treated as in a single-arm phase 2 trial and received the BRAFi vemurafenib (100mg/kg/day) with subsequent addition of lithium carbonate (Li2CO3) containing chow dosed at 0.25% (2.5g/kg) at the indicated time-points. Considering that before exposure to the vemurafenib, HuR heterogeneous expression was already operating at a much higher extent in the shHuR panel than the one expected to be induced by the treatment, excision-biopsies were performed only at the end of the experiment (TP2, no TP1). (B) IHC-based HuR expression distribution in shHuR (blue) and shCtrl (red) xenografts shown as violin plots. (C) Model 2: A375 BRAF-mutated xenografts. Mice were treated as in a double-arm phase 3 trial and assigned to control (blue) or Li2CO3 chow (red) dosed as in (A) and subsequently all received the BRAFi vemurafenib dosed as in (A) at the indicated time-points. Biopsies were performed before initiating the Li2CO3 therapy (TP1j 0 , n=15) and following an average volume-doubling time period of 10 days in a subcohort of mice that did not receive any therapy during that time (TP1j 10 , n=7, purple). Fig. 1 legend). In each panel, the Pearson's correlation r and p-value for testing the hypothesis of no correlation is indicated.",
"metastatic BRAF-mutated melanoma, we conduct here a series of analyses on biopsies obtained from two mouse xenograft models established according to the design of a phase 2 (model 1) and phase 3 (model 2) clinical trials in which biopsy samples would be obtained for translational studies. We focus our IHC-based monitoring on the expression of HuR and its dependent resistance markers, EGFR and pERK. Ex vivo, the increased expression of EGFR or pERK under reversible knockdown of HuR or conversely, their decreased expression under suppression of the HuR Low cells by lithium salts, are clearly detected at the whole cell population level in synchronized cells adapted to, and treated with, a BRAF inhibitor [8]. However, at steady state and under physiologic variation of HuR expression, these changes are difficult to detect, considering that they occur at any moment in a subpopulation of adapting cells. Nevertheless, at the single-cell level, the dependencies between HuR and these resistance markers are expected to change in adapting cells and be detectable in vivo. Conversely, our results predict that these changes will be modulated in tumors concomitantly exposed to lithium salts.",
"In model 1 (Fig. 1A), designed as a single-arm phase 2 trial, all individuals received the BRAFi vemurafenib with subsequent addition of lithium carbonate (Li2CO3) to their regimen. The SK-MEL28 HuR reversible knockdown cells (shHuR) carrying an unstable proportion of HuR Low cells [8], were used to generate a panel of xenografts (n=7) in which the average expression of HuR is variable yet distinguishable from, and inferior to, the average expression of HuR observed in the control (shCtrl) panel (n=8) (Fig. 1B). Based on our previous ex vivo-made sensitive immunocytochemistry analyses, HuR expression is not inducible in these cells as opposed to their shCtrl counterparts in which a positive shift in HuR nuclear and cytoplasmic content is detected upon exposure to therapeutic concentrations of Li2CO3 [8]. This in vivo experimental model is therefore perfectly suited to measure to what extent the dependencies between HuR and both EGFR and pERK are detectable and distinguishable between non-responders' tumors carrying a more heterogeneous expression of HuR and the responders' carrying a more homogeneous expression of HuR. In this model, before treatment initiation, a HuR heterogeneous expression is already operating to a much higher extent in the shHuR panel than the one expected to be induced by the BRAFi; the comparative analyses were therefore performed only on biopsies obtained at the end of the experiment, from tumors exposed to sustained BRAF inhibition (time-point 2, TP2, no time-point 1, TP1).",
"In model 2 (Fig. 1C), designed as a double-arm phase 3 trial, individuals (n=16) were randomly assigned into two arms to receive or not Li2CO3 in addition to the vemurafenib treatment. The A375 BRAF-mutated melanoma cells were used to generate the xenografts. These cells have a high deterministic behavior for simulating tumor relapse following an initial response to BRAF inhibition and are therefore often used as a model of adaptive response to BRAF inhibition [15]. Our ex vivo observations indicate that HuR expression is more heterogeneous in A375 cells having a higher propensity for adaptive response than in cell lines having a lower propensity for adaptive response, e.g., the SK-MEL28 cells used in model 1. Moreover, this expression heterogeneity is highly increasing in A375 cells following exposure to a BRAFi [8]. Therefore, contrary to model 1, a change in the baseline expression dependencies between HuR and both EGFR and pERK is expected to occur in BRAFi-treated tumors. For this reason, the comparative analyses were performed on biopsies obtained both before treatment initiation (n=15, separate cohort, TP1j 0 , see materials & methods) and upon tumor regrowth (TP2). Moreover, the tumor volume is rapidly increasing in this model and might affect the expression dependencies of the markers. Consequently, additional biopsies were performed following an average volumedoubling time period of 10 days in a sub-cohort of individuals (n=7) that did not receive any treatment during that time (TP1j 10 ). The overall outcome of this experiment was previously reported and showed that following the initial response to vemurafenib, relative tumor regrowth was clearly attenuated in the Li2CO3 receiving arm (Fig. 1C) [8].",
"HuR/pERK and HuR/EGFR dependencies were first examined based on the strength of Pearson's correlation between the tumor average expression values of markers (Fig. 2). In model 1 ( Fig. 2A), an overall positive correlation for HuR/pERK and HuR/EGFR was apparent and consistent with FIG. 3. HuR/pERK, HuR/EGFR and HuR/S100 dependencies examined based on the Spearman's rank correlation value of expression of the markers at the single-cell level in tumor biopsies performed as in Fig. 2. For the two models, Fisher transformations were applied on each correlation. In model 1, Wilcoxon rank sum tests were used to compare medians. In model 2, Kruskal-Wallis tests were used to compare the distributions (upper left p-value), when the null hypothesis was rejected post-hoc Tukey-Kramer tests were performed.",
"our ex vivo single-cell observations, in which the adapted HuR High proliferating cells showed the highest expression level of resistance markers [8]. Nevertheless, for HuR/pERK, the strength of correlation was higher in the shHuR panel, and for HuR/EGFR, a positive correlation was only observed in the shHuR panel. In model 2 (Fig. 2B), HuR/EGFR already positively correlated at baseline in TP1j 0 biopsies and a HuR/pERK correlation was induced following exposure to BRAFi in TP2 tumors, compared with baseline. However, the suppression of these correlations/dependencies in TP2Li 2 CO 3 tumors was only apparent for HuR/EGFR. Overall, these results were consistent with the mechanistic model deduced from our ex vivo experimental results in which the dependency of resistance markers toward HuR increases when HuR expression is rendered insufficient: whether experimentally as in model 1 reversible knockdown, or therapeutically following exposure to a BRAFi, as for HuR/pERK in model 2. Yet, the Li2CO3-induced suppression of these dependencies in model 2 was only captured for HuR/EGFR. Moreover, the overall sensitivity of this commonly used approach was undoubtedly insufficient to predict the phenotypic outcome of individual tumors i.e., in terms of predicting in model 2 the therapeutic arm to which they belong.",
"Next, in order to improve our sensitivity to detect nonlinear dependencies, we calculated for each sample the Spearman's (rank) correlation for HuR/pERK and HuR/EGFR at the single-cell level (Fig. 3). Henceforward, the expression of the differentiation marker S100 was also included as a \"negative\" marker of dependency. Indeed, the dependency between HuR and S100 is expected to either be low in the undifferentiated cell lines used in both models or at least not to be positively selected in the adaptive response to BRAF inhibition. In model 1 (Fig. 3A), strikingly, no significant differences in the values were observed between the shHuR and the control panels. In model 2 (Fig. 3B), consistent with the average expression correlations analyses, the coefficient was significantly higher for HuR/pERK upon exposure to the BRAFi in TP2 tumors compared with the baseline TP1j 0 biopsies. Compared with TP2 tumors, here, a significant reduction of was observed in TP2Li 2 CO 3 tumors for both HuR/pERK and HuR/EGFR but was more apparent for the latter. For HuR/S100, as expected no significant differences were observed for values among the samples. These analyses were therefore confirmatory and more sensitive for detecting the Li2CO3-induced suppression of both HuR/pERK and HuR/EGFR dependencies in model 2. However again, they were insufficient in predicting the phenotypic outcome of individual tumors and even in detecting the differences in dependencies in model 1. In this model, the lack of sensitivity was presumably due to an averaging effect between HuR Low and HuR High cells, both types being, according to our ex vivo experimental results, timely involved in these dependencies.",
"MI is a universal metric for quantifying any type rather than just linear dependencies between two variables. Information theory and entropy-based description of MI provide the formalism to define MI between two variables and as",
"where is the entropy associated with each of the variables, i.e. the amount of information gained (or reduced uncertainty) on each of them when measuring them separately, and ( , ) is the joint entropy symmetrically associated with both variables, i.e. the amount of information gained on one of the variables when measuring the other variable. Most importantly, MI is scale-free and its quantification using rank-ordered data (see materials and methods) allows to To calculate surrogates, one hundred assays were conducted by randomly shuffling the intensity of the second marker intensity in the subpopulation of cells used for MI estimate (grey histogram). (B) Tumor growth effect on HuR/pERK and HuR/EGFR MI estimates. Biopsies from baseline (TP1j 0 , green) are compared with identical tumors following a volume-doubling time of 10 days in a subcohort of mice that did not receive any therapy during that time (TP1j 10 , purple). Testing the hypothesis of equal MI under tumor growth was done by using Wilcoxon rank sum tests. (C, D) Sample size effect on HuR/pERK and HuR/EGFR MI estimates in human metastatic melanoma. (C) Example of HuR/pERK co-staining performed on a small intestine metastatic melanoma disease. Note that the region of interest (ROI) was chosen as to be similar to the additional tumor sections that were used for other stainings. (D) The fixed number of cells (percentage of the smallest sample) being used for HuR/pERK and HuR/EGFR MI estimates is changed across eight biopsies of metastatic disease. Biopsies were performed before small-molecule inhibitor therapies were initiated but only half of the patients had a complete response (cream, red and brown color range, n=4), the remaining patients had either partial response or progressive disease (blue color range, n=4) on the biopsied tumor two to three months after treatment initiation (radiologically-assessed response according to RECIST1.1). overcome some of the limitations of semi-quantitative techniques as IHC [16,17]. These include variability in sample preparation and imaging. However, as extensively discussed in the physics literature, MI is highly dependent on the size of the probability distribution and is sample-sizedependent [18]. This is even true for discrete data for which, in order to get a reliable estimate of MI, the number of samples needs to be significantly larger than the cardinality of the underlying distribution. To overcome these limitations, we used a computationally efficient Gaussian kernel estimator (KDE see materials and methods) and applied it throughout this study on a fixed smallest number of cells detected within the samples being compared. With this fixed number, cells were randomly chosen in the one hundred assays which were performed to ensure the stability of each MI estimate.",
"We first demonstrated that in each tumor sample and for each pair of markers, the MI estimates were distinguishable from a \"null\" MI obtained by shuffling one of the two signals across cells (Fig. 4A). An above null MI was quantifiable even for samples having a very low HuR/pERK or HuR/EGFR MI estimate e.g., the TP2Li 2 CO 3 tumors in model 2. Although using a KDE, we then asked if the size of the probability distribution had an impact on the MI estimates. We considered that a natural increase in the probability distribution might occur during tumor growth even if the fixed-size sample is randomly chosen in the tumor section, and estimated the HuR/pERK and HuR/EGFR MI of biopsies obtained in a subcohort of mice that did not receive any treatment during an average volume-doubling time period of 10 days (TP1j 10 ). These MI estimates were not significantly different from the one obtained in biopsies performed on smaller TP1j 0 identical tumors ( Fig. 1C and 4B). Next, we checked if the sample size would affect the MI estimate by varying the number of cells analyzed within tumors. Considering that the xenografts in our mouse models were likely homogeneous in their cell composition, these analyses were carried on biopsies (n=8) obtained from patients with BRAF-mutated metastatic melanoma disease before targeted therapy was initiated. Once treated, these patients had various types of response in their biopsied tumors which were therefore more likely heterogeneous in their cell composition. Yet the change in sample size across all metastatic tumors had no effect in their MI estimates and ranking ( Fig. 4C and D). Overall, we concluded that our copula-transformed/KDEbased estimate of MI was robust enough to overcome both the probability distribution size and the sample size biases.",
"In model 1, MI estimates for both HuR/pERK and HuR/EGFR were significantly higher in the shHuR tumors than in the control panel (Fig. 5A). In contrast, HuR/S100 MI, used here as a control, was slightly but significantly higher in the control panel. Remarkably, HuR/EGFR MI estimates were almost sufficient to individually distinguish the shHuR from the shCtrl tumors. In addition, contrary to the HuR/pERK MI estimates for the 20 th percentile of the highest HuR-expressing cells, these estimates for the 20 th lowest percentile were almost as high as the ones obtained for the total cell population in most shHuR tumors, suggesting that most of the HuR/pERK dependency was operating in the HuR Low cells in these tumors. Overall, the increased HuR/pERK and HuR/EGFR MI-based dependencies observed in shHuR tumors were consistent with the average expression Pearson's coefficient-based analyses discussed in section 3.1, however, MI performed better, particularly in capturing HuR/EGFR dependency in individual tumors. ..nIn model 2 (Fig. 5B), in accordance with 3.1 results, HuR/EGFR MI-determined dependency was already detected at baseline in TP1j 0 biopsies and became significant compared to baseline for HuR/pERK in adapted TP2 tumors. As expected, HuR/S100 MI estimate was low and not affected in BRAFi-treated tumors. Importantly, the Li2CO3 suppression of HuR/EGFR and BRAFi-induced HuR/pERK dependencies, under physiologic expression levels of HuR, was clearly captured here. With the exception of a few mice, MI performed well to distinguish the mice co-treated with Li2CO3 from the ones treated only with the BRAFi.",
"Taken together, these results support the use of MI, rather than the classical metrics used in sections 3.1 and 3.2, for quantifying HuR/pERK and HuR/EGFR dependencies in our therapeutic strategy. The increase in these dependencies under an experimentally induced insufficient expression of HuR occurred similarly, at least for one pair of markers, under BRAFi therapy and were reduced under concomitant Li2CO3 therapy. More importantly, the predictive value of MI at the individual level was far more effective. Ultimately, the validation of MI as a biologic, predictive IHC-based metric of response to combining small-molecule inhibitors with Li2CO3 in BRAF-mutated metastatic melanoma will be carried in trials equivalent to either one of the models developed here.",
"Although extensively used in the literature to elucidate gene regulatory networks [17,19] and quantify cellular signal processing [20,21], MI-based quantification of expression dependencies has not been used, to our knowledge, as an in vivo confirmatory approach for mechanistic studies, nor as a quantitative metric for clinical use. As shown in this work, MI is far more sensitive to change and performs better to predict drug effect than average co-expression or even single-cell-based co-expression correlation coefficients. MI should be particularly used when suspecting complex patterns of dependencies and when dealing with dynamic, reversible dependencies as observed during the adaptive response to targeted therapies in solid malignancies. This approach may help clarify contradictory observations made in this field. As an example, the melanocytic lineage transcription factor MITF has been reported to be either upregulated [22] or downregulated [23,24] in association with an increased level of pERK in melanoma cells adapted to and treated with a MAPK inhibitor. These contradictory observations could either be attributed to the use of different experimental settings including cell types or be related to partial independencies between these markers (in which case the presumed mechanistic effect is at least partially refuted). However, they could as well be related to a heterogeneous, complex, pattern of dependency [25] that could be captured by MI as the one described in this study between HuR and EGFR or pERK.",
"Generally, the in vivo dynamic change in MI between the main regulatory hub and its targets should be first assessed in an experimentally-induced, presumed pathologic expression condition of the targeted hub and subsequently, under its physiologic expression where the incriminated pathologic change of MI is expected to be therapeutically reversed or at least modulated according to the observed phenotypic outcome. Importantly, the integration of the methodology developed here into the translational analytic pipeline used to develop predictive markers of response to therapy in clinical trials is highly feasible. Its potential future use in routine pathology as an automated procedure is therefore predicted."
] | [] | [
"I. INTRODUCTION",
"II. MATERIALS AND METHODS",
"A. Descriptive statistics and mutual information estimation",
"B. Mouse xenografts",
"C. Immunohistochemistry (IHC)-based single-cell automated quantification",
"III. RESULTS",
"A. Average expression correlations",
"B. Single-cell Spearman's correlations",
"C. Mutual information",
"IV. DISCUSSION",
"FIG. 1 .",
"FIG. 4 .",
"FIG. 5 ."
] | [] | [] | [
"A mutual information-based in vivo monitoring of adaptive response to targeted therapies in melanoma",
"A mutual information-based in vivo monitoring of adaptive response to targeted therapies in melanoma"
] | [] |
2,148,877 | 2022-03-30T07:47:11Z | CCBY | https://doi.org/10.1371/journal.pone.0103837 | GOLD | c73c2f285483a537dafb6590f7fe7eced44f7074 | null | null | null | null | 10.1371/journal.pone.0103837 | 1970263046 | 25153150 | 4143169 |
Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line
2014
J T Kwon
Jin S Choi
H Kim
J Jeong
J
Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line
PLoS ONE
981038372014Received May 4, 2014; Accepted July 2, 2014;Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
The F9 cell line, which was derived from a mouse testicular teratoma that originated from pluripotent germ cells, has been used as a model for differentiation. However, it is largely unknown whether F9 cells possess the characteristics of male germ cells. In the present study, we investigated spermatogenic stage-and cell type-specific gene expression in F9 cells. Analysis of previous microarray data showed that a large number of stage-regulated germ cell genes are expressed in F9 cells. Specifically, genes that are prominently expressed in spermatogonia and have transcriptional regulatory functions appear to be enriched in F9 cells. Our in silico and in vitro analyses identified several germ cell-specific or -predominant genes that are expressed in F9 cells. Among them, strong promoter activities were observed in the regions upstream of the spermatogonial genes, Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Tex13 (testis expressed gene 13), in F9 cells. A detailed analysis of the Tex13 promoter allowed us to identify an enhancer and a region that is implicated in germ cell-specificity. We also found that Tex13 expression is regulated by DNA methylation. Finally, analysis of GFP (green fluorescent protein) TEX13 localization revealed that the protein distributes heterogeneously in the cytoplasm and nucleus, suggesting that TEX13 shuttles between these two compartments. Taken together, our results demonstrate that F9 cells express numerous spermatogonial genes and could be used for transcriptional studies focusing on such genes. As an example of this, we use F9 cells to provide comprehensive expressional information about Tex13, and report that this gene appears to encode a germ cell-specific protein that functions in the nucleus during early spermatogenesis.
Introduction
Male germ cell development, or spermatogenesis, is a complex process that involves successive mitotic, meiotic, and post-meiotic phases [1,2]. The tightly regulated nature of this process, which occurs in the seminiferous tubules of testes, indicates that a highly organized network of genes is expressed in germ cells during spermatogenesis. Three levels of control regulate gene expression during spermatogenesis: intrinsic, interactive and extrinsic control [3]. The intrinsic program determines which genes are utilized and when these genes are expressed. The unique feature of this program is germ cell-and stage-specific gene expression. The interactive process, which involves crosstalk between germ cells and somatic cells, is essential for germ cell proliferation and progression. Finally, extrinsic influences, including steroid and peptide hormones, regulate the interactive process. Not all extrinsic regulation act through Sertoli cells. Retinoic acid (RA), the active derivative of vitamin A, is essential for spermatogenesis and has direct effects on germ cell development [4]. Pluripotent/primordial germ cells (PGCs), which colonize the developing gonads by active migration [5,6], turn into gonocytes. The latter differentiate into spermatogonial stem cells (SSCs), which provide a continuous supply of differentiating spermatogonia during spermatogenesis [7]. Signaling and transcriptional regulation are crucial for germ cell pluripotency, survival and differentiation. Misregulation of germ cell pluripotency may lead to tumor formation [8]. Testicular teratomas originate from primordial germ cells [9]. Although teratomas are tumor, they possess the capacity to produce all three germ cell layers. Therefore, testicular teratomas provide a useful tool for investigating the intersection of pluripotency, differentiation and tumorigenesis.
The F9 cell line was isolated as a subline of a testicular teratoma (designated OTT6050) that was established by implanting a sixday-old embryo in the testis of a 129/J mouse [10]. The F9 cell line is a nullipotent cell line that is unable to undergo spontaneous differentiation. However, F9 cells can differentiate into endodermal-like derivatives following treatment with several agents, including retinoic acid. Therefore, the F9 cell line has been used as a model for analyzing the molecular mechanisms of differentiation [11].
Because F9 cells originate from embryonic cells containing primordial germ cells, it is possible that the intrinsic genetic program involving spermatogenic stage-or cell-specific genes is partially active in F9 cells. If this is the case, the F9 cell line could be used as an alternative cell line for investigating germ cell transcription. In fact, the testis-specific promoter region of the human pituitary adenylate cyclase-activating polypeptide gene (PACAP) was previously investigated using F9 cells [12]. To address this issue, we herein used microarray data to analyze the transcripts expressed in F9 cells. We found for the first time that a large number of stage-specific germ cell genes are expressed in F9 cells. Various expressional analyses and reporter assays showed that F9 cells can be used for transcriptional studies of genes that are stage-specifically expressed in early male germ cells. In particular, genes encoding doublesex and mab-3 related transcription factor 1 (Dmrt1), stimulated by retinoic acid gene 8 (Stra8) and testis expressed gene 13 (Tex13) were significantly expressed in F9 cells. Since the functions and transcriptional mechanisms were previously reported for Dmrt1 and Stra8 [35][36][37][38][39][40], we focused on the expression of Tex13 in F9 cells. Our comprehensive analysis of the Tex13 promoter allowed us to identify regions responsible for the germ cell specificity and strong enhancer activity of this promoter. Moreover, Tex13 promoter showed cell-type specific DNA methylation. In addition, we found that Tex13 encodes a potential nucleocytoplasmic shuttling protein. Our study is the first comprehensive and systematic investigation of germ cell genes expressed in F9 cells.
Materials and Methods
Microarray data analysis
We obtained microarray data representing spermatogenic cells, F9 cells and J1 embryonic stem cells from the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/gds/). The GSE4193 dataset contained expression profiles obtained from a purified population of spermatogenic cells [13]; the GSE31280 dataset contained the gene expression profile of F9 cells [14]; and the GSE9978 dataset contained array data obtained from J1 embryonic stem cells [15]. Feature-level data (CEL) files were downloaded and imported into R program for normalization. R is an open source statistical scripting language (http://www. r-project.org). All expressional data were normalized using the GCRMA method [16]. Expressional data obtained from spermatogenic cells (spermatogonia, spermatocytes and spermatids), F9 cells and J1 cells were combined into a microarray dataset. The combined array data were normalized by quantile normalization using the ''normalize.quantiles'' function from R/Bioconductor package. The averages between duplicates derived for each sample were calculated. For each experimental group (Spermatogonia-F9, Spermatocyte-F9 and Spermatid-F9), genes with absolute fold Figure 1. Microarray analysis of genes expressed stage-specifically in male germ cells and F9 cells. A. Heatmaps of the normalized gene expression profiles for each group. A total of 964 genes were expressed more than 1.5-fold higher in both a given germ cell stage and F9 cells compared to other germ cell stages and ESC. Clustering of each group was performed using hierarchical clustering. Up-regulated genes are indicated in red and down-regulated genes are shown in green. Abbreviations: J1, J1 embryonic stem cells; Spg, type B spermatogonia; Spcy, pachytene spermatocyte; Sptd, round spermatid; and F9, F9 cells. B. Mean expression level of all genes in each group. Differences between samples were validated using the Student's t-test. For simplification, the average expression levels between two germ cells with low expression levels were used. Significant differences were observed in the Spg-F9, Spcy-F9 and Sptd-F9 groups (p-value,10E-10). doi:10.1371/journal.pone.0103837.g001 Figure 2. Enriched gene ontology (GO) terms in the spermatogonia-F9 group. A total of 487 genes in the Spg-F9 group were analyzed for the enrichment of GO terms using the DAVID functional annotation chart. Only significant GO terms are shown (p-value,0.05). The pie slices are proportional to the number of genes. doi:10.1371/journal.pone.0103837.g002 changes greater than 1.5 were chosen as differentially expressed genes (DEGs) and subsequently analyzed using the DAVID Functional Annotation Tool for gene ontology (GO) (http://david. abcc.ncifcrf.gov/) [17]. A functional annotation chart is useful for identifying annotation terms that are enriched in the submitted gene list; a smaller p-value indicates increasing significance of the GO term, and fold enrichments of 1.5 and above are considered interesting.
Reverse transcription PCR
To validate our analysis of the available microarray results, we performed reverse-transcription PCR (RT-PCR) using total RNA from testis, F9 cells and NIH3T3 cells. Total RNA was extracted using the TRIzol reagent (Molecular Research Center) according to the manufacturer's protocol, and cDNA was synthesized by random hexamer and oligo(dT) primers using the Omniscript reverse transcriptase (Qiagen). The utilized gene-specific primers are listed in Table S1. PCR was performed for 30 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 1 min 20 s. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was amplified as a control (forward, 59-TGA AGG TCG GAG TCA ACG GAT TTG GT-39 and reverse, 59-CAT GTG GGC CAT GAG GTC CAC CAC-39). The testis-specific expression of the nine tested genes was examined by RT-PCR in eight different mouse tissues (testis, ovary, brain, heart, kidney, lung, liver, and spleen). Specific expression at different stages of spermatogenesis was established using total RNA obtained from testes of prepubertal and adult male mice (ages 8, 10,12,14,16,20,30, and 56 days), and from the testes of W/W v mutant mice, which lack germ cells. All animal investigations were carried out according to the guidelines of the Animal Care and Use of Gwangju Institute of Science and Technology. The protocol was approved by the Animal Care and Use Committee of Gwangju Institute of Science and Technology (Permit number: GIST 2011-13). Sine oculis-related homeobox 5 [22] 26423 Nr5a1
Nuclear receptor subfamily 5, group A, member 1 [23] 14605 Tsc22d3 TSC22 domain family, member 3 [24] 56484 Foxo3 Forkhead box O3 [25] 16478 Jund Jun D proto-oncogene [26] 18024
Nfe2l2
Nuclear factor, erythroid derived 2, like 2 [27] 12578
Cdkn2a
Cyclin-dependent kinase inhibitor 2A [28] 14463
Gata4 GATA binding protein 4 [29] 18128 Notch1 Notch 1 [30] 50796
Dmrt1
Doublesex and mab-3 related transcription factor 1 [31] 20893 Cell culture F9 (CRL-1720) and NIH3T3 cells (CRL-1658) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (F9) or 10% bovine caput serum (NIH3T3), along with 2 mM glutamine, 100 U/ml penicillin and 10 mg/ml streptomycin. The culture vessels for F9 cells were coated with 0.1% gelatin prior to use. Since RA is known to induce the differentiation of F9 cells, the chemical was not included in the media during cultivation. The cells were maintained in a humidified 5% CO 2 atmosphere at 37uC, the cell media were changed every 1-2 days, and the cells were subjected to passage every 3-4 days.
Generation of reporter gene constructs
A luciferase reporter assay system (Promega) was used to measure promoter activity. To construct the 1.5-kb promoter luciferase reporter plasmid for five genes (Dmrt1, Stra8, Tex13, Triml1 and 1700061G19Rik), DNA fragments corresponding to the putative promoters predicted by DBTSS (http://dbtss.hgc.jp./) were prepared by PCR using the pfu DNA polymerase (Enzynomics) with mouse genomic DNA isolated using Dneasy Blood & Tissue kit (Qiagen). The utilized primers are listed in Table S1. Several deleted versions of the Tex13-luciferase reporter plasmids were also prepared. The sequence of each PCR product was confirmed by DNA sequencing (Macrogen), and each fragment of interest was cloned into the multi-cloning site of the promoter-less pGL3-Basic (Promega) plasmid.
DNA transfection and luciferase assay
Cells were plated to 24-well plates at 1.5610 5 cells/plate. After 24 h, when cells were at 50-60% confluence, the Lipofectamine LTX transfection reagent (Invitrogen) was used to transfect 400 ng of the indicated DNA construct together with 8 ng of pRL-TK (Promega), which contains the Renilla luciferase gene driven by the SV40 promoter, and was used an internal control for normalization of transfection efficiency. Twenty-four hours later, whole cell extracts from triplicate wells were assayed. Lysates were prepared using 100 ml of passive lysis buffer (Promega), and luciferase activity was determined from 30 ml of each lysate using the Luciferase Reporter Assay (Promega) and a Centro LB 960 DLReady Micro Plate Illuminometer (Berthold Technologies). Each experiment was repeated at least three times. The obtained promoter activity was normalized against that of the SV40 promoter, and was reported as the fold activity compared to that from pGL3-Basic.
Purification of genomic DNA and bisulfite methylation assay
Genomic DNA (gDNA) was purified from F9 cells and NIH3T3 cells using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol. Promoter methylation was assessed by bisulfite sequencing. In brief, cytosine-to-uracil conversion was performed on 1.0 mg of gDNA using an EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturer's provided protocol. PCR reactions were performed on the converted DNA with promoter specific-primers designed using the online MethPrimer software (http://www.urogene.org/ methprimer/). PCR was performed for 48 cycles of 95uC for 30 s, 48uC for 30 s, and 72uC for 30 s. For bisulfite sequencing, the amplified PCR products were cloned into the pTOP v2 vector (Macrogen). From each cell line (F9 and NIH3T3) a total of 10 independent clones containing each of the desired PCR products were sequenced.
In vitro methylation
The 2402/+20 Tex13 promoter was inserted into pGL3-Basic, and the vector was incubated at 37uC for 4 h with the methyltransferase enzyme, M.SssI (NEB) in the presence of the methyl group donor S-adenosylmethionine (SAM) (NEB). Mockmethylated plasmids were incubated without enzyme but in the presence of SAM. Samples were purified using a PCR purification kit (LaboPass). The mock-methylated and methylated plasmids were subjected to diagnostic digests with HpaII, a methylationsensitive enzyme, to confirm the efficacy of the in vitro methylation.
Localization of recombinant TEX13 in F9 cells
The coding region of mouse Tex13 (NM_031381 in GenBank) was amplified by RT-PCR and subcloned into the N terminus of pEGFP-N2 (Clontech) using EcoRI and BamHI, to generate plasmids expressing the fusion protein GFP-TEX13. Transient transfection of clones was achieved using Lipofectamine LTX transfection reagents and Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 24 h of culture on coverslips, cells were fixed with 4% (v/v) paraformaldehyde and nuclei were labeled with Hoechst 33258 dye (Sigma). Samples were mounted on slides and visualized by microscopes (DMLB; Leica Microsystems and IX81; Olympus). Approximately 100
Results
Identification of germ cell genes expressed in F9 cells
To determine whether F9 cells transcribe genes that undergo stage-specific expression in male germ cells, we analyzed previously deposited microarray gene expression datasets representing type B spermatogonia (Spg), pachytene spermatocyte (Spcy), round spermatid (Sptd), F9 testicular teratoma cells (F9) and J1 embryonic stem cells (ESC). To avoid systematic variations in the analysis, we selected microarray datasets that were all produced using the Affymetrix Mouse Genome 430 2.0 array. Background adjustment of each array dataset was performed using GCRMA (GC Robust Multi-array Average) [16]. Quantile normalization was used to adjust the array data. Since F9 cells were isolated from a teratoma established by implanting an embryo with stem cell properties (pulipotency) in the testis, we used the ESC dataset as a negative control to reduce any influence from the stem cell properties of F9 cells. We selected genes that were up-regulated ($1.5-fold change) in one of the germ cell stages and F9 cells relative to the other germ cell stages and ESC. We found that 487 genes were highly transcribed in spermatogonia and F9 cells (Spg-F9) ( Figure 1A and Table S2), while 308 and 169 genes were up-regulated in spermatocytes and F9 cells (Spcy-F9) ( Table S3) and spermatids and F9 cells (Sptd-F9), respectively (Table S4). Statistical analysis of each group indicated that the expression levels of the selected genes in the appropriate germ cell stage and F9 cells were significantly higher (p,10E-10), than in the other germ cell stages and ESC ( Figure 1B). It should be noted that the Spg-F9 pattern had the largest number of genes among the three groups. Collectively, our in silico results demonstrated that numerous stage-specific germ cell genes (a total of 964 genes) are expressed in F9 cells.
To further investigate the germ cell genes expressed in F9 cells, we performed gene ontology (GO) enrichment analysis using the DAVID functional annotation tool [17]. In this analysis, the enrichment score reflected the degree to which a GO term is overrepresented in the Spg-F9, Spcy-F9 or Sptd-F9 genes, compared to all genome-wide genes. Our results revealed that a number of GO terms were enriched in the Spg-F9 group ( Figure 2), but not in the Spcy-F9 or Sptd-F9 groups. Using the criteria of a fold-enrichment score $1.5 and p,0.05, we found that the Spg-F9-enriched GO terms included transcription regulatory activity, developmental processes, death, biological adhesion, reproductive processes, and reproduction (Table 1). In particular, 57 genes encoding transcription factors were enriched under the GO term transcription regulatory activity; of them, 11 are known to function in germ cell proliferation, germ cell differentiation, and spermatogenesis (Table 2).
Genes known as markers of spermatogonia, including Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Gfra1 (glial cell line derived neurotrophic factor family receptor alpha 1) [18], were found to be expressed in F9 cells ( Figure S1). In addition, various genes expressed in primordial germ cells, including Prdm1 (PR domain containing 1, with ZNF domain) [19], Prdm14 (PR domain containing 14) [20], and Pou5f1 (POU domain, class 5, transcription factor 1) [21], are also predicted to be expressed in F9 cells ( Figure S1).
Identification of testis-specific or -predominant genes expressed in F9 cells
To identify testis-specific or -predominant (collectively called ''testis-preferred'') genes among the germ cell genes found to be expressed in F9 cells, we calculated testis specificity using information from the UniGene database [33], which contains the EST (Expressed sequence tag) expression profile of a given gene in 47 tissues or organs based on transcription per million (TPM, indicating the normalized gene expression level). We identified nine genes as being putatively testis-specific or -predominant, using the criteria of testis specificity $50% or (for genes expressed in fewer than three tissues) 50%. testis specificity $30% (Table 3). The Spg-F9 group contained four testis-specific or -predominant genes: doublesex and mab-3 related transcription factor 1 (Dmrt1); testis expressed gene 13 (Tex13); nuclear receptor subfamily 5, group A, member 1 (Nr5a1); and stimulated by retinoic acid gene 8 (Stra8). The Spcy-F9 group contained a single such gene: Sp2 transcription factor (Sp2). The Sptd-F9 group contained four genes that were predicted to be specifically or predominantly expressed in testis: carboxypeptidase, vitellogeniclike (Cpvl); Fancd2 opposite strand (Fancd2os); tripartite motif family-like 1 (Triml1); and 1700061G19Rik. Interestingly, Dmrt1 and Stra8 are known to be involved in the initiation of meiosis [31], while Nr5a1 is an important gene for reproductive differentiation [23]. The functions of the other listed genes are unknown.
To confirm that these nine genes are expressed in F9 cells and show testis-specific or -predominant expression patterns, we performed in vitro expression analyses. RT-PCR analysis showed that, with the exception of Cpvl, all of the tested genes were transcribed both in testis and F9 cells ( Figure 3A). NIH3T3 cells were used as a negative control for cell type-specific expression. The expression levels of Tex13, Dmrt1, and Stra8 were similar between testis and F9 cells, whereas the other detected genes showed weaker expression in F9 cells than in testis. Next, we examined the tissue distribution of the eight genes confirmed to be expressed in F9 cells ( Figure 3B). With the exception of Nr5a1 and Sp2, all of the genes were expressed specifically or predominantly in testis, which was consistent with our in silico prediction. To investigate the developmental expression patterns of the testisspecific or -predominant genes, we performed RT-PCR using RNA from mouse testis samples obtained at different days after birth ( Figure 3C). We hypothesized that if a given gene is transcribed in germ cells during spermatogenesis, the transcript will appear in the testis at a particular post-partum time point corresponding to a specific stage of spermatogenesis. The expression of all the four genes in the Spg-F9 group started at phosphate dehydrogenase (Gapdh) was included as a loading control. Except for Cpvl, all of the genes were detected in both testis and F9 cells. It should be noted that the two bands for Dmrt1 in NIH3T3 cells are non-specific, based on in silico investigation and an additional PCR analysis (data not shown). B. The tissue distributions of transcripts were assessed by RT-PCR analysis in various tissues of adult male mice. Complementary DNAs from various mouse tissues were amplified by PCR, with Gapdh included as a loading control. Seven genes were found to be testis-specific orpredominant. C. Developmental expression patterns during spermatogenesis. The stage-specific expression of the genes was determined from mouse testes on different days after birth (days 8, 10, 12, 14, 16, 20, 30, and 56). Abbreviations: PL, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; MI, meiotic division I; and MII, meiotic division II. Complementary DNA from germ cell-lacking testes from W/W v mutant mice was also examined. Consistent with our microarray analysis, Tex13, Stra8, Fancd2os, 1700061G19Rik and Triml1 were found to be germ cell-specific. doi:10.1371/journal.pone.0103837.g003 postnatal day 8, corresponding to spermatogonia. In contrast, Fancd2os, 1700061G19Rik and Triml1 from the Sptd-F9 group were first expressed at postnatal days 20 or 30, corresponding to the late spermatocyte and round spermatid stages, respectively. Finally, we examined the germ cell-specific expression of the seven testis-specific or -predominant genes using the germ cell-lacking testes of W/W v (c-kit) mutant mice [34]. The transcripts corresponding to Tex13, Stra8, Fancd2os, 1700061G19Rik and Triml1 were barely detected in the testes of mutant mice ( Figure 3C), suggesting that these genes underwent germ cell-specific expression. Collectively, the results of our in vitro analyses of the in silico-selected genes demonstrated that six genes (Tex13, Dmrt1, Stra8, Fancd2os, 1700061G19Rik and Triml1) show testis-specific or-predominant expression in F9 cells. In particular, Tex13 and Stra8 show germ cell-preferred transcription with abundant expression in F9 cells.
Promoter analysis of germ cell genes in F9 cells
To investigate the expression mechanisms governing the F9 cell-expressed germ cell genes, we performed transient transfection-reporter analyses of Tex13, Dmrt1, Stra8, 1700061G19Rik and Triml1 ( Figure 4A). We were unable to generate a reporter construct for Fancd2os. Upstream regions (,1.5 kb) spanning the transcription start site (TSS) (21500 to + 20 bp) of each gene were cloned into the pGL3-Basic vector, and the dual luciferase assay was used to test for reporter activity in F9 cells ( Figure 4A). We found that the upstream regions of the Spg-F9 genes, Tex13, Stra8 and Dmrt1, exhibited significant promoter activities in F9 cells ( Figure 4A). In particular, the promoters of Tex13 and Stra8 showed strikingly strong reporter activities (214and 76-fold higher, respectively, than that driven by the vector alone). In contrast, and consistent with their weak expression levels in F9 cells ( Figure 3A), the upstream regions of the Sptd-F9 genes (1700061G19Rik and Triml1) did not activate reporter gene expression in F9 cells. Their promoters rather suppressed the reporter activity of the control vector. We also monitored the reporter activity of the promoters of Tex13, Stra8 and Dmrt1 in NIH3T3 cells, and found that the promoters showed significantly lower activities in this somatic cell line compared to F9 cells ( Figure 4B). Thus, our results indicate that F9 cells have active transcriptional machineries for the expression of spermatogonial genes, such as Tex13, Stra8 and Dmrt1.
Characterization of the Tex13 promoter
Previous functional and transcriptional studies on the Dmrt1 and Stra8 genes have been reported [35][36][37][38][39][40]. Dmrt1 is essential for the differentiation of germ cells and Sertoli cells, where it is under the control of distinct transcriptional mechanisms. Stra8 controls the switch from spermatogonial differentiation to meiosis, and its 400-bp promoter is known to direct gene expression in spermatogonial stem cells. In contrast, the expression patterns, expression mechanisms, and functions of Tex13 have not previously been reported. Thus, we further investigated the promoter region of Tex13 in F9 cells. To clarify the regions responsible for Tex13 transcription, we cloned a series of 59-nested deletions in the Tex13 upstream region into the pGL3-Basic vector (21500 to + 20 bp, 2720 to +20 bp, 2402 to +20 bp, 2200 to +20 bp, 2111 to +20 bp, 295 to +20 bp, 282 to +20 bp and 265 to +20 bp) ( Figure 5A). Transient transfection of F9 cells with these constructs revealed that the highest promoter activity (423-fold that of the vector control) was associated with the 2200 to +20 bp region, whereas the lowest degree of significant activity (3-fold that of the vector control) was associated with the 265 to +20 bp region ( Figure 5A). This suggests that the 265 to +20 bp region is a minimal promoter region for Tex13 gene expression, and various additional repressive and enhancer regions are present in the 21500 to +20 bp region. Among the constructs harboring more than the 2200 to +20 bp region, the 21500 to +20 bp and 2720 to +20 bp regions, but not the 2402 to +20 bp region, significantly inhibited the maximal activity of the 2200 to +20 bp region. The degree of repression was similar between the 21500 to +20 bp and 2720 to +20 bp regions, suggesting that the negative regulatory elements are present in the 2720 to 2402 bp region. All constructs smaller than the 2200 to +20 bp region were found to exhibit gradual but significant reductions of promoter activity in F9 cells ( Figure 3A). This suggests that the 2200 to 265 bp region contains enhancer elements for Tex13 transcription. We performed a similar promoter analysis in NIH3T3 cells. In contrast to our findings in F9 cells, the promoter activity of the 2111 to +20 bp region was not reduced in these somatic cells, compared to the activity of the 2200 to +20 bp region. Thus, our results suggest that the 2200 to 2111 bp region might be implicated in F9 cell-and germ cell-preferred expression. Finally, a 19-fold decrease in luciferase activity was observed in F9 cells transiently transfected with the construct spanning the 282 to +20 bp region of the Tex13 promoter. This was the most dramatic reduction in reporter activity observed among the deletion constructs, indicating that a strong enhancer element is present in the region from 295 to 283 bp.
To further investigate this putative enhancer region, we generated constructs corresponding to the 295 to 283 bp region and the 2110 to 0/+20 bp regions lacking the 295 to 283 bp sequence. F9 cells were transfected with these constructs or the intact 2111 to 20 bp region (positive control), and the promoter activities of these constructs were compared ( Figure 5B and 5C). We found that the 295 to 283 bp region alone exhibited very little promoter activity ( Figure 5B), and deletion of the 295 to 283 bp region from the 2111 to 0/+20 bp region dramatically decreased the promoter activity (8-fold reduction) ( Figure 5C). These data confirm that the 295 to 283 bp region is a strong enhancer that is important for Tex13 gene expression, and further indicate that the downstream sequence (the 282 to 0 bp region) is required for the full activity of the 295 to 283 bp region in the Tex13 promoter.
We next performed sequence analysis of the Tex13 promoter. The promoter lacks a typical TATA box. Analysis of the 2200 to 283 bp region using the web-based tool, TFSEARH, revealed the presence of several potential transcription factor binding sites, including those for SRY (sex-determining region Y), CDX1 (caudal type homeobox 1), OCT-1 (POU domain, class 2, transcription factor 1), PRX2 (paired related homeobox 2), STATx (signal transducer and activator of transcription family), ZEB1 (zinc finger E-box binding homeobox 1) and SP1 (transacting transcription factor 1) (see Discussion). Figure 5C summarizes the data obtained from our promoter analyses.
DNA methylation analysis of the Tex13 promoter
Although Tex13 is not expressed in NIH3T3 cells (Figure 3A), the Tex13 promoter exhibited low but significant reporter activity in this somatic cell line ( Figure 4B and 5A). This suggests that an Figure 5. Promoter analysis of the sequence upstream of the mouse Tex13 gene. A. Promoter activity of the murine Tex13 59-flanking sequences. The Tex13 upstream region used for the reporter assay is shown on the left. Various genomic regions were introduced into the promoterless pGL3-Basic vector, and the reporter constructs were transiently transfected into F9 and NIH3T3 cells. Strong promoter activities were observed in F9 cells. The relative promoter activities are represented as the fold increase versus expression from pGL3-Basic. The presented values represent the mean 6 SD of three independent experiments. Statistical significance was determined by the Student's t-test; *p,0.05, **p,0.01. All p-values versus pGL3-Basic were less than 0.01. B. Luciferase reporter analysis of the 295 bp to 283 bp region and the 0 to +20 bp region. The indicated deletion reporter constructs were prepared (left) and transiently transfected into F9 cells. The presented values represent the mean 6 SD of three independent experiments. All p-values versus pGL3-Basic were less than 0.01. Statistical significance was determined by the Student's t-test; **p, 0.01. C. Summary of relative luciferase activity from Tex13 regulatory regions. The minimal promoter region and several enhancer/inhibitor regions are indicated upstream of the mouse Tex13 gene. The locations of putative transcription factor-binding sites between 2206 bp and 280 bp were predicted with the TFSEARCH program (arrow). The direction of each arrow indicates the orientation of the indicated putative element. The sequences of two enhancers are indicated (gray boxes). Binding sites for ZEB1 and CDX1, known to be transcriptional repressors, were predicted to be present in the 2520 to 2508 bp and 2490 to 2482 bp, respectively. doi:10.1371/journal.pone.0103837.g005 Figure 6. Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program (http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (2402/+20)] were in vitro methylated or mock-methylated with SssI methyltransferase, and transfected into F9 cells. Firefly additional mechanism is involved in the germ cell-specific expression of Tex13. Since epigenetic regulation, such as DNA methylation, is closely related to gene expression [41], we examined whether Tex13 undergoes epigenetic regulation. Sequence analysis of the proximal promoter of Tex13 revealed that there are 14 CpG sites in the region from 2383 bp to 228 bp ( Figure 6A). We therefore used bisulfite sequencing to analyze the DNA methylation of this region in F9 and NIH3T3 cells. Our analysis revealed that 47% of these CpG sites were methylated in F9 cells. Interestingly, half of Tex13 promoter in F9 cells were highly methylated, but the others were fully unmethylated ( Figure 6B). In contrast, 85% of the sites were methylated in NIH3T3 cells, with nine CpG sites in the 2141 to 228 bp region showing full methylation in NIH3T3 cells. This result suggests that DNA methylation of the 2141 to 228 bp region of the Tex13 promoter is associated with the expressional silencing of this gene in somatic cells but not in germ cells (i.e., germ cell-specific expression).
To determine whether DNA methylation of the Tex13 promoter induces transcriptional silencing, we performed luciferase reporter analysis of the methylated promoter in F9 cells. We incubated a pGL3-Basic vector construct containing the 2402 to +20 bp region of the Tex13 promoter with SssI (M.SssI) to methylate the cytosines of the 14 CpG sites. Promoter methylation was confirmed by methylation-sensitive restriction digest (data not shown). Compared to the mock-methylated control, the promoter activity of the in vitro-methylated 2402 to +20 bp region was significantly suppressed in F9 cells ( Figure 6C). Taken together, our results suggest that epigenetic regulation may help mediate the gene expression of Tex13.
Expression and localization of the TEX13 protein
Tex13 is predicted to encode a 186-amino acid protein. To explore the characteristics of Tex13 at the protein and cellular levels, we cloned its protein-coding sequence into the pEGFP-N2 vector and transiently expressed GFP-TEX13 proteins in F9 and NIH 3T3 cells ( Figure 7A and 7B). In F9 cells, we observed that GFP-TEX13 localization was heterogeneous with the 47% of the transfected cells showing the protein exclusively in the nucleus and throughout the cytoplasm and nucleus (53%) (Figure 7A and 7B). A similar distribution was also observed in NIH 3T3 cells. The GFP-signal of TEX13 was predominantly located in the nucleus (51%) or evenly distributed both in cytoplasmic and nuclear compartments (39%). The rest of the transfected cells with the GFP-signal showed cytoplasmic localization (10%). Thus, our results suggest that the endogenous TEX13 protein can shuttle between nucleus and cytoplasm.
Discussion
In this study, we investigated the gene expression profile of the F9 mouse testicular teratoma cell line versus male germ cells. Our in silico analyses revealed a total of 964 genes that are stagespecifically transcribed in male germ cells and also expressed in F9 cells. About the half of these genes were up-regulated in spermatogonia (Spg-F9 genes). Interestingly, the enriched GO terms were found only in this group; of them, the most prominently enriched GO term was transcription regulatory activity. A number of the Spg-F9 genes with this GO term are known to function in spermatogonial survival and spermatogenesis. Furthermore, spermatogonial markers and various genes expressed in primordial germ cells are also predicted to be expressed in F9 cells. Thus, F9 cells appear to transcribe genes that are expressed/active in both differentiated spermatogonia and pluripotent germ cell genes.
Genes that are expressed exclusively or predominantly in male germ cells are essential for spermatogenesis. Of the germ cell genes expressed in F9 cells, our in silico and in vitro analyses identified several as having specific or predominant expression in germ cells or testis. One of them, Dmrt1, encodes a protein that is essential in germ cells for the maintenance of embryonic germ cell identity and the development of juvenile spermatogonia [35,36]. DMRT1 also inhibits meiosis in undifferentiated spermatogonia by blocking the retinoic acid-dependent robust expression of Stra8 [31], which is required for meiotic initiation in both sexes. In germ cells lacking STRA8, the mitotic development of germ cells is normal, but they fail to go through meiotic initiation or progression [38,39]. Tex13 is an X-linked gene expressed in spermatogonia [42], but little was known about the expression mechanism or function of this gene prior to this study. Dmrt1, Stra8 and Tex13 are preferentially expressed in spermatogonia compared to the other germ cell stages, whereas Triml1, Fancd2os and 1700061G19Rik are expressed majorly in spermatids. Triml1 is involved in the ubiquitin pathway [43], whereas the expression patterns and functions of Fancd2os and 1700061G19Rik are not yet known. However, these genes are expressed very weakly in F9 cells. Considering its expression pattern and unexplored function, we therefore decided to explore the transcription and function of Tex13, using F9 cells as a model system. Studies on germ cellspecific and -predominant genes have been limited by the lack of stable cell lines expressing such genes. F9 cells could therefore be an alternative tool for investigating the transcriptional regulation of such genes. Using F9 cells, we found that the 1.5-kb regions upstream of Dmrt1, Stra8 and Tex13 exhibited strong reporter activities. Furthermore, we showed for the first time that the Tex13 promoter is located 2200 to 0 bp upstream of the gene. The short sequence spanning the 295 to 283 bp region is responsible for strong promoter activity, but requires the downstream sequences for its activity. Since no known transcription factor is predicted to interact with this region, we speculate that Tex13 transcription might involve the binding of an unknown transcription factor(s) to the 295 to 283 bp region to form a complex with other transcription factors that interact with the downstream region. We also discovered an additional critical region (2200 to 2111 bp) in the proximal promoter of Tex13. Our data suggest that this region is implicated in the F9 and germ cell-preferred transcription of the gene. The SRY, CDX1, OCT-1, PRX2, STAT, ZEB1 and SP1 transcription factors are predicted to bind to the region. Of them, PRX2 is not expressed in spermatogonia [44]; ZEB1 and CDX1 are transcriptional repressors [45,46]. Putative binding sites of these repressors are also located in the repressor region 2720 to 2402 bp. OCT1 is a ubiquitous transcriptional activator [47] that also reportedly functions as a transcriptional repressor in certain genes [48]. Some of the STAT family members are important for PGC formation and spermatogenesis [49,50], while SP1 is known to mediate transcriptional activation and repression in male germ cells [51]. The 2200 to 2111 bp region associated with these transcription factors could therefore be involved in the luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean 6 SD of three independent experiments; **p,0.01 versus mockmethylated Tex13 (2402/+20). doi:10.1371/journal.pone.0103837.g006 transcriptional activation or repression of Tex13 in different cell types. Further studies are needed to determine whether this transcriptional regulation is related to the germ cell-specific expression of Tex13.
Methylation of cytosines at CpG dinucleotides is a well-defined mechanism of epigenetic gene silencing in mammals [41], and promoter methylation has been associated with the testis-or cell type-specific expression of certain genes during spermatogenesis [52,53]. Here, our transient transfection-reporter analysis revealed that the promoter region of Tex13 showed weak but significant activities in NIH3T3 cells, despite the lack of endogenous Tex13 gene expression in these cells. This prompted us to investigate whether Tex13 expression is regulated by DNA methylation. Bisulfite sequencing revealed that the Tex13 promoter is hypermethylated in NIH3T3 cells, but not in F9 cells. We further found that in vitro methylation of the promoter region significantly inhibited the reporter activity. Thus, differential promoter methylation may contribute to the testis-and germ cell-predominant expression of Tex13. Interestingly, half of the F9 genomic DNA samples exhibited hypermethylation in the Tex13 proximal promoter, while the others were completely demethylated. In relation to this, it is noteworthy that embryonal carcinomas and their derived cell lines, including F9 cells, are composed of an undifferentiated stem cell population and a differentiated tumor cell population. The X chromosome is methylated by X-inactive specific transcript (XIST) in differentiated germ cell tumors but not in undifferentiated germ cell tumors, as shown in the case of the androgen receptor gene [54].
Finally, subcellular localization is a critical characteristic used to elucidate protein functions. We found that the localization of GFP-TEX13 was heterogeneous in the cytoplasmic and nuclear compartments, suggesting that endogenous TEX13 can shuttle between the two compartments. The N-terminal region of TEX13 was predicted from sequence analysis to contain a putative nuclear localization sequence (data not shown). Nuclear-cytoplasmic shuttling plays an important role in regulating the activity of several proteins involved in cell cycle progression and proliferation, and signal transduction [55]. It should be noted that the properties of Tex13, including the subcellular localization of its encoded protein, are similar to those of Stra8. It has been reported that STRA8 associates with DNA and possesses transcriptional activity [56], and a mouse knockout study found that STRA8 is crucial for meiotic progression during spermatogenesis [38,39]. Based on our findings, we speculate that TEX13 may act as a transcription factor or cooperate with transcription factors to regulate the expression levels of genes involved in early germ cell development. A previous proteomic study reported the presence of the TEX13 protein in early male germ cells [57]. Further investigations are needed to determine the precise function of TEX13 in male germ cell development.
In conclusion, we herein report a comprehensive analysis of genes transcribed in both F9 cells and male germ cells. Our data suggest that F9 cells can transcribe genes that exhibit stage-specific expression in early germ cells. In particular, we identified a F9 cellexpressed germ cell-specific gene, Tex13, and used the cell line to characterize its promoter and potential epigenetic regulation. Finally, we found that TEX13 is a potential nucleocytoplasmic shuttling protein. Our results collectively establish a basis for future investigations into the transcriptional mechanism of Tex13 and the nuclear function of its encoded protein. Figure S1 Expression profile of genes known to be transcribed in spermatogonia and primordial germ cells. Heatmap of the normalized gene expression profiles of genes is shown. Up-regulated genes are indicated in red and downregulated genes are shown in green. Abbreviations: J1, J1 embryonic stem cells; Spg, type B spermatogonia; Spcy, pachytene spermatocyte; Sptd, round spermatid; and F9, F9 cells. Genes: Plzf, promyelocytic leukemia zinc finger ortholog; Dmrt1, doublesex and mab-3 related transcription factor 1; Stra8, stimulated by retinoic acid gene 8; Gfra1, glial cell line derived neurotrophic factor family receptor alpha 1; Klf2, kruppel-like factor 2; Lin28, RNA-binding protein LIN-28; Nanog, nanog homeobox; Sox2, SRY (sex determining region Y)-box 2; Pou5f1, POU domain, class 5, transcription factor 1; Tcfap2c, transcription factor AP-2, gamma; Prdm14, PR domain containing 14; Prdm1, PR domain containing 1, with ZNF domain. (PDF)
Supporting Information
Figure 3 .
3Tissue distribution and developmental expression patterns of germ cell-specific genes expressed in F9 cells. A. The expression patterns of nine genes predicted to be testis-specific or -predominant, as assessed in testis, F9 cells and NIH3T3 cells. Glyceraldehyde-3-cells were scored per group. Transfection efficiency was 25.1%+/25.1%.
Figure 4 .
4Promoter analysis of five germ cell-specific genes in F9 cells. A. Promoter-luciferase reporter analysis of five germ cell genes identified as being expressed in F9 cells. F9 cells were transfected with luciferase constructs containing 1.5-kb upstream regions spanning the transcription start site (TSS) (21500 to +20 bp) of the indicated genes. The relative promoter activities are represented as the fold increase versus the expression from the promoter-less pGL3-Basic vector. The presented values represent the mean 6 SD of three independent experiments. B. Promoter-luciferase reporter analysis of Dmrt1, Stra8 and Tex13 in NIH3T3 cells. Reporter constructs containing promoters were transiently transfected into NIH3T3 cells. The presented values represent the mean 6 SD of three independent experiments. Statistical significance in A and B was determined by the Student's t-test; *p,0.05 and **p,0.01 versus pGL3-Basic. doi:10.1371/journal.pone.0103837.g004
Table 1 .
1Gene Ontology (GO) terms enriched in the Spg-F9 group.GO term
# of genes
P-value
Fold enrichment
Transcription regulatory activity
57
9.90E-06
1.83
Developmental processes
117
1.70E-08
1.62
Death
22
0.03
1.61
Biological adhesion
23
0.03
1.56
Reproductive processes
25
0.03
1.53
Reproduction
25
0.03
1.52
Cellular component organization
68
0.0053
1.37
Localization
84
0.04
1.21
Biological regulation
213
4.99E-04
1.17
Binding
344
5.36E-10
1.17
Organelles
249
0.002
1.13
Cellular processes
264
0.098
1.08
Cell parts
405
0.04
1.02
Cells
405
0.04
1.02
Unknown
58
-
-
doi:10.1371/journal.pone.0103837.t001
Table 3 .
3Testis-specific genes expressed in F9 cells.Array pattern
Gene
UniGene ID
Testis-specificty (%)
Tissue
Spermatogonia-F9
Dmrt1
Mm.391208
96.88
Embryonic tissue, testis
Tex13
Mm.193025
75
Embryonic tissue, testis
Nr5a1
Mm.31387
51.79
Brain, embryonic tissue, fertilized ovum, lung,
ovary, spleen, testis
Stra8
Mm.5171
34.62
Embryonic tissue, testis
Spermatocyte-F9
Sp2
Mm.155547
62.07
Embryonic tissue, testis, thymus
Spermatid-F9
Cpvl
Mm.158654
100
Testis
Fancd2os
Mm.72959
91.67
Kidney, testis
1700061G19Rik
Mm.160106
85.16
Embryonic tissue, intestine, testis
Triml1
Mm.64542
56
Embryonic tissue, extra embryonic tissue, testis
doi:10.1371/journal.pone.0103837.t003
Table S1
S1Oligonucleotides used for plasmid construction, site-directed mutagenesis and bisulfite sequencing. (XLSX)
Table S2
S2Summary of gene list in Spg-F9. (XLSX)
Table S3
S3Summary of gene list in Spcy-F9. (XLSX)
Table S4
S4Summary of gene list in Sptd-F9. (XLSX)Author Contributions
August 2014 | Volume 9 | Issue 8 | e103837
PLOS ONE | www.plosone.org
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Requirement of Notch 1 and its ligand jagged 2 expressions for spermatogenesis in rat and human testes. T Hayashi, Y Kageyama, K Ishizaka, G Xia, K Kihara, J Androl. 22Hayashi T, Kageyama Y, Ishizaka K, Xia G, Kihara K, et al. (2001) Requirement of Notch 1 and its ligand jagged 2 expressions for spermatogenesis in rat and human testes. J Androl 22: 999-1011.
The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. C K Matson, M W Murphy, M D Griswold, S Yoshida, V J Bardwell, Dev Cell. 19Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, et al. (2010) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev Cell 19: 612-624.
Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. J A Schmidt, M R Avarbock, J W Tobias, R L Brinster, Biol Reprod. 81Schmidt JA, Avarbock MR, Tobias JW, Brinster RL (2009) Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogo- nial stem cell self-renewal in the rat. Biol Reprod 81: 56-66.
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The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. A D Krentz, M W Murphy, S Kim, M S Cook, B Capel, Proc Natl Acad Sci U S A. 106Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, et al. (2009) The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proc Natl Acad Sci U S A 106: 22323-22328.
Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. C S Raymond, M W Murphy, M G O'sullivan, V J Bardwell, D Zarkower, Genes Dev. 14Raymond CS, Murphy MW, O'Sullivan MG, Bardwell VJ, Zarkower D (2000) Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev 14: 2587-2595.
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Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. E L Anderson, A E Baltus, H L Roepers-Gajadien, T J Hassold, D G De Rooij, Proc Natl Acad Sci U S A. 105Anderson EL, Baltus AE, Roepers-Gajadien HL, Hassold TJ, de Rooij DG, et al. (2008) Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. Proc Natl Acad Sci U S A 105: 14976-14980.
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The role of DNA methylation in mammalian epigenetics. P A Jones, D Takai, Science. 293Jones PA, Takai D (2001) The role of DNA methylation in mammalian epigenetics. Science 293: 1068-1070.
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STRA8 shuttles between nucleus and cytoplasm and displays transcriptional activity. M Tedesco, La Sala, G Barbagallo, F , De Felici, M Farini, D , J Biol Chem. 284Tedesco M, La Sala G, Barbagallo F, De Felici M, Farini D (2009) STRA8 shuttles between nucleus and cytoplasm and displays transcriptional activity. J Biol Chem 284: 35781-35793.
Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice. B Zheng, Q Zhou, Y Guo, B Shao, T Zhou, Proteomics. 14Zheng B, Zhou Q, Guo Y, Shao B, Zhou T, et al. (2014) Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice. Proteomics 14: 274-285.
| [
"The F9 cell line, which was derived from a mouse testicular teratoma that originated from pluripotent germ cells, has been used as a model for differentiation. However, it is largely unknown whether F9 cells possess the characteristics of male germ cells. In the present study, we investigated spermatogenic stage-and cell type-specific gene expression in F9 cells. Analysis of previous microarray data showed that a large number of stage-regulated germ cell genes are expressed in F9 cells. Specifically, genes that are prominently expressed in spermatogonia and have transcriptional regulatory functions appear to be enriched in F9 cells. Our in silico and in vitro analyses identified several germ cell-specific or -predominant genes that are expressed in F9 cells. Among them, strong promoter activities were observed in the regions upstream of the spermatogonial genes, Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Tex13 (testis expressed gene 13), in F9 cells. A detailed analysis of the Tex13 promoter allowed us to identify an enhancer and a region that is implicated in germ cell-specificity. We also found that Tex13 expression is regulated by DNA methylation. Finally, analysis of GFP (green fluorescent protein) TEX13 localization revealed that the protein distributes heterogeneously in the cytoplasm and nucleus, suggesting that TEX13 shuttles between these two compartments. Taken together, our results demonstrate that F9 cells express numerous spermatogonial genes and could be used for transcriptional studies focusing on such genes. As an example of this, we use F9 cells to provide comprehensive expressional information about Tex13, and report that this gene appears to encode a germ cell-specific protein that functions in the nucleus during early spermatogenesis."
] | [
"J T Kwon ",
"Jin S Choi ",
"H Kim ",
"J Jeong ",
"J "
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"J",
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] | [
"Kwon",
"Choi",
"Kim",
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] | [
"E M Eddy, ",
"E M Eddy, ",
"E M Eddy, ",
"J Bowles, ",
"P Koopman, ",
"B E Richardson, ",
"R Lehmann, ",
"M Saitou, ",
"M Yamaji, ",
"K Caires, ",
"J Broady, ",
"D Mclean, ",
"= Cy, ",
"Nu, ",
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"J D Heaney, ",
"E L Anderson, ",
"M V Michelson, ",
"J L Zechel, ",
"P A Conrad, ",
"L C Stevens, ",
"L C Stevens, ",
"E Lehtonen, ",
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"P J Park, ",
"L F Zhang, ",
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"K B Laursen, ",
"P M Wong, ",
"L J Gudas, ",
"K B Scotland, ",
"S Chen, ",
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"Y , ",
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"Y Seki, ",
"K Kurimoto, ",
"Y Yabuta, ",
"M Yuasa, ",
"J Kehler, ",
"E Tolkunova, ",
"B Koschorz, ",
"M Pesce, ",
"L Gentile, ",
"P S Sarkar, ",
"S Paul, ",
"J Han, ",
"S Reddy, ",
"T Kato, ",
"M Esaki, ",
"A Matsuzawa, ",
"Y Ikeda, ",
"Y Romero, ",
"M Vuandaba, ",
"P Suarez, ",
"C Grey, ",
"P Calvel, ",
"M J Goertz, ",
"Z Wu, ",
"T D Gallardo, ",
"F K Hamra, ",
"D H Castrillon, ",
"D Thepot, ",
"J B Weitzman, ",
"J Barra, ",
"D Segretain, ",
"M G Stinnakre, ",
"B N Nakamura, ",
"G Lawson, ",
"J Y Chan, ",
"J Banuelos, ",
"M M Cortes, ",
"F Zindy, ",
"J Van Deursen, ",
"G Grosveld, ",
"C J Sherr, ",
"M F Roussel, ",
"I Ketola, ",
"M Anttonen, ",
"T Vaskivuo, ",
"J S Tapanainen, ",
"J Toppari, ",
"T Hayashi, ",
"Y Kageyama, ",
"K Ishizaka, ",
"G Xia, ",
"K Kihara, ",
"C K Matson, ",
"M W Murphy, ",
"M D Griswold, ",
"S Yoshida, ",
"V J Bardwell, ",
"J A Schmidt, ",
"M R Avarbock, ",
"J W Tobias, ",
"R L Brinster, ",
"Y C Chen, ",
"C D Hsiao, ",
"W D Lin, ",
"C M Hu, ",
"P P Hwang, ",
"E N Geissler, ",
"M A Ryan, ",
"D E Housman, ",
"A D Krentz, ",
"M W Murphy, ",
"S Kim, ",
"M S Cook, ",
"B Capel, ",
"C S Raymond, ",
"M W Murphy, ",
"M G O'sullivan, ",
"V J Bardwell, ",
"D Zarkower, ",
"N Lei, ",
"T Karpova, ",
"K I Hornbaker, ",
"D A Rice, ",
"L L Heckert, ",
"E L Anderson, ",
"A E Baltus, ",
"H L Roepers-Gajadien, ",
"T J Hassold, ",
"D G De Rooij, ",
"M Mark, ",
"H Jacobs, ",
"M Oulad-Abdelghani, ",
"C Dennefeld, ",
"B Feret, ",
"G Giuili, ",
"A Tomljenovic, ",
"N Labrecque, ",
"M Oulad-Abdelghani, ",
"M Rassoulzadegan, ",
"P A Jones, ",
"D Takai, ",
"P J Wang, ",
"J R Mccarrey, ",
"F Yang, ",
"D C Page, ",
"L Tian, ",
"X Wu, ",
"Y Lin, ",
"Z Liu, ",
"F Xiong, ",
"K Lee, ",
"J S Park, ",
"Y J Kim, ",
"Soo Lee, ",
"Y S , ",
"Sook Hwang, ",
"T S , ",
"A A Postigo, ",
"D C Dean, ",
"B Rath, ",
"R S Pandey, ",
"P R Debata, ",
"N Maruyama, ",
"P C Supakar, ",
"R A Sturm, ",
"G Das, ",
"W Herr, ",
"A Shakya, ",
"J Kang, ",
"J Chumley, ",
"M A Williams, ",
"D Tantin, ",
"G Herrada, ",
"D J Wolgemuth, ",
"K Murphy, ",
"L Carvajal, ",
"L Medico, ",
"M Pepling, ",
"K Thomas, ",
"J Wu, ",
"D Y Sung, ",
"W Thompson, ",
"M Powell, ",
"Y Hou, ",
"J Yuan, ",
"X Zhou, ",
"X Fu, ",
"H Cheng, ",
"S Sato, ",
"C Maeda, ",
"N Hattori, ",
"S Yagi, ",
"S Tanaka, ",
"L H Looijenga, ",
"A J Gillis, ",
"R J Van Gurp, ",
"A J Verkerk, ",
"J W Oosterhuis, ",
"M Gama-Carvalho, ",
"M Carmo-Fonseca, ",
"M Tedesco, ",
"La Sala, ",
"G Barbagallo, ",
"F , ",
"De Felici, ",
"M Farini, ",
"D , ",
"B Zheng, ",
"Q Zhou, ",
"Y Guo, ",
"B Shao, ",
"T Zhou, "
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"Oulad-Abdelghani",
"Dennefeld",
"Feret",
"Giuili",
"Tomljenovic",
"Labrecque",
"Oulad-Abdelghani",
"Rassoulzadegan",
"Jones",
"Takai",
"Wang",
"Mccarrey",
"Yang",
"Page",
"Tian",
"Wu",
"Lin",
"Liu",
"Xiong",
"Lee",
"Park",
"Kim",
"Lee",
"Hwang",
"Postigo",
"Dean",
"Rath",
"Pandey",
"Debata",
"Maruyama",
"Supakar",
"Sturm",
"Das",
"Herr",
"Shakya",
"Kang",
"Chumley",
"Williams",
"Tantin",
"Herrada",
"Wolgemuth",
"Murphy",
"Carvajal",
"Medico",
"Pepling",
"Thomas",
"Wu",
"Sung",
"Thompson",
"Powell",
"Hou",
"Yuan",
"Zhou",
"Fu",
"Cheng",
"Sato",
"Maeda",
"Hattori",
"Yagi",
"Tanaka",
"Looijenga",
"Gillis",
"Van Gurp",
"Verkerk",
"Oosterhuis",
"Gama-Carvalho",
"Carmo-Fonseca",
"Tedesco",
"Sala",
"Barbagallo",
"Felici",
"Farini",
"Zheng",
"Zhou",
"Guo",
"Shao",
"Zhou"
] | [
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"The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. C K Matson, M W Murphy, M D Griswold, S Yoshida, V J Bardwell, Dev Cell. 19Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, et al. (2010) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev Cell 19: 612-624.",
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"The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. A D Krentz, M W Murphy, S Kim, M S Cook, B Capel, Proc Natl Acad Sci U S A. 106Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, et al. (2009) The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proc Natl Acad Sci U S A 106: 22323-22328.",
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"Chauvinist genes' of male germ cells: gene expression during mouse spermatogenesis",
"Regulation of gene expression during spermatogenesis",
"Male germ cell gene expression",
"Retinoic acid, meiosis and germ cell fate in mammals",
"Mechanisms guiding primordial germ cell migration: strategies from different organisms",
"Germ cell specification in mice: signaling, transcription regulation, and epigenetic consequences",
"Maintaining the male germline: regulation of spermatogonial stem cells",
"Cells transiently expressing GFP-TEX13 fusion proteins were visualized under fluorescent light, and the protein location was determined. Hoechst 33258 dye (blue) was used to stain nuclei. GFP-TEX13 localization was heterogeneous. Scale bar, 20 mm. B. Quantification of the different patterns of TEX13 sub-cellular localization in F9 cells and NIH 3T3 cells",
"Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice",
"Origin of testicular teratomas from primordial germ cells in mice",
"The development of transplantable teratocarcinomas from intratesticular grafts of pre-and postimplantation mouse embryos",
"Teratocarcinoma stem cells as a model for differentiation in the mouse embryo",
"Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) gene",
"Postmeiotic sex chromatin in the male germline of mice",
"Epigenetic regulation by RARalpha maintains ligand-independent transcriptional activity",
"Analysis of Rex1 (zfp42) function in embryonic stem cell differentiation",
"Signal deconvolution based expression-detection and background adjustment for microarray data",
"Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources",
"Distribution of GFRA1-expressing spermatogonia in adult mouse testis",
"Blimp1 is a critical determinant of the germ cell lineage in mice",
"Critical function of Prdm14 for the establishment of the germ cell lineage in mice",
"Oct4 is required for primordial germ cell survival",
"Six5 is required for spermatogenic cell survival and spermiogenesis",
"NR5A1 is required for functional maturation of Sertoli cells during postnatal development",
"The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis",
"Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis",
"Targeted disruption of the murine junD gene results in multiple defects in male reproductive function",
"Knockout of the transcription factor NRF2 disrupts spermatogenesis in an agedependent manner",
"INK4d-deficient mice are fertile despite testicular atrophy",
"Developmental expression and spermatogenic stage specificity of transcription factors GATA-1 and GATA-4 and their cofactors FOG-1 and FOG-2 in the mouse testis",
"Requirement of Notch 1 and its ligand jagged 2 expressions for spermatogenesis in rat and human testes",
"The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells",
"Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat",
"ZooDDD: a cross-species database for digital differential display analysis",
"The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene",
"The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency",
"Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation",
"Distinct transcriptional mechanisms direct expression of the rat Dmrt1 promoter in sertoli cells and germ cells of transgenic mice",
"Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice",
"STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation",
"Murine spermatogonial stem cells: targeted transgene expression and purification in an active state",
"The role of DNA methylation in mammalian epigenetics",
"An abundance of X-linked genes expressed in spermatogonia",
"Characterization and potential function of a novel pre-implantation embryo-specific RING finger protein: TRIML1",
"Differential expression of Prx I and II in mouse testis and their up-regulation by radiation",
"ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation",
"Molecular characterization of senescence marker protein-30 gene promoter: identification of repressor elements and functional nuclear factor binding sites",
"The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain",
"Oct1 is a switchable, bipotential stabilizer of repressed and inducible transcriptional states",
"The mouse transcription factor Stat4 is expressed in haploid male germ cells and is present in the perinuclear theca of spermatozoa",
"Expression of Stat3 in germ cells of developing and adult mouse ovaries and testes",
"SP1 transcription factors in male germ cell development and differentiation",
"DNA demethylation and USF regulate the meiosis-specific expression of the mouse Miwi",
"DNA methylationdependent modulator of Gsg2/Haspin gene expression",
"X inactivation in human testicular tumors. XIST expression and androgen receptor methylation status",
"The rules and roles of nucleocytoplasmic shuttling proteins",
"STRA8 shuttles between nucleus and cytoplasm and displays transcriptional activity",
"Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice"
] | [
"Reprod Fertil Dev",
"Semin Cell Dev Biol",
"Recent Prog Horm Res",
"Development",
"Nat Rev Mol Cell Biol",
"Reproduction",
"J Endocrinol",
"Localization of GFP-TEX13 in F9 cells and NIH 3T3 cells. A",
"Development",
"J Natl Cancer Inst",
"Dev Biol",
"Int J Dev Biol",
"Genes Cells",
"Curr Biol",
"Nucleic Acids Res",
"Dev Dyn",
"J Comput Biol",
"Nat Protoc",
"Reproduction",
"Nature",
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"\nFigure 3 .\n3Tissue distribution and developmental expression patterns of germ cell-specific genes expressed in F9 cells. A. The expression patterns of nine genes predicted to be testis-specific or -predominant, as assessed in testis, F9 cells and NIH3T3 cells. Glyceraldehyde-3-cells were scored per group. Transfection efficiency was 25.1%+/25.1%.",
"\nFigure 4 .\n4Promoter analysis of five germ cell-specific genes in F9 cells. A. Promoter-luciferase reporter analysis of five germ cell genes identified as being expressed in F9 cells. F9 cells were transfected with luciferase constructs containing 1.5-kb upstream regions spanning the transcription start site (TSS) (21500 to +20 bp) of the indicated genes. The relative promoter activities are represented as the fold increase versus the expression from the promoter-less pGL3-Basic vector. The presented values represent the mean 6 SD of three independent experiments. B. Promoter-luciferase reporter analysis of Dmrt1, Stra8 and Tex13 in NIH3T3 cells. Reporter constructs containing promoters were transiently transfected into NIH3T3 cells. The presented values represent the mean 6 SD of three independent experiments. Statistical significance in A and B was determined by the Student's t-test; *p,0.05 and **p,0.01 versus pGL3-Basic. doi:10.1371/journal.pone.0103837.g004",
"\nTable 1 .\n1Gene Ontology (GO) terms enriched in the Spg-F9 group.GO term \n# of genes \nP-value \nFold enrichment \n\nTranscription regulatory activity \n57 \n9.90E-06 \n1.83 \n\nDevelopmental processes \n117 \n1.70E-08 \n1.62 \n\nDeath \n22 \n0.03 \n1.61 \n\nBiological adhesion \n23 \n0.03 \n1.56 \n\nReproductive processes \n25 \n0.03 \n1.53 \n\nReproduction \n25 \n0.03 \n1.52 \n\nCellular component organization \n68 \n0.0053 \n1.37 \n\nLocalization \n84 \n0.04 \n1.21 \n\nBiological regulation \n213 \n4.99E-04 \n1.17 \n\nBinding \n344 \n5.36E-10 \n1.17 \n\nOrganelles \n249 \n0.002 \n1.13 \n\nCellular processes \n264 \n0.098 \n1.08 \n\nCell parts \n405 \n0.04 \n1.02 \n\nCells \n405 \n0.04 \n1.02 \n\nUnknown \n58 \n-\n-\n\ndoi:10.1371/journal.pone.0103837.t001 \n\n",
"\nTable 3 .\n3Testis-specific genes expressed in F9 cells.Array pattern \nGene \nUniGene ID \nTestis-specificty (%) \nTissue \n\nSpermatogonia-F9 \nDmrt1 \nMm.391208 \n96.88 \nEmbryonic tissue, testis \n\nTex13 \nMm.193025 \n75 \nEmbryonic tissue, testis \n\nNr5a1 \nMm.31387 \n51.79 \nBrain, embryonic tissue, fertilized ovum, lung, \novary, spleen, testis \n\nStra8 \nMm.5171 \n34.62 \nEmbryonic tissue, testis \n\nSpermatocyte-F9 \nSp2 \nMm.155547 \n62.07 \nEmbryonic tissue, testis, thymus \n\nSpermatid-F9 \nCpvl \nMm.158654 \n100 \nTestis \n\nFancd2os \nMm.72959 \n91.67 \nKidney, testis \n\n1700061G19Rik \nMm.160106 \n85.16 \nEmbryonic tissue, intestine, testis \n\nTriml1 \nMm.64542 \n56 \nEmbryonic tissue, extra embryonic tissue, testis \n\ndoi:10.1371/journal.pone.0103837.t003 \n",
"\nTable S1\nS1Oligonucleotides used for plasmid construction, site-directed mutagenesis and bisulfite sequencing. (XLSX)",
"\nTable S2\nS2Summary of gene list in Spg-F9. (XLSX)",
"\nTable S3\nS3Summary of gene list in Spcy-F9. (XLSX)",
"\nTable S4\nS4Summary of gene list in Sptd-F9. (XLSX)Author Contributions \n"
] | [
"Tissue distribution and developmental expression patterns of germ cell-specific genes expressed in F9 cells. A. The expression patterns of nine genes predicted to be testis-specific or -predominant, as assessed in testis, F9 cells and NIH3T3 cells. Glyceraldehyde-3-cells were scored per group. Transfection efficiency was 25.1%+/25.1%.",
"Promoter analysis of five germ cell-specific genes in F9 cells. A. Promoter-luciferase reporter analysis of five germ cell genes identified as being expressed in F9 cells. F9 cells were transfected with luciferase constructs containing 1.5-kb upstream regions spanning the transcription start site (TSS) (21500 to +20 bp) of the indicated genes. The relative promoter activities are represented as the fold increase versus the expression from the promoter-less pGL3-Basic vector. The presented values represent the mean 6 SD of three independent experiments. B. Promoter-luciferase reporter analysis of Dmrt1, Stra8 and Tex13 in NIH3T3 cells. Reporter constructs containing promoters were transiently transfected into NIH3T3 cells. The presented values represent the mean 6 SD of three independent experiments. Statistical significance in A and B was determined by the Student's t-test; *p,0.05 and **p,0.01 versus pGL3-Basic. doi:10.1371/journal.pone.0103837.g004",
"Gene Ontology (GO) terms enriched in the Spg-F9 group.",
"Testis-specific genes expressed in F9 cells.",
"Oligonucleotides used for plasmid construction, site-directed mutagenesis and bisulfite sequencing. (XLSX)",
"Summary of gene list in Spg-F9. (XLSX)",
"Summary of gene list in Spcy-F9. (XLSX)",
"Summary of gene list in Sptd-F9. (XLSX)"
] | [
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"Male germ cell development, or spermatogenesis, is a complex process that involves successive mitotic, meiotic, and post-meiotic phases [1,2]. The tightly regulated nature of this process, which occurs in the seminiferous tubules of testes, indicates that a highly organized network of genes is expressed in germ cells during spermatogenesis. Three levels of control regulate gene expression during spermatogenesis: intrinsic, interactive and extrinsic control [3]. The intrinsic program determines which genes are utilized and when these genes are expressed. The unique feature of this program is germ cell-and stage-specific gene expression. The interactive process, which involves crosstalk between germ cells and somatic cells, is essential for germ cell proliferation and progression. Finally, extrinsic influences, including steroid and peptide hormones, regulate the interactive process. Not all extrinsic regulation act through Sertoli cells. Retinoic acid (RA), the active derivative of vitamin A, is essential for spermatogenesis and has direct effects on germ cell development [4]. Pluripotent/primordial germ cells (PGCs), which colonize the developing gonads by active migration [5,6], turn into gonocytes. The latter differentiate into spermatogonial stem cells (SSCs), which provide a continuous supply of differentiating spermatogonia during spermatogenesis [7]. Signaling and transcriptional regulation are crucial for germ cell pluripotency, survival and differentiation. Misregulation of germ cell pluripotency may lead to tumor formation [8]. Testicular teratomas originate from primordial germ cells [9]. Although teratomas are tumor, they possess the capacity to produce all three germ cell layers. Therefore, testicular teratomas provide a useful tool for investigating the intersection of pluripotency, differentiation and tumorigenesis.",
"The F9 cell line was isolated as a subline of a testicular teratoma (designated OTT6050) that was established by implanting a sixday-old embryo in the testis of a 129/J mouse [10]. The F9 cell line is a nullipotent cell line that is unable to undergo spontaneous differentiation. However, F9 cells can differentiate into endodermal-like derivatives following treatment with several agents, including retinoic acid. Therefore, the F9 cell line has been used as a model for analyzing the molecular mechanisms of differentiation [11].",
"Because F9 cells originate from embryonic cells containing primordial germ cells, it is possible that the intrinsic genetic program involving spermatogenic stage-or cell-specific genes is partially active in F9 cells. If this is the case, the F9 cell line could be used as an alternative cell line for investigating germ cell transcription. In fact, the testis-specific promoter region of the human pituitary adenylate cyclase-activating polypeptide gene (PACAP) was previously investigated using F9 cells [12]. To address this issue, we herein used microarray data to analyze the transcripts expressed in F9 cells. We found for the first time that a large number of stage-specific germ cell genes are expressed in F9 cells. Various expressional analyses and reporter assays showed that F9 cells can be used for transcriptional studies of genes that are stage-specifically expressed in early male germ cells. In particular, genes encoding doublesex and mab-3 related transcription factor 1 (Dmrt1), stimulated by retinoic acid gene 8 (Stra8) and testis expressed gene 13 (Tex13) were significantly expressed in F9 cells. Since the functions and transcriptional mechanisms were previously reported for Dmrt1 and Stra8 [35][36][37][38][39][40], we focused on the expression of Tex13 in F9 cells. Our comprehensive analysis of the Tex13 promoter allowed us to identify regions responsible for the germ cell specificity and strong enhancer activity of this promoter. Moreover, Tex13 promoter showed cell-type specific DNA methylation. In addition, we found that Tex13 encodes a potential nucleocytoplasmic shuttling protein. Our study is the first comprehensive and systematic investigation of germ cell genes expressed in F9 cells.",
"We obtained microarray data representing spermatogenic cells, F9 cells and J1 embryonic stem cells from the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/gds/). The GSE4193 dataset contained expression profiles obtained from a purified population of spermatogenic cells [13]; the GSE31280 dataset contained the gene expression profile of F9 cells [14]; and the GSE9978 dataset contained array data obtained from J1 embryonic stem cells [15]. Feature-level data (CEL) files were downloaded and imported into R program for normalization. R is an open source statistical scripting language (http://www. r-project.org). All expressional data were normalized using the GCRMA method [16]. Expressional data obtained from spermatogenic cells (spermatogonia, spermatocytes and spermatids), F9 cells and J1 cells were combined into a microarray dataset. The combined array data were normalized by quantile normalization using the ''normalize.quantiles'' function from R/Bioconductor package. The averages between duplicates derived for each sample were calculated. For each experimental group (Spermatogonia-F9, Spermatocyte-F9 and Spermatid-F9), genes with absolute fold Figure 1. Microarray analysis of genes expressed stage-specifically in male germ cells and F9 cells. A. Heatmaps of the normalized gene expression profiles for each group. A total of 964 genes were expressed more than 1.5-fold higher in both a given germ cell stage and F9 cells compared to other germ cell stages and ESC. Clustering of each group was performed using hierarchical clustering. Up-regulated genes are indicated in red and down-regulated genes are shown in green. Abbreviations: J1, J1 embryonic stem cells; Spg, type B spermatogonia; Spcy, pachytene spermatocyte; Sptd, round spermatid; and F9, F9 cells. B. Mean expression level of all genes in each group. Differences between samples were validated using the Student's t-test. For simplification, the average expression levels between two germ cells with low expression levels were used. Significant differences were observed in the Spg-F9, Spcy-F9 and Sptd-F9 groups (p-value,10E-10). doi:10.1371/journal.pone.0103837.g001 Figure 2. Enriched gene ontology (GO) terms in the spermatogonia-F9 group. A total of 487 genes in the Spg-F9 group were analyzed for the enrichment of GO terms using the DAVID functional annotation chart. Only significant GO terms are shown (p-value,0.05). The pie slices are proportional to the number of genes. doi:10.1371/journal.pone.0103837.g002 changes greater than 1.5 were chosen as differentially expressed genes (DEGs) and subsequently analyzed using the DAVID Functional Annotation Tool for gene ontology (GO) (http://david. abcc.ncifcrf.gov/) [17]. A functional annotation chart is useful for identifying annotation terms that are enriched in the submitted gene list; a smaller p-value indicates increasing significance of the GO term, and fold enrichments of 1.5 and above are considered interesting.",
"To validate our analysis of the available microarray results, we performed reverse-transcription PCR (RT-PCR) using total RNA from testis, F9 cells and NIH3T3 cells. Total RNA was extracted using the TRIzol reagent (Molecular Research Center) according to the manufacturer's protocol, and cDNA was synthesized by random hexamer and oligo(dT) primers using the Omniscript reverse transcriptase (Qiagen). The utilized gene-specific primers are listed in Table S1. PCR was performed for 30 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 1 min 20 s. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was amplified as a control (forward, 59-TGA AGG TCG GAG TCA ACG GAT TTG GT-39 and reverse, 59-CAT GTG GGC CAT GAG GTC CAC CAC-39). The testis-specific expression of the nine tested genes was examined by RT-PCR in eight different mouse tissues (testis, ovary, brain, heart, kidney, lung, liver, and spleen). Specific expression at different stages of spermatogenesis was established using total RNA obtained from testes of prepubertal and adult male mice (ages 8, 10,12,14,16,20,30, and 56 days), and from the testes of W/W v mutant mice, which lack germ cells. All animal investigations were carried out according to the guidelines of the Animal Care and Use of Gwangju Institute of Science and Technology. The protocol was approved by the Animal Care and Use Committee of Gwangju Institute of Science and Technology (Permit number: GIST 2011-13). Sine oculis-related homeobox 5 [22] 26423 Nr5a1",
"Nuclear receptor subfamily 5, group A, member 1 [23] 14605 Tsc22d3 TSC22 domain family, member 3 [24] 56484 Foxo3 Forkhead box O3 [25] 16478 Jund Jun D proto-oncogene [26] 18024",
"Nuclear factor, erythroid derived 2, like 2 [27] 12578",
"Cyclin-dependent kinase inhibitor 2A [28] 14463",
"Gata4 GATA binding protein 4 [29] 18128 Notch1 Notch 1 [30] 50796",
"Doublesex and mab-3 related transcription factor 1 [31] 20893 Cell culture F9 (CRL-1720) and NIH3T3 cells (CRL-1658) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (F9) or 10% bovine caput serum (NIH3T3), along with 2 mM glutamine, 100 U/ml penicillin and 10 mg/ml streptomycin. The culture vessels for F9 cells were coated with 0.1% gelatin prior to use. Since RA is known to induce the differentiation of F9 cells, the chemical was not included in the media during cultivation. The cells were maintained in a humidified 5% CO 2 atmosphere at 37uC, the cell media were changed every 1-2 days, and the cells were subjected to passage every 3-4 days.",
"A luciferase reporter assay system (Promega) was used to measure promoter activity. To construct the 1.5-kb promoter luciferase reporter plasmid for five genes (Dmrt1, Stra8, Tex13, Triml1 and 1700061G19Rik), DNA fragments corresponding to the putative promoters predicted by DBTSS (http://dbtss.hgc.jp./) were prepared by PCR using the pfu DNA polymerase (Enzynomics) with mouse genomic DNA isolated using Dneasy Blood & Tissue kit (Qiagen). The utilized primers are listed in Table S1. Several deleted versions of the Tex13-luciferase reporter plasmids were also prepared. The sequence of each PCR product was confirmed by DNA sequencing (Macrogen), and each fragment of interest was cloned into the multi-cloning site of the promoter-less pGL3-Basic (Promega) plasmid.",
"Cells were plated to 24-well plates at 1.5610 5 cells/plate. After 24 h, when cells were at 50-60% confluence, the Lipofectamine LTX transfection reagent (Invitrogen) was used to transfect 400 ng of the indicated DNA construct together with 8 ng of pRL-TK (Promega), which contains the Renilla luciferase gene driven by the SV40 promoter, and was used an internal control for normalization of transfection efficiency. Twenty-four hours later, whole cell extracts from triplicate wells were assayed. Lysates were prepared using 100 ml of passive lysis buffer (Promega), and luciferase activity was determined from 30 ml of each lysate using the Luciferase Reporter Assay (Promega) and a Centro LB 960 DLReady Micro Plate Illuminometer (Berthold Technologies). Each experiment was repeated at least three times. The obtained promoter activity was normalized against that of the SV40 promoter, and was reported as the fold activity compared to that from pGL3-Basic.",
"Genomic DNA (gDNA) was purified from F9 cells and NIH3T3 cells using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol. Promoter methylation was assessed by bisulfite sequencing. In brief, cytosine-to-uracil conversion was performed on 1.0 mg of gDNA using an EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturer's provided protocol. PCR reactions were performed on the converted DNA with promoter specific-primers designed using the online MethPrimer software (http://www.urogene.org/ methprimer/). PCR was performed for 48 cycles of 95uC for 30 s, 48uC for 30 s, and 72uC for 30 s. For bisulfite sequencing, the amplified PCR products were cloned into the pTOP v2 vector (Macrogen). From each cell line (F9 and NIH3T3) a total of 10 independent clones containing each of the desired PCR products were sequenced.",
"The 2402/+20 Tex13 promoter was inserted into pGL3-Basic, and the vector was incubated at 37uC for 4 h with the methyltransferase enzyme, M.SssI (NEB) in the presence of the methyl group donor S-adenosylmethionine (SAM) (NEB). Mockmethylated plasmids were incubated without enzyme but in the presence of SAM. Samples were purified using a PCR purification kit (LaboPass). The mock-methylated and methylated plasmids were subjected to diagnostic digests with HpaII, a methylationsensitive enzyme, to confirm the efficacy of the in vitro methylation.",
"The coding region of mouse Tex13 (NM_031381 in GenBank) was amplified by RT-PCR and subcloned into the N terminus of pEGFP-N2 (Clontech) using EcoRI and BamHI, to generate plasmids expressing the fusion protein GFP-TEX13. Transient transfection of clones was achieved using Lipofectamine LTX transfection reagents and Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 24 h of culture on coverslips, cells were fixed with 4% (v/v) paraformaldehyde and nuclei were labeled with Hoechst 33258 dye (Sigma). Samples were mounted on slides and visualized by microscopes (DMLB; Leica Microsystems and IX81; Olympus). Approximately 100 ",
"To determine whether F9 cells transcribe genes that undergo stage-specific expression in male germ cells, we analyzed previously deposited microarray gene expression datasets representing type B spermatogonia (Spg), pachytene spermatocyte (Spcy), round spermatid (Sptd), F9 testicular teratoma cells (F9) and J1 embryonic stem cells (ESC). To avoid systematic variations in the analysis, we selected microarray datasets that were all produced using the Affymetrix Mouse Genome 430 2.0 array. Background adjustment of each array dataset was performed using GCRMA (GC Robust Multi-array Average) [16]. Quantile normalization was used to adjust the array data. Since F9 cells were isolated from a teratoma established by implanting an embryo with stem cell properties (pulipotency) in the testis, we used the ESC dataset as a negative control to reduce any influence from the stem cell properties of F9 cells. We selected genes that were up-regulated ($1.5-fold change) in one of the germ cell stages and F9 cells relative to the other germ cell stages and ESC. We found that 487 genes were highly transcribed in spermatogonia and F9 cells (Spg-F9) ( Figure 1A and Table S2), while 308 and 169 genes were up-regulated in spermatocytes and F9 cells (Spcy-F9) ( Table S3) and spermatids and F9 cells (Sptd-F9), respectively (Table S4). Statistical analysis of each group indicated that the expression levels of the selected genes in the appropriate germ cell stage and F9 cells were significantly higher (p,10E-10), than in the other germ cell stages and ESC ( Figure 1B). It should be noted that the Spg-F9 pattern had the largest number of genes among the three groups. Collectively, our in silico results demonstrated that numerous stage-specific germ cell genes (a total of 964 genes) are expressed in F9 cells.",
"To further investigate the germ cell genes expressed in F9 cells, we performed gene ontology (GO) enrichment analysis using the DAVID functional annotation tool [17]. In this analysis, the enrichment score reflected the degree to which a GO term is overrepresented in the Spg-F9, Spcy-F9 or Sptd-F9 genes, compared to all genome-wide genes. Our results revealed that a number of GO terms were enriched in the Spg-F9 group ( Figure 2), but not in the Spcy-F9 or Sptd-F9 groups. Using the criteria of a fold-enrichment score $1.5 and p,0.05, we found that the Spg-F9-enriched GO terms included transcription regulatory activity, developmental processes, death, biological adhesion, reproductive processes, and reproduction (Table 1). In particular, 57 genes encoding transcription factors were enriched under the GO term transcription regulatory activity; of them, 11 are known to function in germ cell proliferation, germ cell differentiation, and spermatogenesis (Table 2).",
"Genes known as markers of spermatogonia, including Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Gfra1 (glial cell line derived neurotrophic factor family receptor alpha 1) [18], were found to be expressed in F9 cells ( Figure S1). In addition, various genes expressed in primordial germ cells, including Prdm1 (PR domain containing 1, with ZNF domain) [19], Prdm14 (PR domain containing 14) [20], and Pou5f1 (POU domain, class 5, transcription factor 1) [21], are also predicted to be expressed in F9 cells ( Figure S1).",
"To identify testis-specific or -predominant (collectively called ''testis-preferred'') genes among the germ cell genes found to be expressed in F9 cells, we calculated testis specificity using information from the UniGene database [33], which contains the EST (Expressed sequence tag) expression profile of a given gene in 47 tissues or organs based on transcription per million (TPM, indicating the normalized gene expression level). We identified nine genes as being putatively testis-specific or -predominant, using the criteria of testis specificity $50% or (for genes expressed in fewer than three tissues) 50%. testis specificity $30% (Table 3). The Spg-F9 group contained four testis-specific or -predominant genes: doublesex and mab-3 related transcription factor 1 (Dmrt1); testis expressed gene 13 (Tex13); nuclear receptor subfamily 5, group A, member 1 (Nr5a1); and stimulated by retinoic acid gene 8 (Stra8). The Spcy-F9 group contained a single such gene: Sp2 transcription factor (Sp2). The Sptd-F9 group contained four genes that were predicted to be specifically or predominantly expressed in testis: carboxypeptidase, vitellogeniclike (Cpvl); Fancd2 opposite strand (Fancd2os); tripartite motif family-like 1 (Triml1); and 1700061G19Rik. Interestingly, Dmrt1 and Stra8 are known to be involved in the initiation of meiosis [31], while Nr5a1 is an important gene for reproductive differentiation [23]. The functions of the other listed genes are unknown.",
"To confirm that these nine genes are expressed in F9 cells and show testis-specific or -predominant expression patterns, we performed in vitro expression analyses. RT-PCR analysis showed that, with the exception of Cpvl, all of the tested genes were transcribed both in testis and F9 cells ( Figure 3A). NIH3T3 cells were used as a negative control for cell type-specific expression. The expression levels of Tex13, Dmrt1, and Stra8 were similar between testis and F9 cells, whereas the other detected genes showed weaker expression in F9 cells than in testis. Next, we examined the tissue distribution of the eight genes confirmed to be expressed in F9 cells ( Figure 3B). With the exception of Nr5a1 and Sp2, all of the genes were expressed specifically or predominantly in testis, which was consistent with our in silico prediction. To investigate the developmental expression patterns of the testisspecific or -predominant genes, we performed RT-PCR using RNA from mouse testis samples obtained at different days after birth ( Figure 3C). We hypothesized that if a given gene is transcribed in germ cells during spermatogenesis, the transcript will appear in the testis at a particular post-partum time point corresponding to a specific stage of spermatogenesis. The expression of all the four genes in the Spg-F9 group started at phosphate dehydrogenase (Gapdh) was included as a loading control. Except for Cpvl, all of the genes were detected in both testis and F9 cells. It should be noted that the two bands for Dmrt1 in NIH3T3 cells are non-specific, based on in silico investigation and an additional PCR analysis (data not shown). B. The tissue distributions of transcripts were assessed by RT-PCR analysis in various tissues of adult male mice. Complementary DNAs from various mouse tissues were amplified by PCR, with Gapdh included as a loading control. Seven genes were found to be testis-specific orpredominant. C. Developmental expression patterns during spermatogenesis. The stage-specific expression of the genes was determined from mouse testes on different days after birth (days 8, 10, 12, 14, 16, 20, 30, and 56). Abbreviations: PL, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; MI, meiotic division I; and MII, meiotic division II. Complementary DNA from germ cell-lacking testes from W/W v mutant mice was also examined. Consistent with our microarray analysis, Tex13, Stra8, Fancd2os, 1700061G19Rik and Triml1 were found to be germ cell-specific. doi:10.1371/journal.pone.0103837.g003 postnatal day 8, corresponding to spermatogonia. In contrast, Fancd2os, 1700061G19Rik and Triml1 from the Sptd-F9 group were first expressed at postnatal days 20 or 30, corresponding to the late spermatocyte and round spermatid stages, respectively. Finally, we examined the germ cell-specific expression of the seven testis-specific or -predominant genes using the germ cell-lacking testes of W/W v (c-kit) mutant mice [34]. The transcripts corresponding to Tex13, Stra8, Fancd2os, 1700061G19Rik and Triml1 were barely detected in the testes of mutant mice ( Figure 3C), suggesting that these genes underwent germ cell-specific expression. Collectively, the results of our in vitro analyses of the in silico-selected genes demonstrated that six genes (Tex13, Dmrt1, Stra8, Fancd2os, 1700061G19Rik and Triml1) show testis-specific or-predominant expression in F9 cells. In particular, Tex13 and Stra8 show germ cell-preferred transcription with abundant expression in F9 cells.",
"To investigate the expression mechanisms governing the F9 cell-expressed germ cell genes, we performed transient transfection-reporter analyses of Tex13, Dmrt1, Stra8, 1700061G19Rik and Triml1 ( Figure 4A). We were unable to generate a reporter construct for Fancd2os. Upstream regions (,1.5 kb) spanning the transcription start site (TSS) (21500 to + 20 bp) of each gene were cloned into the pGL3-Basic vector, and the dual luciferase assay was used to test for reporter activity in F9 cells ( Figure 4A). We found that the upstream regions of the Spg-F9 genes, Tex13, Stra8 and Dmrt1, exhibited significant promoter activities in F9 cells ( Figure 4A). In particular, the promoters of Tex13 and Stra8 showed strikingly strong reporter activities (214and 76-fold higher, respectively, than that driven by the vector alone). In contrast, and consistent with their weak expression levels in F9 cells ( Figure 3A), the upstream regions of the Sptd-F9 genes (1700061G19Rik and Triml1) did not activate reporter gene expression in F9 cells. Their promoters rather suppressed the reporter activity of the control vector. We also monitored the reporter activity of the promoters of Tex13, Stra8 and Dmrt1 in NIH3T3 cells, and found that the promoters showed significantly lower activities in this somatic cell line compared to F9 cells ( Figure 4B). Thus, our results indicate that F9 cells have active transcriptional machineries for the expression of spermatogonial genes, such as Tex13, Stra8 and Dmrt1.",
"Previous functional and transcriptional studies on the Dmrt1 and Stra8 genes have been reported [35][36][37][38][39][40]. Dmrt1 is essential for the differentiation of germ cells and Sertoli cells, where it is under the control of distinct transcriptional mechanisms. Stra8 controls the switch from spermatogonial differentiation to meiosis, and its 400-bp promoter is known to direct gene expression in spermatogonial stem cells. In contrast, the expression patterns, expression mechanisms, and functions of Tex13 have not previously been reported. Thus, we further investigated the promoter region of Tex13 in F9 cells. To clarify the regions responsible for Tex13 transcription, we cloned a series of 59-nested deletions in the Tex13 upstream region into the pGL3-Basic vector (21500 to + 20 bp, 2720 to +20 bp, 2402 to +20 bp, 2200 to +20 bp, 2111 to +20 bp, 295 to +20 bp, 282 to +20 bp and 265 to +20 bp) ( Figure 5A). Transient transfection of F9 cells with these constructs revealed that the highest promoter activity (423-fold that of the vector control) was associated with the 2200 to +20 bp region, whereas the lowest degree of significant activity (3-fold that of the vector control) was associated with the 265 to +20 bp region ( Figure 5A). This suggests that the 265 to +20 bp region is a minimal promoter region for Tex13 gene expression, and various additional repressive and enhancer regions are present in the 21500 to +20 bp region. Among the constructs harboring more than the 2200 to +20 bp region, the 21500 to +20 bp and 2720 to +20 bp regions, but not the 2402 to +20 bp region, significantly inhibited the maximal activity of the 2200 to +20 bp region. The degree of repression was similar between the 21500 to +20 bp and 2720 to +20 bp regions, suggesting that the negative regulatory elements are present in the 2720 to 2402 bp region. All constructs smaller than the 2200 to +20 bp region were found to exhibit gradual but significant reductions of promoter activity in F9 cells ( Figure 3A). This suggests that the 2200 to 265 bp region contains enhancer elements for Tex13 transcription. We performed a similar promoter analysis in NIH3T3 cells. In contrast to our findings in F9 cells, the promoter activity of the 2111 to +20 bp region was not reduced in these somatic cells, compared to the activity of the 2200 to +20 bp region. Thus, our results suggest that the 2200 to 2111 bp region might be implicated in F9 cell-and germ cell-preferred expression. Finally, a 19-fold decrease in luciferase activity was observed in F9 cells transiently transfected with the construct spanning the 282 to +20 bp region of the Tex13 promoter. This was the most dramatic reduction in reporter activity observed among the deletion constructs, indicating that a strong enhancer element is present in the region from 295 to 283 bp.",
"To further investigate this putative enhancer region, we generated constructs corresponding to the 295 to 283 bp region and the 2110 to 0/+20 bp regions lacking the 295 to 283 bp sequence. F9 cells were transfected with these constructs or the intact 2111 to 20 bp region (positive control), and the promoter activities of these constructs were compared ( Figure 5B and 5C). We found that the 295 to 283 bp region alone exhibited very little promoter activity ( Figure 5B), and deletion of the 295 to 283 bp region from the 2111 to 0/+20 bp region dramatically decreased the promoter activity (8-fold reduction) ( Figure 5C). These data confirm that the 295 to 283 bp region is a strong enhancer that is important for Tex13 gene expression, and further indicate that the downstream sequence (the 282 to 0 bp region) is required for the full activity of the 295 to 283 bp region in the Tex13 promoter.",
"We next performed sequence analysis of the Tex13 promoter. The promoter lacks a typical TATA box. Analysis of the 2200 to 283 bp region using the web-based tool, TFSEARH, revealed the presence of several potential transcription factor binding sites, including those for SRY (sex-determining region Y), CDX1 (caudal type homeobox 1), OCT-1 (POU domain, class 2, transcription factor 1), PRX2 (paired related homeobox 2), STATx (signal transducer and activator of transcription family), ZEB1 (zinc finger E-box binding homeobox 1) and SP1 (transacting transcription factor 1) (see Discussion). Figure 5C summarizes the data obtained from our promoter analyses.",
"Although Tex13 is not expressed in NIH3T3 cells (Figure 3A), the Tex13 promoter exhibited low but significant reporter activity in this somatic cell line ( Figure 4B and 5A). This suggests that an Figure 5. Promoter analysis of the sequence upstream of the mouse Tex13 gene. A. Promoter activity of the murine Tex13 59-flanking sequences. The Tex13 upstream region used for the reporter assay is shown on the left. Various genomic regions were introduced into the promoterless pGL3-Basic vector, and the reporter constructs were transiently transfected into F9 and NIH3T3 cells. Strong promoter activities were observed in F9 cells. The relative promoter activities are represented as the fold increase versus expression from pGL3-Basic. The presented values represent the mean 6 SD of three independent experiments. Statistical significance was determined by the Student's t-test; *p,0.05, **p,0.01. All p-values versus pGL3-Basic were less than 0.01. B. Luciferase reporter analysis of the 295 bp to 283 bp region and the 0 to +20 bp region. The indicated deletion reporter constructs were prepared (left) and transiently transfected into F9 cells. The presented values represent the mean 6 SD of three independent experiments. All p-values versus pGL3-Basic were less than 0.01. Statistical significance was determined by the Student's t-test; **p, 0.01. C. Summary of relative luciferase activity from Tex13 regulatory regions. The minimal promoter region and several enhancer/inhibitor regions are indicated upstream of the mouse Tex13 gene. The locations of putative transcription factor-binding sites between 2206 bp and 280 bp were predicted with the TFSEARCH program (arrow). The direction of each arrow indicates the orientation of the indicated putative element. The sequences of two enhancers are indicated (gray boxes). Binding sites for ZEB1 and CDX1, known to be transcriptional repressors, were predicted to be present in the 2520 to 2508 bp and 2490 to 2482 bp, respectively. doi:10.1371/journal.pone.0103837.g005 Figure 6. Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program (http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (2402/+20)] were in vitro methylated or mock-methylated with SssI methyltransferase, and transfected into F9 cells. Firefly additional mechanism is involved in the germ cell-specific expression of Tex13. Since epigenetic regulation, such as DNA methylation, is closely related to gene expression [41], we examined whether Tex13 undergoes epigenetic regulation. Sequence analysis of the proximal promoter of Tex13 revealed that there are 14 CpG sites in the region from 2383 bp to 228 bp ( Figure 6A). We therefore used bisulfite sequencing to analyze the DNA methylation of this region in F9 and NIH3T3 cells. Our analysis revealed that 47% of these CpG sites were methylated in F9 cells. Interestingly, half of Tex13 promoter in F9 cells were highly methylated, but the others were fully unmethylated ( Figure 6B). In contrast, 85% of the sites were methylated in NIH3T3 cells, with nine CpG sites in the 2141 to 228 bp region showing full methylation in NIH3T3 cells. This result suggests that DNA methylation of the 2141 to 228 bp region of the Tex13 promoter is associated with the expressional silencing of this gene in somatic cells but not in germ cells (i.e., germ cell-specific expression).",
"To determine whether DNA methylation of the Tex13 promoter induces transcriptional silencing, we performed luciferase reporter analysis of the methylated promoter in F9 cells. We incubated a pGL3-Basic vector construct containing the 2402 to +20 bp region of the Tex13 promoter with SssI (M.SssI) to methylate the cytosines of the 14 CpG sites. Promoter methylation was confirmed by methylation-sensitive restriction digest (data not shown). Compared to the mock-methylated control, the promoter activity of the in vitro-methylated 2402 to +20 bp region was significantly suppressed in F9 cells ( Figure 6C). Taken together, our results suggest that epigenetic regulation may help mediate the gene expression of Tex13.",
"Tex13 is predicted to encode a 186-amino acid protein. To explore the characteristics of Tex13 at the protein and cellular levels, we cloned its protein-coding sequence into the pEGFP-N2 vector and transiently expressed GFP-TEX13 proteins in F9 and NIH 3T3 cells ( Figure 7A and 7B). In F9 cells, we observed that GFP-TEX13 localization was heterogeneous with the 47% of the transfected cells showing the protein exclusively in the nucleus and throughout the cytoplasm and nucleus (53%) (Figure 7A and 7B). A similar distribution was also observed in NIH 3T3 cells. The GFP-signal of TEX13 was predominantly located in the nucleus (51%) or evenly distributed both in cytoplasmic and nuclear compartments (39%). The rest of the transfected cells with the GFP-signal showed cytoplasmic localization (10%). Thus, our results suggest that the endogenous TEX13 protein can shuttle between nucleus and cytoplasm.",
"In this study, we investigated the gene expression profile of the F9 mouse testicular teratoma cell line versus male germ cells. Our in silico analyses revealed a total of 964 genes that are stagespecifically transcribed in male germ cells and also expressed in F9 cells. About the half of these genes were up-regulated in spermatogonia (Spg-F9 genes). Interestingly, the enriched GO terms were found only in this group; of them, the most prominently enriched GO term was transcription regulatory activity. A number of the Spg-F9 genes with this GO term are known to function in spermatogonial survival and spermatogenesis. Furthermore, spermatogonial markers and various genes expressed in primordial germ cells are also predicted to be expressed in F9 cells. Thus, F9 cells appear to transcribe genes that are expressed/active in both differentiated spermatogonia and pluripotent germ cell genes.",
"Genes that are expressed exclusively or predominantly in male germ cells are essential for spermatogenesis. Of the germ cell genes expressed in F9 cells, our in silico and in vitro analyses identified several as having specific or predominant expression in germ cells or testis. One of them, Dmrt1, encodes a protein that is essential in germ cells for the maintenance of embryonic germ cell identity and the development of juvenile spermatogonia [35,36]. DMRT1 also inhibits meiosis in undifferentiated spermatogonia by blocking the retinoic acid-dependent robust expression of Stra8 [31], which is required for meiotic initiation in both sexes. In germ cells lacking STRA8, the mitotic development of germ cells is normal, but they fail to go through meiotic initiation or progression [38,39]. Tex13 is an X-linked gene expressed in spermatogonia [42], but little was known about the expression mechanism or function of this gene prior to this study. Dmrt1, Stra8 and Tex13 are preferentially expressed in spermatogonia compared to the other germ cell stages, whereas Triml1, Fancd2os and 1700061G19Rik are expressed majorly in spermatids. Triml1 is involved in the ubiquitin pathway [43], whereas the expression patterns and functions of Fancd2os and 1700061G19Rik are not yet known. However, these genes are expressed very weakly in F9 cells. Considering its expression pattern and unexplored function, we therefore decided to explore the transcription and function of Tex13, using F9 cells as a model system. Studies on germ cellspecific and -predominant genes have been limited by the lack of stable cell lines expressing such genes. F9 cells could therefore be an alternative tool for investigating the transcriptional regulation of such genes. Using F9 cells, we found that the 1.5-kb regions upstream of Dmrt1, Stra8 and Tex13 exhibited strong reporter activities. Furthermore, we showed for the first time that the Tex13 promoter is located 2200 to 0 bp upstream of the gene. The short sequence spanning the 295 to 283 bp region is responsible for strong promoter activity, but requires the downstream sequences for its activity. Since no known transcription factor is predicted to interact with this region, we speculate that Tex13 transcription might involve the binding of an unknown transcription factor(s) to the 295 to 283 bp region to form a complex with other transcription factors that interact with the downstream region. We also discovered an additional critical region (2200 to 2111 bp) in the proximal promoter of Tex13. Our data suggest that this region is implicated in the F9 and germ cell-preferred transcription of the gene. The SRY, CDX1, OCT-1, PRX2, STAT, ZEB1 and SP1 transcription factors are predicted to bind to the region. Of them, PRX2 is not expressed in spermatogonia [44]; ZEB1 and CDX1 are transcriptional repressors [45,46]. Putative binding sites of these repressors are also located in the repressor region 2720 to 2402 bp. OCT1 is a ubiquitous transcriptional activator [47] that also reportedly functions as a transcriptional repressor in certain genes [48]. Some of the STAT family members are important for PGC formation and spermatogenesis [49,50], while SP1 is known to mediate transcriptional activation and repression in male germ cells [51]. The 2200 to 2111 bp region associated with these transcription factors could therefore be involved in the luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean 6 SD of three independent experiments; **p,0.01 versus mockmethylated Tex13 (2402/+20). doi:10.1371/journal.pone.0103837.g006 transcriptional activation or repression of Tex13 in different cell types. Further studies are needed to determine whether this transcriptional regulation is related to the germ cell-specific expression of Tex13.",
"Methylation of cytosines at CpG dinucleotides is a well-defined mechanism of epigenetic gene silencing in mammals [41], and promoter methylation has been associated with the testis-or cell type-specific expression of certain genes during spermatogenesis [52,53]. Here, our transient transfection-reporter analysis revealed that the promoter region of Tex13 showed weak but significant activities in NIH3T3 cells, despite the lack of endogenous Tex13 gene expression in these cells. This prompted us to investigate whether Tex13 expression is regulated by DNA methylation. Bisulfite sequencing revealed that the Tex13 promoter is hypermethylated in NIH3T3 cells, but not in F9 cells. We further found that in vitro methylation of the promoter region significantly inhibited the reporter activity. Thus, differential promoter methylation may contribute to the testis-and germ cell-predominant expression of Tex13. Interestingly, half of the F9 genomic DNA samples exhibited hypermethylation in the Tex13 proximal promoter, while the others were completely demethylated. In relation to this, it is noteworthy that embryonal carcinomas and their derived cell lines, including F9 cells, are composed of an undifferentiated stem cell population and a differentiated tumor cell population. The X chromosome is methylated by X-inactive specific transcript (XIST) in differentiated germ cell tumors but not in undifferentiated germ cell tumors, as shown in the case of the androgen receptor gene [54].",
"Finally, subcellular localization is a critical characteristic used to elucidate protein functions. We found that the localization of GFP-TEX13 was heterogeneous in the cytoplasmic and nuclear compartments, suggesting that endogenous TEX13 can shuttle between the two compartments. The N-terminal region of TEX13 was predicted from sequence analysis to contain a putative nuclear localization sequence (data not shown). Nuclear-cytoplasmic shuttling plays an important role in regulating the activity of several proteins involved in cell cycle progression and proliferation, and signal transduction [55]. It should be noted that the properties of Tex13, including the subcellular localization of its encoded protein, are similar to those of Stra8. It has been reported that STRA8 associates with DNA and possesses transcriptional activity [56], and a mouse knockout study found that STRA8 is crucial for meiotic progression during spermatogenesis [38,39]. Based on our findings, we speculate that TEX13 may act as a transcription factor or cooperate with transcription factors to regulate the expression levels of genes involved in early germ cell development. A previous proteomic study reported the presence of the TEX13 protein in early male germ cells [57]. Further investigations are needed to determine the precise function of TEX13 in male germ cell development.",
"In conclusion, we herein report a comprehensive analysis of genes transcribed in both F9 cells and male germ cells. Our data suggest that F9 cells can transcribe genes that exhibit stage-specific expression in early germ cells. In particular, we identified a F9 cellexpressed germ cell-specific gene, Tex13, and used the cell line to characterize its promoter and potential epigenetic regulation. Finally, we found that TEX13 is a potential nucleocytoplasmic shuttling protein. Our results collectively establish a basis for future investigations into the transcriptional mechanism of Tex13 and the nuclear function of its encoded protein. Figure S1 Expression profile of genes known to be transcribed in spermatogonia and primordial germ cells. Heatmap of the normalized gene expression profiles of genes is shown. Up-regulated genes are indicated in red and downregulated genes are shown in green. Abbreviations: J1, J1 embryonic stem cells; Spg, type B spermatogonia; Spcy, pachytene spermatocyte; Sptd, round spermatid; and F9, F9 cells. Genes: Plzf, promyelocytic leukemia zinc finger ortholog; Dmrt1, doublesex and mab-3 related transcription factor 1; Stra8, stimulated by retinoic acid gene 8; Gfra1, glial cell line derived neurotrophic factor family receptor alpha 1; Klf2, kruppel-like factor 2; Lin28, RNA-binding protein LIN-28; Nanog, nanog homeobox; Sox2, SRY (sex determining region Y)-box 2; Pou5f1, POU domain, class 5, transcription factor 1; Tcfap2c, transcription factor AP-2, gamma; Prdm14, PR domain containing 14; Prdm1, PR domain containing 1, with ZNF domain. (PDF) "
] | [] | [
"Introduction",
"Materials and Methods",
"Microarray data analysis",
"Reverse transcription PCR",
"Nfe2l2",
"Cdkn2a",
"Dmrt1",
"Generation of reporter gene constructs",
"DNA transfection and luciferase assay",
"Purification of genomic DNA and bisulfite methylation assay",
"In vitro methylation",
"Localization of recombinant TEX13 in F9 cells",
"Results",
"Identification of germ cell genes expressed in F9 cells",
"Identification of testis-specific or -predominant genes expressed in F9 cells",
"Promoter analysis of germ cell genes in F9 cells",
"Characterization of the Tex13 promoter",
"DNA methylation analysis of the Tex13 promoter",
"Expression and localization of the TEX13 protein",
"Discussion",
"Supporting Information",
"Figure 3 .",
"Figure 4 .",
"Table 1 .",
"Table 3 .",
"Table S1",
"Table S2",
"Table S3",
"Table S4"
] | [
"GO term \n# of genes \nP-value \nFold enrichment \n\nTranscription regulatory activity \n57 \n9.90E-06 \n1.83 \n\nDevelopmental processes \n117 \n1.70E-08 \n1.62 \n\nDeath \n22 \n0.03 \n1.61 \n\nBiological adhesion \n23 \n0.03 \n1.56 \n\nReproductive processes \n25 \n0.03 \n1.53 \n\nReproduction \n25 \n0.03 \n1.52 \n\nCellular component organization \n68 \n0.0053 \n1.37 \n\nLocalization \n84 \n0.04 \n1.21 \n\nBiological regulation \n213 \n4.99E-04 \n1.17 \n\nBinding \n344 \n5.36E-10 \n1.17 \n\nOrganelles \n249 \n0.002 \n1.13 \n\nCellular processes \n264 \n0.098 \n1.08 \n\nCell parts \n405 \n0.04 \n1.02 \n\nCells \n405 \n0.04 \n1.02 \n\nUnknown \n58 \n-\n-\n\ndoi:10.1371/journal.pone.0103837.t001 \n\n",
"Array pattern \nGene \nUniGene ID \nTestis-specificty (%) \nTissue \n\nSpermatogonia-F9 \nDmrt1 \nMm.391208 \n96.88 \nEmbryonic tissue, testis \n\nTex13 \nMm.193025 \n75 \nEmbryonic tissue, testis \n\nNr5a1 \nMm.31387 \n51.79 \nBrain, embryonic tissue, fertilized ovum, lung, \novary, spleen, testis \n\nStra8 \nMm.5171 \n34.62 \nEmbryonic tissue, testis \n\nSpermatocyte-F9 \nSp2 \nMm.155547 \n62.07 \nEmbryonic tissue, testis, thymus \n\nSpermatid-F9 \nCpvl \nMm.158654 \n100 \nTestis \n\nFancd2os \nMm.72959 \n91.67 \nKidney, testis \n\n1700061G19Rik \nMm.160106 \n85.16 \nEmbryonic tissue, intestine, testis \n\nTriml1 \nMm.64542 \n56 \nEmbryonic tissue, extra embryonic tissue, testis \n\ndoi:10.1371/journal.pone.0103837.t003 \n",
"Author Contributions \n"
] | [
"Table S1",
"Table S1",
"Table S2",
"Table S3",
"(Table S4",
"(Table 1",
"(Table 2)",
"(Table 3"
] | [
"Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line",
"Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line"
] | [
"PLoS ONE"
] |
9,059,230 | 2022-03-22T16:51:58Z | CCBY | http://www.oncotarget.com/index.php?journal=oncotarget&op=download&page=article&path[]=20713&path[]=65991 | GOLD | 8a796cafd107c94173749a18090b7891369d1790 | null | null | null | null | 10.18632/oncotarget.20713 | 2751238974 | 29152094 | 5675646 |
Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma
2017
Mari Kogiso
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Lin Qi
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Holly Lindsay
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Yulun Huang
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Department of Neurosurgery
The First Affiliated Hospital
Soochow University
SuzhouChina
Xiumei Zhao
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Department of Ophthalmology
First Affiliated Hospital of Harbin
Medical University
HarbinChina
Zhigang Liu
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Department of Radiotherapy
The Affiliated Cancer Hospital of Xiangya School of Medicine
Hunan Cancer Hospital
Central South University
ChangshaChina
Frank K Braun
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Yuchen Du
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Huiyuan Zhang
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Goeun Bae
Center for Translational Cancer Research
Institute of Biosciences and Technology
Texas A&M College of Medicine
HoustonTXUSA
Sibo Zhao
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Sarah G Injac
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Mary Sobieski
Center for Translational Cancer Research
Institute of Biosciences and Technology
Texas A&M College of Medicine
HoustonTXUSA
David Brunell
Center for Translational Cancer Research
Institute of Biosciences and Technology
Texas A&M College of Medicine
HoustonTXUSA
Vidya Mehta
Department of Pathology
Baylor College of Medicine
Texas Children's Hospital
HoustonTXUSA
Diep Tran
Department of Pathology
Baylor College of Medicine
Texas Children's Hospital
HoustonTXUSA
Jeffrey Murray
Department of Hematology and Oncology
Cook Children's Medical Center
Fort WorthTXUSA
Patricia A Baxter
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Xiao-Jun Yuan
Department of Hematology and Oncology
Xinhua Children's Hospital
ShanghaiChina
Jack M Su
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Adekunle Adesina
Department of Pathology
Baylor College of Medicine
Texas Children's Hospital
HoustonTXUSA
Laszlo Perlaky
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Murali Chintagumpala
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
D Williams Parsons
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Ching C Lau
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Clifford C Stephan
Center for Translational Cancer Research
Institute of Biosciences and Technology
Texas A&M College of Medicine
HoustonTXUSA
Xinyan Lu
Department of Pathology
Northwestern University Feinberg School of Medicine
ChicagoILUSA
Xiao-Nan Li
Department of Pediatrics
Baylor College of Medicine
Texas Children's Cancer Center
Texas Children's Hospital
HoustonTXUSA
Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma
Oncotarget
8502017Received: March 16, 2017 Accepted: August 15, 2017 source are credited.Oncotarget 87455 * These authors have contributed equally to this study Correspondence to: Xiao-Nan Li, Copyright: Kogiso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and Research Paper Oncotarget 87456low grade gliomaorthotopic xenograftcancer stem cellBRAF V600ECDKN2A
To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133 + (<1.5%) and CD15 + (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP -/Vimentin + cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. www.impactjournals.com/oncotarget/ Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP -/Vimentin + cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.
INTRODUCTION
Pediatric low grade gliomas (PLGGs) are slow growing tumors accounting for 1/3 of all childhood brain tumors [1]. Although complete surgical removal results in cure in >90% of patients, some tumors still recur [1][2][3], especially after sub-total resection. Currently, driver(s) of recurrence and malignant progression remain to be elucidated. Mouse models that replicate key biological features of PLGG are highly desired to identify mechanism of recurrence/malignant degeneration and enable preclinical studies of PLGG. We have shown that direct injection of fresh surgical specimens into anatomicallymatched locations in the brains of immunodeficient mice facilitates establishment of clinically-relevant orthotopic xenograft mouse models that replicate the histology, invasive growth, and key genetic features of primary patient tumors [4][5][6][7][8]. The added advantage of patientderived orthotopic xenograft (PDOX) mouse model is that the normal brain responses toward xenograft growth, which is difficult to obtain from patient surgical samples, can be analyzed simultaneously together with brain tumor cells. PDOX mouse models of PLGGs, however, have not been previously established.
Accumulating evidence demonstrates that cancer stem cells (CSCs) play an important role in tumorigenicity, cancer initiation and recurrence [9][10][11][12][13][14]. CD133 and CD15 are two well-characterized cell surface markers that define pediatric glioblastoma and medulloblastoma CSCs [8,9,[14][15][16][17][18]. Despite ongoing controversies about the relative abundance and specificity of these markers [19][20][21], CD133 + brain tumor stem cells are chemotherapy-and radiation-resistant [13,22], and their frequency correlates with adverse survival in adult glioma [23]. In contrast, little is known about CSCs in low grade tumors. Only a few cases have been analyzed for CD133 + cells, revealing variable abundance ranging from undetectable [24] to 37% [25]. The content and function of CD15 + cells in PLGGs is still unknown.
Genetic analysis identified BRAF as a frequent mutation target in PLGGs, including BRAF V600E mutation [26][27][28][29][30][31][32], duplication [33] and gene fusion [28][29][30][31][32][33][34]. BRAF V600E mutation were found in WHO grade II PXA (66%), PXA with anaplasia (65%), grade I GG (18%) and grade I PA (9%) [29]. Homozygous deletions involving the CDKN2A/p14ARF/CDKN2B loci were detected in 60% of PXA [35] and 71% of malignant astrocytomas [36]. These reports suggest contribution of BRAF V600E mutation and CDKN2A deletion to tumor progression and should be targeted. Indeed, multiple novel inhibitors against BRAF V600E mutation have been developed and entered into clinical trials in patients with advanced melanoma, hairy cell leukemia, and thyroid cancers [37][38][39][40]. Developing new PLGG models replicating such druggable mutation would be highly desired not only to understand the functional role of BRAF V600E mutation in driving PLGG recurrence, but also for future examination of drug resistance as has been noted in melanomas [37].
In this report, our goals were to determine if PDOX models can be established from low grade gliomas, whether CSCs are present in PLGG and if their frequencies correlate with in vitro self-renewal, in vivo formation of orthotopic xenografts, and clinical tumor recurrence. To gain insight into in vivo tumor evolution and progression, we examined if the histopathological features and, more importantly, the progression nature of the original patient tumor were replicated in the PDOX tumors during longterm serial subtransplantations in mouse brains, followed by the analysis of the underlying cellular and molecular (e.g. BRAF V600E mutation and CDKN2A deletion) changes in tumor cells and in the host normal brain cells that drove or accompanied the PDOX tumor progression to identify new therapeutic targets.
RESULTS
The overall yields of tumor cells from childhood LGG were low
Despite extensive collaborative effort, the tumor tissues obtained for PLGGs were still limited, frequently less than 3 x 3 x 3 mm 3 (Table 1). Using a combination of mechanical dissociation and combined collagenase/ halogenase enzymatic digestion, we were able to collect viable tumor cells up to 4.3 x 10 6 cells (1.3 x 10 6 ± 1.1 x 10 6 ). The number of assays per PLGG sample was therefore performed depending on the tumor cell availability.
Attempts to establish neurosphere and monolayer cultures from patient tumors
To examine if PLGGs contain cells able to form neurospheres in vitro, dissociated cells from 15 tumors Note: * Tested for neurosphere formation, ¶ Analyzed with FCM for CSC markers, Dx= diagnosis, ICb=Intracerebellar, IC=Intra-cerebral, PA=Pilocytic astrocytoma, AST=astrocytoma, GG=Ganglioglioma, PXA = Pleomorphic Xanthoastrocytoma (Table 1) were plated in serum-free medium containing Neurobasal medium, EGF/bFGF to favor CSC growth [7,8,15]. Sustained neurosphere growth was unsuccessful from any tumor after 19 to 57 (37.6 ± 15.2) days observation. Additionally, 11 patient tumors with >2 x 10 6 cells were incubated in FBS-based media. Again, none attached or expanded in culture. These data suggested that PLGG cells harvested directly from patients did not readily adapt to in vitro growth conditions and failed to proliferate.
Only 1 of 25 PLGGs formed orthotopic xenograft tumors
We tested if rapid return of PLGG cells to anatomically-matched locations in mouse brains would form tumor. 20 cerebellar tumors and 5 cerebral PLGGs were implanted into matching locations in mouse brains either intra-cerebellarly (ICb) or intra-cerebrally (IC) (Table 1) as we described previously. No visible tumor formation was detected in 24 of 25 PLGGs after average of 230 days observation. H&E staining of paraffinembedded brains also did not detect tumors, although scarring and disturbed normal brain structure indicative of previous surgical implantation were seen ( Figure 1A). Subsequent IHC examination revealed reactive astrocytes with strong GFAP positivity surrounding the needle track but did not detect human cells in mouse brains using human-specific MT antibodies, further confirming the lack of xenograft tumor formation ( Figure 1A). Only IC implantation of 3635 pleomorphic xanthoastrocytoma (PXA) patient tumor formed orthotopic xenograft tumors; this model was designated IC-3635PXA and was serially sub-transplanted in mouse brains four times (passage IV) ( Figure 1B). Similar to patient tumor, xenograft cells from passages I to III were positive for human-specific MT in tumor core, invasive foci, and disseminated cells ( Figure 1B), validating the human origin of xenograft tumors.
The PDOX model-initiating patient tumor was pathologically assessed at diagnosis by the local anatomic pathologist and was subsequently reviewed by our institutional neuro-pathologist. The tumor was confirmed to be a pleomorphic xanthoastrocytoma (2016 Central Nervous System (CNS) World Health Organization (WHO) grade II) as it was moderately cellular and infiltrative but with a low proliferation index and no necrosis or anaplasia [41]. Rare sections of the tumor were suspicious for developing vascular proliferation. Pathologic examination of xenograft passage I revealed the presence of a 2016 CNS WHO grade II PXA with no vascular proliferation or necrosis and very low mitotic activity [41]. Xenograft passage II tumors remained grade II but demonstrated increased cellularity, though the proliferation index remained low. At xenograft passage III, tumors had a notable pathologic shift with developing of higher grade features including regional necrosis pseudopalisading necrosis, increased vascular and endothelial proliferation, increased tumor cell infiltration into normal brain. The tumor's proliferative index had increased from <5% to approximately 20%. Xenograft passage IV had even further increases in tumor cellularity and had unequivocally transformed into a high grade tumor.
Tumor take rate of IC-3635PXA increased during serial in vivo sub-transplantations Initial tumor take rate upon implantation of patient tumor into SCID mice was low, as only 2 of 7 (28.5%) mice initially implanted with 3635PXA patient tumor formed tumors. Over in vivo sub-transplantations, tumor take rate steadily increased: 28.5% (2/7) in passage I, 44.4% (4/9) in passage II, 100% (7/7) in passage III, and 88.9% (8/9) in passage IV ( Figure 1B). Presence of normal cells in the patient tumor and the multiple round selection of "pure" and more aggressive clonal cell populations might have played a role.
PDOX tumor cells survived and proliferated in vitro
We also attempted to establish cell lines from IC-3635PXA xenografts. Monolayer cells, labeled Baylor xenograft derived (BXD)-3635PXA-mono, grew in FBSbased media, although the proliferation rate of these cells was slow (21-28 days/passage and reached passage 25 after 586 days). In serum-free media, 3635PXA cells initially formed neurospheres (BXD-3635PXA-NS) but after passage 5 became attached, exhibiting a star-like morphology with occasional formation of small clusters (7-18 days/passage and reached passage 32 after 586 days) ( Figure 1D). PDOX tumor cells may have a better chance of survival in vitro than the patient tumors.
PLGGs contain high levels of CD15 + cells and low levels of CD133 + cells
To determine if lack of putative CSCs was the cause of low xenograft tumor formation, we assessed CD133 and CD15, two common brain tumor CSC markers, in 22 patient tumors using flow cytometry (FCM) ( Figure 2). PLGGs were found to have abundant CD133 -/CD15 + cells (20.7 ± 28.9%) (Figure 2A and 2B). In 3 of 18 (17%) pilocytic astrocytomas (PAs), 1 of 1 (100%) grade II astrocytoma (AST), 2 of 2 (100%) gangliogliomas (GGs), and 1 of 1 (100%) PXA, CD133 -/CD15 + cells accounted for >30% of the total cell population ( Figure 2B), demonstrating CD15 + cells in PLGGs for the first time. Compared with high levels of CD133 + /CD15cells in a childhood GBM xenograft tumor included as positive control (Figure 2A), only low levels (0.46 ± 0.57%) of www.impactjournals.com/oncotarget CD133 + /CD15 -PLGG cells were detected in 22 tumors ( Figure 2B).
We subsequently analyzed CD133 + and/or CD15 + levels in 3635PXA and the resulting xenograft tumors (passages I through III). Similar to other PLGGs, CD133 + / CD15 + and CD133 + /CD15were barely detectable (<1%), while CD133 -/CD15 + cells were the major subpopulation, accounting for 64.1% in patient tumor ( Figure 2B). CD133 -/CD15 + xenograft tumor cells decreased to 9.6% in passage I, 10.3% in passage II and 5.9% in passage III ( Figure 2B), suggesting that they may not be driving in vivo tumor formation.
In order to correlate the content of putative CSC cells with clinical tumor progression, clinical outcomes of 22 patients with PLGG were followed for up to 6 (4.8±1.6) years. Among the four progressive tumors, one GG and one PXA had high CD15 + cells (>60%) but two PAs harbored only 10% and 1% CD15 + cells ( Figure 2B). Altogether, these data suggested that tumorigenicity in mouse brains and clinical progression of PLGGs may not be solely driven by putative CSCs.
BRAF V600E mutation increased during IC-3635PXA progression
Genomic DNA from 3635PXA patient, xenograft tumors and cultured cells was subjected to quantitative mutation detection using BRAF V600E specific pyrosequencing. Compared to 28% in patient tumor, BRAF V600E mutant allele frequency increased in xenografts (67%, 70%, and 67% in passage I, II and III, respectively) ( Figure 3A and 3B). In vitro xenograft tumor cell cultures also had high BRAF mutation frequencies with 66% (p-4), 69% (p-21) in monolayer and 67% (p-5 and p-21) in neurospheres. These observations suggest that BRAF V600E mutation plays an important role in tumor progression, although the molecular mechanisms underlying the increased allele frequency of BRAF mutation frequency remains to be determined.
IC-3635PXA PDOX showed persistent CDKN2A deletion and increasing trisomy 9
Oncogenic BRAF mutation and CDKN2A inactivation characterize a subset of pediatric malignant gliomas [36]. We thus examined if CDKN2A deletion played a role in IC-3635PXA progression. Utilizing Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes ( Figure 4A), we analyzed CDKN2A status in paraffin sections of patient and xenograft tumors and cultured cells derived from IC-3635PXA. In the patient sample, 13% of the cells contained 2 red (R=CDKN2A) and 2 green (G=chromosome 9 centromere) (2R2G in Figure 4B) that are found in normal cells, most patient tumor cells (87%) exhibited homozygous deletion either with disomy chromosome 9 (0R2G) (51%) or trisomy 9 (0R3G) (17.5%). As expected, there were no normal 2R2G human cells in xenograft tumors. In xenograft passage I, homozygous deletion of CDKN2A was found in 77.5% cells with 0R2G, 17% cells with 0R3G, and 5% cells with quadrisomy 9 (0R4G). Subsequent analysis of xenograft tumors revealed a gradual decrease of 0R2G cells with CDKN2A deletion, i.e. 58%, 51%, and 31% in passages II, III and IV, accompanied by increased 0R3G cells from 18% in passage II, to 37% in passage III and 54% in passage IV ( Figure 4C). These data suggested that a sub-population of trisomy chromosome 9 PLGG cells with CDKN2A deletion gained growth advantage over disomy 9 cells.
Cultured xenograft tumor cells also maintained the homozygous CDKN2A deletion ( Figure 4B). The relative abundance of 0R2G decreased from 62.5% in the monolayer cells to 29% in neurospheres while 0R3G and 0R4G cells increased from 17% to 25%, and from 11% to 45%, respectively ( Figure 4C). These data demonstrated that homozygous deletion of CDKN2A, found in 68.5% of 3635PXA patient tumor cells, was well maintained and increased to 100% in the PDOX model and cultured xenograft cells; interestingly, monolayer cells were enriched with disomy 9 cells while neurospheres favored the growth of trisomy and quadrisomy 9 tumor cells.
PDOX tumor evolution was paralleled by loss of GFAP and gain of Vimentin (VIM) expression
To identify cellular changes accompanying increased tumor take rate and the increased BRAF mutation, we performed IHC staining of PDOX tumors ( Figure 1C and Table 2). First, compared with low cell proliferation index (Ki-67) of <2% in patient tumor, xenograft tumors from IC-3635PXA exhibited Ki-67 positivity at 5%-10% in passage I and 5%-15% in passage III. Second, while the majority of patient tumor cells (>95%) were strongly positive (+++) for GFAP, a marker of mature/differentiated glial cells, there was a steady decrease of GFAP + cells in PDOX tumors (26-50%). Third, expression levels of VIM, a marker of intermediate filament associated with poor prognosis and tumor invasion [42][43][44], increased. While high (+++) VIM expression was seen in only a low percentage (~10-30%) of patient tumor cells, nearly all xenograft cells exhibited strong (+++) positivity in passages I and III. In addition to cells in the tumor core, invasive tumor cells (single cells, micro-satellites, and perivascular spread) were also strongly (++++) VIM positive ( Figure 5A). These data suggested that a subpopulation of GFAP -/VIM + cells were enriched during PDOX tumor formation, sub-transplantation, and tumor progression in vivo.
To validate the role of VIM + cells in PLGG invasion and progression, we collected 5 pairs of matched primary and recurrent PLGGs and compared the expression of GFAP and VIM with IHC (Table 3). In 4 of the 5 tumor pairs (#3319, 9036, 1576, 2022), there were no major differences between primary and recurrent tumors, as nearly all cells were positive for GFAP and VIM (Table 3). In recurrent tumor #1614, cells in the tumor core displayed strong (+++) positivity of GFAP and VIM while a small piece of normal tissue demonstrated significant numbers of VIM + /GFAPtumor cells ( Figure 5D, l and o, m and p). This identification of VIM + /GFAPcells in the invasive front of this recurrent tumor was in agreement with our observations in the IC-3635PXA tumors.
In vivo PDOX progression was paralleled by increased perivascular dissemination
We next examined changes in IC-3635PXA cell invasion in order to guide development of new therapies preventing metastasis. In addition to single cell invasion into neighboring normal brain and neural fibers ( Figure 5A, a and b), there was frequent distant dissemination of VIM + PDOX cells via blood vessels deep into normal brain ( Figure 5A, c and d). Using a straight line reticle (eyepiece micrometer), perivascular migration in mouse cerebral was measured. Migration increased from 140 ± 68.3 μm in passage I, to 182 ± 152, 429 ± 226, and 392 ± 237 μm in passages II, III and IV, respectively (P <0.01) ( Figure 5B). Longest migratory distance also increased from 350 μm in passage I, to 570, 1,370 and 1,100 μm in passages II, III and IV, respectively. Starting from passage II, we also observed perivascular invasion into brain stem, presumably due to the close proximity of tumor mass ( Figure 5B). Since the chances of escaping surgical resection is positively correlated with distance of tumor cell invasion into normal brain, such progressive increases of PXA tumor cell migration into distant normal brains suggested perivascular invasion as a potential cause of tumor recurrence and metastasis.
Tumor growth triggered intensive reactive gliosis in normal mouse brain
Since human normal brain tissues must be preserved during surgical resection of brain tumors, little is known about the reaction of normal glial cells to the growth of brain tumors, particularly in areas far away from the tumor mass [45]. Using IC-3635PXA, we examined responses of normal glial cells both adjacent to and distant from the primary tumor to infiltration and dissemination of PXA tumor cells. IHC staining for GFAP (which recognizes both human and mouse astrocytes) detected wide-spread presence of GFAP + astrocytes resembling reactive astrocytes [46]. All GFAP + astrocytes cells were negative for human-specific VIM and MT, confirming the murine origin of these cells. Growth of xenograft tumors triggered gliosis in host mouse brains not only at the tumor-brain interface ( Figure 5C, e-i) but also deep into normal mouse brains beyond the leading edge of tumor invasion, including in the contralateral mouse cerebrum where no human tumor cells were evident ( Figure 5C, f, g, and i). Since dynamic interactions between PLGG cells and microenvironment remain poorly defined, PDOX model IC-3635PXA can be used for future understanding of host responses (molecular mechanisms and biological impacts) toward the growth of PXA.
In vitro anti-tumor activities of BRAF V600E inhibitors
As a proof of principle, we exposed the cultured BXD-3635PXA cells to vincristine (a chemotherapy agents) and a series of inhibitors that target BRAF V600E (n=7), BRAF wild-type and/or RAF genes (n=9) (Supplementary Table 1) and examined their anti-proliferation activities. After 7 days of in vitro treatment (0-10 μM), cell proliferation was suppressed by vincristine (IC50=0.97 μM), 2/7 (28.5%) BRAF V600E inhibitors, PLX-4720 (IC50= 0.13µM) and Dabrefenib (IC50=1.58µM), and 2/9 (22.2%) inhibitors that target BRAF or RAF genes, GDC-0879 (IC50=0.65 μM) and Sorafenib (IC50=1.74 μM) ( Figure 6). The remaining 12 inhibitors, including 5 BRAF V600E inhibitors, were not active during the treatment period. These data suggested that targeting BRAF V600E was effective in suppression BXD-3635PXA cell proliferation. The efficacy of inhibitors, however, was not identical.
DISCUSSION
In this study, we demonstrated that the tumor take rate of PLGGs is very low as only 1 of the 25 tumors formed transplantable orthotopic xenograft tumors in mouse brains. To determine if the lack of cancer stem cells played a role, we analyzed putative cancer stem cells through FCM analysis of dual-stained putative cancer stem cell markers (CD133 and CD15). While the levels of CD133 + cells were much lower (<1%) than a previous report demonstrating 15% CD133 + cells in one childhood PA and 8% in two adult grade II ASTs [24], they appear to be correlated with low tumorigenicity of PLGGs. To validate the role of CD133 + cells in PLGG tumorigenicity, direct implantation of isolated CD133 + PLGG cells should be tested in mouse brains, although low tumor cell abundance coped with low overall tumor cell yields often make it difficult to collect sufficient CD133 + cells for this in vivo assay. Although 7 of the 22 PLGGs expressed high levels (30% -80%) of CD15 + cells, which has been previously detected in medulloblastoma and adult malignant gliomas [16][17][18], only one of these PLGGs (IC-3635PXA) formed xenografts. Since all of the PLGGs were injected into anatomically-matched locations in mouse brains, our results indicated that the functional role of CD15 in PLGGs might be different from that in the high-grade gliomas. Detailed functional validation is needed for existing and new CSC markers of PLGGs.
While the majority of children with LGGs remain tumor-free, a small fraction of PLGGs do recur over time. Malignant transformation, which is rare in pilocytic astrocytomas (2.4% of 288 patients) [47] and more often in grade II PXA (36%) [48,49], might have played a role. In this study, we confirmed that IC-3635PXA replicated the histology and key genetic abnormalities of the original patient tumor. More importantly, we showed that, for the first time, the progressive/metastatic growth of a PXA mouse model replicated the progressive and metastatic nature of the originating patient tumor, as evidenced by the increased tumor take rate, elevated cell proliferation and expanded local invasion and perivascular metastasis over serial in vivo sub-transplantations. Similar to most recurrent and late stage tumors, no surgery or biopsy was performed in 3635PXA patient tumor at the last recurrence, making it difficult to identify the cellular and molecular changes and/or drivers of tumor recurrence. Using our new PDOX model, we were able to detect the cellular drivers of tumor progression, i.e. GFAP -/VIM + tumor cells. Nearly all the xenograft tumor cells became VIM positive starting from passage I with the strongest positivity found in the infiltrating tumor edge. A significant elevation of VIM expression was also confirmed in one paired primary and recurrent PLGGs. Since VIM has been involved in attachment, migration, and cell signaling [50], our results justified additional studies to validate the functional role of GFAP -/VIM + tumor cells in PXA (and other PLGGs) recurrence. TC= tumor core; INV= invasion; Scored intensity as negative (-), low (+), medium (++), strongly positive (+++) and highly strongly positive (++++) and extent of immunopositivity as 0=negative; 1=1-25%; 2=26-50%; 3=51-75%; 4=>75% positive cells. * Microvessel Density (mean ± SD). nd = not done.
Molecularly, we provided new data to support the role of BRAF V600E mutation and CDKN2A deletion in driving PXA progression. Previous studies have shown that BRAF V600E and CDKN2A alterations were less commonly observed in PLGG that did not transform [51], but more frequently detected in secondary high grade gliomas. Unlike KIAA1549-BRAF fusion transcripts which were near exclusively expressed in grade I astrocytomas, BRAF V600E mutation was detected in 22.6% of grade II-IV tumors but in none of the grade I tumors [36]. In secondary high glade gliomas, BRAF V600E and CDKN2A deletion were as high as 39% and 57%, respectively [51]. Since 3635PXA patient tumor contained 13% normal diploid cells, the corrected BRAF V600E mutation rate increased from 28% to 32.18% (i.e., 28/87=32.18%), it is still much lower than that in the xenograft tumors (>67%). Our long-term followup of in vivo growth of IC-3635PXA tumors revealed a clear enrichment of BRAF V600E mutation frequency and detected a novel a shift of diploid chromosome 9 to trisomy 9 in tumor cells bearing CDKN2A deletion. Since copy number of chromosome 9 increased despite CDKN2A deletion, addition studies (preferably in more than 1 models) are needed to examine if CDKN2A deletion is associated with the in vivo progression and in vitro growth of neurospheres, and if amplified chromosome 9 is involved in and/or drive tumor progression in PXA. Since BRAF V600E mutant gliomas often develop acquired resistance to FDA approved small molecule inhibitor [32], our PDOX model would be a powerful tool to conduct preclinical testing of new BRAF V600E targeting inhibitors.
Understanding the mode of tumor invasion should provide new clues to guide the development of new therapies preventing metastasis. In addition to migrating as a single cell and along neural fibers, our data showed that PXA xenograft cells achieve distant dissemination through perivascular spaces far beyond the leading edges of intra-parenchymal invasion. The increased frequencies and distances of perivascular migration, which paralleled the xenograft tumor progression over serial in vivo subtransplantations, would render early radiographic detection and surgical resection nearly impossible. However, since these invasive tumor cells in the perivascular space need to breach the blood brain barrier through disruption of astrocytic endfeet, which envelope vessels, to spread away from tumor core, they might have also damaged the integrity of the local blood brain barrier, potentially making themselves vulnerable to chemotherapies. This newly discovered mode of PXA invasion in vivo has thus provided a therapeutic opportunity to target the distant perivascular invasion that are left behind after surgery and potentially cause tumor recurrences. Using this PDOX model, we also had an opportunity to examine the responses of host (normal) brain cells to the hetero-transplanted PXA tumors, particularly in the areas far away from the tumor mass whereas corresponding studies in patients are not feasible. To the best of our knowledge, the widespread reactive gliosis we observed in the normal mouse brain tissues has not been previously described in human PXA or other types of PLGGs. Reactive gliosis, also known as astrogliosis, refers to the morphological and biochemical changes of astrocytes occurring in association with injury or disease. Given the benign nature of PLGGs, it is surprising to detect such an outbreak of hypertrophic and GFAP + reactive astrocytes [45], which have been found to alter the expression or function of neuronal proteins involved in excitability and may serve as drivers of epileptogenesis in acquired epilepsies [52,53]. Indeed, our 3635PXA patient developed severe epilepsy at tumor recurrence, which highlighted the need and the potential use of our IC-3635PXA model for to decipher the molecular mechanism of reactive gliosis and to identify potential therapeutic targets to improve quality of life by suppressing/ preventing epilepsy.
Since development of new therapy is one of the most important goals of model development, we tested the anti-tumor activities of a series of inhibitors specific to BRAF V600E or to wild-type BRAF or other types of RAF gene/pathways in vitro. In addition to identify a set of 4 inhibitors (2 each for BRAF V600E and BRAF) that suppressed cell proliferation, we also found 5 BRAF V600E inhibitors and 7 BRAF inhibitors that were not active. These findings are interesting as they revealed
GFAP VIM
that differential activities among BRAF V600E and wild-type inhibitors, which not only suggested the need of "personalized" drug testing, but also highlighted the potential use of our model for the examination of underlying mechanisms of action and resistance.
In conclusion, our studies demonstrated that PLGGs has very low tumorigenic capacity in the brains of SCID mice, which is similar to the frequency of tumor progression and recurrence in pediatric patients. Low abundance of CD133 + cells appears to be correlated with such low tumor take rate. The PDOX model IC-3635PXA not only replicated the histological and molecular phenotypes, but also, and more interestingly, evolved in vivo replicating the progressive growth of the originating patient tumor. Using this novel PDOX model, we have identified VIM + GFAPcells as candidate cellular drivers, BRAF V600E mutation and CDKN2A deletion (in cells with trisomy and quadrisomy chromosome 9) as key molecular changes that mediated the progression, invasion and migration of the xenograft tumors over long term in vivo subtransplantations, and showed that a subset of BRAF V600E inhibitors were indeed active in suppression cell proliferation in vitro. This novel model will serve as an important resource to support further biological and pre-clinical studies (such as BRAF V600E mutation) in pediatric PXA.
MATERIALS AND METHODS
Childhood LGG tumor tissues
Freshly resected LGG tumor specimens from 36 children undergoing craniotomy at Texas Children's Hospital and member hospitals of the Texas and Oklahoma Pediatric Neuro-Oncology Consortium were obtained for this study (Table 1). Signed consent was obtained prior to sample acquisition following Institutional Review Board-approved protocols. As described previously [7,8], fresh tumor tissues were washed and dissociated with the Automatic Tissue Dissociator (Miltenyi Biotec), followed by collagenase/halogenase enzymatic digestion.
Tumor #3635 was obtained from a 9 year-old girl receiving subtotal resection of an extensive left temporal tumor. Pathologically, it was diagnosed as PXA with ganglioglioma component (WHO grade II), BRAF V600E mutation and low cell proliferation index (<2%). Two month later, she received more complete subtotal resection. The tumor, however, progressed along with disseminated neuraxis metastasis 5 months from the initial diagnosis. Despite treatment with palliative craniospinal radiatioin along with daily adjuvant temozolomide chemotherapy during radiation therapy, she developed severe epilepsy, post-chemoradiotherapy pancytopenia, speptic shock and passed away 7 months after diagnosis.
Flow cytometry
PLGG tumor cells were labeled with APCconjugated human CD133 antibody and FITC-conjugated human CD15 antibody (Miltenyi Biotec), or isotype control antibodies at 4°C for 15 minutes in FCM buffer comprised of DPBS, 0.5% BSA and 2 mM EDTA. After washing, cells were re-suspended in FCM buffer containing 2 μg/mL propidium iodide (PI) and analyzed with a LSR II flow cytometer and Kaluza Analysis Software Version 1.3 (Beckman Coulter). Dead cells were excluded by PI staining.
In vitro growth of PLGG cells
For neurosphere assays, dissociated PLGG tumor cells were plated at clonal density (1,500 cells/100 μL) and incubated in serum-free media consisting of Neurobasal media, N-2 and B-27 supplements (0.5x each) (Life Technologies), human recombinant basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) (50 ng/mL each) (R&D Systems) [8,54], and 200 units/mL penicillin/streptomycin. Additional cells were seeded in DMEM media supplemented with 10% fetal bovine serum (FBS) and 200 units/mL penicillin/streptomycin. Cells were incubated in 5% CO 2 at 37°C. Media were changed every three days and cell growth examined under phase contrast microscopy.
Direct orthotopic transplantation of patient tumor cells into mouse brain
NOD/SCID mice were bred and housed in a pathogen-free animal facility. All experiments were conducted following an Institutional Animal Care and Use Committee-approved protocol. Mice of both gender, aged 6-8 weeks, were anesthetized with sodium pentobarbital (50 mg/kg, i.p. injections). Tumor cells from 25 PLGGs were re-suspended in DMEM growth medium at 5 x 10 7 live cells/mL and injected (1 x 10 5 cells in 2 μL) orthotopically into mouse brains as described previously [7,8,55]. Cerebellar tumors were implanted into right cerebellum (1 mm to the right of the midline, 1 mm posterior to the lamboidal suture, and 3 mm deep), and cerebral PLGGs into right cerebral hemisphere (1 mm to the right of the midline, 1.5 mm anterior to the lamboidal suture, and 3 mm deep) via a 10 μL26-gauge Hamilton Gastight 1701 syringe needle. Animals were monitored daily for signs of neurological deficits. Mice without neurological deficits after 12 months were euthanized and examined for tumor formation.
Hematoxylin and eosin (H&E) and Immunohistochemical (IHC) Staining
Whole mouse brains were harvested, fixed in zinc formalin, paraffin-embedded, and serially sectioned. H&E staining was performed on every 20 sections. IHC staining was performed using Vectastain Elite ABC kit (Vector Laboratories) or Mouse on Mouse Elite Peroxidase Kit (Vector Laboratories) as described previously [7,8,55]. Primary antibodies included human-specific monoclonal antibodies against mitochondria (MT) (1:150) (EMD Millipore Corporation), Vimentin (VIM) (1:200) (Dako North America,), glial fibrillary acidic protein (GFAP) (1:100) (Abcam), and rabbit anti-von Willebrand Factor (vWF) (1:500) (EMD Millipore Corporation).
Pyrosequencing
BRAF V600E mutation status was examined through pyrosequencing. Following genomic DNA extraction a PCR reaction was set-up (40 cycles, 58°C annealing temperature) using primers Forward 5'-GGCCAAAAATTTAATCAGTGGAA-3' and Reverse 5-Bio-CTTCATAATGCTTG CTCTGATAGG-3'. PCR reaction amplified a 236-bp genomic fragment spanning BRAF codon 600 on exon 15. Pyrosequencing was performed on a PSQ 96 (Qiagen) as previously described www.impactjournals.com/oncotarget [56]. The pyrogram sequence was analyzed using sequencing primer PySeq 5'-CCACTCCATC GAGATT-3' and dispensation order -CTAGCATGCTGT-. Output was recorded as antisense 5'→3' sequence and % A calculated using pyromark analysis software. Incorporation of nonwild type nucleotide at position 7 with allelic frequency >10 was considered positive for mutation.
Florescence in situ hybridization (FISH)
To determine CDKN2A deletion, Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes were applied to paraffin sections from patient and xenograft tumors and cultured xenograft cells. Paraffin sections were de-paraffinized and cultured cells cytospun onto positively charged slides and treated with protease. Slides and probe mixture were co-denatured at 75°C for 5 minutes and placed in the hybrite machine at 37°C overnight. After post-hybridization wash, slides were counterstained with DAPI and examined under florescence microscopy. Data analysis was performed by counting cells using CEP 9 probe labeled with SpectrumGreen, which hybridizes to alpha satellite sequence on chromosome 9.
In vitro drug treatment
Cultured BXD-3635PXA cells in serum-free media were seeded 50 μL per well into 384-well plate using Multidrop dispenser (Thermo Fisher Scientific) and incubated at 37°C for 24 hr before the investigational compounds (50 nL and 5 nL from 10 mM stock, and 50 nL from 0.1 mL stock) were transferred using Echo550, an acoustic liquid handler from Labcyte (San Jose, CA). The plates were then incubated at 37°C in a CO 2 incubator for 7 days. To estimate cell proliferation, 5 μL of CCK8 was added to each well, incubated for 4 hrs before the absorbance was measured at 450 nm using 650 nm as references.
Statistical analysis
Differences between two groups were analyzed with student t test. P values <0.05 were considered significant.
CONFLICTS OF INTEREST
There is no conflict of interests for any author.
Figure 1 :
1Establishment of in vivo and in vitro models of PLGGs. (A) H&E and IHC staining of mouse brain show scars of surgical implantation without tumor formation, disturbed granular layer neurons (b-c, circle), and reactive mouse astrocytes (monoclonal antibodies against GFAP) (e-f). Human tumor cells detected with human-specific antibodies against mitochondria (MT) (h-i). Magnification (x10: a, b, d, e, g, h and x40: c, f, i). (B) H&E stained cross section of IC-3635PXA (top left image). Log-rank analysis of animal survival times during serial sub-transplantation from passage I (P-I) to IV (P-IV) (top right panel). IHC of tumor cells with human-specific MT antibodies (lower panel). (C) Histopathological features of IC-3635PXA xenograft tumors (at passage I and III) compared with patient tumor (magnification: x20). (D) Morphology of cultured PDOX cells in FBS-based media (BXD-3635PXA-mono) and serum-free media supplemented with EGF/bFGF (BXD-3635PXA-NS) from passages 1 (p-1) to 20 (p-20) (magnification: x10). www.impactjournals.com/oncotarget
Figure 2 :
2Analysis of CD133 + and CD15 + cells. (A) Representative graph showing successful double staining of CD133 and CD15 in GBM (left 2 panels) and PLGGs with high (middle panel) and low (right panel) CD15 + cells. (B) Summary graph showing relative abundance of mono-and dual-positive (CD133 and CD15) cells related to tumor cell growth, PDOX formation, pathological grade, years of follow up, and status of progression. Due to limited viable tumor cell yields, not all samples were tested with all assays. "-": not tested; N = did not grow/no progression, Y = grew in culture or in vivo or progressed. www.impactjournals.com/oncotarget
Figure 3 :
3Quantitative analysis of BRAF V600E mutation allele frequency using pyrosequencing. (A) Representative pyrograms indicating A (wild type) to T (mutated) substitution in percent for 3635PXA patient tumor, xenograft passage I as well as in vitro cultured 3635PXA cells (monolayer and neurospheres) are shown. Pyrosquencing was control by including a positive control (HT-29) as well as negative controls with either no mutation (Tonsil) or no DNA. Percent (%) A/T is calculated using pyromark analysis software. (B) Allele frequency of BRAF V600E mutation in patient tumor, xenografts and cultures cells of 3635PXA.
Figure 4 :
4FISH validation of CDKN2A deletion. (A) Location and the coverage of CDKN2A by spectral orange (R = red) on chromosome 9. The alpha satellite on the centromere of chromosome 9 is labeled with SpectrumGreen (G = green) and used as the reference control. (B) Images of FISH hybridization in paraffin sections (patient tumor and xenograft) and in cultured cells (monolayer and neurospheres). Normal cells with two copies of chromosome 9 centromere (green) and two copies of CDKN2A (red) highlighted in circle and labelled as 2R2G. Loss of CDKN2A (no red = 0R) were found in disomy (0R2G), trisomy (0R3G) and quadrosomy (0R4G) tumor cells (arrows). (C) Graph showing the relative percentage of cells with or without CDKN2A deletion. For each sample, 200 cells were counted under high magnification (10 x 60). Compared with the patient tumor, in which CDKN2A were still present in a small fraction of cells (mostly 2R2G), xenograft tumors and both the cultured cells were enriched with homozygous deletion of CDKN2A (0R1G to 0R5G). Note the gradual decrease of disomy chromosome 9 (0R2G) and increase of trisomy 9 (0R3G) (arrows) over in vivo sub-transplantations of IC-3635PXA.
Figure 5 :
5In vivo tumor invasion and host responses detected with IHC. (A) Modes of IC-3635PXA invasion in mouse brains. Tumor cells positively stained with Vimentin (VIM) (arrows, a-c), and blood vessels (bv) with vWF (d). Same area in two consecutive sections was included (c and d, dotted line with dual arrow heads). (B) Images showing long-range perivascular invasion (left panel) and quantitative analysis perivascular migration (right panel) ( ** P< 0.01). Tumor cells positively stained with VIM (arrow heads). (C) IHC showing mutually exclusive positivity between GFAP (marker for mature glial cells) (red arrowheads, e-g) and VIM in tumor mass (Tum) and in invasive satellites and single cells (blue arrowheads, h and i). Note presence of reactive astrocytes in normal brain tissues (g) without presence of tumor cells (j). Matched areas in consecutive sections were used (dotted line with dual arrow heads) for GFAP and VIM staining (magnification x 20). (D) IHC of paired primary and recurrent PA confirming invasive cells to be GFAP-(red arrow, l and m) and VIM + (blue arrowheads, o and p) Magnification (x40: k and n; x20: l, m, o, p).
Figure 6 :
6In vitro drug testing. Cultured 3635PXA cells were exposed to small molecule inhibitors (0.01 to 10 μM) for 7 days and plotted as the fraction of cell killing. (A) Two of the 7 Inhibitors targeting BRAF V600E (left panel) and 2 of the 9 inhibitors against BRAF wild-type and RAF (right panel) were active. Proliferation of the 3635PXA cells were not affected by the remaining agents. (B) Chemotherapy agent Vincristine was included as reference.
FUNDING
This project is funded by National Brain Tumor Society (XN Li), NIH/NCI RO1 CA185402 (XN Li), Cancer Prevention and Research Institute of Texas (CPRIT) RP150032 (XN Li), St Baldrick's Foundation (JM Su), and Sontag Foundation (DW Parsons).
Table 1 :
1Summary of clinical information, tumor cell yield and intra-cranial tumor formation of PLGG tumors No. Tumor ID Age, Gender Dx, WHO GradeTumor cell number
(x 10 6 )
Site of
injection
Tumor formation in
mouse brains
Total Injected/mouse
Total
With Tumor
Table 2 :
2Summary of immunohistochemical characteristics of 3635PXATarget Phenotypes
Marker
Patient Tumor
Xenograft
(passage I)
Xenograft
(passage III)
TC
INV
TC
INV
Proliferation
Ki-67
++
(1-5%)
++
(5-10%)
++
(1-10%)
++
(5-15%)
++
(5-15%)
Astrocyte
GFAP
+++
(4)
+++
(2)
+++
(2)
+++
(2)
+++
(1)
Angiogenesis
vWF *
++
(4.3 ± 1.1 * )
++
(5.8 ± 1.1)
nd
++
(3.9 ± 2.4)
nd
Intermediate Filament
VIM
+++
(2)
++
(4)
+++
(4)
+++
(4)
++++
(4)
Table 3 :
3Expression of GFAP and Vimentin in paired primary and recurrent pilocytic astrocytomasTumor ID
Age /Gender at
diagnosis
Time to
Recurrent (yr)
www.impactjournals.com/oncotarget
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| [
"To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133 + (<1.5%) and CD15 + (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP -/Vimentin + cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. www.impactjournals.com/oncotarget/ Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP -/Vimentin + cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts."
] | [
"Mari Kogiso \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Lin Qi \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Holly Lindsay \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Yulun Huang \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n\nDepartment of Neurosurgery\nThe First Affiliated Hospital\nSoochow University\nSuzhouChina\n",
"Xiumei Zhao \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n\nDepartment of Ophthalmology\nFirst Affiliated Hospital of Harbin\nMedical University\nHarbinChina\n",
"Zhigang Liu \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n\nDepartment of Radiotherapy\nThe Affiliated Cancer Hospital of Xiangya School of Medicine\nHunan Cancer Hospital\nCentral South University\nChangshaChina\n",
"Frank K Braun \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Yuchen Du \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Huiyuan Zhang \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Goeun Bae \nCenter for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA\n",
"Sibo Zhao \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Sarah G Injac \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Mary Sobieski \nCenter for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA\n",
"David Brunell \nCenter for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA\n",
"Vidya Mehta \nDepartment of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA\n",
"Diep Tran \nDepartment of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA\n",
"Jeffrey Murray \nDepartment of Hematology and Oncology\nCook Children's Medical Center\nFort WorthTXUSA\n",
"Patricia A Baxter \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Xiao-Jun Yuan \nDepartment of Hematology and Oncology\nXinhua Children's Hospital\nShanghaiChina\n",
"Jack M Su \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Adekunle Adesina \nDepartment of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA\n",
"Laszlo Perlaky \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Murali Chintagumpala \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"D Williams Parsons \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Ching C Lau \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n",
"Clifford C Stephan \nCenter for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA\n",
"Xinyan Lu \nDepartment of Pathology\nNorthwestern University Feinberg School of Medicine\nChicagoILUSA\n",
"Xiao-Nan Li \nDepartment of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA\n"
] | [
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Neurosurgery\nThe First Affiliated Hospital\nSoochow University\nSuzhouChina",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Ophthalmology\nFirst Affiliated Hospital of Harbin\nMedical University\nHarbinChina",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Radiotherapy\nThe Affiliated Cancer Hospital of Xiangya School of Medicine\nHunan Cancer Hospital\nCentral South University\nChangshaChina",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Center for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Center for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA",
"Center for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA",
"Department of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Hematology and Oncology\nCook Children's Medical Center\nFort WorthTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Hematology and Oncology\nXinhua Children's Hospital\nShanghaiChina",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pathology\nBaylor College of Medicine\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA",
"Center for Translational Cancer Research\nInstitute of Biosciences and Technology\nTexas A&M College of Medicine\nHoustonTXUSA",
"Department of Pathology\nNorthwestern University Feinberg School of Medicine\nChicagoILUSA",
"Department of Pediatrics\nBaylor College of Medicine\nTexas Children's Cancer Center\nTexas Children's Hospital\nHoustonTXUSA"
] | [
"Mari",
"Lin",
"Holly",
"Yulun",
"Xiumei",
"Zhigang",
"Frank",
"K",
"Yuchen",
"Huiyuan",
"Goeun",
"Sibo",
"Sarah",
"G",
"Mary",
"David",
"Vidya",
"Diep",
"Jeffrey",
"Patricia",
"A",
"Xiao-Jun",
"Jack",
"M",
"Adekunle",
"Laszlo",
"Murali",
"D",
"Williams",
"Ching",
"C",
"Clifford",
"C",
"Xinyan",
"Xiao-Nan"
] | [
"Kogiso",
"Qi",
"Lindsay",
"Huang",
"Zhao",
"Liu",
"Braun",
"Du",
"Zhang",
"Bae",
"Zhao",
"Injac",
"Sobieski",
"Brunell",
"Mehta",
"Tran",
"Murray",
"Baxter",
"Yuan",
"Su",
"Adesina",
"Perlaky",
"Chintagumpala",
"Parsons",
"Lau",
"Stephan",
"Lu",
"Li"
] | [
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"G P Zambetti, ",
"D W Ellison, ",
"L E Kun, ",
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"R J Gilbertson, ",
"C E Fuller, ",
"E Marton, ",
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"P Longatti, ",
"J Ivaska, ",
"H M Pallari, ",
"J Nevo, ",
"J E Eriksson, ",
"M Mistry, ",
"N Zhukova, ",
"D Merico, ",
"P Rakopoulos, ",
"R Krishnatry, ",
"M Shago, ",
"J Stavropoulos, ",
"N Alon, ",
"J D Pole, ",
"P N Ray, ",
"V Navickiene, ",
"J Mangerel, ",
"M Remke, ",
"S Robel, ",
"H Sontheimer, ",
"S Robel, ",
"S C Buckingham, ",
"J L Boni, ",
"S L Campbell, ",
"N C Danbolt, ",
"T Riedemann, ",
"B Sutor, ",
"H Sontheimer, ",
"J Lee, ",
"S Kotliarova, ",
"Y Kotliarov, ",
"A Li, ",
"Q Su, ",
"N M Donin, ",
"S Pastorino, ",
"B W Purow, ",
"N Christopher, ",
"W Zhang, ",
"J K Park, ",
"H A Fine, ",
"Q Shu, ",
"B Antalffy, ",
"J M Su, ",
"A Adesina, ",
"C N Ou, ",
"T Pietsch, ",
"S M Blaney, ",
"C C Lau, ",
"X N Li, ",
"H Lindsay, ",
"Y Huang, ",
"Y Du, ",
"F K Braun, ",
"W Y Teo, ",
"M Kogiso, ",
"L Qi, ",
"H Zhang, ",
"S Zhao, ",
"H Mao, ",
"F Lin, ",
"P Baxter, ",
"J M Su, "
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"Combination dabrafenib and trametinib in the management of advanced melanoma with BRAFV600 mutations",
"BRAF V600E mutation in hairy cell leukemia: from bench to bedside",
"The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary",
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"Glia as drivers of abnormal neuronal activity",
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"Valproic Acid prolongs survival time of severe combined immunodeficient mice bearing intracerebellar orthotopic medulloblastoma xenografts",
"Preservation of KIT genotype in a novel pair of patientderived orthotopic xenograft mouse models of metastatic pediatric CNS germinoma"
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"Nat Rev Neurol",
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"Cancer",
"Methods Mol Med",
"Invest New Drugs",
"Cancer Res",
"Neuro Oncol",
"Stem Cells",
"Nature",
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"Proc Natl Acad Sci U S A",
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"Int J Cancer",
"Cancer Res",
"Cancer Res",
"Mol Cancer",
"Clin Cancer Res",
"PLoS One",
"Cancer Res",
"FEBS Open Bio",
"J Clin Invest",
"Acta Neuropathol",
"Acta Neuropathol",
"Clin Cancer Res",
"Neurology",
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"J Clin Invest",
"Clin Transl Med",
"Genes Chromosomes Cancer",
"Cancer Res",
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"Expert Opin Pharmacother",
"Expert Opin Pharmacother",
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"Nat Neurosci",
"J Neurosci",
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"Clin Cancer Res",
"J Neurooncol"
] | [
"\nFigure 1 :\n1Establishment of in vivo and in vitro models of PLGGs. (A) H&E and IHC staining of mouse brain show scars of surgical implantation without tumor formation, disturbed granular layer neurons (b-c, circle), and reactive mouse astrocytes (monoclonal antibodies against GFAP) (e-f). Human tumor cells detected with human-specific antibodies against mitochondria (MT) (h-i). Magnification (x10: a, b, d, e, g, h and x40: c, f, i). (B) H&E stained cross section of IC-3635PXA (top left image). Log-rank analysis of animal survival times during serial sub-transplantation from passage I (P-I) to IV (P-IV) (top right panel). IHC of tumor cells with human-specific MT antibodies (lower panel). (C) Histopathological features of IC-3635PXA xenograft tumors (at passage I and III) compared with patient tumor (magnification: x20). (D) Morphology of cultured PDOX cells in FBS-based media (BXD-3635PXA-mono) and serum-free media supplemented with EGF/bFGF (BXD-3635PXA-NS) from passages 1 (p-1) to 20 (p-20) (magnification: x10). www.impactjournals.com/oncotarget",
"\nFigure 2 :\n2Analysis of CD133 + and CD15 + cells. (A) Representative graph showing successful double staining of CD133 and CD15 in GBM (left 2 panels) and PLGGs with high (middle panel) and low (right panel) CD15 + cells. (B) Summary graph showing relative abundance of mono-and dual-positive (CD133 and CD15) cells related to tumor cell growth, PDOX formation, pathological grade, years of follow up, and status of progression. Due to limited viable tumor cell yields, not all samples were tested with all assays. \"-\": not tested; N = did not grow/no progression, Y = grew in culture or in vivo or progressed. www.impactjournals.com/oncotarget",
"\nFigure 3 :\n3Quantitative analysis of BRAF V600E mutation allele frequency using pyrosequencing. (A) Representative pyrograms indicating A (wild type) to T (mutated) substitution in percent for 3635PXA patient tumor, xenograft passage I as well as in vitro cultured 3635PXA cells (monolayer and neurospheres) are shown. Pyrosquencing was control by including a positive control (HT-29) as well as negative controls with either no mutation (Tonsil) or no DNA. Percent (%) A/T is calculated using pyromark analysis software. (B) Allele frequency of BRAF V600E mutation in patient tumor, xenografts and cultures cells of 3635PXA.",
"\nFigure 4 :\n4FISH validation of CDKN2A deletion. (A) Location and the coverage of CDKN2A by spectral orange (R = red) on chromosome 9. The alpha satellite on the centromere of chromosome 9 is labeled with SpectrumGreen (G = green) and used as the reference control. (B) Images of FISH hybridization in paraffin sections (patient tumor and xenograft) and in cultured cells (monolayer and neurospheres). Normal cells with two copies of chromosome 9 centromere (green) and two copies of CDKN2A (red) highlighted in circle and labelled as 2R2G. Loss of CDKN2A (no red = 0R) were found in disomy (0R2G), trisomy (0R3G) and quadrosomy (0R4G) tumor cells (arrows). (C) Graph showing the relative percentage of cells with or without CDKN2A deletion. For each sample, 200 cells were counted under high magnification (10 x 60). Compared with the patient tumor, in which CDKN2A were still present in a small fraction of cells (mostly 2R2G), xenograft tumors and both the cultured cells were enriched with homozygous deletion of CDKN2A (0R1G to 0R5G). Note the gradual decrease of disomy chromosome 9 (0R2G) and increase of trisomy 9 (0R3G) (arrows) over in vivo sub-transplantations of IC-3635PXA.",
"\nFigure 5 :\n5In vivo tumor invasion and host responses detected with IHC. (A) Modes of IC-3635PXA invasion in mouse brains. Tumor cells positively stained with Vimentin (VIM) (arrows, a-c), and blood vessels (bv) with vWF (d). Same area in two consecutive sections was included (c and d, dotted line with dual arrow heads). (B) Images showing long-range perivascular invasion (left panel) and quantitative analysis perivascular migration (right panel) ( ** P< 0.01). Tumor cells positively stained with VIM (arrow heads). (C) IHC showing mutually exclusive positivity between GFAP (marker for mature glial cells) (red arrowheads, e-g) and VIM in tumor mass (Tum) and in invasive satellites and single cells (blue arrowheads, h and i). Note presence of reactive astrocytes in normal brain tissues (g) without presence of tumor cells (j). Matched areas in consecutive sections were used (dotted line with dual arrow heads) for GFAP and VIM staining (magnification x 20). (D) IHC of paired primary and recurrent PA confirming invasive cells to be GFAP-(red arrow, l and m) and VIM + (blue arrowheads, o and p) Magnification (x40: k and n; x20: l, m, o, p).",
"\nFigure 6 :\n6In vitro drug testing. Cultured 3635PXA cells were exposed to small molecule inhibitors (0.01 to 10 μM) for 7 days and plotted as the fraction of cell killing. (A) Two of the 7 Inhibitors targeting BRAF V600E (left panel) and 2 of the 9 inhibitors against BRAF wild-type and RAF (right panel) were active. Proliferation of the 3635PXA cells were not affected by the remaining agents. (B) Chemotherapy agent Vincristine was included as reference.",
"\nFUNDING\nThis project is funded by National Brain Tumor Society (XN Li), NIH/NCI RO1 CA185402 (XN Li), Cancer Prevention and Research Institute of Texas (CPRIT) RP150032 (XN Li), St Baldrick's Foundation (JM Su), and Sontag Foundation (DW Parsons).",
"\nTable 1 :\n1Summary of clinical information, tumor cell yield and intra-cranial tumor formation of PLGG tumors No. Tumor ID Age, Gender Dx, WHO GradeTumor cell number \n(x 10 6 ) \nSite of \ninjection \n\nTumor formation in \nmouse brains \nTotal Injected/mouse \nTotal \nWith Tumor \n\n",
"\nTable 2 :\n2Summary of immunohistochemical characteristics of 3635PXATarget Phenotypes \nMarker \nPatient Tumor \n\nXenograft \n(passage I) \n\nXenograft \n(passage III) \n\nTC \nINV \nTC \nINV \n\nProliferation \nKi-67 \n++ \n(1-5%) \n\n++ \n(5-10%) \n\n++ \n(1-10%) \n\n++ \n(5-15%) \n\n++ \n(5-15%) \n\nAstrocyte \nGFAP \n+++ \n(4) \n\n+++ \n(2) \n\n+++ \n(2) \n\n+++ \n(2) \n\n+++ \n(1) \n\nAngiogenesis \nvWF * \n++ \n(4.3 ± 1.1 * ) \n\n++ \n(5.8 ± 1.1) \nnd \n++ \n(3.9 ± 2.4) \nnd \n\nIntermediate Filament \nVIM \n+++ \n(2) \n\n++ \n(4) \n\n+++ \n(4) \n\n+++ \n(4) \n\n++++ \n(4) \n\n",
"\nTable 3 :\n3Expression of GFAP and Vimentin in paired primary and recurrent pilocytic astrocytomasTumor ID \nAge /Gender at \ndiagnosis \n\nTime to \nRecurrent (yr) \n\n"
] | [
"Establishment of in vivo and in vitro models of PLGGs. (A) H&E and IHC staining of mouse brain show scars of surgical implantation without tumor formation, disturbed granular layer neurons (b-c, circle), and reactive mouse astrocytes (monoclonal antibodies against GFAP) (e-f). Human tumor cells detected with human-specific antibodies against mitochondria (MT) (h-i). Magnification (x10: a, b, d, e, g, h and x40: c, f, i). (B) H&E stained cross section of IC-3635PXA (top left image). Log-rank analysis of animal survival times during serial sub-transplantation from passage I (P-I) to IV (P-IV) (top right panel). IHC of tumor cells with human-specific MT antibodies (lower panel). (C) Histopathological features of IC-3635PXA xenograft tumors (at passage I and III) compared with patient tumor (magnification: x20). (D) Morphology of cultured PDOX cells in FBS-based media (BXD-3635PXA-mono) and serum-free media supplemented with EGF/bFGF (BXD-3635PXA-NS) from passages 1 (p-1) to 20 (p-20) (magnification: x10). www.impactjournals.com/oncotarget",
"Analysis of CD133 + and CD15 + cells. (A) Representative graph showing successful double staining of CD133 and CD15 in GBM (left 2 panels) and PLGGs with high (middle panel) and low (right panel) CD15 + cells. (B) Summary graph showing relative abundance of mono-and dual-positive (CD133 and CD15) cells related to tumor cell growth, PDOX formation, pathological grade, years of follow up, and status of progression. Due to limited viable tumor cell yields, not all samples were tested with all assays. \"-\": not tested; N = did not grow/no progression, Y = grew in culture or in vivo or progressed. www.impactjournals.com/oncotarget",
"Quantitative analysis of BRAF V600E mutation allele frequency using pyrosequencing. (A) Representative pyrograms indicating A (wild type) to T (mutated) substitution in percent for 3635PXA patient tumor, xenograft passage I as well as in vitro cultured 3635PXA cells (monolayer and neurospheres) are shown. Pyrosquencing was control by including a positive control (HT-29) as well as negative controls with either no mutation (Tonsil) or no DNA. Percent (%) A/T is calculated using pyromark analysis software. (B) Allele frequency of BRAF V600E mutation in patient tumor, xenografts and cultures cells of 3635PXA.",
"FISH validation of CDKN2A deletion. (A) Location and the coverage of CDKN2A by spectral orange (R = red) on chromosome 9. The alpha satellite on the centromere of chromosome 9 is labeled with SpectrumGreen (G = green) and used as the reference control. (B) Images of FISH hybridization in paraffin sections (patient tumor and xenograft) and in cultured cells (monolayer and neurospheres). Normal cells with two copies of chromosome 9 centromere (green) and two copies of CDKN2A (red) highlighted in circle and labelled as 2R2G. Loss of CDKN2A (no red = 0R) were found in disomy (0R2G), trisomy (0R3G) and quadrosomy (0R4G) tumor cells (arrows). (C) Graph showing the relative percentage of cells with or without CDKN2A deletion. For each sample, 200 cells were counted under high magnification (10 x 60). Compared with the patient tumor, in which CDKN2A were still present in a small fraction of cells (mostly 2R2G), xenograft tumors and both the cultured cells were enriched with homozygous deletion of CDKN2A (0R1G to 0R5G). Note the gradual decrease of disomy chromosome 9 (0R2G) and increase of trisomy 9 (0R3G) (arrows) over in vivo sub-transplantations of IC-3635PXA.",
"In vivo tumor invasion and host responses detected with IHC. (A) Modes of IC-3635PXA invasion in mouse brains. Tumor cells positively stained with Vimentin (VIM) (arrows, a-c), and blood vessels (bv) with vWF (d). Same area in two consecutive sections was included (c and d, dotted line with dual arrow heads). (B) Images showing long-range perivascular invasion (left panel) and quantitative analysis perivascular migration (right panel) ( ** P< 0.01). Tumor cells positively stained with VIM (arrow heads). (C) IHC showing mutually exclusive positivity between GFAP (marker for mature glial cells) (red arrowheads, e-g) and VIM in tumor mass (Tum) and in invasive satellites and single cells (blue arrowheads, h and i). Note presence of reactive astrocytes in normal brain tissues (g) without presence of tumor cells (j). Matched areas in consecutive sections were used (dotted line with dual arrow heads) for GFAP and VIM staining (magnification x 20). (D) IHC of paired primary and recurrent PA confirming invasive cells to be GFAP-(red arrow, l and m) and VIM + (blue arrowheads, o and p) Magnification (x40: k and n; x20: l, m, o, p).",
"In vitro drug testing. Cultured 3635PXA cells were exposed to small molecule inhibitors (0.01 to 10 μM) for 7 days and plotted as the fraction of cell killing. (A) Two of the 7 Inhibitors targeting BRAF V600E (left panel) and 2 of the 9 inhibitors against BRAF wild-type and RAF (right panel) were active. Proliferation of the 3635PXA cells were not affected by the remaining agents. (B) Chemotherapy agent Vincristine was included as reference.",
"This project is funded by National Brain Tumor Society (XN Li), NIH/NCI RO1 CA185402 (XN Li), Cancer Prevention and Research Institute of Texas (CPRIT) RP150032 (XN Li), St Baldrick's Foundation (JM Su), and Sontag Foundation (DW Parsons).",
"Summary of clinical information, tumor cell yield and intra-cranial tumor formation of PLGG tumors No. Tumor ID Age, Gender Dx, WHO Grade",
"Summary of immunohistochemical characteristics of 3635PXA",
"Expression of GFAP and Vimentin in paired primary and recurrent pilocytic astrocytomas"
] | [
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"(Figure 2A",
"Figure 2B)",
"Figure 2B",
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"Figure 3A and 3B)",
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"Figure 4B",
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"Figure 4B",
"Figure 4C",
"Figure 1C",
"Figure 5A",
"Figure 5D, l and o, m and p)",
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"Pediatric low grade gliomas (PLGGs) are slow growing tumors accounting for 1/3 of all childhood brain tumors [1]. Although complete surgical removal results in cure in >90% of patients, some tumors still recur [1][2][3], especially after sub-total resection. Currently, driver(s) of recurrence and malignant progression remain to be elucidated. Mouse models that replicate key biological features of PLGG are highly desired to identify mechanism of recurrence/malignant degeneration and enable preclinical studies of PLGG. We have shown that direct injection of fresh surgical specimens into anatomicallymatched locations in the brains of immunodeficient mice facilitates establishment of clinically-relevant orthotopic xenograft mouse models that replicate the histology, invasive growth, and key genetic features of primary patient tumors [4][5][6][7][8]. The added advantage of patientderived orthotopic xenograft (PDOX) mouse model is that the normal brain responses toward xenograft growth, which is difficult to obtain from patient surgical samples, can be analyzed simultaneously together with brain tumor cells. PDOX mouse models of PLGGs, however, have not been previously established.",
"Accumulating evidence demonstrates that cancer stem cells (CSCs) play an important role in tumorigenicity, cancer initiation and recurrence [9][10][11][12][13][14]. CD133 and CD15 are two well-characterized cell surface markers that define pediatric glioblastoma and medulloblastoma CSCs [8,9,[14][15][16][17][18]. Despite ongoing controversies about the relative abundance and specificity of these markers [19][20][21], CD133 + brain tumor stem cells are chemotherapy-and radiation-resistant [13,22], and their frequency correlates with adverse survival in adult glioma [23]. In contrast, little is known about CSCs in low grade tumors. Only a few cases have been analyzed for CD133 + cells, revealing variable abundance ranging from undetectable [24] to 37% [25]. The content and function of CD15 + cells in PLGGs is still unknown.",
"Genetic analysis identified BRAF as a frequent mutation target in PLGGs, including BRAF V600E mutation [26][27][28][29][30][31][32], duplication [33] and gene fusion [28][29][30][31][32][33][34]. BRAF V600E mutation were found in WHO grade II PXA (66%), PXA with anaplasia (65%), grade I GG (18%) and grade I PA (9%) [29]. Homozygous deletions involving the CDKN2A/p14ARF/CDKN2B loci were detected in 60% of PXA [35] and 71% of malignant astrocytomas [36]. These reports suggest contribution of BRAF V600E mutation and CDKN2A deletion to tumor progression and should be targeted. Indeed, multiple novel inhibitors against BRAF V600E mutation have been developed and entered into clinical trials in patients with advanced melanoma, hairy cell leukemia, and thyroid cancers [37][38][39][40]. Developing new PLGG models replicating such druggable mutation would be highly desired not only to understand the functional role of BRAF V600E mutation in driving PLGG recurrence, but also for future examination of drug resistance as has been noted in melanomas [37].",
"In this report, our goals were to determine if PDOX models can be established from low grade gliomas, whether CSCs are present in PLGG and if their frequencies correlate with in vitro self-renewal, in vivo formation of orthotopic xenografts, and clinical tumor recurrence. To gain insight into in vivo tumor evolution and progression, we examined if the histopathological features and, more importantly, the progression nature of the original patient tumor were replicated in the PDOX tumors during longterm serial subtransplantations in mouse brains, followed by the analysis of the underlying cellular and molecular (e.g. BRAF V600E mutation and CDKN2A deletion) changes in tumor cells and in the host normal brain cells that drove or accompanied the PDOX tumor progression to identify new therapeutic targets.",
"Despite extensive collaborative effort, the tumor tissues obtained for PLGGs were still limited, frequently less than 3 x 3 x 3 mm 3 (Table 1). Using a combination of mechanical dissociation and combined collagenase/ halogenase enzymatic digestion, we were able to collect viable tumor cells up to 4.3 x 10 6 cells (1.3 x 10 6 ± 1.1 x 10 6 ). The number of assays per PLGG sample was therefore performed depending on the tumor cell availability.",
"To examine if PLGGs contain cells able to form neurospheres in vitro, dissociated cells from 15 tumors Note: * Tested for neurosphere formation, ¶ Analyzed with FCM for CSC markers, Dx= diagnosis, ICb=Intracerebellar, IC=Intra-cerebral, PA=Pilocytic astrocytoma, AST=astrocytoma, GG=Ganglioglioma, PXA = Pleomorphic Xanthoastrocytoma (Table 1) were plated in serum-free medium containing Neurobasal medium, EGF/bFGF to favor CSC growth [7,8,15]. Sustained neurosphere growth was unsuccessful from any tumor after 19 to 57 (37.6 ± 15.2) days observation. Additionally, 11 patient tumors with >2 x 10 6 cells were incubated in FBS-based media. Again, none attached or expanded in culture. These data suggested that PLGG cells harvested directly from patients did not readily adapt to in vitro growth conditions and failed to proliferate.",
"We tested if rapid return of PLGG cells to anatomically-matched locations in mouse brains would form tumor. 20 cerebellar tumors and 5 cerebral PLGGs were implanted into matching locations in mouse brains either intra-cerebellarly (ICb) or intra-cerebrally (IC) (Table 1) as we described previously. No visible tumor formation was detected in 24 of 25 PLGGs after average of 230 days observation. H&E staining of paraffinembedded brains also did not detect tumors, although scarring and disturbed normal brain structure indicative of previous surgical implantation were seen ( Figure 1A). Subsequent IHC examination revealed reactive astrocytes with strong GFAP positivity surrounding the needle track but did not detect human cells in mouse brains using human-specific MT antibodies, further confirming the lack of xenograft tumor formation ( Figure 1A). Only IC implantation of 3635 pleomorphic xanthoastrocytoma (PXA) patient tumor formed orthotopic xenograft tumors; this model was designated IC-3635PXA and was serially sub-transplanted in mouse brains four times (passage IV) ( Figure 1B). Similar to patient tumor, xenograft cells from passages I to III were positive for human-specific MT in tumor core, invasive foci, and disseminated cells ( Figure 1B), validating the human origin of xenograft tumors.",
"The PDOX model-initiating patient tumor was pathologically assessed at diagnosis by the local anatomic pathologist and was subsequently reviewed by our institutional neuro-pathologist. The tumor was confirmed to be a pleomorphic xanthoastrocytoma (2016 Central Nervous System (CNS) World Health Organization (WHO) grade II) as it was moderately cellular and infiltrative but with a low proliferation index and no necrosis or anaplasia [41]. Rare sections of the tumor were suspicious for developing vascular proliferation. Pathologic examination of xenograft passage I revealed the presence of a 2016 CNS WHO grade II PXA with no vascular proliferation or necrosis and very low mitotic activity [41]. Xenograft passage II tumors remained grade II but demonstrated increased cellularity, though the proliferation index remained low. At xenograft passage III, tumors had a notable pathologic shift with developing of higher grade features including regional necrosis pseudopalisading necrosis, increased vascular and endothelial proliferation, increased tumor cell infiltration into normal brain. The tumor's proliferative index had increased from <5% to approximately 20%. Xenograft passage IV had even further increases in tumor cellularity and had unequivocally transformed into a high grade tumor.",
"Tumor take rate of IC-3635PXA increased during serial in vivo sub-transplantations Initial tumor take rate upon implantation of patient tumor into SCID mice was low, as only 2 of 7 (28.5%) mice initially implanted with 3635PXA patient tumor formed tumors. Over in vivo sub-transplantations, tumor take rate steadily increased: 28.5% (2/7) in passage I, 44.4% (4/9) in passage II, 100% (7/7) in passage III, and 88.9% (8/9) in passage IV ( Figure 1B). Presence of normal cells in the patient tumor and the multiple round selection of \"pure\" and more aggressive clonal cell populations might have played a role.",
"We also attempted to establish cell lines from IC-3635PXA xenografts. Monolayer cells, labeled Baylor xenograft derived (BXD)-3635PXA-mono, grew in FBSbased media, although the proliferation rate of these cells was slow (21-28 days/passage and reached passage 25 after 586 days). In serum-free media, 3635PXA cells initially formed neurospheres (BXD-3635PXA-NS) but after passage 5 became attached, exhibiting a star-like morphology with occasional formation of small clusters (7-18 days/passage and reached passage 32 after 586 days) ( Figure 1D). PDOX tumor cells may have a better chance of survival in vitro than the patient tumors.",
"To determine if lack of putative CSCs was the cause of low xenograft tumor formation, we assessed CD133 and CD15, two common brain tumor CSC markers, in 22 patient tumors using flow cytometry (FCM) ( Figure 2). PLGGs were found to have abundant CD133 -/CD15 + cells (20.7 ± 28.9%) (Figure 2A and 2B). In 3 of 18 (17%) pilocytic astrocytomas (PAs), 1 of 1 (100%) grade II astrocytoma (AST), 2 of 2 (100%) gangliogliomas (GGs), and 1 of 1 (100%) PXA, CD133 -/CD15 + cells accounted for >30% of the total cell population ( Figure 2B), demonstrating CD15 + cells in PLGGs for the first time. Compared with high levels of CD133 + /CD15cells in a childhood GBM xenograft tumor included as positive control (Figure 2A), only low levels (0.46 ± 0.57%) of www.impactjournals.com/oncotarget CD133 + /CD15 -PLGG cells were detected in 22 tumors ( Figure 2B).",
"We subsequently analyzed CD133 + and/or CD15 + levels in 3635PXA and the resulting xenograft tumors (passages I through III). Similar to other PLGGs, CD133 + / CD15 + and CD133 + /CD15were barely detectable (<1%), while CD133 -/CD15 + cells were the major subpopulation, accounting for 64.1% in patient tumor ( Figure 2B). CD133 -/CD15 + xenograft tumor cells decreased to 9.6% in passage I, 10.3% in passage II and 5.9% in passage III ( Figure 2B), suggesting that they may not be driving in vivo tumor formation.",
"In order to correlate the content of putative CSC cells with clinical tumor progression, clinical outcomes of 22 patients with PLGG were followed for up to 6 (4.8±1.6) years. Among the four progressive tumors, one GG and one PXA had high CD15 + cells (>60%) but two PAs harbored only 10% and 1% CD15 + cells ( Figure 2B). Altogether, these data suggested that tumorigenicity in mouse brains and clinical progression of PLGGs may not be solely driven by putative CSCs. ",
"Genomic DNA from 3635PXA patient, xenograft tumors and cultured cells was subjected to quantitative mutation detection using BRAF V600E specific pyrosequencing. Compared to 28% in patient tumor, BRAF V600E mutant allele frequency increased in xenografts (67%, 70%, and 67% in passage I, II and III, respectively) ( Figure 3A and 3B). In vitro xenograft tumor cell cultures also had high BRAF mutation frequencies with 66% (p-4), 69% (p-21) in monolayer and 67% (p-5 and p-21) in neurospheres. These observations suggest that BRAF V600E mutation plays an important role in tumor progression, although the molecular mechanisms underlying the increased allele frequency of BRAF mutation frequency remains to be determined.",
"Oncogenic BRAF mutation and CDKN2A inactivation characterize a subset of pediatric malignant gliomas [36]. We thus examined if CDKN2A deletion played a role in IC-3635PXA progression. Utilizing Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes ( Figure 4A), we analyzed CDKN2A status in paraffin sections of patient and xenograft tumors and cultured cells derived from IC-3635PXA. In the patient sample, 13% of the cells contained 2 red (R=CDKN2A) and 2 green (G=chromosome 9 centromere) (2R2G in Figure 4B) that are found in normal cells, most patient tumor cells (87%) exhibited homozygous deletion either with disomy chromosome 9 (0R2G) (51%) or trisomy 9 (0R3G) (17.5%). As expected, there were no normal 2R2G human cells in xenograft tumors. In xenograft passage I, homozygous deletion of CDKN2A was found in 77.5% cells with 0R2G, 17% cells with 0R3G, and 5% cells with quadrisomy 9 (0R4G). Subsequent analysis of xenograft tumors revealed a gradual decrease of 0R2G cells with CDKN2A deletion, i.e. 58%, 51%, and 31% in passages II, III and IV, accompanied by increased 0R3G cells from 18% in passage II, to 37% in passage III and 54% in passage IV ( Figure 4C). These data suggested that a sub-population of trisomy chromosome 9 PLGG cells with CDKN2A deletion gained growth advantage over disomy 9 cells.",
"Cultured xenograft tumor cells also maintained the homozygous CDKN2A deletion ( Figure 4B). The relative abundance of 0R2G decreased from 62.5% in the monolayer cells to 29% in neurospheres while 0R3G and 0R4G cells increased from 17% to 25%, and from 11% to 45%, respectively ( Figure 4C). These data demonstrated that homozygous deletion of CDKN2A, found in 68.5% of 3635PXA patient tumor cells, was well maintained and increased to 100% in the PDOX model and cultured xenograft cells; interestingly, monolayer cells were enriched with disomy 9 cells while neurospheres favored the growth of trisomy and quadrisomy 9 tumor cells.",
"To identify cellular changes accompanying increased tumor take rate and the increased BRAF mutation, we performed IHC staining of PDOX tumors ( Figure 1C and Table 2). First, compared with low cell proliferation index (Ki-67) of <2% in patient tumor, xenograft tumors from IC-3635PXA exhibited Ki-67 positivity at 5%-10% in passage I and 5%-15% in passage III. Second, while the majority of patient tumor cells (>95%) were strongly positive (+++) for GFAP, a marker of mature/differentiated glial cells, there was a steady decrease of GFAP + cells in PDOX tumors (26-50%). Third, expression levels of VIM, a marker of intermediate filament associated with poor prognosis and tumor invasion [42][43][44], increased. While high (+++) VIM expression was seen in only a low percentage (~10-30%) of patient tumor cells, nearly all xenograft cells exhibited strong (+++) positivity in passages I and III. In addition to cells in the tumor core, invasive tumor cells (single cells, micro-satellites, and perivascular spread) were also strongly (++++) VIM positive ( Figure 5A). These data suggested that a subpopulation of GFAP -/VIM + cells were enriched during PDOX tumor formation, sub-transplantation, and tumor progression in vivo.",
"To validate the role of VIM + cells in PLGG invasion and progression, we collected 5 pairs of matched primary and recurrent PLGGs and compared the expression of GFAP and VIM with IHC (Table 3). In 4 of the 5 tumor pairs (#3319, 9036, 1576, 2022), there were no major differences between primary and recurrent tumors, as nearly all cells were positive for GFAP and VIM (Table 3). In recurrent tumor #1614, cells in the tumor core displayed strong (+++) positivity of GFAP and VIM while a small piece of normal tissue demonstrated significant numbers of VIM + /GFAPtumor cells ( Figure 5D, l and o, m and p). This identification of VIM + /GFAPcells in the invasive front of this recurrent tumor was in agreement with our observations in the IC-3635PXA tumors.",
"We next examined changes in IC-3635PXA cell invasion in order to guide development of new therapies preventing metastasis. In addition to single cell invasion into neighboring normal brain and neural fibers ( Figure 5A, a and b), there was frequent distant dissemination of VIM + PDOX cells via blood vessels deep into normal brain ( Figure 5A, c and d). Using a straight line reticle (eyepiece micrometer), perivascular migration in mouse cerebral was measured. Migration increased from 140 ± 68.3 μm in passage I, to 182 ± 152, 429 ± 226, and 392 ± 237 μm in passages II, III and IV, respectively (P <0.01) ( Figure 5B). Longest migratory distance also increased from 350 μm in passage I, to 570, 1,370 and 1,100 μm in passages II, III and IV, respectively. Starting from passage II, we also observed perivascular invasion into brain stem, presumably due to the close proximity of tumor mass ( Figure 5B). Since the chances of escaping surgical resection is positively correlated with distance of tumor cell invasion into normal brain, such progressive increases of PXA tumor cell migration into distant normal brains suggested perivascular invasion as a potential cause of tumor recurrence and metastasis.",
"Since human normal brain tissues must be preserved during surgical resection of brain tumors, little is known about the reaction of normal glial cells to the growth of brain tumors, particularly in areas far away from the tumor mass [45]. Using IC-3635PXA, we examined responses of normal glial cells both adjacent to and distant from the primary tumor to infiltration and dissemination of PXA tumor cells. IHC staining for GFAP (which recognizes both human and mouse astrocytes) detected wide-spread presence of GFAP + astrocytes resembling reactive astrocytes [46]. All GFAP + astrocytes cells were negative for human-specific VIM and MT, confirming the murine origin of these cells. Growth of xenograft tumors triggered gliosis in host mouse brains not only at the tumor-brain interface ( Figure 5C, e-i) but also deep into normal mouse brains beyond the leading edge of tumor invasion, including in the contralateral mouse cerebrum where no human tumor cells were evident ( Figure 5C, f, g, and i). Since dynamic interactions between PLGG cells and microenvironment remain poorly defined, PDOX model IC-3635PXA can be used for future understanding of host responses (molecular mechanisms and biological impacts) toward the growth of PXA.",
"As a proof of principle, we exposed the cultured BXD-3635PXA cells to vincristine (a chemotherapy agents) and a series of inhibitors that target BRAF V600E (n=7), BRAF wild-type and/or RAF genes (n=9) (Supplementary Table 1) and examined their anti-proliferation activities. After 7 days of in vitro treatment (0-10 μM), cell proliferation was suppressed by vincristine (IC50=0.97 μM), 2/7 (28.5%) BRAF V600E inhibitors, PLX-4720 (IC50= 0.13µM) and Dabrefenib (IC50=1.58µM), and 2/9 (22.2%) inhibitors that target BRAF or RAF genes, GDC-0879 (IC50=0.65 μM) and Sorafenib (IC50=1.74 μM) ( Figure 6). The remaining 12 inhibitors, including 5 BRAF V600E inhibitors, were not active during the treatment period. These data suggested that targeting BRAF V600E was effective in suppression BXD-3635PXA cell proliferation. The efficacy of inhibitors, however, was not identical.",
"In this study, we demonstrated that the tumor take rate of PLGGs is very low as only 1 of the 25 tumors formed transplantable orthotopic xenograft tumors in mouse brains. To determine if the lack of cancer stem cells played a role, we analyzed putative cancer stem cells through FCM analysis of dual-stained putative cancer stem cell markers (CD133 and CD15). While the levels of CD133 + cells were much lower (<1%) than a previous report demonstrating 15% CD133 + cells in one childhood PA and 8% in two adult grade II ASTs [24], they appear to be correlated with low tumorigenicity of PLGGs. To validate the role of CD133 + cells in PLGG tumorigenicity, direct implantation of isolated CD133 + PLGG cells should be tested in mouse brains, although low tumor cell abundance coped with low overall tumor cell yields often make it difficult to collect sufficient CD133 + cells for this in vivo assay. Although 7 of the 22 PLGGs expressed high levels (30% -80%) of CD15 + cells, which has been previously detected in medulloblastoma and adult malignant gliomas [16][17][18], only one of these PLGGs (IC-3635PXA) formed xenografts. Since all of the PLGGs were injected into anatomically-matched locations in mouse brains, our results indicated that the functional role of CD15 in PLGGs might be different from that in the high-grade gliomas. Detailed functional validation is needed for existing and new CSC markers of PLGGs.",
"While the majority of children with LGGs remain tumor-free, a small fraction of PLGGs do recur over time. Malignant transformation, which is rare in pilocytic astrocytomas (2.4% of 288 patients) [47] and more often in grade II PXA (36%) [48,49], might have played a role. In this study, we confirmed that IC-3635PXA replicated the histology and key genetic abnormalities of the original patient tumor. More importantly, we showed that, for the first time, the progressive/metastatic growth of a PXA mouse model replicated the progressive and metastatic nature of the originating patient tumor, as evidenced by the increased tumor take rate, elevated cell proliferation and expanded local invasion and perivascular metastasis over serial in vivo sub-transplantations. Similar to most recurrent and late stage tumors, no surgery or biopsy was performed in 3635PXA patient tumor at the last recurrence, making it difficult to identify the cellular and molecular changes and/or drivers of tumor recurrence. Using our new PDOX model, we were able to detect the cellular drivers of tumor progression, i.e. GFAP -/VIM + tumor cells. Nearly all the xenograft tumor cells became VIM positive starting from passage I with the strongest positivity found in the infiltrating tumor edge. A significant elevation of VIM expression was also confirmed in one paired primary and recurrent PLGGs. Since VIM has been involved in attachment, migration, and cell signaling [50], our results justified additional studies to validate the functional role of GFAP -/VIM + tumor cells in PXA (and other PLGGs) recurrence. TC= tumor core; INV= invasion; Scored intensity as negative (-), low (+), medium (++), strongly positive (+++) and highly strongly positive (++++) and extent of immunopositivity as 0=negative; 1=1-25%; 2=26-50%; 3=51-75%; 4=>75% positive cells. * Microvessel Density (mean ± SD). nd = not done.",
"Molecularly, we provided new data to support the role of BRAF V600E mutation and CDKN2A deletion in driving PXA progression. Previous studies have shown that BRAF V600E and CDKN2A alterations were less commonly observed in PLGG that did not transform [51], but more frequently detected in secondary high grade gliomas. Unlike KIAA1549-BRAF fusion transcripts which were near exclusively expressed in grade I astrocytomas, BRAF V600E mutation was detected in 22.6% of grade II-IV tumors but in none of the grade I tumors [36]. In secondary high glade gliomas, BRAF V600E and CDKN2A deletion were as high as 39% and 57%, respectively [51]. Since 3635PXA patient tumor contained 13% normal diploid cells, the corrected BRAF V600E mutation rate increased from 28% to 32.18% (i.e., 28/87=32.18%), it is still much lower than that in the xenograft tumors (>67%). Our long-term followup of in vivo growth of IC-3635PXA tumors revealed a clear enrichment of BRAF V600E mutation frequency and detected a novel a shift of diploid chromosome 9 to trisomy 9 in tumor cells bearing CDKN2A deletion. Since copy number of chromosome 9 increased despite CDKN2A deletion, addition studies (preferably in more than 1 models) are needed to examine if CDKN2A deletion is associated with the in vivo progression and in vitro growth of neurospheres, and if amplified chromosome 9 is involved in and/or drive tumor progression in PXA. Since BRAF V600E mutant gliomas often develop acquired resistance to FDA approved small molecule inhibitor [32], our PDOX model would be a powerful tool to conduct preclinical testing of new BRAF V600E targeting inhibitors.",
"Understanding the mode of tumor invasion should provide new clues to guide the development of new therapies preventing metastasis. In addition to migrating as a single cell and along neural fibers, our data showed that PXA xenograft cells achieve distant dissemination through perivascular spaces far beyond the leading edges of intra-parenchymal invasion. The increased frequencies and distances of perivascular migration, which paralleled the xenograft tumor progression over serial in vivo subtransplantations, would render early radiographic detection and surgical resection nearly impossible. However, since these invasive tumor cells in the perivascular space need to breach the blood brain barrier through disruption of astrocytic endfeet, which envelope vessels, to spread away from tumor core, they might have also damaged the integrity of the local blood brain barrier, potentially making themselves vulnerable to chemotherapies. This newly discovered mode of PXA invasion in vivo has thus provided a therapeutic opportunity to target the distant perivascular invasion that are left behind after surgery and potentially cause tumor recurrences. Using this PDOX model, we also had an opportunity to examine the responses of host (normal) brain cells to the hetero-transplanted PXA tumors, particularly in the areas far away from the tumor mass whereas corresponding studies in patients are not feasible. To the best of our knowledge, the widespread reactive gliosis we observed in the normal mouse brain tissues has not been previously described in human PXA or other types of PLGGs. Reactive gliosis, also known as astrogliosis, refers to the morphological and biochemical changes of astrocytes occurring in association with injury or disease. Given the benign nature of PLGGs, it is surprising to detect such an outbreak of hypertrophic and GFAP + reactive astrocytes [45], which have been found to alter the expression or function of neuronal proteins involved in excitability and may serve as drivers of epileptogenesis in acquired epilepsies [52,53]. Indeed, our 3635PXA patient developed severe epilepsy at tumor recurrence, which highlighted the need and the potential use of our IC-3635PXA model for to decipher the molecular mechanism of reactive gliosis and to identify potential therapeutic targets to improve quality of life by suppressing/ preventing epilepsy.",
"Since development of new therapy is one of the most important goals of model development, we tested the anti-tumor activities of a series of inhibitors specific to BRAF V600E or to wild-type BRAF or other types of RAF gene/pathways in vitro. In addition to identify a set of 4 inhibitors (2 each for BRAF V600E and BRAF) that suppressed cell proliferation, we also found 5 BRAF V600E inhibitors and 7 BRAF inhibitors that were not active. These findings are interesting as they revealed ",
"that differential activities among BRAF V600E and wild-type inhibitors, which not only suggested the need of \"personalized\" drug testing, but also highlighted the potential use of our model for the examination of underlying mechanisms of action and resistance.",
"In conclusion, our studies demonstrated that PLGGs has very low tumorigenic capacity in the brains of SCID mice, which is similar to the frequency of tumor progression and recurrence in pediatric patients. Low abundance of CD133 + cells appears to be correlated with such low tumor take rate. The PDOX model IC-3635PXA not only replicated the histological and molecular phenotypes, but also, and more interestingly, evolved in vivo replicating the progressive growth of the originating patient tumor. Using this novel PDOX model, we have identified VIM + GFAPcells as candidate cellular drivers, BRAF V600E mutation and CDKN2A deletion (in cells with trisomy and quadrisomy chromosome 9) as key molecular changes that mediated the progression, invasion and migration of the xenograft tumors over long term in vivo subtransplantations, and showed that a subset of BRAF V600E inhibitors were indeed active in suppression cell proliferation in vitro. This novel model will serve as an important resource to support further biological and pre-clinical studies (such as BRAF V600E mutation) in pediatric PXA.",
"Freshly resected LGG tumor specimens from 36 children undergoing craniotomy at Texas Children's Hospital and member hospitals of the Texas and Oklahoma Pediatric Neuro-Oncology Consortium were obtained for this study (Table 1). Signed consent was obtained prior to sample acquisition following Institutional Review Board-approved protocols. As described previously [7,8], fresh tumor tissues were washed and dissociated with the Automatic Tissue Dissociator (Miltenyi Biotec), followed by collagenase/halogenase enzymatic digestion.",
"Tumor #3635 was obtained from a 9 year-old girl receiving subtotal resection of an extensive left temporal tumor. Pathologically, it was diagnosed as PXA with ganglioglioma component (WHO grade II), BRAF V600E mutation and low cell proliferation index (<2%). Two month later, she received more complete subtotal resection. The tumor, however, progressed along with disseminated neuraxis metastasis 5 months from the initial diagnosis. Despite treatment with palliative craniospinal radiatioin along with daily adjuvant temozolomide chemotherapy during radiation therapy, she developed severe epilepsy, post-chemoradiotherapy pancytopenia, speptic shock and passed away 7 months after diagnosis.",
"PLGG tumor cells were labeled with APCconjugated human CD133 antibody and FITC-conjugated human CD15 antibody (Miltenyi Biotec), or isotype control antibodies at 4°C for 15 minutes in FCM buffer comprised of DPBS, 0.5% BSA and 2 mM EDTA. After washing, cells were re-suspended in FCM buffer containing 2 μg/mL propidium iodide (PI) and analyzed with a LSR II flow cytometer and Kaluza Analysis Software Version 1.3 (Beckman Coulter). Dead cells were excluded by PI staining.",
"For neurosphere assays, dissociated PLGG tumor cells were plated at clonal density (1,500 cells/100 μL) and incubated in serum-free media consisting of Neurobasal media, N-2 and B-27 supplements (0.5x each) (Life Technologies), human recombinant basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) (50 ng/mL each) (R&D Systems) [8,54], and 200 units/mL penicillin/streptomycin. Additional cells were seeded in DMEM media supplemented with 10% fetal bovine serum (FBS) and 200 units/mL penicillin/streptomycin. Cells were incubated in 5% CO 2 at 37°C. Media were changed every three days and cell growth examined under phase contrast microscopy.",
"NOD/SCID mice were bred and housed in a pathogen-free animal facility. All experiments were conducted following an Institutional Animal Care and Use Committee-approved protocol. Mice of both gender, aged 6-8 weeks, were anesthetized with sodium pentobarbital (50 mg/kg, i.p. injections). Tumor cells from 25 PLGGs were re-suspended in DMEM growth medium at 5 x 10 7 live cells/mL and injected (1 x 10 5 cells in 2 μL) orthotopically into mouse brains as described previously [7,8,55]. Cerebellar tumors were implanted into right cerebellum (1 mm to the right of the midline, 1 mm posterior to the lamboidal suture, and 3 mm deep), and cerebral PLGGs into right cerebral hemisphere (1 mm to the right of the midline, 1.5 mm anterior to the lamboidal suture, and 3 mm deep) via a 10 μL26-gauge Hamilton Gastight 1701 syringe needle. Animals were monitored daily for signs of neurological deficits. Mice without neurological deficits after 12 months were euthanized and examined for tumor formation.",
"Whole mouse brains were harvested, fixed in zinc formalin, paraffin-embedded, and serially sectioned. H&E staining was performed on every 20 sections. IHC staining was performed using Vectastain Elite ABC kit (Vector Laboratories) or Mouse on Mouse Elite Peroxidase Kit (Vector Laboratories) as described previously [7,8,55]. Primary antibodies included human-specific monoclonal antibodies against mitochondria (MT) (1:150) (EMD Millipore Corporation), Vimentin (VIM) (1:200) (Dako North America,), glial fibrillary acidic protein (GFAP) (1:100) (Abcam), and rabbit anti-von Willebrand Factor (vWF) (1:500) (EMD Millipore Corporation).",
"BRAF V600E mutation status was examined through pyrosequencing. Following genomic DNA extraction a PCR reaction was set-up (40 cycles, 58°C annealing temperature) using primers Forward 5'-GGCCAAAAATTTAATCAGTGGAA-3' and Reverse 5-Bio-CTTCATAATGCTTG CTCTGATAGG-3'. PCR reaction amplified a 236-bp genomic fragment spanning BRAF codon 600 on exon 15. Pyrosequencing was performed on a PSQ 96 (Qiagen) as previously described www.impactjournals.com/oncotarget [56]. The pyrogram sequence was analyzed using sequencing primer PySeq 5'-CCACTCCATC GAGATT-3' and dispensation order -CTAGCATGCTGT-. Output was recorded as antisense 5'→3' sequence and % A calculated using pyromark analysis software. Incorporation of nonwild type nucleotide at position 7 with allelic frequency >10 was considered positive for mutation.",
"To determine CDKN2A deletion, Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes were applied to paraffin sections from patient and xenograft tumors and cultured xenograft cells. Paraffin sections were de-paraffinized and cultured cells cytospun onto positively charged slides and treated with protease. Slides and probe mixture were co-denatured at 75°C for 5 minutes and placed in the hybrite machine at 37°C overnight. After post-hybridization wash, slides were counterstained with DAPI and examined under florescence microscopy. Data analysis was performed by counting cells using CEP 9 probe labeled with SpectrumGreen, which hybridizes to alpha satellite sequence on chromosome 9.",
"Cultured BXD-3635PXA cells in serum-free media were seeded 50 μL per well into 384-well plate using Multidrop dispenser (Thermo Fisher Scientific) and incubated at 37°C for 24 hr before the investigational compounds (50 nL and 5 nL from 10 mM stock, and 50 nL from 0.1 mL stock) were transferred using Echo550, an acoustic liquid handler from Labcyte (San Jose, CA). The plates were then incubated at 37°C in a CO 2 incubator for 7 days. To estimate cell proliferation, 5 μL of CCK8 was added to each well, incubated for 4 hrs before the absorbance was measured at 450 nm using 650 nm as references.",
"Differences between two groups were analyzed with student t test. P values <0.05 were considered significant.",
"There is no conflict of interests for any author. "
] | [] | [
"INTRODUCTION",
"RESULTS",
"The overall yields of tumor cells from childhood LGG were low",
"Attempts to establish neurosphere and monolayer cultures from patient tumors",
"Only 1 of 25 PLGGs formed orthotopic xenograft tumors",
"PDOX tumor cells survived and proliferated in vitro",
"PLGGs contain high levels of CD15 + cells and low levels of CD133 + cells",
"BRAF V600E mutation increased during IC-3635PXA progression",
"IC-3635PXA PDOX showed persistent CDKN2A deletion and increasing trisomy 9",
"PDOX tumor evolution was paralleled by loss of GFAP and gain of Vimentin (VIM) expression",
"In vivo PDOX progression was paralleled by increased perivascular dissemination",
"Tumor growth triggered intensive reactive gliosis in normal mouse brain",
"In vitro anti-tumor activities of BRAF V600E inhibitors",
"DISCUSSION",
"GFAP VIM",
"MATERIALS AND METHODS",
"Childhood LGG tumor tissues",
"Flow cytometry",
"In vitro growth of PLGG cells",
"Direct orthotopic transplantation of patient tumor cells into mouse brain",
"Hematoxylin and eosin (H&E) and Immunohistochemical (IHC) Staining",
"Pyrosequencing",
"Florescence in situ hybridization (FISH)",
"In vitro drug treatment",
"Statistical analysis",
"CONFLICTS OF INTEREST",
"Figure 1 :",
"Figure 2 :",
"Figure 3 :",
"Figure 4 :",
"Figure 5 :",
"Figure 6 :",
"FUNDING",
"Table 1 :",
"Table 2 :",
"Table 3 :"
] | [
"Tumor cell number \n(x 10 6 ) \nSite of \ninjection \n\nTumor formation in \nmouse brains \nTotal Injected/mouse \nTotal \nWith Tumor \n\n",
"Target Phenotypes \nMarker \nPatient Tumor \n\nXenograft \n(passage I) \n\nXenograft \n(passage III) \n\nTC \nINV \nTC \nINV \n\nProliferation \nKi-67 \n++ \n(1-5%) \n\n++ \n(5-10%) \n\n++ \n(1-10%) \n\n++ \n(5-15%) \n\n++ \n(5-15%) \n\nAstrocyte \nGFAP \n+++ \n(4) \n\n+++ \n(2) \n\n+++ \n(2) \n\n+++ \n(2) \n\n+++ \n(1) \n\nAngiogenesis \nvWF * \n++ \n(4.3 ± 1.1 * ) \n\n++ \n(5.8 ± 1.1) \nnd \n++ \n(3.9 ± 2.4) \nnd \n\nIntermediate Filament \nVIM \n+++ \n(2) \n\n++ \n(4) \n\n+++ \n(4) \n\n+++ \n(4) \n\n++++ \n(4) \n\n",
"Tumor ID \nAge /Gender at \ndiagnosis \n\nTime to \nRecurrent (yr) \n\n"
] | [
"(Table 1",
"(Table 1)",
"Table 2",
"(Table 3",
"(Table 3",
"Table 1",
"(Table 1)"
] | [
"Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma",
"Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma"
] | [
"Oncotarget"
] |
235,249,807 | 2022-01-11T21:33:15Z | CCBY | https://www.frontiersin.org/articles/10.3389/fcvm.2021.669975/pdf | GOLD | f0c58db9631816ea41e64455dbd0bcfadbb335ea | null | null | null | null | 10.3389/fcvm.2021.669975 | null | 34136546 | 8202000 |
a section of the journal Frontiers in Cardiovascular Medicine Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress
May 2021
Gianfranco Pintus
Tetsuro Kamiya
Mohamed Ahmed [email protected]
Ayan Rajgarhia
School of Medicine, Children's Mercy Hospital and University of Missouri-Kansas City
Kansas CityMOUnited States
Kameshwar R Ayasolla
Henry Ford Health System
DetroitMIUnited States
Nahla Zaghloul
Neonatal Division
Nemours Children's Hospital
University of Arizona
United States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States
Jorge M Lopez
Da Re
Edmund J Miller
Mohamed Ahmed
Neonatal Division
Nemours Children's Hospital
University of Arizona
United States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States
University of Sharjah
United Arab Emirates
Radha Gopal
Gifu Pharmaceutical University
Japan
University of Pittsburgh
United States
a section of the journal Frontiers in Cardiovascular Medicine Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress
Frontiers in Cardiovascular Medicine | www.frontiersin.org
1669975May 202110.3389/fcvm.2021.669975Specialty section: This article was submitted to Cardiovascular Biologics and Regenerative Medicine, Received: 19 February 2021 Accepted: 16 April 2021ORIGINAL RESEARCH Edited by: Reviewed by: *Correspondence: Citation: Rajgarhia A, Ayasolla KR, Zaghloul N, Lopez Da Re JM, Miller EJ and Ahmed M (2021) Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress. Front. Cardiovasc. Med. 8:669975.hypxoiacardiac fibrosisEC-SODmethylationRASSF1A
Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O 2 ) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.
INTRODUCTION
Cardiac fibrosis can develop following a variety of stimuli, including ischemia, volume overload, pressure overload, and hypoxia (1). A common feature of all these stimuli is the reduced availability of oxygen. Whether from decreased oxygen delivery or from increased oxygen consumption, tissue hypoxia is associated with infiltration of inflammatory cells and activation of resident cells (2). Cardiac fibroblasts, the main resident cells, are activated and transform to myofibroblasts, which are the key driver for the fibrotic response. Other cell types act indirectly by secreting fibrogenic mediators (macrophages, mast cells, lymphocytes, cardiomyocytes, and vascular cells). Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Oxidative stress activates mitogenactivated protein kinases and stress-responsive protein kinases (3). Markers of cardiac fibrosis include collagen I and III, alpha smooth muscle actin (ASMA), and SNAIL (4). Cardiac fibrosis leads to both systolic and diastolic dysfunction and increases cell death and damage by inflammatory mediators. Prognosis depends on the etiology and extent of the disease, with some cases caused by chronic hypoxia showing some reversibility of fibrosis (4).
DNA methylation is an epigenetic modification, which plays an important role in the cellular response to chronic hypoxia and the progression of cardiac fibrosis (2). DNA methylation alters the chromatin structure leading to repression of gene expression. The methylation process is regulated by a family of DNA methyltransferase (DNMT) enzymes. Studies have demonstrated significant increases in the activities of DNMT1 (the enzyme responsible for maintaining the methylation status of daughter cells during cell cycle) and DNMT 3B (de novo methylating enzyme) in response to hypoxia (2). The hypoxiainduced expression of DNMT1 and DNMT 3B is in part regulated by hypoxia-inducible transcription factor 1α (HIF-1α), through specific hypoxic response elements present in the promoter sequence of DNMT1 and 3B (2). Their increased activity correlates positively with the degree of cardiac fibrosis (2). This suggests the role for epigenetic modification in fibrosis secondary to hypoxia (2).
Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene, and alterations in its regulation are frequently seen in cardiac fibrosis (2). RASSF1A inhibits proliferation by inhibiting the accumulation of cyclin D1 and arresting cell division and promotes apoptosis (5).
RASSF1A functions through its effect on downstream proteins such as extracellular signal-regulated kinases (ERK1/2). The Ras/ERK signaling pathway has been recognized as an intracellular signal transduction critically involved in fibroblast proliferation. Ras/ERK1/2 is activated in cardiac fibroblasts by platelet-derived growth factor-BB (PDGF-BB) and promotes cellular proliferation (6). In cardiac myocytes, RASSF1A can prevent hypertrophy through disruption of Ras/Raf-1/ERK MAPK signaling. RASSF1A also activates Mst1 to elicit apoptosis. In cardiac fibroblasts, RASSF1A represses NF-κB transcriptional activity and inhibits TNF-α production and secretion, thereby preventing paracrine-mediated hypertrophic signaling between fibroblasts and myocytes. This mechanism involves multiple cell types and paracrine signaling including calcineurin/NFAT, HDAC/MEF2, and MEK/ERK pathways, which have been elucidated in cardiac myocytes (7).
DNA methylation-mediated silencing of RASSF1A, and subsequent activation of ERK1/2, can lead to activation of fibroblasts and fibroblast proliferation (6). Another contributing mechanism to cardiac dysfunction induced by hypoxia is myofilament modification. Two myosin heavy chain (MHC) isoforms, MHC-α and MHC-β, are expressed in the mouse heart. α-MHC has higher intrinsic adenosine triphosphatase (ATPase) activity and hence contributes to higher contractility, while β-MHC has lower intrinsic ATPase activity and has a greater economy of force maintenance (8). Hypoxia has been shown to cause switching of myosin heavy chains (MHC) from its alpha to beta isoform, thereby decreasing ATPase activity and overall force of contraction (4).
Hypoxia and reactive oxygen species (ROS) play a pivotal role both in the pathogenesis of hypoxia-induced pulmonary hypertension and in the development of cardiac fibrosis (9)(10)(11)(12)(13). The role of ROS, as a trigger of DNA methylation of tumor suppressor gene promoters in carcinogenesis, was shown in previous studies. In human breast cancer, it increases redox concentration (e.g., hydrogen peroxide), induces the overexpression of epigenetic modifiers, including DNMT1 and HDAC1, inhibits gene expression, including tumor suppression; and enhances the expression of epithelial to mesenchymal transition inducer genes, including Snail and Slug (5,14).
The inflammatory response mediated by extracellular reactive oxygen generated from repetitive ischemia/reperfusion in a murine model plays a critical role in the pathogenesis of fibrotic remodeling and ventricular dysfunction (7). Supplementation with vitamins E, C, and A have provided antioxidant protection against cardiomyocyte death and have improved survival in congestive heart failure models and doxorubicin-induced injury (11). Doxorubicin causes cardiotoxicity through the generation of ROS (11). ROS generated by doxorubicin include superoxide, hydrogen peroxide, hydroxyl radical, peroxynitrite, and lipid hydroperoxide, which are messengers for signaling apoptotic cell death (11). In-vitro cardiomyocyte studies suggest that superoxide dismutase-like and glutathione peroxidaselike compounds can protect against free radical production and cellular apoptosis due to doxorubicin (6). EC-SOD is an important antioxidant throughout the cardiovascular system and has been shown to protect the heart from ischemic damage, hypertrophy, and inflammation (15)(16)(17). Studies show that human populations with a mutation in the matrix-binding domain of EC-SOD, which diminishes its affinity for the extracellular matrix, have a higher risk for the development of cardiovascular and ischemic heart disease (4). EC-SOD is important in the prevention of oxidative injury that may contribute to cardiac remodeling, in myocardial infarction models, and alter ex vivo heart function (16,17). EC-SOD overexpression has also been shown to decrease the fibrosis that develops in cardiac tissue secondary to ischemia-reperfusion injury (10,18). However, the specific mechanisms by which EC-SOD protects against fibrosis and tissue damage, in various organs including the lung and heart, remain unclear. Also, the relation between EC-SOD and epigenetic changes has not been fully explored. In this study, we reveal the effects of overexpression of EC-SOD on cardiac fibrosis, epigenetic changes, and myofilament changes due to chronic hypoxic stress.
MATERIALS AND METHODS
In-vivo Studies
All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research. Adult (8-10-week-old) C57BL6 male mice (wild type-WT) and transgenic neonate mice, with an extra copy of the human EC-SOD gene containing a β-actin promoter (TG), were housed in a pathogen-free environment under standard light and dark cycles, with free access to food and water (19). hEC-SOD TG mice with C57BL6 background were studied before, by us and other researchers, and have been well-characterized (19,20). TG mice act and behave similarly under normal conditions (room air), like WT mouse strains as shown in many studies before (19,20).
Animals
The studied animals were divided into three groups, Group A: WT mice housed in room air; Group B: WT mice housed in 10% normobaric oxygen for 21 days using a BioSpherix chamber (Lacona, NY, USA) (WT hypoxic group); and Group C: TG mice housed under the same hypoxic conditions as Group B (18). After 21 days, the animals were euthanized under a surgical plane of anesthesia [Fentanyl/Xylazine (5:1)] (21), and right ventricular tissue was harvested for analysis.
RT-qPCR
Gene expression, within the right ventricular tissue samples, was assessed following tissue disruption and homogenization. RNA was then extracted from the tissue using the AllPrep DNA/RNA extraction kit (Qiagen), according to the manufacturer's instructions. First-strand cDNA synthesis was carried out using SuperScript II RT (Invitrogen). Quantitative real-time PCR primers were designed so that one of each primer pair was exon/exon boundary spanning to ensure that only mature mRNA was amplified. The sequences of the genespecific primers used were ASMA, 5 ′ -aatgagcgtttccgttgc 3 ′ (forward), 5 ′ -atccccgcagactccatac-3 ′ (reverse); collagen 1a 1 (COL1A1), 5 ′ -catgttcagctttgtggacct 3 ′ (forward), 5 ′ gcagctgacttcagggatgt 3 ′ (reverse); collagen 3 a 1 (COL 3A1), 5 ′ tcccctggaatctgtgaatc 3 ′ (forward), 5 ′ tgagtcgaattggggagaat 3 ′ (reverse); α-Myosin Hea vy Chain (Myh6) 5 ′ cgcatcaaggagctcacc-3 ′ (forward), 5 ′ -cctgcagccgcattaagt-3 ′ (reverse); and β-Myosin Heavy Chain (Myh7) 5 ′ -cgcatcaaggagctcacc-3 ′ (forward), 5 ′ -ctgcagccgcagtaggtt-3 ′ (reverse). Q-PCR was performed; amplification and detection were carried out using Roche Applied Science LightCycler 480 PCR Systems with software. The PCR cycling program consisted of 45 three-step cycles of 15 s/95 • C, 1 min/57 • C, 1 s/72 • C. Relative changes in mRNA expression were calculated as fold changes (normalized using Gapdh) by using the comparative Ct ( Ct) method (22).
Immunohistochemistry-Collagen 1 (Cy 3)
Right ventricular tissue, fixed in 4% paraformaldehyde for 24 h, was processed, embedded in paraffin, and subsequently cut into 4-µm-thick sections. The slides then underwent heatmediated antigen retrieval followed by incubation with primary antibody anti-Collagen 1 antibody-3G3 (Abcam ab88147) and secondary antibody AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Lab Inc, Code 115005003). The slides were then analyzed using an Olympus FluoView FV300 Confocal Laser Scanning Microscope (Thermo Fisher Scientific), and the Fiji image processing software (an open-source platform for biological image analysis) was used for analysis of pixel density (23).
Western Blot Analysis
Frozen right ventricular tissues were homogenized, and protein extraction was carried out using a total protein extraction Kit (BioChain Institute, Inc. Hayward, CA). The protein concentration was evaluated using the Modified Lowry Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Samples were prepared for SDS-PAGE in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and processed as previously described (23). Membranes were briefly washed and immediately incubated with the respective primary antibody in 5% BSA with phosphatebuffered saline with Tween 20 (PBST), overnight. Following washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 40-60 min. The membranes were washed and then processed using Amersham ECL detection systems (GE Healthcare, Piscataway, NJ, USA). The membranes were immediately exposed to 8610 Fuji X-Ray Film. The films were assessed using Quantity One 1-D Analysis Software on a GS-800 Calibrated Densitometer. The density of each band is presented as a ratio in comparison to the Actin band density. Primary antibodies were used to detect the following markers: Collagen 1, Alpha Smooth Muscle Actin, SNAIL, DNMT1 and 3b, and HIF1α (each obtained from Abcam, Cambridge, MA); RASSF1A (Origene, Rockville, MD); and ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA).
ROS Assay
Intracellular ROS was assessed using a cell-based assay for measuring hydroxyl, peroxyl, or other reactive oxygen species.
The assay employs the cell-permeable fluorogenic probe 2 ′ ,7 ′dichlorodihydrofluorescein diacetate (DCFH-DA) (Cell Biolabs, San Diego, CA).
In-vitro Analysis
Primary C57BL6 Mouse Cardiac Fibroblasts (MCF) (Cell Biologics, Chicago, IL, Catalog No. C57-6049) were grown to confluence in T75 flasks using the fibroblast growth medium (Cell Biologics, Chicago, IL, Catalog No. M2267), in a humidified incubator (37 • C, 5% CO 2 ). Cells were seeded to six-well tissue culture plates, at a density of 10,000 cells per well, and incubated for 24 h. Next, the cells were transfected with human EC-SOD (hEC-SOD) cDNA inserted into a vector plasmid pcDNA3 (5446 nucleotides; Invitrogen Life Technologies, Carlsbad, CA, USA) as previously described (13)
Methylation Study
Bisulfite chemically converts unmethylated cytosine to uracil but has no effect on methylated cytosine. To determine if promoter region hypermethylation could be responsible for the downregulation of RASSF1A expression, direct bisulfite sequencing on mouse cardiac fibroblasts was performed (17). The methylation ratio (meth-ratio) was calculated using methylated CpG total CpG counts.
RASSF1A Methylation Analysis
Briefly, the genomic DNA from mouse heart was isolated using the TRIzol solution (Invitrogen, Thermo Fisher Scientific Inc), according to the manufacturer's protocols. The methylation status of the RASSF1A promoter region was determined by chemical modification of genomic DNA with sodium bisulfite and methylation-specific PCR. The bisulfite-treated DNA was used as a template for the methylation-specific PCR reaction. Primers for the unmethylated DNA-specific reaction were F, 5V-GGTGTTGAAGTTGTGGTTTG-3V; R, 5V-TATTATACCCAA AACAATACAC-3V. Primers for the methylated DNA-specific reaction were F, 5V-TTTTGCGGTTTCGTTCGTTC-3V; R, 5V-CCCGAAACGTACTACTATAAC-3V. The reactions were incubated at 95 • C for 1 min, 55 • C for 1 min, and 72 • C for 1 min, for 35 cycles. The amplified fragment was confirmed by DNA sequencing. DNA from normal heart was used as a control for unmethylated RASSF1A. The strategies for RASSF1A sequencing and the amplicons map Supplementary #1, #2, respectively.
Statistics
Data was expressed as the mean ± standard error of the mean (SEM). Unless otherwise indicated, a one-way or two-way analysis of variance followed by Bonferroni post-hoc test was used to assess significance (P < 0.05) using GraphPad Prism 8 (GraphPad Software, Inc, La Jolla, CA).
RESULTS
A comparison between WT adult mice and TG mice housed in room air was performed and is shown in Supplementary #3. All molecular testing showed no significant difference between WT and TG under normoxic conditions (room air).
EC-SOD Reduces Cardiac Fibrosis
To examine the effects of EC-SOD overexpression on markers of cardiac fibrosis at the mRNA level, we performed gene expression analysis using RT-PCR. There was a significant decrease in the expression of the cardiac fibrosis markers, Collagen 1 (Col1A1) (Figure 1A), Collagen 3 (Figure 1B), and ASMA ( Figure 1C) in the hypoxic TG, as compared to the hypoxic WT animals (p < 0.05). The gene expression of Collagen 1, Collagen 3, and ASMA in hypoxic TG group was not statistically significantly Frontiers in Cardiovascular Medicine | www.frontiersin.org different from room air controls. These results show that EC-SOD overexpression reduces gene expression of cardiac fibrosis markers to levels comparable to RA groups. To confirm that protein levels of cardiac fibrosis markers showed a similar trend to gene expression level, we performed protein-level assessment using Western blot analysis. There was a significant increase in the cardiac fibrosis marker levels of Collagen 1 (Figure 2A), ASMA (Figure 2B), and SNAIL1 ( Figure 2C) in hypoxic WT animals as compared to hypoxic TG animals (p < 0.05). The protein concentration of these three markers in the hypoxia TG animals was not significantly different from the RA control group. This indicates that EC-SOD overexpression reduces the protein expression of cardiac fibrosis markers to levels comparable to RA groups. Immunohistochemistry staining for Collagen 1 showed a significant decrease in pixel density of Collagen 1 in the hypoxic TG animals, as compared to the hypoxic WT animals (Figures 3A,B) (P < 0.05).
EC-SOD Reduces Epigenetic Modifications-DNA Methylation
To examine the effects of EC-SOD on DNA methylation, the most common form of epigenetic modifications, we transfected cardiac fibroblast with hEC-SOD and subjected them to hypoxia. Quantitative flow cytometry studies, using antibody directed to methylated DNA, revealed a significant increase in global DNA methylation in cardiac fibroblasts subjected to hypoxia, compared to cardiac fibroblasts transfected with hEC-SOD and subjected to the same hypoxic conditions (p < 0.05) (Figure 4), thus showing that in-vitro, EC-SOD significantly decreased global DNA methylation under hypoxic conditions.
To examine if the same results hold true in the invivo environment, we performed protein assessment of DNA methylating enzymes, using Western blot analysis. There was a significant decrease in the DNA methylating enzymes, DNMT1 ( Figure 5A) and DNMT-3b (Figure 5B), in the hypoxic TG animals compared to the hypoxic WT animals (p < 0.05). The levels of HIF1α ( Figure 5C) were also noted to be significantly reduced in the hypoxic TG animals when compared to the WT animals in hypoxia (p < 0.05). However, the levels in the hypoxic TG animals were not significantly different when compared to the TG room air group. Assay of free reactive oxygen species accumulation by DCF assay showed a significant increase of ROS in the WT hypoxic group, with ROS levels being significantly lower in the TG hypoxic group (Figure 5D). Thus, EC-SOD overexpression decreases DNA methylation, HIF1α, and ROS in-vivo under hypoxic conditions.
EC-SOD Ameliorates the Hypoxia-Induced Epigenetic Modifications to RASSF1A Through the Ras/ERK Pathway
We have shown that hypoxia induces epigenetic modifications both in-vitro and in-vivo. RASSF1A is a tumor suppressor gene, frequently involved in cardiac fibrosis. DNA methylationmediated silencing of RASSF1A leads to fibroblast proliferation and cardiac fibrosis. We wanted to examine the effects of EC-SOD overexpression on RASSF1A and further examine the Ras/ERK pathway.
Western blot analysis in the hypoxic WT animals showed a significant reduction in the gene RASSF1A (Figure 6A), as compared to both hypoxic TG animals and RA control groups (p < 0.05). WT hypoxic animals showed a significant increase of ERK phosphorylation in comparison to the RA control group (P < 0.05). The level of ERK 1/2 ( Figure 6B) was significantly reduced in the hypoxic TG animals compared to the hypoxic WT animals. FIGURE 2 | Western blot analysis of markers of fibrosis-Collagen 1, ASMA, and SNAIL. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). WB analysis was used to assess protein levels of Collagen 1 (A), ASMA (B), and SNAIL (C) in the right ventricular tissue. All experiments n = 3. Data represents mean ± SEM. *P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
EC-SOD Prevents Myofilament Changes
Myofilament modification induced by hypoxia causes cardiac dysfunction. Hypoxia can cause switching of Myosin Heavy chains (MHC) from its alpha (high ATP) to beta isoform low ATP, thereby decreasing contractility (Figures 7A,B). Gene expression analysis using RT-PCR showed a significant reduction in the levels of α-MHC and a significant increase in β-HMC in hypoxic WT animals as compared to RA groups and hypoxic TG group (P < 0.05), and this switch was reversed among TG hypoxic group. This provides evidence of EC-SOD overexpression improving cardiac contractility.
RASSF1A Silencing Increase Cardiac Fibroblast Proliferation
To provide further evidence that RASSF1A is involved in cardiac fibroblast proliferation, we incubated mouse cardiac fibroblast (MCF), with SiRNA blocking the expression of RASSF1A and incubated in room air condition, both BrdU assay and cell count studies were performed. BrDU analysis ( Figure 8A) showed a significant increase in cell proliferation post-transfection with SiRNA, inhibiting RASSF1A expression, as compared to control cells also incubated in room air (Figure 8B). There was a significant increase in cell numbers, with SiRNA silencing FIGURE 3 | Immunohistochemistry for Collagen 1. Right ventricular tissue from adult wild-type mice WT and EC-SOD transgenic mice (TG), which were exposed to FiO 2 10% hypoxia for 21 days (WT) and a room air control group (RA) were treated with Cy3 stain to assess for levels of Collagen 1 (A). The images were analyzed using Fiji image processing software to measure pixel density (B). All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
FIGURE 4 | Flow cytometry studies for DNA methylation-In in-vitro
studies using cardiac fibroblasts, DNA methylation levels were assessed in cells exposed to hypoxic conditions (FiO 2 1% for 72 h) and compared to cells transfected with EC-SOD and exposed to hypoxia. Cells cultured in room air were employed as controls. Triplicate wells were analyzed, and the experiments were repeated five times. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
RASSF1A
, as compared to control cells transfected with an empty vector. These findings support that silencing RASSF1A, leads to activation of ERK1/2, which stimulate cell proliferation.
EC-SOD Reduces Cardiac Fibrosis Through Ameliorating DNA Methylation
To elucidate the mechanism of how EC-SOD reduces cardiac fibrosis, we show that mRNA expression levels of DNA methyltransferases and methyl-CpG-binding domain proteins (MBD) were studied to investigate the possible mechanism for the observed methylation differences. Evaluated by RT-PCR normalized to GAPDH, DNMT expression was significantly higher in hypoxic WT than hypoxic TG cardiac cells (Figures 5A,B). The methylation level of the RASSFA1 promotor region (8 amplicons) was significantly higher in the hypoxic WT group vs. hypoxic TG group (Figures 8C,D). This suggests that the methylation pattern of the RASSFA1 promoter region may be attributed to differences in methyl-transferase expression and/or methyl binding proteins at mRNA level.
DISCUSSION
Free radicals play a key role in the pathogenesis of cardiac fibrosis [7]. Previously, it has been reported that the free radical scavenger, EC-SOD, can reduce, as well as reverse, some of the changes seen secondary to chronic hypoxic stress (19,24). In our study, we reported that EC-SOD overexpression leads to a significant decrease in DNA methylation (DNMT1 and DNMT-3b as well as RASSFA1 gene), which is triggered by hypoxia exposure. Among animals subjected to chronic hypoxia, markers of fibrosis were increased significantly including RASSFA1 promoter methylation and SNAIL which is involved in fibromodulation. Each of these changes was significantly reversed when EC-SOD was overexpressed in either in-vitro or in-vivo model. There was a significant increase in collagen 1, Collagen 3, and ASMA in hypoxic WT animals as compared to animals housed in room air (P < 0.05) (Figures 1A-C, 2A-C). Similar data was shown previously using human cardiac tissue [2], and it was shown that the degree of hypoxia was associated with increased expression of Collagen 1 and ASMA. In our animal model, overexpression of EC-SOD offered a significant protective effect, evident by reduction in the above listed fibrotic markers. Our data supports the role of oxidative insult induced by hypoxia, FIGURE 5 | Western blot analysis for DNMT1, 3b, and HIF-1α. Adult mice (WT) and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days. Room air animals were used as a control group (RA). WB analysis was used to assess protein levels of DNMT 1 (A) and DNMT 3b (B); HIF-1α assessment in cardiac fibroblasts (C) in the right ventricular tissue. ROS assay in all studied groups (D). All experiments were carried out in triplicate. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Frontiers in Cardiovascular Medicine | www.frontiersin.org FIGURE 6 | Western blot analysis for RASSF1A and ERK1/2. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG), were exposed to 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). WB analysis was used to assess protein levels of RASSF1A (A) and ERK1/2 (B) in the right ventricular tissue. All experiments were carried out in triplicate. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
FIGURE 7 | (A,B)
RT-PCR for α and β myosin heavy chain. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG), were exposed to 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). Quantitative RT-PCR was used to assess gene expression analysis of α-MHC in the right ventricular tissue. All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
in the pathogenesis of cardiac fibrosis. The dismutation of these free radicals, by overexpression of EC-SOD, leads to reversal of heart pathology as shown by the immunochemistry studies (Figures 3A,B).
Many studies showed the role of SOD and its protective role in animal models with cardiac fibrosis induced by chronic hypoxia, by scavenging free radicals. SOD1 suppressed MCF proliferation and differentiation and reduced the production of collagen type I and III. SOD1 overexpression leads to ROS scavenging and blocking production and block collagen production, suggesting that SOD1 may be a promising therapeutic agent for treating ROS-mediated cardiac fibrosis (25). Mice deficient in SOD2 die of cardiomyopathy within 10 days of birth, whereas heterozygous SOD2(+/-) mice show ultrastructural damage of the myocardium and mitochondria, associated with an increased oxidative stress as well as an activation of apoptotic signaling pathways in the heart (26,27). MnSOD overexpression offers protection against oxidative stress, fibrosis, and apoptosis in the aging heart (28). Serum EC-SOD activity was independently associated with abnormal LV geometry patterns with and without overt HF. Our results indicate that Ec-SOD might be a potential link between LV structure remodeling and the . Proposed mechanism (E). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Frontiers in Cardiovascular Medicine | www.frontiersin.org development of subsequent HF in patients with cardiovascular disease. Extracellular superoxide dismutase is associated with left ventricular geometry and heart failure in patients with cardiovascular disease (29). Under unstressed conditions, EC-SOD deficiency had no effect on myocardial total SOD activity but resulted in small but significant increases in myocardial fibrosis and ventricular mass. EC-SOD deficiency is associated with exacerbated myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction. All these findings indicate that the distribution of EC-SOD in the extracellular space is critically important in protecting the heart against pressure overload (17). In our study, we report an innovative mechanism of overexpression of EC-SOD. In animal models with cardiac fibrosis induced by chronic hypoxia, there is a significant increase of ERK1/2 in the hypoxic animal group in parallel with a significant increase of DNA methylation and a reduction of RASSF1A expression. EC-SOD overexpression reverses this process and leads to significant decreases in DNA methylation (DNMT1 and DNMT-3b as well as RASSFA1 gene), which is triggered by chronic hypoxia exposure.
The role of DNA methylation and epigenetic changes associated with cardiac fibrosis induced by hypoxia was studied previously (15,18). Both DNA methyltransferase enzymes (DNMT1 and DNMT3b), which are regulated by HIF-1α (30) (Figure 5C), are upregulated by chronic hypoxia (Figures 5A,B). Their upregulation was associated with increases in all fibrotic markers examined and a significant reduction in RASSF1A protein synthesis (Figure 6). In addition, the significant increase in the expression of both DNMT enzymes was associated with increased DNA methylation (Figure 6). Epigenetic changes induced by prolonged hypoxia have been shown to contribute to the pro-fibrotic nature of the ischemic environment (2). In normal human lung fibroblasts, there is a significant global hypermethylation detected in hypoxic fibroblasts relative to normoxic controls and is accompanied by increased expression of myofibroblast markers (24).
SNAIL gene expression is a potential target molecule in cardiac fibrosis after ischemia reperfusion (I/R), injury, and or oxidative stress insult (31). The SNAIL gene is best known for its capability to trigger epithelial-to-mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT), which may contribute to myofibroblast formation (30,32). SNAIL is activated by free radicals and mediates the actions of endogenous TGFβ signals that induce EndMT (33). Injection of a selective SNAIL inhibitor remarkably suppressed collagen deposition and cardiac fibrosis in mouse I/R injury and significantly improved cardiac function and reduced SNAIL expression in-vivo (34). SNAIL can recruit multiple chromatin enzymes including LSD1, HDAC1/2, and Suv39H1. These enzymes function in a highly organized manner to generate heterochromatin and promote DNA methyltransferase-mediated DNA methylation at the promoter region (35). Our data showed that a significant increase of SNAIL expression in the WT hypoxic group was attenuated in TG animals, which may lead to a disruption of the connection between SNAIL and these chromatin-modifying enzymes and may represent a therapeutic target for the treatment of cardiac fibrosis.
Hypoxia-induced DNA methylation has been shown to be involved in regulating the process of cardiac fibrosis (2,35). DNA methylation mediated silencing of the RASSF1A gene, which leads to upregulation of ERK1/2 that has been shown to increase cardiac fibrosis in cancer patients under chronic hypoxic stress (6,36). Our data showed a significant increase of ERK1/2 in the hypoxic animal group in parallel with a significant increase of DNA methylation and a reduction of RASSF1A expression ( Figure 6A). In adult cardiomyocytes, the high level of H 2 O 2 is associated with the activation of ERK1/2 MAPK and the stimulation of protein synthesis (37). Increased ERK1/2 activity leads to increased cell proliferation and collagen gene expression in activated cardiac fibroblasts [4]. Dismutation of the free radicals by the activity of EC-SOD leads to a global decrease of DNA methylation, increased RASSF1A protein synthesis, and a significant reduction in phosphorylated ERK1/2 in the transgenic hypoxic animal group (P < 0.05). This finding suggests an additional contributing mechanism to cardiac dysfunction in hypoxia, which is triggered by a change in myosin heavy chain isoform (38). Previously, it has been shown that hypoxia leads to a change in MHC from the α to β isoform, which leads to decreased cardiac contractility (37). Free radicals have been shown to affect this change and scavenging these free radicals by antioxidants can markedly attenuate cardiac fibrosis and improve ventricular ejection fraction and fractional shortening (38). In our study, there was a significant reduction in the levels of α-MHC ( Figure 7A) and significant increase in b-MHC (Figure 7B), in hypoxic WT animals as compared to the hypoxia TG group as shown in the RT-PCT assay. Hypoxia TG animals had both α&β-MHC levels close to RA controls. This critical histological change is crucial in preserving cardiac contractility and function.
We have shown that TG animals that have an additional copy of the EC-SOD gene show a significant reduction in DNA methylation in response to chronic hypoxic stress. While further studies are needed to completely clarify the mechanism, we speculate that this reduction in DNA methylation is through a reduction in the levels of HIF-1α, which is an inducer of DNMT and hence of the process of DNA methylation (6). Our data show a significant reduction in HIF-1α levels in the animals that overexpress EC-SOD (P < 0.05) (Figure 5C). This suggests a mechanism by which DNA methylation leads to cardiac fibrosis. Both these changes were mitigated in our transgenic animals, which overexpress EC-SOD.
To further explore the role of RASSF1A in cardiac fibrosis, its expression was blocked in an in-vitro model using SiRNA. The presence of the SiRNA resulted in a significant increase in fibroblast proliferation (Figures 8A,B). Furthermore, we investigated RASSF1A promoter gene methylation in the three studied groups. Our findings showed a significant increase in RASSF1A promotor region methylation in the hypoxic WT group compared to normoxic animals. This methylation process was reduced by more than 10% among the hypoxic TG animals, which showed a significant reduction of both biochemical and histopathological evidence of cardiac fibrosis.
Epigenetic inactivation of RASSF1A by methylation is a very common event in prostate cancer and might be involved in the progression of the disease (39). In prostate cancer, hypomehtylation of BNIP3 and hypermethylation of both EC-SOD and RASSF1A were observed. These changes were positively associated with oxidative stress and inverse associated with EC-SOD expression. Among patients with prostate cancer, it was found that glutamate carboxypeptidase II genetic variants contribute to increased oxidative stress and prostate cancer risk by modulating the CpG island methylation of Ec-SOD (40).
In summary, our study presents a novel mechanism by which EC-SOD offers cardiac protection against fibrosis induced by chronic or prolonged hypoxia. The data identifies a critical role of EC-SOD in the control of DNA methylation. Our proposed mechanism, illustrated in Figure 8E, suggests that EC-SOD expression will chelate the free radicals, induced by hypoxia; as a result, both HIF-1α and SNAIL gene activation will be decreased and subsequently methylation enzyme activity will decrease and RASSF1A gene expression will not be silenced or decreased due to lack of methylation. RASSF1A expression downregulates the activity of the ERK1/2 pathway which regulates activation of both cardiac fibroblast proliferation and transition of endothelial cells to myofibroblast. Another benefit from using antioxidants is chelating hydrogen peroxide, which will significantly decrease the activation of ERK1/2, as it is triggered directly by hydrogen peroxide concentration. Previously EC-SOD compounds and its mimetics were used in experimental and clinical trials to counteract the oxidative stress which was linked to the pathogenesis of these disorders (41)(42)(43)(44)(45)(46). Further studies of this mechanism could lead to specific inhibition of the pathway in the clinic to significantly reduce cardiac fibrosis and dramatically improve the outcome of this devastating condition.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and use Committee of the Feinstein Institute for Medical Research. Written informed consent was obtained from the owners for the participation of their animals in this study.
AUTHOR CONTRIBUTIONS
MA and AR designed the research. KA, JL, NZ, and AR performed the research and analyzed the data. EM and MA wrote the manuscript. All authors contributed to the article and approved the submitted version.
FUNDING
All funding was provided by institutional support.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2021.669975/full#supplementary-material
FIGURE 1 |
1RT-PCR for markers of fibrosis-Collagen 1, Collagen 3, and ASMA. Adult male C57B6 mice, WT, and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days (WT). The control group was composed of animals housed in room air (RA). Quantitative RT-PCR was used to assess gene expression analysis of Collagen 1 (A), Collagen 3 (B), and ASMA (C) in the right ventricular tissue. All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
FIGURE 8 |
8Cell proliferation studies after transfection with SiRNA for RASSF1A-Cardiac fibroblasts were transfected with SiRNA for RASSF1A and compared to cells placed in RA which served as a control. BrdU cell proliferation studies were undertaken (A), and cells as having underwent cell counts (B). DNA methylation studies of the RASSF1A promotor region as shown in the heat map: RA group (samples 1-4); WT hypoxic group (Samples 6-9); TG hypoxic group (samples 11-15) (C). Quantitative estimation of methylation level among the three studied groups (D)
NY, USA). Cells were maintained in a microenvironment of 37 • C, 1% O 2 , 5% CO 2 , and 100% humidity. To examine the effects of hypoxia ± EC-SOD overexpression on global DNA methylation, both transfected and non-transfected MCF were incubated in hypoxia for 72 h. Control, non-transfected MCF were maintained in 21% oxygen for 72 h. Post culture, MCF cells were fixed in Carnoy's solution prior to 60-min acid hydrolysis in 1 M HCl at 37 • C. Following this DNA denaturation step, cells were then treated with either an anti-5 ′ methylcytidine (5MeC) monoclonal antibody (EpiGentek, Catalog No. A-1014) or a non-specific IgG1 antibody (BD Biosciences, San Jose, CA). IgG1-negative controls were used at the same concentration as the primary antibody. Immunostaining was conducted using an FITC-conjugated rabbit anti-mouse secondary antibody (Thermo Scientific, Catalog No. 31561). The cells were then subjected to flow cytometry (BD Biosciences, San Jose, CA) and the results assessed using CellQuest Pro (BD Biosciences, San Jose, CA).The expression of RASSF1A was reduced by transfection of MCF with small interfering RNA (SiRNA) SiRASSF1A (Thermo Fisher Scientific, catalog no. 185488) located on Chr.9: 107551555-107562267 on Build GRCm38MCF, using Lipofectamine RNAiMAX transfection protocol (Life Technologies). Effectiveness of the transfection was evaluated at 24, 48, 72, and 96 h post transfection with western blot analysis using an antibody specific for RASSF1A (Abcam). Post-transfection studies showed that RASSF1A expression is minimal after 72 h of transfection (data are not shown). This time point was used for cell proliferation assessment in the next step. Cells were housed in a humidified incubator (37 • C, 5% CO 2 ) and compared to control MCF, which were transfected with an empty vector and kept under the same conditions., using the FuGENE kit
(Roche Diagnostics, Indianapolis, IN, USA). Each well received
1 µg DNA/100 µL serum-free medium of the DNA/FuGENE
complex. Control wells received the serum-free medium alone.
Transfected cells were selected using Geneticin (Invitrogen Life
Technologies). Transfection of the fibroblasts was confirmed by
Western blot analysis using an antibody specific for hEC-SOD
(R&D Systems, Minneapolis, MN, US).
Quantitative Flow Cytometry-Effects of Hypoxia on
Global DNA Methylation Profile
A modular incubator chamber (Billups-Rothenberg, Del Mar,
CA, USA) was used for the cell hypoxia studies and a 1% oxygen
atmosphere maintained using an oxygen sensor (BioSpherix,
Lacona, Proliferation Studies-Effects of Blocking RASSF1A
Expression
After 72 h of transfection, MCF proliferation was assessed by
BrdU (5-bromouridine) incorporation, (Roche Diagnostics,
Mannheim). Cell counts were performed using a hemocytometer
24 and 48 h later.
Frontiers in Cardiovascular Medicine | www.frontiersin.org
May 2021 | Volume 8 | Article 669975
Supplementary #1 | Strategy for RASSF1A sequencing.Supplementary #2 | Methylation study design.Supplementary #3 | Comparison between RA WT control group and RA TG groups. (A) Collagen 1: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (B) ASMA: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (C) SNAIL1: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (D) DNMT1: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (E) DNMT 3B: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (F) HIF-1α&β Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (G) RASSF1A: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05). (H) Phosphorylated ERK ½: Comparison between RA WT and RA TG groups, no significant difference (P < 0.05).Conflict of Interest:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Copyright © 2021 Rajgarhia, Ayasolla, Zaghloul, Lopez Da Re, Miller and Ahmed.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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| [
"Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O 2 ) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia."
] | [
"Gianfranco Pintus ",
"Tetsuro Kamiya ",
"Mohamed Ahmed [email protected] ",
"Ayan Rajgarhia \nSchool of Medicine, Children's Mercy Hospital and University of Missouri-Kansas City\nKansas CityMOUnited States\n",
"Kameshwar R Ayasolla \nHenry Ford Health System\nDetroitMIUnited States\n",
"Nahla Zaghloul \nNeonatal Division\nNemours Children's Hospital\nUniversity of Arizona\nUnited States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States\n",
"Jorge M Lopez ",
"Da Re ",
"Edmund J Miller ",
"Mohamed Ahmed \nNeonatal Division\nNemours Children's Hospital\nUniversity of Arizona\nUnited States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States\n",
"\nUniversity of Sharjah\nUnited Arab Emirates\n",
"\nRadha Gopal\nGifu Pharmaceutical University\nJapan\n",
"\nUniversity of Pittsburgh\nUnited States\n"
] | [
"School of Medicine, Children's Mercy Hospital and University of Missouri-Kansas City\nKansas CityMOUnited States",
"Henry Ford Health System\nDetroitMIUnited States",
"Neonatal Division\nNemours Children's Hospital\nUniversity of Arizona\nUnited States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States",
"Neonatal Division\nNemours Children's Hospital\nUniversity of Arizona\nUnited States, 4 Neonatal Division, United States, 5 RDS2 Solutions, Stony BrookTucson, Orlando, Orlando, New YorkAZ, FL, NYUnited States",
"University of Sharjah\nUnited Arab Emirates",
"Radha Gopal\nGifu Pharmaceutical University\nJapan",
"University of Pittsburgh\nUnited States"
] | [
"Gianfranco",
"Tetsuro",
"Mohamed",
"Ayan",
"Kameshwar",
"R",
"Nahla",
"Jorge",
"M",
"Da",
"Edmund",
"J",
"Mohamed"
] | [
"Pintus",
"Kamiya",
"Ahmed",
"Rajgarhia",
"Ayasolla",
"Zaghloul",
"Lopez",
"Re",
"Miller",
"Ahmed"
] | [
"B Swynghedauw, ",
"C J Watson, ",
"P Collier, ",
"I Tea, ",
"R Neary, ",
"J A Watson, ",
"C Robinson, ",
"D A Siwik, ",
"P J Pagano, ",
"W S Colucci, ",
"P Kong, ",
"P Christia, ",
"N Frangogiannis, ",
"L Shivakumar, ",
"J Minna, ",
"T Sakamaki, ",
"R Pestell, ",
"M A White, ",
"H Tao, ",
"J Yang, ",
"Z Chen, ",
"S S Xu, ",
"X Zhou, ",
"H Y Zhang, ",
"J Heineke, ",
"J D Molketin, ",
"C A Dechesne, ",
"P Bouvagnet, ",
"D Walzthony, ",
"J J Leger, ",
"O Dewald, ",
"N G Frangogiannis, ",
"M Zoerlin, ",
"G D Duerr, ",
"C Klemm, ",
"P Knuefermann, ",
"F Gao, ",
"J R Koenitzer, ",
"J M Tobolewski, ",
"D Jiang, ",
"J Liang, ",
"P W Noble, ",
"S V Kalivendi, ",
"S Kotamraju, ",
"H Zhao, ",
"J Joseph, ",
"B Kalyanaraman, ",
"T Kokudo, ",
"Y Suzuki, ",
"Y Yoshimatsu, ",
"T Yamazaki, ",
"T Watabe, ",
"K Miyazono, ",
"G G Konduri, ",
"I Bakhutashvili, ",
"A Eis, ",
"K Pritchard, ",
"N Pradhan, ",
"S Parbin, ",
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"E D Van, ",
"Z Lu, ",
"X Xu, ",
"G Zhu, ",
"X Hu, ",
"T D Oury, ",
"S Sharma, ",
"O Dewald, ",
"J Adrogue, ",
"R L Salazar, ",
"P Razeghi, ",
"J D Crapo, ",
"Z Lu, ",
"X Xu, ",
"X Hu, ",
"G Zhu, ",
"P Zhang, ",
"E D Van Deel, ",
"S O Lim, ",
"J M Gu, ",
"M S Kim, ",
"H S Kim, ",
"Y N Park, ",
"C K Park, ",
"T D Oury, ",
"Y S Ho, ",
"C A Piantadosi, ",
"J D Crapo, ",
"N Zaghloul, ",
"M Nasim, ",
"H Patel, ",
"C Codipilly, ",
"P Marambauk, ",
"S Dewey, ",
"M N Ahmed, ",
"Y Zhang, ",
"C Codipilly, ",
"N Zaghloul, ",
"D Patel, ",
"M Wolin, ",
"S Cikos, ",
"A Bukovska, ",
"J Koppel, ",
"J Schindelin, ",
"I Arganda-Carreras, ",
"E Frise, ",
"V Kaynig, ",
"M Longair, ",
"T Pietzsch, ",
"Y Chu, ",
"A Alwahdani, ",
"S Lida, ",
"D D Lund, ",
"F M Faraci, ",
"D D Heistad, ",
"L G Tan, ",
"J H Xiao, ",
"D L Yu, ",
"L Zhang, ",
"F Zheng, ",
"L Y Guo, ",
"M Strassburger, ",
"W Bloch, ",
"S Sulyok, ",
"J Schuller, ",
"A F Keist, ",
"A Schmidt, ",
"S Sharma, ",
"S Bhattarai, ",
"Ara H Sun, ",
"G St Clair, ",
"D K Bhuiyan, ",
"M S , ",
"H B Kwak, ",
"Y Lee, ",
"J H Kim, ",
"H Van Remmen, ",
"A G Richardson, ",
"J M Lawler, ",
"X Li, ",
"Y Lin, ",
"S Wang, ",
"S Zhou, ",
"J Ju, ",
"X Wang, ",
"M W Luczak, ",
"A Roszak, ",
"P Pawlik, ",
"H Kedzia, ",
"W Kedzia, ",
"B Malkowska-Walczak, ",
"A Barrallo-Gimeno, ",
"M A Nieto, ",
"S W Lee, ",
"J Y Won, ",
"W J Kim, ",
"J Lee, ",
"K H Kim, ",
"S W Youn, ",
"D Medici, ",
"S Potenta, ",
"R Kalluri, ",
"H Wang, ",
"M W Tibbitt, ",
"S J Langer, ",
"L A Leinwand, ",
"K S Anseth, ",
"C M Robinson, ",
"R Neary, ",
"A Levendale, ",
"C J Watson, ",
"J A Baugh, ",
"S Tommasi, ",
"R Dammann, ",
"Z Zhang, ",
"Y Wang, ",
"L Liu, ",
"M Tsark, ",
"K Nakanishi, ",
"Y Nakata, ",
"F Kanazawa, ",
"S I Imamura, ",
"R Matusuoka, ",
"H Osada, ",
"S Arumugam, ",
"S Mito, ",
"R A Thandavarayan, ",
"V V Giridharan, ",
"V Pitchaimani, ",
"V Karuppagounder, ",
"L Liu, ",
"J H Yoon, ",
"R Dammann, ",
"G P Pfeifer, ",
"S Divyya, ",
"S M Naushad, ",
"P V Murthy, ",
"C H R Reddy, ",
"V K Kutala, ",
"W Rosenfeld, ",
"H Evans, ",
"L Concepcion, ",
"R Jhaveri, ",
"H Schaeffer, ",
"A Friedman, ",
"J M Davis, ",
"W N Rosenfeld, ",
"S E Richter, ",
"M R Parad, ",
"I H Gewolb, ",
"A R Spitzer, ",
"G Jadot, ",
"A Vaille, ",
"J Maldonado, ",
"P Vanelle, ",
"D A Siwik, ",
"P J Pagano, ",
"W S Colucci, ",
"J S Rebouças, ",
"I Spasojevi, ",
"I Batinić-Haberle, ",
"R Chen, ",
"Q Zhao, ",
"N Wu, ",
"W Zhong, ",
"Jin X Liu, ",
"C , "
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] | [
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"Vascular effects of the human extracellular superoxide dismutase R213G variant. Y Chu, A Alwahdani, S Lida, D D Lund, F M Faraci, D D Heistad, 10.1161/CIRCULATIONAHA.104.531251Circulation. 112Chu Y, Alwahdani A, Lida S, Lund DD, Faraci FM, Heistad DD, et al. Vascular effects of the human extracellular superoxide dismutase R213G variant. Circulation. (2005) 112:1047- 53. doi: 10.1161/CIRCULATIONAHA.104.531251",
"PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production. L G Tan, J H Xiao, D L Yu, L Zhang, F Zheng, L Y Guo, 10.1186/s12872-015-0103-4BMC Cardiovasc Disord. 15116Tan LG, Xiao JH, Yu DL, Zhang L, Zheng F, Guo LY, et al. PEP- 1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production. BMC Cardiovasc Disord. (2015) 15:116. doi: 10.1186/s12872-015-0103-4",
"Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo. M Strassburger, W Bloch, S Sulyok, J Schuller, A F Keist, A Schmidt, 10.1016/j.freeradbiomed.2005.02.009Free Radic Biol Med. 38Strassburger M, Bloch W, Sulyok S, Schuller J, Keist AF, Schmidt A, et al. Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo. Free Radic Biol Med. (2005) 38:1458-70. doi: 10.1016/j.freeradbiomed.2005.02.009",
"SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy. S Sharma, S Bhattarai, Ara H Sun, G St Clair, D K Bhuiyan, M S , 10.1016/j.redox.2020.101740Redox Biol. 37101740Sharma S, Bhattarai S, Ara H, Sun G, St Clair DK, Bhuiyan MS, et al. SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy. Redox Biol. (2020) 37:101740. doi: 10.1016/j.redox.2020.101740",
"MnSOD overexpression reduces fibrosis and pro-apoptotic signaling in the aging mouse heart. H B Kwak, Y Lee, J H Kim, H Van Remmen, A G Richardson, J M Lawler, 10.1093/gerona/glu090J Gerontol Ser A Biol Sci Med Sci. 70Kwak HB, Lee Y, Kim JH, Van Remmen H, Richardson AG, Lawler JM. MnSOD overexpression reduces fibrosis and pro-apoptotic signaling in the aging mouse heart. J Gerontol Ser A Biol Sci Med Sci. (2015) 70:533- 44. doi: 10.1093/gerona/glu090",
"Extracellular superoxide dismutase is associated with left ventricular geometry and heart failure in patients with cardiovascular disease. X Li, Y Lin, S Wang, S Zhou, J Ju, X Wang, 10.1161/JAHA.120.016862J Am Heart Assoc. 916862Li X, Lin Y, Wang S, Zhou S, Ju J, Wang X, et al. Extracellular superoxide dismutase is associated with left ventricular geometry and heart failure in patients with cardiovascular disease. J Am Heart Assoc. (2020) 9:e016862. doi: 10.1161/JAHA.120.016862",
"Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma. M W Luczak, A Roszak, P Pawlik, H Kedzia, W Kedzia, B Malkowska-Walczak, 10.3892/ijo.2011.1183Int J Oncol. 40Luczak MW, Roszak A, Pawlik P, Kedzia H, Kedzia W, Malkowska-Walczak B, et al. Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma. Int J Oncol. (2012) 40:860-6. doi: 10.3892/ijo.2011.1183",
"The Snail genes as inducers of cell movement and survival: implications in development and cancer. A Barrallo-Gimeno, M A Nieto, 10.1242/dev.01907Development. 132Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development. (2005) 132:3151-61. doi: 10.1242/dev.01907",
"Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis. S W Lee, J Y Won, W J Kim, J Lee, K H Kim, S W Youn, 10.1038/mt.2013.146Mol Ther. 9Lee SW, Won JY, Kim WJ, Lee J, Kim KH, Youn SW, et al. Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis. Mol Ther. (2013) 9:1767- 77. doi: 10.1038/mt.2013.146",
"Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signaling. D Medici, S Potenta, R Kalluri, 10.1042/BJ20101500Biochem J. 437Medici D, Potenta S, Kalluri R. Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signaling. Biochem J. (2011) 437:515-20. doi: 10.1042/BJ20101500",
"Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticityregulated PI3 K/AKT pathway. H Wang, M W Tibbitt, S J Langer, L A Leinwand, K S Anseth, 10.1073/pnas.1306369110Proc Natl Acad Sci. 110Wang H, Tibbitt MW, Langer SJ, Leinwand LA, Anseth KS. Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticity- regulated PI3 K/AKT pathway. Proc Natl Acad Sci USA. (2013) 110:19336- 41. doi: 10.1073/pnas.1306369110",
"Hypoxiainduced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype. C M Robinson, R Neary, A Levendale, C J Watson, J A Baugh, 10.1186/1465-9921-13-74Respir Res. 3174Robinson CM, Neary R, Levendale A, Watson CJ, Baugh JA. Hypoxia- induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype. Respir Res. (2012) 31:74. doi: 10.1186/1465-9921-13-74",
"Tumor susceptibility of Rassf1a knockout mice. S Tommasi, R Dammann, Z Zhang, Y Wang, L Liu, M Tsark, Cancer Res. 65Tommasi S, Dammann R, Zhang Z, Wang Y, Liu L, Tsark M, et al. Tumor susceptibility of Rassf1a knockout mice. Cancer Res. (2005) 65:92-8.",
"Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension. K Nakanishi, Y Nakata, F Kanazawa, S I Imamura, R Matusuoka, H Osada, 10.1002/path.1132J Pathol. 197Nakanishi K, Nakata Y, Kanazawa F, Imamura SI, Matusuoka R, Osada H, et al. Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension. J Pathol. (2002) 197:380-7. doi: 10.1002/path.1132",
"Mulberry leaf diet protects against progression of experimental autoimmune myocarditis to dilated cardiomyopathy via modulation of oxidative stress and MAPK-mediated apoptosis. S Arumugam, S Mito, R A Thandavarayan, V V Giridharan, V Pitchaimani, V Karuppagounder, 10.1111/1755-5922.12029Cardiovasc Ther. 16Arumugam S, Mito S, Thandavarayan RA, Giridharan VV, Pitchaimani V, Karuppagounder V, et al. Mulberry leaf diet protects against progression of experimental autoimmune myocarditis to dilated cardiomyopathy via modulation of oxidative stress and MAPK-mediated apoptosis. Cardiovasc Ther. (2013) 16:352-62. doi: 10.1111/1755-5922.12029",
"Frequent hypermethylation of the RASSF1A gene in prostate cancer. L Liu, J H Yoon, R Dammann, G P Pfeifer, 10.1038/sj.onc.1205814Oncogene. 21Liu L, Yoon JH, Dammann R, Pfeifer GP. Frequent hypermethylation of the RASSF1A gene in prostate cancer. Oncogene. (2002) 21:6835- 40. doi: 10.1038/sj.onc.1205814",
"GCPII modulates oxidative stress and prostate cancer susceptibility through changes in methylation of RASSF1, BNIP3, GSTP1 and Ec-SOD. S Divyya, S M Naushad, P V Murthy, C H R Reddy, V K Kutala, 10.1007/s11033-013-2655-7Mol Biol Rep. 40Divyya S, Naushad SM, Murthy PV, Reddy, C.h.R., Kutala VK. GCPII modulates oxidative stress and prostate cancer susceptibility through changes in methylation of RASSF1, BNIP3, GSTP1 and Ec-SOD. Mol Biol Rep. (2013) 40:5541-50. doi: 10.1007/s11033-013-2655-7",
"Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome. W Rosenfeld, H Evans, L Concepcion, R Jhaveri, H Schaeffer, A Friedman, 10.1016/S0022-3476(84)80307-8J Pediatr. 105Rosenfeld W, Evans H, Concepcion L, Jhaveri R, Schaeffer H, Friedman A. Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome. J Pediatr. (1984) 105:781-5. doi: 10.1016/S0022-3476(84)80307-8",
"Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome. J M Davis, W N Rosenfeld, S E Richter, M R Parad, I H Gewolb, A R Spitzer, 10.1542/peds.100.1.24Pediatrics. 100Davis JM, Rosenfeld WN, Richter SE, Parad MR, Gewolb IH, Spitzer AR, et al. Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome. Pediatrics. (1997) 100:24- 30. doi: 10.1542/peds.100.1.24",
"Clinical pharmacokinetics and delivery of bovine superoxide dismutase. G Jadot, A Vaille, J Maldonado, P Vanelle, 10.2165/00003088-199528010-00003Clin Pharmacokinet. 28Jadot G, Vaille A, Maldonado J, Vanelle P. Clinical pharmacokinetics and delivery of bovine superoxide dismutase. Clin Pharmacokinet. (1995) 28:17- 25. doi: 10.2165/00003088-199528010-00003",
"Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. D A Siwik, P J Pagano, W S Colucci, 10.1152/ajpcell.2001.280.1.C53Am J Physiol Cell Physiol. 280Siwik DA, Pagano PJ, Colucci WS. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol. (2001) 280:C53-60. doi: 10.1152/ajpcell.2001.280.1.C53",
"Quality of potent Mn porphyrin-based SOD mimics and peroxynitrite scavengers for pre-clinical mechanistic/therapeutic purposes. J S Rebouças, I Spasojevi, I Batinić-Haberle, 10.1016/j.jpba.2008.08.005J Pharm Biomed Anal. 48Rebouças JS, Spasojevi,ć I, Batinić-Haberle I. Quality of potent Mn porphyrin-based SOD mimics and peroxynitrite scavengers for pre-clinical mechanistic/therapeutic purposes. J Pharm Biomed Anal. (2008) 48:1046- 9. doi: 10.1016/j.jpba.2008.08.005",
"Pharmacokinetics and safety of PC-SOD, a lecithinized recombinant superoxide dismutase, in healthy Chinese subjects: a phase 1, randomized, placebocontrolled, dose-escalation study. R Chen, Q Zhao, N Wu, W Zhong, Jin X Liu, C , 10.5414/CP203550Int J Clin Pharmacol Ther. 57Chen R, Zhao Q, Wu N, Zhong W, Jin X, Liu C, et al. Pharmacokinetics and safety of PC-SOD, a lecithinized recombinant superoxide dismutase, in healthy Chinese subjects: a phase 1, randomized, placebo- controlled, dose-escalation study. Int J Clin Pharmacol Ther. (2019) 57:596-602. doi: 10.5414/CP203550"
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] | [
"Molecular mechanisms of myocardial remodeling",
"Hypoxia induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype",
"Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts",
"The pathogenesis of cardiac fibrosis",
"The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation",
"DNMT3A silencing RASSF1A promotes cardiac fibrosis through upregulation of ERK1/2",
"Regulation of cardaic hypertrophy by intracellular signalling pathways",
"Visualization of cardiac Ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping",
"Development of murine ischemic cardiomyopathy is associated with a transient inflammatory reaction and depends on reactive oxygen species",
"Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan",
"Doxorubicininduced apoptosis is associated with increased transcription of endothelial nitric-oxide synthase. Effect of antiapoptotic antioxidants and calcium",
"Snail is required for TGFβ-induced endothelialmesenchymal transition of embryonic stem cell derived endothelial cells",
"Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension",
"Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer",
"Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction",
"Induction of antioxidant gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species",
"EC-SOD deficiency exacerbates pressure overload-induced left ventricular hypertrophy and dysfunction",
"Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter",
"Extracellular superoxide dismutase, nitric oxide, and central nervous system O2 toxicity",
"Overexpression of EC-SOD has a protective role against hyperoxia induced brain injury in neonatal mice",
"EC-SOD overexpression can reverse the course of hypoxia-induced pulmonary hypertension in adult mice",
"Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis",
"Fiji: an open-source platform for biological-image analysis",
"Vascular effects of the human extracellular superoxide dismutase R213G variant",
"PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production",
"Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo",
"SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy",
"MnSOD overexpression reduces fibrosis and pro-apoptotic signaling in the aging mouse heart",
"Extracellular superoxide dismutase is associated with left ventricular geometry and heart failure in patients with cardiovascular disease",
"Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma",
"The Snail genes as inducers of cell movement and survival: implications in development and cancer",
"Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis",
"Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signaling",
"Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticityregulated PI3 K/AKT pathway",
"Hypoxiainduced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype",
"Tumor susceptibility of Rassf1a knockout mice",
"Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension",
"Mulberry leaf diet protects against progression of experimental autoimmune myocarditis to dilated cardiomyopathy via modulation of oxidative stress and MAPK-mediated apoptosis",
"Frequent hypermethylation of the RASSF1A gene in prostate cancer",
"GCPII modulates oxidative stress and prostate cancer susceptibility through changes in methylation of RASSF1, BNIP3, GSTP1 and Ec-SOD",
"Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome",
"Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome",
"Clinical pharmacokinetics and delivery of bovine superoxide dismutase",
"Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts",
"Quality of potent Mn porphyrin-based SOD mimics and peroxynitrite scavengers for pre-clinical mechanistic/therapeutic purposes",
"Pharmacokinetics and safety of PC-SOD, a lecithinized recombinant superoxide dismutase, in healthy Chinese subjects: a phase 1, randomized, placebocontrolled, dose-escalation study"
] | [
"Physiol Rev",
"Hum Mol Genet",
"Am J Physiol Cell Physiol",
"Cell Mol Life Sci",
"Mol Cell Biol",
"Toxicology",
"Nat Rev Mol Cell Biol",
"J Cell Biol",
"Proc Natl Acad Sci",
"J Biol Chem",
"J Biol Chem",
"J Cell Sci",
"Am. J. Physiol Heart Circ Physiol",
"Biochim Biophys Acta Mol Basis Dis",
"Free Radic Biol Med",
"Free Radic Biol Med",
"Hypertension",
"Gastroenterology",
"Proc Natl Acad Sci",
"FEBS",
"Mol Med",
"BMC Mol Biol",
"Nat Methods",
"Circulation",
"BMC Cardiovasc Disord",
"Free Radic Biol Med",
"Redox Biol",
"J Gerontol Ser A Biol Sci Med Sci",
"J Am Heart Assoc",
"Int J Oncol",
"Development",
"Mol Ther",
"Biochem J",
"Proc Natl Acad Sci",
"Respir Res",
"Cancer Res",
"J Pathol",
"Cardiovasc Ther",
"Oncogene",
"Mol Biol Rep",
"J Pediatr",
"Pediatrics",
"Clin Pharmacokinet",
"Am J Physiol Cell Physiol",
"J Pharm Biomed Anal",
"Int J Clin Pharmacol Ther"
] | [
"\nFIGURE 1 |\n1RT-PCR for markers of fibrosis-Collagen 1, Collagen 3, and ASMA. Adult male C57B6 mice, WT, and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days (WT). The control group was composed of animals housed in room air (RA). Quantitative RT-PCR was used to assess gene expression analysis of Collagen 1 (A), Collagen 3 (B), and ASMA (C) in the right ventricular tissue. All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"\nFIGURE 8 |\n8Cell proliferation studies after transfection with SiRNA for RASSF1A-Cardiac fibroblasts were transfected with SiRNA for RASSF1A and compared to cells placed in RA which served as a control. BrdU cell proliferation studies were undertaken (A), and cells as having underwent cell counts (B). DNA methylation studies of the RASSF1A promotor region as shown in the heat map: RA group (samples 1-4); WT hypoxic group (Samples 6-9); TG hypoxic group (samples 11-15) (C). Quantitative estimation of methylation level among the three studied groups (D)",
"\n\nNY, USA). Cells were maintained in a microenvironment of 37 • C, 1% O 2 , 5% CO 2 , and 100% humidity. To examine the effects of hypoxia ± EC-SOD overexpression on global DNA methylation, both transfected and non-transfected MCF were incubated in hypoxia for 72 h. Control, non-transfected MCF were maintained in 21% oxygen for 72 h. Post culture, MCF cells were fixed in Carnoy's solution prior to 60-min acid hydrolysis in 1 M HCl at 37 • C. Following this DNA denaturation step, cells were then treated with either an anti-5 ′ methylcytidine (5MeC) monoclonal antibody (EpiGentek, Catalog No. A-1014) or a non-specific IgG1 antibody (BD Biosciences, San Jose, CA). IgG1-negative controls were used at the same concentration as the primary antibody. Immunostaining was conducted using an FITC-conjugated rabbit anti-mouse secondary antibody (Thermo Scientific, Catalog No. 31561). The cells were then subjected to flow cytometry (BD Biosciences, San Jose, CA) and the results assessed using CellQuest Pro (BD Biosciences, San Jose, CA).The expression of RASSF1A was reduced by transfection of MCF with small interfering RNA (SiRNA) SiRASSF1A (Thermo Fisher Scientific, catalog no. 185488) located on Chr.9: 107551555-107562267 on Build GRCm38MCF, using Lipofectamine RNAiMAX transfection protocol (Life Technologies). Effectiveness of the transfection was evaluated at 24, 48, 72, and 96 h post transfection with western blot analysis using an antibody specific for RASSF1A (Abcam). Post-transfection studies showed that RASSF1A expression is minimal after 72 h of transfection (data are not shown). This time point was used for cell proliferation assessment in the next step. Cells were housed in a humidified incubator (37 • C, 5% CO 2 ) and compared to control MCF, which were transfected with an empty vector and kept under the same conditions., using the FuGENE kit \n(Roche Diagnostics, Indianapolis, IN, USA). Each well received \n1 µg DNA/100 µL serum-free medium of the DNA/FuGENE \ncomplex. Control wells received the serum-free medium alone. \nTransfected cells were selected using Geneticin (Invitrogen Life \nTechnologies). Transfection of the fibroblasts was confirmed by \nWestern blot analysis using an antibody specific for hEC-SOD \n(R&D Systems, Minneapolis, MN, US). \n\nQuantitative Flow Cytometry-Effects of Hypoxia on \nGlobal DNA Methylation Profile \n\nA modular incubator chamber (Billups-Rothenberg, Del Mar, \nCA, USA) was used for the cell hypoxia studies and a 1% oxygen \natmosphere maintained using an oxygen sensor (BioSpherix, \nLacona, Proliferation Studies-Effects of Blocking RASSF1A \nExpression \n\nAfter 72 h of transfection, MCF proliferation was assessed by \nBrdU (5-bromouridine) incorporation, (Roche Diagnostics, \n\nMannheim). Cell counts were performed using a hemocytometer \n24 and 48 h later. \n\n"
] | [
"RT-PCR for markers of fibrosis-Collagen 1, Collagen 3, and ASMA. Adult male C57B6 mice, WT, and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days (WT). The control group was composed of animals housed in room air (RA). Quantitative RT-PCR was used to assess gene expression analysis of Collagen 1 (A), Collagen 3 (B), and ASMA (C) in the right ventricular tissue. All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"Cell proliferation studies after transfection with SiRNA for RASSF1A-Cardiac fibroblasts were transfected with SiRNA for RASSF1A and compared to cells placed in RA which served as a control. BrdU cell proliferation studies were undertaken (A), and cells as having underwent cell counts (B). DNA methylation studies of the RASSF1A promotor region as shown in the heat map: RA group (samples 1-4); WT hypoxic group (Samples 6-9); TG hypoxic group (samples 11-15) (C). Quantitative estimation of methylation level among the three studied groups (D)",
"NY, USA). Cells were maintained in a microenvironment of 37 • C, 1% O 2 , 5% CO 2 , and 100% humidity. To examine the effects of hypoxia ± EC-SOD overexpression on global DNA methylation, both transfected and non-transfected MCF were incubated in hypoxia for 72 h. Control, non-transfected MCF were maintained in 21% oxygen for 72 h. Post culture, MCF cells were fixed in Carnoy's solution prior to 60-min acid hydrolysis in 1 M HCl at 37 • C. Following this DNA denaturation step, cells were then treated with either an anti-5 ′ methylcytidine (5MeC) monoclonal antibody (EpiGentek, Catalog No. A-1014) or a non-specific IgG1 antibody (BD Biosciences, San Jose, CA). IgG1-negative controls were used at the same concentration as the primary antibody. Immunostaining was conducted using an FITC-conjugated rabbit anti-mouse secondary antibody (Thermo Scientific, Catalog No. 31561). The cells were then subjected to flow cytometry (BD Biosciences, San Jose, CA) and the results assessed using CellQuest Pro (BD Biosciences, San Jose, CA).The expression of RASSF1A was reduced by transfection of MCF with small interfering RNA (SiRNA) SiRASSF1A (Thermo Fisher Scientific, catalog no. 185488) located on Chr.9: 107551555-107562267 on Build GRCm38MCF, using Lipofectamine RNAiMAX transfection protocol (Life Technologies). Effectiveness of the transfection was evaluated at 24, 48, 72, and 96 h post transfection with western blot analysis using an antibody specific for RASSF1A (Abcam). Post-transfection studies showed that RASSF1A expression is minimal after 72 h of transfection (data are not shown). This time point was used for cell proliferation assessment in the next step. Cells were housed in a humidified incubator (37 • C, 5% CO 2 ) and compared to control MCF, which were transfected with an empty vector and kept under the same conditions."
] | [
"(Figure 1A)",
"(Figure 1B)",
"Figure 1C)",
"(Figure 2A)",
"(Figure 2B)",
"Figure 2C",
"(Figures 3A,B)",
"(Figure 4)",
"Figure 5A",
"(Figure 5B)",
"Figure 5C",
"(Figure 5D)",
"(Figure 6A)",
"Figure 6B",
"(Figures 7A,B)",
"Figure 8A)",
"(Figure 8B",
"(Figures 5A,B)",
"(Figures 8C,D)",
"(Figures 1A-C, 2A-C)",
"(Figures 3A,B)",
"(Figure 5C)",
"(Figures 5A,B)",
"(Figure 6",
"(Figure 6",
"Figure 6A)",
"Figure 7A",
"(Figure 7B)",
"(Figure 5C",
"(Figures 8A,B)",
"Figure 8E"
] | [] | [
"Cardiac fibrosis can develop following a variety of stimuli, including ischemia, volume overload, pressure overload, and hypoxia (1). A common feature of all these stimuli is the reduced availability of oxygen. Whether from decreased oxygen delivery or from increased oxygen consumption, tissue hypoxia is associated with infiltration of inflammatory cells and activation of resident cells (2). Cardiac fibroblasts, the main resident cells, are activated and transform to myofibroblasts, which are the key driver for the fibrotic response. Other cell types act indirectly by secreting fibrogenic mediators (macrophages, mast cells, lymphocytes, cardiomyocytes, and vascular cells). Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Oxidative stress activates mitogenactivated protein kinases and stress-responsive protein kinases (3). Markers of cardiac fibrosis include collagen I and III, alpha smooth muscle actin (ASMA), and SNAIL (4). Cardiac fibrosis leads to both systolic and diastolic dysfunction and increases cell death and damage by inflammatory mediators. Prognosis depends on the etiology and extent of the disease, with some cases caused by chronic hypoxia showing some reversibility of fibrosis (4).",
"DNA methylation is an epigenetic modification, which plays an important role in the cellular response to chronic hypoxia and the progression of cardiac fibrosis (2). DNA methylation alters the chromatin structure leading to repression of gene expression. The methylation process is regulated by a family of DNA methyltransferase (DNMT) enzymes. Studies have demonstrated significant increases in the activities of DNMT1 (the enzyme responsible for maintaining the methylation status of daughter cells during cell cycle) and DNMT 3B (de novo methylating enzyme) in response to hypoxia (2). The hypoxiainduced expression of DNMT1 and DNMT 3B is in part regulated by hypoxia-inducible transcription factor 1α (HIF-1α), through specific hypoxic response elements present in the promoter sequence of DNMT1 and 3B (2). Their increased activity correlates positively with the degree of cardiac fibrosis (2). This suggests the role for epigenetic modification in fibrosis secondary to hypoxia (2).",
"Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene, and alterations in its regulation are frequently seen in cardiac fibrosis (2). RASSF1A inhibits proliferation by inhibiting the accumulation of cyclin D1 and arresting cell division and promotes apoptosis (5).",
"RASSF1A functions through its effect on downstream proteins such as extracellular signal-regulated kinases (ERK1/2). The Ras/ERK signaling pathway has been recognized as an intracellular signal transduction critically involved in fibroblast proliferation. Ras/ERK1/2 is activated in cardiac fibroblasts by platelet-derived growth factor-BB (PDGF-BB) and promotes cellular proliferation (6). In cardiac myocytes, RASSF1A can prevent hypertrophy through disruption of Ras/Raf-1/ERK MAPK signaling. RASSF1A also activates Mst1 to elicit apoptosis. In cardiac fibroblasts, RASSF1A represses NF-κB transcriptional activity and inhibits TNF-α production and secretion, thereby preventing paracrine-mediated hypertrophic signaling between fibroblasts and myocytes. This mechanism involves multiple cell types and paracrine signaling including calcineurin/NFAT, HDAC/MEF2, and MEK/ERK pathways, which have been elucidated in cardiac myocytes (7).",
"DNA methylation-mediated silencing of RASSF1A, and subsequent activation of ERK1/2, can lead to activation of fibroblasts and fibroblast proliferation (6). Another contributing mechanism to cardiac dysfunction induced by hypoxia is myofilament modification. Two myosin heavy chain (MHC) isoforms, MHC-α and MHC-β, are expressed in the mouse heart. α-MHC has higher intrinsic adenosine triphosphatase (ATPase) activity and hence contributes to higher contractility, while β-MHC has lower intrinsic ATPase activity and has a greater economy of force maintenance (8). Hypoxia has been shown to cause switching of myosin heavy chains (MHC) from its alpha to beta isoform, thereby decreasing ATPase activity and overall force of contraction (4).",
"Hypoxia and reactive oxygen species (ROS) play a pivotal role both in the pathogenesis of hypoxia-induced pulmonary hypertension and in the development of cardiac fibrosis (9)(10)(11)(12)(13). The role of ROS, as a trigger of DNA methylation of tumor suppressor gene promoters in carcinogenesis, was shown in previous studies. In human breast cancer, it increases redox concentration (e.g., hydrogen peroxide), induces the overexpression of epigenetic modifiers, including DNMT1 and HDAC1, inhibits gene expression, including tumor suppression; and enhances the expression of epithelial to mesenchymal transition inducer genes, including Snail and Slug (5,14).",
"The inflammatory response mediated by extracellular reactive oxygen generated from repetitive ischemia/reperfusion in a murine model plays a critical role in the pathogenesis of fibrotic remodeling and ventricular dysfunction (7). Supplementation with vitamins E, C, and A have provided antioxidant protection against cardiomyocyte death and have improved survival in congestive heart failure models and doxorubicin-induced injury (11). Doxorubicin causes cardiotoxicity through the generation of ROS (11). ROS generated by doxorubicin include superoxide, hydrogen peroxide, hydroxyl radical, peroxynitrite, and lipid hydroperoxide, which are messengers for signaling apoptotic cell death (11). In-vitro cardiomyocyte studies suggest that superoxide dismutase-like and glutathione peroxidaselike compounds can protect against free radical production and cellular apoptosis due to doxorubicin (6). EC-SOD is an important antioxidant throughout the cardiovascular system and has been shown to protect the heart from ischemic damage, hypertrophy, and inflammation (15)(16)(17). Studies show that human populations with a mutation in the matrix-binding domain of EC-SOD, which diminishes its affinity for the extracellular matrix, have a higher risk for the development of cardiovascular and ischemic heart disease (4). EC-SOD is important in the prevention of oxidative injury that may contribute to cardiac remodeling, in myocardial infarction models, and alter ex vivo heart function (16,17). EC-SOD overexpression has also been shown to decrease the fibrosis that develops in cardiac tissue secondary to ischemia-reperfusion injury (10,18). However, the specific mechanisms by which EC-SOD protects against fibrosis and tissue damage, in various organs including the lung and heart, remain unclear. Also, the relation between EC-SOD and epigenetic changes has not been fully explored. In this study, we reveal the effects of overexpression of EC-SOD on cardiac fibrosis, epigenetic changes, and myofilament changes due to chronic hypoxic stress.",
"All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research. Adult (8-10-week-old) C57BL6 male mice (wild type-WT) and transgenic neonate mice, with an extra copy of the human EC-SOD gene containing a β-actin promoter (TG), were housed in a pathogen-free environment under standard light and dark cycles, with free access to food and water (19). hEC-SOD TG mice with C57BL6 background were studied before, by us and other researchers, and have been well-characterized (19,20). TG mice act and behave similarly under normal conditions (room air), like WT mouse strains as shown in many studies before (19,20).",
"The studied animals were divided into three groups, Group A: WT mice housed in room air; Group B: WT mice housed in 10% normobaric oxygen for 21 days using a BioSpherix chamber (Lacona, NY, USA) (WT hypoxic group); and Group C: TG mice housed under the same hypoxic conditions as Group B (18). After 21 days, the animals were euthanized under a surgical plane of anesthesia [Fentanyl/Xylazine (5:1)] (21), and right ventricular tissue was harvested for analysis.",
"Gene expression, within the right ventricular tissue samples, was assessed following tissue disruption and homogenization. RNA was then extracted from the tissue using the AllPrep DNA/RNA extraction kit (Qiagen), according to the manufacturer's instructions. First-strand cDNA synthesis was carried out using SuperScript II RT (Invitrogen). Quantitative real-time PCR primers were designed so that one of each primer pair was exon/exon boundary spanning to ensure that only mature mRNA was amplified. The sequences of the genespecific primers used were ASMA, 5 ′ -aatgagcgtttccgttgc 3 ′ (forward), 5 ′ -atccccgcagactccatac-3 ′ (reverse); collagen 1a 1 (COL1A1), 5 ′ -catgttcagctttgtggacct 3 ′ (forward), 5 ′ gcagctgacttcagggatgt 3 ′ (reverse); collagen 3 a 1 (COL 3A1), 5 ′ tcccctggaatctgtgaatc 3 ′ (forward), 5 ′ tgagtcgaattggggagaat 3 ′ (reverse); α-Myosin Hea vy Chain (Myh6) 5 ′ cgcatcaaggagctcacc-3 ′ (forward), 5 ′ -cctgcagccgcattaagt-3 ′ (reverse); and β-Myosin Heavy Chain (Myh7) 5 ′ -cgcatcaaggagctcacc-3 ′ (forward), 5 ′ -ctgcagccgcagtaggtt-3 ′ (reverse). Q-PCR was performed; amplification and detection were carried out using Roche Applied Science LightCycler 480 PCR Systems with software. The PCR cycling program consisted of 45 three-step cycles of 15 s/95 • C, 1 min/57 • C, 1 s/72 • C. Relative changes in mRNA expression were calculated as fold changes (normalized using Gapdh) by using the comparative Ct ( Ct) method (22).",
"Right ventricular tissue, fixed in 4% paraformaldehyde for 24 h, was processed, embedded in paraffin, and subsequently cut into 4-µm-thick sections. The slides then underwent heatmediated antigen retrieval followed by incubation with primary antibody anti-Collagen 1 antibody-3G3 (Abcam ab88147) and secondary antibody AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Lab Inc, Code 115005003). The slides were then analyzed using an Olympus FluoView FV300 Confocal Laser Scanning Microscope (Thermo Fisher Scientific), and the Fiji image processing software (an open-source platform for biological image analysis) was used for analysis of pixel density (23).",
"Frozen right ventricular tissues were homogenized, and protein extraction was carried out using a total protein extraction Kit (BioChain Institute, Inc. Hayward, CA). The protein concentration was evaluated using the Modified Lowry Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Samples were prepared for SDS-PAGE in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and processed as previously described (23). Membranes were briefly washed and immediately incubated with the respective primary antibody in 5% BSA with phosphatebuffered saline with Tween 20 (PBST), overnight. Following washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 40-60 min. The membranes were washed and then processed using Amersham ECL detection systems (GE Healthcare, Piscataway, NJ, USA). The membranes were immediately exposed to 8610 Fuji X-Ray Film. The films were assessed using Quantity One 1-D Analysis Software on a GS-800 Calibrated Densitometer. The density of each band is presented as a ratio in comparison to the Actin band density. Primary antibodies were used to detect the following markers: Collagen 1, Alpha Smooth Muscle Actin, SNAIL, DNMT1 and 3b, and HIF1α (each obtained from Abcam, Cambridge, MA); RASSF1A (Origene, Rockville, MD); and ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA).",
"Intracellular ROS was assessed using a cell-based assay for measuring hydroxyl, peroxyl, or other reactive oxygen species.",
"The assay employs the cell-permeable fluorogenic probe 2 ′ ,7 ′dichlorodihydrofluorescein diacetate (DCFH-DA) (Cell Biolabs, San Diego, CA).",
"Primary C57BL6 Mouse Cardiac Fibroblasts (MCF) (Cell Biologics, Chicago, IL, Catalog No. C57-6049) were grown to confluence in T75 flasks using the fibroblast growth medium (Cell Biologics, Chicago, IL, Catalog No. M2267), in a humidified incubator (37 • C, 5% CO 2 ). Cells were seeded to six-well tissue culture plates, at a density of 10,000 cells per well, and incubated for 24 h. Next, the cells were transfected with human EC-SOD (hEC-SOD) cDNA inserted into a vector plasmid pcDNA3 (5446 nucleotides; Invitrogen Life Technologies, Carlsbad, CA, USA) as previously described (13) ",
"Bisulfite chemically converts unmethylated cytosine to uracil but has no effect on methylated cytosine. To determine if promoter region hypermethylation could be responsible for the downregulation of RASSF1A expression, direct bisulfite sequencing on mouse cardiac fibroblasts was performed (17). The methylation ratio (meth-ratio) was calculated using methylated CpG total CpG counts.",
"Briefly, the genomic DNA from mouse heart was isolated using the TRIzol solution (Invitrogen, Thermo Fisher Scientific Inc), according to the manufacturer's protocols. The methylation status of the RASSF1A promoter region was determined by chemical modification of genomic DNA with sodium bisulfite and methylation-specific PCR. The bisulfite-treated DNA was used as a template for the methylation-specific PCR reaction. Primers for the unmethylated DNA-specific reaction were F, 5V-GGTGTTGAAGTTGTGGTTTG-3V; R, 5V-TATTATACCCAA AACAATACAC-3V. Primers for the methylated DNA-specific reaction were F, 5V-TTTTGCGGTTTCGTTCGTTC-3V; R, 5V-CCCGAAACGTACTACTATAAC-3V. The reactions were incubated at 95 • C for 1 min, 55 • C for 1 min, and 72 • C for 1 min, for 35 cycles. The amplified fragment was confirmed by DNA sequencing. DNA from normal heart was used as a control for unmethylated RASSF1A. The strategies for RASSF1A sequencing and the amplicons map Supplementary #1, #2, respectively.",
"Data was expressed as the mean ± standard error of the mean (SEM). Unless otherwise indicated, a one-way or two-way analysis of variance followed by Bonferroni post-hoc test was used to assess significance (P < 0.05) using GraphPad Prism 8 (GraphPad Software, Inc, La Jolla, CA).",
"A comparison between WT adult mice and TG mice housed in room air was performed and is shown in Supplementary #3. All molecular testing showed no significant difference between WT and TG under normoxic conditions (room air).",
"To examine the effects of EC-SOD overexpression on markers of cardiac fibrosis at the mRNA level, we performed gene expression analysis using RT-PCR. There was a significant decrease in the expression of the cardiac fibrosis markers, Collagen 1 (Col1A1) (Figure 1A), Collagen 3 (Figure 1B), and ASMA ( Figure 1C) in the hypoxic TG, as compared to the hypoxic WT animals (p < 0.05). The gene expression of Collagen 1, Collagen 3, and ASMA in hypoxic TG group was not statistically significantly Frontiers in Cardiovascular Medicine | www.frontiersin.org different from room air controls. These results show that EC-SOD overexpression reduces gene expression of cardiac fibrosis markers to levels comparable to RA groups. To confirm that protein levels of cardiac fibrosis markers showed a similar trend to gene expression level, we performed protein-level assessment using Western blot analysis. There was a significant increase in the cardiac fibrosis marker levels of Collagen 1 (Figure 2A), ASMA (Figure 2B), and SNAIL1 ( Figure 2C) in hypoxic WT animals as compared to hypoxic TG animals (p < 0.05). The protein concentration of these three markers in the hypoxia TG animals was not significantly different from the RA control group. This indicates that EC-SOD overexpression reduces the protein expression of cardiac fibrosis markers to levels comparable to RA groups. Immunohistochemistry staining for Collagen 1 showed a significant decrease in pixel density of Collagen 1 in the hypoxic TG animals, as compared to the hypoxic WT animals (Figures 3A,B) (P < 0.05).",
"To examine the effects of EC-SOD on DNA methylation, the most common form of epigenetic modifications, we transfected cardiac fibroblast with hEC-SOD and subjected them to hypoxia. Quantitative flow cytometry studies, using antibody directed to methylated DNA, revealed a significant increase in global DNA methylation in cardiac fibroblasts subjected to hypoxia, compared to cardiac fibroblasts transfected with hEC-SOD and subjected to the same hypoxic conditions (p < 0.05) (Figure 4), thus showing that in-vitro, EC-SOD significantly decreased global DNA methylation under hypoxic conditions.",
"To examine if the same results hold true in the invivo environment, we performed protein assessment of DNA methylating enzymes, using Western blot analysis. There was a significant decrease in the DNA methylating enzymes, DNMT1 ( Figure 5A) and DNMT-3b (Figure 5B), in the hypoxic TG animals compared to the hypoxic WT animals (p < 0.05). The levels of HIF1α ( Figure 5C) were also noted to be significantly reduced in the hypoxic TG animals when compared to the WT animals in hypoxia (p < 0.05). However, the levels in the hypoxic TG animals were not significantly different when compared to the TG room air group. Assay of free reactive oxygen species accumulation by DCF assay showed a significant increase of ROS in the WT hypoxic group, with ROS levels being significantly lower in the TG hypoxic group (Figure 5D). Thus, EC-SOD overexpression decreases DNA methylation, HIF1α, and ROS in-vivo under hypoxic conditions.",
"We have shown that hypoxia induces epigenetic modifications both in-vitro and in-vivo. RASSF1A is a tumor suppressor gene, frequently involved in cardiac fibrosis. DNA methylationmediated silencing of RASSF1A leads to fibroblast proliferation and cardiac fibrosis. We wanted to examine the effects of EC-SOD overexpression on RASSF1A and further examine the Ras/ERK pathway.",
"Western blot analysis in the hypoxic WT animals showed a significant reduction in the gene RASSF1A (Figure 6A), as compared to both hypoxic TG animals and RA control groups (p < 0.05). WT hypoxic animals showed a significant increase of ERK phosphorylation in comparison to the RA control group (P < 0.05). The level of ERK 1/2 ( Figure 6B) was significantly reduced in the hypoxic TG animals compared to the hypoxic WT animals. FIGURE 2 | Western blot analysis of markers of fibrosis-Collagen 1, ASMA, and SNAIL. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). WB analysis was used to assess protein levels of Collagen 1 (A), ASMA (B), and SNAIL (C) in the right ventricular tissue. All experiments n = 3. Data represents mean ± SEM. *P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"Myofilament modification induced by hypoxia causes cardiac dysfunction. Hypoxia can cause switching of Myosin Heavy chains (MHC) from its alpha (high ATP) to beta isoform low ATP, thereby decreasing contractility (Figures 7A,B). Gene expression analysis using RT-PCR showed a significant reduction in the levels of α-MHC and a significant increase in β-HMC in hypoxic WT animals as compared to RA groups and hypoxic TG group (P < 0.05), and this switch was reversed among TG hypoxic group. This provides evidence of EC-SOD overexpression improving cardiac contractility.",
"To provide further evidence that RASSF1A is involved in cardiac fibroblast proliferation, we incubated mouse cardiac fibroblast (MCF), with SiRNA blocking the expression of RASSF1A and incubated in room air condition, both BrdU assay and cell count studies were performed. BrDU analysis ( Figure 8A) showed a significant increase in cell proliferation post-transfection with SiRNA, inhibiting RASSF1A expression, as compared to control cells also incubated in room air (Figure 8B). There was a significant increase in cell numbers, with SiRNA silencing FIGURE 3 | Immunohistochemistry for Collagen 1. Right ventricular tissue from adult wild-type mice WT and EC-SOD transgenic mice (TG), which were exposed to FiO 2 10% hypoxia for 21 days (WT) and a room air control group (RA) were treated with Cy3 stain to assess for levels of Collagen 1 (A). The images were analyzed using Fiji image processing software to measure pixel density (B). All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"studies using cardiac fibroblasts, DNA methylation levels were assessed in cells exposed to hypoxic conditions (FiO 2 1% for 72 h) and compared to cells transfected with EC-SOD and exposed to hypoxia. Cells cultured in room air were employed as controls. Triplicate wells were analyzed, and the experiments were repeated five times. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
", as compared to control cells transfected with an empty vector. These findings support that silencing RASSF1A, leads to activation of ERK1/2, which stimulate cell proliferation.",
"To elucidate the mechanism of how EC-SOD reduces cardiac fibrosis, we show that mRNA expression levels of DNA methyltransferases and methyl-CpG-binding domain proteins (MBD) were studied to investigate the possible mechanism for the observed methylation differences. Evaluated by RT-PCR normalized to GAPDH, DNMT expression was significantly higher in hypoxic WT than hypoxic TG cardiac cells (Figures 5A,B). The methylation level of the RASSFA1 promotor region (8 amplicons) was significantly higher in the hypoxic WT group vs. hypoxic TG group (Figures 8C,D). This suggests that the methylation pattern of the RASSFA1 promoter region may be attributed to differences in methyl-transferase expression and/or methyl binding proteins at mRNA level.",
"Free radicals play a key role in the pathogenesis of cardiac fibrosis [7]. Previously, it has been reported that the free radical scavenger, EC-SOD, can reduce, as well as reverse, some of the changes seen secondary to chronic hypoxic stress (19,24). In our study, we reported that EC-SOD overexpression leads to a significant decrease in DNA methylation (DNMT1 and DNMT-3b as well as RASSFA1 gene), which is triggered by hypoxia exposure. Among animals subjected to chronic hypoxia, markers of fibrosis were increased significantly including RASSFA1 promoter methylation and SNAIL which is involved in fibromodulation. Each of these changes was significantly reversed when EC-SOD was overexpressed in either in-vitro or in-vivo model. There was a significant increase in collagen 1, Collagen 3, and ASMA in hypoxic WT animals as compared to animals housed in room air (P < 0.05) (Figures 1A-C, 2A-C). Similar data was shown previously using human cardiac tissue [2], and it was shown that the degree of hypoxia was associated with increased expression of Collagen 1 and ASMA. In our animal model, overexpression of EC-SOD offered a significant protective effect, evident by reduction in the above listed fibrotic markers. Our data supports the role of oxidative insult induced by hypoxia, FIGURE 5 | Western blot analysis for DNMT1, 3b, and HIF-1α. Adult mice (WT) and EC-SOD transgenic mice (TG) were exposed to FiO 2 10% hypoxia for 21 days. Room air animals were used as a control group (RA). WB analysis was used to assess protein levels of DNMT 1 (A) and DNMT 3b (B); HIF-1α assessment in cardiac fibroblasts (C) in the right ventricular tissue. ROS assay in all studied groups (D). All experiments were carried out in triplicate. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"Frontiers in Cardiovascular Medicine | www.frontiersin.org FIGURE 6 | Western blot analysis for RASSF1A and ERK1/2. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG), were exposed to 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). WB analysis was used to assess protein levels of RASSF1A (A) and ERK1/2 (B) in the right ventricular tissue. All experiments were carried out in triplicate. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"RT-PCR for α and β myosin heavy chain. Adult mice (C57B6) (WT) and EC-SOD transgenic mice (TG), were exposed to 10% hypoxia for 21 days (WT). Room air animals were used as control group (RA). Quantitative RT-PCR was used to assess gene expression analysis of α-MHC in the right ventricular tissue. All experiments n = 5. Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"in the pathogenesis of cardiac fibrosis. The dismutation of these free radicals, by overexpression of EC-SOD, leads to reversal of heart pathology as shown by the immunochemistry studies (Figures 3A,B).",
"Many studies showed the role of SOD and its protective role in animal models with cardiac fibrosis induced by chronic hypoxia, by scavenging free radicals. SOD1 suppressed MCF proliferation and differentiation and reduced the production of collagen type I and III. SOD1 overexpression leads to ROS scavenging and blocking production and block collagen production, suggesting that SOD1 may be a promising therapeutic agent for treating ROS-mediated cardiac fibrosis (25). Mice deficient in SOD2 die of cardiomyopathy within 10 days of birth, whereas heterozygous SOD2(+/-) mice show ultrastructural damage of the myocardium and mitochondria, associated with an increased oxidative stress as well as an activation of apoptotic signaling pathways in the heart (26,27). MnSOD overexpression offers protection against oxidative stress, fibrosis, and apoptosis in the aging heart (28). Serum EC-SOD activity was independently associated with abnormal LV geometry patterns with and without overt HF. Our results indicate that Ec-SOD might be a potential link between LV structure remodeling and the . Proposed mechanism (E). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
"Frontiers in Cardiovascular Medicine | www.frontiersin.org development of subsequent HF in patients with cardiovascular disease. Extracellular superoxide dismutase is associated with left ventricular geometry and heart failure in patients with cardiovascular disease (29). Under unstressed conditions, EC-SOD deficiency had no effect on myocardial total SOD activity but resulted in small but significant increases in myocardial fibrosis and ventricular mass. EC-SOD deficiency is associated with exacerbated myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction. All these findings indicate that the distribution of EC-SOD in the extracellular space is critically important in protecting the heart against pressure overload (17). In our study, we report an innovative mechanism of overexpression of EC-SOD. In animal models with cardiac fibrosis induced by chronic hypoxia, there is a significant increase of ERK1/2 in the hypoxic animal group in parallel with a significant increase of DNA methylation and a reduction of RASSF1A expression. EC-SOD overexpression reverses this process and leads to significant decreases in DNA methylation (DNMT1 and DNMT-3b as well as RASSFA1 gene), which is triggered by chronic hypoxia exposure.",
"The role of DNA methylation and epigenetic changes associated with cardiac fibrosis induced by hypoxia was studied previously (15,18). Both DNA methyltransferase enzymes (DNMT1 and DNMT3b), which are regulated by HIF-1α (30) (Figure 5C), are upregulated by chronic hypoxia (Figures 5A,B). Their upregulation was associated with increases in all fibrotic markers examined and a significant reduction in RASSF1A protein synthesis (Figure 6). In addition, the significant increase in the expression of both DNMT enzymes was associated with increased DNA methylation (Figure 6). Epigenetic changes induced by prolonged hypoxia have been shown to contribute to the pro-fibrotic nature of the ischemic environment (2). In normal human lung fibroblasts, there is a significant global hypermethylation detected in hypoxic fibroblasts relative to normoxic controls and is accompanied by increased expression of myofibroblast markers (24).",
"SNAIL gene expression is a potential target molecule in cardiac fibrosis after ischemia reperfusion (I/R), injury, and or oxidative stress insult (31). The SNAIL gene is best known for its capability to trigger epithelial-to-mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT), which may contribute to myofibroblast formation (30,32). SNAIL is activated by free radicals and mediates the actions of endogenous TGFβ signals that induce EndMT (33). Injection of a selective SNAIL inhibitor remarkably suppressed collagen deposition and cardiac fibrosis in mouse I/R injury and significantly improved cardiac function and reduced SNAIL expression in-vivo (34). SNAIL can recruit multiple chromatin enzymes including LSD1, HDAC1/2, and Suv39H1. These enzymes function in a highly organized manner to generate heterochromatin and promote DNA methyltransferase-mediated DNA methylation at the promoter region (35). Our data showed that a significant increase of SNAIL expression in the WT hypoxic group was attenuated in TG animals, which may lead to a disruption of the connection between SNAIL and these chromatin-modifying enzymes and may represent a therapeutic target for the treatment of cardiac fibrosis.",
"Hypoxia-induced DNA methylation has been shown to be involved in regulating the process of cardiac fibrosis (2,35). DNA methylation mediated silencing of the RASSF1A gene, which leads to upregulation of ERK1/2 that has been shown to increase cardiac fibrosis in cancer patients under chronic hypoxic stress (6,36). Our data showed a significant increase of ERK1/2 in the hypoxic animal group in parallel with a significant increase of DNA methylation and a reduction of RASSF1A expression ( Figure 6A). In adult cardiomyocytes, the high level of H 2 O 2 is associated with the activation of ERK1/2 MAPK and the stimulation of protein synthesis (37). Increased ERK1/2 activity leads to increased cell proliferation and collagen gene expression in activated cardiac fibroblasts [4]. Dismutation of the free radicals by the activity of EC-SOD leads to a global decrease of DNA methylation, increased RASSF1A protein synthesis, and a significant reduction in phosphorylated ERK1/2 in the transgenic hypoxic animal group (P < 0.05). This finding suggests an additional contributing mechanism to cardiac dysfunction in hypoxia, which is triggered by a change in myosin heavy chain isoform (38). Previously, it has been shown that hypoxia leads to a change in MHC from the α to β isoform, which leads to decreased cardiac contractility (37). Free radicals have been shown to affect this change and scavenging these free radicals by antioxidants can markedly attenuate cardiac fibrosis and improve ventricular ejection fraction and fractional shortening (38). In our study, there was a significant reduction in the levels of α-MHC ( Figure 7A) and significant increase in b-MHC (Figure 7B), in hypoxic WT animals as compared to the hypoxia TG group as shown in the RT-PCT assay. Hypoxia TG animals had both α&β-MHC levels close to RA controls. This critical histological change is crucial in preserving cardiac contractility and function.",
"We have shown that TG animals that have an additional copy of the EC-SOD gene show a significant reduction in DNA methylation in response to chronic hypoxic stress. While further studies are needed to completely clarify the mechanism, we speculate that this reduction in DNA methylation is through a reduction in the levels of HIF-1α, which is an inducer of DNMT and hence of the process of DNA methylation (6). Our data show a significant reduction in HIF-1α levels in the animals that overexpress EC-SOD (P < 0.05) (Figure 5C). This suggests a mechanism by which DNA methylation leads to cardiac fibrosis. Both these changes were mitigated in our transgenic animals, which overexpress EC-SOD.",
"To further explore the role of RASSF1A in cardiac fibrosis, its expression was blocked in an in-vitro model using SiRNA. The presence of the SiRNA resulted in a significant increase in fibroblast proliferation (Figures 8A,B). Furthermore, we investigated RASSF1A promoter gene methylation in the three studied groups. Our findings showed a significant increase in RASSF1A promotor region methylation in the hypoxic WT group compared to normoxic animals. This methylation process was reduced by more than 10% among the hypoxic TG animals, which showed a significant reduction of both biochemical and histopathological evidence of cardiac fibrosis.",
"Epigenetic inactivation of RASSF1A by methylation is a very common event in prostate cancer and might be involved in the progression of the disease (39). In prostate cancer, hypomehtylation of BNIP3 and hypermethylation of both EC-SOD and RASSF1A were observed. These changes were positively associated with oxidative stress and inverse associated with EC-SOD expression. Among patients with prostate cancer, it was found that glutamate carboxypeptidase II genetic variants contribute to increased oxidative stress and prostate cancer risk by modulating the CpG island methylation of Ec-SOD (40).",
"In summary, our study presents a novel mechanism by which EC-SOD offers cardiac protection against fibrosis induced by chronic or prolonged hypoxia. The data identifies a critical role of EC-SOD in the control of DNA methylation. Our proposed mechanism, illustrated in Figure 8E, suggests that EC-SOD expression will chelate the free radicals, induced by hypoxia; as a result, both HIF-1α and SNAIL gene activation will be decreased and subsequently methylation enzyme activity will decrease and RASSF1A gene expression will not be silenced or decreased due to lack of methylation. RASSF1A expression downregulates the activity of the ERK1/2 pathway which regulates activation of both cardiac fibroblast proliferation and transition of endothelial cells to myofibroblast. Another benefit from using antioxidants is chelating hydrogen peroxide, which will significantly decrease the activation of ERK1/2, as it is triggered directly by hydrogen peroxide concentration. Previously EC-SOD compounds and its mimetics were used in experimental and clinical trials to counteract the oxidative stress which was linked to the pathogenesis of these disorders (41)(42)(43)(44)(45)(46). Further studies of this mechanism could lead to specific inhibition of the pathway in the clinic to significantly reduce cardiac fibrosis and dramatically improve the outcome of this devastating condition.",
"The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.",
"The animal study was reviewed and approved by Institutional Animal Care and use Committee of the Feinstein Institute for Medical Research. Written informed consent was obtained from the owners for the participation of their animals in this study.",
"MA and AR designed the research. KA, JL, NZ, and AR performed the research and analyzed the data. EM and MA wrote the manuscript. All authors contributed to the article and approved the submitted version.",
"All funding was provided by institutional support.",
"The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2021.669975/full#supplementary-material"
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"In-vivo Studies",
"Animals",
"RT-qPCR",
"Immunohistochemistry-Collagen 1 (Cy 3)",
"Western Blot Analysis",
"ROS Assay",
"In-vitro Analysis",
"Methylation Study",
"RASSF1A Methylation Analysis",
"Statistics",
"RESULTS",
"EC-SOD Reduces Cardiac Fibrosis",
"EC-SOD Reduces Epigenetic Modifications-DNA Methylation",
"EC-SOD Ameliorates the Hypoxia-Induced Epigenetic Modifications to RASSF1A Through the Ras/ERK Pathway",
"EC-SOD Prevents Myofilament Changes",
"RASSF1A Silencing Increase Cardiac Fibroblast Proliferation",
"FIGURE 4 | Flow cytometry studies for DNA methylation-In in-vitro",
"RASSF1A",
"EC-SOD Reduces Cardiac Fibrosis Through Ameliorating DNA Methylation",
"DISCUSSION",
"FIGURE 7 | (A,B)",
"DATA AVAILABILITY STATEMENT",
"ETHICS STATEMENT",
"AUTHOR CONTRIBUTIONS",
"FUNDING",
"SUPPLEMENTARY MATERIAL",
"FIGURE 1 |",
"FIGURE 8 |"
] | [
", using the FuGENE kit \n(Roche Diagnostics, Indianapolis, IN, USA). Each well received \n1 µg DNA/100 µL serum-free medium of the DNA/FuGENE \ncomplex. Control wells received the serum-free medium alone. \nTransfected cells were selected using Geneticin (Invitrogen Life \nTechnologies). Transfection of the fibroblasts was confirmed by \nWestern blot analysis using an antibody specific for hEC-SOD \n(R&D Systems, Minneapolis, MN, US). \n\nQuantitative Flow Cytometry-Effects of Hypoxia on \nGlobal DNA Methylation Profile \n\nA modular incubator chamber (Billups-Rothenberg, Del Mar, \nCA, USA) was used for the cell hypoxia studies and a 1% oxygen \natmosphere maintained using an oxygen sensor (BioSpherix, \nLacona, Proliferation Studies-Effects of Blocking RASSF1A \nExpression \n\nAfter 72 h of transfection, MCF proliferation was assessed by \nBrdU (5-bromouridine) incorporation, (Roche Diagnostics, \n\nMannheim). Cell counts were performed using a hemocytometer \n24 and 48 h later. \n\n"
] | [] | [
"a section of the journal Frontiers in Cardiovascular Medicine Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress",
"a section of the journal Frontiers in Cardiovascular Medicine Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress"
] | [
"Frontiers in Cardiovascular Medicine | www.frontiersin.org"
] |
1,308,182 | 2022-03-31T03:44:11Z | CCBY | https://doi.org/10.1371/journal.pone.0084791 | GOLD | ed21ae4feb4310bd4d2be13bdf13aae19baabfde | null | null | null | null | 10.1371/journal.pone.0084791 | 2045532081 | 24376846 | 3869910 |
Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor, Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis
2013
A Niarchos
M Zouridakis
V Douris
A Georgostathi
D Kalamida
Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor, Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis
PLoS ONE
81284791201310.1371/journal.pone.0084791Received September 20, 2013; Accepted November 25, 2013;Editor: William Phillips, University of Sydney, Australia Copyright: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. *
We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.
Introduction
Chemical transmission of electrical signals from the nervous system to the skeletal muscles is mediated by the muscle nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction [1]. The muscle nAChR is a transmembrane glycoprotein [Molecular mass (Mr) ~290 kDa], consisting of five homologous subunits forming an ionic channel with stoichiometry (α1) 2 β1γδ in embryos or (α1) 2 β1εδ in adults. Muscle nAChR is also the major target of the autoantibodies in most myasthenia gravis (MG) patients [2].
In order to bind the majority of MG autoantibodies and remove them from MG patients' sera, the most appropriate antigen to be used for immunoadsorption columns preparation is the extracellular domain (ECD) of the α subunit of the muscle-type nAChR (α1-ECD). α1-ECD not only contains the main immunogenic region (MIR), which is a major target of the anti-nAChR autoantibodies in MG [3], but also is water soluble [4] and expressed in a much higher yield than the transmembrane, water-insoluble, full-length subunit. For this purpose, in previous studies, human α1-ECD (amino acids 1-210) has been tested for binding of MG autoantibodies in immunoadsorption columns, after expression in E. coli [5] and in the yeast P. pastoris [6].
In the present study, the human α1-ECD was expressed in stably transformed lepidopteran insect cells (i-α1-ECD); an expression system particularly suited for production of soluble proteins [7]. This system has been successfully used for expression of several proteins of mammalian origin, since it has the advantage of conferring posttranslational modifications very similar to the mammalian and of allowing relatively high levels of protein expression [7][8][9]. i-α1-ECD was expressed as a water-soluble monomeric molecule in sufficient quantities for future clinical use. The i-α1-ECD was found to bind more efficiently anti-nAChR monoclonal antibodies (mAbs), as well as autoantibodies from MG patients compared to the homologous protein previously expressed in P. pastoris (y-α1-ECD). Together with far-UV CD studies, i-α1-ECD was proved to acquire a more native conformation and antigenicity compared to y-α1-ECD, and therefore it would be more appropriate for use as a tool for the development of therapeutic approaches in MG.
Materials and Methods
Human α1 nAChR ECD expression in High-Five TM insect cells and in the yeast P. pastoris
For expression in insect cells, a 708 bp fragment of the human α1-ECD cDNA, encoding the first 231 amino acids starting from the N-terminus, was cloned into the BamHI restriction site of plasmid pEIA.PXaMycHis [7]. The recombinant fragment was led by the native signal peptide for secretion (20 a/a) whereas at the C-terminal end, it was fused to a sequence encoding the c-myc epitope and polyhistidine (6xHis) tag, preceded by a factor Xa proteolytic domain. Trichoplusia ni BTI-TN-5B1-4 cells (HighFive TM ; Invitrogen) cultured in ESF921 serum-free medium (Protein Expression Systems) were transfected with Lipofectin TM reagent (Invitrogen) [10] and the stably transfected cell lines were selected as previously described [7]. Static cultures were seeded at a density of 2x10 5 cells/mL, while suspension cultures started from the density of 1x10 6 cells/mL, in Express Five medium (Invitrogen) supplemented with L-glutamine (16 mM), puromycin (15 μg/mL) and gentamycin (50 μg/mL) at 27°C
. When the culture reached a cell density of 5x10 6 cells/mL, it was centrifuged at 600 g for 20 min at 4 °C and the supernatant was harvested for protein purification. For expression in P. pastoris, cDNA was cloned into the pPICZαA vector (Invitrogen) and the protein was expressed in the GS115 strain of P. pastoris, as previously described [4].
Protein purification
After filtration through 0.2 μm filter, the supernatant of either High Five or P. pastoris culture was concentrated using a 10 kDa ultrafiltration system (Ultrasette, Pall Corporation) and dialyzed against 50 mM phosphate buffer (PB), 500 mM NaCl, 10 mM imidazole, pH 8.0, and the protein was purified using Ni-NTA metal affinity chromatography (Qiagen), based on the C-terminal 6×His tag. The washing and elution steps were performed at 40 mM and 150 mM imidazole concentrations, respectively. The ECDs were further purified by gel filtration using a Superose-12 column (Amersham Biosciences) on a FPLC system (AKTApurifier-10) using phosphate-buffered saline (PBS), pH 7.4, at a flow rate of 0.5 mL/min. Fraction volumes were set at 0.5 mL. Protein concentration was determined using the Bradford assay method.
Circular dichroism studies
Far-UV circular dichroism (CD) spectra were recorded at 20°C using a Jasco J-715 spectropolarimeter (Japan Spectroscopic Co.). The scan speed was set at 50 nm/min, the bandwidth at 1 nm, the response time at 2 s, the upper limit of the High Tension voltage at 600 V, the scan ranges at 190-260 nm (far-UV) and the resolution at 0.2 nm. The quartz cell path length was 1 mm and the purified i-α1-ECD (0.3 mg/mL) was dissolved in 10 mM PB, 50 mM sodium fluoride, pH 7.5. The derived spectrum represents the average of ten scans after subtraction of a buffer blank. The secondary structure composition was calculated using the programs CDSSTR, CONTINLL and SELCON3 included in the CDPro software [11].
Dynamic light scattering (DLS) analysis
DLS analysis of purified i-α1-ECD was performed using a Zetasizer NanoS Instrument (Malvern Instruments, UK) and the results were analyzed with DTS v.4.1 software.
SDS-PAGE and western blot analysis
The purified ECDs were analyzed by 12% SDS-PAGE followed by Coomassie staining. For Western blot analysis the protein bands were transferred on a nitrocellulose membrane (Amersham Biosciences), and after blocking in PBS, 5% milk, were probed with the anti-α1 mAb 198 [12] (1:1,000 in PBS, pH 7.4, 0.2% BSA) and subsequently with rabbit anti-rat peroxidase conjugated immunoglobulins (Pierce, dilution 1:1,000 in PBS, pH 7.4, 0.2% BSA). Finally, the bands were observed by incubation in DAB (3,3'-diaminobenzidine) staining buffer (PBS, 0.5 mg/mL DAB, 2 mM NiCl 2 , 0.02% H 2 O 2 ).
Filter Assay for 125 I-α-BTX binding to α1-ECD
Up to 400 ng of purified α1-ECDs were incubated at 4 °C for 3 h with 50,000 cpm of 125 I-α-BTX in a final volume of 50 μL of PBS, 0.2% BSA, pH 7.4. The samples were then diluted with 1 mL of 0.5% Triton X-100 in 20 mM Tris buffer, pH 7.4 (Triton buffer) and immediately filtered through Whatman DE81 anionexchange filters pre-soaked with Triton buffer. The filters were then washed twice with 1 mL of Triton buffer, the bound radioactivity was measured, and after subtracting the nonspecific bound radioactivity, using samples without the α1-ECD, it was expressed as the specifically bound radioactivity (Δcpm). Unpaired t-tests were performed between the bound Δcpm values, scored by i-α1-ECD and y-α1-ECD, for every amount used of each α1-ECD.
Ligand competition experiments
Specific binding of small nicotinic ligands such as nicotine, carbamylcholine, d-tubocurarine and gallamine was studied in competition experiments with 125 I-α-BTX. Various concentrations of the unlabeled ligands, or NaCl as negative control, were added simultaneously with 50,000 cpm of radiolabeled α-bungarotoxin ( 125 I-α-BTX) to 20 ng of α1-ECD in a final volume of 50 μL in PBS buffer, 0.2% BSA, pH 7.4, and the mixture was incubated at 4 °C for 3 h. Bound radioactivity was then measured as above. The residual 125 I-α-BTX binding ability was determined as the ratio (%) of the specific radioactivity (Δcpm) bound in the presence and absence of the unlabeled ligand.
ELISA for mAbs
Wells of microtiter plates (MaxiSorp F96, Nunc) were coated with 40 ng of α1-ECD, and after blocking with PBS, 2% BSA, pH 7.4, 100 μL of various dilutions of mAbs (in PBS pH 7.4, 0.2% BSA) were added to each well, or 10 μL of normal rat serum as the negative control, and the plates were incubated for 3 h at RT. Subsequently 100 μL of peroxidase-conjugated rabbit anti-rat IgG (Pierce, dilution 1:500 in PBS, pH 7.4, 0.2% BSA) were added to each well and incubated for 2 h at RT. Bound mAbs were determined colorimetrically using TMB (3,3´, 5,5´-tetramethylbenzidine) as substrate. The developed color was measured by a microtiter plate reader at 450 nm after addition of 1 M H 2 SO 4 . Unpaired t-tests were performed between the A 450 values, scored by i-α1-ECD and y-α1-ECD, for every volume used of each mAb.
Radioimmunoassay (RIA) for MG patients' sera
40 ng of α1-ECD were labeled with 125 I-α-BTX (50,000 cpm) in a total volume of 45 μL of PBS, 0.2% BSA, pH 7.4, for 3 h at 4 °C. After addition of 1-5 μL of MG patients' sera or normal human serum (NHS) as negative control and PBS, 0.2% BSA, pH 7.4 up to a total volume of 50 μL, the samples were incubated at 4 °C for 15-18 h, and then the immune complexes were precipitated by addition of 25 μL of goat anti-human immunoglobulin antiserum and incubation for 1.5 h at 4 °C. Subsequently, 1 mL of PBS buffer was added, followed by centrifugation (6,000 g, 4 °C, 10 min). The precipitates were washed twice with 1 mL PBS buffer and the precipitated radioactivity was counted. After subtracting the background cpm when using NHS, the precipitated radioactivity was expressed as the ratio (%) of the Δcpm counted in the RIA to the Δcpm counted in the filter assay, when using with the same amount of α1-ECD (40 ng) and 125 I-α-BTX (50,000 cpm). Unpaired t-tests were performed between the % bound precipitated radioactivity values, scored by i-α1-ECD and y-α1-ECD, for every volume used of each serum.
Results
Gel filtration, far-UV CD analysis and dynamic light scattering analysis of i-α1-ECD
The solubility and size of i-α1-ECD were studied by gel filtration, following its first step Ni-NTA metal affinity-based purification, and DLS analysis. As shown in Figure 1A, 125 I-α-BTX binding revealed that i-α1-ECD was expressed exclusively in a monomeric form (Mr ~32 kDa), similarly to the previously studied y-α1-ECD [4]. The first A 280 peak of the chromatograph is attributed to a high molecular mass contaminant, as determined by SDS-PAGE (data not shown). The expression yield of the finally purified i-α1-ECD was estimated by the Bradford assay to be ~0.5 mg/L, thus being 2-fold higher than that described for the y-α1-ECD [4]. The far-UV CD spectrum of i-α1-ECD was characteristic of a β-rich protein, as this presented a positive Cotton effect in the 190-200 nm region and a negative one in the 200-220 nm region (Figure 1B), suggesting a major contribution from β-sheet structure [13]. Deconvolution of the derived spectrum revealed a secondary structure composition of 40% β-sheet structure and 8% αhelical content, while a large proportion of secondary structure is attributed to unordered elements (30%) and to β-turns (22%).
Consistent with gel filtration analysis-based calculated Mr is the DLS analysis ( Figure 1C) for the i-α1-ECD, which showed a hydrodynamic diameter of 4 nm, corresponding to a globular protein of ~30 kDa. Moreover, DLS revealed a significant monodispersity (Calculated polydispersity value of 29%) of the expressed i-α1-ECD molecules, which remained stable in concentrations values of at least 10 mg/mL, a good starting point for crystallization trials.
SDS-PAGE and western blot analysis of i-α1-ECD and y-α1-ECD
Both Coomassie-blue stained SDS-PAGE ( Figure 1D) and western blot analysis (Figure 1E), using the anti-α1 mAb 198, indicated that i-α1-ECD has approximately the same Mr with y-α1-ECD (~32 kDa), which is consistent with that deduced from gel filtration and DLS analysis, but higher than the theoretically predicted based on its primary sequence, probably attributed to glycosylation. Interestingly, i-α1-ECD, in contrast to y-α1-ECD, migrated as a single sharp band, suggesting that its glycosylation is highly homogeneous, an important feature for facilitating crystallization trials for analytical structural studies.
Binding of 125 I-α-BTX and small cholinergic ligands to i-α1-ECD and y-α1-ECD
The ability of both i-α1-ECD and y-α1-ECD to bind 125 I-α-BTX was tested in the filter assay. As shown in Figure 2A, the 125 I-α-BTX binding-ability of i-α1-ECD was somewhat lower (by about one third) than that of y-α1-ECD, which has a K d value of 5 nM for 125 I-α-BTX [4]. To further compare the ligand-binding ability of i-α1-ECD and y-α1-ECD, we then tested the binding of four small cholinergic ligands (d-tubocurarine, nicotine, carbamylcholine and gallamine). Binding of these ligands to both α1-ECDs was assessed indirectly by their ability to inhibit the binding of 125 I-α-BTX in competition experiments. The small agonists carbamylcholine and nicotine did not affect 125 I-α-BTX binding to any of the two ECDs, even at concentrations of up to 10 mM (data not shown). However, the two competitive antagonists, gallamine and d-tubocurarine presented IC 50 values for inhibition of 125 I-α-BTX binding to both y-α1-ECD and i-α1-ECD at ~8 mM and ~10 mM, respectively, without significant difference between the two ECDs ( Figure 2B).
Binding of partially and completely conformationdependent anti-nAChR mAbs to i-α1-ECD and y-α1-ECD
Several anti-nAChR mAbs were tested for binding to both i-α1-ECD and y-α1-ECD, using ELISA ( Figure 3A). mAbs 192, 64 and 35 are known to bind almost exclusively to the native human nAChR, whereas binding of mAb 195 is only partially conformation-dependent [12,14]. It is shown that all mAbs tested, bound at least 2-fold better to i-α1-ECD than y-α1-ECD.
Binding of autoantibodies from MG patients' sera to i-α1-ECD and y-α1-ECD
We then proceeded in comparing the binding abilities of i-α1-ECD and y-α1-ECD to anti-nAChR autoantibodies present in five MG patients' sera, using RIA experiments; three of these sera (sera #1, #2 and #3) have low to moderate titers of anti-nAChR autoantibodies (titers values of 1.9 nM, 16 nM and 15 nM, respectively) and two sera (sera #4, #5) have very high titers (1 and 2 μΜ, respectively). Furthermore, since anti-nAChR autoantibodies are generally highly conformation-dependent and bind to several epitopes on each subunit [15], this set of RIA experiments would also be further indicative of which of both proteins adopts the most appropriate native-like conformation. It was shown that all 5 sera bound much better to i-α1-ECD than to y-α1-ECD ( Figure 3B).
Discussion
Human α1-ECD, was expressed in stably transformed lepidopteran insect cells in order to obtain a recombinant protein with better immunoadsorption potential for future MG therapies. Gel filtration chromatography ( Figure 1A) and DLS analysis ( Figure 2C) indicated that i-α1-ECD was expressed in the form of water-soluble monomer with a molecular weight (Mr~32 kDa) close to the theoretically predicted based on its primary structure (theoretical Mr~28kDa) and of a satisfactory degree of monodispersity. The difference between the calculated and expected Mr is probably attributed to glycosylation. The expression yield of i-α1-ECD (0.5 mg/L) was 2-fold increased compared to that of y-α1-ECD [4]. Moreover, SDS-PAGE ( Figure 1D) and Western blot analysis (Figure 1E), apart from confirming the Mr calculated from gel filtration and DLS analysis, also revealed a more homogeneous glycosylation pattern for the i-α1-ECD conferred by the insect cell expression system. As shown, i-α1-ECD migrated as a sharp band, whereas a smear was observed in the case of y-α1-ECD. Yeast-expressed proteins are known to have very long (and heterogeneous) carbohydrate chains [16], very different from the mammalian-expressed proteins [17]. In contrast, the glycosylation pattern of insect-expressed proteins is more closely related to the mammalian [18]. Interestingly, apart from addressing the question of the improved antigenicity of the i-α1-ECD, which is the main purpose of the current study, the relatively high monodispersity and homogeneous glycosylation of i-α1-ECD render it a promising material for structural studies as well, in order to elucidate the X-ray crystal structure of the human α1-ECD.
Furthermore, we proceeded in assessing the conformation and antigenicity of i-α1-ECD and compared them to y-α1-ECD, using: a) binding of ligands, b) far-UV CD analysis, c) binding of mAbs and d) binding of autoantibodies from MG patients. 125 I-α-BTX was found to have higher binding ability (about 1.5fold increase) for y-α1-ECD than for i-α1-ECD (Figure 2A), probably due to differences in the glycosylation motif conferred . Arrows indicate the peaks of eluted protein markers of known molecular mass. B) Far-UV CD analysis. Deconvolution of the presented spectrum with CDPro software showed 8% α-helical content, 40% β-sheet structure, 22% β-turns and 30% unordered portion. NRMSD (normalized root mean square deviation) value was 0.05, reflecting the accurate analysis of secondary structure. C) DLS analysis. The size distribution by volume of the gel-filtration isolated i-α1-ECD molecules is shown. The calculated hydrodynamic diameter of i-α1-ECD is 4 nm with a polydispersity value of 29%. Protein sample was derived from gel filtration analysis and its concentration was 0.3 mg/mL. SDS-PAGE analysis followed by D) Coomassie Brilliant Blue staining and E) Western blot analysis using the anti-α1 nAChR mAb 198. [4,19]; it is therefore possible that the different glycosylation pattern of i-α1-ECD may have led to the observed 1.5-fold reduced 125 I-α-BTX binding-ability compared to y-α1-ECD. Regarding binding of small cholinergic ligands, these seemed to bind similarly to the two molecules ( Figure 2B).
In order to further assess the native-like conformation of i-α1-ECD we performed far-UV CD studies ( Figure 1B). Interestingly, the α-helical content for the i-α1-ECD is 2-fold higher than that deduced for the y-α1-ECD [20], denoting the probably different folding of the α1-ECD between the two different expression systems. Moreover, taking into account the closer resemblance of the secondary structure composition of the i-α1-ECD to that deduced from the X-ray crystal structures of the homologous AChBP [21,22] and mouse α1-ECD [19], which in all cases is ~8% α-helix and ~45% β-sheet (compared to 8% and 40% for i-α1-ECD and 4.5% and 39.5% for y-α1-ECD [20]), we postulate that α1-ECD probably adopts a more native-like conformation when expressed in insect cells (i-α1-ECD) rather than in yeast (y-α1-ECD).
Since the aforementioned ligand-binding studies provide information only on the conformation of the principal side of the ligand-binding site of muscle nAChR (contributed by the α1-ECD), and since the far-UV CD analysis is only indicative for the secondary structure composition the α1-ECDs under study, we proceeded to testing their binding ability to various anti-nAChR mAbs. Binding ability of these mAbs to the ECDs under study is indicative of their overall conformation, since the tested antibodies are partially or completely conformation-dependent and targeted towards various epitopes of α1-ECD. In addition, high antibody-binding efficiency is our main aim, since we wish to select the best anti-α1 auto-antibody binder for use in antigen-specific future therapies for MG [23]. ELISA experiments showed that all mAbs tested bound much better to i-α1-ECD than y-α1-ECD; in fact, mAb 64 which is an anti-α1 conformation-dependent antibody, in practice bound only to i-α1-ECD ( Figure 3A). These results indicated that i-α1-ECD presents a much improved antigenicity compared to y-α1-ECD, denoting in parallel a more native-like conformation.
Finally, in order to further confirm the superiority of i-α1-ECD in terms of anti-α1 antibody binding, we proceeded in comparing the binding abilities of i-α1-ECD and y-α1-ECD to anti-nAChR autoantibodies present in five MG patients' sera using RIA experiments. i-α1-ECD was found to be more effective than the y-α1-ECD in autoantibody binding, as well ( Figure 3B). Notably, serum 2 (low anti-nAChR-titer) and sera 4 and 5 (high anti-nAChR-titer) showed a similar profile of binding, as they saturated the available antigen at 3 and 5 microliters, while sera 1 and 3 (low anti-nAChR-titer) seemed to be still in a 'linear phase'. This could be due to differences in the percentages of the present anti-α1 antibodies in these sera and in their affinities to the α1-ECDs. Antibody titers were for the intact nAChR and not for the α1 subunit only; therefore it is likely that the differences between sera regarding their titers for the α1-ECDs were smaller than their differences for the intact Up to 400 ng of gel filtration purified y-α1-ECD and i-α1-ECD were incubated with 50,000 cpm of 125 I-α-BTX in the filter assay and the bound radioactivity measured on a γcounter. B) Competition between 125 I-α-BTX and small unlabelled ligands for binding to y-α1-ECD and i-α1-ECD. Inhibition of 125 I-α-BTX-binding by d-tubocurarine and gallamine to both α1-ECDs was assessed by co-incubation competition experiments with unlabelled ligands. Binding of 125 I-α-BTX in the absence of any unlabeled ligand was defined as the 100% of binding. Sodium chloride did not significantly inhibit ligand binding. The values are the mean and standard deviation (±S.D.) from 5 experiments. * Statistically significant differences (unpaired t-test, p<0.05). ** Statistically very significant differences (unpaired t-test, p<0.01). doi: 10.1371/journal.pone.0084791.g002 nAChR and not necessarily proportional. For example, sera 2 and 3 with very similar anti-nAChR titers (16 and 15 nM, respectively) had very different binding efficiency (Figure 3B). Nevertheless sera 4 and 5 with high anti-nAChR titers did bind much more efficiently than sera 1-3 ( Figure 3B). Although sera 2, 4 and 5 (especially #4 and #5) seemed to approach saturation of the available antigen at high serum volumes, nevertheless the differences in binding between the two α1-ECDs (especially at low serum volumes) are apparent.
In conclusion, human α1-ECD expressed in lepidopteran insect cells appears to have improved expression yield, conformation and antigenicity than the corresponding molecule expressed in the yeast P. pastoris. This new recombinant protein (i-α1-ECD) seems to be the most promising human α1 molecule to be used as an effective autoantigen for the development of an antigen-specific therapeutic approach for MG [24]. Figure 3. Binding of antibodies to y-α1-ECD and i-α1-ECD. A) Anti-nAChR mAbs binding. Partially conformation-dependent anti-MIR mAb (mAb 195), strictly conformation-dependent anti-MIR mAbs (mAbs 192 and 35) and mAb 64 (a conformationdependent anti-α1, non anti-MIR mAb), were tested in ELISA experiments showing the binding of the anti-nAChR mAbs at increasing volumes to 40 ng of y-α1-ECD or i-α1-ECD. B) Binding of anti-α1 autoantibodies from MG sera. Five anti-nAChR sera were assayed by RIA for binding to 40 ng of purified y-α1-ECD or i-α1-ECD, labeled with 50,000 cpm 125 I-α-BTX. Sera 1, 2 and 3 have low titers and sera 4 and 5 are of high titer for anti-nAChR autoantibodies. Bound radioactivity to the immunoprecipitates (Yaxis) was expressed as the % ratio to the bound radioactivity when y-α1-ECD or i-α1-ECD filter assayed with 50,000 cpm 125 I-α-BTX. The values are the mean and standard deviation (±S.D.) from 5 experiments. * Statistically significant differences (unpaired ttest, p<0.05). ** Statistically very significant differences (unpaired t-test, p<0.01). doi: 10.1371/journal.pone.0084791.g003
Author Contributions
References
Figure 1 .
1Biochemical and biophysical analysis of i-α1-ECD. A) Gel filtration analysis. The right y axis shows the absorbance at 280 nm (A 280 ) and the left y axis the bound 125 I-α-BTX (Δcpm) in the filter assay, using 20 μL of each gel filtration fraction and 3,000 cpm of 125 I-α-BTX. The values are the mean and standard deviation (±S.D.)
Lane 1 :
1Positions of protein Mr standards; Lane 2: y-α1-ECD; Lane 3: i-α1-ECD. Protein samples were derived from gel filtration chromatography. doi: 10.1371/journal.pone.0084791.g001 by the two different expression systems. Previous studies have shown the critical role of glycosylation of α1 in α-BTX binding
Figure 2 .
2Ligand binding of i-α1-ECD and y-α1-ΕCD. A) 125 I-α-BTX-binding ability.
Conceived and designed the experiments: ST KI KP. Performed the experiments: AN MZ VD AG DK AS. Analyzed the data: AN MZ. Contributed reagents/materials/analysis tools: ST KI. Wrote the manuscript: AN MZ.
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December 2013 | Volume 8 | Issue 12 | e84791
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Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery. V Douris, L Swevers, V Labropoulou, E Andronopoulou, Z Georgoussi, 10.1016/S0065-3527(06)68004-4Adv Virus Res. 68Douris V, Swevers L, Labropoulou V, Andronopoulou E, Georgoussi Z et al. (2006) Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery. Adv Virus Res 68: 113-156. doi:10.1016/S0065-3527(06)68004-4. PubMed: 16997011.
High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. P J Farrell, M Lu, J Prevost, C Brown, L Behie, 10.1002/(SICI)1097-0290(19981220)60:6Biotechnol Bioeng. 60Farrell PJ, Lu M, Prevost J, Brown C, Behie L et al. (1998) High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. Biotechnol Bioeng 60: 656-663. doi:10.1002/(SICI)1097-0290(19981220)60:6. PubMed: 10099475.
Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility. A A Lavdas, R Efrose, V Douris, M Gaitanou, F Papastefanaki, 10.1111/j.1471-4159.2010.07003.xdoi:10.1111/j. 1471-4159.2010.07003J Neurochem. 11520846298Lavdas AA, Efrose R, Douris V, Gaitanou M, Papastefanaki F et al. (2010) Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility. J Neurochem 115: 1137-1149. doi:10.1111/j. 1471-4159.2010.07003.x. PubMed: 20846298.
Use of flow cytometry to rapidly optimize the transfection of animal cells. M B Keith, P J Farrell, K Iatrou, L A Behie, BioTechniques. 2810649786Keith MB, Farrell PJ, Iatrou K, Behie LA (2000) Use of flow cytometry to rapidly optimize the transfection of animal cells. BioTechniques 28: 148-154. PubMed: 10649786.
Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. N Sreerama, R W Woody, 10.1006/abio.2000.4880Anal Biochem. 28711112271Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287: 252-260. doi:10.1006/abio.2000.4880. PubMed: 11112271.
Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. S Tzartos, L Langeberg, S Hochschwender, J Lindstrom, 10.1016/0014-5793(83)80688-7FEBS Lett. 158Tzartos S, Langeberg L, Hochschwender S, Lindstrom J (1983) Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. FEBS Lett 158: 116-118. doi:10.1016/0014-5793(83)80688-7. PubMed: 6862030.
How to study proteins by circular dichroism. S M Kelly, T J Jess, N C Price, 10.1016/j.bbapap.2005.06.005Biochim Biophys Acta. 175116027053Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751: 119-139. doi:10.1016/j.bbapap. 2005.06.005. PubMed: 16027053.
Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity. S Tzartos, L Langeberg, S Hochschwender, L W Swanson, J Lindstrom, 10.1016/0165-5728(86)90105-0J Neuroimmunol. 10Tzartos S, Langeberg L, Hochschwender S, Swanson LW, Lindstrom J (1986) Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity. J Neuroimmunol 10: 235-253. doi: 10.1016/0165-5728(86)90105-0. PubMed: 3484485.
Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. D Kalamida, K Poulas, V Avramopoulou, E Fostieri, G Lagoumintzis, 10.1111/j.1742-4658.2007.05935.xFEBS J. 274Kalamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G et al. (2007) Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J 274: 3799-3845. doi: 10.1111/j.1742-4658.2007.05935.x. PubMed: 17651090.
Protein glycosylation in yeast. M A Kukuruzinska, M L Bergh, B J Jackson, 10.1146/annurev.bi.56.070187.004411Annu Rev Biochem. 563304149Kukuruzinska MA, Bergh ML, Jackson BJ (1987) Protein glycosylation in yeast. Annu Rev Biochem 56: 915-944. doi:10.1146/annurev.bi. 56.070187.004411. PubMed: 3304149.
Mammalian cell gene expression: protein glycosylation. R B Parekh, 10.1016/0958-1669(91)90043-5Curr Opin Biotechnol. 2Parekh RB (1991) Mammalian cell gene expression: protein glycosylation. Curr Opin Biotechnol 2: 730-734. doi: 10.1016/0958-1669(91)90043-5. PubMed: 1367726.
Insect cells as hosts for the expression of recombinant glycoproteins. F Altmann, E Staudacher, I B Wilson, L März, 10.1023/A:1026488408951Glycoconj J. 1610612411Altmann F, Staudacher E, Wilson IB, März L (1999) Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J 16: 109-123. doi:10.1023/A:1026488408951. PubMed: 10612411.
Crystal structure of the extracellular domain of nAChR alpha1 bound to alphabungarotoxin at 1.94 A resolution. C D Dellisanti, Y Yao, J C Stroud, Z Z Wang, L Chen, 10.1038/nn1942Nat Neurosci. 1017643119Dellisanti CD, Yao Y, Stroud JC, Wang ZZ, Chen L (2007) Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha- bungarotoxin at 1.94 A resolution. Nat Neurosci 10: 953-962. doi: 10.1038/nn1942. PubMed: 17643119.
Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure. M Zouridakis, K Kostelidou, A Sotiriadis, C Stergiou, E Eliopoulos, 10.1016/j.ijbiomac.2007.05.012Int J Biol Macromol. 4117659334Zouridakis M, Kostelidou K, Sotiriadis A, Stergiou C, Eliopoulos E et al. (2007) Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure. Int J Biol Macromol 41: 423-429. doi:10.1016/j.ijbiomac.2007.05.012. PubMed: 17659334.
Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. K Brejc, W J Van Dijk, R V Klaassen, M Schuurmans, J Van Der Oost, 10.1038/35077011Nature. 41111357122Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J et al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411: 269-276. doi: 10.1038/35077011. PubMed: 11357122.
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. P H Celie, S E Van Rossum-Fikkert, W J Van Dijk, K Brejc, A B Smit, 10.1016/S0896-6273(04)00115-1Neuron. 41Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB et al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41: 907-914. doi:10.1016/S0896-6273(04)00115-1. PubMed: 15046723.
Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera. S J Tzartos, K Bitzopoulou, Gavra I Kordas, G Jacobson, L , 10.1196/annals.1405.01718567880Ann N Y Acad Sci. 1132Tzartos SJ, Bitzopoulou K, Gavra I, Kordas G, Jacobson L et al. (2008) Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera. Ann N Y Acad Sci 1132: 291-299. doi:10.1196/ annals.1405.017. PubMed: 18567880.
Recent approaches to the development of antigen-specific immunotherapies for myasthenia gravis. G Lagoumintzis, P Zisimopoulou, G Kordas, K Lazaridis, K Poulas, 10.3109/08916930903518099Autoimmunity. 4320187712Lagoumintzis G, Zisimopoulou P, Kordas G, Lazaridis K, Poulas K et al. (2010) Recent approaches to the development of antigen-specific immunotherapies for myasthenia gravis. Autoimmunity 43: 436-445. doi:10.3109/08916930903518099. PubMed: 20187712.
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"We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies."
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"The neuromuscular junction. A G Engel, Handb. Clin Neurol. 91Engel AG (2008) The neuromuscular junction. Handb. Clin Neurol 91: 103-148.",
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"The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis. S J Tzartos, T Barkas, M T Cung, A Kordossi, H Loutrari, 10.3109/08916939109007633Autoimmunity. 81718457Tzartos SJ, Barkas T, Cung MT, Kordossi A, Loutrari H et al. (1991) The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis. Autoimmunity 8: 259-270. doi: 10.3109/08916939109007633. PubMed: 1718457.",
"Expression of soluble ligand-and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding. L Psaridi-Linardaki, A Mamalaki, M Remoundos, S J Tzartos, 10.1074/jbc.M110731200J Biol Chem. 27712015305Psaridi-Linardaki L, Mamalaki A, Remoundos M, Tzartos SJ (2002) Expression of soluble ligand-and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding. J Biol Chem 277: 26980-26986. doi:10.1074/jbc.M110731200. PubMed: 12015305.",
"Antigenspecific apheresis of human anti-acetylcholine receptor autoantibodies from myasthenia gravis patients' sera using Escherichia coli-expressed receptor domains. P Zisimopoulou, G Lagoumintzis, K Poulas, S J Tzartos, 10.1016/j.jneuroim.2008.06.002doi:10.1016/ j.jneuroim.2008.06.002J Neuroimmunol. 20018603305Zisimopoulou P, Lagoumintzis G, Poulas K, Tzartos SJ (2008) Antigen- specific apheresis of human anti-acetylcholine receptor autoantibodies from myasthenia gravis patients' sera using Escherichia coli-expressed receptor domains. J Neuroimmunol 200: 133-141. doi:10.1016/ j.jneuroim.2008.06.002. PubMed: 18603305.",
"Towards antigen-specific apheresis of pathogenic autoantibodies as a further step in the treatment of myasthenia gravis by plasmapheresis. P Zisimopoulou, G Lagoumintzis, K Kostelidou, K Bitzopoulou, G Kordas, 10.1016/j.jneuroim.2008.06.020doi:10.1016/ j.jneuroim.2008.06.020J Neuroimmunol. 20118667243Zisimopoulou P, Lagoumintzis G, Kostelidou K, Bitzopoulou K, Kordas G et al. (2008) Towards antigen-specific apheresis of pathogenic autoantibodies as a further step in the treatment of myasthenia gravis by plasmapheresis. J Neuroimmunol 201-202: 95-103. doi:10.1016/ j.jneuroim.2008.06.020. PubMed: 18667243.",
"Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery. V Douris, L Swevers, V Labropoulou, E Andronopoulou, Z Georgoussi, 10.1016/S0065-3527(06)68004-4Adv Virus Res. 68Douris V, Swevers L, Labropoulou V, Andronopoulou E, Georgoussi Z et al. (2006) Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery. Adv Virus Res 68: 113-156. doi:10.1016/S0065-3527(06)68004-4. PubMed: 16997011.",
"High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. P J Farrell, M Lu, J Prevost, C Brown, L Behie, 10.1002/(SICI)1097-0290(19981220)60:6Biotechnol Bioeng. 60Farrell PJ, Lu M, Prevost J, Brown C, Behie L et al. (1998) High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. Biotechnol Bioeng 60: 656-663. doi:10.1002/(SICI)1097-0290(19981220)60:6. PubMed: 10099475.",
"Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility. A A Lavdas, R Efrose, V Douris, M Gaitanou, F Papastefanaki, 10.1111/j.1471-4159.2010.07003.xdoi:10.1111/j. 1471-4159.2010.07003J Neurochem. 11520846298Lavdas AA, Efrose R, Douris V, Gaitanou M, Papastefanaki F et al. (2010) Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility. J Neurochem 115: 1137-1149. doi:10.1111/j. 1471-4159.2010.07003.x. PubMed: 20846298.",
"Use of flow cytometry to rapidly optimize the transfection of animal cells. M B Keith, P J Farrell, K Iatrou, L A Behie, BioTechniques. 2810649786Keith MB, Farrell PJ, Iatrou K, Behie LA (2000) Use of flow cytometry to rapidly optimize the transfection of animal cells. BioTechniques 28: 148-154. PubMed: 10649786.",
"Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. N Sreerama, R W Woody, 10.1006/abio.2000.4880Anal Biochem. 28711112271Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287: 252-260. doi:10.1006/abio.2000.4880. PubMed: 11112271.",
"Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. S Tzartos, L Langeberg, S Hochschwender, J Lindstrom, 10.1016/0014-5793(83)80688-7FEBS Lett. 158Tzartos S, Langeberg L, Hochschwender S, Lindstrom J (1983) Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. FEBS Lett 158: 116-118. doi:10.1016/0014-5793(83)80688-7. PubMed: 6862030.",
"How to study proteins by circular dichroism. S M Kelly, T J Jess, N C Price, 10.1016/j.bbapap.2005.06.005Biochim Biophys Acta. 175116027053Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751: 119-139. doi:10.1016/j.bbapap. 2005.06.005. PubMed: 16027053.",
"Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity. S Tzartos, L Langeberg, S Hochschwender, L W Swanson, J Lindstrom, 10.1016/0165-5728(86)90105-0J Neuroimmunol. 10Tzartos S, Langeberg L, Hochschwender S, Swanson LW, Lindstrom J (1986) Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity. J Neuroimmunol 10: 235-253. doi: 10.1016/0165-5728(86)90105-0. PubMed: 3484485.",
"Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. D Kalamida, K Poulas, V Avramopoulou, E Fostieri, G Lagoumintzis, 10.1111/j.1742-4658.2007.05935.xFEBS J. 274Kalamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G et al. (2007) Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J 274: 3799-3845. doi: 10.1111/j.1742-4658.2007.05935.x. PubMed: 17651090.",
"Protein glycosylation in yeast. M A Kukuruzinska, M L Bergh, B J Jackson, 10.1146/annurev.bi.56.070187.004411Annu Rev Biochem. 563304149Kukuruzinska MA, Bergh ML, Jackson BJ (1987) Protein glycosylation in yeast. Annu Rev Biochem 56: 915-944. doi:10.1146/annurev.bi. 56.070187.004411. PubMed: 3304149.",
"Mammalian cell gene expression: protein glycosylation. R B Parekh, 10.1016/0958-1669(91)90043-5Curr Opin Biotechnol. 2Parekh RB (1991) Mammalian cell gene expression: protein glycosylation. Curr Opin Biotechnol 2: 730-734. doi: 10.1016/0958-1669(91)90043-5. PubMed: 1367726.",
"Insect cells as hosts for the expression of recombinant glycoproteins. F Altmann, E Staudacher, I B Wilson, L März, 10.1023/A:1026488408951Glycoconj J. 1610612411Altmann F, Staudacher E, Wilson IB, März L (1999) Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J 16: 109-123. doi:10.1023/A:1026488408951. PubMed: 10612411.",
"Crystal structure of the extracellular domain of nAChR alpha1 bound to alphabungarotoxin at 1.94 A resolution. C D Dellisanti, Y Yao, J C Stroud, Z Z Wang, L Chen, 10.1038/nn1942Nat Neurosci. 1017643119Dellisanti CD, Yao Y, Stroud JC, Wang ZZ, Chen L (2007) Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha- bungarotoxin at 1.94 A resolution. Nat Neurosci 10: 953-962. doi: 10.1038/nn1942. PubMed: 17643119.",
"Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure. M Zouridakis, K Kostelidou, A Sotiriadis, C Stergiou, E Eliopoulos, 10.1016/j.ijbiomac.2007.05.012Int J Biol Macromol. 4117659334Zouridakis M, Kostelidou K, Sotiriadis A, Stergiou C, Eliopoulos E et al. (2007) Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure. Int J Biol Macromol 41: 423-429. doi:10.1016/j.ijbiomac.2007.05.012. PubMed: 17659334.",
"Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. K Brejc, W J Van Dijk, R V Klaassen, M Schuurmans, J Van Der Oost, 10.1038/35077011Nature. 41111357122Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J et al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411: 269-276. doi: 10.1038/35077011. PubMed: 11357122.",
"Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. P H Celie, S E Van Rossum-Fikkert, W J Van Dijk, K Brejc, A B Smit, 10.1016/S0896-6273(04)00115-1Neuron. 41Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB et al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41: 907-914. doi:10.1016/S0896-6273(04)00115-1. PubMed: 15046723.",
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"The neuromuscular junction",
"Myasthenia gravis",
"The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis",
"Expression of soluble ligand-and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding",
"Antigenspecific apheresis of human anti-acetylcholine receptor autoantibodies from myasthenia gravis patients' sera using Escherichia coli-expressed receptor domains",
"Towards antigen-specific apheresis of pathogenic autoantibodies as a further step in the treatment of myasthenia gravis by plasmapheresis",
"Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery",
"High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector",
"Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility",
"Use of flow cytometry to rapidly optimize the transfection of animal cells",
"Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set",
"Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor",
"How to study proteins by circular dichroism",
"Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity",
"Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity",
"Protein glycosylation in yeast",
"Mammalian cell gene expression: protein glycosylation",
"Insect cells as hosts for the expression of recombinant glycoproteins",
"Crystal structure of the extracellular domain of nAChR alpha1 bound to alphabungarotoxin at 1.94 A resolution",
"Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure",
"Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors",
"Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures",
"Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera",
"Recent approaches to the development of antigen-specific immunotherapies for myasthenia gravis"
] | [
"Handb. Clin Neurol",
"Orphanet J Rare Dis",
"Autoimmunity",
"J Biol Chem",
"J Neuroimmunol",
"J Neuroimmunol",
"Adv Virus Res",
"Biotechnol Bioeng",
"J Neurochem",
"BioTechniques",
"Anal Biochem",
"FEBS Lett",
"Biochim Biophys Acta",
"J Neuroimmunol",
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"Curr Opin Biotechnol",
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"\nFigure 1 .\n1Biochemical and biophysical analysis of i-α1-ECD. A) Gel filtration analysis. The right y axis shows the absorbance at 280 nm (A 280 ) and the left y axis the bound 125 I-α-BTX (Δcpm) in the filter assay, using 20 μL of each gel filtration fraction and 3,000 cpm of 125 I-α-BTX. The values are the mean and standard deviation (±S.D.)",
"\nLane 1 :\n1Positions of protein Mr standards; Lane 2: y-α1-ECD; Lane 3: i-α1-ECD. Protein samples were derived from gel filtration chromatography. doi: 10.1371/journal.pone.0084791.g001 by the two different expression systems. Previous studies have shown the critical role of glycosylation of α1 in α-BTX binding",
"\nFigure 2 .\n2Ligand binding of i-α1-ECD and y-α1-ΕCD. A) 125 I-α-BTX-binding ability.",
"\n\nConceived and designed the experiments: ST KI KP. Performed the experiments: AN MZ VD AG DK AS. Analyzed the data: AN MZ. Contributed reagents/materials/analysis tools: ST KI. Wrote the manuscript: AN MZ."
] | [
"Biochemical and biophysical analysis of i-α1-ECD. A) Gel filtration analysis. The right y axis shows the absorbance at 280 nm (A 280 ) and the left y axis the bound 125 I-α-BTX (Δcpm) in the filter assay, using 20 μL of each gel filtration fraction and 3,000 cpm of 125 I-α-BTX. The values are the mean and standard deviation (±S.D.)",
"Positions of protein Mr standards; Lane 2: y-α1-ECD; Lane 3: i-α1-ECD. Protein samples were derived from gel filtration chromatography. doi: 10.1371/journal.pone.0084791.g001 by the two different expression systems. Previous studies have shown the critical role of glycosylation of α1 in α-BTX binding",
"Ligand binding of i-α1-ECD and y-α1-ΕCD. A) 125 I-α-BTX-binding ability.",
"Conceived and designed the experiments: ST KI KP. Performed the experiments: AN MZ VD AG DK AS. Analyzed the data: AN MZ. Contributed reagents/materials/analysis tools: ST KI. Wrote the manuscript: AN MZ."
] | [
"Figure 1A",
"(Figure 1B)",
"Figure 1C)",
"Figure 1D",
"(Figure 1E)",
"Figure 2A",
"Figure 2B",
"Figure 3A)",
"Figure 3B",
"Figure 1A)",
"Figure 2C)",
"Figure 1D)",
"(Figure 1E)",
"(Figure 2A)",
"Figure 2B)",
"Figure 1B)",
"Figure 3A)",
"Figure 3B)",
"(Figure 3B",
"Figure 3B",
"Figure 3"
] | [] | [
"Chemical transmission of electrical signals from the nervous system to the skeletal muscles is mediated by the muscle nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction [1]. The muscle nAChR is a transmembrane glycoprotein [Molecular mass (Mr) ~290 kDa], consisting of five homologous subunits forming an ionic channel with stoichiometry (α1) 2 β1γδ in embryos or (α1) 2 β1εδ in adults. Muscle nAChR is also the major target of the autoantibodies in most myasthenia gravis (MG) patients [2].",
"In order to bind the majority of MG autoantibodies and remove them from MG patients' sera, the most appropriate antigen to be used for immunoadsorption columns preparation is the extracellular domain (ECD) of the α subunit of the muscle-type nAChR (α1-ECD). α1-ECD not only contains the main immunogenic region (MIR), which is a major target of the anti-nAChR autoantibodies in MG [3], but also is water soluble [4] and expressed in a much higher yield than the transmembrane, water-insoluble, full-length subunit. For this purpose, in previous studies, human α1-ECD (amino acids 1-210) has been tested for binding of MG autoantibodies in immunoadsorption columns, after expression in E. coli [5] and in the yeast P. pastoris [6].",
"In the present study, the human α1-ECD was expressed in stably transformed lepidopteran insect cells (i-α1-ECD); an expression system particularly suited for production of soluble proteins [7]. This system has been successfully used for expression of several proteins of mammalian origin, since it has the advantage of conferring posttranslational modifications very similar to the mammalian and of allowing relatively high levels of protein expression [7][8][9]. i-α1-ECD was expressed as a water-soluble monomeric molecule in sufficient quantities for future clinical use. The i-α1-ECD was found to bind more efficiently anti-nAChR monoclonal antibodies (mAbs), as well as autoantibodies from MG patients compared to the homologous protein previously expressed in P. pastoris (y-α1-ECD). Together with far-UV CD studies, i-α1-ECD was proved to acquire a more native conformation and antigenicity compared to y-α1-ECD, and therefore it would be more appropriate for use as a tool for the development of therapeutic approaches in MG.",
"For expression in insect cells, a 708 bp fragment of the human α1-ECD cDNA, encoding the first 231 amino acids starting from the N-terminus, was cloned into the BamHI restriction site of plasmid pEIA.PXaMycHis [7]. The recombinant fragment was led by the native signal peptide for secretion (20 a/a) whereas at the C-terminal end, it was fused to a sequence encoding the c-myc epitope and polyhistidine (6xHis) tag, preceded by a factor Xa proteolytic domain. Trichoplusia ni BTI-TN-5B1-4 cells (HighFive TM ; Invitrogen) cultured in ESF921 serum-free medium (Protein Expression Systems) were transfected with Lipofectin TM reagent (Invitrogen) [10] and the stably transfected cell lines were selected as previously described [7]. Static cultures were seeded at a density of 2x10 5 cells/mL, while suspension cultures started from the density of 1x10 6 cells/mL, in Express Five medium (Invitrogen) supplemented with L-glutamine (16 mM), puromycin (15 μg/mL) and gentamycin (50 μg/mL) at 27°C",
". When the culture reached a cell density of 5x10 6 cells/mL, it was centrifuged at 600 g for 20 min at 4 °C and the supernatant was harvested for protein purification. For expression in P. pastoris, cDNA was cloned into the pPICZαA vector (Invitrogen) and the protein was expressed in the GS115 strain of P. pastoris, as previously described [4].",
"After filtration through 0.2 μm filter, the supernatant of either High Five or P. pastoris culture was concentrated using a 10 kDa ultrafiltration system (Ultrasette, Pall Corporation) and dialyzed against 50 mM phosphate buffer (PB), 500 mM NaCl, 10 mM imidazole, pH 8.0, and the protein was purified using Ni-NTA metal affinity chromatography (Qiagen), based on the C-terminal 6×His tag. The washing and elution steps were performed at 40 mM and 150 mM imidazole concentrations, respectively. The ECDs were further purified by gel filtration using a Superose-12 column (Amersham Biosciences) on a FPLC system (AKTApurifier-10) using phosphate-buffered saline (PBS), pH 7.4, at a flow rate of 0.5 mL/min. Fraction volumes were set at 0.5 mL. Protein concentration was determined using the Bradford assay method.",
"Far-UV circular dichroism (CD) spectra were recorded at 20°C using a Jasco J-715 spectropolarimeter (Japan Spectroscopic Co.). The scan speed was set at 50 nm/min, the bandwidth at 1 nm, the response time at 2 s, the upper limit of the High Tension voltage at 600 V, the scan ranges at 190-260 nm (far-UV) and the resolution at 0.2 nm. The quartz cell path length was 1 mm and the purified i-α1-ECD (0.3 mg/mL) was dissolved in 10 mM PB, 50 mM sodium fluoride, pH 7.5. The derived spectrum represents the average of ten scans after subtraction of a buffer blank. The secondary structure composition was calculated using the programs CDSSTR, CONTINLL and SELCON3 included in the CDPro software [11].",
"DLS analysis of purified i-α1-ECD was performed using a Zetasizer NanoS Instrument (Malvern Instruments, UK) and the results were analyzed with DTS v.4.1 software.",
"The purified ECDs were analyzed by 12% SDS-PAGE followed by Coomassie staining. For Western blot analysis the protein bands were transferred on a nitrocellulose membrane (Amersham Biosciences), and after blocking in PBS, 5% milk, were probed with the anti-α1 mAb 198 [12] (1:1,000 in PBS, pH 7.4, 0.2% BSA) and subsequently with rabbit anti-rat peroxidase conjugated immunoglobulins (Pierce, dilution 1:1,000 in PBS, pH 7.4, 0.2% BSA). Finally, the bands were observed by incubation in DAB (3,3'-diaminobenzidine) staining buffer (PBS, 0.5 mg/mL DAB, 2 mM NiCl 2 , 0.02% H 2 O 2 ).",
"Up to 400 ng of purified α1-ECDs were incubated at 4 °C for 3 h with 50,000 cpm of 125 I-α-BTX in a final volume of 50 μL of PBS, 0.2% BSA, pH 7.4. The samples were then diluted with 1 mL of 0.5% Triton X-100 in 20 mM Tris buffer, pH 7.4 (Triton buffer) and immediately filtered through Whatman DE81 anionexchange filters pre-soaked with Triton buffer. The filters were then washed twice with 1 mL of Triton buffer, the bound radioactivity was measured, and after subtracting the nonspecific bound radioactivity, using samples without the α1-ECD, it was expressed as the specifically bound radioactivity (Δcpm). Unpaired t-tests were performed between the bound Δcpm values, scored by i-α1-ECD and y-α1-ECD, for every amount used of each α1-ECD.",
"Specific binding of small nicotinic ligands such as nicotine, carbamylcholine, d-tubocurarine and gallamine was studied in competition experiments with 125 I-α-BTX. Various concentrations of the unlabeled ligands, or NaCl as negative control, were added simultaneously with 50,000 cpm of radiolabeled α-bungarotoxin ( 125 I-α-BTX) to 20 ng of α1-ECD in a final volume of 50 μL in PBS buffer, 0.2% BSA, pH 7.4, and the mixture was incubated at 4 °C for 3 h. Bound radioactivity was then measured as above. The residual 125 I-α-BTX binding ability was determined as the ratio (%) of the specific radioactivity (Δcpm) bound in the presence and absence of the unlabeled ligand.",
"Wells of microtiter plates (MaxiSorp F96, Nunc) were coated with 40 ng of α1-ECD, and after blocking with PBS, 2% BSA, pH 7.4, 100 μL of various dilutions of mAbs (in PBS pH 7.4, 0.2% BSA) were added to each well, or 10 μL of normal rat serum as the negative control, and the plates were incubated for 3 h at RT. Subsequently 100 μL of peroxidase-conjugated rabbit anti-rat IgG (Pierce, dilution 1:500 in PBS, pH 7.4, 0.2% BSA) were added to each well and incubated for 2 h at RT. Bound mAbs were determined colorimetrically using TMB (3,3´, 5,5´-tetramethylbenzidine) as substrate. The developed color was measured by a microtiter plate reader at 450 nm after addition of 1 M H 2 SO 4 . Unpaired t-tests were performed between the A 450 values, scored by i-α1-ECD and y-α1-ECD, for every volume used of each mAb.",
"40 ng of α1-ECD were labeled with 125 I-α-BTX (50,000 cpm) in a total volume of 45 μL of PBS, 0.2% BSA, pH 7.4, for 3 h at 4 °C. After addition of 1-5 μL of MG patients' sera or normal human serum (NHS) as negative control and PBS, 0.2% BSA, pH 7.4 up to a total volume of 50 μL, the samples were incubated at 4 °C for 15-18 h, and then the immune complexes were precipitated by addition of 25 μL of goat anti-human immunoglobulin antiserum and incubation for 1.5 h at 4 °C. Subsequently, 1 mL of PBS buffer was added, followed by centrifugation (6,000 g, 4 °C, 10 min). The precipitates were washed twice with 1 mL PBS buffer and the precipitated radioactivity was counted. After subtracting the background cpm when using NHS, the precipitated radioactivity was expressed as the ratio (%) of the Δcpm counted in the RIA to the Δcpm counted in the filter assay, when using with the same amount of α1-ECD (40 ng) and 125 I-α-BTX (50,000 cpm). Unpaired t-tests were performed between the % bound precipitated radioactivity values, scored by i-α1-ECD and y-α1-ECD, for every volume used of each serum.",
"The solubility and size of i-α1-ECD were studied by gel filtration, following its first step Ni-NTA metal affinity-based purification, and DLS analysis. As shown in Figure 1A, 125 I-α-BTX binding revealed that i-α1-ECD was expressed exclusively in a monomeric form (Mr ~32 kDa), similarly to the previously studied y-α1-ECD [4]. The first A 280 peak of the chromatograph is attributed to a high molecular mass contaminant, as determined by SDS-PAGE (data not shown). The expression yield of the finally purified i-α1-ECD was estimated by the Bradford assay to be ~0.5 mg/L, thus being 2-fold higher than that described for the y-α1-ECD [4]. The far-UV CD spectrum of i-α1-ECD was characteristic of a β-rich protein, as this presented a positive Cotton effect in the 190-200 nm region and a negative one in the 200-220 nm region (Figure 1B), suggesting a major contribution from β-sheet structure [13]. Deconvolution of the derived spectrum revealed a secondary structure composition of 40% β-sheet structure and 8% αhelical content, while a large proportion of secondary structure is attributed to unordered elements (30%) and to β-turns (22%).",
"Consistent with gel filtration analysis-based calculated Mr is the DLS analysis ( Figure 1C) for the i-α1-ECD, which showed a hydrodynamic diameter of 4 nm, corresponding to a globular protein of ~30 kDa. Moreover, DLS revealed a significant monodispersity (Calculated polydispersity value of 29%) of the expressed i-α1-ECD molecules, which remained stable in concentrations values of at least 10 mg/mL, a good starting point for crystallization trials.",
"Both Coomassie-blue stained SDS-PAGE ( Figure 1D) and western blot analysis (Figure 1E), using the anti-α1 mAb 198, indicated that i-α1-ECD has approximately the same Mr with y-α1-ECD (~32 kDa), which is consistent with that deduced from gel filtration and DLS analysis, but higher than the theoretically predicted based on its primary sequence, probably attributed to glycosylation. Interestingly, i-α1-ECD, in contrast to y-α1-ECD, migrated as a single sharp band, suggesting that its glycosylation is highly homogeneous, an important feature for facilitating crystallization trials for analytical structural studies.",
"The ability of both i-α1-ECD and y-α1-ECD to bind 125 I-α-BTX was tested in the filter assay. As shown in Figure 2A, the 125 I-α-BTX binding-ability of i-α1-ECD was somewhat lower (by about one third) than that of y-α1-ECD, which has a K d value of 5 nM for 125 I-α-BTX [4]. To further compare the ligand-binding ability of i-α1-ECD and y-α1-ECD, we then tested the binding of four small cholinergic ligands (d-tubocurarine, nicotine, carbamylcholine and gallamine). Binding of these ligands to both α1-ECDs was assessed indirectly by their ability to inhibit the binding of 125 I-α-BTX in competition experiments. The small agonists carbamylcholine and nicotine did not affect 125 I-α-BTX binding to any of the two ECDs, even at concentrations of up to 10 mM (data not shown). However, the two competitive antagonists, gallamine and d-tubocurarine presented IC 50 values for inhibition of 125 I-α-BTX binding to both y-α1-ECD and i-α1-ECD at ~8 mM and ~10 mM, respectively, without significant difference between the two ECDs ( Figure 2B).",
"Several anti-nAChR mAbs were tested for binding to both i-α1-ECD and y-α1-ECD, using ELISA ( Figure 3A). mAbs 192, 64 and 35 are known to bind almost exclusively to the native human nAChR, whereas binding of mAb 195 is only partially conformation-dependent [12,14]. It is shown that all mAbs tested, bound at least 2-fold better to i-α1-ECD than y-α1-ECD.",
"We then proceeded in comparing the binding abilities of i-α1-ECD and y-α1-ECD to anti-nAChR autoantibodies present in five MG patients' sera, using RIA experiments; three of these sera (sera #1, #2 and #3) have low to moderate titers of anti-nAChR autoantibodies (titers values of 1.9 nM, 16 nM and 15 nM, respectively) and two sera (sera #4, #5) have very high titers (1 and 2 μΜ, respectively). Furthermore, since anti-nAChR autoantibodies are generally highly conformation-dependent and bind to several epitopes on each subunit [15], this set of RIA experiments would also be further indicative of which of both proteins adopts the most appropriate native-like conformation. It was shown that all 5 sera bound much better to i-α1-ECD than to y-α1-ECD ( Figure 3B).",
"Human α1-ECD, was expressed in stably transformed lepidopteran insect cells in order to obtain a recombinant protein with better immunoadsorption potential for future MG therapies. Gel filtration chromatography ( Figure 1A) and DLS analysis ( Figure 2C) indicated that i-α1-ECD was expressed in the form of water-soluble monomer with a molecular weight (Mr~32 kDa) close to the theoretically predicted based on its primary structure (theoretical Mr~28kDa) and of a satisfactory degree of monodispersity. The difference between the calculated and expected Mr is probably attributed to glycosylation. The expression yield of i-α1-ECD (0.5 mg/L) was 2-fold increased compared to that of y-α1-ECD [4]. Moreover, SDS-PAGE ( Figure 1D) and Western blot analysis (Figure 1E), apart from confirming the Mr calculated from gel filtration and DLS analysis, also revealed a more homogeneous glycosylation pattern for the i-α1-ECD conferred by the insect cell expression system. As shown, i-α1-ECD migrated as a sharp band, whereas a smear was observed in the case of y-α1-ECD. Yeast-expressed proteins are known to have very long (and heterogeneous) carbohydrate chains [16], very different from the mammalian-expressed proteins [17]. In contrast, the glycosylation pattern of insect-expressed proteins is more closely related to the mammalian [18]. Interestingly, apart from addressing the question of the improved antigenicity of the i-α1-ECD, which is the main purpose of the current study, the relatively high monodispersity and homogeneous glycosylation of i-α1-ECD render it a promising material for structural studies as well, in order to elucidate the X-ray crystal structure of the human α1-ECD.",
"Furthermore, we proceeded in assessing the conformation and antigenicity of i-α1-ECD and compared them to y-α1-ECD, using: a) binding of ligands, b) far-UV CD analysis, c) binding of mAbs and d) binding of autoantibodies from MG patients. 125 I-α-BTX was found to have higher binding ability (about 1.5fold increase) for y-α1-ECD than for i-α1-ECD (Figure 2A), probably due to differences in the glycosylation motif conferred . Arrows indicate the peaks of eluted protein markers of known molecular mass. B) Far-UV CD analysis. Deconvolution of the presented spectrum with CDPro software showed 8% α-helical content, 40% β-sheet structure, 22% β-turns and 30% unordered portion. NRMSD (normalized root mean square deviation) value was 0.05, reflecting the accurate analysis of secondary structure. C) DLS analysis. The size distribution by volume of the gel-filtration isolated i-α1-ECD molecules is shown. The calculated hydrodynamic diameter of i-α1-ECD is 4 nm with a polydispersity value of 29%. Protein sample was derived from gel filtration analysis and its concentration was 0.3 mg/mL. SDS-PAGE analysis followed by D) Coomassie Brilliant Blue staining and E) Western blot analysis using the anti-α1 nAChR mAb 198. [4,19]; it is therefore possible that the different glycosylation pattern of i-α1-ECD may have led to the observed 1.5-fold reduced 125 I-α-BTX binding-ability compared to y-α1-ECD. Regarding binding of small cholinergic ligands, these seemed to bind similarly to the two molecules ( Figure 2B).",
"In order to further assess the native-like conformation of i-α1-ECD we performed far-UV CD studies ( Figure 1B). Interestingly, the α-helical content for the i-α1-ECD is 2-fold higher than that deduced for the y-α1-ECD [20], denoting the probably different folding of the α1-ECD between the two different expression systems. Moreover, taking into account the closer resemblance of the secondary structure composition of the i-α1-ECD to that deduced from the X-ray crystal structures of the homologous AChBP [21,22] and mouse α1-ECD [19], which in all cases is ~8% α-helix and ~45% β-sheet (compared to 8% and 40% for i-α1-ECD and 4.5% and 39.5% for y-α1-ECD [20]), we postulate that α1-ECD probably adopts a more native-like conformation when expressed in insect cells (i-α1-ECD) rather than in yeast (y-α1-ECD).",
"Since the aforementioned ligand-binding studies provide information only on the conformation of the principal side of the ligand-binding site of muscle nAChR (contributed by the α1-ECD), and since the far-UV CD analysis is only indicative for the secondary structure composition the α1-ECDs under study, we proceeded to testing their binding ability to various anti-nAChR mAbs. Binding ability of these mAbs to the ECDs under study is indicative of their overall conformation, since the tested antibodies are partially or completely conformation-dependent and targeted towards various epitopes of α1-ECD. In addition, high antibody-binding efficiency is our main aim, since we wish to select the best anti-α1 auto-antibody binder for use in antigen-specific future therapies for MG [23]. ELISA experiments showed that all mAbs tested bound much better to i-α1-ECD than y-α1-ECD; in fact, mAb 64 which is an anti-α1 conformation-dependent antibody, in practice bound only to i-α1-ECD ( Figure 3A). These results indicated that i-α1-ECD presents a much improved antigenicity compared to y-α1-ECD, denoting in parallel a more native-like conformation.",
"Finally, in order to further confirm the superiority of i-α1-ECD in terms of anti-α1 antibody binding, we proceeded in comparing the binding abilities of i-α1-ECD and y-α1-ECD to anti-nAChR autoantibodies present in five MG patients' sera using RIA experiments. i-α1-ECD was found to be more effective than the y-α1-ECD in autoantibody binding, as well ( Figure 3B). Notably, serum 2 (low anti-nAChR-titer) and sera 4 and 5 (high anti-nAChR-titer) showed a similar profile of binding, as they saturated the available antigen at 3 and 5 microliters, while sera 1 and 3 (low anti-nAChR-titer) seemed to be still in a 'linear phase'. This could be due to differences in the percentages of the present anti-α1 antibodies in these sera and in their affinities to the α1-ECDs. Antibody titers were for the intact nAChR and not for the α1 subunit only; therefore it is likely that the differences between sera regarding their titers for the α1-ECDs were smaller than their differences for the intact Up to 400 ng of gel filtration purified y-α1-ECD and i-α1-ECD were incubated with 50,000 cpm of 125 I-α-BTX in the filter assay and the bound radioactivity measured on a γcounter. B) Competition between 125 I-α-BTX and small unlabelled ligands for binding to y-α1-ECD and i-α1-ECD. Inhibition of 125 I-α-BTX-binding by d-tubocurarine and gallamine to both α1-ECDs was assessed by co-incubation competition experiments with unlabelled ligands. Binding of 125 I-α-BTX in the absence of any unlabeled ligand was defined as the 100% of binding. Sodium chloride did not significantly inhibit ligand binding. The values are the mean and standard deviation (±S.D.) from 5 experiments. * Statistically significant differences (unpaired t-test, p<0.05). ** Statistically very significant differences (unpaired t-test, p<0.01). doi: 10.1371/journal.pone.0084791.g002 nAChR and not necessarily proportional. For example, sera 2 and 3 with very similar anti-nAChR titers (16 and 15 nM, respectively) had very different binding efficiency (Figure 3B). Nevertheless sera 4 and 5 with high anti-nAChR titers did bind much more efficiently than sera 1-3 ( Figure 3B). Although sera 2, 4 and 5 (especially #4 and #5) seemed to approach saturation of the available antigen at high serum volumes, nevertheless the differences in binding between the two α1-ECDs (especially at low serum volumes) are apparent.",
"In conclusion, human α1-ECD expressed in lepidopteran insect cells appears to have improved expression yield, conformation and antigenicity than the corresponding molecule expressed in the yeast P. pastoris. This new recombinant protein (i-α1-ECD) seems to be the most promising human α1 molecule to be used as an effective autoantigen for the development of an antigen-specific therapeutic approach for MG [24]. Figure 3. Binding of antibodies to y-α1-ECD and i-α1-ECD. A) Anti-nAChR mAbs binding. Partially conformation-dependent anti-MIR mAb (mAb 195), strictly conformation-dependent anti-MIR mAbs (mAbs 192 and 35) and mAb 64 (a conformationdependent anti-α1, non anti-MIR mAb), were tested in ELISA experiments showing the binding of the anti-nAChR mAbs at increasing volumes to 40 ng of y-α1-ECD or i-α1-ECD. B) Binding of anti-α1 autoantibodies from MG sera. Five anti-nAChR sera were assayed by RIA for binding to 40 ng of purified y-α1-ECD or i-α1-ECD, labeled with 50,000 cpm 125 I-α-BTX. Sera 1, 2 and 3 have low titers and sera 4 and 5 are of high titer for anti-nAChR autoantibodies. Bound radioactivity to the immunoprecipitates (Yaxis) was expressed as the % ratio to the bound radioactivity when y-α1-ECD or i-α1-ECD filter assayed with 50,000 cpm 125 I-α-BTX. The values are the mean and standard deviation (±S.D.) from 5 experiments. * Statistically significant differences (unpaired ttest, p<0.05). ** Statistically very significant differences (unpaired t-test, p<0.01). doi: 10.1371/journal.pone.0084791.g003"
] | [] | [
"Introduction",
"Materials and Methods",
"Human α1 nAChR ECD expression in High-Five TM insect cells and in the yeast P. pastoris",
"Protein purification",
"Circular dichroism studies",
"Dynamic light scattering (DLS) analysis",
"SDS-PAGE and western blot analysis",
"Filter Assay for 125 I-α-BTX binding to α1-ECD",
"Ligand competition experiments",
"ELISA for mAbs",
"Radioimmunoassay (RIA) for MG patients' sera",
"Results",
"Gel filtration, far-UV CD analysis and dynamic light scattering analysis of i-α1-ECD",
"SDS-PAGE and western blot analysis of i-α1-ECD and y-α1-ECD",
"Binding of 125 I-α-BTX and small cholinergic ligands to i-α1-ECD and y-α1-ECD",
"Binding of partially and completely conformationdependent anti-nAChR mAbs to i-α1-ECD and y-α1-ECD",
"Binding of autoantibodies from MG patients' sera to i-α1-ECD and y-α1-ECD",
"Discussion",
"Author Contributions",
"References",
"Figure 1 .",
"Lane 1 :",
"Figure 2 ."
] | [] | [] | [
"Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor, Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis",
"Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor, Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis"
] | [
"PLoS ONE"
] |
19,158,949 | 2022-03-14T02:36:30Z | CCBY | https://www.oncotarget.com/article/24680/pdf/ | GOLD | 6828357bdd5c300151cb9e0404241c18c4443bf9 | null | null | null | null | 10.18632/oncotarget.24680 | 2797279288 | 29731967 | 5929410 |
Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy
Published: April 13, 2018
Meng-Chuan Chen
School of Dentistry
Graduated Institute of Dental Science
National Defense Medical Center
TaipeiTaiwan
Wen-Lin Hsu
Department of Radiation Oncology
Buddhist Tzu Chi General Hospital
HualienTaiwan
School of Medicine
Tzu Chi University
HualienTaiwan
Tz-Chong Chou
Cancer Research Center
Buddhist Tzu Chi General Hospital
HualienTaiwan
Institute of Medical Sciences
Tzu Chi University
HualienTaiwan
Department of Biotechnology
Asia University
TaichungTaiwan
China Medical University Hospital
China Medical University
TaichungTaiwan
Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy
Published: April 13, 2018Received: August 05, 2017 Accepted: February 28, 2018Oncotarget 19584 * These authors have contributed equally to this work Correspondence to: Tz-Chong Chou,Antrodia cinnamomealung tumorcancer cachexiamuscle atrophychemotherapy
Skeletal muscle atrophy, the most characteristic feature of cancer cachexia, often occurs in patients with cancer undergoing chemotherapy. Antrodia cinnamomea (AC) a widely used edible medical fungus, exhibits hepatoprotective, anti-inflammatory and anticancer activities. In this study, we investigated whether combined treatment with the ethonolic extract of AC ameliorates cachexia symptoms, especially muscle wasting, in lung tumor-bearing mice treated with chemotherapy. Our results revealed that gemcitabine and cisplatin-induced severe body weight loss and skeletal muscle atrophy in the mice with cancer were greatly attenuated after AC extract administration. The protection may be attributed to the inhibition of skeletal muscle proteolysis by suppressing myostatin and activin release, muscle wasting-related FoxO3/MuRF-1/MAFbx signaling, proteasomal enzyme activity, and pro-inflammatory cytokine production. A significant decrease in insulin-like growth factor 1 (IGF-1) expression and formation was observed in the atrophying muscle of the conventional chemotherapy treatment group (CGC), and this decrease was markedly reversed by AC treatment. Additionally, the anorexia, intestinal injury and dysfunction that occurred in the CGC group were mitigated by AC extract. Taken together, these results demonstrated that the AC extract has a protective effect against chemotherapyinduced muscle atrophy mainly by attenuating muscle proteolysis, pro-inflammatory cytokine production, and anorexia, and activating IGF-1-dependent protein synthesis.
INTRODUCTION
Cancer cachexia is characterized by body weight loss, anorexia, fatigue, inflammation and abnormal metabolism, thereby markedly reducing quality of life and limiting the application of conventional therapy such as chemotherapy [1,2]. More than 80% of patients with advanced cancer suffer from cachexia, and cancer cachexia is estimated to account for at least 20% of deaths in patients with cancer [3,4]. Thus, how to prevent and attenuate the development of cancer cachexia is a key concern during cancer therapy. Muscle wasting, the most prominent phenotypic feature of cancer cachexia, is closely related to the tumor site, size, stage, and treatment type [5]. During cachexia, muscle proteolysis www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 28), pp: [19584][19585][19586][19587][19588][19589][19590][19591][19592][19593][19594][19595][19596] Research Paper www.oncotarget.com is predominately triggered by the ubiquitin proteasome system (UPS). The forkhead box O (FoxO) transcription factor is a key factor for activating the expression of musclespecific ubiquitin conjugating enzymes E3 ligase, F-box (MAFbx)/atrogin-1 and muscle ring finger 1 (MuRF-1). The ubiquitinated target protein substrate can be recognized by the 26S proteasome and then digested to peptides, which in turn leads to muscle protein degradation [6]. Increased ubiquitinated protein expression and proteasome activity have been observed in atrophying muscle [7]. Conversely, mice that are deficient in either MAFbx or MuRF-1 exhibit greater resistance to atrophy [8], suggesting that suppressing the FoxO/MAFbx/MuRF-1/UPS cascade is a promising strategy of preventing the muscle wasting associated with cancer cachexia. The process of cancer cachexia-evoked muscle atrophy is complex and multifactorial; it is mediated by the interplay of tumor factors, host factors, and the interaction between the two [9,10].
To date, chemotherapy remains a widely used option for cancer therapy. Nevertheless, several deleterious effects have been identified after chemotherapy, thereby limiting its application [11]. Currently, combined treatment with chemotherapeutic drugs such as gemcitabine (2', 2'-difluorodeoxycytidine) and cisplatin (cis-diamminedichloroplatinum) is a common regimen for treating metastatic lung cancer. However, many side effects, including nephrotoxicity, gastrointestinal mucosal injury, and severe muscle wasting, have been reported in patients with cancer treated with cisplatin [12,13]. These findings imply that chemotherapy itself can induce muscle atrophy [14]. Although various drugs and supplements have been used, cancer cachexia-evoked skeletal muscle mass loss remains a major problem. Therefore, developing more effective and safe chemotherapeutic adjuvants or nutritional supplements to ameliorate chemotherapy-induced muscle wasting is urgent.
Antrodia cinnamomea (AC), a medical fungus, grows only on the inner cavity of the endemic species Cinnamomum kanehirai, which is a plant native to Taiwan. AC extracts have been demonatrated to possess several beneficial effects, including antioxidant, hepatoprotective, anti-hypertensive, anti-hyperlipidemic, immunomodulatory, anticancer, and anti-inflammatory activities [15,16]. However, the effects of AC extract on cancer cachexia remain unknown. Therefore, the aim of this study was to examine whether AC attenuates these cachectic symptoms, particularly the muscle atrophy, in lung tumor-bearing mice under chemotherapy, and to further investigate the molecular mechanisms involved.
RESULTS
Chemical characteristics of the AC extract and its effects on tumor growth
Data from the HPLC analysis revealed that the ethanolic extract of the patented kinetic bio-activation fruiting body of AC contained seven marker triterpenoid ingredients, namely antcin A (at 74.7 min), antcin B (at 64.4 and 65.4 min), antcin C (at 42.2 and 45.7 min), antcin H (at 45.0 min), antcin K (at 20.1 and 21.0 min), dehydrosulphurenic acid (at 57.4 min) and dehydroeburicoic acid (at 84.9 min) ( Figure 1A). The total amount of the triterpenoids was observed to account for 14.5% (w/w) of the AC extract, and antcin C, antcin H, and antcin K were abundant among these triterpenoids (Table 1). Four groups were used in this study: (1) a normal group; (2) cancer group (tumor-alone group); (3) CGC group: mice treated with a standard diet and an intraperitoneal injection of gemcitabine (1000 mg/m 2 per 3 days) and cisplatin (75 mg/m 2 /week); (4) CGCA group: mice treated with a standard diet plus AC extract (300 mg/kg/day, p.o.) and an intraperitoneal injection of gemcitabine and cisplatin. After orthotopic implantation of LLC2 cells into the lungs for two weeks, the mice were treated with different combinations of drugs for 21 days and were sacrificed to evaluate tumor progression. The number of tumor nodules and the lung weight reflecting the tumor growth were greatly reduced in the CGC and CGCA groups, compared with the cancer alone group. Furthermore, we observed that the anticancer activity of the CGCA group was stronger than that of the CGC group ( Figure 1B).
AC attenuates muscle atrophy and proteasome activity in muscle
At the end of the study, the untreated cancer mice lost 19.0 ± 1.4% of their initial body weight, whereas the normal mice gained body weight. The mice in the CGC and CGCA groups lost 24.2 ± 1.6%, and 18.8 ± 1.8% of their initial weight, respectively ( Figure 2A). During cancer cachexia, body weight loss generally results from skeletal muscle wasting. As expected, the muscle atrophy evaluated through histological examination was parallel with the trend of muscle mass loss in these groups ( Figure 2B). Notably, the CGC group had the most skeletal muscle mass loss, as evidenced by a marked reduction in the weight of the gastrocnemius and soleus muscle, and this loss was inhibited by combined treatment with AC (CGCA) ( Figure 2C). Consistently, a marked increase in muscular proteasome activity, particularly chymotrypsin and trypsin, was observed in the CGC group, and this increase was significantly inhibited by AC treatment ( Figure 2D).
AC inhibits muscle wasting-related signaling pathway
Overproduction of myostatin and activin A observed in the atrophying muscle of the cancer-alone and CGC groups was significantly decreased in the mice in the CGCA group ( Figure 3A). Similarly, the www.oncotarget.com alterations in muscle wasting-related gene expression, including increased levels of ActRIIB, FoxO3, MuRF 1, and MAFbx, as well as decreased expression of p-Akt and p-FoxO3, particularly in the muscle of the CGC group, were markedly reversed through AC treatment ( Figure 3B). The 14-3-3 chaperone protein binds to phosphorylated FoxO3, resulting in FoxO3 degradation, thereby inhibiting downstream MuRF 1 and MAFbx expression [17]. Our results confirmed that the interaction of p-FoxO3 with the 14-3-3 protein determined by an immunoprecipitation assay was increased in the CGCA group, compared with the cancer alone and CGC groups ( Figure 3C). As expected, a marked elevation of FoxO3 transcriptional activity in the CGC group was significantly inhibited by AC treatment ( Figure 3D). Therefore, suppressing FoxO3-mediated processes may contribute to the attenuation of muscle proteolysis by AC.
AC inhibits pro-inflammatory cytokine production and upregulates insulin-like growth factor-1 (IGF-1)
Systemic inflammation is considered a key factor inducing cancer cachexia [18]. A significant elevation of serum levels of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, particularly in the CGC group, was greatly diminished in the CGCA group ( Figure 4A). Notably, combined treatment with the AC extract greatly increased the expression and production of IGF-1, compared with the CGC group ( Figure 4B). These results suggest that inhibiting inflammatory responses and activating IGF-1-dependent process involves the antiatrophic effect of AC.
AC prevents intestinal injury/dysfunction
Severe damage to the intestinal mucosal structure, especially in the CGC group, was ameliorated in the mice in the CGCA group ( Figure 5A). Additionally, impaired digestive enzyme activity, such as leucine amino peptidase (LAP), a digestive enzyme for peptides, amylase (AMYL), a digestive enzyme for sugars, and lipase (LIP), a digestive enzyme for fat, in the cancer-alone and CGC groups, was alleviated after AC administration ( Figure 5B). Notably, AC could mitigate the anorexia observed in the CGC group, as evidenced by an elevation in daily food intake ( Figure 5C).
DISCUSSION
Several tumor and host factors play critical roles in triggering muscle atrophy associated with cancer cachexia by activating muscle proteolytic pathways, impairing protein synthesis, or both [19,20]. The current study demonstrates, for the first time, that combined treatment with the ethanolic extract of AC greatly alleviates gemcitabine and cisplatininduced cachectic symptoms, including body weight loss, skeletal muscle atrophy, intestinal damage and dysfunction, and anorexia in lung tumor-bearing mice; this thus supports the clinical use of this extract. Myostatin belonging to transforming growth factor-α; (TGF-α) superfamily is mostly expressed in skeletal muscle, and it can inhibit skeletal muscle growth by reducing myoblast proliferation and myogenesis [21]. Conversely, blocking the actions of myostatin by using neutralizing antibodies or antagonists remarkably increases muscle size and physical strength [22]. Similarly, activins, members of the TGF-α superfamily, are another muscle atrophying factor. Notably, myostatin and activins can bind the same muscle surface receptor complex that composes of type-II activin receptors (ActRIIA and ActRIIB) and type-I activin receptors (ALK4 and ALK5), which ultimately results in muscle proteolysis through activation of FoxO, especially the FoxO3 isoform. The actions of FoxO are largely controlled by the subcellular localization and the protein degradation of FoxO. When FoxO is phosphorylated and inactivated by Akt, the phosphorylated FoxO is exported from the nucleus into cytoplasma in a chaperone 14-3-3-dependent manner [17]. The 14-3-3 bound phosphorylated FoxO protein is then degraded through proteasome. In response to myostatin and activin, Akt activity is inhibited, leading to the accumulation of the dephospho-FoxO protein, the active form of FoxO. Subsequently, the activated FoxO translocates into the nucleus and enhances the transcription of muscle-specific atrogenic genes, such as MuRF-1 and MAFbx. Previous studies have reported that myostatin and activin signaling in muscle increases in patients with cancer suffering from cachexia and in experimental cancer cachexia [23,24].
In the present study, we demonstrated that the levels of muscle myostatin and activin were significantly reduced in the CGCA group, compared with those in the CGC group. An increase in ActRIIB expression and a decrease in Akt phosphorylation in the CGC group were also reversed by AC treatment, which may be associated with the inhibition of myostatin and activin generation. As expected, treatment with AC increased FoxO3 phosphorylation accompanied by an elevation of the association of 14-3-3 with phospho-FoxO3 in cytoplasma, but it significantly inhibited MuRF-1 and MAFbx expression, and proteasome activity. Collectively, suppressing the myostatin/activins/FoxO3/ MuRF-1/MAFbx signaling pathway may contribute to the antiatrophic effect of AC. Accumulating evidence indicates that systemic inflammation and pro-inflammatory cytokines, including TNF-β, IL-6 and IL-1β, are major factors in promoting muscle atrophy through UPS activation, muscle differentiation and myogenesis inhibition [18], and improvement of anorexia during cancer cachexia [25,26]. Elevated serum levels of pro-inflammatory cytokines were reported in patients with cachexia [27]. However, neutralization of pro-inflammatory cytokines could markedly relieve the cachectic symptoms in an experimental animal model [28]. According to the results that the CGCA group had lower serum levels of NF-β, IL-6, and IL-1β than that in the CGC group, inhibition of pro-inflammatory cytokine formation may also involve the anti-cachectic effects of AC.
In addition to muscle proteolysis, muscle protein generation is another crucial factor in regulating muscle mass. A central role of IGF-1 in enhancing muscle growth by activating PI3K/Akt/mTOR-regulated protein synthesis was demonstrated, previously [29]. Moreover, IGF-1 can activate Akt-induced FoxO phosphorylation, thereby attenuating muscle protein degradation [30]. Therefore, IGF-1 not only enhances protein synthesis but also inhibits muscle proteolysis, suggesting that IGF-1induction is a promising approach to prevent muscle atrophy. A novel finding of this study is that the CGCA group had a higher IGF-1 level in muscle than that of the CGC group. Accordingly, AC-mediated attenuation of muscle mass loss may, at least partly, be attributed to the suppression of inflammation-evoked muscle protein degradation, and enhancement of IGF-1-dependent protein synthesis.
Maintaining the intestinal structure and function is essential for nutritional intake and body growth. Notably, intestinal damage and impaired digestive enzyme activity, particularly in the CGC group, were greatly improved through AC treatment. Thus, increased nutrient absorption and availability due to the attenuation of intestinal injury and dysfunction may in turn enhance appetite, as reflected by increased daily food intake, thereby attenuating body weight loss by AC in this cachectic model. Notably, the inhibition of lung tumor growth through combined treatment with AC was stronger than that observed in the CGC group, suggesting that AC can also enhance the anticancer activity of chemotherapy.
Ergostane-type triterpenoids are the major bioactive components in the fruiting bodies of AC. Among these triterpenoids, antcin C, antcin H, antcin K, and antcin A have been reported to exhibit antiinflammatory and anticancer activities [16,31,32]. The pharmacokinetic and metabolism study demonstrated that several triterpenoids and metabolites were detected in the plasma of rats after oral administration of the ethanol extract of AC (1g/kg). Generally, the ergostane triterpenoids are absorbed and eliminated rapidly, and the pharmacokinetic patterns of Antrodia triterpenoids are closely related to their chemical structure. The highpolarity of antcin H and antcin K were determined to be metabolically stable, and had markedly higher plasma concentrations than antcin B and antcin C [33]. Conversely, antcin A had a low concentration in plasma. Composition analysis revealed that antcin H and antcin K were abundant in the AC extract. Therefore, antcin K and antcin H may be most responsible for the therapeutic effects on cancer cachexia because of their pharmacokinetic parameters, abundance, and biological effects. However, more basic and human studies are required to determine their clinical use. Notably, the hydrogenated metabolites derived from antcin C also revealed a high plasma concentration. However, the biological functions are still unknown, and need further investigation.
In summary, AC administration can attenuate the development of cancer cachexia in lung tumor-bearing mice undergoing chemotherapy. Mechanically, the protective effects of AC against muscle wasting may be associated with the suppression of muscle proteolysis, formation of pro-inflammatory cytokines, activation of IGF-1-regulated protein synthesis, and improvement of intestinal damage and dysfunction ( Figure 6). Overall, AC has potential to ameliorate cachectic symptoms in patients undergoing chemotherapy. www.oncotarget.com with AC inhibits myostatin/activin/FoxO3 signaling and pro-inflammatory cytokine formation, leading to the suppression of MAFbx and MuRF1 expression and proteasome activity in muscle, which in turn attenuates muscle proteolysis. Meanwhile, the AC extract enhances IGF-1 expression and its regulated protein synthesis, and improves anorexia and intestinal damage and dysfunction. Therefore, AC has potential to ameliorate cachectic symptoms under chemotherapy, especially body weight loss. www.oncotarget.com
MATERIALS AND METHODS
Preparation of AC extract and reagents
The fruiting bodies of AC were provided by Balay Biotechnology, Inc. (Taipei, Taiwan) and identified by the Bioresource Collection and Research Center (BCRC, Taiwan). The dried AC powder (30g) was soaked with 0.9 L of distilled water at 80°C-100°C for 1 h. Subsequently, it was extracted with 900 mL of ethanol (95%) for 24 h. The residue was filtered through a Buchner funnel lined with Whatman filter paper; the filtrates were collected and dried using a rotary evaporator (CES-800, Panchum Scientific Corp.) under reduced pressure at 45°C to obtain brownish colored residues with a yield of 7.5 g (25%, w/w) and were stored at -20°C prior to analysis. Antibodies, including anti-TNF-α, anti-IL-1β, anti-IGF-1, anti-MuRF-1, anti-MAFbX-1, and anti-β-actin were purchased from Santa
Cell culture
The Lewis lung carcinoma cell line LLC2 purchased from the Bioresource Collection and Research Center (Taipei, Taiwan) was incubated in Dulbecco's modified Eagle's Medium (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (Thermo Fisher Scientific Inc), 2 mmol/L L-glutamine, and 100 U/ mL penicillin-streptomycin (Gibco, Carlsbad, CA, USA). Cells were maintained in an incubator with room air: CO 2 (95:5, v/v) at 37°C.
Animal model
Seven-week-old male C57B/6 mice weighing approximately 25 g were used for the study. The animal care and experimental procedures were conducted in accordance with the Guiding Principle in the Care and Use of Animals and approved by the Institutional Animal Care and Use Committee of National Defense Medical Center (IACUC 12156). The mice were anaesthetized with an intraperitoneal injection of ketamine HCl/xylazine (100 mg/15 mg body weight per mouse). Anesthetized mice were placed on a platform by their front teeth so that their chests hung vertically beneath them. Light was directed on each mouse's upper chest, on a spot marked by an "X". The mouth was opened using the Exel Safelet IV catheter, and the tongue is gently pulled out using flat forceps. After locating the white light emitted from the trachea, the Exel Safelet IV catheter was slid into the trachea, and the needle was removed. The mouse with the inserted catheter on the platform is moved into a biosafety hood, where the LLC2 cell (1 × 10 6 cells/100 μL) was dispensed into the opening of the catheter [34]. After implantation of cancer cells for 10 days, the mice were divided into four weightmatched groups: (1) the normal group, (2) cancer-alone group, (3) CGC group, and (4) CGCA, as described in the Results section. Each group contains five mice.
Western blotting
Protein samples (100 μg) were separated on a 10% SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk in 5% Tris-buffered saline with Tween 20 (TBST) for 1 h, the membranes were incubated with various appropriately diluted primary antibodies for target genes at 4°C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, and immunoreactivity was visualized using enhanced HRP substrate luminol reagent (Milipore, Billerica, MA, USA).
Proteasome activity assay
Skeletal muscle (gastrocnemius muscle) samples of approximately 5 mg were dissected from mice, and rinsed in ice-cold phosphate-buffered saline to remove blood. A proteasome activity assay for chymotrypsin, trypsin and caspase was performed using a commercially available Proteasome-Glo™ 3-Substrate Systems kit in accordance with the manufacturer's instruction.
Co-immunoprecipitation (Co-IP) assay
Sample lysates (1 mg) of the lung were incubated with anti-14-3-3 antibody in 300 μL of ice-cold lysis buffer containing 50 mM Tris-Cl (pH = 7.5), 150 mM NaCl, 1% Nonidet P-40, and 10% glycerol, and freshly supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific Inc.) containing 1 mM DTT, 1 mM EDTA, and 1 mM PMSF. After rocking for 24 h at 4°C, 60 ml of Protein A magnetic beads (Millipore Corporation, Billerica, MA, USA) was added. The mixtures were incubated overnight at 4°C and washed four times with lysis buffer. The precipitates were boiled at 95°C for 10 min. The eluted proteins were separated on 9% SDS-polyacrylamide gel and detected through Western blot analysis with anti-p-FoxO3a (1:200 dilution). www.oncotarget.com
ELISA assay
The levels of myostatin, activin A, TNF-α, IL-6 and IL-1β were determined using respective ELISA kits from R&D Systems, Inc. (MN, USA).
Histological examination
Intestinal segments were fixed with 10% formaldehyde and embedded in paraffin followed by hematoxylin and eosin staining to evaluate the pathological changes. Intestinal injury was scored according to the histopathological grading system [35] in accordance with the following principle: 0. normal histological findings; 1. mucosa: villus blunting, loss of crypt architecture, sparse inflammatory cell infiltration, vacuolization and edema/normal muscular layer; 2. mucosa: villus blunting with fattened and vacuolated cells, crypt necrosis, intense inflammatory cell infiltration, vacuolization and edema/ normal muscular layer; and 3. mucosa: villus blunting with fattened and vacuolated cells, crypt necrosis, intense inflammatory cell infiltration, vacuolization and edema/muscular edema. To determine the score of tissue injury, histological images were examined by a trained pathologist who was initially blinded to these groups.
Intestinal digestion enzyme activity assay
Intestine extracts were prepared in 0.9% NaCl supplemented with a proteinase inhibitor, and then the major digestive enzyme activity of the intestines, including LAP, LIP, and AMYL, was measured. The biochemical tests were conducted using a Fuji DRI-CHEM 3030 Analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Statistical analysis
The experimental data are expressed as the mean ± standard error of the mean. One-way analysis of variance with a post hoc Bonferroni test was used for statistical analysis. Results were considered significantly different at a P value < 0.05.
Figure 1 :
1The chemical components of the AC extract and its effects on lung tumor growth. The morphology of the fruiting bodies of Antrodia cinnamomea (AC) and the representative HPLC profile of the ethanolic extract of AC (A). The images of tumors, the weight, and the tumor nodules of lungs were measured invarious groups (B). Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05 versus the CGC group.
Figure 2 :
2AC treatment attenuates body weight loss and muscle atrophy. The body weight (A), a representative image of the muscle of limb (B), the weight of gastrocnemius and soleus muscle (C), and the proteasome activity (D) were photographed or measured in various groups. Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com
Figure 3 :
3Effects of AC on muscle atrophy-related mediator formation and gene expression. The formation of myostatin and activin A in muscle (A) and various atrogenic gene expression (B) were determined. The association of p-FoxO3a with 14-3-3 chaperone protein (C) and FoxO3a transcription factor activity (D) in muscle were examined. Data was expressed as mean ±S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com
Figure 4 :
4Effects of AC on serum pro-inflammatory cytokine levels and IGF-1 expression. The serum levels of proinflammatory cytokines (A), and the expression and amount of IGF-1 in muscle of different groups were measured (B). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com
Figure 5 :
5Treatment with AC ameliorates intestinal damage and digestive enzyme dysfunction. The morphological changes and the grading score of the damage in intestines of different groups were evaluated (A). Various intestinal digestive enzyme activity (B) and the daily food intake were measured (C). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com
Figure 6 :
6The proposed mechanisms accounting for the anti-cachectic activity of the AC extract. Combined treatment
Cruz Biotechnology (CA, USA). Anti-Akt, anti-phospho-Akt, anti-phospho-FoxO3a, and anti-FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-myostatin and anti-IL-6 were purchased from GeneTex, Inc. (CA, USA). Horseradish peroxidase (HRP)labeled secondary antibody was obtained from Abcam (Cambridge, MA, USA). Cisplatin and gemcitabine were provided by Eli Lilly (Indianapolis, IN, USA). The enzyme-linked immunosorbent assay (ELISA) kits of myostatin, activin A, IGF-1, TNF-α, IL-6, and IL-1β were purchased from R&D Systems, Inc. (MN, USA). Other reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).
Table 1 :
1The contents of triterpenoid compounds in the ethanol extract of AC determined by LC-MS/MS methodCompound name
ACKNOWLEDGMENTSThis study was supported by the grant from Buddhist Tzu Chi Medical Foundation (TCMMP105-05-01).CONFLICTS OF INTERESTNo potential conflicts of interest were disclosed.
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| [
"Skeletal muscle atrophy, the most characteristic feature of cancer cachexia, often occurs in patients with cancer undergoing chemotherapy. Antrodia cinnamomea (AC) a widely used edible medical fungus, exhibits hepatoprotective, anti-inflammatory and anticancer activities. In this study, we investigated whether combined treatment with the ethonolic extract of AC ameliorates cachexia symptoms, especially muscle wasting, in lung tumor-bearing mice treated with chemotherapy. Our results revealed that gemcitabine and cisplatin-induced severe body weight loss and skeletal muscle atrophy in the mice with cancer were greatly attenuated after AC extract administration. The protection may be attributed to the inhibition of skeletal muscle proteolysis by suppressing myostatin and activin release, muscle wasting-related FoxO3/MuRF-1/MAFbx signaling, proteasomal enzyme activity, and pro-inflammatory cytokine production. A significant decrease in insulin-like growth factor 1 (IGF-1) expression and formation was observed in the atrophying muscle of the conventional chemotherapy treatment group (CGC), and this decrease was markedly reversed by AC treatment. Additionally, the anorexia, intestinal injury and dysfunction that occurred in the CGC group were mitigated by AC extract. Taken together, these results demonstrated that the AC extract has a protective effect against chemotherapyinduced muscle atrophy mainly by attenuating muscle proteolysis, pro-inflammatory cytokine production, and anorexia, and activating IGF-1-dependent protein synthesis."
] | [
"Meng-Chuan Chen \nSchool of Dentistry\nGraduated Institute of Dental Science\nNational Defense Medical Center\nTaipeiTaiwan\n",
"Wen-Lin Hsu \nDepartment of Radiation Oncology\nBuddhist Tzu Chi General Hospital\nHualienTaiwan\n\nSchool of Medicine\nTzu Chi University\nHualienTaiwan\n",
"Tz-Chong Chou \nCancer Research Center\nBuddhist Tzu Chi General Hospital\nHualienTaiwan\n\nInstitute of Medical Sciences\nTzu Chi University\nHualienTaiwan\n\nDepartment of Biotechnology\nAsia University\nTaichungTaiwan\n\nChina Medical University Hospital\nChina Medical University\nTaichungTaiwan\n"
] | [
"School of Dentistry\nGraduated Institute of Dental Science\nNational Defense Medical Center\nTaipeiTaiwan",
"Department of Radiation Oncology\nBuddhist Tzu Chi General Hospital\nHualienTaiwan",
"School of Medicine\nTzu Chi University\nHualienTaiwan",
"Cancer Research Center\nBuddhist Tzu Chi General Hospital\nHualienTaiwan",
"Institute of Medical Sciences\nTzu Chi University\nHualienTaiwan",
"Department of Biotechnology\nAsia University\nTaichungTaiwan",
"China Medical University Hospital\nChina Medical University\nTaichungTaiwan"
] | [
"Meng-Chuan",
"Wen-Lin",
"Tz-Chong"
] | [
"Chen",
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"K Fearon, ",
"F Strasser, ",
"S D Anker, ",
"I Bosaeus, ",
"E Bruera, ",
"R L Fainsinger, ",
"A Jatoi, ",
"C Loprinzi, ",
"N Macdonald, ",
"G Mantovani, ",
"M Davis, ",
"M Muscaritoli, ",
"F Ottery, ",
"E Douglas, ",
"D C Mcmillan, ",
"A Tuca, ",
"P Jimenez-Fonseca, ",
"P Gascón, ",
"C C Coss, ",
"C E Bohl, ",
"J T Dalton, ",
"C L Donohoe, ",
"A M Ryan, ",
"J V Reynolds, ",
"L De Palma, ",
"M Marinelli, ",
"M Pavan, ",
"A Orazi, ",
"S H Lecker, ",
"R T Jagoe, ",
"A Gilbert, ",
"M Gomes, ",
"V Baracos, ",
"J Bailey, ",
"S R Price, ",
"W E Mitch, ",
"A L Goldberg, ",
"S H Kang, ",
"H A Lee, ",
"M Kim, ",
"E Lee, ",
"U D Sohn, ",
"I Kim, ",
"S S Wing, ",
"S H Lecker, ",
"R T Jagoe, ",
"J Zhou, ",
"B Liu, ",
"C Liang, ",
"Y Li, ",
"Y H Song, ",
"V Macdonald, ",
"M H Cohen, ",
"M Rothmann, ",
"Von Der Maase, ",
"H Hayden, ",
"A M , ",
"A Fanzani, ",
"A Zanola, ",
"F Rovetta, ",
"S Rossi, ",
"M F Aleo, ",
"R Barreto, ",
"D L Waning, ",
"H Gao, ",
"Y Liu, ",
"T A Zimmers, ",
"A Bonetto, ",
"Y C Chen, ",
"H O Ho, ",
"C H Su, ",
"M T Sheu, ",
"M Geethangili, ",
"Y M Tzeng, ",
"G Tzivion, ",
"M Dobson, ",
"G Ramakrishnan, ",
"J K Onesti, ",
"D C Guttridge, ",
"M J Tisdale, ",
"K C Fearon, ",
"D J Glass, ",
"D C Guttridge, ",
"Y Elkina, ",
"S Von Haehling, ",
"S D Anker, ",
"J Springer, ",
"P R Chen, ",
"K Lee, ",
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"A Bonetto, ",
"F Penna, ",
"P Costelli, ",
"Di Rienzo, ",
"G Lacitignola, ",
"A Baccino, ",
"F M Ziparo, ",
"V Mercantini, ",
"P , ",
"Rossi Fanelli, ",
"F Muscaritoli, ",
"M , ",
"P Costelli, ",
"M Muscaritoli, ",
"A Bonetto, ",
"F Penna, ",
"P Reffo, ",
"M Bossola, ",
"G Bonelli, ",
"G B Doglietto, ",
"F M Baccino, ",
"Rossi Fanelli, ",
"A A Narsale, ",
"J A Carson, ",
"H J Patel, ",
"B M Patel, ",
"J Pfitzenmaier, ",
"R Vessella, ",
"C S Higano, ",
"J L Noteboom, ",
"D WallaceJr, ",
"Corey E , ",
"M Sharma, ",
"R Kambadur, ",
"S Sriram, ",
"S Lokireddy, ",
"C D Mcfarlane, ",
"C P Velloso, ",
"T N Stitt, ",
"D Drujan, ",
"B A Clarke, ",
"F Panaro, ",
"Y Timofeyva, ",
"W O Kline, ",
"M Gonzalez, ",
"G D Yancopoulos, ",
"D J Glass, ",
"C T Yeh, ",
"Y K Rao, ",
"C J Yao, ",
"C F Yeh, ",
"C H Li, ",
"S E Chuang, ",
"J H Luong, ",
"G M Lai, ",
"Y M Tzeng, ",
"C I Lai, ",
"Y L Chu, ",
"C T Ho, ",
"Y C Su, ",
"Y H Kuo, ",
"L Y Sheen, ",
"X Qiao, ",
"Q Wang, ",
"Ji S Huang, ",
"Y Liu, ",
"K D Zhang, ",
"Z X Bo, ",
"T Tzeng, ",
"Y M Guo, ",
"D A Ye, ",
"M , ",
"M Dupage, ",
"A L Dooley, ",
"T Jacks, ",
"P M Soares, ",
"J M Mota, ",
"A S Gomes, ",
"R B Oliveira, ",
"A M Assreuy, ",
"G A Brito, ",
"A A Santos, ",
"R A Ribeiro, ",
"M H Souza, "
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"Definition and classification of cancer cachexia: an international consensus. K Fearon, F Strasser, S D Anker, I Bosaeus, E Bruera, R L Fainsinger, A Jatoi, C Loprinzi, N Macdonald, G Mantovani, M Davis, M Muscaritoli, F Ottery, 10.1016/S1470-2045Lancet Oncol. 1210Fearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, Jatoi A, Loprinzi C, MacDonald N, Mantovani G, Davis M, Muscaritoli M, Ottery F, et al. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011; 12:489-95. https://doi.org/10.1016/S1470-2045(10)70218-7.",
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"Clinical evaluation and optimal management of cancer cachexia. A Tuca, P Jimenez-Fonseca, P Gascón, 10.1016/j.critrevonc.2013.07.015Crit Rev Oncol Hematol. 88Tuca A, Jimenez-Fonseca P, Gascón P. Clinical evaluation and optimal management of cancer cachexia. Crit Rev Oncol Hematol. 2013; 88:625-36. https://doi.org/10.1016/j. critrevonc.2013.07.015.",
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". 10.1155/2011/601434Gastroenterol Res Pract. Gastroenterol Res Pract. 2011; 2011:601434. https://doi. org/10.1155/2011/601434.",
"Ubiquitin ligases MuRF1 and MAFbx in human skeletal muscle atrophy. L De Palma, M Marinelli, M Pavan, A Orazi, 10.1016/j.jbspin.2007.04.019Joint Bone Spine. 75de Palma L, Marinelli M, Pavan M, Orazi A. Ubiquitin ligases MuRF1 and MAFbx in human skeletal muscle atrophy. Joint Bone Spine. 2008; 75:53-57. https://doi. org/10.1016/j.jbspin.2007.04.019.",
"Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. S H Lecker, R T Jagoe, A Gilbert, M Gomes, V Baracos, J Bailey, S R Price, W E Mitch, A L Goldberg, 10.1096/fj.03-0610comFASEB J. 18Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE, Goldberg AL. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J. 2004; 18:39-51. https://doi.org/10.1096/fj.03-0610com.",
"Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiq uitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome. S H Kang, H A Lee, M Kim, E Lee, U D Sohn, I Kim, 10.1152/ajpendo.00389Am J Physiol Endocrinol Metab. 312Kang SH, Lee HA, Kim M, Lee E, Sohn UD, Kim I. Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiq uitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome. Am J Physiol Endocrinol Metab. 2017; 312:E495-507. https://doi.org/10.1152/ ajpendo.00389.2016.",
"Proteolysis in illnessassociated skeletal muscle atrophy: from pathways to networks. S S Wing, S H Lecker, R T Jagoe, 10.3109/10408363.2011.586171Crit Rev Clin Lab Sci. 48Wing SS, Lecker SH, Jagoe RT. Proteolysis in illness- associated skeletal muscle atrophy: from pathways to networks. Crit Rev Clin Lab Sci. 2011; 48:49-70. https:// doi.org/10.3109/10408363.2011.586171.",
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"Gemcitabine and cisplatin for advanced, metastatic bladder cancer. M H Cohen, M Rothmann, Von Der Maase, H Hayden, A M , 10.1200/JCO.2001.19.4.1229J Clin Oncol. 19Cohen MH, Rothmann M, von der Maase H, Hayden AM. Gemcitabine and cisplatin for advanced, metastatic bladder cancer. J Clin Oncol. 2001; 19:1229-31. https:// doi.org/10.1200/JCO.2001.19.4.1229.",
"Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems. A Fanzani, A Zanola, F Rovetta, S Rossi, M F Aleo, Fanzani A, Zanola A, Rovetta F, Rossi S, Aleo MF. Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems. www.oncotarget.com",
". 10.1016/j.taap.2010.11.003Toxicol Appl Pharmacol. 250Toxicol Appl Pharmacol. 2011; 250:312-21. https://doi. org/10.1016/j.taap.2010.11.003.",
"Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs. R Barreto, D L Waning, H Gao, Y Liu, T A Zimmers, A Bonetto, 10.18632/oncotarget.9779Oncotarget. 7Barreto R, Waning DL, Gao H, Liu Y, Zimmers TA, Bonetto A. Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs. Oncotarget. 2016; 7:43442-60. https://doi. org/10.18632/oncotarget.9779.",
"Anticancer effects of taiwan of fugus camphoratus extracts, isolated compounds and its combinational use. Y C Chen, H O Ho, C H Su, M T Sheu, 10.1016/j.jecm.2010.08.003J Exp Clin Med. 2Chen YC, Ho HO, Su CH, Sheu MT. Anticancer effects of taiwan of fugus camphoratus extracts, isolated compounds and its combinational use. J Exp Clin Med. 2010; 2:274-81. https://doi.org/10.1016/j.jecm.2010.08.003.",
"Review of pharmacological effects of antrodia camphorata and its bioactive compounds. M Geethangili, Y M Tzeng, 10.1093/ecam/nep108Evid Based Complement Alternat Med. Geethangili M, Tzeng YM. Review of pharmacological effects of antrodia camphorata and its bioactive compounds. Evid Based Complement Alternat Med. 2011; 2011:212641. https://doi.org /10.1093/ecam/nep108.",
"FoxO transcription factors; Regulation by AKT and 14-3-3 proteins. G Tzivion, M Dobson, G Ramakrishnan, Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; Regulation by AKT and 14-3-3 proteins.",
". 10.1016/j.bbamcr.2011.06.002Biochim Biophys Acta. 1813Biochim Biophys Acta. 2011; 1813:1938-45. https://doi. org/10.1016/j.bbamcr.2011.06.002.",
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"Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells. C T Yeh, Y K Rao, C J Yao, C F Yeh, C H Li, S E Chuang, J H Luong, G M Lai, Y M Tzeng, 10.1016/j.canlet.2009.05.002Cancer Lett. 285Yeh CT, Rao YK, Yao CJ, Yeh CF, Li CH, Chuang SE, Luong JH, Lai GM, Tzeng YM. Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells. Cancer Lett. 2009; 285:73-79. https://doi.org/10.1016/j.canlet.2009.05.002.",
"Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stressmediated apoptosis in human hepatoma cells. C I Lai, Y L Chu, C T Ho, Y C Su, Y H Kuo, L Y Sheen, 10.1016/j.jtcme.2014.11.026J Tradit Complement Med. 6Lai CI, Chu YL, Ho CT, Su YC, Kuo YH, Sheen LY. Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress- mediated apoptosis in human hepatoma cells. J Tradit Complement Med. 2015; 6:48-56. https://doi.org/10.1016/j. jtcme.2014.11.026.",
"Metabolites identification and multi-component pharmacokinetics of ergostane and lanostane triterpenoids in the anticancer mushroom Antrodia cinnamomea. X Qiao, Q Wang, Ji S Huang, Y Liu, K D Zhang, Z X Bo, T Tzeng, Y M Guo, D A Ye, M , 10.1016/j.jpba.2015.04.010J Pharm Biomed Anal. 111Qiao X, Wang Q, Ji S, Huang Y, Liu KD, Zhang ZX, Bo T, Tzeng YM, Guo DA, Ye M. Metabolites identification and multi-component pharmacokinetics of ergostane and lanostane triterpenoids in the anticancer mushroom Antrodia cinnamomea. J Pharm Biomed Anal. 2015; 111:266-76. https://doi.org/10.1016/j.jpba.2015.04.010.",
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"Gastrointestinal dysmotility in 5-fluorouracil-induced intestinal mucositis outlasts inflammatory process resolution. P M Soares, J M Mota, A S Gomes, R B Oliveira, A M Assreuy, G A Brito, A A Santos, R A Ribeiro, M H Souza, 10.1007/s00280-008-0715-9Cancer Chemother Pharmacol. 63Soares PM, Mota JM, Gomes AS, Oliveira RB, Assreuy AM, Brito GA, Santos AA, Ribeiro RA, Souza MH. Gastrointestinal dysmotility in 5-fluorouracil-induced intestinal mucositis outlasts inflammatory process resolution. Cancer Chemother Pharmacol. 2008; 63:91-98. https://doi.org/10.1007/s00280-008-0715-9."
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"Definition and classification of cancer cachexia: an international consensus",
"Towards a simple objective framework for the investigation and treatment of cancer cachexia: the Glasgow Prognostic Score",
"Clinical evaluation and optimal management of cancer cachexia",
"Cancer cachexia therapy: a key weapon in the fight against cancer",
"Ubiquitin ligases MuRF1 and MAFbx in human skeletal muscle atrophy",
"Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression",
"Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiq uitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome",
"Proteolysis in illnessassociated skeletal muscle atrophy: from pathways to networks",
"Cytokine signaling in skeletal muscle wasting",
"Chemotherapy: managing side effects and safe handling",
"Gemcitabine and cisplatin for advanced, metastatic bladder cancer",
"Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs",
"Anticancer effects of taiwan of fugus camphoratus extracts, isolated compounds and its combinational use",
"Review of pharmacological effects of antrodia camphorata and its bioactive compounds",
"Inflammation based regulation of cancer cachexia",
"Mechanisms of cancer cachexia",
"Cancer cachexia: mediators, signaling, and metabolic pathways",
"The role of myostatin in muscle wasting: an overview",
"INVITED REVIEW: inhibitors of myostatin as methods of en hancing muscle growth and development",
"Changes in myostatin signaling in non-weight-losing cancer patients",
"Role of interleukin-6 in cachexia: therapeutic implications",
"TNF-α and cancer cachexia: molecular insights and clinical implications",
"Elevation of cytokine levels in cachectic patients with prostate carcinoma",
"Molecular targets of cancer cachexia: opportunities for pharmanutritional approaches",
"Regulation of muscle mass by growth hormone and IGF-I",
"The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO trans cription factors",
"Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells",
"Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stressmediated apoptosis in human hepatoma cells",
"Metabolites identification and multi-component pharmacokinetics of ergostane and lanostane triterpenoids in the anticancer mushroom Antrodia cinnamomea",
"Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase",
"Gastrointestinal dysmotility in 5-fluorouracil-induced intestinal mucositis outlasts inflammatory process resolution"
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"Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems",
"Toxicol Appl Pharmacol",
"Oncotarget",
"J Exp Clin Med",
"Evid Based Complement Alternat Med",
"FoxO transcription factors; Regulation by AKT and 14-3-3 proteins",
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"Cell Metab",
"J Cachexia Sarcopenia Muscle",
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"Eur J Clin Invest",
"Curr Opin Support Palliat Care",
"Life Sci",
"Cancer",
"PharmaNutrition",
"Br J Pharmacol",
"Mol Cell",
"Cancer Lett",
"J Tradit Complement Med",
"J Pharm Biomed Anal",
"Nat Protoc",
"Cancer Chemother Pharmacol"
] | [
"\nFigure 1 :\n1The chemical components of the AC extract and its effects on lung tumor growth. The morphology of the fruiting bodies of Antrodia cinnamomea (AC) and the representative HPLC profile of the ethanolic extract of AC (A). The images of tumors, the weight, and the tumor nodules of lungs were measured invarious groups (B). Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05 versus the CGC group.",
"\nFigure 2 :\n2AC treatment attenuates body weight loss and muscle atrophy. The body weight (A), a representative image of the muscle of limb (B), the weight of gastrocnemius and soleus muscle (C), and the proteasome activity (D) were photographed or measured in various groups. Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"\nFigure 3 :\n3Effects of AC on muscle atrophy-related mediator formation and gene expression. The formation of myostatin and activin A in muscle (A) and various atrogenic gene expression (B) were determined. The association of p-FoxO3a with 14-3-3 chaperone protein (C) and FoxO3a transcription factor activity (D) in muscle were examined. Data was expressed as mean ±S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"\nFigure 4 :\n4Effects of AC on serum pro-inflammatory cytokine levels and IGF-1 expression. The serum levels of proinflammatory cytokines (A), and the expression and amount of IGF-1 in muscle of different groups were measured (B). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"\nFigure 5 :\n5Treatment with AC ameliorates intestinal damage and digestive enzyme dysfunction. The morphological changes and the grading score of the damage in intestines of different groups were evaluated (A). Various intestinal digestive enzyme activity (B) and the daily food intake were measured (C). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"\nFigure 6 :\n6The proposed mechanisms accounting for the anti-cachectic activity of the AC extract. Combined treatment",
"\n\nCruz Biotechnology (CA, USA). Anti-Akt, anti-phospho-Akt, anti-phospho-FoxO3a, and anti-FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-myostatin and anti-IL-6 were purchased from GeneTex, Inc. (CA, USA). Horseradish peroxidase (HRP)labeled secondary antibody was obtained from Abcam (Cambridge, MA, USA). Cisplatin and gemcitabine were provided by Eli Lilly (Indianapolis, IN, USA). The enzyme-linked immunosorbent assay (ELISA) kits of myostatin, activin A, IGF-1, TNF-α, IL-6, and IL-1β were purchased from R&D Systems, Inc. (MN, USA). Other reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).",
"\nTable 1 :\n1The contents of triterpenoid compounds in the ethanol extract of AC determined by LC-MS/MS methodCompound name \n"
] | [
"The chemical components of the AC extract and its effects on lung tumor growth. The morphology of the fruiting bodies of Antrodia cinnamomea (AC) and the representative HPLC profile of the ethanolic extract of AC (A). The images of tumors, the weight, and the tumor nodules of lungs were measured invarious groups (B). Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05 versus the CGC group.",
"AC treatment attenuates body weight loss and muscle atrophy. The body weight (A), a representative image of the muscle of limb (B), the weight of gastrocnemius and soleus muscle (C), and the proteasome activity (D) were photographed or measured in various groups. Data was expressed as mean ± S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"Effects of AC on muscle atrophy-related mediator formation and gene expression. The formation of myostatin and activin A in muscle (A) and various atrogenic gene expression (B) were determined. The association of p-FoxO3a with 14-3-3 chaperone protein (C) and FoxO3a transcription factor activity (D) in muscle were examined. Data was expressed as mean ±S.E.M. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"Effects of AC on serum pro-inflammatory cytokine levels and IGF-1 expression. The serum levels of proinflammatory cytokines (A), and the expression and amount of IGF-1 in muscle of different groups were measured (B). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"Treatment with AC ameliorates intestinal damage and digestive enzyme dysfunction. The morphological changes and the grading score of the damage in intestines of different groups were evaluated (A). Various intestinal digestive enzyme activity (B) and the daily food intake were measured (C). Data was expressed as mean ±S.E.M. (n=5). ** P < 0.01, *** P < 0.001 versus the normal group. # P < 0.05, ## P < 0.01 versus the CGC group. www.oncotarget.com",
"The proposed mechanisms accounting for the anti-cachectic activity of the AC extract. Combined treatment",
"Cruz Biotechnology (CA, USA). Anti-Akt, anti-phospho-Akt, anti-phospho-FoxO3a, and anti-FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-myostatin and anti-IL-6 were purchased from GeneTex, Inc. (CA, USA). Horseradish peroxidase (HRP)labeled secondary antibody was obtained from Abcam (Cambridge, MA, USA). Cisplatin and gemcitabine were provided by Eli Lilly (Indianapolis, IN, USA). The enzyme-linked immunosorbent assay (ELISA) kits of myostatin, activin A, IGF-1, TNF-α, IL-6, and IL-1β were purchased from R&D Systems, Inc. (MN, USA). Other reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).",
"The contents of triterpenoid compounds in the ethanol extract of AC determined by LC-MS/MS method"
] | [
"Figure 1A",
"Figure 1B",
"Figure 2A",
"Figure 2B",
"Figure 2C",
"Figure 2D",
"Figure 3A)",
"Figure 3B",
"Figure 3C",
"Figure 3D",
"Figure 4A",
"Figure 4B",
"Figure 5A",
"Figure 5B",
"Figure 5C",
"Figure 6"
] | [] | [
"Cancer cachexia is characterized by body weight loss, anorexia, fatigue, inflammation and abnormal metabolism, thereby markedly reducing quality of life and limiting the application of conventional therapy such as chemotherapy [1,2]. More than 80% of patients with advanced cancer suffer from cachexia, and cancer cachexia is estimated to account for at least 20% of deaths in patients with cancer [3,4]. Thus, how to prevent and attenuate the development of cancer cachexia is a key concern during cancer therapy. Muscle wasting, the most prominent phenotypic feature of cancer cachexia, is closely related to the tumor site, size, stage, and treatment type [5]. During cachexia, muscle proteolysis www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 28), pp: [19584][19585][19586][19587][19588][19589][19590][19591][19592][19593][19594][19595][19596] Research Paper www.oncotarget.com is predominately triggered by the ubiquitin proteasome system (UPS). The forkhead box O (FoxO) transcription factor is a key factor for activating the expression of musclespecific ubiquitin conjugating enzymes E3 ligase, F-box (MAFbx)/atrogin-1 and muscle ring finger 1 (MuRF-1). The ubiquitinated target protein substrate can be recognized by the 26S proteasome and then digested to peptides, which in turn leads to muscle protein degradation [6]. Increased ubiquitinated protein expression and proteasome activity have been observed in atrophying muscle [7]. Conversely, mice that are deficient in either MAFbx or MuRF-1 exhibit greater resistance to atrophy [8], suggesting that suppressing the FoxO/MAFbx/MuRF-1/UPS cascade is a promising strategy of preventing the muscle wasting associated with cancer cachexia. The process of cancer cachexia-evoked muscle atrophy is complex and multifactorial; it is mediated by the interplay of tumor factors, host factors, and the interaction between the two [9,10].",
"To date, chemotherapy remains a widely used option for cancer therapy. Nevertheless, several deleterious effects have been identified after chemotherapy, thereby limiting its application [11]. Currently, combined treatment with chemotherapeutic drugs such as gemcitabine (2', 2'-difluorodeoxycytidine) and cisplatin (cis-diamminedichloroplatinum) is a common regimen for treating metastatic lung cancer. However, many side effects, including nephrotoxicity, gastrointestinal mucosal injury, and severe muscle wasting, have been reported in patients with cancer treated with cisplatin [12,13]. These findings imply that chemotherapy itself can induce muscle atrophy [14]. Although various drugs and supplements have been used, cancer cachexia-evoked skeletal muscle mass loss remains a major problem. Therefore, developing more effective and safe chemotherapeutic adjuvants or nutritional supplements to ameliorate chemotherapy-induced muscle wasting is urgent.",
"Antrodia cinnamomea (AC), a medical fungus, grows only on the inner cavity of the endemic species Cinnamomum kanehirai, which is a plant native to Taiwan. AC extracts have been demonatrated to possess several beneficial effects, including antioxidant, hepatoprotective, anti-hypertensive, anti-hyperlipidemic, immunomodulatory, anticancer, and anti-inflammatory activities [15,16]. However, the effects of AC extract on cancer cachexia remain unknown. Therefore, the aim of this study was to examine whether AC attenuates these cachectic symptoms, particularly the muscle atrophy, in lung tumor-bearing mice under chemotherapy, and to further investigate the molecular mechanisms involved.",
"Data from the HPLC analysis revealed that the ethanolic extract of the patented kinetic bio-activation fruiting body of AC contained seven marker triterpenoid ingredients, namely antcin A (at 74.7 min), antcin B (at 64.4 and 65.4 min), antcin C (at 42.2 and 45.7 min), antcin H (at 45.0 min), antcin K (at 20.1 and 21.0 min), dehydrosulphurenic acid (at 57.4 min) and dehydroeburicoic acid (at 84.9 min) ( Figure 1A). The total amount of the triterpenoids was observed to account for 14.5% (w/w) of the AC extract, and antcin C, antcin H, and antcin K were abundant among these triterpenoids (Table 1). Four groups were used in this study: (1) a normal group; (2) cancer group (tumor-alone group); (3) CGC group: mice treated with a standard diet and an intraperitoneal injection of gemcitabine (1000 mg/m 2 per 3 days) and cisplatin (75 mg/m 2 /week); (4) CGCA group: mice treated with a standard diet plus AC extract (300 mg/kg/day, p.o.) and an intraperitoneal injection of gemcitabine and cisplatin. After orthotopic implantation of LLC2 cells into the lungs for two weeks, the mice were treated with different combinations of drugs for 21 days and were sacrificed to evaluate tumor progression. The number of tumor nodules and the lung weight reflecting the tumor growth were greatly reduced in the CGC and CGCA groups, compared with the cancer alone group. Furthermore, we observed that the anticancer activity of the CGCA group was stronger than that of the CGC group ( Figure 1B).",
"At the end of the study, the untreated cancer mice lost 19.0 ± 1.4% of their initial body weight, whereas the normal mice gained body weight. The mice in the CGC and CGCA groups lost 24.2 ± 1.6%, and 18.8 ± 1.8% of their initial weight, respectively ( Figure 2A). During cancer cachexia, body weight loss generally results from skeletal muscle wasting. As expected, the muscle atrophy evaluated through histological examination was parallel with the trend of muscle mass loss in these groups ( Figure 2B). Notably, the CGC group had the most skeletal muscle mass loss, as evidenced by a marked reduction in the weight of the gastrocnemius and soleus muscle, and this loss was inhibited by combined treatment with AC (CGCA) ( Figure 2C). Consistently, a marked increase in muscular proteasome activity, particularly chymotrypsin and trypsin, was observed in the CGC group, and this increase was significantly inhibited by AC treatment ( Figure 2D).",
"Overproduction of myostatin and activin A observed in the atrophying muscle of the cancer-alone and CGC groups was significantly decreased in the mice in the CGCA group ( Figure 3A). Similarly, the www.oncotarget.com alterations in muscle wasting-related gene expression, including increased levels of ActRIIB, FoxO3, MuRF 1, and MAFbx, as well as decreased expression of p-Akt and p-FoxO3, particularly in the muscle of the CGC group, were markedly reversed through AC treatment ( Figure 3B). The 14-3-3 chaperone protein binds to phosphorylated FoxO3, resulting in FoxO3 degradation, thereby inhibiting downstream MuRF 1 and MAFbx expression [17]. Our results confirmed that the interaction of p-FoxO3 with the 14-3-3 protein determined by an immunoprecipitation assay was increased in the CGCA group, compared with the cancer alone and CGC groups ( Figure 3C). As expected, a marked elevation of FoxO3 transcriptional activity in the CGC group was significantly inhibited by AC treatment ( Figure 3D). Therefore, suppressing FoxO3-mediated processes may contribute to the attenuation of muscle proteolysis by AC.",
"Systemic inflammation is considered a key factor inducing cancer cachexia [18]. A significant elevation of serum levels of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, particularly in the CGC group, was greatly diminished in the CGCA group ( Figure 4A). Notably, combined treatment with the AC extract greatly increased the expression and production of IGF-1, compared with the CGC group ( Figure 4B). These results suggest that inhibiting inflammatory responses and activating IGF-1-dependent process involves the antiatrophic effect of AC.",
"Severe damage to the intestinal mucosal structure, especially in the CGC group, was ameliorated in the mice in the CGCA group ( Figure 5A). Additionally, impaired digestive enzyme activity, such as leucine amino peptidase (LAP), a digestive enzyme for peptides, amylase (AMYL), a digestive enzyme for sugars, and lipase (LIP), a digestive enzyme for fat, in the cancer-alone and CGC groups, was alleviated after AC administration ( Figure 5B). Notably, AC could mitigate the anorexia observed in the CGC group, as evidenced by an elevation in daily food intake ( Figure 5C).",
"Several tumor and host factors play critical roles in triggering muscle atrophy associated with cancer cachexia by activating muscle proteolytic pathways, impairing protein synthesis, or both [19,20]. The current study demonstrates, for the first time, that combined treatment with the ethanolic extract of AC greatly alleviates gemcitabine and cisplatininduced cachectic symptoms, including body weight loss, skeletal muscle atrophy, intestinal damage and dysfunction, and anorexia in lung tumor-bearing mice; this thus supports the clinical use of this extract. Myostatin belonging to transforming growth factor-α; (TGF-α) superfamily is mostly expressed in skeletal muscle, and it can inhibit skeletal muscle growth by reducing myoblast proliferation and myogenesis [21]. Conversely, blocking the actions of myostatin by using neutralizing antibodies or antagonists remarkably increases muscle size and physical strength [22]. Similarly, activins, members of the TGF-α superfamily, are another muscle atrophying factor. Notably, myostatin and activins can bind the same muscle surface receptor complex that composes of type-II activin receptors (ActRIIA and ActRIIB) and type-I activin receptors (ALK4 and ALK5), which ultimately results in muscle proteolysis through activation of FoxO, especially the FoxO3 isoform. The actions of FoxO are largely controlled by the subcellular localization and the protein degradation of FoxO. When FoxO is phosphorylated and inactivated by Akt, the phosphorylated FoxO is exported from the nucleus into cytoplasma in a chaperone 14-3-3-dependent manner [17]. The 14-3-3 bound phosphorylated FoxO protein is then degraded through proteasome. In response to myostatin and activin, Akt activity is inhibited, leading to the accumulation of the dephospho-FoxO protein, the active form of FoxO. Subsequently, the activated FoxO translocates into the nucleus and enhances the transcription of muscle-specific atrogenic genes, such as MuRF-1 and MAFbx. Previous studies have reported that myostatin and activin signaling in muscle increases in patients with cancer suffering from cachexia and in experimental cancer cachexia [23,24].",
"In the present study, we demonstrated that the levels of muscle myostatin and activin were significantly reduced in the CGCA group, compared with those in the CGC group. An increase in ActRIIB expression and a decrease in Akt phosphorylation in the CGC group were also reversed by AC treatment, which may be associated with the inhibition of myostatin and activin generation. As expected, treatment with AC increased FoxO3 phosphorylation accompanied by an elevation of the association of 14-3-3 with phospho-FoxO3 in cytoplasma, but it significantly inhibited MuRF-1 and MAFbx expression, and proteasome activity. Collectively, suppressing the myostatin/activins/FoxO3/ MuRF-1/MAFbx signaling pathway may contribute to the antiatrophic effect of AC. Accumulating evidence indicates that systemic inflammation and pro-inflammatory cytokines, including TNF-β, IL-6 and IL-1β, are major factors in promoting muscle atrophy through UPS activation, muscle differentiation and myogenesis inhibition [18], and improvement of anorexia during cancer cachexia [25,26]. Elevated serum levels of pro-inflammatory cytokines were reported in patients with cachexia [27]. However, neutralization of pro-inflammatory cytokines could markedly relieve the cachectic symptoms in an experimental animal model [28]. According to the results that the CGCA group had lower serum levels of NF-β, IL-6, and IL-1β than that in the CGC group, inhibition of pro-inflammatory cytokine formation may also involve the anti-cachectic effects of AC.",
"In addition to muscle proteolysis, muscle protein generation is another crucial factor in regulating muscle mass. A central role of IGF-1 in enhancing muscle growth by activating PI3K/Akt/mTOR-regulated protein synthesis was demonstrated, previously [29]. Moreover, IGF-1 can activate Akt-induced FoxO phosphorylation, thereby attenuating muscle protein degradation [30]. Therefore, IGF-1 not only enhances protein synthesis but also inhibits muscle proteolysis, suggesting that IGF-1induction is a promising approach to prevent muscle atrophy. A novel finding of this study is that the CGCA group had a higher IGF-1 level in muscle than that of the CGC group. Accordingly, AC-mediated attenuation of muscle mass loss may, at least partly, be attributed to the suppression of inflammation-evoked muscle protein degradation, and enhancement of IGF-1-dependent protein synthesis.",
"Maintaining the intestinal structure and function is essential for nutritional intake and body growth. Notably, intestinal damage and impaired digestive enzyme activity, particularly in the CGC group, were greatly improved through AC treatment. Thus, increased nutrient absorption and availability due to the attenuation of intestinal injury and dysfunction may in turn enhance appetite, as reflected by increased daily food intake, thereby attenuating body weight loss by AC in this cachectic model. Notably, the inhibition of lung tumor growth through combined treatment with AC was stronger than that observed in the CGC group, suggesting that AC can also enhance the anticancer activity of chemotherapy.",
"Ergostane-type triterpenoids are the major bioactive components in the fruiting bodies of AC. Among these triterpenoids, antcin C, antcin H, antcin K, and antcin A have been reported to exhibit antiinflammatory and anticancer activities [16,31,32]. The pharmacokinetic and metabolism study demonstrated that several triterpenoids and metabolites were detected in the plasma of rats after oral administration of the ethanol extract of AC (1g/kg). Generally, the ergostane triterpenoids are absorbed and eliminated rapidly, and the pharmacokinetic patterns of Antrodia triterpenoids are closely related to their chemical structure. The highpolarity of antcin H and antcin K were determined to be metabolically stable, and had markedly higher plasma concentrations than antcin B and antcin C [33]. Conversely, antcin A had a low concentration in plasma. Composition analysis revealed that antcin H and antcin K were abundant in the AC extract. Therefore, antcin K and antcin H may be most responsible for the therapeutic effects on cancer cachexia because of their pharmacokinetic parameters, abundance, and biological effects. However, more basic and human studies are required to determine their clinical use. Notably, the hydrogenated metabolites derived from antcin C also revealed a high plasma concentration. However, the biological functions are still unknown, and need further investigation.",
"In summary, AC administration can attenuate the development of cancer cachexia in lung tumor-bearing mice undergoing chemotherapy. Mechanically, the protective effects of AC against muscle wasting may be associated with the suppression of muscle proteolysis, formation of pro-inflammatory cytokines, activation of IGF-1-regulated protein synthesis, and improvement of intestinal damage and dysfunction ( Figure 6). Overall, AC has potential to ameliorate cachectic symptoms in patients undergoing chemotherapy. www.oncotarget.com with AC inhibits myostatin/activin/FoxO3 signaling and pro-inflammatory cytokine formation, leading to the suppression of MAFbx and MuRF1 expression and proteasome activity in muscle, which in turn attenuates muscle proteolysis. Meanwhile, the AC extract enhances IGF-1 expression and its regulated protein synthesis, and improves anorexia and intestinal damage and dysfunction. Therefore, AC has potential to ameliorate cachectic symptoms under chemotherapy, especially body weight loss. www.oncotarget.com",
"The fruiting bodies of AC were provided by Balay Biotechnology, Inc. (Taipei, Taiwan) and identified by the Bioresource Collection and Research Center (BCRC, Taiwan). The dried AC powder (30g) was soaked with 0.9 L of distilled water at 80°C-100°C for 1 h. Subsequently, it was extracted with 900 mL of ethanol (95%) for 24 h. The residue was filtered through a Buchner funnel lined with Whatman filter paper; the filtrates were collected and dried using a rotary evaporator (CES-800, Panchum Scientific Corp.) under reduced pressure at 45°C to obtain brownish colored residues with a yield of 7.5 g (25%, w/w) and were stored at -20°C prior to analysis. Antibodies, including anti-TNF-α, anti-IL-1β, anti-IGF-1, anti-MuRF-1, anti-MAFbX-1, and anti-β-actin were purchased from Santa ",
"The Lewis lung carcinoma cell line LLC2 purchased from the Bioresource Collection and Research Center (Taipei, Taiwan) was incubated in Dulbecco's modified Eagle's Medium (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (Thermo Fisher Scientific Inc), 2 mmol/L L-glutamine, and 100 U/ mL penicillin-streptomycin (Gibco, Carlsbad, CA, USA). Cells were maintained in an incubator with room air: CO 2 (95:5, v/v) at 37°C.",
"Seven-week-old male C57B/6 mice weighing approximately 25 g were used for the study. The animal care and experimental procedures were conducted in accordance with the Guiding Principle in the Care and Use of Animals and approved by the Institutional Animal Care and Use Committee of National Defense Medical Center (IACUC 12156). The mice were anaesthetized with an intraperitoneal injection of ketamine HCl/xylazine (100 mg/15 mg body weight per mouse). Anesthetized mice were placed on a platform by their front teeth so that their chests hung vertically beneath them. Light was directed on each mouse's upper chest, on a spot marked by an \"X\". The mouth was opened using the Exel Safelet IV catheter, and the tongue is gently pulled out using flat forceps. After locating the white light emitted from the trachea, the Exel Safelet IV catheter was slid into the trachea, and the needle was removed. The mouse with the inserted catheter on the platform is moved into a biosafety hood, where the LLC2 cell (1 × 10 6 cells/100 μL) was dispensed into the opening of the catheter [34]. After implantation of cancer cells for 10 days, the mice were divided into four weightmatched groups: (1) the normal group, (2) cancer-alone group, (3) CGC group, and (4) CGCA, as described in the Results section. Each group contains five mice.",
"Protein samples (100 μg) were separated on a 10% SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk in 5% Tris-buffered saline with Tween 20 (TBST) for 1 h, the membranes were incubated with various appropriately diluted primary antibodies for target genes at 4°C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, and immunoreactivity was visualized using enhanced HRP substrate luminol reagent (Milipore, Billerica, MA, USA).",
"Skeletal muscle (gastrocnemius muscle) samples of approximately 5 mg were dissected from mice, and rinsed in ice-cold phosphate-buffered saline to remove blood. A proteasome activity assay for chymotrypsin, trypsin and caspase was performed using a commercially available Proteasome-Glo™ 3-Substrate Systems kit in accordance with the manufacturer's instruction.",
"Sample lysates (1 mg) of the lung were incubated with anti-14-3-3 antibody in 300 μL of ice-cold lysis buffer containing 50 mM Tris-Cl (pH = 7.5), 150 mM NaCl, 1% Nonidet P-40, and 10% glycerol, and freshly supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific Inc.) containing 1 mM DTT, 1 mM EDTA, and 1 mM PMSF. After rocking for 24 h at 4°C, 60 ml of Protein A magnetic beads (Millipore Corporation, Billerica, MA, USA) was added. The mixtures were incubated overnight at 4°C and washed four times with lysis buffer. The precipitates were boiled at 95°C for 10 min. The eluted proteins were separated on 9% SDS-polyacrylamide gel and detected through Western blot analysis with anti-p-FoxO3a (1:200 dilution). www.oncotarget.com",
"The levels of myostatin, activin A, TNF-α, IL-6 and IL-1β were determined using respective ELISA kits from R&D Systems, Inc. (MN, USA).",
"Intestinal segments were fixed with 10% formaldehyde and embedded in paraffin followed by hematoxylin and eosin staining to evaluate the pathological changes. Intestinal injury was scored according to the histopathological grading system [35] in accordance with the following principle: 0. normal histological findings; 1. mucosa: villus blunting, loss of crypt architecture, sparse inflammatory cell infiltration, vacuolization and edema/normal muscular layer; 2. mucosa: villus blunting with fattened and vacuolated cells, crypt necrosis, intense inflammatory cell infiltration, vacuolization and edema/ normal muscular layer; and 3. mucosa: villus blunting with fattened and vacuolated cells, crypt necrosis, intense inflammatory cell infiltration, vacuolization and edema/muscular edema. To determine the score of tissue injury, histological images were examined by a trained pathologist who was initially blinded to these groups.",
"Intestine extracts were prepared in 0.9% NaCl supplemented with a proteinase inhibitor, and then the major digestive enzyme activity of the intestines, including LAP, LIP, and AMYL, was measured. The biochemical tests were conducted using a Fuji DRI-CHEM 3030 Analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).",
"The experimental data are expressed as the mean ± standard error of the mean. One-way analysis of variance with a post hoc Bonferroni test was used for statistical analysis. Results were considered significantly different at a P value < 0.05."
] | [] | [
"INTRODUCTION",
"RESULTS",
"Chemical characteristics of the AC extract and its effects on tumor growth",
"AC attenuates muscle atrophy and proteasome activity in muscle",
"AC inhibits muscle wasting-related signaling pathway",
"AC inhibits pro-inflammatory cytokine production and upregulates insulin-like growth factor-1 (IGF-1)",
"AC prevents intestinal injury/dysfunction",
"DISCUSSION",
"MATERIALS AND METHODS",
"Preparation of AC extract and reagents",
"Cell culture",
"Animal model",
"Western blotting",
"Proteasome activity assay",
"Co-immunoprecipitation (Co-IP) assay",
"ELISA assay",
"Histological examination",
"Intestinal digestion enzyme activity assay",
"Statistical analysis",
"Figure 1 :",
"Figure 2 :",
"Figure 3 :",
"Figure 4 :",
"Figure 5 :",
"Figure 6 :",
"Table 1 :"
] | [
"Compound name \n"
] | [
"(Table 1)"
] | [
"Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy",
"Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy"
] | [] |
51,899,340 | 2022-03-01T16:45:09Z | CCBY | https://thyroidresearchjournal.biomedcentral.com/track/pdf/10.1186/s13044-018-0055-8 | GOLD | b29a5e43d408cedefea3b3d51ce3bada1edd1240 | null | null | null | null | 10.1186/s13044-018-0055-8 | 2887659392 | 30083234 | 6069891 |
A rare malignant thyroid carcinosarcoma with aggressive behavior and DICER1 gene mutation: a case report with literature review
Jing Yang
Carmen Sarita-Reyes
David Kindelberger
Qing Zhao
A rare malignant thyroid carcinosarcoma with aggressive behavior and DICER1 gene mutation: a case report with literature review
10.1186/s13044-018-0055-8C A S E R E P O R T Open Access
Background: Malignant biphasic tumor also known as carcinosarcoma is an uncommon neoplasm that is composed of both malignant epithelial and mesenchymal components. Most reported cases of carcinosarcoma affect the female genital tract; however, other sites including head and neck, lung, and breast have been described. Carcinosarcoma of the thyroid is an extremely rare and aggressive malignancy with an ominous clinical course similar to anaplastic carcinoma. Case presentation: We report a case of a 45-year-old female who was found to have a biphasic thyroid carcinosarcoma. Her clinical course declined significantly shortly after she underwent a total thyroidectomy and she developed distant metastases to the lungs. Histopathological features of the primary and metastatic tumor were identical. The tumor is composed of an intimately intermixed epithelial component of poorly differentiated follicular thyroid carcinoma and a spindle cell sarcoma with rhabdomyosarcoma differentiation. Molecular analysis using a next-generation sequencing based assay revealed a DICER1 (E1705K) point mutation in neoplastic cells. Conclusion: To our knowledge, the E1705K point mutation within the DICER1 gene is the first reported mutation in carcinosarcoma of the thyroid. A comprehensive review of the relevant literature is also included for discussion.
Background
Thyroid carcinosarcoma is a rare and aggressive malignant thyroid tumor [1]. These tumors are usually found to infiltrate the surrounding soft tissue at the time of diagnosis. The overall survival rate for these patients is only a few months and most of the cases occur in women who are older than 50 years of age. To date, less than 30 cases of thyroid carcinosarcoma have been reported in the literature (Table 1) [1][2][3][4][5][6][7][8]. According to the latest World Health Organization (WHO 2017) classification for thyroid tumors, carcinosarcoma is considered to be a variant of anaplastic carcinoma [9]. Recently, Agrawal et al. proposed that, with the presence of both malignant epithelial and mesenchymal cells, 'thyroid carcinosarcoma' should be considered a distinct entity from anaplastic carcinoma [6].
The pathogenesis of thyroid carcinosarcoma is not fully understood. Carcinosarcoma of the thyroid has been suggested to originate from both malignant epithelial (carcinoma) and mesenchymal (sarcoma) elements of the thyroid [1,6]. Positive immunohistochemical (IHC) staining for thyroglobulin in carcinomatous cells, and positive staining for vimentin in mesenchymal cells supports a diagnosis of carcinosarcoma [1]. In contrast, studies from anaplastic thyroid carcinoma suggested a monoclonal origin of the tumor [2]. The sarcoma-like morphology of the tumor is thought to be de-differentiated from the epithelial component during the process of carcinogenesis.
In 2013, Agrawal et al. reviewed 25 cases reported in the literature as carcinosarcoma of the thyroid. Since then, there have been two additional cases reported. Among the reported cases, most were described as fibrosarcoma or osteosarcoma with co-existing differentiated thyroid carcinoma, such as follicular or papillary carcinoma [1][2][3][4][5][6][7][8]. In most of the cases, the tumors behaved aggressively, with death from disease occurring within 2 months to 26 months following diagnosis [1][2][3][4][5][6][7][8]. No specific molecular signatures or genetic alteration have been reported. The current report presents a case of a young female patient with a biphasic thyroid carcinosarcoma. Next generation sequencing (NGS) analysis revealed a novel point mutation in the DICER1 gene (E1705K) in neoplastic cells.
Case presentation
A 45-year-old woman presented to our hospital with multiple lung nodules. She had a history of poorly differentiated thyroid carcinoma, diagnosed 7 months prior to admission, at an outside hospital. The patient was healthy otherwise and reported no radiation exposure or any family history of thyroid cancer. The initial work-up at the time of discovery of the right thyroid nodule included fine needle aspiration and core biopsy, with findings consistent with poorly differentiated thyroid carcinoma. The patient then underwent a total thyroidectomy and central neck lymph node dissection. The pathologic diagnosis from the outside hospital reported a 2.8 × 2.4 × 1.1 cm tumor in the right thyroid without extrathyroidal extension or lymph node metastasis. However, both capsular invasion and extensive vascular space invasion were noted. Based on the tumor size, tumor extension and lymph node status, the tumor was designated as Stage II (pT2 pN0 pMx). IHC staining showed that the tumor cells were positive for thyroglobulin and thyroid transcription factor 1 (TTF1). An immunostain for p53 was also performed at the outside hospital and showed a small focus (< 1 cm) with p53 positivity, suggesting a diagnosis of anaplastic thyroid carcinoma.
At our institution, the diagnosis was revised, based on review of both the primary thyroid tumor and the current lung metastases. Both tumors were remarkable for biphasic malignant components: the carcinoma and the sarcoma. The carcinoma component showed a poorly differentiated microfollicular type thyroid carcinoma, composed of sheets and islands of tightly packed thyroid follicles with dense colloid. The tumor nuclei were small and round with vesicular chromatin, resembling those of typical poorly differentiated follicular thyroid carcinoma. Admixed with the epithelial component were malignant spindle cells with small round blue cell type morphology. Focally, rhabdomyosarcoma-like cells with eosinophilic cytoplasm were appreciated. No heterologous cartilage or bone components were identified. The IHC staining performed at the outside hospital showed that the thyroid carcinoma (epithelial) component was positive for thyroglobulin, PAX8 and TTF1 (Fig. 1). The sarcoma (spindled) component was negative for all thyroid carcinoma markers (TTF-1, thyroglobulin and PAX8), but was positive for vimentin and focally positive for myogenin (supporting skeletal muscle differentiation) consistent with mesenchymal differentiation. Interestingly, the foci of vascular space invasion contained both epithelial and mesenchymal components as well.
The patient received Taxol with Carboplatin for 7 weeks followed by radiation therapy. Her thyroglobulin level rose from 1.2 ng/mL to 25.40 ng/mL 5 months after completion of the chemo-radiation therapy, suggesting progression of the disease. A follow-up CT scan of the chest showed multiple newly developed nodules (ranging from 1 to 2 cm) in the right lung, highly suspicious for metastases. The patient underwent a right thoracotomy, right lung resection/metastasectomy. The surgery was uneventful with negative resection margins. However, the patient's general condition deteriorated and she succumbed to the disease 4 months later.
Histological examination of the lung nodules revealed similar tumor morphology and tumor differentiation when compared to the original thyroid tumor, which is somewhat unusual for a biphasic carcinosarcoma (Fig. 2). Tumor necrosis was also present. Mutational analysis using a next-generation sequencing based assay showed that the neoplastic cells from the lung metastasis were devoid of genomic alterations for known thyroid cancers, including BRAF, RAS family (KRAS, NRAS and HRAS), EGFR, PTEN, TERT, PI3Kinase or RET. BRAF or RAS family are known as the most commonly altered genes in papillary thyroid cancers. Other molecular mutations reported in the development of anaplastic thyroid carcinoma include p53, PAX8/PPAR gamma rearrangement [10]. None of the mentioned gene mutations were identified in our patient. However, an interesting finding in this case is the presence of a point mutation in DICER1 (E1705K) that has previously been associated with differentiated thyroid carcinoma [11,12]. Whether the DICER1 (E1705K) mutation is the underlying genetic event leading to the initiation of tumorigenesis or is downstream to other gene alterations in tumor development is largely unknown. Additional mutations of unknown significance were also detected in this tumor including FLCN (R239H), POLD1 (Q684H) and SYK (R217L). These variants have not been adequately characterized in the scientific literature and their prognostic and therapeutic significance is unclear.
Discussion and conclusions
Thyroid carcinosarcoma is a very aggressive malignant tumor with a clinical course similar to that of anaplastic thyroid carcinoma [4]. All reported thyroid carcinosarcoma cases have resulted in patients only surviving a few months after initial diagnosis [1][2][3][4][5][6][7][8]. To our knowledge, there is no previously published molecular analysis of thyroid carcinosarcoma. This is the first report to describe potential gene alterations identified using next generation sequencing. The DICER1 protein is a member of the ribonuclease III (RNase III) family that plays an important role in the post-transcriptional regulation of gene expression. Heterozygous germline DICER mutations are described in the so-called DICER1 syndrome, which leads to a predisposition to develop a variety of tumors in children, including pleuropulmonary blastoma, cystic nephroma, rhabdomyosarcoma, ovarian Sertoli-Leydig tumors and multinodular goiter. A recent case study reported that a germline DICER1 mutation (S1814 L) is associated with an increased risk of thyroid follicular carcinoma [11]. A somatic DICER1 hotspot mutation at E1705K was reported in a case of anaplastic sarcoma of the kidney and in approximately 60% of Sertoli-Leydig cell tumors [12,13]. The presence of this point mutation of DICER1 at E1705K in thyroid carcinosarcoma has never been reported. The presence of a DICER1 mutation in this case provides further evidence that development of the carcinosarcoma may not be a random occurrence, but is a likely due to a specific genetic alteration. Due to the highly aggressive nature of thyroid carcinosarcomas, the identification of specific genetic signatures for these tumors becomes critical in identifying effective treatments. However, there are still many questions regarding the pathogenesis and tumorigenesis of thyroid carcinosarcoma. Further studies will be of great benefit for the diagnosis and treatment.
Other mutations that were identified in this tumor include POLD1 (Q684H), FLCN (R239H) and SYK (R217L). POLD1 (Q684H) is a recently described mutation that had been reported in a patient with colorectal cancer [14]. Germline mutations in the folliculin (FLCN) gene, encoding the folliculin tumor-suppressor protein, are reported to be associated with Birt Hogg Dubé syndrome [15]. Spleen tyrosine kinase (SYK) is an essential enzyme required for signaling involving multiple classes of immune receptors [16]. SYK functions as a modulator of tumorigenesis and has been reported in association with leukemia and breast cancers [16]. The clinical significance of FLCN (R239H) and SYK (R217L) mutations in thyroid carcinosarcoma is unknown.
The differential diagnosis of thyroid carcinosarcoma includes anaplastic carcinoma. P53 point mutations are present in 60-80% of anaplastic thyroid carcinomas. Patients develop anaplastic carcinoma from a pre or coexistent differentiated carcinoma after a multistep process of dedifferentiation associated with loss of the p53 oncogene suppressor. Due to the similarities between anaplastic carcinoma and carcinosarcoma of the thyroid gland, it has been proposed that p53 mutation may contribute to the pathogenesis of carcinosarcoma. In our patient, a less than 1 cm focus within the anaplastic area showed p53 IHC positivity. No P53 mutation was detected by next generation sequencing. Molecular pathogenetic mechanisms of p53 involvement in the transformation of carcinosarcoma are not well understood. Other mutations reported in anaplastic thyroid carcinoma include RAS, BRAF, PTEN, TERT, and PIK3kinase which were not identified in this patient. Thus, the diagnosis of anaplastic carcinoma is not supported by our studies.
The clinical course of thyroid carcinosarcoma is similar to anaplastic carcinoma. Some case reports have recommended following the standard treatment approach for anaplastic carcinoma. Multimodality treatment for anaplastic carcinoma, consisting of radical surgery followed by radiotherapy and chemotherapy is reported to be associated with better clinical outcomes. However, there is no uniform consensus about the treatment approach for thyroid carcinosarcoma due to its very low incidence, its aggressive nature with poor prognosis, and the consequent lack of large clinical series. Most reported cases were treated with total or subtotal thyroidectomy. Adjuvant chemotherapy, radiation therapy and immunotherapy have not proven to be beneficial. However, NGS performed on collected thyroid carcinosarcoma cases could be of great benefit for the identification of targeted treatments.
In conclusion, the present study reports a rare case of primary thyroid carcinosarcoma with metastasis to the lung in a 45-year-old female patient who ultimately succumbed to the disease after receiving surgeries for primary and metastatic tumors and adjuvant chemoradiation therapy. Her total survival time was 11 months from the time of diagnosis and her total disease free survival was 7 months. A DICER1 (E1705K) gene mutation was identified in this patient. Primary thyroid carcinosarcoma is extremely rare and can be diagnostically challenging.
Immunohistochemical staining may be useful for establishing a diagnosis and for distinguishing the disease from anaplastic carcinoma. Although the overall survival is dismal despite aggressive treatment, careful evaluation and the use of NGS to detect specific gene alterations may lead to the development of effective targeted therapies.
Abbreviations BRAF: B-Raf Proto-Oncogene; DICER1: Ribonuclease III; EGFR: Epidermal growth factor receptor; IHC: Immunohistochemical; NGS: Next generation sequencing; PAX8: Paired box gene 8; PIK3kinase: Phosphatidylinositol-4, 5bisphosphate 3-kinase; RET: Proto-oncogene encodes a receptor tyrosine kinase; TTF1: Thyroid transcription factor 1; WHO: World health organization
Fig. 1 Fig. 2
12Immunohistochemical stains demonstrated biphasic components. The carcinoma component (a) showed positivity for thyroglobulin (b). The sarcoma component (c) showed positivity for myogenin (d). All pictures are at 200× Histological features of primary thyroid cancer and metastatic lung nodules. The epithelial component in the thyroid cancer (a) is morphologically similar to the epithelial component in the lung nodule (d). The sarcomatous component is composed of spindle cells both in the thyroid cancer (b) and in the lung nodule (e). There is vascular invasion in the thyroid cancer (c). Low power review of a small lung nodule (40×) in (f)
Table 1
1Summary of all reported cases of thyroid carcinosarcoma in English literatureAuthor; year; Ref [#]
Morphology
Metastasis
Treatment
Survival (months)
Rasheed; 2017; [8]
Follicular carcinoma with sarcoma
none
total thyroidectomy, chemotherapy
3
Ekici; 2015; [7]
Papillary carcinoma with sarcoma
none
total thyroidectomy
2
Agrawal; 2013; [6]
Papillary carcinoma with sarcoma
lymph nodes
total thyroidectomy, right neck dissection
12
Naqiyah; 2010; [5]
Follicular carcinoma with sarcoma
none
total thyroidectomy, radiation
8
Guiffrida; 2000; [1]
Follicular carcinoma with sarcoma
bilateral lung,
lymph nodes
total thyroidectomy lymph node dissection
and chemoradiation therapy
6
Al-Sobhi; 1997; [4]
Follicular carcinoma with sarcoma
lung
total thyroidectomy, radiation
8
Cooper; 1989; [3]
Follicular carcinoma with
chondrosarcoma
N/A
subtotal thyroidectomy
5
Donnell; 1987; [2]
Follicular carcinoma with
osteosarcoma and chondrosarcoma
lung
subtotal thyroidectomy
26
AcknowledgementsNot applicable.FundingNot applicable.Availability of data and materialsData sharing is not applicable to this article.Author's contributions All authors were directly involved in the care of this patient. JY and QZ contributed to the acquisition of data, writing and revision of the manuscript; all authors have read and approved the final version of the manuscript for publication.Ethics approval and consent to participateThis case report was exempt from the Institutional Review Board standards at Boston University.Consent for publicationConsent was not obtained from this deceased patient but the presented data are anonymized and risk of identification is minimal.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Received: 17 May 2018 Accepted: 16 July 2018
Thyroid carcinosarcoma, a rare and aggressive histotype: a case report. D Giuffrida, M Attard, L Marasa, F Ferrau, F Marletta, Ann Oncol. 11Giuffrida D, Attard M, Marasa L, Ferrau F, Marletta F, et al. Thyroid carcinosarcoma, a rare and aggressive histotype: a case report. Ann Oncol. 2000;11:1497-9.
Thyroid carcinosarcoma. C Donnell, W Pollock, W Sybers, Arch Pathol Lab Med. 111Donnell C, Pollock W, Sybers W. Thyroid carcinosarcoma. Arch Pathol Lab Med. 1987;111:1169-72.
Thyroid carcinosarcoma. A case report. K Cooper, E Barker, S. Afr J Surg. 27Cooper K, Barker E. Thyroid carcinosarcoma. A case report. S. Afr J Surg. 1989;27:192-3.
Management of thyroid carcinosarcoma. S Al-Sobhi, F Novosolov, U Sabançi, Surgery. 122Al-Sobhi S, Novosolov F, Sabançi U, et al. Management of thyroid carcinosarcoma. Surgery. 1997;122:548-52.
Carcinosarcoma of the thyroid: a case report. I Naqiyah, A N Zulkarnaen, M Rohaizak, S Das, Hippokratia. 14Naqiyah I, Zulkarnaen AN, Rohaizak M, Das S. Carcinosarcoma of the thyroid: a case report. Hippokratia. 2010;14:141-2.
Carcinosarcoma thyroid: an unusual morphology with a review of the literature. M Agrawal, S G Uppin, S Challa, A K Prayaga, South Asian J Cancer. 2226Agrawal M, Uppin SG, Challa S, Prayaga AK. Carcinosarcoma thyroid: an unusual morphology with a review of the literature. South Asian J Cancer. 2013;2:226.
Carcinosarcoma of the thyroid gland. Case Rep Surg. M Ekici, C Kocak, Z Bayhan, 10.1155/2015/494383Ekici M, Kocak C, Bayhan Z, et al. Carcinosarcoma of the thyroid gland. Case Rep Surg; 2015. https://doi.org/10.1155/2015/494383.
Sporadic carcinosarcoma of thyroid gland resistant to chemotherapy: a case report. R Rasheed, N Saeed, A Naqvi, S Rasheed, J Endocrinol. Thyroid Res. 1Rasheed R, Saeed N, Naqvi A, Rasheed S, et al. Sporadic carcinosarcoma of thyroid gland resistant to chemotherapy: a case report. J Endocrinol. Thyroid Res. 2017;1:1-3.
World Health Organization classification of tumors, pathology and genetics of tumors of endocrine organs. A El-Naggar, Z Baloch, C Eng, IARC PressLyon, FranceAnaplastic thyroid carcinomaEl-Naggar A, Baloch Z, Eng C, et al. Anaplastic thyroid carcinoma. In: Lloyd RV, Osamura RY, Kloppel G, Rosai J, editors. World Health Organization classification of tumors, pathology and genetics of tumors of endocrine organs. Lyon, France: IARC Press; 2017. p. 104-6.
Molecular pathogenesis and mechanisms of thyroid cancer. M Xing, Nat Rev Cancer. 13Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 2013;13:184-99.
DICER1 mutations and differentiated thyroid carcinoma: evidence of a direct association. M Rutter, P Jha, K Schultz, J Clin Endocrinol Metab. 101Rutter M, Jha P, Schultz K, et al. DICER1 mutations and differentiated thyroid carcinoma: evidence of a direct association. J Clin Endocrinol Metab. 2016; 101:1-5.
Metachronous anaplastic sarcoma of the kidney and thyroid follicular carcinoma as manifestations of DICER1 abnormalities. M Yoshida, S Hamanoue, M Seki, Hum Pathol. 61Yoshida M, Hamanoue S, Seki M, et al. Metachronous anaplastic sarcoma of the kidney and thyroid follicular carcinoma as manifestations of DICER1 abnormalities. Hum Pathol. 2017;61:205-9.
A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors. N Conlon, A Schultheis, S Piscuoglio, Mod Pathol. 28Conlon N, Schultheis A, Piscuoglio S, et al. A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors. Mod Pathol. 2015;28:1603-12.
Targeted sequencing of established and candidate colorectal cancer genes in the colon cancer family registry cohort. L Raskin, Y Guo, L Du, Oncotarget. 8Raskin L, Guo Y, Du L, et al. Targeted sequencing of established and candidate colorectal cancer genes in the colon cancer family registry cohort. Oncotarget. 2017;8:93450-63.
FLCN: the causative gene for Birt-Hogg-Dubé syndrome. L Schmidt, W Linehan, Gene. 640Schmidt L, Linehan W. FLCN: the causative gene for Birt-Hogg-Dubé syndrome. Gene. 2018;640:28-42.
Calling in SYK: SYK's dual role as a tumor promoter and tumor suppressor in cancer. M Krisenko, R Geahlen, Biochim Biophys Acta. Krisenko M, Geahlen R. Calling in SYK: SYK's dual role as a tumor promoter and tumor suppressor in cancer. Biochim Biophys Acta. 1853;2015:254-63.
| [
"Background: Malignant biphasic tumor also known as carcinosarcoma is an uncommon neoplasm that is composed of both malignant epithelial and mesenchymal components. Most reported cases of carcinosarcoma affect the female genital tract; however, other sites including head and neck, lung, and breast have been described. Carcinosarcoma of the thyroid is an extremely rare and aggressive malignancy with an ominous clinical course similar to anaplastic carcinoma. Case presentation: We report a case of a 45-year-old female who was found to have a biphasic thyroid carcinosarcoma. Her clinical course declined significantly shortly after she underwent a total thyroidectomy and she developed distant metastases to the lungs. Histopathological features of the primary and metastatic tumor were identical. The tumor is composed of an intimately intermixed epithelial component of poorly differentiated follicular thyroid carcinoma and a spindle cell sarcoma with rhabdomyosarcoma differentiation. Molecular analysis using a next-generation sequencing based assay revealed a DICER1 (E1705K) point mutation in neoplastic cells. Conclusion: To our knowledge, the E1705K point mutation within the DICER1 gene is the first reported mutation in carcinosarcoma of the thyroid. A comprehensive review of the relevant literature is also included for discussion."
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"Thyroid carcinosarcoma, a rare and aggressive histotype: a case report. D Giuffrida, M Attard, L Marasa, F Ferrau, F Marletta, Ann Oncol. 11Giuffrida D, Attard M, Marasa L, Ferrau F, Marletta F, et al. Thyroid carcinosarcoma, a rare and aggressive histotype: a case report. Ann Oncol. 2000;11:1497-9.",
"Thyroid carcinosarcoma. C Donnell, W Pollock, W Sybers, Arch Pathol Lab Med. 111Donnell C, Pollock W, Sybers W. Thyroid carcinosarcoma. Arch Pathol Lab Med. 1987;111:1169-72.",
"Thyroid carcinosarcoma. A case report. K Cooper, E Barker, S. Afr J Surg. 27Cooper K, Barker E. Thyroid carcinosarcoma. A case report. S. Afr J Surg. 1989;27:192-3.",
"Management of thyroid carcinosarcoma. S Al-Sobhi, F Novosolov, U Sabançi, Surgery. 122Al-Sobhi S, Novosolov F, Sabançi U, et al. Management of thyroid carcinosarcoma. Surgery. 1997;122:548-52.",
"Carcinosarcoma of the thyroid: a case report. I Naqiyah, A N Zulkarnaen, M Rohaizak, S Das, Hippokratia. 14Naqiyah I, Zulkarnaen AN, Rohaizak M, Das S. Carcinosarcoma of the thyroid: a case report. Hippokratia. 2010;14:141-2.",
"Carcinosarcoma thyroid: an unusual morphology with a review of the literature. M Agrawal, S G Uppin, S Challa, A K Prayaga, South Asian J Cancer. 2226Agrawal M, Uppin SG, Challa S, Prayaga AK. Carcinosarcoma thyroid: an unusual morphology with a review of the literature. South Asian J Cancer. 2013;2:226.",
"Carcinosarcoma of the thyroid gland. Case Rep Surg. M Ekici, C Kocak, Z Bayhan, 10.1155/2015/494383Ekici M, Kocak C, Bayhan Z, et al. Carcinosarcoma of the thyroid gland. Case Rep Surg; 2015. https://doi.org/10.1155/2015/494383.",
"Sporadic carcinosarcoma of thyroid gland resistant to chemotherapy: a case report. R Rasheed, N Saeed, A Naqvi, S Rasheed, J Endocrinol. Thyroid Res. 1Rasheed R, Saeed N, Naqvi A, Rasheed S, et al. Sporadic carcinosarcoma of thyroid gland resistant to chemotherapy: a case report. J Endocrinol. Thyroid Res. 2017;1:1-3.",
"World Health Organization classification of tumors, pathology and genetics of tumors of endocrine organs. A El-Naggar, Z Baloch, C Eng, IARC PressLyon, FranceAnaplastic thyroid carcinomaEl-Naggar A, Baloch Z, Eng C, et al. Anaplastic thyroid carcinoma. In: Lloyd RV, Osamura RY, Kloppel G, Rosai J, editors. World Health Organization classification of tumors, pathology and genetics of tumors of endocrine organs. Lyon, France: IARC Press; 2017. p. 104-6.",
"Molecular pathogenesis and mechanisms of thyroid cancer. M Xing, Nat Rev Cancer. 13Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 2013;13:184-99.",
"DICER1 mutations and differentiated thyroid carcinoma: evidence of a direct association. M Rutter, P Jha, K Schultz, J Clin Endocrinol Metab. 101Rutter M, Jha P, Schultz K, et al. DICER1 mutations and differentiated thyroid carcinoma: evidence of a direct association. J Clin Endocrinol Metab. 2016; 101:1-5.",
"Metachronous anaplastic sarcoma of the kidney and thyroid follicular carcinoma as manifestations of DICER1 abnormalities. M Yoshida, S Hamanoue, M Seki, Hum Pathol. 61Yoshida M, Hamanoue S, Seki M, et al. Metachronous anaplastic sarcoma of the kidney and thyroid follicular carcinoma as manifestations of DICER1 abnormalities. Hum Pathol. 2017;61:205-9.",
"A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors. N Conlon, A Schultheis, S Piscuoglio, Mod Pathol. 28Conlon N, Schultheis A, Piscuoglio S, et al. A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors. Mod Pathol. 2015;28:1603-12.",
"Targeted sequencing of established and candidate colorectal cancer genes in the colon cancer family registry cohort. L Raskin, Y Guo, L Du, Oncotarget. 8Raskin L, Guo Y, Du L, et al. Targeted sequencing of established and candidate colorectal cancer genes in the colon cancer family registry cohort. Oncotarget. 2017;8:93450-63.",
"FLCN: the causative gene for Birt-Hogg-Dubé syndrome. L Schmidt, W Linehan, Gene. 640Schmidt L, Linehan W. FLCN: the causative gene for Birt-Hogg-Dubé syndrome. Gene. 2018;640:28-42.",
"Calling in SYK: SYK's dual role as a tumor promoter and tumor suppressor in cancer. M Krisenko, R Geahlen, Biochim Biophys Acta. Krisenko M, Geahlen R. Calling in SYK: SYK's dual role as a tumor promoter and tumor suppressor in cancer. Biochim Biophys Acta. 1853;2015:254-63."
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"Thyroid carcinosarcoma, a rare and aggressive histotype: a case report",
"Thyroid carcinosarcoma",
"Thyroid carcinosarcoma. A case report",
"Management of thyroid carcinosarcoma",
"Carcinosarcoma of the thyroid: a case report",
"Carcinosarcoma thyroid: an unusual morphology with a review of the literature",
"Sporadic carcinosarcoma of thyroid gland resistant to chemotherapy: a case report",
"Molecular pathogenesis and mechanisms of thyroid cancer",
"DICER1 mutations and differentiated thyroid carcinoma: evidence of a direct association",
"Metachronous anaplastic sarcoma of the kidney and thyroid follicular carcinoma as manifestations of DICER1 abnormalities",
"A survey of DICER1 hotspot mutations in ovarian and testicular sex cord-stromal tumors",
"Targeted sequencing of established and candidate colorectal cancer genes in the colon cancer family registry cohort",
"FLCN: the causative gene for Birt-Hogg-Dubé syndrome",
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] | [
"\nFig. 1 Fig. 2\n12Immunohistochemical stains demonstrated biphasic components. The carcinoma component (a) showed positivity for thyroglobulin (b). The sarcoma component (c) showed positivity for myogenin (d). All pictures are at 200× Histological features of primary thyroid cancer and metastatic lung nodules. The epithelial component in the thyroid cancer (a) is morphologically similar to the epithelial component in the lung nodule (d). The sarcomatous component is composed of spindle cells both in the thyroid cancer (b) and in the lung nodule (e). There is vascular invasion in the thyroid cancer (c). Low power review of a small lung nodule (40×) in (f)",
"\nTable 1\n1Summary of all reported cases of thyroid carcinosarcoma in English literatureAuthor; year; Ref [#] \nMorphology \nMetastasis \nTreatment \nSurvival (months) \n\nRasheed; 2017; [8] \nFollicular carcinoma with sarcoma \nnone \ntotal thyroidectomy, chemotherapy \n3 \n\nEkici; 2015; [7] \nPapillary carcinoma with sarcoma \nnone \ntotal thyroidectomy \n2 \n\nAgrawal; 2013; [6] \nPapillary carcinoma with sarcoma \nlymph nodes \ntotal thyroidectomy, right neck dissection \n12 \n\nNaqiyah; 2010; [5] \nFollicular carcinoma with sarcoma \nnone \ntotal thyroidectomy, radiation \n8 \n\nGuiffrida; 2000; [1] \nFollicular carcinoma with sarcoma \nbilateral lung, \nlymph nodes \n\ntotal thyroidectomy lymph node dissection \nand chemoradiation therapy \n\n6 \n\nAl-Sobhi; 1997; [4] \nFollicular carcinoma with sarcoma \nlung \ntotal thyroidectomy, radiation \n8 \n\nCooper; 1989; [3] \nFollicular carcinoma with \nchondrosarcoma \n\nN/A \nsubtotal thyroidectomy \n5 \n\nDonnell; 1987; [2] \nFollicular carcinoma with \nosteosarcoma and chondrosarcoma \n\nlung \nsubtotal thyroidectomy \n26 \n"
] | [
"Immunohistochemical stains demonstrated biphasic components. The carcinoma component (a) showed positivity for thyroglobulin (b). The sarcoma component (c) showed positivity for myogenin (d). All pictures are at 200× Histological features of primary thyroid cancer and metastatic lung nodules. The epithelial component in the thyroid cancer (a) is morphologically similar to the epithelial component in the lung nodule (d). The sarcomatous component is composed of spindle cells both in the thyroid cancer (b) and in the lung nodule (e). There is vascular invasion in the thyroid cancer (c). Low power review of a small lung nodule (40×) in (f)",
"Summary of all reported cases of thyroid carcinosarcoma in English literature"
] | [
"(Fig. 1",
"(Fig. 2)"
] | [] | [
"Thyroid carcinosarcoma is a rare and aggressive malignant thyroid tumor [1]. These tumors are usually found to infiltrate the surrounding soft tissue at the time of diagnosis. The overall survival rate for these patients is only a few months and most of the cases occur in women who are older than 50 years of age. To date, less than 30 cases of thyroid carcinosarcoma have been reported in the literature (Table 1) [1][2][3][4][5][6][7][8]. According to the latest World Health Organization (WHO 2017) classification for thyroid tumors, carcinosarcoma is considered to be a variant of anaplastic carcinoma [9]. Recently, Agrawal et al. proposed that, with the presence of both malignant epithelial and mesenchymal cells, 'thyroid carcinosarcoma' should be considered a distinct entity from anaplastic carcinoma [6].",
"The pathogenesis of thyroid carcinosarcoma is not fully understood. Carcinosarcoma of the thyroid has been suggested to originate from both malignant epithelial (carcinoma) and mesenchymal (sarcoma) elements of the thyroid [1,6]. Positive immunohistochemical (IHC) staining for thyroglobulin in carcinomatous cells, and positive staining for vimentin in mesenchymal cells supports a diagnosis of carcinosarcoma [1]. In contrast, studies from anaplastic thyroid carcinoma suggested a monoclonal origin of the tumor [2]. The sarcoma-like morphology of the tumor is thought to be de-differentiated from the epithelial component during the process of carcinogenesis.",
"In 2013, Agrawal et al. reviewed 25 cases reported in the literature as carcinosarcoma of the thyroid. Since then, there have been two additional cases reported. Among the reported cases, most were described as fibrosarcoma or osteosarcoma with co-existing differentiated thyroid carcinoma, such as follicular or papillary carcinoma [1][2][3][4][5][6][7][8]. In most of the cases, the tumors behaved aggressively, with death from disease occurring within 2 months to 26 months following diagnosis [1][2][3][4][5][6][7][8]. No specific molecular signatures or genetic alteration have been reported. The current report presents a case of a young female patient with a biphasic thyroid carcinosarcoma. Next generation sequencing (NGS) analysis revealed a novel point mutation in the DICER1 gene (E1705K) in neoplastic cells.",
"A 45-year-old woman presented to our hospital with multiple lung nodules. She had a history of poorly differentiated thyroid carcinoma, diagnosed 7 months prior to admission, at an outside hospital. The patient was healthy otherwise and reported no radiation exposure or any family history of thyroid cancer. The initial work-up at the time of discovery of the right thyroid nodule included fine needle aspiration and core biopsy, with findings consistent with poorly differentiated thyroid carcinoma. The patient then underwent a total thyroidectomy and central neck lymph node dissection. The pathologic diagnosis from the outside hospital reported a 2.8 × 2.4 × 1.1 cm tumor in the right thyroid without extrathyroidal extension or lymph node metastasis. However, both capsular invasion and extensive vascular space invasion were noted. Based on the tumor size, tumor extension and lymph node status, the tumor was designated as Stage II (pT2 pN0 pMx). IHC staining showed that the tumor cells were positive for thyroglobulin and thyroid transcription factor 1 (TTF1). An immunostain for p53 was also performed at the outside hospital and showed a small focus (< 1 cm) with p53 positivity, suggesting a diagnosis of anaplastic thyroid carcinoma.",
"At our institution, the diagnosis was revised, based on review of both the primary thyroid tumor and the current lung metastases. Both tumors were remarkable for biphasic malignant components: the carcinoma and the sarcoma. The carcinoma component showed a poorly differentiated microfollicular type thyroid carcinoma, composed of sheets and islands of tightly packed thyroid follicles with dense colloid. The tumor nuclei were small and round with vesicular chromatin, resembling those of typical poorly differentiated follicular thyroid carcinoma. Admixed with the epithelial component were malignant spindle cells with small round blue cell type morphology. Focally, rhabdomyosarcoma-like cells with eosinophilic cytoplasm were appreciated. No heterologous cartilage or bone components were identified. The IHC staining performed at the outside hospital showed that the thyroid carcinoma (epithelial) component was positive for thyroglobulin, PAX8 and TTF1 (Fig. 1). The sarcoma (spindled) component was negative for all thyroid carcinoma markers (TTF-1, thyroglobulin and PAX8), but was positive for vimentin and focally positive for myogenin (supporting skeletal muscle differentiation) consistent with mesenchymal differentiation. Interestingly, the foci of vascular space invasion contained both epithelial and mesenchymal components as well.",
"The patient received Taxol with Carboplatin for 7 weeks followed by radiation therapy. Her thyroglobulin level rose from 1.2 ng/mL to 25.40 ng/mL 5 months after completion of the chemo-radiation therapy, suggesting progression of the disease. A follow-up CT scan of the chest showed multiple newly developed nodules (ranging from 1 to 2 cm) in the right lung, highly suspicious for metastases. The patient underwent a right thoracotomy, right lung resection/metastasectomy. The surgery was uneventful with negative resection margins. However, the patient's general condition deteriorated and she succumbed to the disease 4 months later.",
"Histological examination of the lung nodules revealed similar tumor morphology and tumor differentiation when compared to the original thyroid tumor, which is somewhat unusual for a biphasic carcinosarcoma (Fig. 2). Tumor necrosis was also present. Mutational analysis using a next-generation sequencing based assay showed that the neoplastic cells from the lung metastasis were devoid of genomic alterations for known thyroid cancers, including BRAF, RAS family (KRAS, NRAS and HRAS), EGFR, PTEN, TERT, PI3Kinase or RET. BRAF or RAS family are known as the most commonly altered genes in papillary thyroid cancers. Other molecular mutations reported in the development of anaplastic thyroid carcinoma include p53, PAX8/PPAR gamma rearrangement [10]. None of the mentioned gene mutations were identified in our patient. However, an interesting finding in this case is the presence of a point mutation in DICER1 (E1705K) that has previously been associated with differentiated thyroid carcinoma [11,12]. Whether the DICER1 (E1705K) mutation is the underlying genetic event leading to the initiation of tumorigenesis or is downstream to other gene alterations in tumor development is largely unknown. Additional mutations of unknown significance were also detected in this tumor including FLCN (R239H), POLD1 (Q684H) and SYK (R217L). These variants have not been adequately characterized in the scientific literature and their prognostic and therapeutic significance is unclear.",
"Thyroid carcinosarcoma is a very aggressive malignant tumor with a clinical course similar to that of anaplastic thyroid carcinoma [4]. All reported thyroid carcinosarcoma cases have resulted in patients only surviving a few months after initial diagnosis [1][2][3][4][5][6][7][8]. To our knowledge, there is no previously published molecular analysis of thyroid carcinosarcoma. This is the first report to describe potential gene alterations identified using next generation sequencing. The DICER1 protein is a member of the ribonuclease III (RNase III) family that plays an important role in the post-transcriptional regulation of gene expression. Heterozygous germline DICER mutations are described in the so-called DICER1 syndrome, which leads to a predisposition to develop a variety of tumors in children, including pleuropulmonary blastoma, cystic nephroma, rhabdomyosarcoma, ovarian Sertoli-Leydig tumors and multinodular goiter. A recent case study reported that a germline DICER1 mutation (S1814 L) is associated with an increased risk of thyroid follicular carcinoma [11]. A somatic DICER1 hotspot mutation at E1705K was reported in a case of anaplastic sarcoma of the kidney and in approximately 60% of Sertoli-Leydig cell tumors [12,13]. The presence of this point mutation of DICER1 at E1705K in thyroid carcinosarcoma has never been reported. The presence of a DICER1 mutation in this case provides further evidence that development of the carcinosarcoma may not be a random occurrence, but is a likely due to a specific genetic alteration. Due to the highly aggressive nature of thyroid carcinosarcomas, the identification of specific genetic signatures for these tumors becomes critical in identifying effective treatments. However, there are still many questions regarding the pathogenesis and tumorigenesis of thyroid carcinosarcoma. Further studies will be of great benefit for the diagnosis and treatment.",
"Other mutations that were identified in this tumor include POLD1 (Q684H), FLCN (R239H) and SYK (R217L). POLD1 (Q684H) is a recently described mutation that had been reported in a patient with colorectal cancer [14]. Germline mutations in the folliculin (FLCN) gene, encoding the folliculin tumor-suppressor protein, are reported to be associated with Birt Hogg Dubé syndrome [15]. Spleen tyrosine kinase (SYK) is an essential enzyme required for signaling involving multiple classes of immune receptors [16]. SYK functions as a modulator of tumorigenesis and has been reported in association with leukemia and breast cancers [16]. The clinical significance of FLCN (R239H) and SYK (R217L) mutations in thyroid carcinosarcoma is unknown.",
"The differential diagnosis of thyroid carcinosarcoma includes anaplastic carcinoma. P53 point mutations are present in 60-80% of anaplastic thyroid carcinomas. Patients develop anaplastic carcinoma from a pre or coexistent differentiated carcinoma after a multistep process of dedifferentiation associated with loss of the p53 oncogene suppressor. Due to the similarities between anaplastic carcinoma and carcinosarcoma of the thyroid gland, it has been proposed that p53 mutation may contribute to the pathogenesis of carcinosarcoma. In our patient, a less than 1 cm focus within the anaplastic area showed p53 IHC positivity. No P53 mutation was detected by next generation sequencing. Molecular pathogenetic mechanisms of p53 involvement in the transformation of carcinosarcoma are not well understood. Other mutations reported in anaplastic thyroid carcinoma include RAS, BRAF, PTEN, TERT, and PIK3kinase which were not identified in this patient. Thus, the diagnosis of anaplastic carcinoma is not supported by our studies.",
"The clinical course of thyroid carcinosarcoma is similar to anaplastic carcinoma. Some case reports have recommended following the standard treatment approach for anaplastic carcinoma. Multimodality treatment for anaplastic carcinoma, consisting of radical surgery followed by radiotherapy and chemotherapy is reported to be associated with better clinical outcomes. However, there is no uniform consensus about the treatment approach for thyroid carcinosarcoma due to its very low incidence, its aggressive nature with poor prognosis, and the consequent lack of large clinical series. Most reported cases were treated with total or subtotal thyroidectomy. Adjuvant chemotherapy, radiation therapy and immunotherapy have not proven to be beneficial. However, NGS performed on collected thyroid carcinosarcoma cases could be of great benefit for the identification of targeted treatments.",
"In conclusion, the present study reports a rare case of primary thyroid carcinosarcoma with metastasis to the lung in a 45-year-old female patient who ultimately succumbed to the disease after receiving surgeries for primary and metastatic tumors and adjuvant chemoradiation therapy. Her total survival time was 11 months from the time of diagnosis and her total disease free survival was 7 months. A DICER1 (E1705K) gene mutation was identified in this patient. Primary thyroid carcinosarcoma is extremely rare and can be diagnostically challenging.",
"Immunohistochemical staining may be useful for establishing a diagnosis and for distinguishing the disease from anaplastic carcinoma. Although the overall survival is dismal despite aggressive treatment, careful evaluation and the use of NGS to detect specific gene alterations may lead to the development of effective targeted therapies.",
"Abbreviations BRAF: B-Raf Proto-Oncogene; DICER1: Ribonuclease III; EGFR: Epidermal growth factor receptor; IHC: Immunohistochemical; NGS: Next generation sequencing; PAX8: Paired box gene 8; PIK3kinase: Phosphatidylinositol-4, 5bisphosphate 3-kinase; RET: Proto-oncogene encodes a receptor tyrosine kinase; TTF1: Thyroid transcription factor 1; WHO: World health organization"
] | [] | [
"Background",
"Case presentation",
"Discussion and conclusions",
"Fig. 1 Fig. 2",
"Table 1"
] | [
"Author; year; Ref [#] \nMorphology \nMetastasis \nTreatment \nSurvival (months) \n\nRasheed; 2017; [8] \nFollicular carcinoma with sarcoma \nnone \ntotal thyroidectomy, chemotherapy \n3 \n\nEkici; 2015; [7] \nPapillary carcinoma with sarcoma \nnone \ntotal thyroidectomy \n2 \n\nAgrawal; 2013; [6] \nPapillary carcinoma with sarcoma \nlymph nodes \ntotal thyroidectomy, right neck dissection \n12 \n\nNaqiyah; 2010; [5] \nFollicular carcinoma with sarcoma \nnone \ntotal thyroidectomy, radiation \n8 \n\nGuiffrida; 2000; [1] \nFollicular carcinoma with sarcoma \nbilateral lung, \nlymph nodes \n\ntotal thyroidectomy lymph node dissection \nand chemoradiation therapy \n\n6 \n\nAl-Sobhi; 1997; [4] \nFollicular carcinoma with sarcoma \nlung \ntotal thyroidectomy, radiation \n8 \n\nCooper; 1989; [3] \nFollicular carcinoma with \nchondrosarcoma \n\nN/A \nsubtotal thyroidectomy \n5 \n\nDonnell; 1987; [2] \nFollicular carcinoma with \nosteosarcoma and chondrosarcoma \n\nlung \nsubtotal thyroidectomy \n26 \n"
] | [
"(Table 1"
] | [
"A rare malignant thyroid carcinosarcoma with aggressive behavior and DICER1 gene mutation: a case report with literature review",
"A rare malignant thyroid carcinosarcoma with aggressive behavior and DICER1 gene mutation: a case report with literature review"
] | [] |
220,908,706 | 2022-01-23T01:46:18Z | CCBY | https://doi.org/10.1016/j.exer.2020.108150 | HYBRID | 684fd4552d6a8c2e0246580f57092e0d2a87ad9a | null | null | null | null | 10.1016/j.exer.2020.108150 | 3045745587 | 32735797 | null |
A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts
2020
Pål A Olsvik
Faculty of Biosciences and Aquaculture
Nord University
BodøNorway
Institute of Marine Research
Nordnes, BergenNorway
Roderick Nigel Finn
Department of Biological Sciences
University of Bergen
BergenNorway
IRTA-Institute of Biotechnology and Biomedicine (IBB)
Universitat Autònoma de Barcelona
08193Cerdanyola del VallèsBellaterraSpain
Sofie C Remø
Institute of Marine Research
Nordnes, BergenNorway
PerG Fjelldal
Institute of Marine Research
Nordnes, BergenNorway
François Chauvigné
IRTA-Institute of Biotechnology and Biomedicine (IBB)
Universitat Autònoma de Barcelona
08193Cerdanyola del VallèsBellaterraSpain
Kevin A Glover
Institute of Marine Research
Nordnes, BergenNorway
Department of Biological Sciences
University of Bergen
BergenNorway
Tom Hansen
Institute of Marine Research
Nordnes, BergenNorway
Rune Waagbø
Institute of Marine Research
Nordnes, BergenNorway
Department of Biological Sciences
University of Bergen
BergenNorway
A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts
Experimental Eye Research
199108150202010.1016/j.exer.2020.1081500014-4835/ A R T I C L E I N F OTriploid Atlantic salmon Cataracts Transcriptional responses RNA-seq
A B S T R A C TTo avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.
Introduction
Recent years have seen renewed efforts to establish commercial farming of triploid Atlantic salmon (Salmo salar) -hereafter referred to as salmon (Benfey, 2016;Stien et al., 2019). Farming triploid salmon has two major advantages. The first is that triploid females do not mature sexually and can diverge energy into somatic growth (Piferrer et al., 2009). The second is related to the fact that domesticated salmon escapees and genetic interactions with wild conspecifics represents one of the most significant environmental challenges to salmon aquaculture . Rearing sterile triploid fish reduced this threat, and is an effective way to mitigate further genetic interactions.
Although production of triploid salmon has potential benefits, the global Atlantic salmon aquaculture industry is still primarily based upon rearing diploid fish. While there are several reasons for this, in part, this is due to the fact that triploid salmon often show poor performance. For example, in comparison with diploid salmon, they display differences in physiology, behavior and morphology, with increased prevalence of vertebral deformity and ocular cataracts as two of the most severe disorders (Wall and Richards, 1992;Piferrer et al., 2009;Taranger et al., 2010;Taylor et al., 2015;Sambraus et al., 2017). Cataracts are defined as the loss of transparency of the lens and can appear both as reversible osmotic cataracts and permanent cataracts, which can have multiple causes (Hejtmancik, 2008). In farmed salmon, cataract formation has been linked to genetic predispositions and several nutritional and environmental factors (reviewed by Bjerkås et al., 2006). Cataract has been observed in both freshwater and seawater, however, farmed salmon are particularly prone to cataract development during the smolt transition from fresh to saltwater (Waagbø et al., 1998;Breck and Sveier, 2001;Breck et al., 2005a;Remø et al., 2014) and during periods of rapid growth Breck and Sveier, 2001;Waagbø et al., 1996Waagbø et al., , 2010Remø et al., 2014Remø et al., , 2017. Increased prevalence of cataracts in triploid fish is not well understood but may partly rely on altered metabolism due to differences in cellular morphology (Benfey, 1999).
A sub-optimal level of dietary histidine is currently considered the most important causative factor for cataract development in farmed Atlantic salmon (Breck et al., 2003(Breck et al., , 2005bRemø et al., 2014Remø et al., , 2017Waagbø et al., 2010). Taylor et al. (2015) investigated the preventive effects of dietary histidine supplementation in triploid Atlantic salmon during seawater grow-out. Although the severity was higher in triploids compared to diploids irrespective of diet, applying a high histidine diet mitigated further cataract development in triploids. Similarly, dietary histidine supplementation reduced the severity of cataracts in diploid and triploid yearling smolt, but also with a higher severity in triploids compared to diploids at the highest dietary level (Sambraus et al., 2017). The cataract preventative effect of dietary histidine has been attributed to the functional roles of histidine and the derivative N-acetyl-histidine (NAH) as buffer component (Breck et al., 2005a), osmolyte (Rhodes et al., 2010) and possibly antioxidant (Remø et al., 2014), therefore being important to maintain cell integrity and water balance. The lens concentration of NAH was lower in triploids compared to diploids given the same high histidine diet (Sambraus et al., 2017), suggesting that the triploid lens may be more vulnerable to cataract development, possibly due to lower protection of the triploid lens through lower ability to synthesize NAH, or a higher requirement to maintain water balance in the lens. The latter might be linked to larger cell size in triploids (Wu et al., 2010). Thus, differences in susceptibility to cataracts, as well as the apparent higher requirement of histidine to mitigate (but not eliminate) cataract development in triploids, may be hypothesized to be due to alterations or weakness in the lens of triploids.
Thus far, no attempts have been conducted to evaluate the mechanisms behind increased prevalence of cataracts in triploid fish at the molecular level. Relatively few genome-wide examinations of the molecular mechanisms behind cataract formation have been performed on healthy and cataractous lenses in vertebrates (Sousounis and Tsonis, 2012), possibly due to the biased lens transcriptome, where the expression of structural genes, such as crystallins, predominates over genes that regulate cell function and phenotype (Wistow, 2006;Manthey et al., 2014). Global transcriptional examinations of the mammalian cataractous lens have revealed differential regulation on numerous types of genes, including crystallins and heat shock proteins, cytochrome oxidases, growth factors, metalloproteinases and collagen, as well as various transcription factors (Wistow et al., 2002;Wride et al., 2003;Hawse et al., 2003;Mansergh et al., 2004;Medvedovic et al., 2006;Hejtmancik, 2008;Shiels et al., 2010;Hejtmancik, 2017, 2019). In Atlantic salmon, Tröße et al. (2009) used a 16K salmonid microarray to screen for transcriptional responses to histidine related cataracts in lenses of Atlantic salmon and reported differences in genes encoding proteins linked to lipid metabolism, carbohydrate metabolism, and protein degradation. Among the significantly differentially regulated genes were gamma crystallin M2 (homolog to mammalian crygb), lens fiber membrane lim2, secreted protein, acidic, cysteine-rich (sparc), metallothionein B (mt-b), heat-shock cognate 70 (hsc70a), calpain (capns1), Na/K ATPase alpha subunit isoform 1c (atpa1c) and fatty acid binding protein 2 (fabp2), of which several have been linked to cataracts before.
In the present study, we used transcriptomics (RNA-seq) to examine why triploid fish are more prone to cataract development than diploid fish. To do so, we compared the transcriptional patterns in the lens of diploid and triploid Atlantic salmon originating from both a domesticated strain and a wild population, with and without mature cataract, as assessed by a slit-lamp biomicroscope.
Materials and methods
Experimental animals and set-up
The salmon used in this experiment originated from females (f) and males (m) from the domesticated Mowi strain (M) crossed with females and males from a wild population in the river Figgjo (F) in November 2011. Eight groups were made as diploid and triploid of the systematic breeding of the farmed and wild strains: mM × fM, mM × fF, mF × fF and mF × fM. The offspring groups were start-fed with a commercial feed (Skretting, Stavanger, Norway) at March 26th , 2012 and were held at 12 • C water temperature from start feeding to mid-summer. Thereafter, the groups were reared at ambient temperature. Fish were reared under continuous light from start feeding to October 1st, followed by a simulated natural photoperiod to initiate parr-smolt transformation. Experimental groups were held separately in eight tanks until November 27th , 2012, when they were individually passive integrated transponder-tagged (PIT-tags, Electronic I, Inc., Dallas, TX, USA) and the groups distributed equally into three replicate tanks. Fish were transferred to seawater at May 10th, 2013. In sea, the fish were fed Skretting Spirit 75-50A. The experiment was terminated October 16th , 2013.
Fish were sampled as post smolts in seawater at a mean body weight of 143 ± 8 g (n = 46). Upon sampling, the fish were inspected for cataracts, and weight and length measured. From each fish, two lenses and heart tissues were dissected and immediately frozen on liquid nitrogen. The left lens was used for transcriptome de novo assembly and transcriptomics.
Cataract determination
Cataract assessment was performed on anaesthetized fish by use of a Kowa SL-15 slit-lamp biomicroscope (Kowa, Tokyo, Japan). The type, position and severity of the observed cataractous changes were determined according to Wall and Richards (1992), but with a maximum severity extended from 3 to 4 per eye to match the amplitude of the macroscopic scale (microscopic cataract score 0: absent, 1: slight, 2: moderate, 3: severe, 4: total cataract).
Histidine determination
Heart tissue from the sampled fish was used as status organ for histidine and NAH (Remø et al., 2014). NAH and free histidine in the heart tissue were analyzed by reverse phase HPLC and UV detection at 210 nm, with modifications according to Breck et al. (2005b).
RNA isolation
Lens tissue was thoroughly homogenized before RNA extraction using a Precellys 24 homogenizer and ceramic beads CK28 (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted using the BioRobot EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany), treated with DNase according to the manufacturer's instructions and eluted in 50 μL RNase-free MilliQ H 2 O. RNA quality and integrity were assessed with the NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the RNA integrity of the lens samples. The RNA integrity number (RIN) of RNA extracted for transcriptome assembly (2N: n = 10, 3N: n = 10) and RNA-seq (2N: n = 12, 3N: n = 14) were 8.5 ± 0.0 (n = 20) and 8.2 ± 0.1 (n = 26), respectively (mean ± SEM). of analysis, transcriptome de novo assembly had to be conducted using Illumina paired-end reads before RNA-seq analyses. RNA extracted from 10 diploid and 10 triploid lenses (n = 20) was mixed and used to generate the assembly. Transcriptome de novo assembly was conducted using the short reads assembling software Trinity as described by Grabherr et al. (2011). Trinity combines three independent software modules, Inchworm, Chrysalis, and Butterfly, to process the RNA-seq reads into unigenes. The output sequences were aligned to the databases of NR, NT, SwissProt, KEGG, COG and GO using Blastx, and the best aligning result was used to decide sequence direction. Sequence direction of unigenes not aligned to any of the above-mentioned databases was determined with the ESTScan software (Iseli et al., 1999).
RNA-seq analysis
Direct RNA sequencing (RNA-seq) was used to screen for differentially expressed genes (DEGs) in lenses of both diploid and triploid individuals. As the two strains used here are known to display divergent transcription patterns (Bicskei et al., 2014(Bicskei et al., , 2016, fish from both groups were randomly mixed and pooled prior to any analysis. Individual left lenses from 26 salmon were used for RNA-seq examination (2N-: n = 6, 2N+: n = 6, 3N-: n = 6, 3N+: n = 8). Poly (A) mRNA was isolated using magnetic beads with oligo (dT) from total RNA obtained from the lens samples. Fragmentation buffer was added to shred mRNA to short reads. Using these short fragments (about 200 bp) as templates, random hexamer primers were applied to synthesize first-strand cDNA. Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. QiaQuick PCR extraction kit (Qiagen) was used to purify short double-stranded cDNA fragments according to manufacturer's instructions. These fragments were then resolved with EB buffer for end reparation, added poly (A), and ligated to the sequencing adapters. After agarose gel electrophoresis, the suitable fragments were selected for PCR amplification as templates. Finally, the libraries were sequenced using Illumina HiSeq™ 2000 (San Diego, CA, USA).
The de novo lens transcriptome described above was thereafter used as a reference for alignment of the RNA-seq data. Unigenes were annotated with Blastx alignment using an e-value cut-off of 10 − 5 between unigenes and the databases of NR, NT, SwissProt, KEGG, COG and GO. The NOISeq software package (Tarazona et al., 2011) was used to screen for differentially expressed genes (DEGs). NOISeq is a novel non-parametric method for the identification of DEGs, which shows a good performance when compared to other differential expression methods, like Fisher's Exact Test, edgeR, DESeq and baySeq. All RNA-seq work was performed by staff at the Beijing Genome Institute (BGI, Hong Kong).
Ploidy verification
Fish from each ploidy were sampled and measured for erythrocyte diameter to verify their ploidy status (Benfey et al., 1984). Blood smears were used to measure the relative diameters of 10 erythrocytes per fish (Image-Pro Plus, version 4.0, Media Cybernetics Silver Spring). The triploid fish had significantly (22%) larger blood cells than diploid fish.
Statistics
To calculate differential expression, NOISeq default settings were used (Tarazona et al., 2011). NOISeq empirically models the noise distribution of count changes by contrasting fold-change differences (M) and absolute expression differences (D) for all the features in samples within the same condition. This reference distribution is used to assess whether the M-D values computed between two conditions for a given gene is likely to be part of the noise or represent a true differential expression. Instead of using a false discovery rate (FDR) or a q-value cut-off, the NOISeq method calculates a differential expression probability value. A gene is declared as differentially expressed if this probability is higher than q. The threshold q is set to 0.8 by default, since this value is equivalent to an odd of 4:1 (the gene in 4 times more likely to be differentially expressed than not). In this work we used a log2 M-value cut-off of ≥2 (fold-change ≥2). For genes not expressed in some samples, the gene expression value (D) of 0.001 was used. Functional pathway analyses, including prediction of activation and inhibition of upstream transcription factors and downstream effects, were generated through the use of QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.qiagen.com/ingenuity). Since IPA only can map mammalian homolog entries, identifiers were obtained with Blast alignment against the RefSeq databases (cut-off E10 − 5 ) and assuming orthologous genes have the same function. A limited number of fish-specific genes with no mammalian homologs were for this reason not included in the IPA pathway analysis. This may have skewed the interpretation of the transcriptomic data.
Data availability
The RNA-seq dataset discussed in this publication has been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE153933 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE153933).
Results
Growth and heart histidine levels
Growth of the farmed and wild stocks is reported in detail by Harvey et al. (2017), and therefore not reported here. At the present sampling, there was no significant difference in weight between the diploid and triploid fish. Fig. 1 shows the concentrations of L-histidine and N-acetyl-L-histidine (NAH) analyzed in salmon heart as a measure on the ambient histidine status. Two-way ANOVA analysis showed that there was a significant effect of cataract on L-histidine (Fig. 1A, p = 0.016), while there were no significant effects of either cataract or ploidy on NAH (Fig. 1B). Posthoc tests showed reduced levels of L-histidine (p = 0.019) and increased levels of N-acetyl-L-histidine (p = 0.046) in diploid fish with cataract.
Cataract status
The mean cataract scores of the left lenses used to generate the lens transcriptome (2N + had score 2.5 and 3N + had score 1.5, while both 2N-and 3N-had score 0) and the right lenses used for the RNA-seq analysis (both 2N+ and 3N + had score 3, while both 2N-and 3Nhad score 0) are shown in Fig. 2A and B.
Lens de novo transcriptome assembly
Total RNA extracted from lenses of 20 domesticated and wild salmon, 10 of which were diploid and 10 were triploid, was mixed ( Fig. 2A), sequenced and used to assemble the lens transcriptome. Using the Illumina HiSeq 2000 platform, a total of 68,403,252 raw reads and 63,098,790 clean reads were sequenced. The clean reads were assembled into 78,306 contigs with mean length of 284 nucleotides (nts). Of these, 29,177 contigs with mean length of 659 nts mapped to UniGene entries. Distinct clusters, which contained highly similar UniGene entries (more than 70% that may come from the same gene or homologous genes), were 7391. Distinct singletons representing a single UniGene were 21,786. Using blastx and blastn, a cut-off of 10 − 5 and the following priority order, 15,711,21,084,14,445,11,680,5231,11,327 UniGene entries were functionally annotated to the NR, NT, Swiss-Prot, KEGG, COG and GO databases, respectively. UniGene entries aligned to a higher-priority database were not aligned to a lower-priority database. In total, 22,160 out of the 29,177 UniGene entries were given a functional annotation in these databases.
For protein coding region prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 15,428. The number of predicted CDS, Unigene entries that could not be aligned to any database and were scanned by ESTScan, was 701. The total number of CDS was 16,129. According to the microsatellite analysis conducted with the MicroSAtellite (MISA) software and using Unigenes as reference, there were 7274 simple sequence repeat (SSR) in the transcriptome. Heterozygous analysis using SOAPsnp, a member of the Short Oligonucleotide Analysis Package (SOAP), revealed 23,759 single- nucleotide polymorphisms (SNPs) in the transcriptome.
A summary of the NR annotation is shown in Fig. S1 (Supplementary File 1). Fig. S1A shows E-value distribution, while similarity distribution is shown in Fig. S1B. Fig. S1C shows the species distribution of UniGene entries annotated to the NR database. Most UniGene entries mapped to Atlantic salmon, followed by hits against Nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and other fish species. Gene ontology (GO) annotation of the NR unigenes was obtained with the Blast2GO and WEGO software's (Conesa et al., 2005;Ye et al., 2006). Fig. S2 shows the major GOs from the salmon lens transcriptome, divided into the three ontologies biological process, molecular function and cellular component. Of the more specialized molecular process worth mentioning is the antioxidant activity, while the biological function GO annotation indicate that the lens cells are relatively metabolic active.
Differentially expressed genes (DEGs)
To search for differentially expressed genes (DEGs) in diploid and triploid salmon with and without cataracts (Fig. 2B), the left lens from 26 individual fish were selected for RNA-seq analysis. The selection was based on cataract score (score 0 vs 3) and ploidy (2N vs 3N). A total of 634,610,512 single-end reads were sequenced with the Illumina HiSeq™ 2000 system. In average, 24,023,481 ± 199,810 single-end reads were sequenced per sample (n = 26, mean ± SEM). Average total reads mapped to the in-house made lens transcriptome were 17,882,297 ± 161,561 (n = 26, mean ± SEM), representing 74.4% of the total reads. As expected for fish, some contigs had redundant annotations.
Using the default NOISeq setting for calculation of differential expression (q ≥ 0.8 and log 2 ≥ 1), the comparison between diploid fish without and with cataracts (2N-vs 2N+) showed that 182 DEGs were more highly expressed in 2N-lenses and 25 DEGs were more highly expressed in 2N + lenses (Fig. 2C). For the comparison between triploid fish without and with cataracts (3N-vs 3N+), 74 DEGs were more highly expressed in 3N-and 78 DEGs were more highly expressed in 3N+. Comparison of healthy diploid lenses vs healthy triploid lenses (2N-vs 3N-) yielded 107 DEGs, with 93 genes more highly expressed in 2N-, and 14 more highly expressed in 3N-. Comparison of cataractous diploid lenses vs cataractous triploid lenses (2N + vs 3N+) yielded 165 significant DEGs, with 9 genes more highly expressed in 2N+, and 156 genes more highly expressed in 3N+. All significant DEGs in the four comparisons, including fold changes, significance levels and best annotation, which were used in downstream functional analyses, are shown in Supplementary File 2. Annotations were given to about 52% of the DEGs.
Very few DEGs with unique expression were found in the lenses from the four treatment groups. Fig. 2D shows a Venn diagram of the number of unique and shared DEGs determined with a four-way comparison. There were 4 unique DEGs in the 2N-group, 17 in the 2N + group,15 in the 3N-group and 245 in the 3N + group. 98.7% of the DEGs were shared between all treatment groups. Most of these unique DEGs were expressed only in one or a few of the lenses from their respective group. Annotations of unique DEGs are shown in Supplementary File 3.
Functional analysis
Two pathway analysis methods, KEGG and IPA pathway analysis, were employed for functional analysis of DEGs in cataractous lenses from diploid and triploid fish. KEGG pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways in DEGs by comparison to the whole genome. Table 1 shows the most significant KEGG pathways from the four comparisons based on a q-value cut-off of 0.05. The top three pathways in both diploid and triploid fish with cataracts were "Phototransduction", "Carbohydrate digestion and absorption" and "Proximal tubule bicarbonate reclamation". Interestingly, for the phototransduction pathway (KEGG pathway ko04744), the significant DEGs linked to this system, 13 DEGs in the diploid fish and 17 DEGs in the triploid fish, (DEGs only found in triploid fish were gnb1, arrb1 and arr3), were all upregulated in the diploid cataractous lens (Fig. 3A) and down-regulated in the triploid cataractous lens (Fig. 3B). As expected, direct comparisons between diploid and triploid lens from fish without and with cataract gives similar patterns. The "ECM-receptor interaction" and "PPAR signaling" pathways were two the most significantly affected KEGG entries based on a direct comparison of DEGs in diploid and triploid salmon with cataracts not listed in the other comparisons.
IPA Core analysis and the IPA Compare function were used for evaluation of biological processes, pathways and networks. In order to use IPA, all identifiers must be recognized as mammalian homologs. Some fish-specific genes obviously cannot be given human ortholog names recognized by IPA, and thus were omitted from the IPA-Core analysis. About 52% of the DEGs from the four gene lists were given automatic annotation as described above (Supplementary File 2). In addition, all unknown DEGs were manually aligned against the core nucleotide and EST databases, and given annotation based on hits against NCBI Unigene entries (Blastn cut-off E10-5). This way, 64.4% of the DEGs used for the functional analysis had IPA identifiers. Table 2 shows annotated salmon genes with human identifiers used in these functional analyses which were significantly differently expressed according to the four comparisons (2N + vs 2N-, 3N + vs. 3N-, 3N + vs 2N+ and 3N-vs 2N-). Highlighted in the table are cataract-linked genes that are differentially regulated in various mice knockout models (data obtained from the iSyTE (integrated Systems Tool for Eye gene discovery) database (URL: http://research.bioinformatics.udel.edu/iSyTE).
Impact of cataracts
To get an idea of the mechanistic basis for cataract development in the salmon lens and the impact of ploidy, we used IPA Core Analysis with the predicted upstream regulators function and the categorical annotations of disease or function to search for differences in the four comparisons described above. By sorting with an activation z-score >2 and p-value of overlap <10.5, IPA Core Analysis predicted six upstream regulators that may explain the observed DEGs in lenses of diploid salmon with cataracts. These were CRX, GTF2IRD1, HIF1A, EDN1, hexachlorobenzene and EPO (Supplementary File 4). The dataset for the most significant transcriptional regulator, CRX with a z-score of 2.43 and a p-value of overlap of 8,35E-19, was made up of the DEGs arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. For GTF2IRD1, which had a z-score of 2.10 and p-value of overlap of 2,89E-10, the dataset consisted of arr3, gnat1, gnat2, opn1lw, opn1sw, pdc and rho. For the disease of functional annotation, the analysis predicted eight categories with a z-score above 2 and p-value >10-5. These were "Cellular Movement, Immune Cell Trafficking-leukocyte migration", "Cellular Movement-cell movement", "Cellular Movement, Hematological System Development and Function, Immune Cell Trafficking-cell movement of leukocytes", "Cellular Movement-migration of cells", "Cell Death and Survival-cell viability", "Cell Death and Survival-cell survival", "Tissue Morphology-quantity of cells" and "Cellular Movement-migration of brain cancer cell lines".
In the lenses of triploid salmon with cataracts, five upstream regulators had a predicted activation state based on the same cut-off as described above. These were CRX, GTF2IRD1, beta-estradiol, trichostatin A and decitabine (Supplementary File 5). CRX, the most significantly regulator with a z-score of − 2.73 and a p-value of overlap of 8,90E-20, was predicted affected based on the same DEGs as in diploid fish, i.e. arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. The predicted activation state for GTF2IRD1 (z-score: − 2.10, p-value of overlap: 6,33E-11) in lenses of triploid salmon was based on the same DEGs as in diploid fish. Using the same cut-off, no disease or function categories had a predicted activation state in the lenses of triploid salmon. By comparing the transcriptional patterns in cataractous lenses of diploid and triploid salmon indirectly (IPA Compare Analysis of 2Nversus 2N+ and 3N-versus 3N+), the most pronounced differences were seen for the "IL8" and "Production of nitric oxide and reactive oxygen species in macrophage" canonical pathways (data not shown). These pathways had higher activation z-scores in lenses of the diploid fish compared to the triploid fish. A predicted regulator network generated from the comparison of DEGs in cataractous lenses of diploid and triploid salmon is shown in Fig. 4. This network, which had a consistency score of 13.87 and was based on target DEGs apoe, clu, gal, igfbp2, junb, krt18, lep, mmp2, plaur, rbp4 and snca, and on upstream regulators AGT, CREB1, ERK, HIF1A and P38 MAPK, predicted that synthesis of nitric oxide and chemotaxis of cells might be different in cataractous lenses of diploid and triploid salmon. Based on analysis of predicted upstream regulators and hierarchical clustering, the most pronounced difference in triploid fish was seen for CRX, GTF2IRD1, SRC and RHO (Table 3). Interestingly, these upstream regulators were predicted activated in diploid fish (positive z-score) and predicted inhibited in triploid fish (negative z-score) with cataracts. Fig. 5 shows the molecules in these four networks. Except krt18 and ckm in the SRC network, all genes in these networks were up-regulated by cataracts in diploid fish and downregulated in triploid fish.
Impact of ploidy
Comparison of the transcriptional patterns in lenses from diploid and triploid salmon without cataracts revealed two upstream regulators with predicted activation scores above 2 and p-values of overlap >10-4 (Supplementary File 6). According to the IPA Core Analysis, both the transcription regulator CRX and the chemical drug trichostatin A were predicted activated with z-scores of 2.55 and 2.40, respectively. Targeted DEGs for CRX were gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho, while cdh1, hba1/hba2, hbb, hbz, ndrg1 and slc1a2 made up the dataset for the trichostatin A prediction.
A comparison of transcriptional patterns in lenses from diploid and triploid fish without cataracts showed that two disease or function annotations had prediction scores above 2 and a p-value of overlap >10-5 (Supplementary File 6). "Organ degeneration" (z-score 2.19) and "Degeneration of cells" (z-score 2.19) showed significant differential prediction scores in lenses of triploid and diploid salmon without cataracts. The "Organ degeneration" z-score was based on differential transcription of prph2, rho, crb1, slc1a2, pde6g, gngt1, rpe65, rcvrn, ca1, gnat1, opn1mw (includes others) and ca2, whereas the "Degeneration of cells" z-score was based on differential transcription of rho, slc1a2, pde6g, gngt1, rpe65, gnat1 and prph2. All these genes were more highly expressed in lenses of diploid fish compared to triploid fish. This could reflect differential transcription in non-cataractous lenses from diploid and triploid salmon, or indicate that mechanisms leading to cataracts The direct comparison of transcriptional patterns in lenses of diploid and triploid salmon with cataracts yielded five predicted upstream regulators with activation z-score >2 and p-value of overlap >10-5 (Supplementary File 7). These were CRX, GTF2IRD1, beta-estradiol, trichostatin A and decitabine. Targeted DEGs in the most significant regulator (transcription regulator CRX, p-value of overlap 8, 90E-20) were arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. A significant result for the transcription regulator GTF2IRD1 was based on the DEGs arr3, gnat1, gnat2, opn1lw, opn1sw, pdc and rho. Five categories with disease or functional annotation had predicted activation state based on z-score >2 and p-value of overlap >10-5 (Supplementary File 7). These were "DNA Replication, Recombination, and Repair, Nucleic Acid Metabolism, Small Molecule Biochemistryhydrolysis of nucleotide", "Behavior-", "Cellular-Movement-migration of blood cells", "Cellular Movement-cell movement" and "Organismal Development-size of body".
Discussion
This is the first study to investigate the transcriptomics of salmon lenses in diploid and triploid salmon with and without cataracts. Functional analysis showed that retina-associated genes were differentially affected in diploid and triploid fish. Predicted differential effects of NO-induced oxidative stress, modified cytoskeleton stability and lipid metabolism, possibly affecting cellular metabolism, indicate that the triploid lens might be more vulnerable to cataract due to altered protein degradation and turnover.
Overall, this study indicates that the transcriptional patterns in the lenses of diploid and triploid Atlantic salmon are very similar. This is consistent with the results from a recent study, which showed that the vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon (Oncorhynchus kisutch) (Christensen et al., 2019). Similar results have been shown for other fish species (Chatchaiphan et al., 2017). At the protein level there also appears to be small differences in expression between diploid and triploid salmon (Nuez-Ortin et al., 2017). Relatively few significant DEGs were found in the current dataset. Most of the significant DEGs in the cataractous triploid lenses were higher expressed compared to the cataractous diploid lens (156 vs 9). In healthy lenses the pattern was opposite, with more of the significant DEGs being lower expressed in the diploid lenses (93 vs 14). According to the functional analysis, the most distinct difference between diploid and triploid cataractous lenses in transcript levels were seen for genes encoding proteins involved in the phototransduction pathway. Whether this reflects a direct effect of ploidy on the transcription of these genes is unknown.
The N3+ vs N2+ comparison list contained a gene associated with heat shock protein (HSP) activity, e.g. hsp47/serpinh1. Furthermore, two heat shock protein genes, annotated to hspa8 and hspa8b, were upregulated in diploid cataractous lenses but not in diploid noncataractous lenses. These findings potentially suggest a different ability to handle damaged proteins and protein turnover. Crystallins, watersoluble structural protein found in the lens and the cornea of the eye accounting for the transparency, are relatively similar to HSPs, and have similar chaperone activity (Wang and Spector, 1995;Slingsby et al., 2013). "Protein digestion and absorption" (KEGG pathway ko04974) was the second most significantly affected pathway, after phototransduction, in triploid cataractous lenses compared to diploid cataractous lenses, according to the functional analysis. In humans, ROS-generated protein oxidation may lead to cataract formation in the aged lens (Taylor and Davies, 1987). In addition to oxidative stress and the inflammatory response, an unfolded protein response is known to be activated in age-related ocular disorders such as cataracts (Lenox et al., 2015). Histidine has been shown to stimulate the proteasome and thereby protein degradation and turnover (Hamel et al., 2003;Breck et al., 2005a). With diminished antioxidant capacity and decreased proteolytic capabilities, the triploid lens may be less efficient in clearance of damaged proteins. Taken together, the results from the current study indicate that the higher susceptibility to cataract development in triploid vs diploid salmon may in part rely on how well the cells handle damaged proteins.
The heart histidine and NAH levels observed in the present study represent normal values obtained from a commercial salmon smolt feed (Remø et al., 2014). According to the factorial analysis, the histidine concentration was related to cataract status and not to ploidy, while NAH status did not indicate differences with neither cataract status nor ploidy. The result confirms the higher sensitivity of histidine relative to NAH status in heart tissue, but more importantly, a corresponding lens histidine status to the diploid cataract group and both the triploid groups indicates suboptimal conditions for salmon smolts (Remø et al., 2014). The present groups of salmon would therefore be prone to cataract development.
At the molecular level, functional analysis predicted that the upstream regulators cone-rod homeobox (CRX), GTF2I repeat domain- Table 2 Annotated salmon genes with human orthologs used in the IPA functional analyses. Genes common for all four comparisons were all up-regulated in diploid cataractous lenses and down-regulated in triploid cataractous lenses. Genes in bold are differential regulated in mice cataract mutants (>2.0 fold) according to the iSyTE database (Kakrana et al., 2018). Underlined are genes that are differentially regulated by more than one mutant type.
Comparison
Genes agbl4, anxa2, anxa5, apoc1, apoe, arl14ep, arr3, arrb2, atp1a3, atp1a4, aurka, btbd17, c18orf25, c4orf33, ca1, ca2, cars, ccl28, cdca8, ckmt2, clu, col11a1, cyb5r1, deptor, dnajc3, egfl7, f11r, gal, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, hba1, hbb, hbz, homer2, hspa8, ifitm5, igfbp2, junb, krt18, ldhb, lep, linc00998, lurap1, lztr1, mlkl, mllt11, mmp2, ndrg1, ndrg4, nmt1, opn1lw, opn1mw2, opn1sw, parp15, pdc, pde6g, plaur, ppdpf, ppp2ca, prdm9, prph2, rab32, rasd2, rbp4, rcvrn, rho, rpe65, rtbdn, s100a1, s100p, slc27a2, smarcd1, snca, specc1, stc1, tmsb10, tpd52l1, trpm7, wwp2, znf391, znf501 anxa5, arl14ep, arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cars, ccl28, ckb, bckm, ckmt2, clu, cndp2, cntnap5, dbnl, defb4b, erbb2ip, f11r, fabp4, fabp7 cntnap5, coch, col2a1, col9a1, col9a2, dnase1l3, eef1a1, fabp4, fabp7, gja1, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, igsf11, lcn1, ldhb, lrrn1, mgst3, nbl1, ndrg1, ndufa4, opn1lw, opn1mw2, opn1sw, pdc, pde6g, pkm, plaur, ppdpf, prph2, ptma, pygm, rbp4, rcvrn, rgmb, rho, rpe65, rtbdn, serpinh1, sirt1, slc39a11, snca, sparc, spon1, stmn1, suclg1, trpm7, ube2j1 3N-vs 2N-arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cdh1, ckm, ckmt2, crb1, crygs, cyp2c19, erbb2ip, exosc2, glra1, glul, gnat1, gnat2, gnb3, gngt1, gngt2, harbi1, hba1, hbb, hbz, igsf11, ldhb, mvp, ndrg1, opn1lw, opn1mw2, opn1sw, pdc, pde6g, plaur, polr3c, prph2, rbp4, rcvrn, rho, rpe65, rps12, rtbdn, slc1a2, stc1, suclg1, trpm7 Common in 2N+ and 3N+ anxa2,anxa5,arl14ep,arrb2,atp1a3,atp1a4,btbd17,ca1,ca2,cars,ccl28,ckmt2,clu,f11r,gal,glul,gnat1,gnat2,gnb3,gngt1,gngt2,hamp,harbi1,ifitm5,igfbp2,junb,krt18,ldhb,lep,mlkl,ndrg1,nmt1,opn1lw,opn1mw2,opn1sw,pdc,pde6g,plaur,prdm9,prph2,rcvrn,rho,rpe65,rtbdn,s100a1,s100p,slc27a2,snca,tmsb10,trpm7,znf391,znf501 Common for all 4 comparisons arrb2,atp1a3,atp1a4,btbd17,ca1,ca2,ckmt2,glul,gnat1,gnat2,gnb3,gngt1,gngt2,harbi1,ldhb,ndrg1,opn1lw,opn1mw2,opn1sw,pdc,pde6g,plaur,prph2,rcvrn,rho,rpe65,rtbdn,trpm7 P.A. Olsvik et al. containing 1 (GTF2IRD1), SRC proto-oncogene, non-receptor tyrosine kinase (SRC) and rhodopsin (RHO) could explain the differences observed in lens transcript levels between cataractous diploid and triploid fish. All these upstream regulators were predicted to be activated in cataractous lenses of diploid fish and inhibited in cataractous lenses of triploid fish. Expression of visual pigment-like proteins has been described in extraretinal tissue (Shichida and Yamashita, 2003). Differential expression of retina-associated genes has also been documented in zebrafish (Danio rerio) cataractous lens cells (Posner et al., 2019). Since the lenses in the current study were extracted through an incision in the cornea, without contacting retinal tissues, it seems unlikely that such visual pigment-like proteins were derived from retinal contamination. CRX is a photoreceptor-specific transcription factor, which plays a role in the differentiation of photoreceptor cells, controlling the maintenance of normal cone and rod function (GeneCards database). While not directly linked to cataracts, mutations in the gene encoding CRX have been linked to severe dystrophy of the human retina (Weleber et al., 1993). GTF2IRD1 function as a transcription factor under the control of retinoblastoma protein, and may be a transcription regulator involved in cell-cycle progression and skeletal muscle differentiation (GeneCards database). In human stromal cells, vitamin E treatment has been shown to down-regulate GTF2IRD1, suggesting a link to the lens antioxidative defense. GTF2IRD1 also respond to chemical exposure. For example, down-regulation of GTF2IRD1 has been shown in human liver cells after exposure to benzo(a)pyrene (Jennen et al., 2010), while the peroxisome proliferator-activated receptor alpha (PPARα) agonist pirinixic acid increases GTF2IRD1 mRNA levels in mice liver (Sanderson et al., 2008). The SRC proto-oncogene may play a role in cell growth and participates in signaling pathways that control a broad spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation (GeneCards database). Of interest for lens damage, SRC plays an important role in the regulation of cytoskeletal organization through phosphorylation mechanisms (Gene-Cards database). Recent findings suggest that accumulation of crystallin proteins, a prerequisite for refractive properties and transparency of the lens, in part is controlled by post-transcriptional mechanisms rather than by differential gene transcription (mRNA synthesis) (Terrell et al., 2015). This may explain why we did not see any differentially expressed crystallin genes in the current study. RHO is a photoreceptor primarily expressed in rod cells in the retina required for image-forming vision at low light intensity (GeneCards database). Of nutrients and essential elements, Zn is known to affect stability and folding of RHO (Stojanovic et al., 2004;Gleim et al., 2009), indicating that Zn imbalance might impact RHO activity in cataractous triploid lenses. Zn deficiency has been linked to cataract development in rainbow trout (Ketola, 1979). The functional implication of differential expression of RHO-associated transcripts in lens cells is unknown, as RHO protein is primarily expressed in rod photoreceptors in the retina. In agreement with the current examination, down-regulation of rho has been observed in the cataractous zebrafish lens (Posner et al., 2019). The observed effect on rho may be linked to dysregulation of vitamin A1 and A2 in the lens endothelial cells outside the retina (Enright et al., 2015), or alternatively may reflect signaling interactions between the lens and retina through hyaloid capillaries (Dhakal et al., 2015). Lower activation z-scores for pathways linked to synthesis of nitric oxide (NO) and chemotaxis in triploid fish, suggests that their lens cells may be more prone to oxidative damage and chemotactic cell movement than their diploid counterparts. It is well known that NO has a role in cataract formation in the mammalian lens (Ito et al., 2001;Ornek et al., 2003;Chamberlain et al., 2008). Oxidative stress is an important factor in the development of cataracts for both animals and humans (Ottonello et al., 2000;Williams, 2006). High concentrations of reactive oxygen species (ROS), produced from both endogenous and exogenous sources, cause oxidative damage to cellular constituents that results in interrupted physiological functions and oxidative stress-associated diseases such as cataracts (Lou, 2003). The decreased protein turnover towards the nucleus makes lenses especially vulnerable to increased ROS production in the epithelial cells (reviewed by Brennan and Kantorow, 2009). In human age-related cataracts, oxidation of membrane proteins has been found to precede the development of cataract formation (Wang and Spector, 1995). The accumulation of oxidized proteins further results in loss of cell function, apoptosis and necrosis (Brennan and Kantorow, 2009). Lens NAH has been suggested to be an important intracellular antioxidant in the salmon lens and may contribute to the cataract mitigating effect of dietary histidine (Remø et al., 2011). The present results suggest a higher susceptibility to oxidative stress in Fig. 5. IPA Compare Analysis of transcripts differentially regulated in cataractous lenses of triploid salmon compared to diploid salmon (2N + vs 3N+). The figure shows genes associated with four predicted regulators that were activated in diploid fish but inhibited in triploid fish, based on hierarchical clustering and z-score using Upstream Analysis. A) Cone-rod homeobox (CRX), B) GTF21 repeat domain containing 1 (GTF21RD1), C) rhodopsin (RHO) and D) SRC proto-oncogene, nonreceptor tyrosine kinase (SRC).
2N + vs 2N-(diploid cataract) ada,3N + vs 3N-(triploid cataract) anxa2,
triploid lenses, which may be hypothesized to be an underlying factor for the higher prevalence of cataracts compared to diploids, as well as the lower lens NAH status observed when reared under similar conditions in the studies by Taylor et al. (2015) and Sambraus et al. (2017). Likewise, cataract and chemotactic activity have been extensively studied over the years (Rosenbaum et al., 1987;Schneider et al., 2012). Several of the predicted upstream regulators for this network, angiotensinogen (AGT), CAMP responsive element binding protein 1 (CREB1), extracellular signal-regulated kinase (ERK), HIF1A and P38 mitogen-activated protein kinase (P38 MAPK), have been linked to cataract formation (AGT: Taube et al., 2012;Ji et al., 2015;CREB1: Weng et al., 2008;ERK: Iyengar et al., 2007;HIF1A: Chen et al., 2014;P38 MAPK: Bai et al., 2015). Follow-up studies should look at how NO induce ROS and oxidative stress in the triploid fish lens, as well as the involvement of chemotaxis in the development of cataract in triploid salmon.
Two pathways, "ECM-receptor interaction" and "PPAR signaling pathway", were among the most significantly affected KEGG entries based on a direct comparison of DEGs in diploid and triploid salmon lenses with cataracts. These were not listed in the other comparisons. The ECM-receptor interaction pathway, including collagen, type II, alpha 1 (col2a1) and secreted protein, acidic, cysteine-rich (osteonectin) sparc, has been linked to disorders of the eye characterized by early onset cataract (Bradshaw, 2009). SPARC is a key lens-and cataract-associated protein (Shiels et al., 2010;Sousounis and Tsonis, 2012;Terrell et al., 2015). SPARC is important for normal cellular proliferation and differentiation and is involved in maintaining lens transparency as shown for mice (Gilmour et al., 1998) and humans (Yan et al., 2000). SPARC is also one of at least 13 proteins harboring mutations that have been associated with a lens or cataract phenotype in mice but not yet in humans (Shiels et al., 2010). In Atlantic salmon, SPARC was suggested to be an "early" up-regulated marker for cataract development (Tröße et al., 2009). Lower expression of sparc suggests that the cataractous triploid lenses might have impaired circulation of fluids, ions, and small molecules, possibly resulting in depolarized membrane resting voltage as shown in mice (Greiling et al., 2009). Cartilage extracellular matrix (ECM) is composed of type II collagen, fibrous proteins and proteoglycans, hyaluronic acid and chondroitin sulfate (Gao et al., 2014). The finding indicates a differential regulated mechanism linked to cytoskeleton disruption and NO-induced oxidative stress (Gao et al., 2014). Differential regulation of PPARs, which are transcription factors in control of many cellular processes, indicate an effect on lipid metabolism in the lens. An effect on lipid/cholesterol transport, previously reported in age-related cataract in humans (Utheim et al., 2008), is suggested by differential expression of apolipoprotein E (apoe). APOE is a major apoprotein that is essential for the normal catabolism of triglyceride-rich lipoprotein constituents (Genecards database), indicating a differential effect on lipoprotein metabolism. Apoe, together with sparc, was among the differentially regulated genes in cataractous lenses of Atlantic salmon fed a low-histidine diet compared to a high-histidine diet (Tröße et al., 2009) and had a lower expression level in the lens of Atlantic salmon fed plant oils compared to fish oils (Remø et al., 2011).
Only five genes from the Cat-Map gene list, an online chromosome map and reference database for cataract in humans and mice (Shiels et al., 2010), showed overlap with the current gene list of cataractous diploid and triploid lenses from salmon (direct comparison). These were, in addition to sparc and apoe, col2a1, gap junction protein alpha 1, 43 kDa (gja1) and retinal pigment epithelium-specific protein 65 kDa (rpe65). Gap junction proteins, also called connexins, are constituents of gap junctions, channels specialized in cell-cell contacts that provide direct intracellular communication. They allow passive diffusion of molecules up to 1 kDa, including nutrients, metabolites (glucose), ions and second messengers (Genecards database). They are especially important for nutrition and intercellular communication in the avascular lens (Hejtmancik, 2008). Mutations in gap junction proteins such as GJA1, present in the lens epithelium, have been linked to human cataracts (Hejtmancik, 2008). RPE65 is a protein located in the retinal pigment epithelium and involved in the production of 11-cis retinal and in visual pigment regeneration (Genecards database). Finally, RPE65 has been associated with leber congenital amaurosis (LCA), a severe dystrophy of the retina (Weleber et al., 1993). No genes associated with Mendelian (inherited) cataracts or cataracts caused by mutations in transcription factors or metabolic enzymes in humans (Shiels and Hejtmancik, 2019) were on the significant lists in this study. Several of the genes that were differentially expressed in cataractous lenses of triploid salmon have previously been documented to be affected by mutations in the mouse lens (Table 2). By comparing our significant genes with the responses of mammalian orthologs with lens defects or cataract as listed in the iSyTE database (Kakrana et al., 2018), it appears that several may be potential candidate markers for follow-up studies in salmon. Apart from the CBP:p300 E9.5 mutation, which seems to down-regulate many of these genes in mice (iSyTE database), several gene knockout mutation types impact the expression of genes from our lists.
Hsp47, also called serpinh1, was one of the genes that were lower expressed in cataractous triploid lenses than in cataractous diploid lenses. HSP47, localized in the endoplasmic reticulum, plays a role in collagen biosynthesis as a collagen-specific molecular chaperone (Genecards database). Heat shock proteins, found throughout the various tissues of the eye, protect and maintain cell viability under stressful conditions such as those occurring during thermal and oxidative challenges chiefly by refolding and stabilizing proteins (Urbak and Vorum, 2010). In the human eye, HSP47 has been suggested to aid the control of pro-collagen under stressful conditions and is induced by corneal structure damage (Urbak and Vorum, 2010). In the salmon lens, increased expression of hsp70 has been shown after short-term handling stress (30 min), indicating that HSPs are transcriptionally controlled and act to protect the cells after stress-induced protein misfolding (Tröße et al., 2010). Lower expression of hsp47 in triploid lenses suggests a poorer ability to facilitate proper folding of proteins. It may be speculated that this is linked to the synthesis, accumulation, repair or breakdown of crystallins or other structural proteins, responsible for lens transparency (Hejtmancik, 2008). Crystallins make up about 80-90% of the soluble proteins in the lens (Hejtmancik, 2008). Mutations in crystalline genes is one of the major reasons for human cataract, and improper ability of chaperones to correct for misfolding or protein damage may render the triploid lens more vulnerable to imbalances responsible for cataract formation in the salmon lens.
When studying the lens transcriptome, it is important to note that the eye lens mostly consists of fiber cells without nuclei and organelles (Bassnett, 2002). With transcription restricted to metabolically active lens epithelial cells and young fiber cells (Hejtmancik et al., 2015), transcriptional differences between diploid and triploid cataractous fish lenses may generally be small. Furthermore, triploid salmon in general differ from diploids by containing fewer and larger cells in most organs (Swarup, 1959;Small and Benfey, 1987), possibly impacting transcriptional differences.
With 74.4% of the total reads mapped to the novel in-house made lens transcriptome, the mapping degree was similar to using a fully sequenced genome as reference. In total however, only 52% of the significant DEGs were annotated using the described pipeline. With manual annotation of all unknowns, about 64% of the DEGs were assigned annotation for IPA functional evaluation. The reason for this relatively poor annotation level is unknown. A good mapping score combined with a poor annotation level might suggest that the lens transcriptome contains a relatively high number of novel transcripts. Among the most strongly differentially regulated genes in both diploid and triploid salmon with cataracts was the CXC chemokine cxcf1a. This is a fishspecific chemokine with no mammalian ortholog (Chen et al., 2013), so its function was not included in the IPA functional analysis. This illustrates one of the limitations studying cataract mode of action in non-model fish species.
In conclusion, this study shows only moderate differences in lens mRNA levels between diploid and triploid Atlantic salmon with score-3 cataract, and very few DEGs with unique expression. Several retina related genes were differentially expressed in the diploid and triploid lenses. The study indicates that the triploid lens may be more vulnerable to cataract than the diploid lens due to predicted effects of protein degradation and turnover, NO-induced oxidative stress, modified cytoskeleton stability and lipid metabolism, possibly linked to repair and compensation mechanisms. Overall, this study suggests that cataract formation is associated with modest changes in gene expression levels, and that transcriptional controls to a large degree regulate gene expression levels independent of chromosomal number in salmon.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Fig. 1 .
1Heart concentrations of A) L-histidine and B) N-acetyl-L-histidine in diploid and triploid Atlantic salmon. Significance levels of two-factor analyses are shown in figures (2-way ANOVA). Capped lines indicate significance levels of direct comparisons of the compounds in 2N and 3N fish (uncorrected Fisher's test, P < 0.05). ns = not significant.
Fig. 2 .
2Cataract score of the Atlantic salmon lenses used to A) generate a lens transcriptome and B) quantification of DEGs. C) Number of DEGs in lenses of diploid (2N) and triploid (3N) Atlantic salmon without (− ) and with cataracts (+). Exact numbers of genes are given above the individual bars. D) Venn diagram showing the degree of overlap of DEGs in the four treatment groups based on a four-way comparison of genes.
Fig. 3 .
3Six DEGs encoding protein involved in phototransduction (KEGG pathway ko04744) were up-regulated in cataractous lenses from diploid (2N+) Atlantic salmon (A) and down-regulated in cataractous lenses from triploid (3N+) Atlantic salmon (B). Border color indicates up-regulated genes (red) and down-regulated genes (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) may be affected in some of the lenses even without visible signs of damage.
Fig. 4 .
4Predicted upstream regulator network with highest consistency score based on IPA Compare Analysis, generated by comparison of DEGs in cataractous lenses of diploid and triploid Atlantic salmon (2N + vs 3N+). Up-regulated and down-regulated genes are highlighted in red and green, while predicted activated and inhibited regulators and relationships are highlighted in orange and blue, respectively. The color depth is correlated to the fold change. Solid lines show direct interactions between two gene products, while dashed lines represent indirect interactions among genes shown in the network. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1
1KEGG pathway enrichment analysis of DEGs.#
Treatment comparison/Pathway
DEGs with pathway
annotation
All genes with pathway
annotation
p-value
Q-value
Pathway
ID
2N-vs 2N+
72
11680
1
Phototransduction
13 (18.06%)
67 (0.57%)
1.161234e-16
9.638242e-
15
ko04744
2
Carbohydrate digestion and absorption
8 (11.11%)
39 (0.33%)
7.384585e-11
3.064603e-
09
ko04973
3
Proximal tubule bicarbonate reclamation
7 (9.72%)
27 (0.23%)
2.019998e-10
5.588661e-
09
ko04964
4
Mineral absorption
6 (8.33%)
48 (0.41%)
4.43982e-07
7.370101e-
06
ko04978
5
Glutamatergic synapse
7 (9.72%)
95 (0.81%)
1.803348e-06
1.663088e-
05
ko04724
6
Aldosterone-regulated sodium reabsorption
5 (6.94%)
35 (0.3%)
2.173693e-06
1.804165e-
05
ko04960
7
Chemokine signaling pathway
9 (12.5%)
231 (1.98%)
1.141543e-05
7.895672e-
05
ko04062
8
Dopaminergic synapse
8 (11.11%)
178 (1.52%)
1.295758e-05
8.272916e-
05
ko04728
9
GABAergic synapse
6 (8.33%)
86 (0.74%)
1.414778e-05
8.387612e-
05
ko04727
10
Endocrine and other factor-regulated calcium
reabsorption
5 (6.94%)
70 (0.6%)
6.855952e-05
3.793627e-
04
ko04961
11
Retrograde endocannabinoid signaling
5 (6.94%)
77 (0.66%)
0.0001082454
5.615230e-
04
ko04723
12
Cholinergic synapse
5 (6.94%)
86 (0.74%)
0.0001827854
8.924228e-
04
ko04725
13
Serotonergic synapse
5 (6.94%)
92 (0.79%)
0.0002508305
1.095733e-
03
ko04726
14
Protein digestion and absorption
5 (6.94%)
122 (1.04%)
0.0009166398
3.732900e-
03
ko04974
15
Cardiac muscle contraction
5 (6.94%)
140 (1.2%)
0.001692799
6.108796e-
03
ko04260
16
MAPK signaling pathway
6 (8.33%)
302 (2.59%)
0.01064676
3.398773e-
02
ko04010
17
Endocytosis
6 (8.33%)
324 (2.77%)
0.01466203
4.507217e-
02
ko04144
1
3N-vs 3N+
63
11680
Proximal tubule bicarbonate reclamation
7 (11.11%)
27 (0.23%)
7.690174e-11
5.998336e-
09
ko04964
2
Carbohydrate digestion and absorption
6 (9.52%)
39 (0.33%)
5.481106e-08
1.654731e-
06
ko04973
3
Phototransduction
7 (11.11%)
67 (0.57%)
6.364351e-08
1.654731e-
06
ko04744
4
Mineral absorption
6 (9.52%)
48 (0.41%)
1.985252e-07
3.871241e-
06
ko04978
5
Aldosterone-regulated sodium reabsorption
5 (7.94%)
35 (0.3%)
1.113271e-06
1.010844e-
05
ko04960
6
GABAergic synapse
6 (9.52%)
86 (0.74%)
6.486509e-06
4.599525e-
05
ko04727
7
Glutamatergic synapse
6 (9.52%)
95 (0.81%)
1.154713e-05
7.505634e-
05
ko04724
8
Endocrine and other factor-regulated calcium
reabsorption
5 (7.94%)
70 (0.6%)
3.590785e-05
2.154471e-
04
ko04961
9
Protein digestion and absorption
6 (9.52%)
122 (1.04%)
4.796446e-05
2.672306e-
04
ko04974
10
Retrograde endocannabinoid signaling
5 (7.94%)
77 (0.66%)
5.694637e-05
2.961211e-
04
ko04723
11
Cholinergic synapse
5 (7.94%)
86 (0.74%)
9.671235e-05
4.714727e-
04
ko04725
12
Serotonergic synapse
5 (7.94%)
92 (0.79%)
0.0001332214
6.112511e-
04
ko04726
13
Chemokine signaling pathway
7 (11.11%)
231 (1.98%)
0.0002331504
1.010318e-
03
ko04062
14
Dopaminergic synapse
6 (9.52%)
178 (1.52%)
0.0003808976
1.555533e-
03
ko04728
15
Arginine and proline metabolism
4 (6.35%)
67 (0.57%)
0.00045676
1.696537e-
03
ko00330
16
Cardiac muscle contraction
5 (7.94%)
140 (1.2%)
0.0009267016
3.142727e-
03
ko04260
17
PPAR signaling pathway
3 (4.76%)
90 (0.77%)
0.01259403
3.929337e-
02
ko03320
1
2N-vs 3N-
45
11680
Phototransduction
9 (20%)
67 (0.57%)
2.900818e-12
1.885532e-
10
ko04744
(continued on next page)
P.A. Olsvik et al.
Table 1 (
1continued )
#
Treatment comparison/Pathway
DEGs with pathway
annotation
All genes with pathway
annotation
p-value
Q-value
Pathway
ID
2N-vs 2N+
72
11680
2
Carbohydrate digestion and absorption
7 (15.56%)
39 (0.33%)
1.084661e-10
3.525148e-
09
ko04973
3
Proximal tubule bicarbonate reclamation
6 (13.33%)
27 (0.23%)
6.445801e-10
1.396590e-
08
ko04964
4
Aldosterone-regulated sodium reabsorption
5 (11.11%)
35 (0.3%)
2.011263e-07
2.614642e-
06
ko04960
5
Mineral absorption
5 (11.11%)
48 (0.41%)
1.022166e-06
9.305999e-
06
ko04978
6
Serotonergic synapse
6 (13.33%)
92 (0.79%)
1.288523e-06
9.305999e-
06
ko04726
7
Chemokine signaling pathway
8 (17.78%)
231 (1.98%)
2.378076e-06
1.405227e-
05
ko04062
8
Protein digestion and absorption
6 (13.33%)
122 (1.04%)
6.701316e-06
3.150043e-
05
ko04974
9
Endocrine and other factor-regulated calcium
reabsorption
5 (11.11%)
70 (0.6%)
6.784707e-06
3.150043e-
05
ko04961
10
Retrograde endocannabinoid signaling
5 (11.11%)
77 (0.66%)
1.085652e-05
4.704492e-
05
ko04723
11
Cholinergic synapse
5 (11.11%)
86 (0.74%)
1.865061e-05
7.131116e-
05
ko04725
12
GABAergic synapse
5 (11.11%)
86 (0.74%)
1.865061e-05
7.131116e-
05
ko04727
13
Glutamatergic synapse
5 (11.11%)
95 (0.81%)
3.024149e-05
1.092054e-
04
ko04724
14
Dopaminergic synapse
6 (13.33%)
178 (1.52%)
5.726626e-05
1.959109e-
04
ko04728
15
Cardiac muscle contraction
5 (11.11%)
140 (1.2%)
0.0001913906
5.923995e-
04
ko04260
16
Retinol metabolism
2 (4.44%)
19 (0.16%)
0.002380689
6.447699e-
03
ko00830
17
Neuroactive ligand-receptor interaction
2 (4.44%)
52 (0.45%)
0.01703389
4.258472e-
02
ko04080
1
2N + vs 3N+
68
11680
Phototransduction
14 (20.59%)
67 (0.57%)
9.57472e-19
5.170349e-
17
ko04744
2
Protein digestion and absorption
16 (23.53%)
122 (1.04%)
6.816708e-18
1.840511e-
16
ko04974
3
Carbohydrate digestion and absorption
8 (11.76%)
39 (0.33%)
4.604135e-11
6.215582e-
10
ko04973
4
Proximal tubule bicarbonate reclamation
7 (10.29%)
27 (0.23%)
1.337376e-10
1.444366e-
09
ko04964
5
Dopaminergic synapse
9 (13.24%)
178 (1.52%)
8.241675e-07
5.454431e-
06
ko04728
6
Aldosterone-regulated sodium reabsorption
5 (7.35%)
35 (0.3%)
1.633423e-06
8.820484e-
06
ko04960
7
ECM-receptor interaction
8 (11.76%)
146 (1.25%)
1.929870e-06
8.874621e-
06
ko04512
8
Chemokine signaling pathway
9 (13.24%)
231 (1.98%)
7.078719e-06
2.940391e-
05
ko04062
9
Mineral absorption
5 (7.35%)
48 (0.41%)
8.12637e-06
3.134457e-
05
ko04978
10
GABAergic synapse
6 (8.82%)
86 (0.74%)
1.014675e-05
3.652830e-
05
ko04727
11
Glutamatergic synapse
6 (8.82%)
95 (0.81%)
1.800377e-05
6.076272e-
05
ko04724
12
Endocrine and other factor-regulated calcium
reabsorption
5 (7.35%)
70 (0.6%)
5.203389e-05
1.652841e-
04
ko04961
13
Retrograde endocannabinoid signaling
5 (7.35%)
77 (0.66%)
8.231658e-05
2.339524e-
04
ko04723
14
Cholinergic synapse
5 (7.35%)
86 (0.74%)
0.000139355
3.762585e-
04
ko04725
15
Serotonergic synapse
5 (7.35%)
92 (0.79%)
0.0001915559
4.925723e-
04
ko04726
16
Arginine and proline metabolism
4 (5.88%)
67 (0.57%)
0.0006112079
1.500238e-
03
ko00330
17
Cardiac muscle contraction
5 (7.35%)
140 (1.2%)
0.001310250
2.948062e-
03
ko04260
18
PPAR signaling pathway
4 (5.88%)
90 (0.77%)
0.001843347
3.828490e-
03
ko03320
19
Focal adhesion
8 (11.76%)
418 (3.58%)
0.002838214
5.676428e-
03
ko04510
P.A. Olsvik et al.
Table 3
3Comparison of predicted upstream regulators in lenses of diploid and triploid Atlantic salmon. Hierarchical clustering based on z-scores as determined with IPA Compare Analysis.Upstream regulator
2N + vs 2N-
3N + vs 3N-
CRX
2.73
− 2.55
GTF2IRD1
2.10
− 1.85
SRC
1.70
− 1.73
RHO
1.89
− 1.63
decitabine
2.77
2.20
IL6
2.25
3.06
P38 MAPK
2.11
1.44
OSM
1.23
2.36
NFkB (complex)
1.34
1.95
APP
1.25
1.94
hexachlorobenzene
2.0
1.98
cisplatin
1.77
1.86
STAT3
1.76
2.20
thioacetamide
1.96
2.21
lipopolysaccharide
2.12
2.32
pioglitazone
− 1.92
curcumin
− 0.73
− 1.71
U0126
− 1.96
− 1.29
LY294002
− 1.67
− 1.67
N-acetyl-L-cysteine
− 1.67
− 0.44
MYC
− 1.40
0.30
CSF2
− 1.48
PD98059
− 1.82
− 0.01
sirolimus
− 2.0
ESR2
− 1.94
0.15
cyclosporin A
1.07
1.39
nitrofurantoin
0.65
1.48
dihydrotestosterone
0.78
1.26
bucladesine
1.27
1.19
beta-estradiol
1.41
1.15
P.A. Olsvik et al.
AcknowledgementWe like to acknowledge the valuable laboratory help from Leikny Fjeldstad (IMR). This project was funded by the Norwegian Research Council (grant number 224816/E40). The fish used in this study were produced in the Norwegian Research Council project INTERACT (grant number 200510).Appendix A. Supplementary dataSupplementary data to this article can be found online at https://doi. org/10.1016/j.exer.2020.108150.
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Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes. A M Terrell, D Anand, S F Smith, C A Dang, S M Waters, M Pathania, D C Beebe, S A Lachke, Exp. Eye Res. 131Terrell, A.M., Anand, D., Smith, S.F., Dang, C.A., Waters, S.M., Pathania, M., Beebe, D.C., Lachke, S.A., 2015. Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes. Exp. Eye Res. 131, 42-55.
Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L). C Tröße, R Waagbø, O Breck, A K Stavrum, K Petersen, P A Olsvik, Mol. Vision. 15Tröße, C., Waagbø, R., Breck, O., Stavrum, A.K., Petersen, K., Olsvik, P.A., 2009. Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L). Mol. Vision 15, 1332-1350.
Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage. C Tröße, R Waagbø, O Breck, P A Olsvik, Fish Physiol. Biochem. 36Tröße, C., Waagbø, R., Breck, O., Olsvik, P.A., 2010. Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage. Fish Physiol. Biochem. 36, 1217-1225.
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Apolipoprotein E genotype and risk for development of cataract and age-related macular degeneration. O A Utheim, J S Ritland, T P Utheim, T Espeseth, S Lydersen, H Rootwelt, S O Semb, T Elsas, Acta Ophthalmol. 86Utheim, O.A., Ritland, J.S., Utheim, T.P., Espeseth, T., Lydersen, S., Rootwelt, H., Semb, S.O., Elsas, T., 2008. Apolipoprotein E genotype and risk for development of cataract and age-related macular degeneration. Acta Ophthalmol. 86, 401-403.
Nutritional status assessed in groups of smolting Atlantic salmon, Salmo salar L., developing cataracts. R Waagbø, E Bjerkås, H Sveier, O Breck, E Bjørnestad, A Maage, J. Fish. Dis. 19Waagbø, R., Bjerkås, E., Sveier, H., Breck, O., Bjørnestad, E., Maage, A., 1996. Nutritional status assessed in groups of smolting Atlantic salmon, Salmo salar L., developing cataracts. J. Fish. Dis. 19, 365-373.
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Dietary histidine supplementation prevents cataract development in adult Atlantic salmon, Salmo salar L., in seawater. R Waagbø, C Troße, W Koppe, R Fontanillas, O Breck, Br. J. Nutr. 104Waagbø, R., Troße, C., Koppe, W., Fontanillas, R., Breck, O., 2010. Dietary histidine supplementation prevents cataract development in adult Atlantic salmon, Salmo salar L., in seawater. Br. J. Nutr. 104, 1460-1470.
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Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. J Weng, J Luo, X Cheng, C Jin, X Zhou, J Qu, L Tu, D Ai, D Li, J Wang, J F Martin, B A Amendt, M Liu, Proc. Natl. Acad. Sci. U. S. A. 105Weng, J., Luo, J., Cheng, X., Jin, C., Zhou, X., Qu, J., Tu, L., Ai, D., Li, D., Wang, J., Martin, J.F., Amendt, B.A., Liu, M., 2008. Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. Proc. Natl. Acad. Sci. U. S. A. 105, 6081-6086.
Oxidation, antioxidants and cataract formation: a literature review. D L Williams, Vet. Ophtamol. 9Williams, D.L., 2006. Oxidation, antioxidants and cataract formation: a literature review. Vet. Ophtamol. 9, 292-298.
Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. G Wistow, S L Bernstein, M K Wyatt, A Behal, J W Touchman, G Bouffard, D Smith, K Peterson, Mol. Vision. 8Wistow, G., Bernstein, S.L., Wyatt, M.K., Behal, A., Touchman, J.W., Bouffard, G., Smith, D., Peterson, K., 2002. Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. Mol. Vision 8, 171-184.
The NEIBank project for ocular genomics: data-mining gene expression in human and rodent eye tissues. G Wistow, Prog. Retin. Eye Res. 25Wistow, G., 2006. The NEIBank project for ocular genomics: data-mining gene expression in human and rodent eye tissues. Prog. Retin. Eye Res. 25, 43-77.
Expression profiling and gene discovery in the mouse lens. M A Wride, F C Mansergh, S Adams, R Everitt, S E Minnema, D E Rancourt, M J Evans, Mol. Vision. 9Wride, M.A., Mansergh, F.C., Adams, S., Everitt, R., Minnema, S.E., Rancourt, D.E., Evans, M.J., 2003. Expression profiling and gene discovery in the mouse lens. Mol. Vision 9, 360-396.
Control of transcription by cell size. C Y Wu, P A Rolfe, D K Gifford, G R Fink, PLoS Biol. 81000523Wu, C.Y., Rolfe, P.A., Gifford, D.K., Fink, G.R., 2010. Control of transcription by cell size. PLoS Biol. 8, e1000523.
Expression and characterization of SPARC in human lens and in the aqueous and vitreous humors. Q Yan, J I Clark, E H Sage, Exp. Eye Res. 71Yan, Q., Clark, J.I., Sage, E.H., 2000. Expression and characterization of SPARC in human lens and in the aqueous and vitreous humors. Exp. Eye Res. 71, 81-90.
WEGO: a web tool for plotting GO annotations. J Ye, L Fang, H Zheng, Y Zhang, J Chen, Z Zhang, J Wang, S Li, R Li, L Bolund, J Wang, Nucleic Acids Res. 34Ye, J., Fang, L., Zheng, H., Zhang, Y., Chen, J., Zhang, Z., Wang, J., Li, S., Li, R., Bolund, L., Wang, J., 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293-W297.
| [
"A B S T R A C TTo avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon."
] | [
"Pål A Olsvik \nFaculty of Biosciences and Aquaculture\nNord University\nBodøNorway\n\nInstitute of Marine Research\nNordnes, BergenNorway\n",
"Roderick Nigel Finn \nDepartment of Biological Sciences\nUniversity of Bergen\nBergenNorway\n\nIRTA-Institute of Biotechnology and Biomedicine (IBB)\nUniversitat Autònoma de Barcelona\n08193Cerdanyola del VallèsBellaterraSpain\n",
"Sofie C Remø \nInstitute of Marine Research\nNordnes, BergenNorway\n",
"PerG Fjelldal \nInstitute of Marine Research\nNordnes, BergenNorway\n",
"François Chauvigné \nIRTA-Institute of Biotechnology and Biomedicine (IBB)\nUniversitat Autònoma de Barcelona\n08193Cerdanyola del VallèsBellaterraSpain\n",
"Kevin A Glover \nInstitute of Marine Research\nNordnes, BergenNorway\n\nDepartment of Biological Sciences\nUniversity of Bergen\nBergenNorway\n",
"Tom Hansen \nInstitute of Marine Research\nNordnes, BergenNorway\n",
"Rune Waagbø \nInstitute of Marine Research\nNordnes, BergenNorway\n\nDepartment of Biological Sciences\nUniversity of Bergen\nBergenNorway\n"
] | [
"Faculty of Biosciences and Aquaculture\nNord University\nBodøNorway",
"Institute of Marine Research\nNordnes, BergenNorway",
"Department of Biological Sciences\nUniversity of Bergen\nBergenNorway",
"IRTA-Institute of Biotechnology and Biomedicine (IBB)\nUniversitat Autònoma de Barcelona\n08193Cerdanyola del VallèsBellaterraSpain",
"Institute of Marine Research\nNordnes, BergenNorway",
"Institute of Marine Research\nNordnes, BergenNorway",
"IRTA-Institute of Biotechnology and Biomedicine (IBB)\nUniversitat Autònoma de Barcelona\n08193Cerdanyola del VallèsBellaterraSpain",
"Institute of Marine Research\nNordnes, BergenNorway",
"Department of Biological Sciences\nUniversity of Bergen\nBergenNorway",
"Institute of Marine Research\nNordnes, BergenNorway",
"Institute of Marine Research\nNordnes, BergenNorway",
"Department of Biological Sciences\nUniversity of Bergen\nBergenNorway"
] | [
"Pål",
"A",
"Roderick",
"Nigel",
"Sofie",
"C",
"G",
"François",
"Kevin",
"A",
"Tom",
"Rune"
] | [
"Olsvik",
"Finn",
"Remø",
"Fjelldal",
"Chauvigné",
"Glover",
"Hansen",
"Waagbø"
] | [
"J Bai, ",
"Y Zheng, ",
"L Dong, ",
"X Cai, ",
"G Wang, ",
"P Liu, ",
"S Bassnett, ",
"T J Benfey, ",
"A M Sutterlin, ",
"R J Thompson, ",
"T J Benfey, ",
"T J Benfey, ",
"B Bicskei, ",
"J E Bron, ",
"K A Glover, ",
"J B Taggart, ",
"B Bicskei, ",
"J B Taggart, ",
"K A Glover, ",
"J E Bron, ",
"E Bjerkås, ",
"R Waagbø, ",
"H Sveier, ",
"I Bjerkås, ",
"E Bjørnestad, ",
"A Maage, ",
"E Bjerkås, ",
"O Breck, ",
"R Waagbø, ",
"A D Bradshaw, ",
"O Breck, ",
"H Sveier, ",
"O Breck, ",
"E Bjerkås, ",
"P Campbell, ",
"P Arnesen, ",
"P Haldorsen, ",
"R Waagbø, ",
"O Breck, ",
"E Bjerkas, ",
"J Sanderson, ",
"R Waagbo, ",
"P Campbell, ",
"O Breck, ",
"E Bjerkås, ",
"P Campbell, ",
"J D Rhodes, ",
"J Sanderson, ",
"R Waagbø, ",
"L A Brennan, ",
"M Kantorow, ",
"C G Chamberlain, ",
"K J Mansfield, ",
"A Cerra, ",
"S Chatchaiphan, ",
"P Srisapoome, ",
"J H Kim, ",
"R H Devlin, ",
"U Na-Nakorn, ",
"J Chen, ",
"Q Xu, ",
"T Wang, ",
"B Collet, ",
"Y Corripio-Miyar, ",
"S Bird, ",
"P Xie, ",
"P Nie, ",
"C J Secombes, ",
"J Zou, ",
"Y Chen, ",
"Q Wu, ",
"A Miao, ",
"Y Jiang, ",
"X Wu, ",
"Z Wang, ",
"F Wu, ",
"Y Lu, ",
"A Conesa, ",
"S Gotz, ",
"J M Garcia-Gomez, ",
"J Terol, ",
"M Talon, ",
"M Robles, ",
"K A Christensen, ",
"D Sakhrani, ",
"E B Rondeau, ",
"J Richards, ",
"B F Koop, ",
"R H Devlin, ",
"S Dhakal, ",
"C B Stevens, ",
"M Sebbagh, ",
"O Weiss, ",
"R A Frey, ",
"S Adamson, ",
"E A Shelden, ",
"A Inbal, ",
"D L Stenkamp, ",
"J M Enright, ",
"M B Toomey, ",
"S Y Sato, ",
"S E Temple, ",
"J R Allen, ",
"R Fujiwara, ",
"V M Kramlinger, ",
"L D Nagy, ",
"K M Johnson, ",
"Y Xiao, ",
"M J How, ",
"S L Johnson, ",
"N W Roberts, ",
"V J Kefalov, ",
"F P Guengerich, ",
"J C Corbo, ",
"Y Gao, ",
"S Liu, ",
"J Huang, ",
"W Guo, ",
"J Chen, ",
"L Zhang, ",
"B Zhao, ",
"J Peng, ",
"A Wang, ",
"Y Wang, ",
"W Xu, ",
"S Lu, ",
"M Yuan, ",
"Q Guo, ",
"D T Gilmour, ",
"G J Lyon, ",
"M B Carlton, ",
"J R Sanes, ",
"J M Cunningham, ",
"J R Anderson, ",
"B L Hogan, ",
"M J Evans, ",
"W H Colledge, ",
"S Gleim, ",
"A Stojanovic, ",
"E Arehart, ",
"D Byington, ",
"J Hwa, ",
"K A Glover, ",
"M F Solberg, ",
"P Mcginnity, ",
"K Hindar, ",
"E Verspoor, ",
"M W Coulson, ",
"M M Hansen, ",
"H Araki, ",
"Ø Skaala, ",
"T Svåsand, ",
"M G Grabherr, ",
"B J Haas, ",
"M Yassour, ",
"J Z Levin, ",
"D A Thompson, ",
"I Amit, ",
"X Adiconis, ",
"L Fan, ",
"R Raychowdhury, ",
"Q D Zeng, ",
"Z H Chen, ",
"E Mauceli, ",
"N Hacohen, ",
"A Gnirke, ",
"N Rhind, ",
"F Di Palma, ",
"B W Birren, ",
"C Nusbaum, ",
"K Lindblad-Toh, ",
"N Friedman, ",
"A Regev, ",
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"B Stone, ",
"J I Clark, ",
"F G Hamel, ",
"J L Upward, ",
"G L Siford, ",
"W C Duckworth, ",
"A C Harvey, ",
"P G Fjelldal, ",
"M F Solberg, ",
"T Hansen, ",
"K A Glover, ",
"J R Hawse, ",
"J F Hejtmancik, ",
"Q Huang, ",
"N L Sheets, ",
"D A Hosack, ",
"R A Lempicki, ",
"J Horwitz, ",
"M Kantorow, ",
"J F Hejtmancik, ",
"J F Hejtmancik, ",
"S A Riazuddin, ",
"R Mcgreal, ",
"W Liu, ",
"A Cvekl, ",
"A Shiels, ",
"C Iseli, ",
"C V Jongeneel, ",
"P Bucher, ",
"Y Ito, ",
"T Nabekura, ",
"M Takeda, ",
"M Nakao, ",
"M Terao, ",
"R Hori, ",
"M Tomohiro, ",
"L Iyengar, ",
"Q Wang, ",
"J E Rasko, ",
"J W Mcavoy, ",
"F J Lovicu, ",
"D G Jennen, ",
"C Magkoufopoulou, ",
"H B Ketelslegers, ",
"M H Van Herwijnen, ",
"J C Kleinjans, ",
"J H Van Delft, ",
"Y Ji, ",
"X Rong, ",
"H Ye, ",
"K Zhang, ",
"Y Lu, ",
"A Kakrana, ",
"A Yang, ",
"D Anand, ",
"D Djordjevic, ",
"D Ramachandruni, ",
"A Singh, ",
"H Huang, ",
"J W K Ho, ",
"S A Lachke, ",
"H G Ketola, ",
"A R Lenox, ",
"Y Bhootada, ",
"O Gorbatyuk, ",
"R Fullard, ",
"M Gorbatyuk, ",
"M F Lou, ",
"F C Mansergh, ",
"M A Wride, ",
"V E Walker, ",
"S Adams, ",
"S M Hunter, ",
"M J Evans, ",
"A L Manthey, ",
"A M Terrell, ",
"S A Lachke, ",
"S W Polson, ",
"M K Duncan, ",
"M Medvedovic, ",
"C R Tomlinson, ",
"M K Call, ",
"M Grogg, ",
"P A Tsonis, ",
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"C G Carter, ",
"R Wilson, ",
"I R Cooke, ",
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"P D Nichols, ",
"K Ornek, ",
"F Karel, ",
"Z Buyukbingol, ",
"S Ottonello, ",
"C Foronia, ",
"A Cartab, ",
"S Petruccob, ",
"G Maraini, ",
"F Piferrer, ",
"A Beaumont, ",
"J.-C Falguiere, ",
"M Flajshans, ",
"P Haffray, ",
"L Colombo, ",
"M Posner, ",
"M S Mcdonald, ",
"K L Murray, ",
"A J Kiss, ",
"S C Remø, ",
"P A Olsvik, ",
"B E Torstensen, ",
"H Amlund, ",
"O Breck, ",
"R Waagbø, ",
"S C Remø, ",
"E M Hevrøy, ",
"P A Olsvik, ",
"R Fontanillas, ",
"O Breck, ",
"R Waagbø, ",
"S C Remø, ",
"E M Hevrøy, ",
"O Breck, ",
"P A Olsvik, ",
"R Waagbø, ",
"J D Rhodes, ",
"O Breck, ",
"R Waagbø, ",
"E Bjerkås, ",
"J Sanderson, ",
"J T Rosenbaum, ",
"J R Samples, ",
"B Seymour, ",
"L Langlois, ",
"L David, ",
"F Sambraus, ",
"P G Fjelldal, ",
"S C Remø, ",
"E M Hevrøy, ",
"T O Nilsen, ",
"A Thorsen, ",
"T J Hansen, ",
"R Waagbø, ",
"L M Sanderson, ",
"P J De Groot, ",
"G J Hooiveld, ",
"A Koppen, ",
"E Kalkhoven, ",
"M Muller, ",
"S Kersten, ",
"E H Schneider, ",
"J D Weaver, ",
"S S Gaur, ",
"B K Tripathi, ",
"A J Jesaitis, ",
"P S Zelenka, ",
"J L Gao, ",
"P M Murphy, ",
"Y Shichida, ",
"T Yamashita, ",
"A Shiels, ",
"T M Bennett, ",
"J F Hejtmancik, ",
"A Shiels, ",
"J F Hejtmancik, ",
"A Shiels, ",
"J F Hejtmancik, ",
"C Slingsby, ",
"G J Wistow, ",
"A R Clark, ",
"S A Small, ",
"T J Benfey, ",
"K Sousounis, ",
"P A Tsonis, ",
"L H Stien, ",
"P A Saeter, ",
"T Kristiansen, ",
"P G Fjelldal, ",
"F Sambraus, ",
"A Stojanovic, ",
"J Stitham, ",
"J Hwa, ",
"H Swarup, ",
"G L Taranger, ",
"M Carrillo, ",
"R W Schulz, ",
"P Fontaine, ",
"S Zanuy, ",
"A Felip, ",
"F A Weltzien, ",
"S Dufour, ",
"O Karlsen, ",
"B Norberg, ",
"E Andersson, ",
"T Hansen, ",
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"J Hanrieder, ",
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"A Alm, ",
"J Bergquist, ",
"A Taylor, ",
"K J A Davies, ",
"J F Taylor, ",
"R Waagbø, ",
"M Diez-Padrisa, ",
"P Campbell, ",
"J Walton, ",
"D Hunter, ",
"C Matthew, ",
"H Migaud, ",
"A M Terrell, ",
"D Anand, ",
"S F Smith, ",
"C A Dang, ",
"S M Waters, ",
"M Pathania, ",
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"S A Lachke, ",
"C Tröße, ",
"R Waagbø, ",
"O Breck, ",
"A K Stavrum, ",
"K Petersen, ",
"P A Olsvik, ",
"C Tröße, ",
"R Waagbø, ",
"O Breck, ",
"P A Olsvik, ",
"L Urbak, ",
"H Vorum, ",
"O A Utheim, ",
"J S Ritland, ",
"T P Utheim, ",
"T Espeseth, ",
"S Lydersen, ",
"H Rootwelt, ",
"S O Semb, ",
"T Elsas, ",
"R Waagbø, ",
"E Bjerkås, ",
"H Sveier, ",
"O Breck, ",
"E Bjørnestad, ",
"A Maage, ",
"R Waagbø, ",
"H Sveier, ",
"O Breck, ",
"E Bjørnestad, ",
"A Maage, ",
"E Bjerkås, ",
"R Waagbø, ",
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"W Koppe, ",
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"G Bouffard, ",
"D Smith, ",
"K Peterson, ",
"G Wistow, ",
"M A Wride, ",
"F C Mansergh, ",
"S Adams, ",
"R Everitt, ",
"S E Minnema, ",
"D E Rancourt, ",
"M J Evans, ",
"C Y Wu, ",
"P A Rolfe, ",
"D K Gifford, ",
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"J Chen, ",
"Z Zhang, ",
"J Wang, ",
"S Li, ",
"R Li, ",
"L Bolund, ",
"J Wang, "
] | [
"J",
"Y",
"L",
"X",
"G",
"P",
"S",
"T",
"J",
"A",
"M",
"R",
"J",
"T",
"J",
"T",
"J",
"B",
"J",
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"Inhibition of p38 mitogenactivated protein kinase phosphorylation decreases H(2)O(2)-induced apoptosis in human lens epithelial cells. J Bai, Y Zheng, L Dong, X Cai, G Wang, P Liu, Graefes Arch. Clin. Exp. Ophthalmol. 253Bai, J., Zheng, Y., Dong, L., Cai, X., Wang, G., Liu, P., 2015. Inhibition of p38 mitogen- activated protein kinase phosphorylation decreases H(2)O(2)-induced apoptosis in human lens epithelial cells. Graefes Arch. Clin. Exp. Ophthalmol. 253, 1933-1940.",
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"The physiology and behavior of triploid fishes. T J Benfey, Rev. Fish. Sci. 7Benfey, T.J., 1999. The physiology and behavior of triploid fishes. Rev. Fish. Sci. 7, 39-67.",
"Effectiveness of triploidy as a management tool for reproductive containment of farmed fish: Atlantic salmon (Salmo salar) as a case study. T J Benfey, Rev. Aquacult. 83Benfey, T.J., 2016. Effectiveness of triploidy as a management tool for reproductive containment of farmed fish: Atlantic salmon (Salmo salar) as a case study. Rev. Aquacult. 8 (3), 264-282.",
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"327",
"(Conesa et al., 2005;",
"Ye et al., 2006)",
"clu, gal, igfbp2, junb, krt18, lep, mmp2, plaur, rbp4",
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"2N+ and 3N+ anxa2,",
"anxa5,",
"arl14ep,",
"arrb2,",
"atp1a3,",
"atp1a4,",
"btbd17,",
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"rtbdn,",
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"ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences",
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"Comparison of HepG2 and HepaRG by whole-genome gene expression analysis for the purpose of chemical hazard identification",
"Proteomic analysis of aqueous humor proteins associated with cataract development",
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"Development of novel filtering criteria to analyze RNA-sequencing data obtained from the murine ocular lens during embryogenesis",
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"Triploid Atlantic salmon shows similar performance, fatty acid composition and proteome response to diploids during early freshwater rearing",
"May nitric oxide molecule have a role in the pathogenesis of human cataract? Exp",
"Oxidative stress and age-related cataract",
"Polyploid fish and shellfish: production, biology and applications to aquaculture for performance improvement and genetic containment",
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"Dietary histidine requirement to reduce the risk and severity of cataracts is higher than the requirement for growth in Atlantic salmon smolts, independently of the dietary lipid source",
"Lens metabolomic profiling as a tool to understand cataractogenesis in Atlantic salmon and rainbow trout reared at optimum and high temperature",
"N-acetylhistidine, a novel osmolyte in the lens of Atlantic salmon (Salmo salar L.)",
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"Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage",
"Heat shock proteins in the human eye",
"Apolipoprotein E genotype and risk for development of cataract and age-related macular degeneration",
"Nutritional status assessed in groups of smolting Atlantic salmon, Salmo salar L., developing cataracts",
"Cataract formation in smolting Atlantic salmon, Salmo salar, fed low and high energy diets",
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"Expression profiling and gene discovery in the mouse lens",
"Control of transcription by cell size",
"Expression and characterization of SPARC in human lens and in the aqueous and vitreous humors",
"WEGO: a web tool for plotting GO annotations"
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"\nFig. 1 .\n1Heart concentrations of A) L-histidine and B) N-acetyl-L-histidine in diploid and triploid Atlantic salmon. Significance levels of two-factor analyses are shown in figures (2-way ANOVA). Capped lines indicate significance levels of direct comparisons of the compounds in 2N and 3N fish (uncorrected Fisher's test, P < 0.05). ns = not significant.",
"\nFig. 2 .\n2Cataract score of the Atlantic salmon lenses used to A) generate a lens transcriptome and B) quantification of DEGs. C) Number of DEGs in lenses of diploid (2N) and triploid (3N) Atlantic salmon without (− ) and with cataracts (+). Exact numbers of genes are given above the individual bars. D) Venn diagram showing the degree of overlap of DEGs in the four treatment groups based on a four-way comparison of genes.",
"\nFig. 3 .\n3Six DEGs encoding protein involved in phototransduction (KEGG pathway ko04744) were up-regulated in cataractous lenses from diploid (2N+) Atlantic salmon (A) and down-regulated in cataractous lenses from triploid (3N+) Atlantic salmon (B). Border color indicates up-regulated genes (red) and down-regulated genes (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) may be affected in some of the lenses even without visible signs of damage.",
"\nFig. 4 .\n4Predicted upstream regulator network with highest consistency score based on IPA Compare Analysis, generated by comparison of DEGs in cataractous lenses of diploid and triploid Atlantic salmon (2N + vs 3N+). Up-regulated and down-regulated genes are highlighted in red and green, while predicted activated and inhibited regulators and relationships are highlighted in orange and blue, respectively. The color depth is correlated to the fold change. Solid lines show direct interactions between two gene products, while dashed lines represent indirect interactions among genes shown in the network. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)",
"\nTable 1\n1KEGG pathway enrichment analysis of DEGs.# \nTreatment comparison/Pathway \nDEGs with pathway \nannotation \n\nAll genes with pathway \nannotation \n\np-value \nQ-value \nPathway \nID \n\n2N-vs 2N+ \n72 \n11680 \n\n1 \nPhototransduction \n13 (18.06%) \n67 (0.57%) \n1.161234e-16 \n9.638242e-\n15 \n\nko04744 \n\n2 \nCarbohydrate digestion and absorption \n8 (11.11%) \n39 (0.33%) \n7.384585e-11 \n3.064603e-\n09 \n\nko04973 \n\n3 \nProximal tubule bicarbonate reclamation \n7 (9.72%) \n27 (0.23%) \n2.019998e-10 \n5.588661e-\n09 \n\nko04964 \n\n4 \nMineral absorption \n6 (8.33%) \n48 (0.41%) \n4.43982e-07 \n7.370101e-\n06 \n\nko04978 \n\n5 \nGlutamatergic synapse \n7 (9.72%) \n95 (0.81%) \n1.803348e-06 \n1.663088e-\n05 \n\nko04724 \n\n6 \nAldosterone-regulated sodium reabsorption \n5 (6.94%) \n35 (0.3%) \n2.173693e-06 \n1.804165e-\n05 \n\nko04960 \n\n7 \nChemokine signaling pathway \n9 (12.5%) \n231 (1.98%) \n1.141543e-05 \n7.895672e-\n05 \n\nko04062 \n\n8 \nDopaminergic synapse \n8 (11.11%) \n178 (1.52%) \n1.295758e-05 \n8.272916e-\n05 \n\nko04728 \n\n9 \nGABAergic synapse \n6 (8.33%) \n86 (0.74%) \n1.414778e-05 \n8.387612e-\n05 \n\nko04727 \n\n10 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (6.94%) \n70 (0.6%) \n6.855952e-05 \n3.793627e-\n04 \n\nko04961 \n\n11 \nRetrograde endocannabinoid signaling \n5 (6.94%) \n77 (0.66%) \n0.0001082454 \n5.615230e-\n04 \n\nko04723 \n\n12 \nCholinergic synapse \n5 (6.94%) \n86 (0.74%) \n0.0001827854 \n8.924228e-\n04 \n\nko04725 \n\n13 \nSerotonergic synapse \n5 (6.94%) \n92 (0.79%) \n0.0002508305 \n1.095733e-\n03 \n\nko04726 \n\n14 \nProtein digestion and absorption \n5 (6.94%) \n122 (1.04%) \n0.0009166398 \n3.732900e-\n03 \n\nko04974 \n\n15 \nCardiac muscle contraction \n5 (6.94%) \n140 (1.2%) \n0.001692799 \n6.108796e-\n03 \n\nko04260 \n\n16 \nMAPK signaling pathway \n6 (8.33%) \n302 (2.59%) \n0.01064676 \n3.398773e-\n02 \n\nko04010 \n\n17 \nEndocytosis \n6 (8.33%) \n324 (2.77%) \n0.01466203 \n4.507217e-\n02 \n\nko04144 \n\n1 \n3N-vs 3N+ \n63 \n11680 \nProximal tubule bicarbonate reclamation \n7 (11.11%) \n27 (0.23%) \n7.690174e-11 \n5.998336e-\n09 \n\nko04964 \n\n2 \nCarbohydrate digestion and absorption \n6 (9.52%) \n39 (0.33%) \n5.481106e-08 \n1.654731e-\n06 \n\nko04973 \n\n3 \nPhototransduction \n7 (11.11%) \n67 (0.57%) \n6.364351e-08 \n1.654731e-\n06 \n\nko04744 \n\n4 \nMineral absorption \n6 (9.52%) \n48 (0.41%) \n1.985252e-07 \n3.871241e-\n06 \n\nko04978 \n\n5 \nAldosterone-regulated sodium reabsorption \n5 (7.94%) \n35 (0.3%) \n1.113271e-06 \n1.010844e-\n05 \n\nko04960 \n\n6 \nGABAergic synapse \n6 (9.52%) \n86 (0.74%) \n6.486509e-06 \n4.599525e-\n05 \n\nko04727 \n\n7 \nGlutamatergic synapse \n6 (9.52%) \n95 (0.81%) \n1.154713e-05 \n7.505634e-\n05 \n\nko04724 \n\n8 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (7.94%) \n70 (0.6%) \n3.590785e-05 \n2.154471e-\n04 \n\nko04961 \n\n9 \nProtein digestion and absorption \n6 (9.52%) \n122 (1.04%) \n4.796446e-05 \n2.672306e-\n04 \n\nko04974 \n\n10 \nRetrograde endocannabinoid signaling \n5 (7.94%) \n77 (0.66%) \n5.694637e-05 \n2.961211e-\n04 \n\nko04723 \n\n11 \nCholinergic synapse \n5 (7.94%) \n86 (0.74%) \n9.671235e-05 \n4.714727e-\n04 \n\nko04725 \n\n12 \nSerotonergic synapse \n5 (7.94%) \n92 (0.79%) \n0.0001332214 \n6.112511e-\n04 \n\nko04726 \n\n13 \nChemokine signaling pathway \n7 (11.11%) \n231 (1.98%) \n0.0002331504 \n1.010318e-\n03 \n\nko04062 \n\n14 \nDopaminergic synapse \n6 (9.52%) \n178 (1.52%) \n0.0003808976 \n1.555533e-\n03 \n\nko04728 \n\n15 \nArginine and proline metabolism \n4 (6.35%) \n67 (0.57%) \n0.00045676 \n1.696537e-\n03 \n\nko00330 \n\n16 \nCardiac muscle contraction \n5 (7.94%) \n140 (1.2%) \n0.0009267016 \n3.142727e-\n03 \n\nko04260 \n\n17 \nPPAR signaling pathway \n3 (4.76%) \n90 (0.77%) \n0.01259403 \n3.929337e-\n02 \n\nko03320 \n\n1 \n2N-vs 3N-\n45 \n11680 \nPhototransduction \n9 (20%) \n67 (0.57%) \n2.900818e-12 \n1.885532e-\n10 \n\nko04744 \n\n(continued on next page) \n\nP.A. Olsvik et al. \n",
"\nTable 1 (\n1continued ) \n\n# \nTreatment comparison/Pathway \nDEGs with pathway \nannotation \n\nAll genes with pathway \nannotation \n\np-value \nQ-value \nPathway \nID \n\n2N-vs 2N+ \n72 \n11680 \n\n2 \nCarbohydrate digestion and absorption \n7 (15.56%) \n39 (0.33%) \n1.084661e-10 \n3.525148e-\n09 \n\nko04973 \n\n3 \nProximal tubule bicarbonate reclamation \n6 (13.33%) \n27 (0.23%) \n6.445801e-10 \n1.396590e-\n08 \n\nko04964 \n\n4 \nAldosterone-regulated sodium reabsorption \n5 (11.11%) \n35 (0.3%) \n2.011263e-07 \n2.614642e-\n06 \n\nko04960 \n\n5 \nMineral absorption \n5 (11.11%) \n48 (0.41%) \n1.022166e-06 \n9.305999e-\n06 \n\nko04978 \n\n6 \nSerotonergic synapse \n6 (13.33%) \n92 (0.79%) \n1.288523e-06 \n9.305999e-\n06 \n\nko04726 \n\n7 \nChemokine signaling pathway \n8 (17.78%) \n231 (1.98%) \n2.378076e-06 \n1.405227e-\n05 \n\nko04062 \n\n8 \nProtein digestion and absorption \n6 (13.33%) \n122 (1.04%) \n6.701316e-06 \n3.150043e-\n05 \n\nko04974 \n\n9 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (11.11%) \n70 (0.6%) \n6.784707e-06 \n3.150043e-\n05 \n\nko04961 \n\n10 \nRetrograde endocannabinoid signaling \n5 (11.11%) \n77 (0.66%) \n1.085652e-05 \n4.704492e-\n05 \n\nko04723 \n\n11 \nCholinergic synapse \n5 (11.11%) \n86 (0.74%) \n1.865061e-05 \n7.131116e-\n05 \n\nko04725 \n\n12 \nGABAergic synapse \n5 (11.11%) \n86 (0.74%) \n1.865061e-05 \n7.131116e-\n05 \n\nko04727 \n\n13 \nGlutamatergic synapse \n5 (11.11%) \n95 (0.81%) \n3.024149e-05 \n1.092054e-\n04 \n\nko04724 \n\n14 \nDopaminergic synapse \n6 (13.33%) \n178 (1.52%) \n5.726626e-05 \n1.959109e-\n04 \n\nko04728 \n\n15 \nCardiac muscle contraction \n5 (11.11%) \n140 (1.2%) \n0.0001913906 \n5.923995e-\n04 \n\nko04260 \n\n16 \nRetinol metabolism \n2 (4.44%) \n19 (0.16%) \n0.002380689 \n6.447699e-\n03 \n\nko00830 \n\n17 \nNeuroactive ligand-receptor interaction \n2 (4.44%) \n52 (0.45%) \n0.01703389 \n4.258472e-\n02 \n\nko04080 \n\n1 \n2N + vs 3N+ \n68 \n11680 \nPhototransduction \n14 (20.59%) \n67 (0.57%) \n9.57472e-19 \n5.170349e-\n17 \n\nko04744 \n\n2 \nProtein digestion and absorption \n16 (23.53%) \n122 (1.04%) \n6.816708e-18 \n1.840511e-\n16 \n\nko04974 \n\n3 \nCarbohydrate digestion and absorption \n8 (11.76%) \n39 (0.33%) \n4.604135e-11 \n6.215582e-\n10 \n\nko04973 \n\n4 \nProximal tubule bicarbonate reclamation \n7 (10.29%) \n27 (0.23%) \n1.337376e-10 \n1.444366e-\n09 \n\nko04964 \n\n5 \nDopaminergic synapse \n9 (13.24%) \n178 (1.52%) \n8.241675e-07 \n5.454431e-\n06 \n\nko04728 \n\n6 \nAldosterone-regulated sodium reabsorption \n5 (7.35%) \n35 (0.3%) \n1.633423e-06 \n8.820484e-\n06 \n\nko04960 \n\n7 \nECM-receptor interaction \n8 (11.76%) \n146 (1.25%) \n1.929870e-06 \n8.874621e-\n06 \n\nko04512 \n\n8 \nChemokine signaling pathway \n9 (13.24%) \n231 (1.98%) \n7.078719e-06 \n2.940391e-\n05 \n\nko04062 \n\n9 \nMineral absorption \n5 (7.35%) \n48 (0.41%) \n8.12637e-06 \n3.134457e-\n05 \n\nko04978 \n\n10 \nGABAergic synapse \n6 (8.82%) \n86 (0.74%) \n1.014675e-05 \n3.652830e-\n05 \n\nko04727 \n\n11 \nGlutamatergic synapse \n6 (8.82%) \n95 (0.81%) \n1.800377e-05 \n6.076272e-\n05 \n\nko04724 \n\n12 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (7.35%) \n70 (0.6%) \n5.203389e-05 \n1.652841e-\n04 \n\nko04961 \n\n13 \nRetrograde endocannabinoid signaling \n5 (7.35%) \n77 (0.66%) \n8.231658e-05 \n2.339524e-\n04 \n\nko04723 \n\n14 \nCholinergic synapse \n5 (7.35%) \n86 (0.74%) \n0.000139355 \n3.762585e-\n04 \n\nko04725 \n\n15 \nSerotonergic synapse \n5 (7.35%) \n92 (0.79%) \n0.0001915559 \n4.925723e-\n04 \n\nko04726 \n\n16 \nArginine and proline metabolism \n4 (5.88%) \n67 (0.57%) \n0.0006112079 \n1.500238e-\n03 \n\nko00330 \n\n17 \nCardiac muscle contraction \n5 (7.35%) \n140 (1.2%) \n0.001310250 \n2.948062e-\n03 \n\nko04260 \n\n18 \nPPAR signaling pathway \n4 (5.88%) \n90 (0.77%) \n0.001843347 \n3.828490e-\n03 \n\nko03320 \n\n19 \nFocal adhesion \n8 (11.76%) \n418 (3.58%) \n0.002838214 \n5.676428e-\n03 \n\nko04510 \n\nP.A. Olsvik et al. \n",
"\nTable 3\n3Comparison of predicted upstream regulators in lenses of diploid and triploid Atlantic salmon. Hierarchical clustering based on z-scores as determined with IPA Compare Analysis.Upstream regulator \n2N + vs 2N-\n3N + vs 3N-\n\nCRX \n2.73 \n− 2.55 \nGTF2IRD1 \n2.10 \n− 1.85 \nSRC \n1.70 \n− 1.73 \nRHO \n1.89 \n− 1.63 \ndecitabine \n2.77 \n2.20 \nIL6 \n2.25 \n3.06 \nP38 MAPK \n2.11 \n1.44 \nOSM \n1.23 \n2.36 \nNFkB (complex) \n1.34 \n1.95 \nAPP \n1.25 \n1.94 \nhexachlorobenzene \n2.0 \n1.98 \ncisplatin \n1.77 \n1.86 \nSTAT3 \n1.76 \n2.20 \nthioacetamide \n1.96 \n2.21 \nlipopolysaccharide \n2.12 \n2.32 \npioglitazone \n− 1.92 \ncurcumin \n− 0.73 \n− 1.71 \nU0126 \n− 1.96 \n− 1.29 \nLY294002 \n− 1.67 \n− 1.67 \nN-acetyl-L-cysteine \n− 1.67 \n− 0.44 \nMYC \n− 1.40 \n0.30 \nCSF2 \n− 1.48 \nPD98059 \n− 1.82 \n− 0.01 \nsirolimus \n− 2.0 \nESR2 \n− 1.94 \n0.15 \ncyclosporin A \n1.07 \n1.39 \nnitrofurantoin \n0.65 \n1.48 \ndihydrotestosterone \n0.78 \n1.26 \nbucladesine \n1.27 \n1.19 \nbeta-estradiol \n1.41 \n1.15 \n"
] | [
"Heart concentrations of A) L-histidine and B) N-acetyl-L-histidine in diploid and triploid Atlantic salmon. Significance levels of two-factor analyses are shown in figures (2-way ANOVA). Capped lines indicate significance levels of direct comparisons of the compounds in 2N and 3N fish (uncorrected Fisher's test, P < 0.05). ns = not significant.",
"Cataract score of the Atlantic salmon lenses used to A) generate a lens transcriptome and B) quantification of DEGs. C) Number of DEGs in lenses of diploid (2N) and triploid (3N) Atlantic salmon without (− ) and with cataracts (+). Exact numbers of genes are given above the individual bars. D) Venn diagram showing the degree of overlap of DEGs in the four treatment groups based on a four-way comparison of genes.",
"Six DEGs encoding protein involved in phototransduction (KEGG pathway ko04744) were up-regulated in cataractous lenses from diploid (2N+) Atlantic salmon (A) and down-regulated in cataractous lenses from triploid (3N+) Atlantic salmon (B). Border color indicates up-regulated genes (red) and down-regulated genes (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) may be affected in some of the lenses even without visible signs of damage.",
"Predicted upstream regulator network with highest consistency score based on IPA Compare Analysis, generated by comparison of DEGs in cataractous lenses of diploid and triploid Atlantic salmon (2N + vs 3N+). Up-regulated and down-regulated genes are highlighted in red and green, while predicted activated and inhibited regulators and relationships are highlighted in orange and blue, respectively. The color depth is correlated to the fold change. Solid lines show direct interactions between two gene products, while dashed lines represent indirect interactions among genes shown in the network. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)",
"KEGG pathway enrichment analysis of DEGs.",
"Comparison of predicted upstream regulators in lenses of diploid and triploid Atlantic salmon. Hierarchical clustering based on z-scores as determined with IPA Compare Analysis."
] | [
"Fig. 1",
"(Fig. 1A",
"(Fig. 1B)",
"Fig. 2A",
"Fig. 2A)",
"Fig. S1",
"Fig. S1A",
"Fig. S1B",
"Fig. S1C",
"Fig. S2",
"(Fig. 2B)",
"(Fig. 2C",
"Fig. 2D",
"(Fig. 3A)",
"(Fig. 3B",
"Fig. 4",
"Fig. 5",
"(Supplementary File 7)",
"agbl4, anxa2, anxa5, apoc1, apoe, arl14ep, arr3, arrb2, atp1a3, atp1a4, aurka, btbd17, c18orf25, c4orf33, ca1, ca2, cars, ccl28, cdca8, ckmt2, clu, col11a1, cyb5r1, deptor, dnajc3, egfl7, f11r, gal, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, hba1, hbb, hbz, homer2, hspa8, ifitm5, igfbp2, junb, krt18, ldhb, lep, linc00998, lurap1, lztr1, mlkl, mllt11, mmp2, ndrg1, ndrg4, nmt1, opn1lw, opn1mw2, opn1sw, parp15, pdc, pde6g, plaur, ppdpf, ppp2ca, prdm9, prph2, rab32, rasd2, rbp4, rcvrn, rho, rpe65, rtbdn, s100a1, s100p, slc27a2, smarcd1, snca, specc1, stc1, tmsb10, tpd52l1, trpm7",
"anxa5, arl14ep, arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cars, ccl28, ckb, bckm, ckmt2, clu, cndp2, cntnap5, dbnl, defb4b, erbb2ip, f11r, fabp4, fabp7",
"cntnap5, coch, col2a1, col9a1, col9a2, dnase1l3, eef1a1, fabp4, fabp7, gja1, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, igsf11, lcn1, ldhb, lrrn1, mgst3, nbl1, ndrg1, ndufa4, opn1lw, opn1mw2, opn1sw, pdc, pde6g, pkm, plaur, ppdpf, prph2, ptma, pygm, rbp4, rcvrn, rgmb, rho, rpe65, rtbdn, serpinh1, sirt1, slc39a11, snca, sparc, spon1, stmn1, suclg1, trpm7, ube2j1 3N-vs 2N-arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cdh1, ckm, ckmt2, crb1, crygs, cyp2c19, erbb2ip, exosc2, glra1, glul, gnat1, gnat2, gnb3, gngt1, gngt2, harbi1, hba1, hbb, hbz, igsf11, ldhb, mvp, ndrg1, opn1lw, opn1mw2, opn1sw, pdc, pde6g, plaur, polr3c, prph2, rbp4, rcvrn, rho, rpe65, rps12, rtbdn, slc1a2, stc1, suclg1",
"Fig. 5"
] | [
"2N + vs 2N-(diploid cataract) ada,",
"3N + vs 3N-(triploid cataract) anxa2,"
] | [
"Recent years have seen renewed efforts to establish commercial farming of triploid Atlantic salmon (Salmo salar) -hereafter referred to as salmon (Benfey, 2016;Stien et al., 2019). Farming triploid salmon has two major advantages. The first is that triploid females do not mature sexually and can diverge energy into somatic growth (Piferrer et al., 2009). The second is related to the fact that domesticated salmon escapees and genetic interactions with wild conspecifics represents one of the most significant environmental challenges to salmon aquaculture . Rearing sterile triploid fish reduced this threat, and is an effective way to mitigate further genetic interactions.",
"Although production of triploid salmon has potential benefits, the global Atlantic salmon aquaculture industry is still primarily based upon rearing diploid fish. While there are several reasons for this, in part, this is due to the fact that triploid salmon often show poor performance. For example, in comparison with diploid salmon, they display differences in physiology, behavior and morphology, with increased prevalence of vertebral deformity and ocular cataracts as two of the most severe disorders (Wall and Richards, 1992;Piferrer et al., 2009;Taranger et al., 2010;Taylor et al., 2015;Sambraus et al., 2017). Cataracts are defined as the loss of transparency of the lens and can appear both as reversible osmotic cataracts and permanent cataracts, which can have multiple causes (Hejtmancik, 2008). In farmed salmon, cataract formation has been linked to genetic predispositions and several nutritional and environmental factors (reviewed by Bjerkås et al., 2006). Cataract has been observed in both freshwater and seawater, however, farmed salmon are particularly prone to cataract development during the smolt transition from fresh to saltwater (Waagbø et al., 1998;Breck and Sveier, 2001;Breck et al., 2005a;Remø et al., 2014) and during periods of rapid growth Breck and Sveier, 2001;Waagbø et al., 1996Waagbø et al., , 2010Remø et al., 2014Remø et al., , 2017. Increased prevalence of cataracts in triploid fish is not well understood but may partly rely on altered metabolism due to differences in cellular morphology (Benfey, 1999).",
"A sub-optimal level of dietary histidine is currently considered the most important causative factor for cataract development in farmed Atlantic salmon (Breck et al., 2003(Breck et al., , 2005bRemø et al., 2014Remø et al., , 2017Waagbø et al., 2010). Taylor et al. (2015) investigated the preventive effects of dietary histidine supplementation in triploid Atlantic salmon during seawater grow-out. Although the severity was higher in triploids compared to diploids irrespective of diet, applying a high histidine diet mitigated further cataract development in triploids. Similarly, dietary histidine supplementation reduced the severity of cataracts in diploid and triploid yearling smolt, but also with a higher severity in triploids compared to diploids at the highest dietary level (Sambraus et al., 2017). The cataract preventative effect of dietary histidine has been attributed to the functional roles of histidine and the derivative N-acetyl-histidine (NAH) as buffer component (Breck et al., 2005a), osmolyte (Rhodes et al., 2010) and possibly antioxidant (Remø et al., 2014), therefore being important to maintain cell integrity and water balance. The lens concentration of NAH was lower in triploids compared to diploids given the same high histidine diet (Sambraus et al., 2017), suggesting that the triploid lens may be more vulnerable to cataract development, possibly due to lower protection of the triploid lens through lower ability to synthesize NAH, or a higher requirement to maintain water balance in the lens. The latter might be linked to larger cell size in triploids (Wu et al., 2010). Thus, differences in susceptibility to cataracts, as well as the apparent higher requirement of histidine to mitigate (but not eliminate) cataract development in triploids, may be hypothesized to be due to alterations or weakness in the lens of triploids.",
"Thus far, no attempts have been conducted to evaluate the mechanisms behind increased prevalence of cataracts in triploid fish at the molecular level. Relatively few genome-wide examinations of the molecular mechanisms behind cataract formation have been performed on healthy and cataractous lenses in vertebrates (Sousounis and Tsonis, 2012), possibly due to the biased lens transcriptome, where the expression of structural genes, such as crystallins, predominates over genes that regulate cell function and phenotype (Wistow, 2006;Manthey et al., 2014). Global transcriptional examinations of the mammalian cataractous lens have revealed differential regulation on numerous types of genes, including crystallins and heat shock proteins, cytochrome oxidases, growth factors, metalloproteinases and collagen, as well as various transcription factors (Wistow et al., 2002;Wride et al., 2003;Hawse et al., 2003;Mansergh et al., 2004;Medvedovic et al., 2006;Hejtmancik, 2008;Shiels et al., 2010;Hejtmancik, 2017, 2019). In Atlantic salmon, Tröße et al. (2009) used a 16K salmonid microarray to screen for transcriptional responses to histidine related cataracts in lenses of Atlantic salmon and reported differences in genes encoding proteins linked to lipid metabolism, carbohydrate metabolism, and protein degradation. Among the significantly differentially regulated genes were gamma crystallin M2 (homolog to mammalian crygb), lens fiber membrane lim2, secreted protein, acidic, cysteine-rich (sparc), metallothionein B (mt-b), heat-shock cognate 70 (hsc70a), calpain (capns1), Na/K ATPase alpha subunit isoform 1c (atpa1c) and fatty acid binding protein 2 (fabp2), of which several have been linked to cataracts before.",
"In the present study, we used transcriptomics (RNA-seq) to examine why triploid fish are more prone to cataract development than diploid fish. To do so, we compared the transcriptional patterns in the lens of diploid and triploid Atlantic salmon originating from both a domesticated strain and a wild population, with and without mature cataract, as assessed by a slit-lamp biomicroscope.",
"The salmon used in this experiment originated from females (f) and males (m) from the domesticated Mowi strain (M) crossed with females and males from a wild population in the river Figgjo (F) in November 2011. Eight groups were made as diploid and triploid of the systematic breeding of the farmed and wild strains: mM × fM, mM × fF, mF × fF and mF × fM. The offspring groups were start-fed with a commercial feed (Skretting, Stavanger, Norway) at March 26th , 2012 and were held at 12 • C water temperature from start feeding to mid-summer. Thereafter, the groups were reared at ambient temperature. Fish were reared under continuous light from start feeding to October 1st, followed by a simulated natural photoperiod to initiate parr-smolt transformation. Experimental groups were held separately in eight tanks until November 27th , 2012, when they were individually passive integrated transponder-tagged (PIT-tags, Electronic I, Inc., Dallas, TX, USA) and the groups distributed equally into three replicate tanks. Fish were transferred to seawater at May 10th, 2013. In sea, the fish were fed Skretting Spirit 75-50A. The experiment was terminated October 16th , 2013.",
"Fish were sampled as post smolts in seawater at a mean body weight of 143 ± 8 g (n = 46). Upon sampling, the fish were inspected for cataracts, and weight and length measured. From each fish, two lenses and heart tissues were dissected and immediately frozen on liquid nitrogen. The left lens was used for transcriptome de novo assembly and transcriptomics.",
"Cataract assessment was performed on anaesthetized fish by use of a Kowa SL-15 slit-lamp biomicroscope (Kowa, Tokyo, Japan). The type, position and severity of the observed cataractous changes were determined according to Wall and Richards (1992), but with a maximum severity extended from 3 to 4 per eye to match the amplitude of the macroscopic scale (microscopic cataract score 0: absent, 1: slight, 2: moderate, 3: severe, 4: total cataract).",
"Heart tissue from the sampled fish was used as status organ for histidine and NAH (Remø et al., 2014). NAH and free histidine in the heart tissue were analyzed by reverse phase HPLC and UV detection at 210 nm, with modifications according to Breck et al. (2005b).",
"Lens tissue was thoroughly homogenized before RNA extraction using a Precellys 24 homogenizer and ceramic beads CK28 (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted using the BioRobot EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany), treated with DNase according to the manufacturer's instructions and eluted in 50 μL RNase-free MilliQ H 2 O. RNA quality and integrity were assessed with the NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the RNA integrity of the lens samples. The RNA integrity number (RIN) of RNA extracted for transcriptome assembly (2N: n = 10, 3N: n = 10) and RNA-seq (2N: n = 12, 3N: n = 14) were 8.5 ± 0.0 (n = 20) and 8.2 ± 0.1 (n = 26), respectively (mean ± SEM). of analysis, transcriptome de novo assembly had to be conducted using Illumina paired-end reads before RNA-seq analyses. RNA extracted from 10 diploid and 10 triploid lenses (n = 20) was mixed and used to generate the assembly. Transcriptome de novo assembly was conducted using the short reads assembling software Trinity as described by Grabherr et al. (2011). Trinity combines three independent software modules, Inchworm, Chrysalis, and Butterfly, to process the RNA-seq reads into unigenes. The output sequences were aligned to the databases of NR, NT, SwissProt, KEGG, COG and GO using Blastx, and the best aligning result was used to decide sequence direction. Sequence direction of unigenes not aligned to any of the above-mentioned databases was determined with the ESTScan software (Iseli et al., 1999).",
"Direct RNA sequencing (RNA-seq) was used to screen for differentially expressed genes (DEGs) in lenses of both diploid and triploid individuals. As the two strains used here are known to display divergent transcription patterns (Bicskei et al., 2014(Bicskei et al., , 2016, fish from both groups were randomly mixed and pooled prior to any analysis. Individual left lenses from 26 salmon were used for RNA-seq examination (2N-: n = 6, 2N+: n = 6, 3N-: n = 6, 3N+: n = 8). Poly (A) mRNA was isolated using magnetic beads with oligo (dT) from total RNA obtained from the lens samples. Fragmentation buffer was added to shred mRNA to short reads. Using these short fragments (about 200 bp) as templates, random hexamer primers were applied to synthesize first-strand cDNA. Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. QiaQuick PCR extraction kit (Qiagen) was used to purify short double-stranded cDNA fragments according to manufacturer's instructions. These fragments were then resolved with EB buffer for end reparation, added poly (A), and ligated to the sequencing adapters. After agarose gel electrophoresis, the suitable fragments were selected for PCR amplification as templates. Finally, the libraries were sequenced using Illumina HiSeq™ 2000 (San Diego, CA, USA).",
"The de novo lens transcriptome described above was thereafter used as a reference for alignment of the RNA-seq data. Unigenes were annotated with Blastx alignment using an e-value cut-off of 10 − 5 between unigenes and the databases of NR, NT, SwissProt, KEGG, COG and GO. The NOISeq software package (Tarazona et al., 2011) was used to screen for differentially expressed genes (DEGs). NOISeq is a novel non-parametric method for the identification of DEGs, which shows a good performance when compared to other differential expression methods, like Fisher's Exact Test, edgeR, DESeq and baySeq. All RNA-seq work was performed by staff at the Beijing Genome Institute (BGI, Hong Kong).",
"Fish from each ploidy were sampled and measured for erythrocyte diameter to verify their ploidy status (Benfey et al., 1984). Blood smears were used to measure the relative diameters of 10 erythrocytes per fish (Image-Pro Plus, version 4.0, Media Cybernetics Silver Spring). The triploid fish had significantly (22%) larger blood cells than diploid fish.",
"To calculate differential expression, NOISeq default settings were used (Tarazona et al., 2011). NOISeq empirically models the noise distribution of count changes by contrasting fold-change differences (M) and absolute expression differences (D) for all the features in samples within the same condition. This reference distribution is used to assess whether the M-D values computed between two conditions for a given gene is likely to be part of the noise or represent a true differential expression. Instead of using a false discovery rate (FDR) or a q-value cut-off, the NOISeq method calculates a differential expression probability value. A gene is declared as differentially expressed if this probability is higher than q. The threshold q is set to 0.8 by default, since this value is equivalent to an odd of 4:1 (the gene in 4 times more likely to be differentially expressed than not). In this work we used a log2 M-value cut-off of ≥2 (fold-change ≥2). For genes not expressed in some samples, the gene expression value (D) of 0.001 was used. Functional pathway analyses, including prediction of activation and inhibition of upstream transcription factors and downstream effects, were generated through the use of QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.qiagen.com/ingenuity). Since IPA only can map mammalian homolog entries, identifiers were obtained with Blast alignment against the RefSeq databases (cut-off E10 − 5 ) and assuming orthologous genes have the same function. A limited number of fish-specific genes with no mammalian homologs were for this reason not included in the IPA pathway analysis. This may have skewed the interpretation of the transcriptomic data.",
"The RNA-seq dataset discussed in this publication has been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE153933 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE153933).",
"Growth of the farmed and wild stocks is reported in detail by Harvey et al. (2017), and therefore not reported here. At the present sampling, there was no significant difference in weight between the diploid and triploid fish. Fig. 1 shows the concentrations of L-histidine and N-acetyl-L-histidine (NAH) analyzed in salmon heart as a measure on the ambient histidine status. Two-way ANOVA analysis showed that there was a significant effect of cataract on L-histidine (Fig. 1A, p = 0.016), while there were no significant effects of either cataract or ploidy on NAH (Fig. 1B). Posthoc tests showed reduced levels of L-histidine (p = 0.019) and increased levels of N-acetyl-L-histidine (p = 0.046) in diploid fish with cataract.",
"The mean cataract scores of the left lenses used to generate the lens transcriptome (2N + had score 2.5 and 3N + had score 1.5, while both 2N-and 3N-had score 0) and the right lenses used for the RNA-seq analysis (both 2N+ and 3N + had score 3, while both 2N-and 3Nhad score 0) are shown in Fig. 2A and B.",
"Total RNA extracted from lenses of 20 domesticated and wild salmon, 10 of which were diploid and 10 were triploid, was mixed ( Fig. 2A), sequenced and used to assemble the lens transcriptome. Using the Illumina HiSeq 2000 platform, a total of 68,403,252 raw reads and 63,098,790 clean reads were sequenced. The clean reads were assembled into 78,306 contigs with mean length of 284 nucleotides (nts). Of these, 29,177 contigs with mean length of 659 nts mapped to UniGene entries. Distinct clusters, which contained highly similar UniGene entries (more than 70% that may come from the same gene or homologous genes), were 7391. Distinct singletons representing a single UniGene were 21,786. Using blastx and blastn, a cut-off of 10 − 5 and the following priority order, 15,711,21,084,14,445,11,680,5231,11,327 UniGene entries were functionally annotated to the NR, NT, Swiss-Prot, KEGG, COG and GO databases, respectively. UniGene entries aligned to a higher-priority database were not aligned to a lower-priority database. In total, 22,160 out of the 29,177 UniGene entries were given a functional annotation in these databases.",
"For protein coding region prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 15,428. The number of predicted CDS, Unigene entries that could not be aligned to any database and were scanned by ESTScan, was 701. The total number of CDS was 16,129. According to the microsatellite analysis conducted with the MicroSAtellite (MISA) software and using Unigenes as reference, there were 7274 simple sequence repeat (SSR) in the transcriptome. Heterozygous analysis using SOAPsnp, a member of the Short Oligonucleotide Analysis Package (SOAP), revealed 23,759 single- nucleotide polymorphisms (SNPs) in the transcriptome.",
"A summary of the NR annotation is shown in Fig. S1 (Supplementary File 1). Fig. S1A shows E-value distribution, while similarity distribution is shown in Fig. S1B. Fig. S1C shows the species distribution of UniGene entries annotated to the NR database. Most UniGene entries mapped to Atlantic salmon, followed by hits against Nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and other fish species. Gene ontology (GO) annotation of the NR unigenes was obtained with the Blast2GO and WEGO software's (Conesa et al., 2005;Ye et al., 2006). Fig. S2 shows the major GOs from the salmon lens transcriptome, divided into the three ontologies biological process, molecular function and cellular component. Of the more specialized molecular process worth mentioning is the antioxidant activity, while the biological function GO annotation indicate that the lens cells are relatively metabolic active.",
"To search for differentially expressed genes (DEGs) in diploid and triploid salmon with and without cataracts (Fig. 2B), the left lens from 26 individual fish were selected for RNA-seq analysis. The selection was based on cataract score (score 0 vs 3) and ploidy (2N vs 3N). A total of 634,610,512 single-end reads were sequenced with the Illumina HiSeq™ 2000 system. In average, 24,023,481 ± 199,810 single-end reads were sequenced per sample (n = 26, mean ± SEM). Average total reads mapped to the in-house made lens transcriptome were 17,882,297 ± 161,561 (n = 26, mean ± SEM), representing 74.4% of the total reads. As expected for fish, some contigs had redundant annotations.",
"Using the default NOISeq setting for calculation of differential expression (q ≥ 0.8 and log 2 ≥ 1), the comparison between diploid fish without and with cataracts (2N-vs 2N+) showed that 182 DEGs were more highly expressed in 2N-lenses and 25 DEGs were more highly expressed in 2N + lenses (Fig. 2C). For the comparison between triploid fish without and with cataracts (3N-vs 3N+), 74 DEGs were more highly expressed in 3N-and 78 DEGs were more highly expressed in 3N+. Comparison of healthy diploid lenses vs healthy triploid lenses (2N-vs 3N-) yielded 107 DEGs, with 93 genes more highly expressed in 2N-, and 14 more highly expressed in 3N-. Comparison of cataractous diploid lenses vs cataractous triploid lenses (2N + vs 3N+) yielded 165 significant DEGs, with 9 genes more highly expressed in 2N+, and 156 genes more highly expressed in 3N+. All significant DEGs in the four comparisons, including fold changes, significance levels and best annotation, which were used in downstream functional analyses, are shown in Supplementary File 2. Annotations were given to about 52% of the DEGs.",
"Very few DEGs with unique expression were found in the lenses from the four treatment groups. Fig. 2D shows a Venn diagram of the number of unique and shared DEGs determined with a four-way comparison. There were 4 unique DEGs in the 2N-group, 17 in the 2N + group,15 in the 3N-group and 245 in the 3N + group. 98.7% of the DEGs were shared between all treatment groups. Most of these unique DEGs were expressed only in one or a few of the lenses from their respective group. Annotations of unique DEGs are shown in Supplementary File 3.",
"Two pathway analysis methods, KEGG and IPA pathway analysis, were employed for functional analysis of DEGs in cataractous lenses from diploid and triploid fish. KEGG pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways in DEGs by comparison to the whole genome. Table 1 shows the most significant KEGG pathways from the four comparisons based on a q-value cut-off of 0.05. The top three pathways in both diploid and triploid fish with cataracts were \"Phototransduction\", \"Carbohydrate digestion and absorption\" and \"Proximal tubule bicarbonate reclamation\". Interestingly, for the phototransduction pathway (KEGG pathway ko04744), the significant DEGs linked to this system, 13 DEGs in the diploid fish and 17 DEGs in the triploid fish, (DEGs only found in triploid fish were gnb1, arrb1 and arr3), were all upregulated in the diploid cataractous lens (Fig. 3A) and down-regulated in the triploid cataractous lens (Fig. 3B). As expected, direct comparisons between diploid and triploid lens from fish without and with cataract gives similar patterns. The \"ECM-receptor interaction\" and \"PPAR signaling\" pathways were two the most significantly affected KEGG entries based on a direct comparison of DEGs in diploid and triploid salmon with cataracts not listed in the other comparisons.",
"IPA Core analysis and the IPA Compare function were used for evaluation of biological processes, pathways and networks. In order to use IPA, all identifiers must be recognized as mammalian homologs. Some fish-specific genes obviously cannot be given human ortholog names recognized by IPA, and thus were omitted from the IPA-Core analysis. About 52% of the DEGs from the four gene lists were given automatic annotation as described above (Supplementary File 2). In addition, all unknown DEGs were manually aligned against the core nucleotide and EST databases, and given annotation based on hits against NCBI Unigene entries (Blastn cut-off E10-5). This way, 64.4% of the DEGs used for the functional analysis had IPA identifiers. Table 2 shows annotated salmon genes with human identifiers used in these functional analyses which were significantly differently expressed according to the four comparisons (2N + vs 2N-, 3N + vs. 3N-, 3N + vs 2N+ and 3N-vs 2N-). Highlighted in the table are cataract-linked genes that are differentially regulated in various mice knockout models (data obtained from the iSyTE (integrated Systems Tool for Eye gene discovery) database (URL: http://research.bioinformatics.udel.edu/iSyTE).",
"To get an idea of the mechanistic basis for cataract development in the salmon lens and the impact of ploidy, we used IPA Core Analysis with the predicted upstream regulators function and the categorical annotations of disease or function to search for differences in the four comparisons described above. By sorting with an activation z-score >2 and p-value of overlap <10.5, IPA Core Analysis predicted six upstream regulators that may explain the observed DEGs in lenses of diploid salmon with cataracts. These were CRX, GTF2IRD1, HIF1A, EDN1, hexachlorobenzene and EPO (Supplementary File 4). The dataset for the most significant transcriptional regulator, CRX with a z-score of 2.43 and a p-value of overlap of 8,35E-19, was made up of the DEGs arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. For GTF2IRD1, which had a z-score of 2.10 and p-value of overlap of 2,89E-10, the dataset consisted of arr3, gnat1, gnat2, opn1lw, opn1sw, pdc and rho. For the disease of functional annotation, the analysis predicted eight categories with a z-score above 2 and p-value >10-5. These were \"Cellular Movement, Immune Cell Trafficking-leukocyte migration\", \"Cellular Movement-cell movement\", \"Cellular Movement, Hematological System Development and Function, Immune Cell Trafficking-cell movement of leukocytes\", \"Cellular Movement-migration of cells\", \"Cell Death and Survival-cell viability\", \"Cell Death and Survival-cell survival\", \"Tissue Morphology-quantity of cells\" and \"Cellular Movement-migration of brain cancer cell lines\".",
"In the lenses of triploid salmon with cataracts, five upstream regulators had a predicted activation state based on the same cut-off as described above. These were CRX, GTF2IRD1, beta-estradiol, trichostatin A and decitabine (Supplementary File 5). CRX, the most significantly regulator with a z-score of − 2.73 and a p-value of overlap of 8,90E-20, was predicted affected based on the same DEGs as in diploid fish, i.e. arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. The predicted activation state for GTF2IRD1 (z-score: − 2.10, p-value of overlap: 6,33E-11) in lenses of triploid salmon was based on the same DEGs as in diploid fish. Using the same cut-off, no disease or function categories had a predicted activation state in the lenses of triploid salmon. By comparing the transcriptional patterns in cataractous lenses of diploid and triploid salmon indirectly (IPA Compare Analysis of 2Nversus 2N+ and 3N-versus 3N+), the most pronounced differences were seen for the \"IL8\" and \"Production of nitric oxide and reactive oxygen species in macrophage\" canonical pathways (data not shown). These pathways had higher activation z-scores in lenses of the diploid fish compared to the triploid fish. A predicted regulator network generated from the comparison of DEGs in cataractous lenses of diploid and triploid salmon is shown in Fig. 4. This network, which had a consistency score of 13.87 and was based on target DEGs apoe, clu, gal, igfbp2, junb, krt18, lep, mmp2, plaur, rbp4 and snca, and on upstream regulators AGT, CREB1, ERK, HIF1A and P38 MAPK, predicted that synthesis of nitric oxide and chemotaxis of cells might be different in cataractous lenses of diploid and triploid salmon. Based on analysis of predicted upstream regulators and hierarchical clustering, the most pronounced difference in triploid fish was seen for CRX, GTF2IRD1, SRC and RHO (Table 3). Interestingly, these upstream regulators were predicted activated in diploid fish (positive z-score) and predicted inhibited in triploid fish (negative z-score) with cataracts. Fig. 5 shows the molecules in these four networks. Except krt18 and ckm in the SRC network, all genes in these networks were up-regulated by cataracts in diploid fish and downregulated in triploid fish.",
"Comparison of the transcriptional patterns in lenses from diploid and triploid salmon without cataracts revealed two upstream regulators with predicted activation scores above 2 and p-values of overlap >10-4 (Supplementary File 6). According to the IPA Core Analysis, both the transcription regulator CRX and the chemical drug trichostatin A were predicted activated with z-scores of 2.55 and 2.40, respectively. Targeted DEGs for CRX were gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho, while cdh1, hba1/hba2, hbb, hbz, ndrg1 and slc1a2 made up the dataset for the trichostatin A prediction.",
"A comparison of transcriptional patterns in lenses from diploid and triploid fish without cataracts showed that two disease or function annotations had prediction scores above 2 and a p-value of overlap >10-5 (Supplementary File 6). \"Organ degeneration\" (z-score 2.19) and \"Degeneration of cells\" (z-score 2.19) showed significant differential prediction scores in lenses of triploid and diploid salmon without cataracts. The \"Organ degeneration\" z-score was based on differential transcription of prph2, rho, crb1, slc1a2, pde6g, gngt1, rpe65, rcvrn, ca1, gnat1, opn1mw (includes others) and ca2, whereas the \"Degeneration of cells\" z-score was based on differential transcription of rho, slc1a2, pde6g, gngt1, rpe65, gnat1 and prph2. All these genes were more highly expressed in lenses of diploid fish compared to triploid fish. This could reflect differential transcription in non-cataractous lenses from diploid and triploid salmon, or indicate that mechanisms leading to cataracts The direct comparison of transcriptional patterns in lenses of diploid and triploid salmon with cataracts yielded five predicted upstream regulators with activation z-score >2 and p-value of overlap >10-5 (Supplementary File 7). These were CRX, GTF2IRD1, beta-estradiol, trichostatin A and decitabine. Targeted DEGs in the most significant regulator (transcription regulator CRX, p-value of overlap 8, 90E-20) were arr3, gnat1, gnat2, opn1lw, opn1sw, pdc, pde6g, prph2, rcvrn and rho. A significant result for the transcription regulator GTF2IRD1 was based on the DEGs arr3, gnat1, gnat2, opn1lw, opn1sw, pdc and rho. Five categories with disease or functional annotation had predicted activation state based on z-score >2 and p-value of overlap >10-5 (Supplementary File 7). These were \"DNA Replication, Recombination, and Repair, Nucleic Acid Metabolism, Small Molecule Biochemistryhydrolysis of nucleotide\", \"Behavior-\", \"Cellular-Movement-migration of blood cells\", \"Cellular Movement-cell movement\" and \"Organismal Development-size of body\".",
"This is the first study to investigate the transcriptomics of salmon lenses in diploid and triploid salmon with and without cataracts. Functional analysis showed that retina-associated genes were differentially affected in diploid and triploid fish. Predicted differential effects of NO-induced oxidative stress, modified cytoskeleton stability and lipid metabolism, possibly affecting cellular metabolism, indicate that the triploid lens might be more vulnerable to cataract due to altered protein degradation and turnover.",
"Overall, this study indicates that the transcriptional patterns in the lenses of diploid and triploid Atlantic salmon are very similar. This is consistent with the results from a recent study, which showed that the vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon (Oncorhynchus kisutch) (Christensen et al., 2019). Similar results have been shown for other fish species (Chatchaiphan et al., 2017). At the protein level there also appears to be small differences in expression between diploid and triploid salmon (Nuez-Ortin et al., 2017). Relatively few significant DEGs were found in the current dataset. Most of the significant DEGs in the cataractous triploid lenses were higher expressed compared to the cataractous diploid lens (156 vs 9). In healthy lenses the pattern was opposite, with more of the significant DEGs being lower expressed in the diploid lenses (93 vs 14). According to the functional analysis, the most distinct difference between diploid and triploid cataractous lenses in transcript levels were seen for genes encoding proteins involved in the phototransduction pathway. Whether this reflects a direct effect of ploidy on the transcription of these genes is unknown.",
"The N3+ vs N2+ comparison list contained a gene associated with heat shock protein (HSP) activity, e.g. hsp47/serpinh1. Furthermore, two heat shock protein genes, annotated to hspa8 and hspa8b, were upregulated in diploid cataractous lenses but not in diploid noncataractous lenses. These findings potentially suggest a different ability to handle damaged proteins and protein turnover. Crystallins, watersoluble structural protein found in the lens and the cornea of the eye accounting for the transparency, are relatively similar to HSPs, and have similar chaperone activity (Wang and Spector, 1995;Slingsby et al., 2013). \"Protein digestion and absorption\" (KEGG pathway ko04974) was the second most significantly affected pathway, after phototransduction, in triploid cataractous lenses compared to diploid cataractous lenses, according to the functional analysis. In humans, ROS-generated protein oxidation may lead to cataract formation in the aged lens (Taylor and Davies, 1987). In addition to oxidative stress and the inflammatory response, an unfolded protein response is known to be activated in age-related ocular disorders such as cataracts (Lenox et al., 2015). Histidine has been shown to stimulate the proteasome and thereby protein degradation and turnover (Hamel et al., 2003;Breck et al., 2005a). With diminished antioxidant capacity and decreased proteolytic capabilities, the triploid lens may be less efficient in clearance of damaged proteins. Taken together, the results from the current study indicate that the higher susceptibility to cataract development in triploid vs diploid salmon may in part rely on how well the cells handle damaged proteins.",
"The heart histidine and NAH levels observed in the present study represent normal values obtained from a commercial salmon smolt feed (Remø et al., 2014). According to the factorial analysis, the histidine concentration was related to cataract status and not to ploidy, while NAH status did not indicate differences with neither cataract status nor ploidy. The result confirms the higher sensitivity of histidine relative to NAH status in heart tissue, but more importantly, a corresponding lens histidine status to the diploid cataract group and both the triploid groups indicates suboptimal conditions for salmon smolts (Remø et al., 2014). The present groups of salmon would therefore be prone to cataract development.",
"At the molecular level, functional analysis predicted that the upstream regulators cone-rod homeobox (CRX), GTF2I repeat domain- Table 2 Annotated salmon genes with human orthologs used in the IPA functional analyses. Genes common for all four comparisons were all up-regulated in diploid cataractous lenses and down-regulated in triploid cataractous lenses. Genes in bold are differential regulated in mice cataract mutants (>2.0 fold) according to the iSyTE database (Kakrana et al., 2018). Underlined are genes that are differentially regulated by more than one mutant type.",
"Genes agbl4, anxa2, anxa5, apoc1, apoe, arl14ep, arr3, arrb2, atp1a3, atp1a4, aurka, btbd17, c18orf25, c4orf33, ca1, ca2, cars, ccl28, cdca8, ckmt2, clu, col11a1, cyb5r1, deptor, dnajc3, egfl7, f11r, gal, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, hba1, hbb, hbz, homer2, hspa8, ifitm5, igfbp2, junb, krt18, ldhb, lep, linc00998, lurap1, lztr1, mlkl, mllt11, mmp2, ndrg1, ndrg4, nmt1, opn1lw, opn1mw2, opn1sw, parp15, pdc, pde6g, plaur, ppdpf, ppp2ca, prdm9, prph2, rab32, rasd2, rbp4, rcvrn, rho, rpe65, rtbdn, s100a1, s100p, slc27a2, smarcd1, snca, specc1, stc1, tmsb10, tpd52l1, trpm7, wwp2, znf391, znf501 anxa5, arl14ep, arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cars, ccl28, ckb, bckm, ckmt2, clu, cndp2, cntnap5, dbnl, defb4b, erbb2ip, f11r, fabp4, fabp7 cntnap5, coch, col2a1, col9a1, col9a2, dnase1l3, eef1a1, fabp4, fabp7, gja1, glul, gnat1, gnat2, gnb1, gnb3, gnb5, gngt1, gngt2, hamp, harbi1, igsf11, lcn1, ldhb, lrrn1, mgst3, nbl1, ndrg1, ndufa4, opn1lw, opn1mw2, opn1sw, pdc, pde6g, pkm, plaur, ppdpf, prph2, ptma, pygm, rbp4, rcvrn, rgmb, rho, rpe65, rtbdn, serpinh1, sirt1, slc39a11, snca, sparc, spon1, stmn1, suclg1, trpm7, ube2j1 3N-vs 2N-arrb2, atp1a3, atp1a4, btbd17, ca1, ca2, cdh1, ckm, ckmt2, crb1, crygs, cyp2c19, erbb2ip, exosc2, glra1, glul, gnat1, gnat2, gnb3, gngt1, gngt2, harbi1, hba1, hbb, hbz, igsf11, ldhb, mvp, ndrg1, opn1lw, opn1mw2, opn1sw, pdc, pde6g, plaur, polr3c, prph2, rbp4, rcvrn, rho, rpe65, rps12, rtbdn, slc1a2, stc1, suclg1, trpm7 Common in 2N+ and 3N+ anxa2,anxa5,arl14ep,arrb2,atp1a3,atp1a4,btbd17,ca1,ca2,cars,ccl28,ckmt2,clu,f11r,gal,glul,gnat1,gnat2,gnb3,gngt1,gngt2,hamp,harbi1,ifitm5,igfbp2,junb,krt18,ldhb,lep,mlkl,ndrg1,nmt1,opn1lw,opn1mw2,opn1sw,pdc,pde6g,plaur,prdm9,prph2,rcvrn,rho,rpe65,rtbdn,s100a1,s100p,slc27a2,snca,tmsb10,trpm7,znf391,znf501 Common for all 4 comparisons arrb2,atp1a3,atp1a4,btbd17,ca1,ca2,ckmt2,glul,gnat1,gnat2,gnb3,gngt1,gngt2,harbi1,ldhb,ndrg1,opn1lw,opn1mw2,opn1sw,pdc,pde6g,plaur,prph2,rcvrn,rho,rpe65,rtbdn,trpm7 P.A. Olsvik et al. containing 1 (GTF2IRD1), SRC proto-oncogene, non-receptor tyrosine kinase (SRC) and rhodopsin (RHO) could explain the differences observed in lens transcript levels between cataractous diploid and triploid fish. All these upstream regulators were predicted to be activated in cataractous lenses of diploid fish and inhibited in cataractous lenses of triploid fish. Expression of visual pigment-like proteins has been described in extraretinal tissue (Shichida and Yamashita, 2003). Differential expression of retina-associated genes has also been documented in zebrafish (Danio rerio) cataractous lens cells (Posner et al., 2019). Since the lenses in the current study were extracted through an incision in the cornea, without contacting retinal tissues, it seems unlikely that such visual pigment-like proteins were derived from retinal contamination. CRX is a photoreceptor-specific transcription factor, which plays a role in the differentiation of photoreceptor cells, controlling the maintenance of normal cone and rod function (GeneCards database). While not directly linked to cataracts, mutations in the gene encoding CRX have been linked to severe dystrophy of the human retina (Weleber et al., 1993). GTF2IRD1 function as a transcription factor under the control of retinoblastoma protein, and may be a transcription regulator involved in cell-cycle progression and skeletal muscle differentiation (GeneCards database). In human stromal cells, vitamin E treatment has been shown to down-regulate GTF2IRD1, suggesting a link to the lens antioxidative defense. GTF2IRD1 also respond to chemical exposure. For example, down-regulation of GTF2IRD1 has been shown in human liver cells after exposure to benzo(a)pyrene (Jennen et al., 2010), while the peroxisome proliferator-activated receptor alpha (PPARα) agonist pirinixic acid increases GTF2IRD1 mRNA levels in mice liver (Sanderson et al., 2008). The SRC proto-oncogene may play a role in cell growth and participates in signaling pathways that control a broad spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation (GeneCards database). Of interest for lens damage, SRC plays an important role in the regulation of cytoskeletal organization through phosphorylation mechanisms (Gene-Cards database). Recent findings suggest that accumulation of crystallin proteins, a prerequisite for refractive properties and transparency of the lens, in part is controlled by post-transcriptional mechanisms rather than by differential gene transcription (mRNA synthesis) (Terrell et al., 2015). This may explain why we did not see any differentially expressed crystallin genes in the current study. RHO is a photoreceptor primarily expressed in rod cells in the retina required for image-forming vision at low light intensity (GeneCards database). Of nutrients and essential elements, Zn is known to affect stability and folding of RHO (Stojanovic et al., 2004;Gleim et al., 2009), indicating that Zn imbalance might impact RHO activity in cataractous triploid lenses. Zn deficiency has been linked to cataract development in rainbow trout (Ketola, 1979). The functional implication of differential expression of RHO-associated transcripts in lens cells is unknown, as RHO protein is primarily expressed in rod photoreceptors in the retina. In agreement with the current examination, down-regulation of rho has been observed in the cataractous zebrafish lens (Posner et al., 2019). The observed effect on rho may be linked to dysregulation of vitamin A1 and A2 in the lens endothelial cells outside the retina (Enright et al., 2015), or alternatively may reflect signaling interactions between the lens and retina through hyaloid capillaries (Dhakal et al., 2015). Lower activation z-scores for pathways linked to synthesis of nitric oxide (NO) and chemotaxis in triploid fish, suggests that their lens cells may be more prone to oxidative damage and chemotactic cell movement than their diploid counterparts. It is well known that NO has a role in cataract formation in the mammalian lens (Ito et al., 2001;Ornek et al., 2003;Chamberlain et al., 2008). Oxidative stress is an important factor in the development of cataracts for both animals and humans (Ottonello et al., 2000;Williams, 2006). High concentrations of reactive oxygen species (ROS), produced from both endogenous and exogenous sources, cause oxidative damage to cellular constituents that results in interrupted physiological functions and oxidative stress-associated diseases such as cataracts (Lou, 2003). The decreased protein turnover towards the nucleus makes lenses especially vulnerable to increased ROS production in the epithelial cells (reviewed by Brennan and Kantorow, 2009). In human age-related cataracts, oxidation of membrane proteins has been found to precede the development of cataract formation (Wang and Spector, 1995). The accumulation of oxidized proteins further results in loss of cell function, apoptosis and necrosis (Brennan and Kantorow, 2009). Lens NAH has been suggested to be an important intracellular antioxidant in the salmon lens and may contribute to the cataract mitigating effect of dietary histidine (Remø et al., 2011). The present results suggest a higher susceptibility to oxidative stress in Fig. 5. IPA Compare Analysis of transcripts differentially regulated in cataractous lenses of triploid salmon compared to diploid salmon (2N + vs 3N+). The figure shows genes associated with four predicted regulators that were activated in diploid fish but inhibited in triploid fish, based on hierarchical clustering and z-score using Upstream Analysis. A) Cone-rod homeobox (CRX), B) GTF21 repeat domain containing 1 (GTF21RD1), C) rhodopsin (RHO) and D) SRC proto-oncogene, nonreceptor tyrosine kinase (SRC).",
"triploid lenses, which may be hypothesized to be an underlying factor for the higher prevalence of cataracts compared to diploids, as well as the lower lens NAH status observed when reared under similar conditions in the studies by Taylor et al. (2015) and Sambraus et al. (2017). Likewise, cataract and chemotactic activity have been extensively studied over the years (Rosenbaum et al., 1987;Schneider et al., 2012). Several of the predicted upstream regulators for this network, angiotensinogen (AGT), CAMP responsive element binding protein 1 (CREB1), extracellular signal-regulated kinase (ERK), HIF1A and P38 mitogen-activated protein kinase (P38 MAPK), have been linked to cataract formation (AGT: Taube et al., 2012;Ji et al., 2015;CREB1: Weng et al., 2008;ERK: Iyengar et al., 2007;HIF1A: Chen et al., 2014;P38 MAPK: Bai et al., 2015). Follow-up studies should look at how NO induce ROS and oxidative stress in the triploid fish lens, as well as the involvement of chemotaxis in the development of cataract in triploid salmon.",
"Two pathways, \"ECM-receptor interaction\" and \"PPAR signaling pathway\", were among the most significantly affected KEGG entries based on a direct comparison of DEGs in diploid and triploid salmon lenses with cataracts. These were not listed in the other comparisons. The ECM-receptor interaction pathway, including collagen, type II, alpha 1 (col2a1) and secreted protein, acidic, cysteine-rich (osteonectin) sparc, has been linked to disorders of the eye characterized by early onset cataract (Bradshaw, 2009). SPARC is a key lens-and cataract-associated protein (Shiels et al., 2010;Sousounis and Tsonis, 2012;Terrell et al., 2015). SPARC is important for normal cellular proliferation and differentiation and is involved in maintaining lens transparency as shown for mice (Gilmour et al., 1998) and humans (Yan et al., 2000). SPARC is also one of at least 13 proteins harboring mutations that have been associated with a lens or cataract phenotype in mice but not yet in humans (Shiels et al., 2010). In Atlantic salmon, SPARC was suggested to be an \"early\" up-regulated marker for cataract development (Tröße et al., 2009). Lower expression of sparc suggests that the cataractous triploid lenses might have impaired circulation of fluids, ions, and small molecules, possibly resulting in depolarized membrane resting voltage as shown in mice (Greiling et al., 2009). Cartilage extracellular matrix (ECM) is composed of type II collagen, fibrous proteins and proteoglycans, hyaluronic acid and chondroitin sulfate (Gao et al., 2014). The finding indicates a differential regulated mechanism linked to cytoskeleton disruption and NO-induced oxidative stress (Gao et al., 2014). Differential regulation of PPARs, which are transcription factors in control of many cellular processes, indicate an effect on lipid metabolism in the lens. An effect on lipid/cholesterol transport, previously reported in age-related cataract in humans (Utheim et al., 2008), is suggested by differential expression of apolipoprotein E (apoe). APOE is a major apoprotein that is essential for the normal catabolism of triglyceride-rich lipoprotein constituents (Genecards database), indicating a differential effect on lipoprotein metabolism. Apoe, together with sparc, was among the differentially regulated genes in cataractous lenses of Atlantic salmon fed a low-histidine diet compared to a high-histidine diet (Tröße et al., 2009) and had a lower expression level in the lens of Atlantic salmon fed plant oils compared to fish oils (Remø et al., 2011).",
"Only five genes from the Cat-Map gene list, an online chromosome map and reference database for cataract in humans and mice (Shiels et al., 2010), showed overlap with the current gene list of cataractous diploid and triploid lenses from salmon (direct comparison). These were, in addition to sparc and apoe, col2a1, gap junction protein alpha 1, 43 kDa (gja1) and retinal pigment epithelium-specific protein 65 kDa (rpe65). Gap junction proteins, also called connexins, are constituents of gap junctions, channels specialized in cell-cell contacts that provide direct intracellular communication. They allow passive diffusion of molecules up to 1 kDa, including nutrients, metabolites (glucose), ions and second messengers (Genecards database). They are especially important for nutrition and intercellular communication in the avascular lens (Hejtmancik, 2008). Mutations in gap junction proteins such as GJA1, present in the lens epithelium, have been linked to human cataracts (Hejtmancik, 2008). RPE65 is a protein located in the retinal pigment epithelium and involved in the production of 11-cis retinal and in visual pigment regeneration (Genecards database). Finally, RPE65 has been associated with leber congenital amaurosis (LCA), a severe dystrophy of the retina (Weleber et al., 1993). No genes associated with Mendelian (inherited) cataracts or cataracts caused by mutations in transcription factors or metabolic enzymes in humans (Shiels and Hejtmancik, 2019) were on the significant lists in this study. Several of the genes that were differentially expressed in cataractous lenses of triploid salmon have previously been documented to be affected by mutations in the mouse lens (Table 2). By comparing our significant genes with the responses of mammalian orthologs with lens defects or cataract as listed in the iSyTE database (Kakrana et al., 2018), it appears that several may be potential candidate markers for follow-up studies in salmon. Apart from the CBP:p300 E9.5 mutation, which seems to down-regulate many of these genes in mice (iSyTE database), several gene knockout mutation types impact the expression of genes from our lists.",
"Hsp47, also called serpinh1, was one of the genes that were lower expressed in cataractous triploid lenses than in cataractous diploid lenses. HSP47, localized in the endoplasmic reticulum, plays a role in collagen biosynthesis as a collagen-specific molecular chaperone (Genecards database). Heat shock proteins, found throughout the various tissues of the eye, protect and maintain cell viability under stressful conditions such as those occurring during thermal and oxidative challenges chiefly by refolding and stabilizing proteins (Urbak and Vorum, 2010). In the human eye, HSP47 has been suggested to aid the control of pro-collagen under stressful conditions and is induced by corneal structure damage (Urbak and Vorum, 2010). In the salmon lens, increased expression of hsp70 has been shown after short-term handling stress (30 min), indicating that HSPs are transcriptionally controlled and act to protect the cells after stress-induced protein misfolding (Tröße et al., 2010). Lower expression of hsp47 in triploid lenses suggests a poorer ability to facilitate proper folding of proteins. It may be speculated that this is linked to the synthesis, accumulation, repair or breakdown of crystallins or other structural proteins, responsible for lens transparency (Hejtmancik, 2008). Crystallins make up about 80-90% of the soluble proteins in the lens (Hejtmancik, 2008). Mutations in crystalline genes is one of the major reasons for human cataract, and improper ability of chaperones to correct for misfolding or protein damage may render the triploid lens more vulnerable to imbalances responsible for cataract formation in the salmon lens.",
"When studying the lens transcriptome, it is important to note that the eye lens mostly consists of fiber cells without nuclei and organelles (Bassnett, 2002). With transcription restricted to metabolically active lens epithelial cells and young fiber cells (Hejtmancik et al., 2015), transcriptional differences between diploid and triploid cataractous fish lenses may generally be small. Furthermore, triploid salmon in general differ from diploids by containing fewer and larger cells in most organs (Swarup, 1959;Small and Benfey, 1987), possibly impacting transcriptional differences.",
"With 74.4% of the total reads mapped to the novel in-house made lens transcriptome, the mapping degree was similar to using a fully sequenced genome as reference. In total however, only 52% of the significant DEGs were annotated using the described pipeline. With manual annotation of all unknowns, about 64% of the DEGs were assigned annotation for IPA functional evaluation. The reason for this relatively poor annotation level is unknown. A good mapping score combined with a poor annotation level might suggest that the lens transcriptome contains a relatively high number of novel transcripts. Among the most strongly differentially regulated genes in both diploid and triploid salmon with cataracts was the CXC chemokine cxcf1a. This is a fishspecific chemokine with no mammalian ortholog (Chen et al., 2013), so its function was not included in the IPA functional analysis. This illustrates one of the limitations studying cataract mode of action in non-model fish species.",
"In conclusion, this study shows only moderate differences in lens mRNA levels between diploid and triploid Atlantic salmon with score-3 cataract, and very few DEGs with unique expression. Several retina related genes were differentially expressed in the diploid and triploid lenses. The study indicates that the triploid lens may be more vulnerable to cataract than the diploid lens due to predicted effects of protein degradation and turnover, NO-induced oxidative stress, modified cytoskeleton stability and lipid metabolism, possibly linked to repair and compensation mechanisms. Overall, this study suggests that cataract formation is associated with modest changes in gene expression levels, and that transcriptional controls to a large degree regulate gene expression levels independent of chromosomal number in salmon.",
"The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."
] | [] | [
"Introduction",
"Materials and methods",
"Experimental animals and set-up",
"Cataract determination",
"Histidine determination",
"RNA isolation",
"RNA-seq analysis",
"Ploidy verification",
"Statistics",
"Data availability",
"Results",
"Growth and heart histidine levels",
"Cataract status",
"Lens de novo transcriptome assembly",
"Differentially expressed genes (DEGs)",
"Functional analysis",
"Impact of cataracts",
"Impact of ploidy",
"Discussion",
"Comparison",
"Declaration of competing interest",
"Fig. 1 .",
"Fig. 2 .",
"Fig. 3 .",
"Fig. 4 .",
"Table 1",
"Table 1 (",
"Table 3"
] | [
"# \nTreatment comparison/Pathway \nDEGs with pathway \nannotation \n\nAll genes with pathway \nannotation \n\np-value \nQ-value \nPathway \nID \n\n2N-vs 2N+ \n72 \n11680 \n\n1 \nPhototransduction \n13 (18.06%) \n67 (0.57%) \n1.161234e-16 \n9.638242e-\n15 \n\nko04744 \n\n2 \nCarbohydrate digestion and absorption \n8 (11.11%) \n39 (0.33%) \n7.384585e-11 \n3.064603e-\n09 \n\nko04973 \n\n3 \nProximal tubule bicarbonate reclamation \n7 (9.72%) \n27 (0.23%) \n2.019998e-10 \n5.588661e-\n09 \n\nko04964 \n\n4 \nMineral absorption \n6 (8.33%) \n48 (0.41%) \n4.43982e-07 \n7.370101e-\n06 \n\nko04978 \n\n5 \nGlutamatergic synapse \n7 (9.72%) \n95 (0.81%) \n1.803348e-06 \n1.663088e-\n05 \n\nko04724 \n\n6 \nAldosterone-regulated sodium reabsorption \n5 (6.94%) \n35 (0.3%) \n2.173693e-06 \n1.804165e-\n05 \n\nko04960 \n\n7 \nChemokine signaling pathway \n9 (12.5%) \n231 (1.98%) \n1.141543e-05 \n7.895672e-\n05 \n\nko04062 \n\n8 \nDopaminergic synapse \n8 (11.11%) \n178 (1.52%) \n1.295758e-05 \n8.272916e-\n05 \n\nko04728 \n\n9 \nGABAergic synapse \n6 (8.33%) \n86 (0.74%) \n1.414778e-05 \n8.387612e-\n05 \n\nko04727 \n\n10 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (6.94%) \n70 (0.6%) \n6.855952e-05 \n3.793627e-\n04 \n\nko04961 \n\n11 \nRetrograde endocannabinoid signaling \n5 (6.94%) \n77 (0.66%) \n0.0001082454 \n5.615230e-\n04 \n\nko04723 \n\n12 \nCholinergic synapse \n5 (6.94%) \n86 (0.74%) \n0.0001827854 \n8.924228e-\n04 \n\nko04725 \n\n13 \nSerotonergic synapse \n5 (6.94%) \n92 (0.79%) \n0.0002508305 \n1.095733e-\n03 \n\nko04726 \n\n14 \nProtein digestion and absorption \n5 (6.94%) \n122 (1.04%) \n0.0009166398 \n3.732900e-\n03 \n\nko04974 \n\n15 \nCardiac muscle contraction \n5 (6.94%) \n140 (1.2%) \n0.001692799 \n6.108796e-\n03 \n\nko04260 \n\n16 \nMAPK signaling pathway \n6 (8.33%) \n302 (2.59%) \n0.01064676 \n3.398773e-\n02 \n\nko04010 \n\n17 \nEndocytosis \n6 (8.33%) \n324 (2.77%) \n0.01466203 \n4.507217e-\n02 \n\nko04144 \n\n1 \n3N-vs 3N+ \n63 \n11680 \nProximal tubule bicarbonate reclamation \n7 (11.11%) \n27 (0.23%) \n7.690174e-11 \n5.998336e-\n09 \n\nko04964 \n\n2 \nCarbohydrate digestion and absorption \n6 (9.52%) \n39 (0.33%) \n5.481106e-08 \n1.654731e-\n06 \n\nko04973 \n\n3 \nPhototransduction \n7 (11.11%) \n67 (0.57%) \n6.364351e-08 \n1.654731e-\n06 \n\nko04744 \n\n4 \nMineral absorption \n6 (9.52%) \n48 (0.41%) \n1.985252e-07 \n3.871241e-\n06 \n\nko04978 \n\n5 \nAldosterone-regulated sodium reabsorption \n5 (7.94%) \n35 (0.3%) \n1.113271e-06 \n1.010844e-\n05 \n\nko04960 \n\n6 \nGABAergic synapse \n6 (9.52%) \n86 (0.74%) \n6.486509e-06 \n4.599525e-\n05 \n\nko04727 \n\n7 \nGlutamatergic synapse \n6 (9.52%) \n95 (0.81%) \n1.154713e-05 \n7.505634e-\n05 \n\nko04724 \n\n8 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (7.94%) \n70 (0.6%) \n3.590785e-05 \n2.154471e-\n04 \n\nko04961 \n\n9 \nProtein digestion and absorption \n6 (9.52%) \n122 (1.04%) \n4.796446e-05 \n2.672306e-\n04 \n\nko04974 \n\n10 \nRetrograde endocannabinoid signaling \n5 (7.94%) \n77 (0.66%) \n5.694637e-05 \n2.961211e-\n04 \n\nko04723 \n\n11 \nCholinergic synapse \n5 (7.94%) \n86 (0.74%) \n9.671235e-05 \n4.714727e-\n04 \n\nko04725 \n\n12 \nSerotonergic synapse \n5 (7.94%) \n92 (0.79%) \n0.0001332214 \n6.112511e-\n04 \n\nko04726 \n\n13 \nChemokine signaling pathway \n7 (11.11%) \n231 (1.98%) \n0.0002331504 \n1.010318e-\n03 \n\nko04062 \n\n14 \nDopaminergic synapse \n6 (9.52%) \n178 (1.52%) \n0.0003808976 \n1.555533e-\n03 \n\nko04728 \n\n15 \nArginine and proline metabolism \n4 (6.35%) \n67 (0.57%) \n0.00045676 \n1.696537e-\n03 \n\nko00330 \n\n16 \nCardiac muscle contraction \n5 (7.94%) \n140 (1.2%) \n0.0009267016 \n3.142727e-\n03 \n\nko04260 \n\n17 \nPPAR signaling pathway \n3 (4.76%) \n90 (0.77%) \n0.01259403 \n3.929337e-\n02 \n\nko03320 \n\n1 \n2N-vs 3N-\n45 \n11680 \nPhototransduction \n9 (20%) \n67 (0.57%) \n2.900818e-12 \n1.885532e-\n10 \n\nko04744 \n\n(continued on next page) \n\nP.A. Olsvik et al. \n",
"continued ) \n\n# \nTreatment comparison/Pathway \nDEGs with pathway \nannotation \n\nAll genes with pathway \nannotation \n\np-value \nQ-value \nPathway \nID \n\n2N-vs 2N+ \n72 \n11680 \n\n2 \nCarbohydrate digestion and absorption \n7 (15.56%) \n39 (0.33%) \n1.084661e-10 \n3.525148e-\n09 \n\nko04973 \n\n3 \nProximal tubule bicarbonate reclamation \n6 (13.33%) \n27 (0.23%) \n6.445801e-10 \n1.396590e-\n08 \n\nko04964 \n\n4 \nAldosterone-regulated sodium reabsorption \n5 (11.11%) \n35 (0.3%) \n2.011263e-07 \n2.614642e-\n06 \n\nko04960 \n\n5 \nMineral absorption \n5 (11.11%) \n48 (0.41%) \n1.022166e-06 \n9.305999e-\n06 \n\nko04978 \n\n6 \nSerotonergic synapse \n6 (13.33%) \n92 (0.79%) \n1.288523e-06 \n9.305999e-\n06 \n\nko04726 \n\n7 \nChemokine signaling pathway \n8 (17.78%) \n231 (1.98%) \n2.378076e-06 \n1.405227e-\n05 \n\nko04062 \n\n8 \nProtein digestion and absorption \n6 (13.33%) \n122 (1.04%) \n6.701316e-06 \n3.150043e-\n05 \n\nko04974 \n\n9 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (11.11%) \n70 (0.6%) \n6.784707e-06 \n3.150043e-\n05 \n\nko04961 \n\n10 \nRetrograde endocannabinoid signaling \n5 (11.11%) \n77 (0.66%) \n1.085652e-05 \n4.704492e-\n05 \n\nko04723 \n\n11 \nCholinergic synapse \n5 (11.11%) \n86 (0.74%) \n1.865061e-05 \n7.131116e-\n05 \n\nko04725 \n\n12 \nGABAergic synapse \n5 (11.11%) \n86 (0.74%) \n1.865061e-05 \n7.131116e-\n05 \n\nko04727 \n\n13 \nGlutamatergic synapse \n5 (11.11%) \n95 (0.81%) \n3.024149e-05 \n1.092054e-\n04 \n\nko04724 \n\n14 \nDopaminergic synapse \n6 (13.33%) \n178 (1.52%) \n5.726626e-05 \n1.959109e-\n04 \n\nko04728 \n\n15 \nCardiac muscle contraction \n5 (11.11%) \n140 (1.2%) \n0.0001913906 \n5.923995e-\n04 \n\nko04260 \n\n16 \nRetinol metabolism \n2 (4.44%) \n19 (0.16%) \n0.002380689 \n6.447699e-\n03 \n\nko00830 \n\n17 \nNeuroactive ligand-receptor interaction \n2 (4.44%) \n52 (0.45%) \n0.01703389 \n4.258472e-\n02 \n\nko04080 \n\n1 \n2N + vs 3N+ \n68 \n11680 \nPhototransduction \n14 (20.59%) \n67 (0.57%) \n9.57472e-19 \n5.170349e-\n17 \n\nko04744 \n\n2 \nProtein digestion and absorption \n16 (23.53%) \n122 (1.04%) \n6.816708e-18 \n1.840511e-\n16 \n\nko04974 \n\n3 \nCarbohydrate digestion and absorption \n8 (11.76%) \n39 (0.33%) \n4.604135e-11 \n6.215582e-\n10 \n\nko04973 \n\n4 \nProximal tubule bicarbonate reclamation \n7 (10.29%) \n27 (0.23%) \n1.337376e-10 \n1.444366e-\n09 \n\nko04964 \n\n5 \nDopaminergic synapse \n9 (13.24%) \n178 (1.52%) \n8.241675e-07 \n5.454431e-\n06 \n\nko04728 \n\n6 \nAldosterone-regulated sodium reabsorption \n5 (7.35%) \n35 (0.3%) \n1.633423e-06 \n8.820484e-\n06 \n\nko04960 \n\n7 \nECM-receptor interaction \n8 (11.76%) \n146 (1.25%) \n1.929870e-06 \n8.874621e-\n06 \n\nko04512 \n\n8 \nChemokine signaling pathway \n9 (13.24%) \n231 (1.98%) \n7.078719e-06 \n2.940391e-\n05 \n\nko04062 \n\n9 \nMineral absorption \n5 (7.35%) \n48 (0.41%) \n8.12637e-06 \n3.134457e-\n05 \n\nko04978 \n\n10 \nGABAergic synapse \n6 (8.82%) \n86 (0.74%) \n1.014675e-05 \n3.652830e-\n05 \n\nko04727 \n\n11 \nGlutamatergic synapse \n6 (8.82%) \n95 (0.81%) \n1.800377e-05 \n6.076272e-\n05 \n\nko04724 \n\n12 \nEndocrine and other factor-regulated calcium \nreabsorption \n\n5 (7.35%) \n70 (0.6%) \n5.203389e-05 \n1.652841e-\n04 \n\nko04961 \n\n13 \nRetrograde endocannabinoid signaling \n5 (7.35%) \n77 (0.66%) \n8.231658e-05 \n2.339524e-\n04 \n\nko04723 \n\n14 \nCholinergic synapse \n5 (7.35%) \n86 (0.74%) \n0.000139355 \n3.762585e-\n04 \n\nko04725 \n\n15 \nSerotonergic synapse \n5 (7.35%) \n92 (0.79%) \n0.0001915559 \n4.925723e-\n04 \n\nko04726 \n\n16 \nArginine and proline metabolism \n4 (5.88%) \n67 (0.57%) \n0.0006112079 \n1.500238e-\n03 \n\nko00330 \n\n17 \nCardiac muscle contraction \n5 (7.35%) \n140 (1.2%) \n0.001310250 \n2.948062e-\n03 \n\nko04260 \n\n18 \nPPAR signaling pathway \n4 (5.88%) \n90 (0.77%) \n0.001843347 \n3.828490e-\n03 \n\nko03320 \n\n19 \nFocal adhesion \n8 (11.76%) \n418 (3.58%) \n0.002838214 \n5.676428e-\n03 \n\nko04510 \n\nP.A. Olsvik et al. \n",
"Upstream regulator \n2N + vs 2N-\n3N + vs 3N-\n\nCRX \n2.73 \n− 2.55 \nGTF2IRD1 \n2.10 \n− 1.85 \nSRC \n1.70 \n− 1.73 \nRHO \n1.89 \n− 1.63 \ndecitabine \n2.77 \n2.20 \nIL6 \n2.25 \n3.06 \nP38 MAPK \n2.11 \n1.44 \nOSM \n1.23 \n2.36 \nNFkB (complex) \n1.34 \n1.95 \nAPP \n1.25 \n1.94 \nhexachlorobenzene \n2.0 \n1.98 \ncisplatin \n1.77 \n1.86 \nSTAT3 \n1.76 \n2.20 \nthioacetamide \n1.96 \n2.21 \nlipopolysaccharide \n2.12 \n2.32 \npioglitazone \n− 1.92 \ncurcumin \n− 0.73 \n− 1.71 \nU0126 \n− 1.96 \n− 1.29 \nLY294002 \n− 1.67 \n− 1.67 \nN-acetyl-L-cysteine \n− 1.67 \n− 0.44 \nMYC \n− 1.40 \n0.30 \nCSF2 \n− 1.48 \nPD98059 \n− 1.82 \n− 0.01 \nsirolimus \n− 2.0 \nESR2 \n− 1.94 \n0.15 \ncyclosporin A \n1.07 \n1.39 \nnitrofurantoin \n0.65 \n1.48 \ndihydrotestosterone \n0.78 \n1.26 \nbucladesine \n1.27 \n1.19 \nbeta-estradiol \n1.41 \n1.15 \n"
] | [
"Table 1",
"Table 2",
"(Table 3)",
"Table 2",
"(Table 2"
] | [
"A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts",
"A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts"
] | [
"Experimental Eye Research"
] |
12,351,017 | 2022-03-20T03:40:35Z | CCBYNC | https://doi.org/10.3349/ymj.2012.53.6.1165 | GOLD | 58394b1277732eed90b6a5b8e962b4767f5c585b | null | null | null | null | 10.3349/ymj.2012.53.6.1165 | 3024948094 | 23074118 | 3481382 |
Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy
6 November 2012. 2012
Do-Hee Kim
Tae-Hyun Yoo
Soon Ha Lee
Hye Young Kang
SeungBo Young Nam
Jae Kwak
Jwa-Kyung Kim
Jung Tak Park
Seung Hyeok Han
Shin-Wook Kang
DrShin-Wook Kang
Department of Internal Medicine
Brain Korea
Project for Medical Science
Department of Internal Medicine
Yonsei University College of Medicine
SeoulKorea
Yonsei University College of Medicine
50 Yonsei-ro, Seodaemun-gu120-752SeoulKorea
Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy
Yonsei Med J
5366 November 2012. 201210.3349/ymj.2012.53.6.1165Received: March 30, 2012 Revised: May 16, 2012 Accepted: May 17, 20121165 Original Article Corresponding author: *Do-Hee Kim and Tae-Hyun Yoo contributed equally to this work. • The authors have no financial conflicts of interest.Gamma linolenic acidexperimental diabetic nephropathyanti-in- flammatoryanti-fibrotic
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Purpose: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. Materials and Methods: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 µM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. Results: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. Conclusion: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.
INTRODUCTION
Diabetic nephropathy, the leading cause of end-stage renal disease worldwide, is characterized pathologically by cellular hypertrophy and increased extracellular matrix (ECM) accumulation. 1 The ECM accumulation in diabetic nephropathy results in mesangial expansion, tubulointerstitial fibrosis, and irreversible deterioration of renal function. 2 Even though previous studies have shown that ECM ac-
MATERIALS AND METHODS Animals
All animal studies were conducted using an approved protocol the committee for the care and use of laboratory animals of Yonsei University College of Medicine. Sprague-Dawley rats weighing 250-280 g were intraperitoneally injected with either a diluent [n=16, control (C)] or 65 mg/ kg streptozotocin [n=16, diabetes (DM)]. Diabetes was confirmed by tail vein blood glucose levels on the third post-injection day. After confirming diabetes, eight rats each from the C and DM groups were treated with 450 mg/kg/day of evening primrose oil (EPO, a generous gift from Dalim Biotech, Seoul, Korea) by gavage (C+GLA or DM+ GLA) for three months. EPO contained 8-10% GLA, and the amount of EPO used in this study provided an approximate GLA dose of 40 mg/kg/day. Rats were housed in a temperaturecontrolled room and given free access to water and standard laboratory chow during the three-month study period. Body weight and serum glucose level were checked monthly, and kidney weight and 24-hour urinary albumin excretion were checked at the time of sacrifice. Blood glucose was measured by a glucometer, and 24-hour urinary albumin excretion was determined by enzyme-linked immunosorbent assay (ELISA) (Nephrat II, Exocell, Inc., Philadelphia, PA, USA).
Cell culture
Primary culture of glomerular mesangial cells was performed as previously described. 17 Identification of mesangial cells was performed by their characteristic stellate appearance in culture and confirmed by immunofluorescent microscopy for the presence of actin, myosin, and Thy-1 antigen, as well as the absence of factor VIII and cytokeratin (Synbiotics, San Diego, CA, USA). Mesangial and NRK-52E cells and immortalized rat tubular epithelial cells, were maintained, respectively, in RPMI 1640 and DMEM medium supplemented with 5% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 26 mM NaHCO 3 , and grown at 37ºC in humidified 5% CO 2 in air. Subconfluent mesangial cells and NRK-52E cells were serum restricted for 24 hours, after which the medium was replaced by serum-free medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without 10 or 100 μM GLA (Sigma Chemical Co., St. Louis, MO, USA). In addition, both of the cells were cumulation under diabetic conditions is attributable to hyperglycemia per se, advanced glycation end-products, hemodynamic changes, and local growth factors such as angiotensin II (AII) and transforming growth factor (TGF)-β1, 3 the precise molecular and cellular mechanisms responsible for this have yet to be resolved.
Recently, accumulating evidence has suggested that the inflammatory process also plays an important role in the pathogenesis of diabetic nephropathy. 4 Infiltration of inflammatory cells in glomeruli and renal tubulointerstitium is commonly seen in both human diabetic patients and experimental diabetic animals. 5,6 In addition, intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), which mediates the recruitment and infiltration of monocytes/macrophages, have been shown to be involved in the pathogenesis of diabetic nephropathy. 7,8 Based on these findings, modulation of the inflammatory process is considered to be a potential means of preventing the development and progression of diabetic nephropathy, and some immunosuppressive agents and anti-inflammatory drugs have been found to be beneficial in diabetic nephropathy. 9,10 Nevertheless, chronic use of these drugs in the clinical field is not appropriate due to many systemic side effects. Therefore, safe agents for chronic treatment of diabetic nephropathy are undoubtedly needed.
Polyunsaturated fatty acids (PUFAs), which exist in high concentrations in cell membranes as structural phospholipids, are essential to cell integrity and viability. 11,12 There are two classes of PUFAs: ω-3 and ω-6, designated according to their carbon ring structure. 12 γ-linolenic acid (GLA), a member of ω-6 PUFA, is produced from linoleic acid by the enzyme δ-6 desaturase, and is elongated to dihomogamma linolenic acid (DGLA). [11][12][13] In a previous study, GLA was shown to abrogate renal fibrosis in a 5/6 nephrectomy model, 14 and other investigations have demonstrated that GLA treatment improved autoimmune diseases and diabetic neuropathy via an anti-inflammatory mechanism. 15,16 As mentioned earlier, since the inflammatory process is also involved in the pathogenesis of diabetic nephropathy, there is a possibility that GLA may ameliorate diabetic nephropathy, but this has never been explored.
In this study, therefore, we investigated the effects of GLA in experimental diabetic kidneys as well as in high glucosestimulated mesangial cells and tubular epithelial cells in regards to inflammation and ECM synthesis. GG. CCG-3'; and 18s, sense 5'-AGTCCCTGCCCTTTGT ACACA-3', antisense 5'-GATCCGAGGGCCTCACTA AAC-3'. cDNAs from 25 ng RNA of the renal cortical tissue or cultured cells per reaction tube were used for amplification.
Using the ABI PRISM ® 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), PCR was performed with a total volume of 20 μL in each well, containing 10 μL of SYBR Green ® PCR Master Mix (Applied Biosystems), 5 μL of cDNA, and 5 pM sense and antisense primers. Primer concentrations were determined by preliminary experiments that analyzed the optimal concentrations of each primer. Each sample was run in triplicate in separate tubes. The PCR conditions were as follows: 35 cycles of denaturation at 94.5ºC for 30 sec, annealing at 60ºC for 30 sec, and extension at 72ºC for one minute. Initial heating at 95ºC for nine minutes and final extension at 72ºC for seven minutes were performed for all PCRs.
After real-time PCR, the temperature was increased from 60 to 95ºC at a rate of 2ºC/min to construct a melting curve. A control without cDNA was run in parallel with each assay. The cDNA content of each specimen was determined using a comparative C T method with 2 -∆∆T . The results are given as relative expressions of ICAM-1, MCP-1, and fibronectin normalized to the expression of the 18s housekeeping gene.
Western blot analysis
The renal cortical tissue and cultured cells harvested from plates were lysed in sodium dodecyl sulfate (SDS) sample buffer [2% sodium dodecyl sulfate, 10 mM Tris-HCl, pH 6.8, 10% (vol/vol) glycerol], treated with Laemmli sample buffer, heated at 100ºC for five minutes, and electrophoresed in an 8% acrylamide denaturing SDS-polyacrylamide gel. Proteins were then transferred to a Hybond-ECL membrane using a Hoeffer semidry blotting apparatus (Hoeffer Instruments, San Francisco, CA, USA). The membrane was then incubated in blocking buffer A (1×PBS, 0.1% Tween-20, and 8% nonfat milk) at room temperature for one hour, followed by an overnight incubation at 4ºC in a 1 : 1000 dilution of polyclonal antibodies to ICAM-1 (R&D systems, Minneapolis, MN, USA), fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or β-actin (Sigma Chemical Co., St. Louis, MO, USA). The membrane was then washed once for 15 minutes and twice for five minutes in 1×PBS with 0.1% Tween-20. Next, the membrane was incubated in buffer A containing a 1 : 1000 treated by 10 or 100 μM linoleic acid (Sigma Chemical Co., St. Louis, MO, USA). At 24 hours after the media change, the cells were harvested and conditioned culture media were collected.
Total RNA extraction
Total RNA from the renal cortical tissue was extracted as previously described. 17 Briefly, 100 μL of RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA) was added to the renal cortical tissues, which were lysed by freezing and thawing three times. Another 700 μL of RNA STAT-60 reagent was then added and the mixture was vortexed and stored for five minutes at room temperature. Next, 160 μL of chloroform was added and the mixture was shaken vigorously for 30 seconds. After three minutes, the mixture was centrifuged at 12000×g for 15 minutes at 4ºC and the upper aqueous phase containing the extracted RNA was transferred to a new tube. RNA was precipitated from the aqueous phase by adding 400 μL of isopropanol and then pelleted by centrifugation at 12000×g for 30 minutes at 4ºC. The RNA precipitate was washed with 70% ice-ethanol, dried using a Speed Vac, and dissolved in DEPC-treated distilled water. RNA yield and quality were assessed based on spectrophotometric measurements at wavelengths of 260 and 280 nm. Total RNA from the mesangial and NRK-52E cells was similarly extracted.
Reverse transcription
First strand cDNA was synthesized using a Boehringer Mannheim cDNA synthesis kit (Boehringer Mannheim GmbH, Mannheim, Germany). Two μg of total RNA extracted from the renal cortex and cultured cells were reverse transcribed using 10 μM random hexanucleotide primer, 1 mM dNTP, 8 mM MgCl 2 , 30 mM KCl, 50 mM Tris-HCl, pH 8.5, 0.2 mM dithiothreitol, 25 U RNase inhibitor, and 40 U AMV reverse transcriptase. The mixture was incubated at 30ºC for 10 minutes and 42ºC for one hour, followed by inactivation of the enzyme at 99ºC for five minutes.
Real-time polymerase chain reaction (real-time PCR)
The primers used for ICAM-1, MCP-1, fibronectin, and 18s amplification were as follows: ICAM-1, sense 5'-AGGTA TCCATCCATCCCAC-3', antisense 5'-GCCGAGG TTCTCGTCTTC-3'; MCP-1, sense 5'-TCTCTTCCTCC ACCACTATGCA-3', antisense 5'-GGCTGAGACAGC ACGTGGAT-3'; fibronectin, sense 5'-TGACAACTGCCG TAGACCTGG-3', antisense 5'-TACTGGTTGTAGGTGT respectively, and by a digital image analyzer (MetaMorph version 4.6r5, Universal Imaging Corp., Downingtown, PA, USA) as previously described. 17 The degree of staining was semi-quantitated on a scale of 0-4+. The staining score was obtained by multiplying the intensity of staining by the percentage of glomeruli or tubulointerstitium staining for that intensity, and these numbers were then added for each experimental animal to give the staining score [Σ=(Intensity of staining)×(% of glomeruli or tubulointerstitium with that intensity)]. The number of ED-1 positive cells was counted in at least 20 glomeruli and 20 fields of the tubulointerstitium/section under ×400 magnification.
Statistical analysis
All values are expressed as the mean±standard error of the mean. Statistical analysis was performed using the statistical package SPSS for Windows Ver. 11.0 (SPSS, Inc., Chicago, IL, USA). Results were analyzed using the Kruskal-Wallis non-parametric test for multiple comparisons. Significant differences on the Kruskal-Wallis test were further confirmed by the Mann-Whitney U test. p-values less than 0.05 were considered statistically significant.
RESULTS
Animal studies
Animal data All the animals gained weight over the three-month experimental period, but body weight was highest among the C rats (593±11 g). The ratio of kidney to body weight in the DM rats (1.17±0.15%) was significantly higher than those of the C (0.58±0.05%), C+GLA (0.53±0.06%) (p<0.01), and DM+GLA (0.88±0.11) (p<0.05) rats. The mean blood glucose levels of C, C+GLA, DM, and DM+GLA rats were 104.1±3.9, 97.6±3.5, 489.5±14.0, and 474.0±13.0 mg/dL, dilution of horseradish peroxidase-linked donkey anti-goat IgG (Amersham Life Science, Inc., Arlington Heights, IL, USA). The washes were repeated, and the membrane was developed with a chemiluminescent agent (ECL; Amersham Life Science, Inc., Arlington Heights, IL, USA). The band densities were measured using TINA image software (Raytest, Straubenhardt, Germany).
Measurement of MCP-1 by ELISA
The levels of MCP-1 in the renal cortical tissue and culture media were determined using a commercial ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's protocol. The kit for rat MCP-1 was species-specific and sensitive up to 750 pg/mL. All concentrations of MCP-1 were normalized to the total protein amount.
Pathology
For immunohistochemical staining, slices of kidney were snap-frozen in optimal cutting temperature solution and 4 μm sections of tissues were utilized. Slides were fixed in acetone for 10 minutes, air dried at room temperature for 10 minutes, and blocked with 10% donkey serum at room temperature for 20 minutes. For ICAM-1, fibronectin and ED-1 staining, the primary polyclonal antibody to ICAM-1, the extracellular domain of fibronectin (Chemicon International, Inc., Billerica, MA, USA) or ED-1 (Chemicon International, Inc., Billerica, MA, USA), respectively, was diluted to 1 : 100 with 2% casein in BSA and was applied for overnight incubation at room temperature. After washing, a secondary donkey anti-goat antibody was added for 20 minutes and the slides were then washed and incubated with a tertiary PAP complex for 20 minutes. DAB was added for two minutes and the slides were counterstained with hematoxylin. A semi-quantitative score for measuring the intensity of ICAM-1 and fibronectin staining within the glomeruli and tubulointerstitial area was determined by examining at least 20 glomeruli under ×400 magnification and 20 tubulointerstitial fields under ×200 magnification, 1 and fibronectin mRNA expression in DM rats (p<0.05) (Fig. 1).
Effect of GLA on renal cortical MCP-1 levels and ICAM-1 and fibronectin protein expression
The levels of renal MCP-1 assessed by ELISA were also significantly higher in DM than in C rats (563.5±42.9 vs. 287.1±22.3 ng/μg, p<0.01), and the increase in MCP-1 levels in DM rats was significantly ameliorated by GLA treatment (354.9±31.3 ng/μg, p<0.05) (Fig. 2). Renal ICAM-1 and fibronectin protein expression assessed by Western blot was also significantly increased in DM rats relative to C and C+GLA rats (p<0.01), and GLA treatment significantly abrogated these increases in DM rats (p<0.05) (Fig. 3). In addition, immunohistochemical staining for ICAM-1 and fibronectin confirmed the real-time PCR and Western blot findings. There were significant increases in glomerular and tubulointerstitial ICAM-1 and fibronectin staining in the respectively (p<0.01). Compared to the C group (0.35±0.07 mg/day), 24-hour urinary albumin excretion at three months was significantly higher in DM rats (2.51±0.28 mg/day) (p<0.01), and GLA treatment significantly reduced albuminuria in DM rats (1.11±0.12 mg/day) (p<0.05) ( Table 1).
Effect of GLA on renal cortical MCP-1, ICAM-1, and fibronectin mRNA expression Renal MCP-1 mRNA expression assessed by real-time PCR was significantly higher in DM than in C rats (p<0.01), and this increase in MCP-1 mRNA expression was significantly inhibited by administration of GLA (p<0.05). The MCP-1 mRNA/18s rRNA ratio was 2.1-fold higher in DM than in C kidneys, and GLA treatment significantly abrogated this increase by 65.7%. The ratios of ICAM-1 and fibronectin mRNA/18s rRNA were also significantly higher in DM compared to C and C+GLA kidneys (p<0.01), and GLA treatment significantly attenuated increases in renal ICAM- Fig. 1. Renal MCP-1, ICAM-1, and fibronectin mRNA/18s rRNA ratios in control (C), C+gamma linolenic acid (GLA), diabetic (DM), and DM+GLA rats. There was a 2.1-fold increase in MCP-1 mRNA/18s rRNA, a 1.8-fold increase in ICAM-1 mRNA/18s rRNA, and a 2.7-fold increase in fibronectin mRNA/18s rRNA ratios in DM rats compared to C rats, and GLA treatment significantly abrogated these increases in mRNA/18s rRNA ratios in DM rats. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. MCP-1, monocyte chemoattractant protein-1; ICAM-1, intracellular adhesion molecule-1. NRK-52E cells exposed to HG medium (p<0.01), respectively, and GLA treatment significantly ameliorated these increases in a dose-dependent manner (p<0.05) (Fig. 5). The levels of MCP-1 protein in conditioned culture media assessed by ELISA showed a similar pattern to the mRNA expression (Fig. 5). However, linoleic acid did not induce significant changes in these patterns (data not shown).
Effect of GLA on ICAM-1 and fibronectin mRNA expression
High glucose significantly induced ICAM-1 and fibronectin mRNA expression in mesangial and NRK-52E cells. Compared to NG cells, the ICAM-1 mRNA/18s rRNA ratios were 2.2-and 1.8-fold higher in HG-stimulated mesangial cells and tubular epithelial cells (p<0.01), respectively, and these increases were significantly attenuated by 60.9% and 62.3%, respectively, with 10 μM GLA treatment, and by 70.7% and 76.1%, respectively, with 100 μM GLA treatment (Fig. 6). Fibronectin mRNA/18s rRNA ratios were significantly increased in HG-stimulated mesangial and NRK-52E cells relative to NG cells by 154.4% and 122.2% (p<0.01), respectively, and these increases were significant-DM group compared to the C and C+GLA groups, and these increases in DM rats were significantly ameliorated by the administration of GLA (Fig. 4).
Effect of GLA on macrophage accumulation
The number of macrophages within glomeruli and tubulointerstitium assessed by immunohistochemical staining with ED-1 antibody was significantly higher in DM rats than in C rats (27.8±4.3 vs. 3.6±0.8, p<0.01), and GLA treatment significantly abrogated the number of ED-1-positive cells in DM rats (10.1±1.2) (p<0.05) (Fig. 4).
Cell culture studies
Effect of GLA on MCP-1 mRNA and protein expression MCP-1 mRNA expression assessed by real-time PCR was significantly increased in HG-stimulated mesangial cells and NRK-52E cells (p<0.01), and this increase in MCP-1 mRNA expression was significantly abrogated by GLA treatment (p<0.05). Compared to NG cells, the MCP-1 mRNA/18s rRNA ratios were 2.0-and 2.1-fold higher in mesangial and Fig. 4. Immunohistochemical staining for glomerular and tubulointerstitial ICAM-1, fibronectin, and ED-1 (as a marker of macrophage) in C, C+GLA, DM, and DM+GLA rats. Glomerular (A) and tubulointerstitial (B) ICAM-1 and fibronectin staining was significantly increased in DM rats compared to C rats, and GLA treatment significantly inhibited these increases in DM rats. The number of ED-1-positive cells was significantly higher in DM rats than in C rats, and GLA treatment significantly abrogated the number of glomerular and tubulointerstitial macrophages in DM rats. (C) IHC scores for ICAM-1 and fibronectin within the glomeruli and the tubulointerstitial area were significantly higher in DM rats relative to C rats, and GLA treatment significantly attenuated these increas- ed by inhibiting increases in MCP-1 and ICAM-1 expression under diabetic conditions. Even though the diabetic milieu per se, hemodynamic changes, and local growth factors such as AII and TGF-β are considered mediators in the pathogenesis of diabetic nephropathy, 3,6 recent studies suggest that an inflammatory mechanism may also contribute to the development of diabetic nephropathy based on the pathological findings of inflammatory cell infiltration in diabetic kidneys. 4 Monocytes/macrophages are the major inflammatory cells found in diabetic kidneys. 5,17 They are extravasculated from the bloodstream and attracted to the target tissue through a process mediated by various chemokines and adhesion molecules such as MCP-1 and ICAM-1. 7,18 In the kidneys, MCP-1 is expressed in mesangial cells and tubular epithelial cells and is known to be involved in the pathogenesis of various renal diseases, including diabetic nephropathy. 7,19 Previous studies have demonstrated that plasma MCP-1 levels are ly abrogated by GLA in a dose-dependent manner (p<0.05) (Fig. 6).
Effect of GLA on ICAM-1 and fibronectin protein expression
ICAM-1 and fibronectin protein expression showed similar patterns to their mRNA expressions. GLA also significantly inhibited HG-induced ICAM-1 and fibronectin protein expression in cultured mesangial cells and NRK-52E cells (Fig. 7).
DISCUSSION
In the present study, we demonstrated that GLA has a renoprotective effect via its anti-inflammatory and anti-fibrotic actions in experimental diabetic nephropathy. In addition, the results of this study suggested that the anti-inflammatory effect of GLA under diabetic conditions is partly mediat- FAs are beneficial to one's overall health and a number of various diseases. 13 In cases of kidney disease, PUFAs have been reported to have beneficial effects on IgA nephropathy, 27 chronic renal failure, and diabetic nephropathy via antioxidant, anti-inflammation, and anti-fibrotic mechanisms. 13,28 In contrast, the effect of GLA, a member of the ω-6 PUFA family, on kidney diseases has been less explored. Ingram ,et al. 14 observed that administration of borage oil (BO), a rich source of GLA, was effective in a rat 5/6-renal-ablation model. BO prevented increases in blood pressure and proteinuria, rising plasma cholesterol levels, and declining glomerular filtration rates. In addition, glomerular macrophage infiltration, mesangial expansion, and glomerulosclerosis were attenuated in BO-treated rats compared to the control diet group. Meanwhile, since these glomerular changes are also characteristic of diabetic nephropathy, the activities of δ-5 and δ-6 desaturase are decreased along with low levels of GLA and DGLA in diabetes, 29 and since GLA has proven useful in diabetic neuropathy, 16,30 supplementation of GLA and/or DGLA may also be of benefit in diabetic nephropathy. However, the effect of ω-6 PUFA has never been elucidated in diabetic nephropathy. The results of the present study showed for the first time that GLA inhibits inflammatory cell infiltration and ECM accumulation in experimental diabetic kidneys, suggesting the usefulness of GLA in patients with diabetic nephropathy.
Even though the underlying mechanisms of the anti-inflammatory effect of GLA in this study are not completely understood, several plausible explanations can be proposed. First, PUFAs, including GLA, are known to serve as endogenous ligands of peroxisome proliferator activated receptors (PPARs) and to bind and activate all PPARs isoforms. 31 Additionally, 15-HETE, one of the metabolites of GLA, has been reported to upregulate nuclear PPAR-γ expression. 32 PPARs participate in regulating inflammatory responses by inhibiting monocyte expression of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α. 33 Furthermore, PPAR-γ attenuates the nuclear factor-κB-mediated transcriptional activation of proinflammatory genes. 33 Recent studies have also demonstrated that PPAR-γ agonist exerts a renoprotective effect through an anti-inflammatory mechanism in diabetic nephropathy. 34 Taken together, GLA, as a ligand of PPARs and its metabolite, as well as an upregulator of PPAR-γ, could induce an anti-inflammatory effect in diabetic nephropathy. Second, a small amount of DGLA can be converted to prostacyclin (PGI2) and prostaglandin E1 (PGE1) by δ-5 desaturases. Because PGI2 and PGE1 inhib-higher in type 1 diabetes with microalbuminuria 20 and that urinary levels of MCP-1 are higher in accordance with the extent of albuminuria. 7 Renal expression of ICAM-1, a cell surface glycoprotein that plays a major role in the regulation of interactions with immune cells and whose expression is upregulated at sites of inflammation, 8,21 is also known to be increased in experimental type 1 and type 2 diabetic animals. 17,21 These findings suggest that MCP-1 and ICAM-1 may play an important role in the pathogenesis of diabetic nephropathy by inducing inflammatory cell infiltration. 7,8 Once the recruited monocytes/macrophages are activated, they release lysosomal enzymes, nitric oxide, reactive oxygen species, platelet-derived growth factor (PDGF), tumor necrosis factor-α, interleukin (IL)-1, and TGF-β, in turn promoting renal injury. 5,6,22 PDGF stimulates fibroblast proliferation 6 and IL-1 induces the expression of TGF-β, the most well-known profibrotic cytokine, in fibroblasts. 5 In experimental diabetic nephropathy, various anti-inflammatory agents inhibited not only inflammatory cell infiltration by abrogating increases in MCP-1 and ICAM-1 expression but also ameliorated ECM accumulation. 17,23 In addition, renal fibrosis was significantly inhibited along with less inflammatory cell infiltration in MCP-1-and ICAM-1-deficient diabetic mice. 24,25 Taken together, this suggests that the inhibition of inflammatory cell recruitment may lead to an attenuation of ECM accumulation. In this study, we demonstrated that MCP-1 and ICAM-1 expression were increased in experimental diabetic nephropathy, which were associated with glomerular and tubulointerstitial fibrosis, and in high glucose-stimulated mesangial cells and tubular epithelial cells, and these increases under diabetic conditions were inhibited by GLA treatment. In addition, GLA reduced the ratio of kidney/body weight in diabetic rats, suggesting GLA prevents diabetes-induced kidney enlargement. A recent study also demonstrated that prevention of glomerular hypertrophy ameliorates the development of diabetic nephropathy, including proteinuria and podocytopenia. 26 Taken together, the anti-inflammatory, anti-fibrotic, and anti-hypertrophic effects of GLA in diabetic nephropathy may be partly attributable to the suppression of MCP-1 and ICAM-1 expression by GLA, by which inflammatory cell infiltration is abrogated, in turn ameliorating ECM accumulation.
PUFAs are important constituents of all cell membranes. Since PUFAs are not synthesized in humans, they can only be obtained by diet. 11 There are two classes of PUFAs: ω-3 and ω-6, designated according to their carbon ring structure; 12 and accumulating evidence has shown that these PU-it platelet aggregation, and PGI2 analogue abrogates glomerular hyperfiltration and macrophage infiltration in diabetic kidneys, 35 the effect of GLA may be in part attributed to these consequences. Third, 15-HETE markedly inhibits the generation of leukotriene, which is a potent pro-inflammatory mediator, via stimulating adhesion molecule expression and macrophage infiltration. 36 Collectively, the anti-inflammatory effect of GLA seems to be attributable to the modulation of biological cascades at multiple sites by itself and/or its metabolites. Prolonged use of anti-inflammatory drugs may be harmful and is not appropriate for long-term use due to many systemic side effects in patients with chronic metabolic disturbances such as diabetic nephropathy. However, since the activities of both δ-5 and δ-6 desaturase are already reduced in diabetes and the toxic effects of GLA as a medicinal oil have not been reported, chronic administration of GLA, even at a high dosage, may not induce accumulation of its metabolites and thus would not be harmful.
In summary, the results of the present study demonstrate that GLA exerts anti-inflammatory and anti-fibrotic effects in experimental diabetic nephropathy and in high glucosestimulated renal cells, suggesting that GLA supplementation could be a valuable therapeutic option for the treatment of diabetic nephropathy.
Fig. 3 .
3Renal ICAM-1 and fibronectin protein expression in C, C+GLA, DM, and DM+GLA rats. There was a 3.2-fold increase in ICAM-1 and a 3.7-fold increase in fibronectin protein expression in DM rats compared to C rats, and these increases were significantly attenuated by administration of GLA. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes.
Fig. 2 . 1
21Renal MCP-1 protein levels in C, C+GLA, DM, and DM+GLA rats. There was a 2.0-fold increase in renal MCP-1 protein levels in DM rats compared to C rats, and this increase in the DM rats was significantly ameliorated by GLA treatment. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. C, control; GLA, γ-linolenic acid; DM, diabetes; MCP-
Fig. 6 .
6es in DM rats (×400). *p<0.05 vs. other groups. † p<0.01 vs. C and C+GLA groups. ‡ p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes. ICAM-1 and fibronectin mRNA/18s rRNA ratios in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and H+10 or 100 μM GLA (HG+GLA). There were 2.2-and 1.8-fold increases in ICAM-1 mRNA/18s rRNA ratios in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and these increases in ICAM-1 mRNA/18s rRNA ratios were significantly abrogated by administration of GLA. There were 2.5-fold and 2.2-fold increases in fibronectin mRNA/18s rRNA ratios in HGstimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA treatment significantly attenuated these increases in fibronectin mRNA/18s rRNA ratios in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.
Fig. 5 .
5MCP-1 mRNA/18s rRNA ratios and secreted MCP-1 protein levels in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and HG+10 or 100 μM GLA (HG+GLA). There were 2.0-and 2.1-fold increases in MCP-1 mRNA/18s rRNA ratios in the HG-stimulated mesangial and NRK-52E cells, respectively, compared to the NG cells, and these increases in MCP-1 mRNA expression were significantly ameliorated by GLA treatment in a dose-dependent manner. There were 2.4-and 3.6-fold increases in MCP-1 levels in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA significantly attenuated these increases in MCP-1 levels in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. MCP-1, monocyte chemoattractant protein-1; GLA,
Fig. 7 .
7Representative Western blots of ICAM-1 and fibronectin in cultured mesangial cells (A) and NRK-52E cells (B). There were significant increases in ICAM-1 and fibronectin protein expression in HG-stimulated cells as compared to NG cells, and these increases were significantly ameliorated with GLA treatment. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.
Table 1 .
1Animal Data for the Four Groups UAE, urinary albumin excretion; GLA, γ-linolenic acid; C, control; DM, diabetes. *p<0.01 vs. C and C+GLA group. † p<0.05 vs. DM group.C (n=8)
C+GLA (n=8)
DM (n=8)
DM+GLA (n=8)
Body Wt (g)
593±11
584±14
328± 9*
335±8*
Kidney Wt/Body Wt (%)
0.58±0.05
0.53±0.06
1.17±0.15*
0.88±0.11 †
Blood glucose (mg/dL)
104.1± 3.9
97.6± 3.5
489.5±14.0*
474.0±13.0*
24-hr UAE (mg/day)
0.35±0.07
0.31±0.09
2.51±0.28*
1.11±0.12 †
Wt, weight;
Yonsei Med J http://www.eymj.org Volume 53 Number 6 November 2012
ACKNOWLEDGEMENTSThis study was supported by a faculty research grant of Yonsei University College of Medicine for 2008 (6-2008-0105).
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Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium. S Takata, M Matsubara, P G Allen, P A Janmey, C N Serhan, H R Brady, J Clin Invest. 93Takata S, Matsubara M, Allen PG, Janmey PA, Serhan CN, Brady HR. Remodeling of neutrophil phospholipids with 15(S)-hy- droxyeicosatetraenoic acid inhibits leukotriene B4-induced neutro- phil migration across endothelium. J Clin Invest 1994;93:499-508.
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Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Y Kikuchi, T Imakiire, M Yamada, T Saigusa, T Hyodo, N Hyodo, Nephrol Dial Transplant. 20Kikuchi Y, Imakiire T, Yamada M, Saigusa T, Hyodo T, Hyodo N, et al. Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Nephrol Dial Transplant 2005;20:1573-81.
Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. F Y Chow, D J Nikolic-Paterson, E Ozols, R C Atkins, G H Tesch, J Am Soc Nephrol. 16Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH. Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. J Am Soc Nephrol 2005;16:1711-22.
Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice. F Y Chow, D J Nikolic-Paterson, F Y Ma, E Ozols, B J Rollins, G H Tesch, Diabetologia. 50Chow FY, Nikolic-Paterson DJ, Ma FY, Ozols E, Rollins BJ, Tesch GH. Monocyte chemoattractant protein-1-induced tissue inflam- mation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice. Diabetologia 2007;50:471-80.
Translationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes. D K Kim, B Y Nam, J J Li, J T Park, S H Lee, D H Kim, Diabetologia. 55Kim DK, Nam BY, Li JJ, Park JT, Lee SH, Kim DH, et al. Trans- lationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes. Diabetologia 2012;55:1205-17.
Management of IgA nephropathy: evidencebased recommendations. L Nolin, M Courteau, Kidney Int Suppl. 70Nolin L, Courteau M. Management of IgA nephropathy: evidence- based recommendations. Kidney Int Suppl 1999;70:S56-62.
Omega-3 polyunsaturated fatty acids in the treatment of kidney disease. R G Fassett, G C Gobe, J M Peake, J S Coombes, Am J Kidney Dis. 56Fassett RG, Gobe GC, Peake JM, Coombes JS. Omega-3 polyun- saturated fatty acids in the treatment of kidney disease. Am J Kid- ney Dis 2010;56:728-42.
Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease. U N Das, Prostaglandins Leukot Essent Fatty Acids. 52Das UN. Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease. Prosta- glandins Leukot Essent Fatty Acids 1995;52:387-91.
| [
"This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Purpose: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. Materials and Methods: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 µM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. Results: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. Conclusion: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy."
] | [
"Do-Hee Kim ",
"Tae-Hyun Yoo ",
"Soon Ha Lee ",
"Hye Young Kang ",
"SeungBo Young Nam ",
"Jae Kwak ",
"Jwa-Kyung Kim ",
"Jung Tak Park ",
"Seung Hyeok Han ",
"Shin-Wook Kang ",
"DrShin-Wook Kang ",
"\nDepartment of Internal Medicine\nBrain Korea\n",
"\nProject for Medical Science\nDepartment of Internal Medicine\nYonsei University College of Medicine\nSeoulKorea\n",
"\nYonsei University College of Medicine\n50 Yonsei-ro, Seodaemun-gu120-752SeoulKorea\n"
] | [
"Department of Internal Medicine\nBrain Korea",
"Project for Medical Science\nDepartment of Internal Medicine\nYonsei University College of Medicine\nSeoulKorea",
"Yonsei University College of Medicine\n50 Yonsei-ro, Seodaemun-gu120-752SeoulKorea"
] | [
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"Tae-Hyun",
"Soon",
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"Hye",
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"Young",
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"Jwa-Kyung",
"Jung",
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"Seung",
"Hyeok",
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] | [
"B A Young, ",
"R J Johnson, ",
"C E Alpers, ",
"E Eng, ",
"K Gordon, ",
"J Floege, ",
"N Banba, ",
"T Nakamura, ",
"M Matsumura, ",
"H Kuroda, ",
"Y Hattori, ",
"K Kasai, ",
"H Sugimoto, ",
"K Shikata, ",
"K Hirata, ",
"K Akiyama, ",
"M Matsuda, ",
"M Kushiro, ",
"R Utimura, ",
"C K Fujihara, ",
"A L Mattar, ",
"D M Malheiros, ",
"I L Noronha, ",
"R Zatz, ",
"R Nasrallah, ",
"S J Robertson, ",
"R L Hébert, ",
"P Benatti, ",
"G Peluso, ",
"R Nicolai, ",
"M Calvani, ",
"P H Buist, ",
"Y Y Fan, ",
"R S Chapkin, ",
"A J Ingram, ",
"A Parbtani, ",
"W F Clark, ",
"E Spanner, ",
"M W Huff, ",
"D J Philbrick, ",
"R B Zurier, ",
"R G Rossetti, ",
"E W Jacobson, ",
"D M Demarco, ",
"N Y Liu, ",
"J E Temming, ",
"H Keen, ",
"J Payan, ",
"J Allawi, ",
"J Walker, ",
"G A Jamal, ",
"A I Weir, ",
"J J Li, ",
"S H Lee, ",
"D K Kim, ",
"Jin R Jung, ",
"D S Kwak, ",
"S J , ",
"G T Huang, ",
"L Eckmann, ",
"T C Savidge, ",
"M F Kagnoff, ",
"B H Rovin, ",
"T Yoshiumura, ",
"L Tan, ",
"F Chiarelli, ",
"F Cipollone, ",
"A Mohn, ",
"M Marini, ",
"A Iezzi, ",
"M Fazia, ",
"T M Coimbra, ",
"U Janssen, ",
"H J Gröne, ",
"T Ostendorf, ",
"U Kunter, ",
"H Schmidt, ",
"E S Yeo, ",
"J Y Hwang, ",
"J E Park, ",
"Y J Choi, ",
"K B Huh, ",
"W Y Kim, ",
"G Wolf, ",
"F N Ziyadeh, ",
"S Chen, ",
"B Jim, ",
"F N Ziyadeh, ",
"A Solini, ",
"C Iacobini, ",
"C Ricci, ",
"P Chiozzi, ",
"L Amadio, ",
"F Pricci, ",
"J F Navarro-González, ",
"C Mora-Fernández, ",
"T Furuta, ",
"T Saito, ",
"T Ootaka, ",
"J Soma, ",
"K Obara, ",
"K Abe, ",
"L Hounsom, ",
"D F Horrobin, ",
"H Tritschler, ",
"R Corder, ",
"D R Tomlinson, ",
"B M Forman, ",
"J Chen, ",
"R M Evans, ",
"H Pham, ",
"T Banerjee, ",
"G M Nalbandian, ",
"V A Ziboh, ",
"R A Daynes, ",
"D C Jones, ",
"Y Zhang, ",
"Y Guan, ",
"M Watanabe, ",
"H Nakashima, ",
"S Mochizuki, ",
"Y Abe, ",
"A Ishimura, ",
"K Ito, ",
"S Takata, ",
"M Matsubara, ",
"P G Allen, ",
"P A Janmey, ",
"C N Serhan, ",
"H R Brady, ",
"Y Kikuchi, ",
"T Imakiire, ",
"M Yamada, ",
"T Saigusa, ",
"T Hyodo, ",
"N Hyodo, ",
"F Y Chow, ",
"D J Nikolic-Paterson, ",
"E Ozols, ",
"R C Atkins, ",
"G H Tesch, ",
"F Y Chow, ",
"D J Nikolic-Paterson, ",
"F Y Ma, ",
"E Ozols, ",
"B J Rollins, ",
"G H Tesch, ",
"D K Kim, ",
"B Y Nam, ",
"J J Li, ",
"J T Park, ",
"S H Lee, ",
"D H Kim, ",
"L Nolin, ",
"M Courteau, ",
"R G Fassett, ",
"G C Gobe, ",
"J M Peake, ",
"J S Coombes, ",
"U N Das, "
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] | [
"Cellular events in the evolution of experimental diabetic nephropathy. B A Young, R J Johnson, C E Alpers, E Eng, K Gordon, J Floege, Kidney Int. 47Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, et al. Cellular events in the evolution of experimental diabetic ne- phropathy. Kidney Int 1995;47:935-44.",
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"Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. B M Forman, J Chen, R M Evans, Proc Natl Acad Sci U S A. 94Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyun- saturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A 1997;94:4312-7.",
"Activation of peroxisome proliferator-activated receptor (PPAR)-gamma by 15S-hydroxyeicosatrienoic acid parallels growth suppression of androgen-dependent prostatic adenocarcinoma cells. H Pham, T Banerjee, G M Nalbandian, V A Ziboh, Cancer Lett. 189Pham H, Banerjee T, Nalbandian GM, Ziboh VA. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma by 15S-hydroxyeicosatrienoic acid parallels growth suppression of androgen-dependent prostatic adenocarcinoma cells. Cancer Lett 2003;189:17-25.",
"Emerging roles of PPARs in inflammation and immunity. R A Daynes, D C Jones, Nat Rev Immunol. 2Daynes RA, Jones DC. Emerging roles of PPARs in inflammation and immunity. Nat Rev Immunol 2002;2:748-59.",
"PPAR-gamma agonists and diabetic nephropathy. Y Zhang, Y Guan, Curr Diab Rep. 5Zhang Y, Guan Y. PPAR-gamma agonists and diabetic nephropa- thy. Curr Diab Rep 2005;5:470-5.",
"Amelioration of diabetic nephropathy in OLETF rats by prostaglandin I(2) analog, beraprost sodium. M Watanabe, H Nakashima, S Mochizuki, Y Abe, A Ishimura, K Ito, Am J Nephrol. 30Watanabe M, Nakashima H, Mochizuki S, Abe Y, Ishimura A, Ito K, et al. Amelioration of diabetic nephropathy in OLETF rats by prostaglandin I(2) analog, beraprost sodium. Am J Nephrol 2009;30:1-11.",
"Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium. S Takata, M Matsubara, P G Allen, P A Janmey, C N Serhan, H R Brady, J Clin Invest. 93Takata S, Matsubara M, Allen PG, Janmey PA, Serhan CN, Brady HR. Remodeling of neutrophil phospholipids with 15(S)-hy- droxyeicosatetraenoic acid inhibits leukotriene B4-induced neutro- phil migration across endothelium. J Clin Invest 1994;93:499-508.",
"tients with type 2 diabetes. Yonsei Med J. 51tients with type 2 diabetes. Yonsei Med J 2010;51:519-25.",
"Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Y Kikuchi, T Imakiire, M Yamada, T Saigusa, T Hyodo, N Hyodo, Nephrol Dial Transplant. 20Kikuchi Y, Imakiire T, Yamada M, Saigusa T, Hyodo T, Hyodo N, et al. Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Nephrol Dial Transplant 2005;20:1573-81.",
"Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. F Y Chow, D J Nikolic-Paterson, E Ozols, R C Atkins, G H Tesch, J Am Soc Nephrol. 16Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH. Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice. J Am Soc Nephrol 2005;16:1711-22.",
"Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice. F Y Chow, D J Nikolic-Paterson, F Y Ma, E Ozols, B J Rollins, G H Tesch, Diabetologia. 50Chow FY, Nikolic-Paterson DJ, Ma FY, Ozols E, Rollins BJ, Tesch GH. Monocyte chemoattractant protein-1-induced tissue inflam- mation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice. Diabetologia 2007;50:471-80.",
"Translationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes. D K Kim, B Y Nam, J J Li, J T Park, S H Lee, D H Kim, Diabetologia. 55Kim DK, Nam BY, Li JJ, Park JT, Lee SH, Kim DH, et al. Trans- lationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes. Diabetologia 2012;55:1205-17.",
"Management of IgA nephropathy: evidencebased recommendations. L Nolin, M Courteau, Kidney Int Suppl. 70Nolin L, Courteau M. Management of IgA nephropathy: evidence- based recommendations. Kidney Int Suppl 1999;70:S56-62.",
"Omega-3 polyunsaturated fatty acids in the treatment of kidney disease. R G Fassett, G C Gobe, J M Peake, J S Coombes, Am J Kidney Dis. 56Fassett RG, Gobe GC, Peake JM, Coombes JS. Omega-3 polyun- saturated fatty acids in the treatment of kidney disease. Am J Kid- ney Dis 2010;56:728-42.",
"Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease. U N Das, Prostaglandins Leukot Essent Fatty Acids. 52Das UN. Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease. Prosta- glandins Leukot Essent Fatty Acids 1995;52:387-91."
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] | [
"Cellular events in the evolution of experimental diabetic nephropathy",
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"Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties",
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"gamma-Linolenic acid treatment of rheumatoid arthritis. A randomized, placebo-controlled trial",
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"Colchicine attenuates inflammatory cell infiltration and extracellular matrix accumulation in diabetic nephropathy",
"Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion",
"Cytokine-induced production of monocyte chemoattractant protein-1 by cultured human mesangial cells",
"Circulating monocyte chemoattractant protein-1 and early development of nephropathy in type 1 diabetes",
"Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes",
"Molecular mechanisms of diabetic renal hypertrophy",
"Diabetic nephropathy and transforming growth factor-beta: transforming our view of glomerulosclerosis and fibrosis build-up",
"Purinergic modulation of mesangial extracellular matrix production: role in diabetic and other glomerular diseases",
"The role of inflammatory cytokines in diabetic nephropathy",
"The role of macrophages in diabetic glomerulosclerosis",
"A lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neuropathy",
"Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta",
"Activation of peroxisome proliferator-activated receptor (PPAR)-gamma by 15S-hydroxyeicosatrienoic acid parallels growth suppression of androgen-dependent prostatic adenocarcinoma cells",
"Emerging roles of PPARs in inflammation and immunity",
"PPAR-gamma agonists and diabetic nephropathy",
"Amelioration of diabetic nephropathy in OLETF rats by prostaglandin I(2) analog, beraprost sodium",
"Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium",
"tients with type 2 diabetes",
"Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats",
"Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice",
"Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice",
"Translationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes",
"Management of IgA nephropathy: evidencebased recommendations",
"Omega-3 polyunsaturated fatty acids in the treatment of kidney disease",
"Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease"
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"\nFig. 3 .\n3Renal ICAM-1 and fibronectin protein expression in C, C+GLA, DM, and DM+GLA rats. There was a 3.2-fold increase in ICAM-1 and a 3.7-fold increase in fibronectin protein expression in DM rats compared to C rats, and these increases were significantly attenuated by administration of GLA. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes.",
"\nFig. 2 . 1\n21Renal MCP-1 protein levels in C, C+GLA, DM, and DM+GLA rats. There was a 2.0-fold increase in renal MCP-1 protein levels in DM rats compared to C rats, and this increase in the DM rats was significantly ameliorated by GLA treatment. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. C, control; GLA, γ-linolenic acid; DM, diabetes; MCP-",
"\nFig. 6 .\n6es in DM rats (×400). *p<0.05 vs. other groups. † p<0.01 vs. C and C+GLA groups. ‡ p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes. ICAM-1 and fibronectin mRNA/18s rRNA ratios in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and H+10 or 100 μM GLA (HG+GLA). There were 2.2-and 1.8-fold increases in ICAM-1 mRNA/18s rRNA ratios in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and these increases in ICAM-1 mRNA/18s rRNA ratios were significantly abrogated by administration of GLA. There were 2.5-fold and 2.2-fold increases in fibronectin mRNA/18s rRNA ratios in HGstimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA treatment significantly attenuated these increases in fibronectin mRNA/18s rRNA ratios in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.",
"\nFig. 5 .\n5MCP-1 mRNA/18s rRNA ratios and secreted MCP-1 protein levels in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and HG+10 or 100 μM GLA (HG+GLA). There were 2.0-and 2.1-fold increases in MCP-1 mRNA/18s rRNA ratios in the HG-stimulated mesangial and NRK-52E cells, respectively, compared to the NG cells, and these increases in MCP-1 mRNA expression were significantly ameliorated by GLA treatment in a dose-dependent manner. There were 2.4-and 3.6-fold increases in MCP-1 levels in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA significantly attenuated these increases in MCP-1 levels in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. MCP-1, monocyte chemoattractant protein-1; GLA,",
"\nFig. 7 .\n7Representative Western blots of ICAM-1 and fibronectin in cultured mesangial cells (A) and NRK-52E cells (B). There were significant increases in ICAM-1 and fibronectin protein expression in HG-stimulated cells as compared to NG cells, and these increases were significantly ameliorated with GLA treatment. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.",
"\nTable 1 .\n1Animal Data for the Four Groups UAE, urinary albumin excretion; GLA, γ-linolenic acid; C, control; DM, diabetes. *p<0.01 vs. C and C+GLA group. † p<0.05 vs. DM group.C (n=8) \nC+GLA (n=8) \nDM (n=8) \nDM+GLA (n=8) \nBody Wt (g) \n593±11 \n584±14 \n328± 9* \n335±8* \nKidney Wt/Body Wt (%) \n0.58±0.05 \n0.53±0.06 \n1.17±0.15* \n0.88±0.11 † \nBlood glucose (mg/dL) \n104.1± 3.9 \n97.6± 3.5 \n489.5±14.0* \n474.0±13.0* \n24-hr UAE (mg/day) \n0.35±0.07 \n0.31±0.09 \n2.51±0.28* \n1.11±0.12 † \nWt, weight; "
] | [
"Renal ICAM-1 and fibronectin protein expression in C, C+GLA, DM, and DM+GLA rats. There was a 3.2-fold increase in ICAM-1 and a 3.7-fold increase in fibronectin protein expression in DM rats compared to C rats, and these increases were significantly attenuated by administration of GLA. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes.",
"Renal MCP-1 protein levels in C, C+GLA, DM, and DM+GLA rats. There was a 2.0-fold increase in renal MCP-1 protein levels in DM rats compared to C rats, and this increase in the DM rats was significantly ameliorated by GLA treatment. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. C, control; GLA, γ-linolenic acid; DM, diabetes; MCP-",
"es in DM rats (×400). *p<0.05 vs. other groups. † p<0.01 vs. C and C+GLA groups. ‡ p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes. ICAM-1 and fibronectin mRNA/18s rRNA ratios in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and H+10 or 100 μM GLA (HG+GLA). There were 2.2-and 1.8-fold increases in ICAM-1 mRNA/18s rRNA ratios in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and these increases in ICAM-1 mRNA/18s rRNA ratios were significantly abrogated by administration of GLA. There were 2.5-fold and 2.2-fold increases in fibronectin mRNA/18s rRNA ratios in HGstimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA treatment significantly attenuated these increases in fibronectin mRNA/18s rRNA ratios in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.",
"MCP-1 mRNA/18s rRNA ratios and secreted MCP-1 protein levels in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 μM GLA (NG+GLA), 30 mM glucose (HG), and HG+10 or 100 μM GLA (HG+GLA). There were 2.0-and 2.1-fold increases in MCP-1 mRNA/18s rRNA ratios in the HG-stimulated mesangial and NRK-52E cells, respectively, compared to the NG cells, and these increases in MCP-1 mRNA expression were significantly ameliorated by GLA treatment in a dose-dependent manner. There were 2.4-and 3.6-fold increases in MCP-1 levels in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA significantly attenuated these increases in MCP-1 levels in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. MCP-1, monocyte chemoattractant protein-1; GLA,",
"Representative Western blots of ICAM-1 and fibronectin in cultured mesangial cells (A) and NRK-52E cells (B). There were significant increases in ICAM-1 and fibronectin protein expression in HG-stimulated cells as compared to NG cells, and these increases were significantly ameliorated with GLA treatment. *p<0.01 vs. NG, NG+M, and NG+GLA groups. † p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.",
"Animal Data for the Four Groups UAE, urinary albumin excretion; GLA, γ-linolenic acid; C, control; DM, diabetes. *p<0.01 vs. C and C+GLA group. † p<0.05 vs. DM group."
] | [
"(Fig. 1)",
"(Fig. 2)",
"(Fig. 3",
"Fig. 1",
"(Fig. 5)",
"(Fig. 5)",
"(Fig. 6",
"(Fig. 4)",
"(Fig. 4)",
"Fig. 4",
"(Fig. 6)",
"(Fig. 7)"
] | [] | [
"Diabetic nephropathy, the leading cause of end-stage renal disease worldwide, is characterized pathologically by cellular hypertrophy and increased extracellular matrix (ECM) accumulation. 1 The ECM accumulation in diabetic nephropathy results in mesangial expansion, tubulointerstitial fibrosis, and irreversible deterioration of renal function. 2 Even though previous studies have shown that ECM ac-",
"All animal studies were conducted using an approved protocol the committee for the care and use of laboratory animals of Yonsei University College of Medicine. Sprague-Dawley rats weighing 250-280 g were intraperitoneally injected with either a diluent [n=16, control (C)] or 65 mg/ kg streptozotocin [n=16, diabetes (DM)]. Diabetes was confirmed by tail vein blood glucose levels on the third post-injection day. After confirming diabetes, eight rats each from the C and DM groups were treated with 450 mg/kg/day of evening primrose oil (EPO, a generous gift from Dalim Biotech, Seoul, Korea) by gavage (C+GLA or DM+ GLA) for three months. EPO contained 8-10% GLA, and the amount of EPO used in this study provided an approximate GLA dose of 40 mg/kg/day. Rats were housed in a temperaturecontrolled room and given free access to water and standard laboratory chow during the three-month study period. Body weight and serum glucose level were checked monthly, and kidney weight and 24-hour urinary albumin excretion were checked at the time of sacrifice. Blood glucose was measured by a glucometer, and 24-hour urinary albumin excretion was determined by enzyme-linked immunosorbent assay (ELISA) (Nephrat II, Exocell, Inc., Philadelphia, PA, USA).",
"Primary culture of glomerular mesangial cells was performed as previously described. 17 Identification of mesangial cells was performed by their characteristic stellate appearance in culture and confirmed by immunofluorescent microscopy for the presence of actin, myosin, and Thy-1 antigen, as well as the absence of factor VIII and cytokeratin (Synbiotics, San Diego, CA, USA). Mesangial and NRK-52E cells and immortalized rat tubular epithelial cells, were maintained, respectively, in RPMI 1640 and DMEM medium supplemented with 5% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 26 mM NaHCO 3 , and grown at 37ºC in humidified 5% CO 2 in air. Subconfluent mesangial cells and NRK-52E cells were serum restricted for 24 hours, after which the medium was replaced by serum-free medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without 10 or 100 μM GLA (Sigma Chemical Co., St. Louis, MO, USA). In addition, both of the cells were cumulation under diabetic conditions is attributable to hyperglycemia per se, advanced glycation end-products, hemodynamic changes, and local growth factors such as angiotensin II (AII) and transforming growth factor (TGF)-β1, 3 the precise molecular and cellular mechanisms responsible for this have yet to be resolved.",
"Recently, accumulating evidence has suggested that the inflammatory process also plays an important role in the pathogenesis of diabetic nephropathy. 4 Infiltration of inflammatory cells in glomeruli and renal tubulointerstitium is commonly seen in both human diabetic patients and experimental diabetic animals. 5,6 In addition, intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), which mediates the recruitment and infiltration of monocytes/macrophages, have been shown to be involved in the pathogenesis of diabetic nephropathy. 7,8 Based on these findings, modulation of the inflammatory process is considered to be a potential means of preventing the development and progression of diabetic nephropathy, and some immunosuppressive agents and anti-inflammatory drugs have been found to be beneficial in diabetic nephropathy. 9,10 Nevertheless, chronic use of these drugs in the clinical field is not appropriate due to many systemic side effects. Therefore, safe agents for chronic treatment of diabetic nephropathy are undoubtedly needed.",
"Polyunsaturated fatty acids (PUFAs), which exist in high concentrations in cell membranes as structural phospholipids, are essential to cell integrity and viability. 11,12 There are two classes of PUFAs: ω-3 and ω-6, designated according to their carbon ring structure. 12 γ-linolenic acid (GLA), a member of ω-6 PUFA, is produced from linoleic acid by the enzyme δ-6 desaturase, and is elongated to dihomogamma linolenic acid (DGLA). [11][12][13] In a previous study, GLA was shown to abrogate renal fibrosis in a 5/6 nephrectomy model, 14 and other investigations have demonstrated that GLA treatment improved autoimmune diseases and diabetic neuropathy via an anti-inflammatory mechanism. 15,16 As mentioned earlier, since the inflammatory process is also involved in the pathogenesis of diabetic nephropathy, there is a possibility that GLA may ameliorate diabetic nephropathy, but this has never been explored.",
"In this study, therefore, we investigated the effects of GLA in experimental diabetic kidneys as well as in high glucosestimulated mesangial cells and tubular epithelial cells in regards to inflammation and ECM synthesis. GG. CCG-3'; and 18s, sense 5'-AGTCCCTGCCCTTTGT ACACA-3', antisense 5'-GATCCGAGGGCCTCACTA AAC-3'. cDNAs from 25 ng RNA of the renal cortical tissue or cultured cells per reaction tube were used for amplification.",
"Using the ABI PRISM ® 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), PCR was performed with a total volume of 20 μL in each well, containing 10 μL of SYBR Green ® PCR Master Mix (Applied Biosystems), 5 μL of cDNA, and 5 pM sense and antisense primers. Primer concentrations were determined by preliminary experiments that analyzed the optimal concentrations of each primer. Each sample was run in triplicate in separate tubes. The PCR conditions were as follows: 35 cycles of denaturation at 94.5ºC for 30 sec, annealing at 60ºC for 30 sec, and extension at 72ºC for one minute. Initial heating at 95ºC for nine minutes and final extension at 72ºC for seven minutes were performed for all PCRs.",
"After real-time PCR, the temperature was increased from 60 to 95ºC at a rate of 2ºC/min to construct a melting curve. A control without cDNA was run in parallel with each assay. The cDNA content of each specimen was determined using a comparative C T method with 2 -∆∆T . The results are given as relative expressions of ICAM-1, MCP-1, and fibronectin normalized to the expression of the 18s housekeeping gene.",
"The renal cortical tissue and cultured cells harvested from plates were lysed in sodium dodecyl sulfate (SDS) sample buffer [2% sodium dodecyl sulfate, 10 mM Tris-HCl, pH 6.8, 10% (vol/vol) glycerol], treated with Laemmli sample buffer, heated at 100ºC for five minutes, and electrophoresed in an 8% acrylamide denaturing SDS-polyacrylamide gel. Proteins were then transferred to a Hybond-ECL membrane using a Hoeffer semidry blotting apparatus (Hoeffer Instruments, San Francisco, CA, USA). The membrane was then incubated in blocking buffer A (1×PBS, 0.1% Tween-20, and 8% nonfat milk) at room temperature for one hour, followed by an overnight incubation at 4ºC in a 1 : 1000 dilution of polyclonal antibodies to ICAM-1 (R&D systems, Minneapolis, MN, USA), fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or β-actin (Sigma Chemical Co., St. Louis, MO, USA). The membrane was then washed once for 15 minutes and twice for five minutes in 1×PBS with 0.1% Tween-20. Next, the membrane was incubated in buffer A containing a 1 : 1000 treated by 10 or 100 μM linoleic acid (Sigma Chemical Co., St. Louis, MO, USA). At 24 hours after the media change, the cells were harvested and conditioned culture media were collected.",
"Total RNA from the renal cortical tissue was extracted as previously described. 17 Briefly, 100 μL of RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA) was added to the renal cortical tissues, which were lysed by freezing and thawing three times. Another 700 μL of RNA STAT-60 reagent was then added and the mixture was vortexed and stored for five minutes at room temperature. Next, 160 μL of chloroform was added and the mixture was shaken vigorously for 30 seconds. After three minutes, the mixture was centrifuged at 12000×g for 15 minutes at 4ºC and the upper aqueous phase containing the extracted RNA was transferred to a new tube. RNA was precipitated from the aqueous phase by adding 400 μL of isopropanol and then pelleted by centrifugation at 12000×g for 30 minutes at 4ºC. The RNA precipitate was washed with 70% ice-ethanol, dried using a Speed Vac, and dissolved in DEPC-treated distilled water. RNA yield and quality were assessed based on spectrophotometric measurements at wavelengths of 260 and 280 nm. Total RNA from the mesangial and NRK-52E cells was similarly extracted.",
"First strand cDNA was synthesized using a Boehringer Mannheim cDNA synthesis kit (Boehringer Mannheim GmbH, Mannheim, Germany). Two μg of total RNA extracted from the renal cortex and cultured cells were reverse transcribed using 10 μM random hexanucleotide primer, 1 mM dNTP, 8 mM MgCl 2 , 30 mM KCl, 50 mM Tris-HCl, pH 8.5, 0.2 mM dithiothreitol, 25 U RNase inhibitor, and 40 U AMV reverse transcriptase. The mixture was incubated at 30ºC for 10 minutes and 42ºC for one hour, followed by inactivation of the enzyme at 99ºC for five minutes.",
"The primers used for ICAM-1, MCP-1, fibronectin, and 18s amplification were as follows: ICAM-1, sense 5'-AGGTA TCCATCCATCCCAC-3', antisense 5'-GCCGAGG TTCTCGTCTTC-3'; MCP-1, sense 5'-TCTCTTCCTCC ACCACTATGCA-3', antisense 5'-GGCTGAGACAGC ACGTGGAT-3'; fibronectin, sense 5'-TGACAACTGCCG TAGACCTGG-3', antisense 5'-TACTGGTTGTAGGTGT respectively, and by a digital image analyzer (MetaMorph version 4.6r5, Universal Imaging Corp., Downingtown, PA, USA) as previously described. 17 The degree of staining was semi-quantitated on a scale of 0-4+. The staining score was obtained by multiplying the intensity of staining by the percentage of glomeruli or tubulointerstitium staining for that intensity, and these numbers were then added for each experimental animal to give the staining score [Σ=(Intensity of staining)×(% of glomeruli or tubulointerstitium with that intensity)]. The number of ED-1 positive cells was counted in at least 20 glomeruli and 20 fields of the tubulointerstitium/section under ×400 magnification.",
"All values are expressed as the mean±standard error of the mean. Statistical analysis was performed using the statistical package SPSS for Windows Ver. 11.0 (SPSS, Inc., Chicago, IL, USA). Results were analyzed using the Kruskal-Wallis non-parametric test for multiple comparisons. Significant differences on the Kruskal-Wallis test were further confirmed by the Mann-Whitney U test. p-values less than 0.05 were considered statistically significant.",
"Animal data All the animals gained weight over the three-month experimental period, but body weight was highest among the C rats (593±11 g). The ratio of kidney to body weight in the DM rats (1.17±0.15%) was significantly higher than those of the C (0.58±0.05%), C+GLA (0.53±0.06%) (p<0.01), and DM+GLA (0.88±0.11) (p<0.05) rats. The mean blood glucose levels of C, C+GLA, DM, and DM+GLA rats were 104.1±3.9, 97.6±3.5, 489.5±14.0, and 474.0±13.0 mg/dL, dilution of horseradish peroxidase-linked donkey anti-goat IgG (Amersham Life Science, Inc., Arlington Heights, IL, USA). The washes were repeated, and the membrane was developed with a chemiluminescent agent (ECL; Amersham Life Science, Inc., Arlington Heights, IL, USA). The band densities were measured using TINA image software (Raytest, Straubenhardt, Germany).",
"The levels of MCP-1 in the renal cortical tissue and culture media were determined using a commercial ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's protocol. The kit for rat MCP-1 was species-specific and sensitive up to 750 pg/mL. All concentrations of MCP-1 were normalized to the total protein amount.",
"For immunohistochemical staining, slices of kidney were snap-frozen in optimal cutting temperature solution and 4 μm sections of tissues were utilized. Slides were fixed in acetone for 10 minutes, air dried at room temperature for 10 minutes, and blocked with 10% donkey serum at room temperature for 20 minutes. For ICAM-1, fibronectin and ED-1 staining, the primary polyclonal antibody to ICAM-1, the extracellular domain of fibronectin (Chemicon International, Inc., Billerica, MA, USA) or ED-1 (Chemicon International, Inc., Billerica, MA, USA), respectively, was diluted to 1 : 100 with 2% casein in BSA and was applied for overnight incubation at room temperature. After washing, a secondary donkey anti-goat antibody was added for 20 minutes and the slides were then washed and incubated with a tertiary PAP complex for 20 minutes. DAB was added for two minutes and the slides were counterstained with hematoxylin. A semi-quantitative score for measuring the intensity of ICAM-1 and fibronectin staining within the glomeruli and tubulointerstitial area was determined by examining at least 20 glomeruli under ×400 magnification and 20 tubulointerstitial fields under ×200 magnification, 1 and fibronectin mRNA expression in DM rats (p<0.05) (Fig. 1).",
"The levels of renal MCP-1 assessed by ELISA were also significantly higher in DM than in C rats (563.5±42.9 vs. 287.1±22.3 ng/μg, p<0.01), and the increase in MCP-1 levels in DM rats was significantly ameliorated by GLA treatment (354.9±31.3 ng/μg, p<0.05) (Fig. 2). Renal ICAM-1 and fibronectin protein expression assessed by Western blot was also significantly increased in DM rats relative to C and C+GLA rats (p<0.01), and GLA treatment significantly abrogated these increases in DM rats (p<0.05) (Fig. 3). In addition, immunohistochemical staining for ICAM-1 and fibronectin confirmed the real-time PCR and Western blot findings. There were significant increases in glomerular and tubulointerstitial ICAM-1 and fibronectin staining in the respectively (p<0.01). Compared to the C group (0.35±0.07 mg/day), 24-hour urinary albumin excretion at three months was significantly higher in DM rats (2.51±0.28 mg/day) (p<0.01), and GLA treatment significantly reduced albuminuria in DM rats (1.11±0.12 mg/day) (p<0.05) ( Table 1).",
"Effect of GLA on renal cortical MCP-1, ICAM-1, and fibronectin mRNA expression Renal MCP-1 mRNA expression assessed by real-time PCR was significantly higher in DM than in C rats (p<0.01), and this increase in MCP-1 mRNA expression was significantly inhibited by administration of GLA (p<0.05). The MCP-1 mRNA/18s rRNA ratio was 2.1-fold higher in DM than in C kidneys, and GLA treatment significantly abrogated this increase by 65.7%. The ratios of ICAM-1 and fibronectin mRNA/18s rRNA were also significantly higher in DM compared to C and C+GLA kidneys (p<0.01), and GLA treatment significantly attenuated increases in renal ICAM- Fig. 1. Renal MCP-1, ICAM-1, and fibronectin mRNA/18s rRNA ratios in control (C), C+gamma linolenic acid (GLA), diabetic (DM), and DM+GLA rats. There was a 2.1-fold increase in MCP-1 mRNA/18s rRNA, a 1.8-fold increase in ICAM-1 mRNA/18s rRNA, and a 2.7-fold increase in fibronectin mRNA/18s rRNA ratios in DM rats compared to C rats, and GLA treatment significantly abrogated these increases in mRNA/18s rRNA ratios in DM rats. *p<0.01 vs. C and C+GLA groups. † p<0.05 vs. DM group. MCP-1, monocyte chemoattractant protein-1; ICAM-1, intracellular adhesion molecule-1. NRK-52E cells exposed to HG medium (p<0.01), respectively, and GLA treatment significantly ameliorated these increases in a dose-dependent manner (p<0.05) (Fig. 5). The levels of MCP-1 protein in conditioned culture media assessed by ELISA showed a similar pattern to the mRNA expression (Fig. 5). However, linoleic acid did not induce significant changes in these patterns (data not shown).",
"High glucose significantly induced ICAM-1 and fibronectin mRNA expression in mesangial and NRK-52E cells. Compared to NG cells, the ICAM-1 mRNA/18s rRNA ratios were 2.2-and 1.8-fold higher in HG-stimulated mesangial cells and tubular epithelial cells (p<0.01), respectively, and these increases were significantly attenuated by 60.9% and 62.3%, respectively, with 10 μM GLA treatment, and by 70.7% and 76.1%, respectively, with 100 μM GLA treatment (Fig. 6). Fibronectin mRNA/18s rRNA ratios were significantly increased in HG-stimulated mesangial and NRK-52E cells relative to NG cells by 154.4% and 122.2% (p<0.01), respectively, and these increases were significant-DM group compared to the C and C+GLA groups, and these increases in DM rats were significantly ameliorated by the administration of GLA (Fig. 4).",
"The number of macrophages within glomeruli and tubulointerstitium assessed by immunohistochemical staining with ED-1 antibody was significantly higher in DM rats than in C rats (27.8±4.3 vs. 3.6±0.8, p<0.01), and GLA treatment significantly abrogated the number of ED-1-positive cells in DM rats (10.1±1.2) (p<0.05) (Fig. 4).",
"Effect of GLA on MCP-1 mRNA and protein expression MCP-1 mRNA expression assessed by real-time PCR was significantly increased in HG-stimulated mesangial cells and NRK-52E cells (p<0.01), and this increase in MCP-1 mRNA expression was significantly abrogated by GLA treatment (p<0.05). Compared to NG cells, the MCP-1 mRNA/18s rRNA ratios were 2.0-and 2.1-fold higher in mesangial and Fig. 4. Immunohistochemical staining for glomerular and tubulointerstitial ICAM-1, fibronectin, and ED-1 (as a marker of macrophage) in C, C+GLA, DM, and DM+GLA rats. Glomerular (A) and tubulointerstitial (B) ICAM-1 and fibronectin staining was significantly increased in DM rats compared to C rats, and GLA treatment significantly inhibited these increases in DM rats. The number of ED-1-positive cells was significantly higher in DM rats than in C rats, and GLA treatment significantly abrogated the number of glomerular and tubulointerstitial macrophages in DM rats. (C) IHC scores for ICAM-1 and fibronectin within the glomeruli and the tubulointerstitial area were significantly higher in DM rats relative to C rats, and GLA treatment significantly attenuated these increas- ed by inhibiting increases in MCP-1 and ICAM-1 expression under diabetic conditions. Even though the diabetic milieu per se, hemodynamic changes, and local growth factors such as AII and TGF-β are considered mediators in the pathogenesis of diabetic nephropathy, 3,6 recent studies suggest that an inflammatory mechanism may also contribute to the development of diabetic nephropathy based on the pathological findings of inflammatory cell infiltration in diabetic kidneys. 4 Monocytes/macrophages are the major inflammatory cells found in diabetic kidneys. 5,17 They are extravasculated from the bloodstream and attracted to the target tissue through a process mediated by various chemokines and adhesion molecules such as MCP-1 and ICAM-1. 7,18 In the kidneys, MCP-1 is expressed in mesangial cells and tubular epithelial cells and is known to be involved in the pathogenesis of various renal diseases, including diabetic nephropathy. 7,19 Previous studies have demonstrated that plasma MCP-1 levels are ly abrogated by GLA in a dose-dependent manner (p<0.05) (Fig. 6).",
"ICAM-1 and fibronectin protein expression showed similar patterns to their mRNA expressions. GLA also significantly inhibited HG-induced ICAM-1 and fibronectin protein expression in cultured mesangial cells and NRK-52E cells (Fig. 7).",
"In the present study, we demonstrated that GLA has a renoprotective effect via its anti-inflammatory and anti-fibrotic actions in experimental diabetic nephropathy. In addition, the results of this study suggested that the anti-inflammatory effect of GLA under diabetic conditions is partly mediat- FAs are beneficial to one's overall health and a number of various diseases. 13 In cases of kidney disease, PUFAs have been reported to have beneficial effects on IgA nephropathy, 27 chronic renal failure, and diabetic nephropathy via antioxidant, anti-inflammation, and anti-fibrotic mechanisms. 13,28 In contrast, the effect of GLA, a member of the ω-6 PUFA family, on kidney diseases has been less explored. Ingram ,et al. 14 observed that administration of borage oil (BO), a rich source of GLA, was effective in a rat 5/6-renal-ablation model. BO prevented increases in blood pressure and proteinuria, rising plasma cholesterol levels, and declining glomerular filtration rates. In addition, glomerular macrophage infiltration, mesangial expansion, and glomerulosclerosis were attenuated in BO-treated rats compared to the control diet group. Meanwhile, since these glomerular changes are also characteristic of diabetic nephropathy, the activities of δ-5 and δ-6 desaturase are decreased along with low levels of GLA and DGLA in diabetes, 29 and since GLA has proven useful in diabetic neuropathy, 16,30 supplementation of GLA and/or DGLA may also be of benefit in diabetic nephropathy. However, the effect of ω-6 PUFA has never been elucidated in diabetic nephropathy. The results of the present study showed for the first time that GLA inhibits inflammatory cell infiltration and ECM accumulation in experimental diabetic kidneys, suggesting the usefulness of GLA in patients with diabetic nephropathy.",
"Even though the underlying mechanisms of the anti-inflammatory effect of GLA in this study are not completely understood, several plausible explanations can be proposed. First, PUFAs, including GLA, are known to serve as endogenous ligands of peroxisome proliferator activated receptors (PPARs) and to bind and activate all PPARs isoforms. 31 Additionally, 15-HETE, one of the metabolites of GLA, has been reported to upregulate nuclear PPAR-γ expression. 32 PPARs participate in regulating inflammatory responses by inhibiting monocyte expression of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α. 33 Furthermore, PPAR-γ attenuates the nuclear factor-κB-mediated transcriptional activation of proinflammatory genes. 33 Recent studies have also demonstrated that PPAR-γ agonist exerts a renoprotective effect through an anti-inflammatory mechanism in diabetic nephropathy. 34 Taken together, GLA, as a ligand of PPARs and its metabolite, as well as an upregulator of PPAR-γ, could induce an anti-inflammatory effect in diabetic nephropathy. Second, a small amount of DGLA can be converted to prostacyclin (PGI2) and prostaglandin E1 (PGE1) by δ-5 desaturases. Because PGI2 and PGE1 inhib-higher in type 1 diabetes with microalbuminuria 20 and that urinary levels of MCP-1 are higher in accordance with the extent of albuminuria. 7 Renal expression of ICAM-1, a cell surface glycoprotein that plays a major role in the regulation of interactions with immune cells and whose expression is upregulated at sites of inflammation, 8,21 is also known to be increased in experimental type 1 and type 2 diabetic animals. 17,21 These findings suggest that MCP-1 and ICAM-1 may play an important role in the pathogenesis of diabetic nephropathy by inducing inflammatory cell infiltration. 7,8 Once the recruited monocytes/macrophages are activated, they release lysosomal enzymes, nitric oxide, reactive oxygen species, platelet-derived growth factor (PDGF), tumor necrosis factor-α, interleukin (IL)-1, and TGF-β, in turn promoting renal injury. 5,6,22 PDGF stimulates fibroblast proliferation 6 and IL-1 induces the expression of TGF-β, the most well-known profibrotic cytokine, in fibroblasts. 5 In experimental diabetic nephropathy, various anti-inflammatory agents inhibited not only inflammatory cell infiltration by abrogating increases in MCP-1 and ICAM-1 expression but also ameliorated ECM accumulation. 17,23 In addition, renal fibrosis was significantly inhibited along with less inflammatory cell infiltration in MCP-1-and ICAM-1-deficient diabetic mice. 24,25 Taken together, this suggests that the inhibition of inflammatory cell recruitment may lead to an attenuation of ECM accumulation. In this study, we demonstrated that MCP-1 and ICAM-1 expression were increased in experimental diabetic nephropathy, which were associated with glomerular and tubulointerstitial fibrosis, and in high glucose-stimulated mesangial cells and tubular epithelial cells, and these increases under diabetic conditions were inhibited by GLA treatment. In addition, GLA reduced the ratio of kidney/body weight in diabetic rats, suggesting GLA prevents diabetes-induced kidney enlargement. A recent study also demonstrated that prevention of glomerular hypertrophy ameliorates the development of diabetic nephropathy, including proteinuria and podocytopenia. 26 Taken together, the anti-inflammatory, anti-fibrotic, and anti-hypertrophic effects of GLA in diabetic nephropathy may be partly attributable to the suppression of MCP-1 and ICAM-1 expression by GLA, by which inflammatory cell infiltration is abrogated, in turn ameliorating ECM accumulation.",
"PUFAs are important constituents of all cell membranes. Since PUFAs are not synthesized in humans, they can only be obtained by diet. 11 There are two classes of PUFAs: ω-3 and ω-6, designated according to their carbon ring structure; 12 and accumulating evidence has shown that these PU-it platelet aggregation, and PGI2 analogue abrogates glomerular hyperfiltration and macrophage infiltration in diabetic kidneys, 35 the effect of GLA may be in part attributed to these consequences. Third, 15-HETE markedly inhibits the generation of leukotriene, which is a potent pro-inflammatory mediator, via stimulating adhesion molecule expression and macrophage infiltration. 36 Collectively, the anti-inflammatory effect of GLA seems to be attributable to the modulation of biological cascades at multiple sites by itself and/or its metabolites. Prolonged use of anti-inflammatory drugs may be harmful and is not appropriate for long-term use due to many systemic side effects in patients with chronic metabolic disturbances such as diabetic nephropathy. However, since the activities of both δ-5 and δ-6 desaturase are already reduced in diabetes and the toxic effects of GLA as a medicinal oil have not been reported, chronic administration of GLA, even at a high dosage, may not induce accumulation of its metabolites and thus would not be harmful.",
"In summary, the results of the present study demonstrate that GLA exerts anti-inflammatory and anti-fibrotic effects in experimental diabetic nephropathy and in high glucosestimulated renal cells, suggesting that GLA supplementation could be a valuable therapeutic option for the treatment of diabetic nephropathy."
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS Animals",
"Cell culture",
"Western blot analysis",
"Total RNA extraction",
"Reverse transcription",
"Real-time polymerase chain reaction (real-time PCR)",
"Statistical analysis",
"RESULTS",
"Animal studies",
"Measurement of MCP-1 by ELISA",
"Pathology",
"Effect of GLA on renal cortical MCP-1 levels and ICAM-1 and fibronectin protein expression",
"Effect of GLA on ICAM-1 and fibronectin mRNA expression",
"Effect of GLA on macrophage accumulation",
"Cell culture studies",
"Effect of GLA on ICAM-1 and fibronectin protein expression",
"DISCUSSION",
"Fig. 3 .",
"Fig. 2 . 1",
"Fig. 6 .",
"Fig. 5 .",
"Fig. 7 .",
"Table 1 ."
] | [
"C (n=8) \nC+GLA (n=8) \nDM (n=8) \nDM+GLA (n=8) \nBody Wt (g) \n593±11 \n584±14 \n328± 9* \n335±8* \nKidney Wt/Body Wt (%) \n0.58±0.05 \n0.53±0.06 \n1.17±0.15* \n0.88±0.11 † \nBlood glucose (mg/dL) \n104.1± 3.9 \n97.6± 3.5 \n489.5±14.0* \n474.0±13.0* \n24-hr UAE (mg/day) \n0.35±0.07 \n0.31±0.09 \n2.51±0.28* \n1.11±0.12 † \nWt, weight; "
] | [
"Table 1)"
] | [
"Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy",
"Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy"
] | [
"Yonsei Med J"
] |
237,307,079 | 2022-01-10T08:43:42Z | CCBY | https://stemcellres.biomedcentral.com/track/pdf/10.1186/s13287-021-02448-w | GOLD | de7691151c9b2f18402eb10bf887be332f012a52 | null | null | null | null | 10.1186/s13287-021-02448-w | null | 34433490 | 8390253 |
Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells
Akiko Tanuma-Takahashi
Momoko Inoue
Kazuhiro Kajiwara
Ryo Takagi
Ayumi Yamaguchi
Osamu Samura
Hidenori Akutsu
Haruhiko Sago
Tohru Kiyono
Aikou Okamoto
Akihiro Umezawa
Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells
10.1186/s13287-021-02448-wR E S E A R C H Open Access
Background: An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). Methods: Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21-and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. Results: Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. Conclusions: Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.
Background
Down syndrome patients have an extra copy of chromosome 21, which causes health-related problems and agerelated disorders. Trisomy 21 results in the triplication of over 400 genes, which makes clarification of the precise mechanisms of some phenotypes difficult in Down syndrome [1]. The complications of Down syndrome are numerous; clinical manifestations include mental retardation, congenital heart disease, impaired cognition, premature aging, metabolic problems, and leukemia [2,3]. Trisomy 21 is also associated with an increased incidence of several dermatological conditions. Immunological disturbances and/or barrier dysfunction of skin are associated with psoriasis, atopic dermatitis, and cutaneous infections. Keratodermatoses and abnormality of elastic fibers also contribute to dermatological disorders such as anetoderma and keratosis pilaris in Down syndrome patients [4][5][6]. Genetic signatures of these dermatological diseases have been reported using keratinocytes derived from patient-specific iPSCs [7].
A Down syndrome mouse model exhibits hyperproliferation of trisomy 21-keratinocytes in the skin [8]. Cell culture models of trisomy 21 are important for development of conventional therapy for Down syndrome. However, few studies have focused on differences in epidermal characteristics between Down syndrome and euploid cells in vitro. Generation of disease-specific induced pluripotent stem cells (iPSC) allows an alternative paradigm for modeling human genetic disease in vitro [9,10]. Directed differentiation from these iPSC theoretically enables researchers to derive any given diseaseaffected human cell-type [11]. Disease-specific iPSCs, therefore, serve as a powerful tool for investigating the mechanisms of Down syndrome [9,12,13].
The differences in genetic backgrounds of individuals has hindered the assessment the phenotypic disparities between Down syndrome cells and healthy control cells. Paired disomic/trisomic iPSCs derived from the same trisomic individual by insertion of a transgene into a chromosome 21 has allowed investigation of trisomy 21dependent gene expression and the impact of an extra copy of chromosome 21 without the confounding effects of a different genetic background [13][14][15][16]. The artificial selection of disomic clones possibly results in epigenetic changes that add to the transcriptional differences between the trisomic and disomic iPSCs [17]. In the previous study, we have obtained revertant cells with normal chromosome 21 diploids from the trisomy 21 iPSCs during long-term cultivation [18]. Autonomously rescued disomic cells serve as a good control for trisomic cells. Both disomy 21 iPSC and trisomy 21 iPSC have been shown to differentiate into neural stem cells [18].
In this study, we successfully extended the lifespan of trisomy-rescued iPSC-derived keratinocytes to more than 34 population doublings over a period of 160 days with maintenance of keratinocytic phenotypes. In addition, keratinocytes derived from autonomous trisomy-rescued iPSCs regained capability of stratification and were suitable for manufacturing three-dimensional skin with keratinocytic function.
Methods
Cells
iPSCs from Down syndrome patients were cultivated as previously described [19]. Trisomy 21 iPSCs (T21-iPSCs) and rescued disomy 21 iPSCs (D21-iPSCs) were maintained in Essential 8 (E8) medium (Life Technologies, catalog number (#) A1517001) onto vitronectin (VTN) (Life Technologies, #A14700)-coated dishes and passaged using 0.5 mM EDTA in phosphate buffered saline (PBS) [18]. Normal human epidermal keratinocytes (LONZA, #192906) were also cultured in defined keratinocyte serum-free medium (DKSFM) (Invitrogen, #10744-019) supplemented with 10 μM Y-27632 on type I collagen (Advanced Biomatrix, #5005-B) and fibronectin (Sigma-Aldrich, #F0895-1MG)-coated dishes. HDK1-K4DT, which is a normal human keratinocyte line immortalized with TERT, a mutant form of CDK4 and cyclin D1 by lentivirus-mediated gene transfer, was cultured in keratinocyte serum-free medium (Invitrogen, #17005042, low calcium concentration) [20].
Real-Time qPCR
RNA was extracted from cells using the RNeasy Plus Mini kit (Qiagen, #74134). An aliquot of total RNA was reverse transcribed using an oligo (dT) primer (Invitrogen, #18418-020). For the thermal cycle reactions, the cDNA template was amplified (Applied Biosystems Quantstudio 12K Flex Real-Time PCR System) with gene-specific primer sets (see Additional file 1: Table S1) using the Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, #11733-046) under the following reaction conditions: 40 cycles of PCR (95°C for 15 s and 60°C for 1 min) after an initial denaturation (95°C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. mRNA levels were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. RT-qPCR analyses for expression of epithelial markers were showed as means of percent of GAPDH (%GAPDH) with the standard deviation (SD).
Immunocytochemical analysis
Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After washing with PBS and treatment with 0.1% Triton X-100 (Sigma-Aldrich, # T8787-100 ML) for 10 min at room temperature, the cells were incubated with Protein Block Serum-Free Ready-to-use (Dako, # X0909) for 30 min at room temperature. This was followed by reaction with primary antibodies in blocking buffer for 60 min at room temperature. After washing with PBS, the cells were incubated with fluorescently conjugated secondary antibodies; anti-rabbit or anti-mouse immunoglobulin G (IgG) bound to Alexa 488 or 546 (1:1,000) was incubated in blocking buffer for 30 min at room temperature. The nuclei were stained with DAPI (Biotium, # 40043). All images were captured using confocal microscopy (LSM 510 and LSM 510 MERA laser scanning microscope; Carl Zeiss, Germany). The fluorescent cells were quantified by hybrid cell counting using a digital microscope (BZ-X 710; Keyence Corp., Osaka, Japan). We measured the number of positive cells in at least six fields to obtain a total of more than 450 cells. Antibody information is provided in Additional file 2: table S2a.
Immunohistochemical analysis
Immunohistochemistry was performed as previously described [21]. Paraffin sections were deparaffinized and heated in antigen retrieval solution (Nichirei, #415211) for 20 min. After washing with PBS, samples were placed in 3% hydrogen peroxide/methanol for 5 min to block endogenous peroxidase. The sections were then incubated at room temperature for 90 min in primary antibodies diluted with antibody diluent. Antibody information is provided in Additional file 2: table S2b. The sections were then washed three times with PBS and incubated with peroxidase-labeled goat anti-mouse or anti-rabbit antibodies (Nichirei, #424151) at room temperature for 45 min. After washing with PBS, they were incubated in 3,3′diaminobenzidin (Muto pure chemicals, #40651) for 5-10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.
Differentiation of iPSCs into keratinocytes
The induction of differentiation into keratinocytes was carried out as previously described [19]. We subcultured small clumps of undifferentiated iPSC on VTN-coated 100-mm dish in E8 medium on day 0. iPSCs were cultured in DKSFM (Invitrogen, #10744-019) supplemented with 1 μM all-trans RA (Wako, #182-01111) and 10 ng/mL bone morphogenetic protein 4 (BMP4) (R&D systems, #314-BP-010/CF) from day 1 to day 4. After 4 days, iPSCs were maintained in DKSFM supplemented with 20 ng/mL epidermal growth factor (EGF) (R&D systems, #236-EG-200) for 10 days, then passaged to a 100-mm dish coated with 0.03 mg/mL type I collagen (Advanced Biomatrix, #5005-B) and 0.01 mg/mL fibronectin (Sigma-Aldrich, #F0895-1MG), and maintained in DKSFM supplemented with 20 ng/mL EGF and 10 μM Y-27632 (Wako, #251-00514). Cells were subcultured when approximately 80 to 90% confluence. Cells were rinsed with PBS and incubated with 0.25w/v% trypsin-1mmol/L EDTA (Wako, #209-16941) at 37°C for 5 min for passaging. Cells were passaged at 0.3 × 10 5 cells/cm 2 to a new plate coated with type I collagen and fibronectin and enriched by rapid adherence to fibronectin and type I collagen-coated dished for 15 min at 37°C. Nonadherent cells were removed and rapidly attached cells were cultured. The medium was changed every 2 or 3 days. All cells were maintained at 37°C in a humidified incubator with 5% CO 2 .
Propagation of keratinocytes derived from iPSCs
For proliferation of iPSC-derived keratinocytes, we employed specific culture conditions suitable for keratinocytes [22][23][24].
iPSC-derived keratinocytes were cultured in ESTEM-EP medium (GlycoTechnica Ltd., Japan) [Dulbecco's modified Eagle's medium (DMEM) +F12 (3:1) medium supplemented with FBS, hydrocortisone, adenine, EGF, cholera toxin, insulin, A83-01, nicotinamide, SB202190, Y-27632, and penicillin/streptomycin] onto irradiated mouse embryo fibroblasts (MEF). The medium was changed every 2 or 3 days until the cells were reached 80 to 90% confluence. Cells were passaged at 0.1-0.25 × 10 5 cells/cm 2 to a new plate of irradiated MEF feeder cells. The cell sheets were retrieved from the dish with 10 mg/mL dispase (Wako, #383-02281) or harvested using a cell scraper. The sheets in iPGell (Genostaff, #PG20-1) were prepared for immunohistochemistry. The cell numbers were recorded at each passage using the Countess automated cell counter and population doublings were determined.
Dispase-based keratinocytic selection
Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Coculture of iPSC-derived keratinocytes and MEF under culture condition B were rinsed with PBS and incubated with 10 mg/mL dispase at 37°C for 5-8 min, with close monitoring by phase microscopy. When non-keratinocytes began to detach by tapping the dish, dispase was inactivated, and the solution containing non-keratinocytes and feeder cells were removed by aspiration. Stratified colonies remained tightly adherent. The remaining colonies were rinsed with PBS and incubated with trypsin-EDTA solution at 37°C for about 3 min. The cells were passaged to a new plate of irradiated MEF feeder cells at a density of 0.1-0.25 × 10 5 cells/cm 2 .
Generation of a 3D skin equivalent
Three-dimensional skin was generated according to a previously described protocol [25]. Type I collagen (Koken, #IPC-50) and 1 × 10 6 human foreskin fibroblasts (HFFs) were mixed and poured into an untreated 60-mm Petri dish (Falcon, #351007) while cooling and allowed to gel at 37°C for 1 h to prepare the dermal equivalent. The collagen gel was detached from the surface of the Petri dish, and the contraction of the gel was facilitated. The medium was changed every 2 or 3 days for 7 days. iPSC-derived keratinocytes were plated at 0.2 × 10 6 cells inside in a glass ring (IWAKI, #RING-12) on the surface of the contracted collagen gel, which was plated onto polyethylene terephthalate membranes (Corning, #35-3493). iPSC-derived keratinocytes were grown in ESTEM-EP medium for 1 day, following which they were exposed to air in a 1:1 mixture of ESTEM-EP medium and DMEM supplemented with 10% FBS (medium for HFFs), in which the Ca 2+ concentration was adjusted to 0.9 mM. The medium was changed every 2 or 3 days. Multilayered 3D cultures of keratinocytes were obtained by day 14.
Statistical analysis
The results are expressed as means with SD. Comparisons between the two groups were evaluated with the unpaired Student's t test. For three groups, a one-way analysis of variance (ANOVA) with a Bonferroni correction was used. Statistical analysis was performed using Stata 14.0 (StataCorp LP, College Station, Texas, USA). P values less than 0.05 were considered to be statistically significant.
Results
Rescued disomy 21 iPSCs, but not trisomy 21 iPSCs, are likely to differentiate into keratinocytes
We employed a standard protocol for differentiation of T21-iPSCs and D21-iPSCs into keratinocytes (Fig. 1a). The differentiated cells derived from T21-and D21-iPSCs, i.e., keratinocyte progenitor cells, resembled normal human epidermal keratinocytes (NHEK) at passage 2 ( Fig. 1b). Gene expression analysis was performed using iPSC-derived keratinocytes at passage 2 and/or passage 3. Immunostaining analysis revealed that both keratinocytes derived from T21-iPSC (T21-KC) and D21-iPSC#1 (D21-KC#1) were positive for epithelial markers, i.e., KERATIN 14 (KRT14), KRT10, involucrin, and loricrin (Fig. 1c). T21-KCs at passage 5 had a significantly low number of KRT14-positive cells, compared to HDK1-K4DT and D21-KC at passage 5 (Fig. 1d). The number of Involucrin-positive cells in both T21-and D21-KCs was low, compared with NHEK (Fig. 1c). T21-KCs also contained non-keratinocytes such as iPSC-like colonies and cells and exhibited loss of nuclear-cytoplasmic boundaries at passage 3 (see Additional file 3: Fig. S1A). It was extremely difficult to maintain T21-KCs with keratinocyte morphology. In contrast, D21-KC#1 and #2 showed keratinocytic morphology and KRT14 reactivity in most cells, like NHEK cells ( Fig. 1c; Additional file 3: Fig. S1B). The expression of the OCT3/4 and NANOG genes was significantly suppressed after differentiation of T21-iPSCs and D21-iPSCs (Fig. 1e, f). The basal cell marker KRT14, the epithelial progenitor marker TP63, and the keratinizing cell marker Filaggrin were induced in T21-KC and D21-KC#1 ( Fig. 1g-i). The expression of KRT14 and TP63 was relatively increased in D21-KCs, compared with T21-KCs (Fig. 1g, h). Filaggrin expression was relatively high in T21-KCs (Fig. 1i). The expression of KRT14 and TP63 were unchanged in D21-KC#1 (Fig. j, k). T21-KC, D21-KC#1, and D21-KC#2 had keratinocytic morphology stopped dividing at 4 to 5 population doublings (PDs) (Fig. 1l). These results imply that T21-and D21-iPSCs have a distinct propensity for keratinocytic differentiation.
A combination of serum, Y-27632, and feeder cells allows propagation and sheet formation of D21-KCs
To escape cell senescence and form epidermal sheets, we altered the cultivation conditions (Fig. 2a). T21-KCs, D21-KC#1, and D21-KC#2 recovered the ability to grow and proliferated as colonies on MEFs (Fig. 2b). The differentiated cells under these culture conditions were defined as mature keratinocytes. We analyzed these cells 1-2 weeks after the change in cultivation conditions. T21-KCs showed multilayer structures with a high nucleus/cytoplasm (N/C) ratio and chromatin aggregation (Fig. 2c). In contrast, D21-KC#1 and D21-KC#2 cells showed an epithelial structure with basal, spinous, and granular layers (Fig. 2d). Uniformly thick multilayersheets were obtained by dispase treatment (Fig. 2e). Immunostaining of D21-KC stratified epithelium revealed layer-dependent reactivity to KRT14, basement membrane integrin β4, the suprabasal marker KRT10, and spinous and granular layer markers involucrin (Fig. 2f). Loricrin, a major component of granular layers, was also detected in the KRT14-negative cells. In contrast to D21-KC#1 and D21-KC#2 cells, T21-KC expressed OCT3/4 in some cells and failed to express KRT10 and KRT14. These results suggest that the autonomously rescued disomy 21 keratinocytes produced epithelial structures. Along with the immunocytochemical analysis, RT-qPCR analysis revealed a lack of expression of epithelial markers KRT14, TP63, and Involucrin in T21-KCs (Fig. 2g). D21-KC#1, but not T21-KCs, significantly increased in the epithelial markers after the change in culture conditions. These significant increases in keratinocytic marker expression are probably due to culture condition B, which is suitable for keratinocyte proliferation and maturation (Fig. 2a). The expression of OCT3/4 and NANOG diminished in both T21-and D21-KCs. Cytological analysis showed that T21-KCs, D21-KC#1, and D21-KC#2 treated with condition A showed variable cell sizes with a low N/C ratios (see Additional file 4: Fig. S2).
Trisomy 21-and rescued disomy 21-iPSCs escape from senescence for more than 160 days
The proliferative capability of T21-KC, D21-KC#1, and D21-KC#2 was restored after the change in cultivation conditions (Fig. 3). T21-KC cells continued to proliferate rapidly and had fewer cells that showed keratinocytic morphology. In contrast, D21-KC#1 and D21-KC#2 exhibited stable growth and maintained keratinocytic morphology for a period of more than 160 days. D21-KC#1 and D21-KC#2 showed growth cessation at 34 and 52 PDs, respectively. The reason for the differences in growth cessation is unclear but may be related to the differentiation competency of these cells.
Dispase-based purification of iPSC-derived keratinocytes improves epidermal sheet formation
After long-term cultivation, D21-KC#1 cell cultures contained both keratinocytes and non-keratinocytes (Fig. 4a). (Fig. 4b).
D21-KC#1 keratinocyte-like cells expressed KRT14 in
To separate keratinocytelike cells from non-keratinocyte-like cells, we used differential reactivity of these cells to dispase, i.e., tight or loose cell-dish adhesion: Stratified colonies are relatively resistant to dispase and non-keratinocytes are easily detached after dispase treatment (Fig. 4c). This treatment was applied to D21-KC#1 cells at passage 7 to passage 9 (Fig. 4d). The ratio of keratinocytes increased at each passage by microscopic observation with a phase-contrast microscope. KRT14 increased, TP63 decreased, and Involucrin remained unchanged at each passage ( Fig. 4e-g). D21-KC#1 was immunocytochemically positive for epidermal markers, i.e., KRT14, KRT10, involucrin, and loricrin, at passage 13 (Fig. 4h). D21-KCs were able to reproducibly generate epidermal sheets (Fig. 4i). T21-KCs became fragmented upon dispase treatment; thus, we decided not to use dispase for keratinocytic purification. We picked up colonies with keratinocyte morphology at each passage from passage 6 to passage 8 (see Additional file 5: Fig. S3A, B). Keratinocyte-like cells were microscopically observed at passage 8; however, these cells did not express KRT14 and TP63 and epithelial markers were absent (Fig. 4e, f, h). The lack of keratinocyte marker expression may be caused by a gene dosage imbalance due to trisomy 21.
3D skin can be manufactured from disomy 21 iPSCderived keratinocytes
The dispase-based selection at each passage helped maintain the proliferation of KRT14 positive cells and stratified cell sheets were manufactured in plate cultures of D21-KC#1. We therefore investigated whether disomy 21 iPSC-derived keratinocytes could manufacture 3D skin. To this end, we generated artificial dermis by combining human cultured dermal cells and type I collagen in a Petri dish, and overlaid D21-KC#1 cells in a glass ring. D21-KC#1 cells on the artificial dermis were then cultivated in an air-liquid interface for 2 weeks to accelerate epidermal differentiation. The 3D skin from D21-KC#1 was generated with irregular layer formation and expressed KRT14, KRT10, involucrin, loricrin, and integrin β4 (Fig. 5a, b). Laminin 5 was not detected at the dermal-epidermal junction.
Discussion
Differentiation incompetence in T21-iPSCs and its restoration in D21-iPSCs
Restoration of differentiation and growth of trisomyrescued iPSCs is a focus of our laboratory. This restoration or normalization may simply be attributable to a gene dosage effect, i.e., a change from 3 (aneuploid) to 2 copies (euploid). The Down syndrome phenotype is generally derived from the increased expression levels of dosagesensitive genes, and most of the triplicated genes show upregulated expression that is compatible with gene dosage (i.e., at close to 1.5-fold increase) [15,26]. The expression levels of the genes for APP (Alzheimer's disease marker), DYRK1A, DSCR1 (Down syndrome critical region 1), ETS2, and SOD1, all of which are located on chromosome 21, are dysregulated in trisomic cells. Although a gene dosage imbalance is the main molecular mechanism, extensive dysregulation of euploid genes are associated with Down syndrome phenotypes [26,27]. An extra copy of chromosome 21 also alters gene expression across every chromosome, not just chromosome 21 [9,28]. The altered expression levels decrease to normal levels in trisomyrescued undifferentiated iPSCs; in other words, revertant cells regain the gene expression levels of intact iPSCs [18]. These normalized gene expression levels may restore keratinocytic differentiation capability in trisomy-rescued iPSCs. The detailed mechanism of keratinocytic restoration in trisomy 21 cells remains unclear, and additional analysis may elucidate the mechanism underlying epidermal abnormalities in Down syndrome. The ability of trisomy 21-stem/progenitor cells to differentiate also depends on the target organs or cells [14,15,29,30]. The downregulated genes in T21-iPSCs reveal significant enrichment for genes involved in embryonic and tissue morphogenesis [15]. In addition, differentiated ectoderm germ structures are not found in T21-iPSC-derived teratomas [31]. In another study, lack of pluripotency in T21-iPSCs with reduced expression of HERVH might explain the lack of T21-iPSC differentiation compared to disomic iPSCs [15]. These reports may explain the inability of to T21-iPSCs differentiate, i.e., a lack of KRT14 marker and the partial expression of OCT3/4 in T21-KCs, and increased population of nonkeratinocytes after the long-term culture. It is noted that keratinocytic differentiation of D21-iPSCs was achieved at Fig. 3 Differentiating cells from T21-iPSCs and the rescued disomy (D21)-iPSCs shows proliferative capability for more than 160 days. The numbers of cells were recorded at each passage using an automated cell counter and population doublings were determined. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10 (2) from two or three independent experiments passage 106. This implies that the differentiation capability of trisomy-rescued cells is extremely stable for a period of more than 500 days.
Uncontrolled growth of T21-KCs and normalized growth of D21-KCs
Accelerated growth of differentiated cells from T21-iPSCs into keratinocytes was unexpected because the incidence of solid tumors, such as dermatological tumors, is decreased in Down syndrome patients [32,33]. Candidate genes such as DYRK1A, PIGP, and RCAN1 are related to keratinocyte hyperproliferation in a Down syndrome mouse model [8]. Triplication of Usp16, located on chromosome 21, reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts [34]. Fig. 4 Purification of iPSC-derived keratinocytes, based on differential reactions to dispase. a Phase-contrast photomicrographs of keratinocytes derived from D21-KC#1 at passage 5. Two types of colonies could be observed: stratified keratinocyte colonies (left) and non-keratinocyte-like cell colonies (right). Scale bars, 200 μm. b Immunocytochemical analysis of D21-KC#1 colony with epithelial markers, i.e., KRT14 and KRT10. Scale bars, 200 μm. c Selection of keratinocytes with dispase digestion. Non-keratinocytes (non-KC) detached earlier than keratinocytes with dispase treatment. Nonkeratinocytes and feeder cells were removed at each passage. Keratinocytes were then passaged. d Phase-contrast photomicrographs of D21-KC#1 during the dispase selection. There were insular colonies, which were formed by stratified epithelial cells, and another area contained non-keratinocytelike cells. Dispase-based selection at each passage caused non-keratinocyte-like cells to decrease. Scale bars, 500 μm. e Real-time qPCR analysis of KRT14 at each passage. Values are shown as means ± SD from two or three independent experiments. f Real-time qPCR analysis of TP63 at each passage. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of Involucrin at each passage. Values are shown as means ± SD from two or three independent experiments. h Immunocytochemical analysis of epithelial markers, i.e., KRT14, KRT10, involucrin, and loricrin after colony isolations (T21-KC) at passage 7 and dispase-based selection (D21-KC#1) at passage 13. Scale bars, 100 μm. i Epithelial sheets of keratinocyte-like cells derived from D21-iPSC#1 at passage 8. Cell sheets were harvested by dispase treatment. Scale bar, 1 cm Overexpression of patched-1, a receptor that represses the mitogenic sonic hedgehog pathway, may be associated with proliferation impairment in skin [35][36][37][38]. Moreover, proliferation of trisomy 21-fibroblasts and trisomy 21-iPSCs decrease due to oxidative stress and protein aggregation [39,40]. Neurogenesis-related genes such as DYRK1A and RCAN1, craniofacial defect-related genes such as DYRK1A, ETS2, and RCAN1, and tumor suppressor-related genes such as DYRK1A, ETS2, and RCAN1 were also reported in Down syndrome [26]. In our study, an increased growth rate, an increased nucleus/cytoplasm ratio, and aggregated chromatin of T21-KCs may have resulted from susceptibility to growth stimulation. Skin tumor growth and hyperkeratosis were detected in Ts1Rhr mice model of Down syndrome [8]. We have also reported accelerated growth of T21derived neural stem cells [18]. These increases in growth of other cell types are indeed compatible with that of T21-KCs in this study.
Psoriasis is one of the dermatological abnormalities observed in Down syndrome and is characterized by hyperproliferation and defective differentiation of keratinocytes. Altered gene expression such as upregulation of IFN genes is linked to hyperproliferation of KCs derived from psoriasis patient-specific iPSCs [7]. The autoimmune abnormalities and altered sensitivity to stimuli in Down syndrome may be linked to the excessive proliferation of T21-KCs and reduced differentiation of T21-iPSCs.
In addition to growth rate, senescence or aging of iPSC-derived keratinocytes is a concern. Human primary keratinocytes in serum-free and chemically defined media senesce around 15-20 PDs [41,42]. iPSC-derived keratinocytes exhibit growth arrest when cultured in feeder-free, serum-free medium containing EGF and Y-27632 around passage 4 or 5 [19]. Combination of Rho kinase inhibitor and feeder cells induces conditional reprograming and immortalization of human epithelial cells without the use of viral infection or genetic modification [22][23][24]. In line with these reports, D21-KC survival and proliferation was achieved with a combination of a Rho kinase inhibitor and feeder cells. The successful extension of the D21-KC lifespan to more than 34 population doublings over the period of 160 days is surprising, since extended lifespan of T21-KCs was not accompanied by keratinocytic phenotypes. The trisomy-rescued D21-KCs with a longer lifespan have the ability to stratify, which is essential for manufacturing 3D skin with keratinocytic functions.
Conclusion
To study the impact of trisomy 21 on keratinocytic function, we provide an iPSC-derived model using trisomy 21 iPSC and autonomous trisomy-rescued iPSCs. Our results suggest that there is impairment in keratinocytic differentiation in trisomy 21 iPSCs. In contrast, propagation of trisomy-rescued iPSC-derived keratinocytes with purification and manufacturing of stratified epithelial sheets and 3D skin imply a restoration of keratinocytic functions. Further investigation of the influence of the extra copy of chromosome 21 may help determine the underlying causes of Down syndrome phenotypes and lead to the generation of a Down syndrome model of dermatological abnormalities.
Fig. 1
1Keratinocytic differentiation of Down syndrome-iPSCs and autonomously rescued disomy 21-iPSCs. a Protocol for keratinocytic differentiation from amniotic fluid-iPSCs. DKSFM, defined keratinocyte serum-free medium; RA, retinoic acid; BMP4, bone morphogenetic protein 4; VTN, vitronectin; E8, Essential 8 medium; EGF, epidermal growth factor. b Phase-contrast photomicrograph of normal human epidermal keratinocytes (NHEK) at passage 3, and keratinocytes derived from T21-iPSCs (T21-KC), D21-iPSC#1 (D21-KC#1) and D21-iPSC#2 (D21-KC#2) at passage 2 (day 25). T21-KC, D21-KC#1, and D21-KC#2 exhibited keratinocyte-like morphology. Scale bars, 200 μm. c Immunocytochemical analysis of T21-KC at day 28 and D21-KC#1 at day 34 with the antibodies to epithelial markers, i.e., KRT14, KRT10, involucrin and loricrin. Scale bars, 100 μm. d Immunocytochemistry with the epithelial stem cell marker KRT14. The percentage of KRT-positive cells is shown for HDK1-K4DT (control), T21-KCs at day 56, D21-KC#1 at day 55, and D21-KC#2 at day 57. Scale bars, 100 μm. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni correction. e Real-time qPCR analysis of OCT3/4 in T21-KCs at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from three independent experiments. f Real-time qPCR analysis of NANOG in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of KRT14 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of KRT14 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.42). Values are shown as means ± SD from three independent experiments. h Realtime qPCR analysis of TP63 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of TP63 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.31). Values are shown as means ± SD from three independent experiments. i Real-time qPCR analysis of Filaggrin in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of Filaggrin was relatively high in T21-KCs, compared with D21-KCs (p-value, 0.34). Values are shown as means ± SD from three independent experiments. j Real-time qPCR analysis of KRT14 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. k Real-time qPCR analysis of TP63 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. l Growth of iPSC-derived keratinocytes. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10(2). Phase-contrast photomicrograph of D21-KC#1 at senescence is shown most cells of the stratified colonies and KRT10 at the periphery of the colonies
Fig. 2
2Sheet formation and characterization of keratinocytes derived from T21-and D21-iPSC. a Schematic diagram of the experimental design. Keratinocytes derived from iPSCs were cultivated under culture condition A followed by condition B for the purpose of propagation of the lifespan. As the growth rate decreased, the culture conditions were changed from A to B. In the culture condition A, keratinocytes were maintained in DKSFM supplemented with 20 ng/ mL EGF and 10 μM Y-27632 at a plate coated with type I collagen and fibronectin. In culture condition B, keratinocytes were cultured in the ESTEM-EP medium supplemented with 10 μM Y-27632 on irradiated mouse embryo fibroblasts (MEF).b Phase-contrast photomicrographs of keratinocytes derived from T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4. c Thin section of T21-KC at day 42 in iPGell. Hematoxylin and eosin (HE) stain. d Thin section of D21-KC#1 at day 50 and D21-KC#2 at day 48 in iPGell. HE stain. e Fabrication of D21-KC#1 epithelial sheet after dispase treatment. (Left panel) Histology of the sheet. HE stain. Scale bar, 100 μm. (Right panel) Macroscopic view of the sheet. Scale bar, 1 cm. f Immunohistochemical analysis of skin, HDK1-K4DT, T21-KCs at day 42, D21-KC#1 at day 50, and D21-KC#2 at day 48 with antibodies to pan-cytokeratins (Pan-CK), KRT14, P63, KRT10, integrin β4, involucrin, loricrin, filaggrin, and OCT3/4. D21-KC exhibited terminal differentiation and formed structured cell sheets. The expression patterns of these markers in intact skin are shown for reference. g Real-time qPCR analysis of epithelial markers (KRT14, TP63, Involucrin) and pluripotent markers (OCT3/4, NANOG) in T21-KC at day 47, D21-KC#1 at day 49, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. *p < 0.05
Fig. 5
5Three-dimensional (3D) cultured skin equivalents from D21-KC#1. a Histology of the 3D keratinocytes from D21-iPSCs. HE stain. Scale bar, 200 μm. b Immunohistochemical analysis of the expression and location of epithelial markers, i.e., KRT14, KRT10, involucrin, loricrin, integrin β4, and laminin 5. The expression patterns of these markers in intact skin are shown for reference. Scale bars, 200 μm
Tanuma-Takahashi et al. Stem Cell Research & Therapy (2021) 12:476
AcknowledgementsWe would like to express our sincere thanks to M. Ichinose for providing expert technical assistance, to E. Suzuki and K. Saito for secretarial work, and to Catherine Ketcham for English language review.Supplementary InformationThe online version contains supplementary material available at https://doi. org/10.1186/s13287-021-02448-w.Additional file 1:Table S1. List of primers Additional file 2:Table S2. List of antibodies for immunochemistry Additional file 3:Figure S1. Phase-contrast photomicrographs of T21-KC and D21-KC#1 at passage 3 (Related toFig. 1Additional file 4:Figure S2. Characterization of keratinocyte derived from iPSCs (Related toFig. 2). Left panels: Phase-contrast photomicrographs of HDK1-K4DT (normal human keratinocytes) at passage 14, T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4 in a defined keratinocyte serum-free medium (DKSFM), i.e., culture condition A. Right panels: Thin sections of T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4. These cells did not adhere each other because of low-calcium medium (DKSFM). HE stain.Additional file 5Figure S3. Colonial isolation of T21-KC (Related toFig. 4). A Phase-contrast photomicrographs of T21-KC before colony isolation. B Phase-contrast photomicrographs of T21-KC after three colony isolations. After three times of isolations from passage 6 to passage 8, keratinocyte-like-cells were observed all over the dish. However,Fig. 4Hshowed that cytokeratins were not expressed in these cells.Authors' contributionsAT and AU designed experiments. AT, MI, and KK performed experiments. AT, RT, and AU analyzed data. MI, KK, and TK contributed reagents and materials. AT, MI, KK, RT, AY, OS, HA, HS, TK, AO, and AU discussed the data and manuscript. AU and AT wrote this manuscript. The authors read and approved the final manuscript.FundingThis study was supported by a grant from KAKENHI (grant number 19K17317).Availability of data and materialsThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.DeclarationsEthics approval and consent to participate This study was fully compliant with the Ethical Guidelines for Clinical Studies (Ministry of Health, Labor, and Welfare). The protocol for using human cells in this study was approved by Institutional Review Board of National Center for Child Health and Development Research Institute of Japan.Consent for publicationNot applicable.Competing interestsThe author Akihiro Umezawa is the Associate Editor of the journal but was not involved in the peer review process of this article. The other authors declare that they have no competing interests.
Induced pluripotent stem cells; T21-iPSCs: Trisomy 21-induced pluripotent stem cells; D21-iPSCs: Autonomous rescued disomy 21-iPSCs. 3Threedimensional; E8: Essential 8; VTN: Vitronectin; DKSFM: Defined keratinocyte serum-free medium. GAPDH: Glyceraldehyde-3-phosphate dehydrogenaseiPSCs: Induced pluripotent stem cells; T21-iPSCs: Trisomy 21-induced pluripo- tent stem cells; D21-iPSCs: Autonomous rescued disomy 21-iPSCs; 3D: Three- dimensional; E8: Essential 8; VTN: Vitronectin; DKSFM: Defined keratinocyte serum-free medium; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase;
SD: Standard deviation. Bone morphogenetic protein. 4SD: Standard deviation; BMP4: Bone morphogenetic protein 4;
EGF: Epidermal growth factor; DMEM: Dulbecco's modified Eagle's medium. EGF: Epidermal growth factor; DMEM: Dulbecco's modified Eagle's medium;
MEF: Mouse embryo fibroblasts; HFFs: Human foreskin fibroblasts. MEF: Mouse embryo fibroblasts; HFFs: Human foreskin fibroblasts;
NHEK: Normal human epidermal keratinocytes. KRT: KERATINNHEK: Normal human epidermal keratinocytes; KRT: KERATIN;
KC: Keratinocytes; T21-KC: Keratinocytes derived from T21-iPSC. D21-KC: Keratinocytes derived from D21-iPSCKC: Keratinocytes; T21-KC: Keratinocytes derived from T21-iPSC; D21- KC: Keratinocytes derived from D21-iPSC;
. N/C, Nucleus/cytoplasm. N/C: Nucleus/cytoplasm;
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Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro. J C Chiang, J Jiang, P E Newburger, J B Lawrence, 10.1038/s41467-018-07630-yNat Commun. 915180Chiang JC, Jiang J, Newburger PE, Lawrence JB. Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro. Nat Commun. 2018;9(1):5180. https://doi. org/10.1038/s41467-018-07630-y.
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DNA methylation dynamics in human induced pluripotent stem cells over time. K Nishino, M Toyoda, M Yamazaki-Inoue, Y Fukawatase, E Chikazawa, H Sakaguchi, 10.1371/journal.pgen.1002085PLoS Genet. 751002085Nishino K, Toyoda M, Yamazaki-Inoue M, Fukawatase Y, Chikazawa E, Sakaguchi H, et al. DNA methylation dynamics in human induced pluripotent stem cells over time. PLoS Genet. 2011;7(5):e1002085. https:// doi.org/10.1371/journal.pgen.1002085.
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Fetal Therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells. K Kajiwara, T Tanemoto, S Wada, J Karibe, N Ihara, Y Ikemoto, 10.1016/j.stemcr.2017.05.013Stem Cell Reports. 86Kajiwara K, Tanemoto T, Wada S, Karibe J, Ihara N, Ikemoto Y, et al. Fetal Therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells. Stem Cell Reports. 2017;8(6):1701-13. https://doi.org/10.1016/j.stemcr.2017.05.013.
The E1 protein of human papillomavirus type 16 is dispensable for maintenance replication of the viral genome. N Egawa, T Nakahara, S Ohno, M Narisawa-Saito, T Yugawa, M Fujita, 10.1128/JVI.06450-11J Virol. 866Egawa N, Nakahara T, Ohno S, Narisawa-Saito M, Yugawa T, Fujita M, et al. The E1 protein of human papillomavirus type 16 is dispensable for maintenance replication of the viral genome. J Virol. 2012;86(6):3276-83. https://doi.org/10.1128/JVI.06450-11.
Efficiency of human epiphyseal chondrocytes with differential replication numbers for cellular therapy products. M Nasu, S Takayama, A Umezawa, Biomed Res Int. 6437658Nasu M, Takayama S, Umezawa A. Efficiency of human epiphyseal chondrocytes with differential replication numbers for cellular therapy products. Biomed Res Int. 2016;2016:6437658.
Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. S Chapman, X Liu, C Meyers, R Schlegel, A A Mcbride, 10.1172/JCI42297J Clin Invest. 1207Chapman S, Liu X, Meyers C, Schlegel R, McBride AA. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest. 2010; 120(7):2619-26. https://doi.org/10.1172/JCI42297.
ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. X Liu, V Ory, S Chapman, H Yuan, C Albanese, B Kallakury, 10.1016/j.ajpath.2011.10.036Am J Pathol. 1802Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol. 2012;180(2):599-607. https://doi.org/10.1016/j.a jpath.2011.10.036.
Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells. N Palechor-Ceron, F A Suprynowicz, G Upadhyay, A Dakic, T Minas, V Simic, 10.1016/j.ajpath.2013.08.009Am J Pathol. 1836Palechor-Ceron N, Suprynowicz FA, Upadhyay G, Dakic A, Minas T, Simic V, et al. Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells. Am J Pathol. 2013;183(6):1862-70. https://doi.org/10.1016/j.ajpath.2013.08.009.
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Publisher's Note. Publisher's Note
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| [
"Background: An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). Methods: Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21-and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. Results: Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. Conclusions: Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions."
] | [
"Akiko Tanuma-Takahashi ",
"Momoko Inoue ",
"Kazuhiro Kajiwara ",
"Ryo Takagi ",
"Ayumi Yamaguchi ",
"Osamu Samura ",
"Hidenori Akutsu ",
"Haruhiko Sago ",
"Tohru Kiyono ",
"Aikou Okamoto ",
"Akihiro Umezawa "
] | [] | [
"Akiko",
"Momoko",
"Kazuhiro",
"Ryo",
"Ayumi",
"Osamu",
"Hidenori",
"Haruhiko",
"Tohru",
"Aikou",
"Akihiro"
] | [
"Tanuma-Takahashi",
"Inoue",
"Kajiwara",
"Takagi",
"Yamaguchi",
"Samura",
"Akutsu",
"Sago",
"Kiyono",
"Okamoto",
"Umezawa"
] | [
"N/C, ",
"Pds, ",
"C Down Epstein, ",
"Syndrome, ",
"N J Roizen, ",
"D Patterson, ",
"V Madan, ",
"J Williams, ",
"J T Lear, ",
"A J Esbensen, ",
"R Sureshbabu, ",
"R Kumari, ",
"S Ranugha, ",
"R Sathyamoorthy, ",
"C Udayashankar, ",
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"E J Wolvetang, ",
"X Mou, ",
"Y Wu, ",
"H Cao, ",
"Q Meng, ",
"Q Wang, ",
"C Sun, ",
"S Irion, ",
"M C Nostro, ",
"S J Kattman, ",
"G M Keller, ",
"A L Brigida, ",
"D Siniscalco, ",
"J C Chiang, ",
"J Jiang, ",
"P E Newburger, ",
"J B Lawrence, ",
"L B Li, ",
"K H Chang, ",
"P R Wang, ",
"R K Hirata, ",
"T Papayannopoulou, ",
"D W Russell, ",
"P K Gonzales, ",
"C M Roberts, ",
"V Fonte, ",
"C Jacobsen, ",
"G H Stein, ",
"C D Link, ",
"J Jiang, ",
"Y Jing, ",
"G J Cost, ",
"J C Chiang, ",
"H J Kolpa, ",
"A M Cotton, ",
"K Nishino, ",
"M Toyoda, ",
"M Yamazaki-Inoue, ",
"Y Fukawatase, ",
"E Chikazawa, ",
"H Sakaguchi, ",
"M Inoue, ",
"K Kajiwara, ",
"A Yamaguchi, ",
"T Kiyono, ",
"O Samura, ",
"H Akutsu, ",
"K Kajiwara, ",
"T Tanemoto, ",
"S Wada, ",
"J Karibe, ",
"N Ihara, ",
"Y Ikemoto, ",
"N Egawa, ",
"T Nakahara, ",
"S Ohno, ",
"M Narisawa-Saito, ",
"T Yugawa, ",
"M Fujita, ",
"M Nasu, ",
"S Takayama, ",
"A Umezawa, ",
"S Chapman, ",
"X Liu, ",
"C Meyers, ",
"R Schlegel, ",
"A A Mcbride, ",
"X Liu, ",
"V Ory, ",
"S Chapman, ",
"H Yuan, ",
"C Albanese, ",
"B Kallakury, ",
"N Palechor-Ceron, ",
"F A Suprynowicz, ",
"G Upadhyay, ",
"A Dakic, ",
"T Minas, ",
"V Simic, ",
"M Tsunenaga, ",
"Y Kohno, ",
"I Horii, ",
"S Yasumoto, ",
"N H Huh, ",
"T Tachikawa, ",
"B Liu, ",
"S Filippi, ",
"A Roy, ",
"I Roberts, ",
"S E Antonarakis, ",
"R Lyle, ",
"E T Dermitzakis, ",
"A Reymond, ",
"S Deutsch, ",
"A Letourneau, ",
"F A Santoni, ",
"X Bonilla, ",
"M R Sailani, ",
"D Gonzalez, ",
"J Kind, ",
"I Roberts, ",
"S Izraeli, ",
"J P Weick, ",
"D L Held, ",
"Bonadurer Gf 3rd, ",
"M E Doers, ",
"Y Liu, ",
"C Maguire, ",
"Y Hibaoui, ",
"I Grad, ",
"A Letourneau, ",
"M R Sailani, ",
"S Dahoun, ",
"F A Santoni, ",
"H Hasle, ",
"I H Clemmensen, ",
"M Mikkelsen, ",
"H Hasle, ",
"J M Friedman, ",
"J H Olsen, ",
"S A Rasmussen, ",
"M Adorno, ",
"S Sikandar, ",
"S S Mitra, ",
"A Kuo, ",
"Nicolis Di Robilant, ",
"B Haro-Acosta, ",
"V , ",
"C Fuchs, ",
"E Ciani, ",
"S Guidi, ",
"S Trazzi, ",
"R Bartesaghi, ",
"H Ikehara, ",
"K Fujii, ",
"T Miyashita, ",
"Y Ikemoto, ",
"M Nagamine, ",
"N Shimojo, ",
"Y Ikemoto, ",
"Y Takayama, ",
"K Fujii, ",
"M Masuda, ",
"C Kato, ",
"H Hatsuse, ",
"Y Ikemoto, ",
"T Miyashita, ",
"M Nasu, ",
"H Hatsuse, ",
"K Kajiwara, ",
"K Fujii, ",
"A Gimeno, ",
"J L Garcia-Gimenez, ",
"Audi L Toran, ",
"N Andaluz, ",
"P Dasi, ",
"F , ",
"N Nawa, ",
"K Hirata, ",
"K Kawatani, ",
"T Nambara, ",
"S Omori, ",
"K Banno, ",
"M Choi, ",
"C Lee, ",
"T Kiyono, ",
"S A Foster, ",
"J I Koop, ",
"J K Mcdougall, ",
"D A Galloway, ",
"A J Klingelhutz, "
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"Koop",
"Mcdougall",
"Galloway",
"Klingelhutz"
] | [
"Induced pluripotent stem cells; T21-iPSCs: Trisomy 21-induced pluripotent stem cells; D21-iPSCs: Autonomous rescued disomy 21-iPSCs. 3Threedimensional; E8: Essential 8; VTN: Vitronectin; DKSFM: Defined keratinocyte serum-free medium. GAPDH: Glyceraldehyde-3-phosphate dehydrogenaseiPSCs: Induced pluripotent stem cells; T21-iPSCs: Trisomy 21-induced pluripo- tent stem cells; D21-iPSCs: Autonomous rescued disomy 21-iPSCs; 3D: Three- dimensional; E8: Essential 8; VTN: Vitronectin; DKSFM: Defined keratinocyte serum-free medium; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase;",
"SD: Standard deviation. Bone morphogenetic protein. 4SD: Standard deviation; BMP4: Bone morphogenetic protein 4;",
"EGF: Epidermal growth factor; DMEM: Dulbecco's modified Eagle's medium. EGF: Epidermal growth factor; DMEM: Dulbecco's modified Eagle's medium;",
"MEF: Mouse embryo fibroblasts; HFFs: Human foreskin fibroblasts. MEF: Mouse embryo fibroblasts; HFFs: Human foreskin fibroblasts;",
"NHEK: Normal human epidermal keratinocytes. KRT: KERATINNHEK: Normal human epidermal keratinocytes; KRT: KERATIN;",
"KC: Keratinocytes; T21-KC: Keratinocytes derived from T21-iPSC. D21-KC: Keratinocytes derived from D21-iPSCKC: Keratinocytes; T21-KC: Keratinocytes derived from T21-iPSC; D21- KC: Keratinocytes derived from D21-iPSC;",
". N/C, Nucleus/cytoplasm. N/C: Nucleus/cytoplasm;",
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"Publisher's Note. Publisher's Note",
"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."
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"SD: Standard deviation",
"Population doublings; HE: Hematoxylin and eosin References 1. Antonarakis SE. Down syndrome and the complexity of genome dosage imbalance",
"Dermatological manifestations of Down's syndrome",
"Phenotypic and dermatological manifestations in Down Syndrome",
"Keratinocytes derived from patient-specific induced pluripotent stem cells recapitulate the genetic signature of psoriasis disease",
"Increased skin tumor incidence and keratinocyte hyper-proliferation in a mouse model of Down syndrome",
"Concise review: new paradigms for Down syndrome research using induced pluripotent stem cells: tackling complex human genetic disease",
"Generation of diseasespecific induced pluripotent stem cells from patients with different karyotypes of Down syndrome",
"Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications",
"Induced pluripotent stem cells as a cellular model for studying Down Syndrome",
"Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro",
"Trisomy correction in Down syndrome induced pluripotent stem cells",
"Transcriptome analysis of genetically matched human induced pluripotent stem cells disomic or trisomic for chromosome 21",
"Translating dosage compensation to trisomy 21",
"DNA methylation dynamics in human induced pluripotent stem cells over time",
"Autonomous trisomic rescue of Down syndrome cells",
"Fetal Therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells",
"The E1 protein of human papillomavirus type 16 is dispensable for maintenance replication of the viral genome",
"Efficiency of human epiphyseal chondrocytes with differential replication numbers for cellular therapy products",
"Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor",
"ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells",
"Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells",
"Growth and differentiation properties of normal and transformed human keratinocytes in organotypic culture",
"Stem and progenitor cell dysfunction in human trisomies",
"Chromosome 21 and down syndrome: from genomics to pathophysiology",
"Domains of genome-wide gene expression dysregulation in Down's syndrome",
"Haematopoietic development and leukaemia in Down syndrome",
"Deficits in human trisomy 21 iPSCs and neurons",
"Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21",
"Low risk of solid tumors in persons with Down syndrome",
"Usp16 contributes to somatic stem-cell defects in Down's syndrome",
"Early-occurring proliferation defects in peripheral tissues of the Ts65Dn mouse model of Down syndrome are associated with patched1 over expression",
"Establishment of a Gorlin syndrome model from induced neural progenitor cells exhibiting constitutive GLI1 expression and high sensitivity to inhibition by smoothened (SMO)",
"Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome",
"Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1",
"Decreased cell proliferation and higher oxidative stress in fibroblasts from Down Syndrome fetuses. Preliminary study",
"Elimination of protein aggregates prevents premature senescence in human trisomy 21 fibroblasts",
"Immortalization of primary keratinocytes and its application to skin research",
"Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells"
] | [
"Induced pluripotent stem cells; T21-iPSCs: Trisomy 21-induced pluripotent stem cells; D21-iPSCs: Autonomous rescued disomy 21-iPSCs",
"Bone morphogenetic protein",
"EGF: Epidermal growth factor; DMEM: Dulbecco's modified Eagle's medium",
"MEF: Mouse embryo fibroblasts; HFFs: Human foreskin fibroblasts",
"NHEK: Normal human epidermal keratinocytes",
"KC: Keratinocytes; T21-KC: Keratinocytes derived from T21-iPSC",
"Nucleus/cytoplasm",
"Nat Rev Genet",
"The metabolic and molecular bases of inherited disease",
"Down's syndrome. The Lancet",
"Clinical and Experimental Dermatology",
"Health conditions associated with aging and end of life of adults with Down syndrome",
"Dermatol Online J",
"Stem Cells Dev",
"PLoS One",
"Stem Cells Transl Med",
"Stem Cell Res Ther",
"Cold Spring Harb Symp Quant Biol",
"J Stem Cells Regen Med",
"Nat Commun",
"Cell Stem Cell",
"PLoS One",
"Nature",
"PLoS Genet",
"Lab Invest",
"Stem Cell Reports",
"J Virol",
"Biomed Res Int",
"J Clin Invest",
"Am J Pathol",
"Am J Pathol",
"Jpn J Cancer Res",
"EMBO Rep",
"Nat Rev Genet",
"Nature",
"Br J Haematol",
"Proc Natl Acad Sci U S A",
"EMBO Mol Med",
"Risks of leukaemia and solid tumours in individuals with Down's syndrome. The Lancet",
"Genet Med",
"Nature",
"Lab Invest",
"Lab Invest",
"J Med Genet",
"Aging (Albany NY)",
"Biochim Biophys Acta",
"PLoS One",
"Biomol Ther (Seoul)",
"Nature",
"Publisher's Note",
"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations"
] | [
"\nFig. 1\n1Keratinocytic differentiation of Down syndrome-iPSCs and autonomously rescued disomy 21-iPSCs. a Protocol for keratinocytic differentiation from amniotic fluid-iPSCs. DKSFM, defined keratinocyte serum-free medium; RA, retinoic acid; BMP4, bone morphogenetic protein 4; VTN, vitronectin; E8, Essential 8 medium; EGF, epidermal growth factor. b Phase-contrast photomicrograph of normal human epidermal keratinocytes (NHEK) at passage 3, and keratinocytes derived from T21-iPSCs (T21-KC), D21-iPSC#1 (D21-KC#1) and D21-iPSC#2 (D21-KC#2) at passage 2 (day 25). T21-KC, D21-KC#1, and D21-KC#2 exhibited keratinocyte-like morphology. Scale bars, 200 μm. c Immunocytochemical analysis of T21-KC at day 28 and D21-KC#1 at day 34 with the antibodies to epithelial markers, i.e., KRT14, KRT10, involucrin and loricrin. Scale bars, 100 μm. d Immunocytochemistry with the epithelial stem cell marker KRT14. The percentage of KRT-positive cells is shown for HDK1-K4DT (control), T21-KCs at day 56, D21-KC#1 at day 55, and D21-KC#2 at day 57. Scale bars, 100 μm. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni correction. e Real-time qPCR analysis of OCT3/4 in T21-KCs at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from three independent experiments. f Real-time qPCR analysis of NANOG in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of KRT14 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of KRT14 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.42). Values are shown as means ± SD from three independent experiments. h Realtime qPCR analysis of TP63 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of TP63 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.31). Values are shown as means ± SD from three independent experiments. i Real-time qPCR analysis of Filaggrin in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of Filaggrin was relatively high in T21-KCs, compared with D21-KCs (p-value, 0.34). Values are shown as means ± SD from three independent experiments. j Real-time qPCR analysis of KRT14 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. k Real-time qPCR analysis of TP63 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. l Growth of iPSC-derived keratinocytes. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10(2). Phase-contrast photomicrograph of D21-KC#1 at senescence is shown most cells of the stratified colonies and KRT10 at the periphery of the colonies",
"\nFig. 2\n2Sheet formation and characterization of keratinocytes derived from T21-and D21-iPSC. a Schematic diagram of the experimental design. Keratinocytes derived from iPSCs were cultivated under culture condition A followed by condition B for the purpose of propagation of the lifespan. As the growth rate decreased, the culture conditions were changed from A to B. In the culture condition A, keratinocytes were maintained in DKSFM supplemented with 20 ng/ mL EGF and 10 μM Y-27632 at a plate coated with type I collagen and fibronectin. In culture condition B, keratinocytes were cultured in the ESTEM-EP medium supplemented with 10 μM Y-27632 on irradiated mouse embryo fibroblasts (MEF).b Phase-contrast photomicrographs of keratinocytes derived from T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4. c Thin section of T21-KC at day 42 in iPGell. Hematoxylin and eosin (HE) stain. d Thin section of D21-KC#1 at day 50 and D21-KC#2 at day 48 in iPGell. HE stain. e Fabrication of D21-KC#1 epithelial sheet after dispase treatment. (Left panel) Histology of the sheet. HE stain. Scale bar, 100 μm. (Right panel) Macroscopic view of the sheet. Scale bar, 1 cm. f Immunohistochemical analysis of skin, HDK1-K4DT, T21-KCs at day 42, D21-KC#1 at day 50, and D21-KC#2 at day 48 with antibodies to pan-cytokeratins (Pan-CK), KRT14, P63, KRT10, integrin β4, involucrin, loricrin, filaggrin, and OCT3/4. D21-KC exhibited terminal differentiation and formed structured cell sheets. The expression patterns of these markers in intact skin are shown for reference. g Real-time qPCR analysis of epithelial markers (KRT14, TP63, Involucrin) and pluripotent markers (OCT3/4, NANOG) in T21-KC at day 47, D21-KC#1 at day 49, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. *p < 0.05",
"\nFig. 5\n5Three-dimensional (3D) cultured skin equivalents from D21-KC#1. a Histology of the 3D keratinocytes from D21-iPSCs. HE stain. Scale bar, 200 μm. b Immunohistochemical analysis of the expression and location of epithelial markers, i.e., KRT14, KRT10, involucrin, loricrin, integrin β4, and laminin 5. The expression patterns of these markers in intact skin are shown for reference. Scale bars, 200 μm"
] | [
"Keratinocytic differentiation of Down syndrome-iPSCs and autonomously rescued disomy 21-iPSCs. a Protocol for keratinocytic differentiation from amniotic fluid-iPSCs. DKSFM, defined keratinocyte serum-free medium; RA, retinoic acid; BMP4, bone morphogenetic protein 4; VTN, vitronectin; E8, Essential 8 medium; EGF, epidermal growth factor. b Phase-contrast photomicrograph of normal human epidermal keratinocytes (NHEK) at passage 3, and keratinocytes derived from T21-iPSCs (T21-KC), D21-iPSC#1 (D21-KC#1) and D21-iPSC#2 (D21-KC#2) at passage 2 (day 25). T21-KC, D21-KC#1, and D21-KC#2 exhibited keratinocyte-like morphology. Scale bars, 200 μm. c Immunocytochemical analysis of T21-KC at day 28 and D21-KC#1 at day 34 with the antibodies to epithelial markers, i.e., KRT14, KRT10, involucrin and loricrin. Scale bars, 100 μm. d Immunocytochemistry with the epithelial stem cell marker KRT14. The percentage of KRT-positive cells is shown for HDK1-K4DT (control), T21-KCs at day 56, D21-KC#1 at day 55, and D21-KC#2 at day 57. Scale bars, 100 μm. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni correction. e Real-time qPCR analysis of OCT3/4 in T21-KCs at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from three independent experiments. f Real-time qPCR analysis of NANOG in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of KRT14 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of KRT14 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.42). Values are shown as means ± SD from three independent experiments. h Realtime qPCR analysis of TP63 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of TP63 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.31). Values are shown as means ± SD from three independent experiments. i Real-time qPCR analysis of Filaggrin in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of Filaggrin was relatively high in T21-KCs, compared with D21-KCs (p-value, 0.34). Values are shown as means ± SD from three independent experiments. j Real-time qPCR analysis of KRT14 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. k Real-time qPCR analysis of TP63 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. l Growth of iPSC-derived keratinocytes. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10(2). Phase-contrast photomicrograph of D21-KC#1 at senescence is shown most cells of the stratified colonies and KRT10 at the periphery of the colonies",
"Sheet formation and characterization of keratinocytes derived from T21-and D21-iPSC. a Schematic diagram of the experimental design. Keratinocytes derived from iPSCs were cultivated under culture condition A followed by condition B for the purpose of propagation of the lifespan. As the growth rate decreased, the culture conditions were changed from A to B. In the culture condition A, keratinocytes were maintained in DKSFM supplemented with 20 ng/ mL EGF and 10 μM Y-27632 at a plate coated with type I collagen and fibronectin. In culture condition B, keratinocytes were cultured in the ESTEM-EP medium supplemented with 10 μM Y-27632 on irradiated mouse embryo fibroblasts (MEF).b Phase-contrast photomicrographs of keratinocytes derived from T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4. c Thin section of T21-KC at day 42 in iPGell. Hematoxylin and eosin (HE) stain. d Thin section of D21-KC#1 at day 50 and D21-KC#2 at day 48 in iPGell. HE stain. e Fabrication of D21-KC#1 epithelial sheet after dispase treatment. (Left panel) Histology of the sheet. HE stain. Scale bar, 100 μm. (Right panel) Macroscopic view of the sheet. Scale bar, 1 cm. f Immunohistochemical analysis of skin, HDK1-K4DT, T21-KCs at day 42, D21-KC#1 at day 50, and D21-KC#2 at day 48 with antibodies to pan-cytokeratins (Pan-CK), KRT14, P63, KRT10, integrin β4, involucrin, loricrin, filaggrin, and OCT3/4. D21-KC exhibited terminal differentiation and formed structured cell sheets. The expression patterns of these markers in intact skin are shown for reference. g Real-time qPCR analysis of epithelial markers (KRT14, TP63, Involucrin) and pluripotent markers (OCT3/4, NANOG) in T21-KC at day 47, D21-KC#1 at day 49, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. *p < 0.05",
"Three-dimensional (3D) cultured skin equivalents from D21-KC#1. a Histology of the 3D keratinocytes from D21-iPSCs. HE stain. Scale bar, 200 μm. b Immunohistochemical analysis of the expression and location of epithelial markers, i.e., KRT14, KRT10, involucrin, loricrin, integrin β4, and laminin 5. The expression patterns of these markers in intact skin are shown for reference. Scale bars, 200 μm"
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] | [] | [
"Down syndrome patients have an extra copy of chromosome 21, which causes health-related problems and agerelated disorders. Trisomy 21 results in the triplication of over 400 genes, which makes clarification of the precise mechanisms of some phenotypes difficult in Down syndrome [1]. The complications of Down syndrome are numerous; clinical manifestations include mental retardation, congenital heart disease, impaired cognition, premature aging, metabolic problems, and leukemia [2,3]. Trisomy 21 is also associated with an increased incidence of several dermatological conditions. Immunological disturbances and/or barrier dysfunction of skin are associated with psoriasis, atopic dermatitis, and cutaneous infections. Keratodermatoses and abnormality of elastic fibers also contribute to dermatological disorders such as anetoderma and keratosis pilaris in Down syndrome patients [4][5][6]. Genetic signatures of these dermatological diseases have been reported using keratinocytes derived from patient-specific iPSCs [7].",
"A Down syndrome mouse model exhibits hyperproliferation of trisomy 21-keratinocytes in the skin [8]. Cell culture models of trisomy 21 are important for development of conventional therapy for Down syndrome. However, few studies have focused on differences in epidermal characteristics between Down syndrome and euploid cells in vitro. Generation of disease-specific induced pluripotent stem cells (iPSC) allows an alternative paradigm for modeling human genetic disease in vitro [9,10]. Directed differentiation from these iPSC theoretically enables researchers to derive any given diseaseaffected human cell-type [11]. Disease-specific iPSCs, therefore, serve as a powerful tool for investigating the mechanisms of Down syndrome [9,12,13].",
"The differences in genetic backgrounds of individuals has hindered the assessment the phenotypic disparities between Down syndrome cells and healthy control cells. Paired disomic/trisomic iPSCs derived from the same trisomic individual by insertion of a transgene into a chromosome 21 has allowed investigation of trisomy 21dependent gene expression and the impact of an extra copy of chromosome 21 without the confounding effects of a different genetic background [13][14][15][16]. The artificial selection of disomic clones possibly results in epigenetic changes that add to the transcriptional differences between the trisomic and disomic iPSCs [17]. In the previous study, we have obtained revertant cells with normal chromosome 21 diploids from the trisomy 21 iPSCs during long-term cultivation [18]. Autonomously rescued disomic cells serve as a good control for trisomic cells. Both disomy 21 iPSC and trisomy 21 iPSC have been shown to differentiate into neural stem cells [18].",
"In this study, we successfully extended the lifespan of trisomy-rescued iPSC-derived keratinocytes to more than 34 population doublings over a period of 160 days with maintenance of keratinocytic phenotypes. In addition, keratinocytes derived from autonomous trisomy-rescued iPSCs regained capability of stratification and were suitable for manufacturing three-dimensional skin with keratinocytic function.",
"iPSCs from Down syndrome patients were cultivated as previously described [19]. Trisomy 21 iPSCs (T21-iPSCs) and rescued disomy 21 iPSCs (D21-iPSCs) were maintained in Essential 8 (E8) medium (Life Technologies, catalog number (#) A1517001) onto vitronectin (VTN) (Life Technologies, #A14700)-coated dishes and passaged using 0.5 mM EDTA in phosphate buffered saline (PBS) [18]. Normal human epidermal keratinocytes (LONZA, #192906) were also cultured in defined keratinocyte serum-free medium (DKSFM) (Invitrogen, #10744-019) supplemented with 10 μM Y-27632 on type I collagen (Advanced Biomatrix, #5005-B) and fibronectin (Sigma-Aldrich, #F0895-1MG)-coated dishes. HDK1-K4DT, which is a normal human keratinocyte line immortalized with TERT, a mutant form of CDK4 and cyclin D1 by lentivirus-mediated gene transfer, was cultured in keratinocyte serum-free medium (Invitrogen, #17005042, low calcium concentration) [20].",
"RNA was extracted from cells using the RNeasy Plus Mini kit (Qiagen, #74134). An aliquot of total RNA was reverse transcribed using an oligo (dT) primer (Invitrogen, #18418-020). For the thermal cycle reactions, the cDNA template was amplified (Applied Biosystems Quantstudio 12K Flex Real-Time PCR System) with gene-specific primer sets (see Additional file 1: Table S1) using the Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, #11733-046) under the following reaction conditions: 40 cycles of PCR (95°C for 15 s and 60°C for 1 min) after an initial denaturation (95°C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. mRNA levels were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. RT-qPCR analyses for expression of epithelial markers were showed as means of percent of GAPDH (%GAPDH) with the standard deviation (SD).",
"Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After washing with PBS and treatment with 0.1% Triton X-100 (Sigma-Aldrich, # T8787-100 ML) for 10 min at room temperature, the cells were incubated with Protein Block Serum-Free Ready-to-use (Dako, # X0909) for 30 min at room temperature. This was followed by reaction with primary antibodies in blocking buffer for 60 min at room temperature. After washing with PBS, the cells were incubated with fluorescently conjugated secondary antibodies; anti-rabbit or anti-mouse immunoglobulin G (IgG) bound to Alexa 488 or 546 (1:1,000) was incubated in blocking buffer for 30 min at room temperature. The nuclei were stained with DAPI (Biotium, # 40043). All images were captured using confocal microscopy (LSM 510 and LSM 510 MERA laser scanning microscope; Carl Zeiss, Germany). The fluorescent cells were quantified by hybrid cell counting using a digital microscope (BZ-X 710; Keyence Corp., Osaka, Japan). We measured the number of positive cells in at least six fields to obtain a total of more than 450 cells. Antibody information is provided in Additional file 2: table S2a.",
"Immunohistochemistry was performed as previously described [21]. Paraffin sections were deparaffinized and heated in antigen retrieval solution (Nichirei, #415211) for 20 min. After washing with PBS, samples were placed in 3% hydrogen peroxide/methanol for 5 min to block endogenous peroxidase. The sections were then incubated at room temperature for 90 min in primary antibodies diluted with antibody diluent. Antibody information is provided in Additional file 2: table S2b. The sections were then washed three times with PBS and incubated with peroxidase-labeled goat anti-mouse or anti-rabbit antibodies (Nichirei, #424151) at room temperature for 45 min. After washing with PBS, they were incubated in 3,3′diaminobenzidin (Muto pure chemicals, #40651) for 5-10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.",
"The induction of differentiation into keratinocytes was carried out as previously described [19]. We subcultured small clumps of undifferentiated iPSC on VTN-coated 100-mm dish in E8 medium on day 0. iPSCs were cultured in DKSFM (Invitrogen, #10744-019) supplemented with 1 μM all-trans RA (Wako, #182-01111) and 10 ng/mL bone morphogenetic protein 4 (BMP4) (R&D systems, #314-BP-010/CF) from day 1 to day 4. After 4 days, iPSCs were maintained in DKSFM supplemented with 20 ng/mL epidermal growth factor (EGF) (R&D systems, #236-EG-200) for 10 days, then passaged to a 100-mm dish coated with 0.03 mg/mL type I collagen (Advanced Biomatrix, #5005-B) and 0.01 mg/mL fibronectin (Sigma-Aldrich, #F0895-1MG), and maintained in DKSFM supplemented with 20 ng/mL EGF and 10 μM Y-27632 (Wako, #251-00514). Cells were subcultured when approximately 80 to 90% confluence. Cells were rinsed with PBS and incubated with 0.25w/v% trypsin-1mmol/L EDTA (Wako, #209-16941) at 37°C for 5 min for passaging. Cells were passaged at 0.3 × 10 5 cells/cm 2 to a new plate coated with type I collagen and fibronectin and enriched by rapid adherence to fibronectin and type I collagen-coated dished for 15 min at 37°C. Nonadherent cells were removed and rapidly attached cells were cultured. The medium was changed every 2 or 3 days. All cells were maintained at 37°C in a humidified incubator with 5% CO 2 .",
"For proliferation of iPSC-derived keratinocytes, we employed specific culture conditions suitable for keratinocytes [22][23][24].",
"iPSC-derived keratinocytes were cultured in ESTEM-EP medium (GlycoTechnica Ltd., Japan) [Dulbecco's modified Eagle's medium (DMEM) +F12 (3:1) medium supplemented with FBS, hydrocortisone, adenine, EGF, cholera toxin, insulin, A83-01, nicotinamide, SB202190, Y-27632, and penicillin/streptomycin] onto irradiated mouse embryo fibroblasts (MEF). The medium was changed every 2 or 3 days until the cells were reached 80 to 90% confluence. Cells were passaged at 0.1-0.25 × 10 5 cells/cm 2 to a new plate of irradiated MEF feeder cells. The cell sheets were retrieved from the dish with 10 mg/mL dispase (Wako, #383-02281) or harvested using a cell scraper. The sheets in iPGell (Genostaff, #PG20-1) were prepared for immunohistochemistry. The cell numbers were recorded at each passage using the Countess automated cell counter and population doublings were determined.",
"Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Coculture of iPSC-derived keratinocytes and MEF under culture condition B were rinsed with PBS and incubated with 10 mg/mL dispase at 37°C for 5-8 min, with close monitoring by phase microscopy. When non-keratinocytes began to detach by tapping the dish, dispase was inactivated, and the solution containing non-keratinocytes and feeder cells were removed by aspiration. Stratified colonies remained tightly adherent. The remaining colonies were rinsed with PBS and incubated with trypsin-EDTA solution at 37°C for about 3 min. The cells were passaged to a new plate of irradiated MEF feeder cells at a density of 0.1-0.25 × 10 5 cells/cm 2 .",
"Three-dimensional skin was generated according to a previously described protocol [25]. Type I collagen (Koken, #IPC-50) and 1 × 10 6 human foreskin fibroblasts (HFFs) were mixed and poured into an untreated 60-mm Petri dish (Falcon, #351007) while cooling and allowed to gel at 37°C for 1 h to prepare the dermal equivalent. The collagen gel was detached from the surface of the Petri dish, and the contraction of the gel was facilitated. The medium was changed every 2 or 3 days for 7 days. iPSC-derived keratinocytes were plated at 0.2 × 10 6 cells inside in a glass ring (IWAKI, #RING-12) on the surface of the contracted collagen gel, which was plated onto polyethylene terephthalate membranes (Corning, #35-3493). iPSC-derived keratinocytes were grown in ESTEM-EP medium for 1 day, following which they were exposed to air in a 1:1 mixture of ESTEM-EP medium and DMEM supplemented with 10% FBS (medium for HFFs), in which the Ca 2+ concentration was adjusted to 0.9 mM. The medium was changed every 2 or 3 days. Multilayered 3D cultures of keratinocytes were obtained by day 14.",
"The results are expressed as means with SD. Comparisons between the two groups were evaluated with the unpaired Student's t test. For three groups, a one-way analysis of variance (ANOVA) with a Bonferroni correction was used. Statistical analysis was performed using Stata 14.0 (StataCorp LP, College Station, Texas, USA). P values less than 0.05 were considered to be statistically significant.",
"Rescued disomy 21 iPSCs, but not trisomy 21 iPSCs, are likely to differentiate into keratinocytes",
"We employed a standard protocol for differentiation of T21-iPSCs and D21-iPSCs into keratinocytes (Fig. 1a). The differentiated cells derived from T21-and D21-iPSCs, i.e., keratinocyte progenitor cells, resembled normal human epidermal keratinocytes (NHEK) at passage 2 ( Fig. 1b). Gene expression analysis was performed using iPSC-derived keratinocytes at passage 2 and/or passage 3. Immunostaining analysis revealed that both keratinocytes derived from T21-iPSC (T21-KC) and D21-iPSC#1 (D21-KC#1) were positive for epithelial markers, i.e., KERATIN 14 (KRT14), KRT10, involucrin, and loricrin (Fig. 1c). T21-KCs at passage 5 had a significantly low number of KRT14-positive cells, compared to HDK1-K4DT and D21-KC at passage 5 (Fig. 1d). The number of Involucrin-positive cells in both T21-and D21-KCs was low, compared with NHEK (Fig. 1c). T21-KCs also contained non-keratinocytes such as iPSC-like colonies and cells and exhibited loss of nuclear-cytoplasmic boundaries at passage 3 (see Additional file 3: Fig. S1A). It was extremely difficult to maintain T21-KCs with keratinocyte morphology. In contrast, D21-KC#1 and #2 showed keratinocytic morphology and KRT14 reactivity in most cells, like NHEK cells ( Fig. 1c; Additional file 3: Fig. S1B). The expression of the OCT3/4 and NANOG genes was significantly suppressed after differentiation of T21-iPSCs and D21-iPSCs (Fig. 1e, f). The basal cell marker KRT14, the epithelial progenitor marker TP63, and the keratinizing cell marker Filaggrin were induced in T21-KC and D21-KC#1 ( Fig. 1g-i). The expression of KRT14 and TP63 was relatively increased in D21-KCs, compared with T21-KCs (Fig. 1g, h). Filaggrin expression was relatively high in T21-KCs (Fig. 1i). The expression of KRT14 and TP63 were unchanged in D21-KC#1 (Fig. j, k). T21-KC, D21-KC#1, and D21-KC#2 had keratinocytic morphology stopped dividing at 4 to 5 population doublings (PDs) (Fig. 1l). These results imply that T21-and D21-iPSCs have a distinct propensity for keratinocytic differentiation.",
"A combination of serum, Y-27632, and feeder cells allows propagation and sheet formation of D21-KCs",
"To escape cell senescence and form epidermal sheets, we altered the cultivation conditions (Fig. 2a). T21-KCs, D21-KC#1, and D21-KC#2 recovered the ability to grow and proliferated as colonies on MEFs (Fig. 2b). The differentiated cells under these culture conditions were defined as mature keratinocytes. We analyzed these cells 1-2 weeks after the change in cultivation conditions. T21-KCs showed multilayer structures with a high nucleus/cytoplasm (N/C) ratio and chromatin aggregation (Fig. 2c). In contrast, D21-KC#1 and D21-KC#2 cells showed an epithelial structure with basal, spinous, and granular layers (Fig. 2d). Uniformly thick multilayersheets were obtained by dispase treatment (Fig. 2e). Immunostaining of D21-KC stratified epithelium revealed layer-dependent reactivity to KRT14, basement membrane integrin β4, the suprabasal marker KRT10, and spinous and granular layer markers involucrin (Fig. 2f). Loricrin, a major component of granular layers, was also detected in the KRT14-negative cells. In contrast to D21-KC#1 and D21-KC#2 cells, T21-KC expressed OCT3/4 in some cells and failed to express KRT10 and KRT14. These results suggest that the autonomously rescued disomy 21 keratinocytes produced epithelial structures. Along with the immunocytochemical analysis, RT-qPCR analysis revealed a lack of expression of epithelial markers KRT14, TP63, and Involucrin in T21-KCs (Fig. 2g). D21-KC#1, but not T21-KCs, significantly increased in the epithelial markers after the change in culture conditions. These significant increases in keratinocytic marker expression are probably due to culture condition B, which is suitable for keratinocyte proliferation and maturation (Fig. 2a). The expression of OCT3/4 and NANOG diminished in both T21-and D21-KCs. Cytological analysis showed that T21-KCs, D21-KC#1, and D21-KC#2 treated with condition A showed variable cell sizes with a low N/C ratios (see Additional file 4: Fig. S2).",
"Trisomy 21-and rescued disomy 21-iPSCs escape from senescence for more than 160 days",
"The proliferative capability of T21-KC, D21-KC#1, and D21-KC#2 was restored after the change in cultivation conditions (Fig. 3). T21-KC cells continued to proliferate rapidly and had fewer cells that showed keratinocytic morphology. In contrast, D21-KC#1 and D21-KC#2 exhibited stable growth and maintained keratinocytic morphology for a period of more than 160 days. D21-KC#1 and D21-KC#2 showed growth cessation at 34 and 52 PDs, respectively. The reason for the differences in growth cessation is unclear but may be related to the differentiation competency of these cells.",
"After long-term cultivation, D21-KC#1 cell cultures contained both keratinocytes and non-keratinocytes (Fig. 4a). (Fig. 4b).",
"To separate keratinocytelike cells from non-keratinocyte-like cells, we used differential reactivity of these cells to dispase, i.e., tight or loose cell-dish adhesion: Stratified colonies are relatively resistant to dispase and non-keratinocytes are easily detached after dispase treatment (Fig. 4c). This treatment was applied to D21-KC#1 cells at passage 7 to passage 9 (Fig. 4d). The ratio of keratinocytes increased at each passage by microscopic observation with a phase-contrast microscope. KRT14 increased, TP63 decreased, and Involucrin remained unchanged at each passage ( Fig. 4e-g). D21-KC#1 was immunocytochemically positive for epidermal markers, i.e., KRT14, KRT10, involucrin, and loricrin, at passage 13 (Fig. 4h). D21-KCs were able to reproducibly generate epidermal sheets (Fig. 4i). T21-KCs became fragmented upon dispase treatment; thus, we decided not to use dispase for keratinocytic purification. We picked up colonies with keratinocyte morphology at each passage from passage 6 to passage 8 (see Additional file 5: Fig. S3A, B). Keratinocyte-like cells were microscopically observed at passage 8; however, these cells did not express KRT14 and TP63 and epithelial markers were absent (Fig. 4e, f, h). The lack of keratinocyte marker expression may be caused by a gene dosage imbalance due to trisomy 21.",
"The dispase-based selection at each passage helped maintain the proliferation of KRT14 positive cells and stratified cell sheets were manufactured in plate cultures of D21-KC#1. We therefore investigated whether disomy 21 iPSC-derived keratinocytes could manufacture 3D skin. To this end, we generated artificial dermis by combining human cultured dermal cells and type I collagen in a Petri dish, and overlaid D21-KC#1 cells in a glass ring. D21-KC#1 cells on the artificial dermis were then cultivated in an air-liquid interface for 2 weeks to accelerate epidermal differentiation. The 3D skin from D21-KC#1 was generated with irregular layer formation and expressed KRT14, KRT10, involucrin, loricrin, and integrin β4 (Fig. 5a, b). Laminin 5 was not detected at the dermal-epidermal junction.",
"Differentiation incompetence in T21-iPSCs and its restoration in D21-iPSCs",
"Restoration of differentiation and growth of trisomyrescued iPSCs is a focus of our laboratory. This restoration or normalization may simply be attributable to a gene dosage effect, i.e., a change from 3 (aneuploid) to 2 copies (euploid). The Down syndrome phenotype is generally derived from the increased expression levels of dosagesensitive genes, and most of the triplicated genes show upregulated expression that is compatible with gene dosage (i.e., at close to 1.5-fold increase) [15,26]. The expression levels of the genes for APP (Alzheimer's disease marker), DYRK1A, DSCR1 (Down syndrome critical region 1), ETS2, and SOD1, all of which are located on chromosome 21, are dysregulated in trisomic cells. Although a gene dosage imbalance is the main molecular mechanism, extensive dysregulation of euploid genes are associated with Down syndrome phenotypes [26,27]. An extra copy of chromosome 21 also alters gene expression across every chromosome, not just chromosome 21 [9,28]. The altered expression levels decrease to normal levels in trisomyrescued undifferentiated iPSCs; in other words, revertant cells regain the gene expression levels of intact iPSCs [18]. These normalized gene expression levels may restore keratinocytic differentiation capability in trisomy-rescued iPSCs. The detailed mechanism of keratinocytic restoration in trisomy 21 cells remains unclear, and additional analysis may elucidate the mechanism underlying epidermal abnormalities in Down syndrome. The ability of trisomy 21-stem/progenitor cells to differentiate also depends on the target organs or cells [14,15,29,30]. The downregulated genes in T21-iPSCs reveal significant enrichment for genes involved in embryonic and tissue morphogenesis [15]. In addition, differentiated ectoderm germ structures are not found in T21-iPSC-derived teratomas [31]. In another study, lack of pluripotency in T21-iPSCs with reduced expression of HERVH might explain the lack of T21-iPSC differentiation compared to disomic iPSCs [15]. These reports may explain the inability of to T21-iPSCs differentiate, i.e., a lack of KRT14 marker and the partial expression of OCT3/4 in T21-KCs, and increased population of nonkeratinocytes after the long-term culture. It is noted that keratinocytic differentiation of D21-iPSCs was achieved at Fig. 3 Differentiating cells from T21-iPSCs and the rescued disomy (D21)-iPSCs shows proliferative capability for more than 160 days. The numbers of cells were recorded at each passage using an automated cell counter and population doublings were determined. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10 (2) from two or three independent experiments passage 106. This implies that the differentiation capability of trisomy-rescued cells is extremely stable for a period of more than 500 days.",
"Accelerated growth of differentiated cells from T21-iPSCs into keratinocytes was unexpected because the incidence of solid tumors, such as dermatological tumors, is decreased in Down syndrome patients [32,33]. Candidate genes such as DYRK1A, PIGP, and RCAN1 are related to keratinocyte hyperproliferation in a Down syndrome mouse model [8]. Triplication of Usp16, located on chromosome 21, reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts [34]. Fig. 4 Purification of iPSC-derived keratinocytes, based on differential reactions to dispase. a Phase-contrast photomicrographs of keratinocytes derived from D21-KC#1 at passage 5. Two types of colonies could be observed: stratified keratinocyte colonies (left) and non-keratinocyte-like cell colonies (right). Scale bars, 200 μm. b Immunocytochemical analysis of D21-KC#1 colony with epithelial markers, i.e., KRT14 and KRT10. Scale bars, 200 μm. c Selection of keratinocytes with dispase digestion. Non-keratinocytes (non-KC) detached earlier than keratinocytes with dispase treatment. Nonkeratinocytes and feeder cells were removed at each passage. Keratinocytes were then passaged. d Phase-contrast photomicrographs of D21-KC#1 during the dispase selection. There were insular colonies, which were formed by stratified epithelial cells, and another area contained non-keratinocytelike cells. Dispase-based selection at each passage caused non-keratinocyte-like cells to decrease. Scale bars, 500 μm. e Real-time qPCR analysis of KRT14 at each passage. Values are shown as means ± SD from two or three independent experiments. f Real-time qPCR analysis of TP63 at each passage. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of Involucrin at each passage. Values are shown as means ± SD from two or three independent experiments. h Immunocytochemical analysis of epithelial markers, i.e., KRT14, KRT10, involucrin, and loricrin after colony isolations (T21-KC) at passage 7 and dispase-based selection (D21-KC#1) at passage 13. Scale bars, 100 μm. i Epithelial sheets of keratinocyte-like cells derived from D21-iPSC#1 at passage 8. Cell sheets were harvested by dispase treatment. Scale bar, 1 cm Overexpression of patched-1, a receptor that represses the mitogenic sonic hedgehog pathway, may be associated with proliferation impairment in skin [35][36][37][38]. Moreover, proliferation of trisomy 21-fibroblasts and trisomy 21-iPSCs decrease due to oxidative stress and protein aggregation [39,40]. Neurogenesis-related genes such as DYRK1A and RCAN1, craniofacial defect-related genes such as DYRK1A, ETS2, and RCAN1, and tumor suppressor-related genes such as DYRK1A, ETS2, and RCAN1 were also reported in Down syndrome [26]. In our study, an increased growth rate, an increased nucleus/cytoplasm ratio, and aggregated chromatin of T21-KCs may have resulted from susceptibility to growth stimulation. Skin tumor growth and hyperkeratosis were detected in Ts1Rhr mice model of Down syndrome [8]. We have also reported accelerated growth of T21derived neural stem cells [18]. These increases in growth of other cell types are indeed compatible with that of T21-KCs in this study.",
"Psoriasis is one of the dermatological abnormalities observed in Down syndrome and is characterized by hyperproliferation and defective differentiation of keratinocytes. Altered gene expression such as upregulation of IFN genes is linked to hyperproliferation of KCs derived from psoriasis patient-specific iPSCs [7]. The autoimmune abnormalities and altered sensitivity to stimuli in Down syndrome may be linked to the excessive proliferation of T21-KCs and reduced differentiation of T21-iPSCs.",
"In addition to growth rate, senescence or aging of iPSC-derived keratinocytes is a concern. Human primary keratinocytes in serum-free and chemically defined media senesce around 15-20 PDs [41,42]. iPSC-derived keratinocytes exhibit growth arrest when cultured in feeder-free, serum-free medium containing EGF and Y-27632 around passage 4 or 5 [19]. Combination of Rho kinase inhibitor and feeder cells induces conditional reprograming and immortalization of human epithelial cells without the use of viral infection or genetic modification [22][23][24]. In line with these reports, D21-KC survival and proliferation was achieved with a combination of a Rho kinase inhibitor and feeder cells. The successful extension of the D21-KC lifespan to more than 34 population doublings over the period of 160 days is surprising, since extended lifespan of T21-KCs was not accompanied by keratinocytic phenotypes. The trisomy-rescued D21-KCs with a longer lifespan have the ability to stratify, which is essential for manufacturing 3D skin with keratinocytic functions.",
"To study the impact of trisomy 21 on keratinocytic function, we provide an iPSC-derived model using trisomy 21 iPSC and autonomous trisomy-rescued iPSCs. Our results suggest that there is impairment in keratinocytic differentiation in trisomy 21 iPSCs. In contrast, propagation of trisomy-rescued iPSC-derived keratinocytes with purification and manufacturing of stratified epithelial sheets and 3D skin imply a restoration of keratinocytic functions. Further investigation of the influence of the extra copy of chromosome 21 may help determine the underlying causes of Down syndrome phenotypes and lead to the generation of a Down syndrome model of dermatological abnormalities. "
] | [] | [
"Background",
"Methods",
"Cells",
"Real-Time qPCR",
"Immunocytochemical analysis",
"Immunohistochemical analysis",
"Differentiation of iPSCs into keratinocytes",
"Propagation of keratinocytes derived from iPSCs",
"Dispase-based keratinocytic selection",
"Generation of a 3D skin equivalent",
"Statistical analysis",
"Results",
"Dispase-based purification of iPSC-derived keratinocytes improves epidermal sheet formation",
"D21-KC#1 keratinocyte-like cells expressed KRT14 in",
"3D skin can be manufactured from disomy 21 iPSCderived keratinocytes",
"Discussion",
"Uncontrolled growth of T21-KCs and normalized growth of D21-KCs",
"Conclusion",
"Fig. 1",
"Fig. 2",
"Fig. 5"
] | [] | [
"Table S1"
] | [
"Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells",
"Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells"
] | [] |
232,016,698 | 2022-01-15T02:47:08Z | CCBYNCND | https://www.jstage.jst.go.jp/article/tox/34/1/34_2020-0061/_pdf | GOLD | e9bcbe1552d1579d01704b25e47707ffad434c09 | null | null | null | null | 10.1293/tox.2020-0061 | null | 33627948 | 7890165 |
Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma
2021
Yasuhiro Shimada
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Tomoharu Naito
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Takuo Hayashi
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Tsuyoshi Saito
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Yoshiyuki Suehara
Department of Orthopaedic Surgery
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Chihaya Kakinuma
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Yuji Nozaki
Biotechnical Center
SLC, Inc
3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan
Hisayoshi Takagi
Biotechnical Center
SLC, Inc
3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan
Takashi Yao
Department of Human Pathology
School of Medicine
Juntendo University
1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan
Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma
J Toxicol Pathol
34202110.1293/tox.2020-0061Original Articlepatient-derived xenograft (PDX)pleomorphic leiomyosarcoma (PLMS)mouse model
Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas. (
Introduction
Cell-based assay systems are the primary tools used for drug discovery owing to their simplicity, which facilitates assessment of the effects of drugs on cells cultured in vitro. Although in vivo tumor models established using tumor cell lines obtained from immunodeficient mice (nude and SCID) have been used to mimic human cancer pathology 1 , in many cases, the cell characteristics and tumor formation differ from those of the original tumor, owing to the different microenvironments, including the immune system and cancer stroma in host animals [2][3][4] . Therefore, translational studies that use patient-derived tissues are required for the development of more effective cancer drugs. Some patient-derived xenograft (PDX) models have been established on the basis of this consideration 5 . These models are increasingly being used as tools for drug development in pre-clinical settings and have been shown to recapitulate the histology and behavior of the cancers from which they are derived 6 . Furthermore, studies conducted to aid clinical decision-making have shown high concordance between individual PDX and patient responses to therapy 7 . Although these findings are encouraging, the role of this approach in sarcoma-derived models and in the context of genomic drug-matching strategies remains undefined. This has thus created an opportunity to evaluate the utility of PDX models as clinical predictors to direct the use of chemo-and targeted therapies in combination with comprehensive genomic and epigenetic analyses for patients with sarcomas. Moreover, the National Cancer Institute recommends the screening of anti-cancer drugs using PDX models, given that unlike cell lines, the tissue structure and gene expression patterns of PDX models more closely resemble those of patients 8 . Thus, PDX models are predicted to emerge as the mainstream approach for drug discovery, and represent a valuable support tool for investigator-initiated clinical trials.
However, owing to the low efficiency of engraftment on host tissue and slow growth rate 9, 10 , a PDX model suitable for all tumor types, particularly sarcomas, is currently unavailable. In this study, we aimed to establish a PDX model for certain types of sarcoma, using SCID mice, for drug discovery. We describe the development of a novel PDX model of pleomorphic leiomyosarcoma (PLMS), a morphological variant of leiomyosarcoma with aggressive clinical behavior 11 , which shows histological and immunohistological features similar to those of the original PLMS in mice. Our case model may aid in elucidating the biological characteristics of PLMS and in developing novel candidate drugs for the treatment of this type of sarcoma.
Materials and Methods
Preparation of patient-derived tumor samples
Tumor tissue samples were obtained with informed consent from a 71-year-old man who underwent surgery at the Juntendo University Hospital. The tumor specimens originating from his thigh were preserved in minimum essential medium (Cat. #51200038; GIBCO, Grand Island, NY) supplemented with antibiotics (penicillin) under refrigerated conditions. The specimens were subsequently washed twice in Dulbecco's modified Eagle's medium without fetal bovine serum and minced at 1 mm 3 per sample block on ice using a razor before transplantation into the mice. This study was approved by the ethics committee of Juntendo University (approval code: 2017040).
Animals
C.B-17/IcrHsd-Prkdc scid mice (Japan SLC, Shizuoka, Japan) were obtained at 6 weeks of age and maintained under specific pathogen-free conditions. The mice were used for the experiments at 7 weeks of age. All experiments and procedures for the care and treatment of the animals used in the present study were performed in accordance with the requirements of the Juntendo University Animal Care and Experimentation Committee (Experimental Protocol: No. 2017040; Juntendo University, Tokyo, Japan) and the SLC Animal Care and Experimentation Committee (Experimental Protocols: No. BT18098 and No. 18099).
Preparation of a PDX mouse model
Twenty mice were subcutaneously transplanted with tumor tissues on day 0. Tumor specimens were subcutaneously injected into the right flank region of each animal using a transplant needle (φ2.5 × L85 mm, Cat. #KN-391-25; Natsume Seisakusho, Tokyo, Japan). The tumor diameter and body weight were measured twice per week. To calculate the tumor volume, both long and short diameters (mm) were measured using a Solar Digital Caliper (Cat. #1-3255-02; AZ-One, Osaka, Japan). The formula used to calculate tumor volume was as follows: tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) × 0.5. The volumes of tumor were measured until reaching 2,000 mm 3 , in line with experimental ethics. Animals with moribundity [marked reduction in body weight, hypothermia, significant drop in temperature, and significant exhaustion (crouching position)] were euthanized immediately by bloodletting under isoflurane anesthesia (induction: 4.0%; maintenance: 1.0-3.0%) without any other pain 12 . Prolifer-ated tumor nodules were collected at the time of autopsy.
Histological examination
All removed tissues were fixed in 10% neutral buffered formalin. Tissue slices were routinely processed for paraffin embedding and sectioned at 3 μm thickness. The sections were stained with hematoxylin and eosin and observed under a light microscope.
Immunohistochemistry (IHC)
The PDX specimens were fixed with 10% formaldehyde, embedded in paraffin blocks, cut into 4-μm-thick sections, and mounted on glass slides. Staining of the sections was performed following standard methods described previously 13 . The sections were then incubated overnight at 4°C and thereafter at room temperature for 30 min with mouse monoclonal anti-Caldesmon (clone: h-CD; Dakopatts, Glostrup, Denmark), anti-human muscle actin (M-actin) (clone: HHF35; Dakopatts), or anti-smooth muscle actin (SMA) (clone: 1A4; Dakopatts) antibodies at dilutions of 1:1, 1:100, and 1:200, respectively, in phosphate-buffered saline containing 1% bovine serum albumin.
Results
Establishment of a soft tissue sarcoma PDX model
In our experiments, three of the 10 soft tissue tumor specimens derived from the patient were subcutaneously engrafted on the mice, one of which subsequently proliferated, with the size of the tumor increasing at each passage (Fig. 1). This soft tissue tumor was identified as a type of high-grade PLMS. Most of the transplanted mice showed massive tumor growth and enlargement (Fig. 1). Moreover, tumor progression continued to persist at each passage. In contrast, the PDX tumor models displayed enhanced growth rate with successive in vivo passages. Following transplantation, the time until harvesting decreased from 55 days at passage 0 to 50 days at passage 1, 40 days at passage 2, and 35 days at passage 3 ( Fig. 1). Finally, after re-transplantation, all the tumor foci proliferated and their volumes increased.
Histological features of the patient's tumor
Histologically, the tumor was characterized by fascicular proliferation of spindle-shaped cells. Tumor cells with anomalous giant nuclei were also scattered throughout the lesion ( Fig. 2A). IHC revealed that the tumor cells were positive for SMA (Fig. 2B). Additionally, these tumor cells showed an MIB-1 index of approximately 80%. On the basis of the histological features and IHC findings, the patient was diagnosed as having a PLMS.
Histopathological characteristics of the original PLMS were maintained at each passage in mice
The storiform pattern and fibrotic elements of the patient-derived PLMS were observed at all passages in transplanted mice. The nuclei showed atypical anisokaryosis, nuclear pleomorphism with coarse chromatin, and mitotic figures, and the tissues were also characterized by small foci of fascicles consisting of smooth muscle tumor cells. A mucoid matrix was also found in the same field. The tumor tissues in mice sampled at each successive passaged showed no significant differences in structure (Fig. 3, upper panel). Therefore, structural characteristics at the third passage were histologically similar to those of the primary patientderived tumors (Fig. 3, upper panel).
Immunohistochemical analysis of PDX tumor samples at each passage
PDX tumor samples obtained at each passage were immunologically stained for Caldesmon, M-actin, and SMA to confirm differentiation to smooth muscle in the original patient-derived leiomyosarcoma tumor. We observed that the PDX tumor was derived from the leiomyosarcoma, as indicated by the positive staining for all smooth musclespecific markers (Fig. 3). In particular, positive staining for SMA was diffuse and significant (Fig. 3, lower panel). With respect to the expression in PDX tumors through several passages, we detected no significant differences in the levels of Caldesmon, M-actin, and SMA staining among multiple passages (data not shown).
Discussion
Although in vivo sarcoma PDX models represent promising tools for drug development and elucidation of underlying pathological mechanisms, it is difficult to establish sarcoma models in vivo. owing to the low reproducibility of tumor restructuring and the significantly low rate of tumor engraftment in experimental mice 9,10 . Although our PDX mouse model also showed a low rate of tumor engraftment (approximately 10%), we succeeded in establishing a PDX derived from a very rare type of tumor, PLMS. We believe that the following procedural details may have contribute to our success in establishing the PLMS PDX model. 1. Following surgery, the specimens were optimally processed prior to implantation in the mice. This optimization shortened the timeline from surgery to implantation of the specimen in mice, thereby contributing to specimen preservation. 2. The specimens were rapidly preserved during cold acclimation under refrigeration and optimal medium conditions. 3. The specimens that underwent rapid washout and cutting comprised an optimum tumor tissue for engrafting onto the subcutaneous site in the mice. Collectively, these procedures would have contributed to the prevention of sample degradation and maintenance of high cell viability. Whereas tumor tissue bricks characterized by a tumor microenvironment with scaffold stroma ensure successful engraftment of sarcoma specimens on xenograft models that mimic the characteristics of PLMS, it is also conceivable that high malignancy contributes to the success rate of PDX engraft-ment. Although the detailed mechanisms have yet to be sufficiently clarified, we hypothesize that the high frequency of cell division and proliferation in this type of sarcoma is conducive to good engraftment and time-dependent tumor growth in mice.
Microscopically, conventional leiomyosarcomas show a characteristic histology of smooth muscle differentiation with a low to intermediate grade potential for malignancy. In contrast, PLMS shows histological features of high-grade spindle cell sarcoma, such as variable, large, and atypical cells, and multinuclear giant cells, whilst partially retaining the features of conventional LMS, and also show distinct myogenic differentiation, as confirmed by IHC 11 . Although pleomorphic and conventional leiomyosarcomatous areas are typically intermixed within a tumor, the demarcation may be distinct or gradual in some cases 14 . Furthermore, it has been reported that PLMS contain myxoid or focally collagenous stroma, which resemble those of storiformpleomorphic undifferentiated sarcomas 15 . The characteristics of the PDX in our model were closely comparable to the typical characteristics of PLMS, thereby confirming the establishment of an original tumor-like PLMS PDX model. However, the tumor growth rate and commencement of tumor proliferation were clearly accelerated on tumor passaging in each mouse model. In this regard, it is conceivable that a tumor microenvironment characterized by the incorporation of interstitial stroma and host animal angiogenesis created an isolated niche for human tissue-resident sarcoma foci that favored tumor survival and proliferation at successive passage. Accordingly, tumors might be adopted in the host environment and undergo immune accommodation as a foreign body. In contrast, tumor-specific marker expres- sion and histological structure remained unchanged. Therefore, the important point to note would be the tumor niche environment for tumor growth changes. Improvements in tumor engraftment and growth rate are major factors necessary for the establishment of a rare cancer PDX model. Although the specific conditions required for PDX remain unclear, we speculate that establishing a basal scaffold will be essential for enhancing human carcinogenesis in future xenograft models.
In summary, we succeeded in developing a PLMS PDX mouse model that mimicked the original human tissue structure at the subcutaneous site. Patients with PLMS have poor prognoses, and novel systemic therapies are being investigated to improve the survival of afflicted patients. We anticipate that our PDX model will make a valuable contribution to future drug development for PLMS through drug efficacy screening.
Disclosure of Potential Conflicts of Interest:
There are no known conflicts of interest associated with this study.
Fig. 1 .
1Graph showing tumor volume after transplantation at each passage. Autopsy tumor samples obtained from the patient were minced (1 mm 3 /tumor piece) and subcutaneously transplanted into SCID mice (first inoculation: passage 0). After some tumors had reached a volume of 2,000 mm 3 , the tumor nodule was removed and sub-transplanted into the next normal SCID mice (passage 1). Sub-transplantation was conducted continually at passage 3. Volumetric measurements of xenografted tumors were performed using an external caliper, as described in the Materials and Methods section. Tumor nodules were collected at the time of necropsy and subjected to histological and immunohistochemical analyses.
Fig. 2 .
2Histological characteristics of the tumor derived from the patient. The tumor was characterized by fascicular proliferation of spindleshaped cells with scattered giant cells (A). Immunohistochemically, the tumor cells were positive for myogenic markers, including smooth muscle actin (SMA) (B). The black bar indicates 100 μm.
Fig. 3 .
3Histological and immunohistochemical analyses of a patient-derived xenograft (PDX) at each passage. Tumor nodules were fixed in formalin-containing bottles, and tumor sections were prepared on slides. The slides were stained with hematoxylin and eosin (H&E) solution, as described in the Materials and Methods section. All sections containing tumor tissue were microscopically examined, and representative images at each passage are shown (upper panels). The expression of Caldesmon (CALD, CDM), M-actin, and smooth muscle actin (SMA) in tumors collected from recipient mice engrafted with PDX were measured using immunohistochemical staining. Staining for SMA is shown at each passage (lower panels). The black bar indicates 100 μm for sections stained with H&E, whereas the white bar indicates 50 μm for sections stained with SMA.
Acknowledgment:We are grateful for the considerable support provided by Dr. Tamami Higuchi.
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Current and future horizons of patient-derived xenograft models in colorectal cancer translational research. Cancers (Basel). A Inoue, A K Deem, S Kopetz, T P Heffernan, G F Draetta, A Carugo, 10.3390/cancers11091321E1321. 201911Cross-RefInoue A, Deem AK, Kopetz S, Heffernan TP, Draetta GF, and Carugo A. Current and future horizons of patient-de- rived xenograft models in colorectal cancer translational re- search. Cancers (Basel). 11: E1321. 2019. [Medline] [Cross- Ref]
Examining the utility of patient-derived xenograft mouse models. S Aparicio, M Hidalgo, A L Kung, 10.1038/nrc3944Nat Rev Cancer. 15CrossRefAparicio S, Hidalgo M, and Kung AL. Examining the util- ity of patient-derived xenograft mouse models. Nat Rev Cancer. 15: 311-316. 2015. [Medline] [CrossRef]
Translational value of mouse models in oncology drug development. S E Gould, M R Junttila, F J De Sauvage, 10.1038/nm.3853Nat Med. 21CrossRefGould SE, Junttila MR, and de Sauvage FJ. Translational value of mouse models in oncology drug development. Nat Med. 21: 431-439. 2015. [Medline] [CrossRef]
Patient-derived xenograft models: an emerging platform for translational cancer research. M Hidalgo, F Amant, A V Biankin, E Budinská, A T Byrne, C Caldas, R B Clarke, S De Jong, J Jonkers, G M Maelandsmo, S Roman-Roman, J Seoane, L Trusolino, A Villanueva, 10.1158/2159-8290.CD-14-0001Cancer Discov. 4CrossRefHidalgo M, Amant F, Biankin AV, Budinská E, Byrne AT, Caldas C, Clarke RB, de Jong S, Jonkers J, Maelandsmo GM, Roman-Roman S, Seoane J, Trusolino L, and Vil- lanueva A. Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov. 4: 998-1013. 2014. [Medline] [CrossRef]
US cancer institute to overhaul tumour cell lines. H Ledford, 10.1038/nature.2016.19364Nature. 530391CrossRefLedford H. US cancer institute to overhaul tumour cell lines. Nature. 530: 391. 2016. [Medline] [CrossRef]
Cancer cell lines for drug discovery and development. J L Wilding, W F Bodmer, 10.1158/0008-5472.CAN-13-2971Cancer Res. 74CrossRefWilding JL, and Bodmer WF. Cancer cell lines for drug dis- covery and development. Cancer Res. 74: 2377-2384. 2014. [Medline] [CrossRef]
Small animal models for the study of bone sarcoma pathogenesis:characteristics, therapeutic interests and limitations. C Jacques, N Renema, F Lezot, B Ory, C R Walkley, A E Grigoriadis, Heymann D , 10.1016/j.jbo.2018.02.004J Bone Oncol. 12CrossRefJacques C, Renema N, Lezot F, Ory B, Walkley CR, Grigo- riadis AE, and Heymann D. Small animal models for the study of bone sarcoma pathogenesis:characteristics, thera- peutic interests and limitations. J Bone Oncol. 12: 7-13. 2018. [Medline] [CrossRef]
Pleomorphic leiomyosarcoma: clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma. Y Oda, K Miyajima, K Kawaguchi, S Tamiya, Y Oshiro, Y Hachitanda, M Oya, Y Iwamoto, M Tsuneyoshi, 10.1097/00000478-200108000-00007Am J Surg Pathol. 25Medline. CrossRefOda Y, Miyajima K, Kawaguchi K, Tamiya S, Oshiro Y, Hachitanda Y, Oya M, Iwamoto Y, and Tsuneyoshi M. Pleo- morphic leiomyosarcoma: clinicopathologic and immuno- histochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous his- tiocytoma. Am J Surg Pathol. 25: 1030-1038. 2001. [Med- line] [CrossRef]
Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research. S Gargiulo, A Greco, M Gramanzini, S Esposito, A Affuso, A Brunetti, G Vesce, ILAR J. 53CrossRefGargiulo S, Greco A, Gramanzini M, Esposito S, Affuso A, Brunetti A, and Vesce G. Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical re- search. ILAR J. 53: E55-E69. 2012. [Medline] [CrossRef]
Galectin-4, a novel predictor for lymph node metastasis in lung adenocarcinoma. T Hayashi, T Saito, T Fujimura, K Hara, K Takamochi, K Mitani, R Mineki, S Kazuno, S Oh, T Ueno, K Suzuki, T Yao, 10.1371/journal.pone.0081883e81883. 2013PLoS One. 8CrossRefHayashi T, Saito T, Fujimura T, Hara K, Takamochi K, Mi- tani K, Mineki R, Kazuno S, Oh S, Ueno T, Suzuki K, and Yao T. Galectin-4, a novel predictor for lymph node metas- tasis in lung adenocarcinoma. PLoS One. 8: e81883. 2013. [Medline] [CrossRef]
. M Sun, J G Liu, Q Y Weng, L Yu, Wang J , Pleomorphic and dedifferentiated leiomyosarcoma: a clinicopathologic analysisSun M, Liu JG, Weng QY, Yu L, and Wang J. [Pleomorphic and dedifferentiated leiomyosarcoma: a clinicopathologic analysis].
. Zhonghua Bing Li Xue Za Zhi. 47in ChineseZhonghua Bing Li Xue Za Zhi. 47: 87-93. 2018. (in Chinese) [Medline]
Storiformpleomorphic type of multifocal malignant fibrous histiocytoma of the lumbar spine. A Akpinar, C O Ozdemir, N Ucler, H M Inan, 10.12659/AJCR.891290Am J Case Rep. 15CrossRefAkpinar A, Ozdemir CO, Ucler N, and Inan HM. Storiform- pleomorphic type of multifocal malignant fibrous histiocy- toma of the lumbar spine. Am J Case Rep. 15: 565-568. 2014. [Medline] [CrossRef]
| [
"Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas. ("
] | [
"Yasuhiro Shimada \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Tomoharu Naito \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Takuo Hayashi \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Tsuyoshi Saito \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Yoshiyuki Suehara \nDepartment of Orthopaedic Surgery\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Chihaya Kakinuma \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n",
"Yuji Nozaki \nBiotechnical Center\nSLC, Inc\n3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan\n",
"Hisayoshi Takagi \nBiotechnical Center\nSLC, Inc\n3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan\n",
"Takashi Yao \nDepartment of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan\n"
] | [
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Department of Orthopaedic Surgery\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan",
"Biotechnical Center\nSLC, Inc\n3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan",
"Biotechnical Center\nSLC, Inc\n3-5-1 Aoihigashi, Naka-ku433-8114HamamatsuShizuokaJapan, Japan",
"Department of Human Pathology\nSchool of Medicine\nJuntendo University\n1-1-19 Hongo, Bunkyo-ku113-0033TokyoJapan"
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"Yasuhiro",
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"I M Meraz, ",
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"F Meng, ",
"R Shao, ",
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"B Fang, ",
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"Q Y Weng, ",
"L Yu, ",
"Wang J , ",
"A Akpinar, ",
"C O Ozdemir, ",
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"Oya",
"Iwamoto",
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"Inan"
] | [
"New immunodeficient (nudescid, beige-scid) mice as excellent recipients of human skin grafts containing intraepidermal neoplasms. Y Takizawa, T Saida, Y Tokuda, S Dohi, Y L Wang, K Urano, K Hioki, Y Ueyama, 10.1007/s004030050182Arch Dermatol Res. 289CrossRefTakizawa Y, Saida T, Tokuda Y, Dohi S, Wang YL, Urano K, Hioki K, and Ueyama Y. New immunodeficient (nude- scid, beige-scid) mice as excellent recipients of human skin grafts containing intraepidermal neoplasms. Arch Derma- tol Res. 289: 213-218. 1997. [Medline] [CrossRef]",
"Characteristics of in vivo model systems for ovarian cancer studies. Diagnostics (Basel). P Tudrej, K A Kujawa, A J Cortez, K M Lisowska, 10.3390/diagnostics9030120E120. 20199Cross-RefTudrej P, Kujawa KA, Cortez AJ, and Lisowska KM. Char- acteristics of in vivo model systems for ovarian cancer stud- ies. Diagnostics (Basel). 9: E120. 2019. [Medline] [Cross- Ref]",
"An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses. I M Meraz, M Majidi, F Meng, R Shao, M J Ha, S Neri, B Fang, S H Lin, P T Tinkey, E J Shpall, J Morris, J A Roth, 10.1158/2326-6066.CIR-18-0874Cancer Immunol Res. 7CrossRefMeraz IM, Majidi M, Meng F, Shao R, Ha MJ, Neri S, Fang B, Lin SH, Tinkey PT, Shpall EJ, Morris J, and Roth JA. An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses. Cancer Immunol Res. 7: 1267-1279. 2019. [Medline] [CrossRef]",
"Current and future horizons of patient-derived xenograft models in colorectal cancer translational research. Cancers (Basel). A Inoue, A K Deem, S Kopetz, T P Heffernan, G F Draetta, A Carugo, 10.3390/cancers11091321E1321. 201911Cross-RefInoue A, Deem AK, Kopetz S, Heffernan TP, Draetta GF, and Carugo A. Current and future horizons of patient-de- rived xenograft models in colorectal cancer translational re- search. Cancers (Basel). 11: E1321. 2019. [Medline] [Cross- Ref]",
"Examining the utility of patient-derived xenograft mouse models. S Aparicio, M Hidalgo, A L Kung, 10.1038/nrc3944Nat Rev Cancer. 15CrossRefAparicio S, Hidalgo M, and Kung AL. Examining the util- ity of patient-derived xenograft mouse models. Nat Rev Cancer. 15: 311-316. 2015. [Medline] [CrossRef]",
"Translational value of mouse models in oncology drug development. S E Gould, M R Junttila, F J De Sauvage, 10.1038/nm.3853Nat Med. 21CrossRefGould SE, Junttila MR, and de Sauvage FJ. Translational value of mouse models in oncology drug development. Nat Med. 21: 431-439. 2015. [Medline] [CrossRef]",
"Patient-derived xenograft models: an emerging platform for translational cancer research. M Hidalgo, F Amant, A V Biankin, E Budinská, A T Byrne, C Caldas, R B Clarke, S De Jong, J Jonkers, G M Maelandsmo, S Roman-Roman, J Seoane, L Trusolino, A Villanueva, 10.1158/2159-8290.CD-14-0001Cancer Discov. 4CrossRefHidalgo M, Amant F, Biankin AV, Budinská E, Byrne AT, Caldas C, Clarke RB, de Jong S, Jonkers J, Maelandsmo GM, Roman-Roman S, Seoane J, Trusolino L, and Vil- lanueva A. Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov. 4: 998-1013. 2014. [Medline] [CrossRef]",
"US cancer institute to overhaul tumour cell lines. H Ledford, 10.1038/nature.2016.19364Nature. 530391CrossRefLedford H. US cancer institute to overhaul tumour cell lines. Nature. 530: 391. 2016. [Medline] [CrossRef]",
"Cancer cell lines for drug discovery and development. J L Wilding, W F Bodmer, 10.1158/0008-5472.CAN-13-2971Cancer Res. 74CrossRefWilding JL, and Bodmer WF. Cancer cell lines for drug dis- covery and development. Cancer Res. 74: 2377-2384. 2014. [Medline] [CrossRef]",
"Small animal models for the study of bone sarcoma pathogenesis:characteristics, therapeutic interests and limitations. C Jacques, N Renema, F Lezot, B Ory, C R Walkley, A E Grigoriadis, Heymann D , 10.1016/j.jbo.2018.02.004J Bone Oncol. 12CrossRefJacques C, Renema N, Lezot F, Ory B, Walkley CR, Grigo- riadis AE, and Heymann D. Small animal models for the study of bone sarcoma pathogenesis:characteristics, thera- peutic interests and limitations. J Bone Oncol. 12: 7-13. 2018. [Medline] [CrossRef]",
"Pleomorphic leiomyosarcoma: clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma. Y Oda, K Miyajima, K Kawaguchi, S Tamiya, Y Oshiro, Y Hachitanda, M Oya, Y Iwamoto, M Tsuneyoshi, 10.1097/00000478-200108000-00007Am J Surg Pathol. 25Medline. CrossRefOda Y, Miyajima K, Kawaguchi K, Tamiya S, Oshiro Y, Hachitanda Y, Oya M, Iwamoto Y, and Tsuneyoshi M. Pleo- morphic leiomyosarcoma: clinicopathologic and immuno- histochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous his- tiocytoma. Am J Surg Pathol. 25: 1030-1038. 2001. [Med- line] [CrossRef]",
"Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research. S Gargiulo, A Greco, M Gramanzini, S Esposito, A Affuso, A Brunetti, G Vesce, ILAR J. 53CrossRefGargiulo S, Greco A, Gramanzini M, Esposito S, Affuso A, Brunetti A, and Vesce G. Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical re- search. ILAR J. 53: E55-E69. 2012. [Medline] [CrossRef]",
"Galectin-4, a novel predictor for lymph node metastasis in lung adenocarcinoma. T Hayashi, T Saito, T Fujimura, K Hara, K Takamochi, K Mitani, R Mineki, S Kazuno, S Oh, T Ueno, K Suzuki, T Yao, 10.1371/journal.pone.0081883e81883. 2013PLoS One. 8CrossRefHayashi T, Saito T, Fujimura T, Hara K, Takamochi K, Mi- tani K, Mineki R, Kazuno S, Oh S, Ueno T, Suzuki K, and Yao T. Galectin-4, a novel predictor for lymph node metas- tasis in lung adenocarcinoma. PLoS One. 8: e81883. 2013. [Medline] [CrossRef]",
". M Sun, J G Liu, Q Y Weng, L Yu, Wang J , Pleomorphic and dedifferentiated leiomyosarcoma: a clinicopathologic analysisSun M, Liu JG, Weng QY, Yu L, and Wang J. [Pleomorphic and dedifferentiated leiomyosarcoma: a clinicopathologic analysis].",
". Zhonghua Bing Li Xue Za Zhi. 47in ChineseZhonghua Bing Li Xue Za Zhi. 47: 87-93. 2018. (in Chinese) [Medline]",
"Storiformpleomorphic type of multifocal malignant fibrous histiocytoma of the lumbar spine. A Akpinar, C O Ozdemir, N Ucler, H M Inan, 10.12659/AJCR.891290Am J Case Rep. 15CrossRefAkpinar A, Ozdemir CO, Ucler N, and Inan HM. Storiform- pleomorphic type of multifocal malignant fibrous histiocy- toma of the lumbar spine. Am J Case Rep. 15: 565-568. 2014. [Medline] [CrossRef]"
] | [
"[2]",
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"New immunodeficient (nudescid, beige-scid) mice as excellent recipients of human skin grafts containing intraepidermal neoplasms",
"An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses",
"Examining the utility of patient-derived xenograft mouse models",
"Translational value of mouse models in oncology drug development",
"Patient-derived xenograft models: an emerging platform for translational cancer research",
"US cancer institute to overhaul tumour cell lines",
"Cancer cell lines for drug discovery and development",
"Small animal models for the study of bone sarcoma pathogenesis:characteristics, therapeutic interests and limitations",
"Pleomorphic leiomyosarcoma: clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma",
"Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research",
"Galectin-4, a novel predictor for lymph node metastasis in lung adenocarcinoma",
"Storiformpleomorphic type of multifocal malignant fibrous histiocytoma of the lumbar spine"
] | [
"Arch Dermatol Res",
"Characteristics of in vivo model systems for ovarian cancer studies. Diagnostics (Basel)",
"Cancer Immunol Res",
"Current and future horizons of patient-derived xenograft models in colorectal cancer translational research. Cancers (Basel)",
"Nat Rev Cancer",
"Nat Med",
"Cancer Discov",
"Nature",
"Cancer Res",
"J Bone Oncol",
"Am J Surg Pathol",
"ILAR J",
"PLoS One",
"Zhonghua Bing Li Xue Za Zhi",
"Am J Case Rep"
] | [
"\nFig. 1 .\n1Graph showing tumor volume after transplantation at each passage. Autopsy tumor samples obtained from the patient were minced (1 mm 3 /tumor piece) and subcutaneously transplanted into SCID mice (first inoculation: passage 0). After some tumors had reached a volume of 2,000 mm 3 , the tumor nodule was removed and sub-transplanted into the next normal SCID mice (passage 1). Sub-transplantation was conducted continually at passage 3. Volumetric measurements of xenografted tumors were performed using an external caliper, as described in the Materials and Methods section. Tumor nodules were collected at the time of necropsy and subjected to histological and immunohistochemical analyses.",
"\nFig. 2 .\n2Histological characteristics of the tumor derived from the patient. The tumor was characterized by fascicular proliferation of spindleshaped cells with scattered giant cells (A). Immunohistochemically, the tumor cells were positive for myogenic markers, including smooth muscle actin (SMA) (B). The black bar indicates 100 μm.",
"\nFig. 3 .\n3Histological and immunohistochemical analyses of a patient-derived xenograft (PDX) at each passage. Tumor nodules were fixed in formalin-containing bottles, and tumor sections were prepared on slides. The slides were stained with hematoxylin and eosin (H&E) solution, as described in the Materials and Methods section. All sections containing tumor tissue were microscopically examined, and representative images at each passage are shown (upper panels). The expression of Caldesmon (CALD, CDM), M-actin, and smooth muscle actin (SMA) in tumors collected from recipient mice engrafted with PDX were measured using immunohistochemical staining. Staining for SMA is shown at each passage (lower panels). The black bar indicates 100 μm for sections stained with H&E, whereas the white bar indicates 50 μm for sections stained with SMA."
] | [
"Graph showing tumor volume after transplantation at each passage. Autopsy tumor samples obtained from the patient were minced (1 mm 3 /tumor piece) and subcutaneously transplanted into SCID mice (first inoculation: passage 0). After some tumors had reached a volume of 2,000 mm 3 , the tumor nodule was removed and sub-transplanted into the next normal SCID mice (passage 1). Sub-transplantation was conducted continually at passage 3. Volumetric measurements of xenografted tumors were performed using an external caliper, as described in the Materials and Methods section. Tumor nodules were collected at the time of necropsy and subjected to histological and immunohistochemical analyses.",
"Histological characteristics of the tumor derived from the patient. The tumor was characterized by fascicular proliferation of spindleshaped cells with scattered giant cells (A). Immunohistochemically, the tumor cells were positive for myogenic markers, including smooth muscle actin (SMA) (B). The black bar indicates 100 μm.",
"Histological and immunohistochemical analyses of a patient-derived xenograft (PDX) at each passage. Tumor nodules were fixed in formalin-containing bottles, and tumor sections were prepared on slides. The slides were stained with hematoxylin and eosin (H&E) solution, as described in the Materials and Methods section. All sections containing tumor tissue were microscopically examined, and representative images at each passage are shown (upper panels). The expression of Caldesmon (CALD, CDM), M-actin, and smooth muscle actin (SMA) in tumors collected from recipient mice engrafted with PDX were measured using immunohistochemical staining. Staining for SMA is shown at each passage (lower panels). The black bar indicates 100 μm for sections stained with H&E, whereas the white bar indicates 50 μm for sections stained with SMA."
] | [
"(Fig. 1)",
"(Fig. 1)",
"Fig. 1)",
"Fig. 2A)",
"(Fig. 2B)",
"(Fig. 3, upper panel)",
"(Fig. 3, upper panel)",
"(Fig. 3)",
"(Fig. 3, lower panel)"
] | [] | [
"Cell-based assay systems are the primary tools used for drug discovery owing to their simplicity, which facilitates assessment of the effects of drugs on cells cultured in vitro. Although in vivo tumor models established using tumor cell lines obtained from immunodeficient mice (nude and SCID) have been used to mimic human cancer pathology 1 , in many cases, the cell characteristics and tumor formation differ from those of the original tumor, owing to the different microenvironments, including the immune system and cancer stroma in host animals [2][3][4] . Therefore, translational studies that use patient-derived tissues are required for the development of more effective cancer drugs. Some patient-derived xenograft (PDX) models have been established on the basis of this consideration 5 . These models are increasingly being used as tools for drug development in pre-clinical settings and have been shown to recapitulate the histology and behavior of the cancers from which they are derived 6 . Furthermore, studies conducted to aid clinical decision-making have shown high concordance between individual PDX and patient responses to therapy 7 . Although these findings are encouraging, the role of this approach in sarcoma-derived models and in the context of genomic drug-matching strategies remains undefined. This has thus created an opportunity to evaluate the utility of PDX models as clinical predictors to direct the use of chemo-and targeted therapies in combination with comprehensive genomic and epigenetic analyses for patients with sarcomas. Moreover, the National Cancer Institute recommends the screening of anti-cancer drugs using PDX models, given that unlike cell lines, the tissue structure and gene expression patterns of PDX models more closely resemble those of patients 8 . Thus, PDX models are predicted to emerge as the mainstream approach for drug discovery, and represent a valuable support tool for investigator-initiated clinical trials.",
"However, owing to the low efficiency of engraftment on host tissue and slow growth rate 9, 10 , a PDX model suitable for all tumor types, particularly sarcomas, is currently unavailable. In this study, we aimed to establish a PDX model for certain types of sarcoma, using SCID mice, for drug discovery. We describe the development of a novel PDX model of pleomorphic leiomyosarcoma (PLMS), a morphological variant of leiomyosarcoma with aggressive clinical behavior 11 , which shows histological and immunohistological features similar to those of the original PLMS in mice. Our case model may aid in elucidating the biological characteristics of PLMS and in developing novel candidate drugs for the treatment of this type of sarcoma.",
"Tumor tissue samples were obtained with informed consent from a 71-year-old man who underwent surgery at the Juntendo University Hospital. The tumor specimens originating from his thigh were preserved in minimum essential medium (Cat. #51200038; GIBCO, Grand Island, NY) supplemented with antibiotics (penicillin) under refrigerated conditions. The specimens were subsequently washed twice in Dulbecco's modified Eagle's medium without fetal bovine serum and minced at 1 mm 3 per sample block on ice using a razor before transplantation into the mice. This study was approved by the ethics committee of Juntendo University (approval code: 2017040).",
"C.B-17/IcrHsd-Prkdc scid mice (Japan SLC, Shizuoka, Japan) were obtained at 6 weeks of age and maintained under specific pathogen-free conditions. The mice were used for the experiments at 7 weeks of age. All experiments and procedures for the care and treatment of the animals used in the present study were performed in accordance with the requirements of the Juntendo University Animal Care and Experimentation Committee (Experimental Protocol: No. 2017040; Juntendo University, Tokyo, Japan) and the SLC Animal Care and Experimentation Committee (Experimental Protocols: No. BT18098 and No. 18099).",
"Twenty mice were subcutaneously transplanted with tumor tissues on day 0. Tumor specimens were subcutaneously injected into the right flank region of each animal using a transplant needle (φ2.5 × L85 mm, Cat. #KN-391-25; Natsume Seisakusho, Tokyo, Japan). The tumor diameter and body weight were measured twice per week. To calculate the tumor volume, both long and short diameters (mm) were measured using a Solar Digital Caliper (Cat. #1-3255-02; AZ-One, Osaka, Japan). The formula used to calculate tumor volume was as follows: tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) × 0.5. The volumes of tumor were measured until reaching 2,000 mm 3 , in line with experimental ethics. Animals with moribundity [marked reduction in body weight, hypothermia, significant drop in temperature, and significant exhaustion (crouching position)] were euthanized immediately by bloodletting under isoflurane anesthesia (induction: 4.0%; maintenance: 1.0-3.0%) without any other pain 12 . Prolifer-ated tumor nodules were collected at the time of autopsy.",
"All removed tissues were fixed in 10% neutral buffered formalin. Tissue slices were routinely processed for paraffin embedding and sectioned at 3 μm thickness. The sections were stained with hematoxylin and eosin and observed under a light microscope.",
"The PDX specimens were fixed with 10% formaldehyde, embedded in paraffin blocks, cut into 4-μm-thick sections, and mounted on glass slides. Staining of the sections was performed following standard methods described previously 13 . The sections were then incubated overnight at 4°C and thereafter at room temperature for 30 min with mouse monoclonal anti-Caldesmon (clone: h-CD; Dakopatts, Glostrup, Denmark), anti-human muscle actin (M-actin) (clone: HHF35; Dakopatts), or anti-smooth muscle actin (SMA) (clone: 1A4; Dakopatts) antibodies at dilutions of 1:1, 1:100, and 1:200, respectively, in phosphate-buffered saline containing 1% bovine serum albumin.",
"In our experiments, three of the 10 soft tissue tumor specimens derived from the patient were subcutaneously engrafted on the mice, one of which subsequently proliferated, with the size of the tumor increasing at each passage (Fig. 1). This soft tissue tumor was identified as a type of high-grade PLMS. Most of the transplanted mice showed massive tumor growth and enlargement (Fig. 1). Moreover, tumor progression continued to persist at each passage. In contrast, the PDX tumor models displayed enhanced growth rate with successive in vivo passages. Following transplantation, the time until harvesting decreased from 55 days at passage 0 to 50 days at passage 1, 40 days at passage 2, and 35 days at passage 3 ( Fig. 1). Finally, after re-transplantation, all the tumor foci proliferated and their volumes increased.",
"Histologically, the tumor was characterized by fascicular proliferation of spindle-shaped cells. Tumor cells with anomalous giant nuclei were also scattered throughout the lesion ( Fig. 2A). IHC revealed that the tumor cells were positive for SMA (Fig. 2B). Additionally, these tumor cells showed an MIB-1 index of approximately 80%. On the basis of the histological features and IHC findings, the patient was diagnosed as having a PLMS.",
"The storiform pattern and fibrotic elements of the patient-derived PLMS were observed at all passages in transplanted mice. The nuclei showed atypical anisokaryosis, nuclear pleomorphism with coarse chromatin, and mitotic figures, and the tissues were also characterized by small foci of fascicles consisting of smooth muscle tumor cells. A mucoid matrix was also found in the same field. The tumor tissues in mice sampled at each successive passaged showed no significant differences in structure (Fig. 3, upper panel). Therefore, structural characteristics at the third passage were histologically similar to those of the primary patientderived tumors (Fig. 3, upper panel).",
"PDX tumor samples obtained at each passage were immunologically stained for Caldesmon, M-actin, and SMA to confirm differentiation to smooth muscle in the original patient-derived leiomyosarcoma tumor. We observed that the PDX tumor was derived from the leiomyosarcoma, as indicated by the positive staining for all smooth musclespecific markers (Fig. 3). In particular, positive staining for SMA was diffuse and significant (Fig. 3, lower panel). With respect to the expression in PDX tumors through several passages, we detected no significant differences in the levels of Caldesmon, M-actin, and SMA staining among multiple passages (data not shown).",
"Although in vivo sarcoma PDX models represent promising tools for drug development and elucidation of underlying pathological mechanisms, it is difficult to establish sarcoma models in vivo. owing to the low reproducibility of tumor restructuring and the significantly low rate of tumor engraftment in experimental mice 9,10 . Although our PDX mouse model also showed a low rate of tumor engraftment (approximately 10%), we succeeded in establishing a PDX derived from a very rare type of tumor, PLMS. We believe that the following procedural details may have contribute to our success in establishing the PLMS PDX model. 1. Following surgery, the specimens were optimally processed prior to implantation in the mice. This optimization shortened the timeline from surgery to implantation of the specimen in mice, thereby contributing to specimen preservation. 2. The specimens were rapidly preserved during cold acclimation under refrigeration and optimal medium conditions. 3. The specimens that underwent rapid washout and cutting comprised an optimum tumor tissue for engrafting onto the subcutaneous site in the mice. Collectively, these procedures would have contributed to the prevention of sample degradation and maintenance of high cell viability. Whereas tumor tissue bricks characterized by a tumor microenvironment with scaffold stroma ensure successful engraftment of sarcoma specimens on xenograft models that mimic the characteristics of PLMS, it is also conceivable that high malignancy contributes to the success rate of PDX engraft-ment. Although the detailed mechanisms have yet to be sufficiently clarified, we hypothesize that the high frequency of cell division and proliferation in this type of sarcoma is conducive to good engraftment and time-dependent tumor growth in mice.",
"Microscopically, conventional leiomyosarcomas show a characteristic histology of smooth muscle differentiation with a low to intermediate grade potential for malignancy. In contrast, PLMS shows histological features of high-grade spindle cell sarcoma, such as variable, large, and atypical cells, and multinuclear giant cells, whilst partially retaining the features of conventional LMS, and also show distinct myogenic differentiation, as confirmed by IHC 11 . Although pleomorphic and conventional leiomyosarcomatous areas are typically intermixed within a tumor, the demarcation may be distinct or gradual in some cases 14 . Furthermore, it has been reported that PLMS contain myxoid or focally collagenous stroma, which resemble those of storiformpleomorphic undifferentiated sarcomas 15 . The characteristics of the PDX in our model were closely comparable to the typical characteristics of PLMS, thereby confirming the establishment of an original tumor-like PLMS PDX model. However, the tumor growth rate and commencement of tumor proliferation were clearly accelerated on tumor passaging in each mouse model. In this regard, it is conceivable that a tumor microenvironment characterized by the incorporation of interstitial stroma and host animal angiogenesis created an isolated niche for human tissue-resident sarcoma foci that favored tumor survival and proliferation at successive passage. Accordingly, tumors might be adopted in the host environment and undergo immune accommodation as a foreign body. In contrast, tumor-specific marker expres- sion and histological structure remained unchanged. Therefore, the important point to note would be the tumor niche environment for tumor growth changes. Improvements in tumor engraftment and growth rate are major factors necessary for the establishment of a rare cancer PDX model. Although the specific conditions required for PDX remain unclear, we speculate that establishing a basal scaffold will be essential for enhancing human carcinogenesis in future xenograft models.",
"In summary, we succeeded in developing a PLMS PDX mouse model that mimicked the original human tissue structure at the subcutaneous site. Patients with PLMS have poor prognoses, and novel systemic therapies are being investigated to improve the survival of afflicted patients. We anticipate that our PDX model will make a valuable contribution to future drug development for PLMS through drug efficacy screening.",
"There are no known conflicts of interest associated with this study."
] | [] | [
"Introduction",
"Materials and Methods",
"Preparation of patient-derived tumor samples",
"Animals",
"Preparation of a PDX mouse model",
"Histological examination",
"Immunohistochemistry (IHC)",
"Results",
"Establishment of a soft tissue sarcoma PDX model",
"Histological features of the patient's tumor",
"Histopathological characteristics of the original PLMS were maintained at each passage in mice",
"Immunohistochemical analysis of PDX tumor samples at each passage",
"Discussion",
"Disclosure of Potential Conflicts of Interest:",
"Fig. 1 .",
"Fig. 2 .",
"Fig. 3 ."
] | [] | [] | [
"Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma",
"Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma"
] | [
"J Toxicol Pathol"
] |
44,181,715 | 2022-03-02T23:25:18Z | CCBY | https://www.oncotarget.com/article/25116/pdf/ | GOLD | 3da05367da7f24161798c20b5f9f70a5bbf9c110 | null | null | null | null | 10.18632/oncotarget.25116 | 2948066676 | 29861845 | 5982777 |
Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment
2018
Aparna Karthikeyan
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Neelima Gupta
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Carol Tang
Department of Research
National Neuroscience Institute
Singapore
Duke-NUS Medical School
Singapore
Division of Cellular and Molecular Research
National Cancer Centre
Singapore
Karthik Mallilankaraman
Department of Physiology
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Maskomani Silambarasan
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Meng Shi
School of Biological Sciences
Nanyang Technological University
Singapore
Lei Lu
School of Biological Sciences
Nanyang Technological University
Singapore
Beng Ti Ang
Department of Physiology
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Duke-NUS Medical School
Singapore
Department of Neurosurgery
National Neuroscience Institute
Singapore
Singapore Institute for Clinical Sciences
A*STARSingapore
Eng-Ang Ling
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
S Thameem Dheen
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment
Oncotarget
9382018Received: October 05, 2017 Accepted: March 19, 2018Oncotarget 24950 Correspondence to: S. Thameem Dheen, Research Paper Oncotarget 24951microgliagliomaTGFβSMAD4microRNA-146a
Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFβ level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell linebased model, increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFβ signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned www.oncotarget.com medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.
INTRODUCTION
Gliomas are malignant brain tumors with a wide range of clinical features. Gliomas arise from the glial cells of the brain, progressing through the benign stage (WHO Grade I) to highly malignant (WHO Grades II to IV) stages. Higher grades of glioma are heterogeneous in nature, consisting of neoplastic cells, glioma-like stem cells, extensive vasculature and immune cells [1][2][3]. Microglia, the resident immune cells of the brain, form a significant portion of the infiltrating immune cell population in a tumor, making up to one-third of the tumor mass in some higher grade tumors [4,5]. As the first responders to any injury, insult or infection in the central nervous system (CNS), microglia become activated, secrete a variety of pro-inflammatory cytokines and exhibit phagocytic activity to clear tissue debris [6]. Subsequently, microglia facilitate repair and regeneration of the affected region in the CNS via release of growth factors and anti-inflammatory cytokines [7,8]. Activated microglia exhibit neurotoxic and neuroprotective roles in neuropathology and based on their functions, microglia are categorized as the classical (pro-inflammatory) phenotype and the alternative (anti-inflammatory) phenotype [9,10]. Microglial function in glioma tumors is an alternative form of activation wherein microglia secrete cytokines and chemokines that are gliomagenic and support the growth of the tumor [11,12]. However, recent studies suggest that tumor-associated microglia express genes that are distinct from either activation state [13,14], thus emphasizing the complex nature of tumor-associated microglia and its roles in a glioma microenvironment. This tumorigenic nature of microglia in glioma tumors may be attributed to molecular and epigenetic pathways that are altered by signaling molecules released from cancerous cells in the microenvironment.
Neoplastic cells within a tumor secrete a number of soluble cytokines, chemokines and growth factors that affect microglial motility, proliferation and phagocytosis [15,16]. A key signaling molecule that is highly enriched in the glioma microenvironment is the Transforming Growth Factor-beta (TGFβ) which activates the TGFβ pathway that is mediated by SMAD2 and 3, substrates for the TGFβ family of receptors. Upon binding of the TGFβ ligand to its receptor, the SMAD2/3 complex is phosphorylated and coupled with the common mediator SMAD4, translocated to the nucleus where the complex regulates the transcription of TGFβ responsive genes [17]. TGFβ is a known inhibitor of cell cycle progression [18] and thus, functions as a tumor suppressor in the early stages of certain cancers. On the contrary, TGFβ signaling can be pro-tumorigenic by inducing genes that promote tumorigenic aspects of glioma progression such as angiogenesis [19], metastasis [20,21] and epithelialmesenchymal transition [22]. Hyperactive TGFβ signaling is associated with certain subtypes of glioblastoma tumors, such as the mesenchymal subset and contributes to aggressiveness of the tumor and poor prognosis in patients [23][24][25]. In tumors with activated TGFβ signaling such as hepatocellular cancer, elevated SMAD4 has been shown to mediate tumor promoting signaling [26], while in other cancers such as pancreatic cancer, deletion of SMAD4 is associated with tumor progression and metastasis [27,28]. Therapeutic approaches using TGFβ antagonists and oligonucleotides coding anti-sense TGFβ2 have proven successful in reversal of TGFβ-aided immunosuppression in glioma [29,30]. However, systemic inhibition of TGFβ pathway can lead to unfavorable effects as TGFβ is involved in several cellular signaling pathways. This led us to investigate alternate specific mechanisms by which the TGFβ signaling pathways can be disrupted to attenuate the tumor supportive phenotype of microglia. Moreover, the role of SMAD4 in microglial functions in gliomas has been poorly understood and hence, this study is aimed to understand the role of SMAD4 in tumor-associated microglia in mediating tumor progression.
In addition to altered signaling pathways, activated microglia in different neuropathologies exhibit dysregulated epigenetic mechanisms such as chromatin modifications, changes in gene-specific histone acetylation and methylation and differential microRNA (miRNA) expression [31,32]. In particular, miRNAs have emerged as a central class of epigenetic mediators that post-transcriptionally regulate gene expression [33]. Dysregulation of miRNAs in activated microglia has been shown to contribute to development and progression of neurodegenerative diseases and brain injuries [33]. A global miRNA microarray analysis of activated primary microglial cells identified several miRNAs that were differentially expressed in activated microglia. The micro RNA 146a (miR-146a) was found to be upregulated in activated microglia as compared to control microglia (unpublished data). MiR-146a, which is enriched in activated macrophages and microglia [34], has been shown to target and suppress mediators of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling pathway in activated microglia and astrocytes, thereby functioning as a negative feedback regulator of microglial activation [35,36]. In addition, miR-146a was reported to target Notch1 in glioma cells www.oncotarget.com and further inhibit the process of gliomagenesis by suppressing migration and proliferation of cancer cells [37]. Further, our bioinformatics analysis predicted miR-146a to target SMAD4. Given the important role of miR-146a in microglia activation and gliomagenesis and its putative effect on SMAD4, this study attempted to understand the role of miR-146a and its putative target SMAD4 in microglia functions in tumor progression in glioma environment.
It was hypothesized that altered molecular and epigenetic mechanisms regulate the tumor supportive behavior of glioma-associated microglia. In this study, SMAD4, a mediator of the TGFβ signaling pathway was upregulated in microglia exposed to glioma conditioned medium and was found to be robustly expressed in microglia associated with human glioblastoma tissues. Stable loss of SMAD4 in microglia decreased expression level of a tumor promoter, MMP9, which resulted in decreased migratory potential of microglia in a transwell migration assay. In addition, miR-146a which was predicted to target SMAD4, was downregulated in microglia exposed to glioma conditioned medium treatment and regulated the expression levels of tumor supportive gene MMP9 in microglia. Overall, the present study implicates the role of miR-146a-SMAD4 in regulating microglial functions in glioma tumors.
RESULTS
Glioma conditioned medium induced phosphorylation of SMAD2 and SMAD3 in microglia
To understand the effect of glioma microenvironment on microglia, BV2 microglial cells were cultured in glioma conditioned medium (GCM) derived from C6 glioma cells. The concentration of TGFβ in the GCM was assessed to be ~5ng/ml as compared to undetectable levels of TGFβ in serum containing medium ( Figure 1A). First, the expression of total and phosphorylated SMAD2/3, which mediates the TGFβ signaling, was determined in GCM-treated microglia. Western blot analysis showed an increase in the phosphorylated levels of SMAD2 and SMAD3 in GCM treated microglia as compared to control microglia ( Figure 1B, 1C). Upon phosphorylation of SMAD2 and SMAD3, the complex has been shown to bind to SMAD4 and translocate to the nucleus [38]. Confocal imaging revealed a co-localization of the pSMAD2/3 with SMAD4 in nuclei of microglial cells treated with GCM ( Figure 1D). Further, immunocytochemistry revealed increased co-localization of the SMAD4 and total SMAD2/3 in microglia treated with GCM as compared to control cells ( Figure 1E), suggesting that the TGFβ pathway is activated in GCMtreated microglia.
Microglia treated with GCM show increased expression of SMAD4 and tumor supportive genes
As SMAD2/3 forms a complex with SMAD4 to regulate TGFβ responsive genes, the effect of glioma microenvironment on SMAD4 expression in microglia was determined by western blot and immunocytochemistry. Upregulation of SMAD4 protein expression was clearly evident in microglia treated with GCM as compared to that of control (Figure 2A, 2B). Concomitantly, GCM treated microglial cells showed an upregulation in proteins involved in TGFβ signaling pathway such as matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGFa) (Figure 2A, 2B), suggesting that glioma-associated microglia exhibit upregulated expression of tumor supportive factors.
SMAD4 is expressed in microglia associated with human glioblastoma samples
In light of the above results, we sought to validate the expression of SMAD4 in human glioblastoma tissue to better understand its role in glioma pathogenesis. An analysis of the expression profiling data of glioblastoma samples in The Cancer Genome Atlas database using Oncomine software showed a significant increase (3.271 fold) in the expression of SMAD4 (Reporter ID: 202527_s_at) mRNA in glioblastoma tissues (n=542) when compared with non-malignant brain tissue samples available in the database ( Figure 3A) [39].
In the present study, double immunofluorescence analysis was performed to determine SMAD4 protein expression in glioblastoma tissues ( Figure 3B). Confocal imaging showed expression of SMAD4 in Iba1-positive microglia, as well as non-microglial cells in different tissue samples ( Figure 3B). The Iba1-immunoreactive cells appeared to be an amoeboid or rounded phenotype, indicative of activated state of the cell type. High magnification images show evident nuclear expression of SMAD4 in Iba1 positive microglia ( Figure 3B). Quantitative analysis of Iba1-positive cell bodies revealed a high percentage of microglia in the tumor samples ( Figure 3C).
SMAD4 regulates the expression of tumor supportive factor MMP9
In order to ascertain the role of SMAD4 in microglia, stable knockdown of SMAD4 in microglia was carried out by transduction of small-hairpin RNA (shRNA) against the Smad4 gene. ShRNA-mediated silencing of the Smad4 gene resulted in 90% decrease in mRNA levels of Smad4 ( Figure 4A) and 80% decrease in the protein level of SMAD4 ( Figure 4A, 4C). A downregulation of MMP9 at mRNA and protein level was observed in microglia www.oncotarget.com Western Blot (B) shows an increase in phosphorylated form of pSMAD2/3 in microglia treated with GCM when compared with control. Histogram depicts densitometric quantification of pSMAD2/3 normalized against total SMAD2/3 expression (C) Data represent mean±SD, (n=4), Students t-test, * p<0.05, ** p<0.01. Confocal images show pSMAD2/3 (green, D), total SMAD2/3 expression (green, E) and SMAD4 expression (red, D, E) in BV2 microglia (indicated by arrows). Cell nuclei are labelled with DAPI (blue). GCM increases immunofluorescence intensity of pSMAD2/3 as compared to control cells (D). Immunocolocalization of pSMAD2/3, total SMAD2/3 and SMAD4 reveals that pSMAD2/3 and total SMAD2/3 colocalize with SMAD4 in microglia treated with GCM (D, E). Scale bar=30μm. www.oncotarget.com Western blot analysis shows an increase in SMAD4 protein levels and tumor supportive genes, VEGFa and MMP9 in BV2 microglia upon GCM treatment (A). Histogram depicts densitometric quantification of protein levels of SMAD4, VEGFa and MMP9 in microglia exposed to GCM (B). Data represent Mean±SD, (n=3-5), Students t-test, * p<0.05. after knockdown of SMAD4 compared to cells transduced with the empty vector, which served as a negative control ( Figure 4B, 4C). This suggests that SMAD4 regulates the expression of tumor supportive factor, MMP9 in microglia.
ShRNA-mediated silencing of SMAD4 suppresses the migration of microglia towards glioma conditioned medium
The transwell migration assay was performed, wherein microglial cells were seeded in serum-free medium in the upper chamber of a transwell insert and allowed to migrate towards the lower chamber containing GCM or chemoattractants such as TGFβ and EGF, to assess the migratory potential of microglia towards GCM ( Figure 5A-5D). There is a significant increase in the number of microglial cells migrating towards GCM ( Figure 5B, 5E) in the lower chamber as compared to the number of microglial cells migrating to serum containing medium which served as a control ( Figure 5A). In addition, microglial cells showed increased migration towards chambers containing soluble TGFβ and EGF ( Figure 5C, 5D, 5E) as compared to control. Further, the role of SMAD4 in the migration of microglia towards GCM was studied. A significant decrease in the number of shSMAD4 cells migrating towards the serum containing medium in the lower chamber, was observed as compared to control cells which were transfected with empty vector ( Figure 5F, 5G, 5J). This indicates that SMAD4 plays a significant role in the migratory potential of microglia. On the other hand, upon exposure to GCM, there was an increase in the number of migrating control and shSMAD4 microglial cells compared to that of control and shSMAD4 microglial cells, respectively. However, the increase in migration of shSMAD4 microglial cells exposed to GCM was significantly less than that of control microglial cells show an increase in the number of migrated microglial cells (purple) after exposure to GCM (B), TGFβ (C) and EGF (D) as compared to cells exposed to serum containing medium (A). Histogram depicts fold change in migrating cells exposed to GCM, TGFβ and EGF as compared with control group (E). Data represent mean±SD, * p<0.05, ** p<0.01, (n=3). There was a decrease in the number of migrated shSMAD4 microglial cells (purple) in response to GCM and serum containing medium (I, G) as compared to negative control (Neg Cont) microglial cells exposed to GCM and serum containing medium (H, F). Histogram depicts fold change in migrating cells (J). Data represent mean±SD, (n=3), * p<0.05, ** p<0.01. www.oncotarget.com exposed to GCM ( Figure 5H, 5I, 5J). In addition, GCMinduced migration index of both control and shSMAD4 microglial cells appear to be comparable ( Figure 5J), suggesting that factors involving TGFβ pathway present in GCM could play a role in migration of microglia.
Microglial conditioned medium from shSMAD4 cells inhibits glioma viability
Microglial cells have been shown to promote glioma progression through secretion of factors that aid in tumor growth. In the present study, the viability of glioma cells in response to conditioned medium from microglia was evaluated using an MTS assay and alamar blue assay. The results indicate that there is a significant increase in cell viability ( Figure 6A, 6B) of glioma cells after 24, 48 and 72h of treatment with medium derived from control microglial cells, confirming that microglial cells secrete factors that promote glioma cell growth. In order to determine the effect of knockdown of SMAD4 in microglia on glioma cell viability, glioma cells were treated with conditioned medium derived from shSMAD4 microglial cells. The results indicate that there is a decrease in viability of glioma cells when treated with medium from SMAD4 knockdown microglial cells ( Figure 6C), suggesting that suppression of SMAD4 in microglia decreases the viability of glioma cells. This was further confirmed with an alamar blue assay wherein a significant decrease in reduction of alamar blue was observed in vitro after treatment of glioma cells with medium derived from shSMAD4 microglial cells as compared to the corresponding control ( Figure 6D).
MiR-146a regulates SMAD4 expression in GCM treated microglia
In order to delineate the epigenetic mechanisms that regulate SMAD4 expression in glioma-associated microglia, we examined the 3'UTR of the Smad4 mRNA sequence to identify miRNA binding sites. TargetScan algorithms software (http://www.targetscan.org/mmu_71/) revealed putative complimentary binding site for miR-146a in the 3'UTR of Smad4 ( Figure 7A). Given the role of miR-146a in activation of microglia [36,40,41], its role in the tumor supportive phenotype of microglia was determined. The quantitative RT-PCR analysis showed that the expression level of miR-146a-5p was significantly decreased in glioma-associated microglia as compared to control cells ( Figure 7B). This is concurrent with an increase in mRNA and protein levels of SMAD4 ( Figure 7B, 2A), demonstrating an inverse relationship between miR-146a and SMAD4 in microglia.
Further, 3'UTR luciferase assay was performed to confirm that SMAD4 is a target of miR-146a. BV2 microglial cells were transfected with a luciferase vector containing the 3'UTR of the mouse Smad4 gene together with miR-146a overexpression (mimics) or scrambled probes. A significant decrease in the luciferase activity in BV2 microglia was observed upon co-transfection of the mimics and the luciferase vector, indicating that the miR-146a binds to the 3'UTR of Smad4 in luciferase vector ( Figure 7C). Transfection efficiency of the miRNA overexpression and inhibition was determined by examining the levels of the miR-146a in microglial cells after transfection of mimics and inhibitors. Overexpression of miR-146a mimics was observed to increase miR-146a levels in microglia by nearly 400-fold while inhibition of the miR-146a resulted in a ~50% decrease in miR-146a levels ( Figure 7D). The mRNA expression of SMAD4 after overexpression and inhibition of the miR-146a was analyzed. MiR-146a mimic transfection resulted in decrease in mRNA and protein expression levels of SMAD4 and conversely, inhibition of miRNA function resulted in an increase in the mRNA and protein levels of SMAD4 ( Figure 7E, 7F, 7G) in microglia, suggesting that miR-146a directly targets SMAD4 in microglia.
In order to understand the functional relationship between miR-146a and SMAD4 in GCM-induced microglia, loss-of and gain-of function experiments were carried out. MiR-146a overexpression was found to significantly decrease SMAD4 expression at protein levels in microglia treated with GCM, as determined by western blot (Figure 8A, 8C). In contrast, inhibition of the miR-146a resulted in a marginal increase in the protein levels of SMAD4 in microglia treated with GCM as revealed by western blot analyses ( Figure 8B, 8D). Immunocytochemistry analysis also revealed that miR-146a overexpression with mimics decreased the expression of SMAD4 in microglia treated with or without GCM as compared to that transfected with scrambled probes ( Figure 8E). This suggests that miR-146a regulates SMAD4 expression in glioma-associated microglia.
MiR-146a regulates the expression of MMP9 in microglia and suppresses migration of microglia towards GCM
Microglia treated with GCM showed an induction of tumor promoter gene MMP9, concomitant with an increase in SMAD4 level. In order to determine if miR-146a regulates tumor supportive gene expression in microglia, expression of MMP9 upon overexpression and knockdown of miR-146a was analyzed. Overexpression of the miR-146a in microglia resulted in a significant suppression of MMP9 protein expression while inhibition of miR-146a was found to increase MMP9 expression ( Figure 9A, 9B). In addition, overexpression of miR-146a in microglia resulted in a significant decrease in migration of microglia towards GCM in a transwell migration assay ( Figure 9C-9G), indicating that miR-146a suppresses microglial migration through regulation of SMAD4 and its downstream gene MMP9. www.oncotarget.com Histogram depicts a significant decrease in the levels of miR-146a and a significant increase in mRNA levels of SMAD4, indicating an inverse relationship between SMAD4 and miR-146a in microglia treated with GCM (B). Histogram shows a significant decrease in the luciferase activity in cells co-transfected with miR-146a mimic and luciferase vector as compared to cells co-transfected with scrambled control miRNA and luciferase vector, indicating that miR-146a targets SMAD4 (C). Data represent mean±SD, (n=3), Students t-test, * p<0.05. MiRNA mimics and inhibitor transfection efficiency was verified using qRT-PCR. Histogram shows an about 400-fold increase in miR-146a levels in microglia after mimic transfection and about 50% decrease in miR-146a levels after inhibitor transfection (D). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Histogram shows a decrease in the mRNA levels of SMAD4 upon overexpression of miR-146a and conversely an increase in SMAD4 mRNA upon inhibition of miR-146a (E). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Western blot (F) and densitometry analysis (G) confirm that overexpression of miR-146a suppresses SMAD4 protein levels and inhibition of miR-146a increases SMAD4 protein level as compared to scrambled probe transfected cells. Data represent mean±SD, (n=3), Students t-test, * p<0.05, ** p<0.01. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com
miR-146a overexpression in microglia suppresses glioma viability and growth
To determine the effect of miR-146a overexpression in microglia on glioma cell viability, glioma cells were treated with conditioned medium derived from miR-146a overexpressed microglial cells. The MTS results indicate that there is a decreased viability of glioma cells across different time points after treatment with miR-146a overexpressed microglia-derived medium as compared with that of scrambled control transfected cells ( Figure 10A). This was further confirmed with an alamar blue assay wherein a significant decrease in alamar blue reduction was observed in vitro after treatment of glioma cells with medium from miR-146a overexpressed microglial cells ( Figure 10B), indicating that overexpression of miR-146a in microglia decreased the viability of glioma cells.
DISCUSSION
It is well documented that microglia play diverse roles, either detrimental or beneficial, during CNS pathology [6]. In the normal healthy brain, microglia monitor the brain microenvironment for pathogens and injury and are involved in functions such as neuronal synapse formation, maintenance and pruning [42][43][44]. Upon activation, the microglia with processes rapidly transform into amoeboid phenotype and are involved in phagocytosis of debris in brain parenchyma [45]. In the present study, microglia within gliomas exhibit primarily amoeboid phenotype, suggesting that they are activated, probably in response to the glioma secretome which consists of signaling molecules released by neoplastic and non-neoplastic cells such as vascular endothelial cells, astrocytes and cancer stem cells of the tumor that lie in quantification (B, D). Data represent mean±SD, (n=5), * p<0.05. Immunofluorescence labelling shows that miR-146a overexpression attenuated SMAD4 expression (red) in BV2 microglia nuclei with or without GCM treatment as compared to cells transfected with scrambled miRNA (Neg Cont) probes (E). DAPI-nuclei staining, blue. Scale bar=30μm. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com spatial proximity to microglial cells. This is supported by recent experimental evidence, which showed that microglia exposed to glioma conditioned medium in vitro and microglia associated with glioma tumors in mice models in vivo exhibit an amoeboid phenotype that is characteristic of a state of activation [46][47][48]. Further, the glioma tumors analyzed in the present study showed a high percentage of Iba1-positive microglial cells, with certain glioblastoma tumors hosting nearly 25%-50% of microglial cells in the tumor mass. A higher frequency of microglial cells in the tumor may be attributed to migration of microglia in the brain parenchyma in response to factors released by the glioma cells as can be seen in the in vitro migration assay. Several studies have shown that soluble factors such as EGF [49,50] and TGFβ [51] serve as potent chemotactic factors in tumors and may promote migration of microglia towards the tumor as observed in the present study.
In addition to its role as a chemoattractant, the TGFβ ligand activates an anti-inflammatory signaling pathway in microglia, exerting an opposing effect on pro-inflammatory signaling that is widely known to be neurotoxic to brain tissue [52,53]. TGFβ has been shown to act on microglia in an autocrine manner and maintain microglial quiescence [54]. It has also been shown that microglia-derived TGFβ enhanced the invasiveness and tumorigenicity of the glioma cells and siRNA-mediated knockdown of TGFβ Receptor II in glioma cells disrupted this tumor promoting effect of TGFβ [30]. The present study shows increased levels of total and pSMAD2/3, which mediate TGFβ signaling pathway, in microglia after GCM treatment in vitro. While the roles of SMAD2/3 have been widely studied in microglia [55,56], the role of SMAD4 in microglial activation, specifically in context of the TGFβ signaling has remained unclear. There is evidence showing that SMAD4 is upregulated in LPS- activated microglia and acts as a negative feedback inhibitor of NFκB, a pro-inflammatory signaling response in the activated microglia [57]. In the present study, glioma-associated microglia expressed SMAD4 in human glioblastoma tumors in vivo and microglia exposed to GCM showed increased expression of SMAD4 in vitro. This was further supported by an analysis of the data in The Cancer Genome Atlas which revealed an upregulated SMAD4 level in glioblastoma tumors as compared to normal brain samples. Further, shRNA-mediated silencing of SMAD4 in microglia was associated with a decrease in the expression of MMP9, an extracellular matrix metalloproteinase and suppression of microglial migration. This has been evidenced in hepatocellular carcinoma wherein knockdown of SMAD4 reduced migratory capacity and colony formation ability of the cancer cells [26]. SMAD4 has also been shown to control endodermal cell migration during embryonic development through regulation of extracellular matrix modelling enzymes, MMP9 and MMP14 [58]. In addition, SMAD4 silencing in pancreatic tumor cells and keratinocytes has been shown to abolish TGFβ induced migration [59], therefore highlighting a vital role for SMAD4 in microglial migration.
Microglial cells are highly secretory in nature. In the present study, microglia-derived growth factors in the conditioned medium were found to promote glioma cell viability in vitro. This is an important finding as it may lead to a novel therapeutic strategy which may focus on suppressing microglia-mediated tumorigenesis. Moreover, this study demonstrates that glioma-associated microglia promote tumorigenesis through SMAD4 expression, since glioma cells treated with conditioned medium derived from shSMAD4 microglial cells showed decreased viability. This decreased viability of glioma cells is also possible via SMAD4-induced negative regulation of NF-κB pathway in microglia, since recent studies have shown that SMAD4 knockdown in microglia induced pro-inflammatory cytokine, IL-6 in an NF-κB dependent manner [57]. Overall, these results indicate that the possible interaction between SMAD4 and NF-κB in glioma-associated microglia may determine the tumor progression. The epigenetic regulation of SMAD4 was investigated, as these mechanisms provide an additional layer of post-transcriptional control. In this study, SMAD4 was found to be targeted by miR-146a in microglia. MiR-146a, a miRNA enriched in immune cells, is known to be upregulated in activated microglia and macrophages in pathological conditions such as infection [41], ischemic stroke [60] and Alzheimer's disease [40,61]. Several studies have shown that miR-146a modulates the innate immune response of activated microglial cells through regulation of the pro-inflammatory transcription factor, NFκB [41,62]. Recently, the miRNA-146b, which shows sequence similarity with miR-146a has been shown to inhibit glioma growth in vitro through modulation of its target EGFR [63]. Downregulation of miR-146a in gliomaassociated microglia as observed in the present study may favour tumorigenesis through increased expression of SMAD4 and its downstream genes which are involved in tumor progression. This also indicates that the gliomaassociated microglia do not exhibit activated microglial phenotype as observed in other neuropathologies such as Alzheimer's and stroke. Functional studies further confirmed that miR-146a is a negative regulator of tumorigenic gene expression in microglia via its target SMAD4, as its overexpression in microglia resulted in suppression of MMP9, which is a tumorigenic factor that promotes migration of microglia towards GCM and the glioma viability in vitro.
As the immune cell of the brain, microglia show complex phenotypes in the brain tumor and did not reject or phagocytose the tumor cells. Instead, they conglomerate within the tumor core and support the tumor progression. It is thus imperative to study microglial function in context of different molecular and genetic subtypes of glioblastoma. In this regard, this study shows robust expression of SMAD4 in glioblastoma tumors and in glioma-associated microglia. It has also been demonstrated that SMAD4 which was found to be post-transcriptionally regulated by miR-146a, regulates the migration of microglial cells in response to glioma conditioned medium. This study further established that miR-146a suppresses tumorigenic gene, MMP9 in gliomaassociated microglia and glioma cell viability through its target SMAD4 (Figure 11). Further in vivo studies are required to evaluate the therapeutic effect of miR-146a and SMAD4 on glioma growth and progression.
MATERIALS AND METHODS
Human tissue samples
Graded brain tumor specimens were obtained with written informed consent, as part of a study protocol approved by the SingHealth Centralised Institutional Review Board A and the National Healthcare Group Domain-Specific Review Board A ( Table 1). All protocols were approved by the Institutional Review Board, National University of Singapore. Use of glioblastoma human tissues was reviewed and approved by the National University of Singapore Institutional Review Board (NUS-IRB Reference Code: B-16-049E).
Immunofluorescence
Human tumor sections were fixed, frozen, sectioned (7μm thick) and mounted onto slides. Tissue sections were blocked in 3% bovine serum albumin (BSA) solution in phosphate-buffered-saline (PBS) containing 0.3% Triton-X (TX). Primary antibody against the full length human SMAD4 protein was a kind gift from Dr. Lu Lei from the Nanyang Technological University, Singapore. Sections were incubated with primary antibodies in 1% BSA overnight at 4ºC at the following concentrations: SMAD4-1:300, Iba1-1:50 (Abcam, ab5076). Further, sections were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:5000) and mounted with a fluorescent mounting medium. Fluorescence images were captured using a confocal microscope (Olympus, FV1000 Fluoview). Iba1 positive cell bodies showing a well-defined DAPI stained nuclei were manually counted across 4 fields of the tumor section and plotted as a percentage of total DAPI cell nuclei.
Cell culture
BV2 murine microglial cells and C6 rat astrocytoma cells obtained from American Tissue type Culture Collection (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a 5% CO 2 incubator at 37ºC and regularly passaged with Trypsin-EDTA solution to allow healthy growth of cells.
Preparation of glioma conditioned medium (GCM)
To mimic a glioma microenvironment in vitro, microglial BV2 cells were treated with conditioned medium from the C6 cell line [64,65]. Briefly, C6 cells were seeded in 10cm culture dishes at a density of 2X10 6 cells. Cells were allowed to settle overnight and DMEM supplemented with 10% FBS was added to the culture the next day. Culture supernatant was collected after 48h and filtered through a 0.22μm filter to remove cellular debris. GCM was stored at -80ºC and freeze-thaw cycles were minimized.
Generation of SMAD4 knockdown stable cells in microglia
Stable knockdown of SMAD4 was performed by lentiviral mediated transduction of SMAD4 specific shRNA in BV2 microglial cells. Microglial cells were transduced with 4 shRNA individual clones (Dharmacon, GE Healthcare) against the SMAD4 gene (Accession Number: NM_008540). Selection pressure was applied 48h after transduction using puromycin at the concentration of 2μg/ml. Cells were maintained in puromycin containing medium for 6-10 days and expanded [66]. Efficiency of knockdown in microglia was confirmed using western blotting analysis and the shSMAD4 clone that induced maximal knockdown, shSMAD4_2, was selected for further analysis (Supplementary Figure 1).
RNA extraction, cDNA conversion and quantitative real-time PCR (miRNA)
RNA was isolated from the BV2 cells after transfection or treatment, using miRNeasy Mini Kit The qRT-PCR for gene expression was performed using Fast SYBR Green Master Mix (ThermoFisher Scientific, Cat No: 4385614) with 1:10 ratio dilution of cDNA in nuclease-free water. Primer sequences for PCR reactions are given in Table 2. Fold change between control and experimental groups was calculated as per 2 -ΔΔCt method [67]. All PCR reactions were carried out in a real time Applied Biosystems PCR system (Life Technologies, Model No: 7900HT).
Migration assay
The Transwell migration assay was carried out to assess the migratory potential of microglia towards GCM, TGFβ and EGF. 40,000 microglial cells were seeded in the upper chamber of the Transwell migration insert (Corning, Cat No:3422) and was placed in the lower chamber containing glioma conditioned medium and medium containing TGFβ (PeproTech, Cat No 100-21C) and human recombinant EGF (Life Technologies, PHG0311).
The cells were allowed to migrate for 15h. Inserts were fixed in 100% methanol for 10min and stained for visualization using 0.5% cresyl violet solution. Cells on the upper membrane of the insert were removed using a cotton swab. 5 random fields from 3 biological replicates were quantified and the results are plotted as fold change between treatment and control groups.
Transfection of miRNA mimics and inhibitors
BV2 cells were plated at a density of 2X10 5 in 6-well plates and gain-of and loss-of-function studies of miR-146a-5p was carried out using miRNA mimics (Ambion,
Luciferase assay
A luciferase assay was performed to verify if miR-146a targets SMAD4 mRNA. BV2 microglial cells were plated at a density of 20,000 cells in 24 well plates. The luciferase vector containing the 3`UTR of SMAD4 was commercially purchased from GeneCopoeia (Cat No: MmiT027594). Cells were co-transfected with mimics and scrambled probes (30nm) and luciferase vector (1000ng) and the medium containing secreted luciferase was collected at 24h. The Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia Cat No: SPDA-D010) was used to determine luminescence of secreted Gaussia luciferase (GLuc). A secondary reporter, secreted alkaline phosphatase (SEAP), served as an internal control. GLuc/SEAP ratio was determined to measure the luminescence output of the transfected sample.
Immunocytochemistry
BV2 microglial cells were grown on poly-L-lysine coated coverslips. Following transfection and/or treatment, cells were fixed with 4% paraformaldehyde for 15min at room temperature. Permeabilization of cell membranes was achieved using 0.1% Triton-X containing PBS. Following this, the slides were blocked using 5% normal goat serum and incubated overnight at 4ºC with the following antibodies-SMAD4-1:100 (Santa Cruz, sc-7966), pSMAD2/3-1:200 (Cell signaling technology, Cat No: 8828) and SMAD2/3-1:200 (Cell Signaling Technology, Cat No: 8685). Following this, cells were incubated with fluorophore tagged secondary antibodies: anti-rabbit Cy3 (Sigma, Cat No: c2306), anti-mouse Cy3 (Sigma, Cat No: C2181) and FITC conjugated lectin (Sigma, Cat No: L0401) was used as a marker for microglial cells. Cell nuclei were counterstained with DAPI for visualization. Fluorescence images were captured using a confocal microscope (Olympus, FV1000 Fluoview).
ELISA based quantification of TGFβ in GCM
TGFβ in the C6 glioma conditioned medium (GCM) was quantified using the Quantikine ELISA Kit (RND Systems, MB100B) as per the manufacturer's instructions. Briefly, latent TGFβ1 was activated to its immunoreactive form by addition of 20μl of 1M HCl to 100μl of the GCM, and subsequently neutralized by adding 20μl of 1.2N NaOH. The TGFβ1 standard was reconstituted and serially diluted using the diluent solution provided in the kit. 50μl of GCM and the TGFβ1 standard samples were added to the TGF-β1 antibody pre-coated ELISA plate and incubated for 2h. Subsequently, the samples were discarded, and the wells were thoroughly washed using wash buffer. Next, 100μl of the TGFβ1 conjugate was added to each well and incubated for 2h. Following washing, the plate was incubated with substrate solution, and reaction was stopped using the stop solution provided in the kit. The optical density was measured at 450nm using a plate reader.
MTS and alamar blue cell viability assay
In order to evaluate the effect of SMAD4 knockdown in microglia on glioma cell viability, an MTS and alamar blue assay was performed. Conditioned medium was collected from control microglia (microglial CM) and from microglia after knockdown of SMAD4 (shSMAD4-CM) or miR-146a overexpression (MiR-146a OE-CM). Conditioned medium from microglia transduced with the empty vector (Neg Control CM (stables)) and the scrambled probes (Neg Control CM (miRNA)) served as controls. About 10,000 of C6 glioma cells were seeded in 96-well plates and treated with the conditioned medium from different groups. Post 24, 48 and 72h of treatment, 20μl of MTS reagent (Promega, Cat No. G358C) was added to the cells and incubated at 37°C for 2h. Following this, absorbance was read at 490nm in a 96-well plate reader. The alamar blue assay was performed by adding 10μl of the alamar blue reagent (ThermoFisher Scientific, Cat No. DAL1100) to the cells after treatment with conditioned medium. Absorbance was read at 570 nm and normalized against absorbance at 600nm. The results are plotted as a percentage of alamar blue reduction.
Statistical analysis
Data from at least three biological replicates were analyzed using GraphPad Prism and Microsoft Excel software and represented as mean ± S.D. In comparing 2 experimental groups, the Student's t-test was used. Multiple groups were analyzed using one-way or two-way ANOVA tests, followed by post-hoc Tukey's and Sidak's test. Data sets were considered significant at p<0.05.
Figure 1 :
1GCM induces TGFβ signaling pathway in microglia. ELISA assay quantification revealed that GCM contains ~5ng/ml of TGFβ (A).
Figure 2 :
2SMAD4 and tumor supportive genes MMP9 and VEGFa are upregulated in glioma associated microglia.
Figure 3 :
3SMAD4 is expressed in microglia associated with human glioblastoma samples. An analysis of the TCGA profiling data reveals a significant increase in SMAD4 mRNA levels in brain glioblastoma tissue as compared to normal non-malignant tissue (A). Panel shows expression of SMAD4 in human glioblastoma tumor tissue (GB 34) (B). Note that there is evident expression of SMAD4 (red) expression in Iba1 positive microglial cells (green). Cell nuclei are labelled with DAPI (blue). Scale=30μm (low magnification), Scale=10μm (high magnification). Table depicts the percentage of Iba1 positive microglial cells per tumor (C). www.oncotarget.com
Figure 4 :
4SMAD4 regulates expression of tumor supportive factor MMP9 in microglia. Quantitative RT-PCR and western blot analyses shows a decrease in mRNA and protein expression of SMAD4 in shSMAD4 BV2 cells as compared to negative control (A, C). Data represent Mean±SD, (n=3), Students t-test, *** p<0.001. Quantitative RT-PCR and western blot analyses show that shRNAmediated knockdown of SMAD4 resulted in a decrease in the mRNA and protein expression of MMP9 (B, C). Mean±SD, (n=4), Students t-test, * p<0.05.
Figure 5 :
5shRNA-mediated knockdown of SMAD4 suppresses microglial migration. Trans-well membrane image panels
Figure 6 :
6Effect of conditioned medium derived from shSMAD4 knockdown microglial cells on glioma cell viability.Histogram depicts MTS assay absorbance values at 24, 48 and 72h after treatment of glioma cells with conditioned medium derived from microglia. A significant increase in glioma viability was observed after treatment with microglia conditioned medium at all time points (A). This was further confirmed using alamar blue viability assay wherein an increase in % of reduction of alamar blue was observed in glioma cells treated with microglia conditioned medium at all time points (B). Histogram further shows the MTS assay absorbance values at 24, 48 and 72h treatment of glioma cells with conditioned medium derived from shSMAD4 microglial cells (C). A decrease in absorbance values indicates a decrease in viability of glioma cells after treatment with conditioned medium from shSMAD4 cells as compared to glioma cells treated with negative control medium (C). In addition, alamar blue assay results (D) show a decrease in percentage of alamar blue reduction in glioma cells treated with medium from shSMAD4 microglia as compared to glioma cells treated with negative control medium. Data represent Mean±SD, (n=4), *** p<0.001, **** p<0.0001. www.oncotarget.com
Figure 7 :
7MiR-146a is downregulated in GCM treated microglia and targets SMAD4. TargetScan software predicted that miR-146a putatively binds to 3'UTR of SMAD4 mRNA (A).
Figure 8 :
8MiR-146a regulates SMAD4 in GCM-treated microglia. Western blot analyses show that transfection of microglial cells with miR-146a mimics suppressed the GCM induced induction of SMAD4 protein (A, C) Data represent mean±SD, (n=3), * p<0.05. MiR-146a inhibition increases the protein levels of SMAD4 upon GCM treatment of microglia as seen by western blot analyses and densitometry
Figure 9 :
9MiR-146a alters expression of tumor supportive gene MMP9 in glioma associated microglia and suppressed microglial migration towards GCM. Overexpression of miR-146a suppressed expression of MMP9 at the protein level and conversely, inhibition of miR-146a in microglia was found to increase levels of MMP9 as depicted by immunoblotting and densitometry analysis (A, B). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Image panel shows a decrease in the number of migrated microglial cells (purple) after overexpression of miR-146a in response to GCM as compared to Neg Cont cells (C-F). Histogram depicts fold change in migrating cells (G). Data represent mean±SD, (n=3), ** p<0.01. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com
Figure 10 :
10Effect of conditioned medium derived from miR-146a overexpression in microglia on viability of glioma cells. Histogram depicts MTS assay absorbance values 24, 48 and 72h treatment of glioma cells with conditioned medium derived from miR-146a overexpression in microglia. A decrease in absorbance values at all time points indicates an inhibition of viability of glioma cells after treatment with medium from miR-146a overexpressed cells as compared to glioma cells treated with negative control (scrambled miRNA) transfected medium (A). Further, alamar blue assay results show a decrease in percentage of alamar blue reduction, indicating a suppression of glioma cell viability upon treatment with medium from miR-146a overexpressed microglia as compared to glioma cells treated with scrambled control transfected medium (B). Data represent Mean±SD, (n=4), ** p<0.01, **** p<0.0001. www.oncotarget.com
Figure 11 :
11SMAD4, regulated by microRNA-146a, promotes microglial cell migration and tumor progression in glioma environment. Glioma cells secrete a number of factors including TGFβ (A) that activates the TGFβ pathway in microglia associated with glioma. Glioma conditioned medium was found to induce phosphorylation of SMAD2/3 complex and increase the level of SMAD4 (B) in microglia. Concurrently, miR-146a which was found to target SMAD4, was downregulated in glioma associated microglia (C). Downregulation of miR-146a in glioma-associated microglia increases the levels of tumor supportive factors (D), including SMAD4 and MMP9 which promote glioma progression and microglial migration. www.oncotarget.com
(Qiagen, Cat No: 217004) as per the manufacturer's instructions. RNA isolate (10ng) was used for conversion of miRNA to cDNA using Universal cDNA Synthesis Kit II (Exiqon, Cat No: 203301). Quantification of miRNA expression in control and GCM treated groups was carried out using ExiLENT SYBR ® Green master mix (Exiqon, Cat No: 203421). LNA-modified miR-146a-5p primers (Exiqon, Cat No: 204688) and small nuclear U6 RNA primers (Exiqon, Cat No: 203907) were used for miRNA real time PCR. cDNA conversion and quantitative Real-time PCR (mRNA) The total RNA (2000ng) was converted to cDNA for gene expression analysis using a master mix consisting of M-MLV Reverse transcriptase enzyme (Promega, Cat No: M170A), M-MLV Reverse Transcriptase 5X Reaction Buffer (Promega, Cat No: M531A), dNTP (Promega, Cat No: U1511) and RNasin ® Ribonuclease inhibitor (Promega, Cat No: N2111) in a 25μl reaction volume.
Table 1 :
1Tumor sample grade and typeSample Name
Tumor Type
Tumor Grade
GB-21
Glioblastoma
grade IV
GB-22
Glioblastoma
grade IV
GBO-24
Glioblastoma with oligodendroglial component
grade IV
GB-25
Glioblastoma
grade IV
GBO-26
Glioblastoma with oligodendroglioma
grade IV
GB-28
Glioblastoma
grade IV
GB-34
Glioblastoma
grade IV
Table 2 :
2Primer sequencesGene
Forward Primer
Reverse Primer
SMAD4
TCCAACACCCGCCAAGTAAT
GCTGGCTGAGCAGTAAATCC
MMP9
GCGTGTCTGGAGATTCGACTT
TATCCACGCGAATGACGCT
β-Actin
GGATTCCATACCCAAGAAGGA
GGATTCCATACCCAAGAAGGA
Cat No: 4464066) and miRNA inhibitors (Exiqon, Cat No: 4100679-001). BV2 cells were also transfected with scrambled mimics (Ambion, Cat No: 4464058) or inhibitors (Exiqon, Cat No: 199006-001) as negative controls. The mimic and inhibitors complexes for transfection were prepared using X-tremeGENE siRNA Transfection Reagent (Roche, Cat No: 04476093001) in Opti-MEM medium at concentrations of 20 and 40nM, respectively. RNA and protein analyses were performed at 48h and 72h post transfection, respectively. Protein extracts from BV2 microglial cells were obtained using a cocktail of M-PER ™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Cat No: 78501), Halt ™ Protease Inhibitor (ThermoFisher Scientific, Cat No: 78430) and phosphatase inhibitor (ThermoFisher Scientific, Cat No: 78427). Protein quantification was performed by Bradford assay using the Bio-Rad protein Assay Kit (Bio-Rad, Cat No: 5000001). Total protein lysate (30μg) was denatured at 95ºC for 10min. Proteins were loaded onto a 10% SDS-polyacrylamide gel (PAGE) electrophoresis setup and transferred to membranes (Bio-Rad, Cat No: 162-0177). Blocking of non-specific sites on the membrane was done using 5% milk or bovine serum albumin (BSA). Membranes were incubated overnight at 4ºC with primary antibodies as follows: SMAD4-1:1000 (Cell Signaling Technology, Cat No: 9515), VEGFa-1:500 (SantaCruz, Cat No: sc-152) and MMP9-1:2000 (EMD Millipore, Cat No: AB19016) and subsequently incubated with horseradish peroxidase conjugated secondary antibodies (ThermoFisher Scientific, Cat No: 31430, Cat No: 31460) 1h. Pico Chemiluminescent substrate (ThermoFisher Scientific, Cat No: 37070) was used to develop the blots and the protein expression level was quantified densitometrically (Bio-Rad Quantity One ® 1-D Analysis Software, Cat No: 1709600).Western blotting
ACKNOWLEDGMENTSThe authors would like to acknowledge Ms. Ashwini Karanth for some technical assistance in the project.CONFLICTS OF INTERESTThe authors declare no conflicts of interest.
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"Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFβ level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell linebased model, increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFβ signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned www.oncotarget.com medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability."
] | [
"Aparna Karthikeyan \nDepartment of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n",
"Neelima Gupta \nDepartment of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n",
"Carol Tang \nDepartment of Research\nNational Neuroscience Institute\nSingapore\n\nDuke-NUS Medical School\nSingapore\n\nDivision of Cellular and Molecular Research\nNational Cancer Centre\nSingapore\n",
"Karthik Mallilankaraman \nDepartment of Physiology\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n",
"Maskomani Silambarasan \nDepartment of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n",
"Meng Shi \nSchool of Biological Sciences\nNanyang Technological University\nSingapore\n",
"Lei Lu \nSchool of Biological Sciences\nNanyang Technological University\nSingapore\n",
"Beng Ti Ang \nDepartment of Physiology\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n\nDuke-NUS Medical School\nSingapore\n\nDepartment of Neurosurgery\nNational Neuroscience Institute\nSingapore\n\nSingapore Institute for Clinical Sciences\nA*STARSingapore\n",
"Eng-Ang Ling \nDepartment of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n",
"S Thameem Dheen \nDepartment of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore\n"
] | [
"Department of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"Department of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"Department of Research\nNational Neuroscience Institute\nSingapore",
"Duke-NUS Medical School\nSingapore",
"Division of Cellular and Molecular Research\nNational Cancer Centre\nSingapore",
"Department of Physiology\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"Department of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"School of Biological Sciences\nNanyang Technological University\nSingapore",
"School of Biological Sciences\nNanyang Technological University\nSingapore",
"Department of Physiology\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"Duke-NUS Medical School\nSingapore",
"Department of Neurosurgery\nNational Neuroscience Institute\nSingapore",
"Singapore Institute for Clinical Sciences\nA*STARSingapore",
"Department of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore",
"Department of Anatomy\nYong Loo Lin School of Medicine\nNational University of Singapore\nSingapore"
] | [
"Aparna",
"Neelima",
"Carol",
"Karthik",
"Maskomani",
"Meng",
"Lei",
"Beng",
"Eng-Ang",
"S",
"Thameem"
] | [
"Karthikeyan",
"Gupta",
"Tang",
"Mallilankaraman",
"Silambarasan",
"Shi",
"Lu",
"Ti Ang",
"Ling",
"Dheen"
] | [
"Y Shi, ",
"Y F Ping, ",
"X Zhang, ",
"X W Bian, ",
"M Erreni, ",
"G Solinas, ",
"P Brescia, ",
"D Osti, ",
"F Zunino, ",
"P Colombo, ",
"A Destro, ",
"M Roncalli, ",
"A Mantovani, ",
"R Draghi, ",
"D Levi, ",
"Y Rodriguez, ",
"R Baena, ",
"P Gaetani, ",
"A Svensson, ",
"I Özen, ",
"G Genové, ",
"G Paul, ",
"J Bengzon, ",
"M B Graeber, ",
"B W Scheithauer, ",
"G W Kreutzberg, ",
"W Roggendorf, ",
"S Strupp, ",
"W Paulus, ",
"S T Dheen, ",
"C Kaur, ",
"E A Ling, ",
"I Francos-Quijorna, ",
"J Amo-Aparicio, ",
"A Martinez-Muriana, ",
"R López-Vales, ",
"V E Miron, ",
"A Boyd, ",
"J W Zhao, ",
"T J Yuen, ",
"J M Ruckh, ",
"J L Shadrach, ",
"P Van Wijngaarden, ",
"A J Wagers, ",
"A Williams, ",
"R J Franklin, ",
"A Kumar, ",
"D M Alvarez-Croda, ",
"B A Stoica, ",
"A I Faden, ",
"D J Loane, ",
"Y Tang, ",
"W Le, ",
"A Grimaldi, ",
"D 'alessandro, ",
"G Golia, ",
"M T Grössinger, ",
"E M , ",
"Di Angelantonio, ",
"S Ragozzino, ",
"D Santoro, ",
"A Esposito, ",
"V Wulff, ",
"H Catalano, ",
"M Limatola, ",
"C , ",
"M Prosniak, ",
"L A Harshyne, ",
"D W Andrews, ",
"L C Kenyon, ",
"K Bedelbaeva, ",
"T V Apanasovich, ",
"E Heber-Katz, ",
"M T Curtis, ",
"P Cotzia, ",
"D C Hooper, ",
"F Szulzewsky, ",
"A Pelz, ",
"X Feng, ",
"M Synowitz, ",
"D Markovic, ",
"T Langmann, ",
"I R Holtman, ",
"X Wang, ",
"B J Eggen, ",
"H W Boddeke, ",
"D Hambardzumyan, ",
"S A Wolf, ",
"H Kettenmann, ",
"K Gabrusiewicz, ",
"B Rodriguez, ",
"J Wei, ",
"Y Hashimoto, ",
"L M Healy, ",
"S N Maiti, ",
"G Thomas, ",
"S Zhou, ",
"Q Wang, ",
"A Elakkad, ",
"B D Liebelt, ",
"N K Yaghi, ",
"R Ezhilarasan, ",
"A Ellert-Miklaszewska, ",
"M Dabrowski, ",
"M Lipko, ",
"M Sliwa, ",
"M Maleszewska, ",
"B Kaminska, ",
"D Hambardzumyan, ",
"D H Gutmann, ",
"H Kettenmann, ",
"L Attisano, ",
"J L Wrana, ",
"K S Chung, ",
"S H Cho, ",
"J S Shin, ",
"D H Kim, ",
"J H Choi, ",
"S Y Choi, ",
"Y K Rhee, ",
"H D Hong, ",
"K T Lee, ",
"K E Craven, ",
"J Gore, ",
"J L Wilson, ",
"M Korc, ",
"J H Yuan, ",
"F Yang, ",
"F Wang, ",
"J Z Ma, ",
"Y J Guo, ",
"Q F Tao, ",
"F Liu, ",
"W Pan, ",
"T T Wang, ",
"C C Zhou, ",
"S B Wang, ",
"Y Z Wang, ",
"Y Yang, ",
"Y L Juang, ",
"Y M Jeng, ",
"C L Chen, ",
"H C Lien, ",
"I Tarhoni, ",
"G C Lobato, ",
"C Fhied, ",
"J Borgia, ",
"M Pergande, ",
"Y W Chai, ",
"A Bruna, ",
"R S Darken, ",
"F Rojo, ",
"A Ocaña, ",
"S Peñuelas, ",
"A Arias, ",
"R Paris, ",
"A Tortosa, ",
"J Mora, ",
"J Baselga, ",
"J Seoane, ",
"P J Eichhorn, ",
"L Rodón, ",
"A Gonzàlez-Juncà, ",
"A Dirac, ",
"M Gili, ",
"E Martínez-Sáez, ",
"Aura C Barba, ",
"I Peg, ",
"V Prat, ",
"A Cuartas, ",
"I Jimenez, ",
"J García-Dorado, ",
"D , ",
"R G Verhaak, ",
"K A Hoadley, ",
"E Purdom, ",
"V Wang, ",
"Y Qi, ",
"M D Wilkerson, ",
"C R Miller, ",
"L Ding, ",
"T Golub, ",
"J P Mesirov, ",
"G Alexe, ",
"M Lawrence, ",
"O' Kelly, ",
"M , ",
"P T FullertonJr, ",
"C J Creighton, ",
"M M Matzuk, ",
"N Bardeesy, ",
"K H Cheng, ",
"J H Berger, ",
"G C Chu, ",
"J Pahler, ",
"P Olson, ",
"A F Hezel, ",
"J Horner, ",
"G Y Lauwers, ",
"D Hanahan, ",
"R A Depinho, ",
"J V Joseph, ",
"V Balasubramaniyan, ",
"A Walenkamp, ",
"F A Kruyt, ",
"A Wesolowska, ",
"A Kwiatkowska, ",
"L Slomnicki, ",
"M Dembinski, ",
"A Master, ",
"M Sliwa, ",
"K Franciszkiewicz, ",
"S Chouaib, ",
"B Kaminska, ",
"S P Jadhav, ",
"S P Kamath, ",
"M Choolani, ",
"J Lu, ",
"S T Dheen, ",
"R Patnala, ",
"T V Arumugam, ",
"N Gupta, ",
"S T Dheen, ",
"A Karthikeyan, ",
"R Patnala, ",
"S P Jadhav, ",
"L Eng-Ang, ",
"S T Dheen, ",
"K D Taganov, ",
"M P Boldin, ",
"K J Chang, ",
"D Baltimore, ",
"A Iyer, ",
"E Zurolo, ",
"A Prabowo, ",
"K Fluiter, ",
"W G Spliet, ",
"P C Van Rijen, ",
"J A Gorter, ",
"E Aronica, ",
"R Saba, ",
"D L Sorensen, ",
"S A Booth, ",
"J Mei, ",
"R Bachoo, ",
"C L Zhang, ",
"J Massagué, ",
"J Seoane, ",
"D Wotton, ",
"D R Rhodes, ",
"J Yu, ",
"K Shanker, ",
"N Deshpande, ",
"R Varambally, ",
"D Ghosh, ",
"T Barrette, ",
"A Pandey, ",
"A M Chinnaiyan, ",
"W J Lukiw, ",
"Y Zhao, ",
"J G Cui, ",
"R Saba, ",
"S Gushue, ",
"R L Huzarewich, ",
"K Manguiat, ",
"S Medina, ",
"C Robertson, ",
"S A Booth, ",
"C N Parkhurst, ",
"G Yang, ",
"I Ninan, ",
"J N Savas, ",
"Yates Jr 3rd, ",
"J J Lafaille, ",
"B L Hempstead, ",
"D R Littman, ",
"W B Gan, ",
"D P Schafer, ",
"E K Lehrman, ",
"A G Kautzman, ",
"R Koyama, ",
"A R Mardinly, ",
"R Yamasaki, ",
"R M Ransohoff, ",
"M E Greenberg, ",
"R C Paolicelli, ",
"G Bolasco, ",
"F Pagani, ",
"L Maggi, ",
"M Scianni, ",
"P Panzanelli, ",
"M Giustetto, ",
"T A Ferreira, ",
"E Guiducci, ",
"L Dumas, ",
"D Ragozzino, ",
"C T Gross, ",
"C Kaur, ",
"G Rathnasamy, ",
"E A Ling, ",
"H Zhai, ",
"F L Heppner, ",
"S E Tsirka, ",
"F F Resende, ",
"X Bai, ",
"Del Bel, ",
"E A Kirchhoff, ",
"F Scheller, ",
"A Titze-De-Almeida, ",
"R , ",
"N Richter, ",
"S Wendt, ",
"P B Georgieva, ",
"D Hambardzumyan, ",
"C Nolte, ",
"H Kettenmann, ",
"Y Zheng, ",
"W Yang, ",
"K Aldape, ",
"J He, ",
"Z Lu, ",
"C Nolte, ",
"F Kirchhoff, ",
"H Kettenmann, ",
"De Simone, ",
"R Ambrosini, ",
"E Carnevale, ",
"D , ",
"Ajmone-Cat , ",
"M A Minghetti, ",
"L , ",
"A Suzumura, ",
"M Sawada, ",
"H Yamamoto, ",
"T Marunouchi, ",
"D M Norden, ",
"A M Fenn, ",
"A Dugan, ",
"J P Godbout, ",
"B Spittau, ",
"L Wullkopf, ",
"X Zhou, ",
"J Rilka, ",
"D Pfeifer, ",
"K Krieglstein, ",
"Y Le, ",
"P Iribarren, ",
"W Gong, ",
"Y Cui, ",
"X Zhang, ",
"J M Wang, ",
"J E Tichauer, ",
"Von Bernhardi, ",
"R , ",
"X Liu, ",
"Y Qin, ",
"A Dai, ",
"Y Zhang, ",
"H Xue, ",
"H Ni, ",
"L Han, ",
"L Zhu, ",
"D Yuan, ",
"T Tao, ",
"M Cao, ",
"I Costello, ",
"C A Biondi, ",
"J M Taylor, ",
"E K Bikoff, ",
"E J Robertson, ",
"L Levy, ",
"C S Hill, ",
"H Kong, ",
"A Omran, ",
"M U Ashhab, ",
"N Gan, ",
"J Peng, ",
"F He, ",
"L Wu, ",
"X Deng, ",
"F Yin, ",
"P N Alexandrov, ",
"P Dua, ",
"W J Lukiw, ",
"M Deng, ",
"G Du, ",
"J Zhao, ",
"X Du, ",
"M Katakowski, ",
"B Buller, ",
"X Zheng, ",
"Y Lu, ",
"T Rogers, ",
"O Osobamiro, ",
"W Shu, ",
"F Jiang, ",
"M Chopp, ",
"Y J Kim, ",
"S Y Hwang, ",
"J S Hwang, ",
"J W Lee, ",
"E S Oh, ",
"I O Han, ",
"X Shen, ",
"M A Burguillos, ",
"A M Osman, ",
"J Frijhoff, ",
"A Carrillo-Jiménez, ",
"S Kanatani, ",
"M Augsten, ",
"D Saidi, ",
"J Rodhe, ",
"E Kavanagh, ",
"A Rongvaux, ",
"V Rraklli, ",
"U Nyman, ",
"K Mallilankaraman, ",
"C Cárdenas, ",
"P J Doonan, ",
"H C Chandramoorthy, ",
"K M Irrinki, ",
"T Golenár, ",
"G Csordás, ",
"P Madireddi, ",
"J Yang, ",
"M Müller, ",
"R Miller, ",
"J E Kolesar, ",
"J Molgó, ",
"K J Livak, ",
"T D Schmittgen, "
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"Hambardzumyan",
"Wolf",
"Kettenmann",
"Gabrusiewicz",
"Rodriguez",
"Wei",
"Hashimoto",
"Healy",
"Maiti",
"Thomas",
"Zhou",
"Wang",
"Elakkad",
"Liebelt",
"Yaghi",
"Ezhilarasan",
"Ellert-Miklaszewska",
"Dabrowski",
"Lipko",
"Sliwa",
"Maleszewska",
"Kaminska",
"Hambardzumyan",
"Gutmann",
"Kettenmann",
"Attisano",
"Wrana",
"Chung",
"Cho",
"Shin",
"Kim",
"Choi",
"Choi",
"Rhee",
"Hong",
"Lee",
"Craven",
"Gore",
"Wilson",
"Korc",
"Yuan",
"Yang",
"Wang",
"Ma",
"Guo",
"Tao",
"Liu",
"Pan",
"Wang",
"Zhou",
"Wang",
"Wang",
"Yang",
"Juang",
"Jeng",
"Chen",
"Lien",
"Tarhoni",
"Lobato",
"Fhied",
"Borgia",
"Pergande",
"Chai",
"Bruna",
"Darken",
"Rojo",
"Ocaña",
"Peñuelas",
"Arias",
"Paris",
"Tortosa",
"Mora",
"Baselga",
"Seoane",
"Eichhorn",
"Rodón",
"Gonzàlez-Juncà",
"Dirac",
"Gili",
"Martínez-Sáez",
"Barba",
"Peg",
"Prat",
"Cuartas",
"Jimenez",
"García-Dorado",
"Verhaak",
"Hoadley",
"Purdom",
"Wang",
"Qi",
"Wilkerson",
"Miller",
"Ding",
"Golub",
"Mesirov",
"Alexe",
"Lawrence",
"Kelly",
"Fullerton",
"Creighton",
"Matzuk",
"Bardeesy",
"Cheng",
"Berger",
"Chu",
"Pahler",
"Olson",
"Hezel",
"Horner",
"Lauwers",
"Hanahan",
"Depinho",
"Joseph",
"Balasubramaniyan",
"Walenkamp",
"Kruyt",
"Wesolowska",
"Kwiatkowska",
"Slomnicki",
"Dembinski",
"Master",
"Sliwa",
"Franciszkiewicz",
"Chouaib",
"Kaminska",
"Jadhav",
"Kamath",
"Choolani",
"Lu",
"Dheen",
"Patnala",
"Arumugam",
"Gupta",
"Dheen",
"Karthikeyan",
"Patnala",
"Jadhav",
"Eng-Ang",
"Dheen",
"Taganov",
"Boldin",
"Chang",
"Baltimore",
"Iyer",
"Zurolo",
"Prabowo",
"Fluiter",
"Spliet",
"Van Rijen",
"Gorter",
"Aronica",
"Saba",
"Sorensen",
"Booth",
"Mei",
"Bachoo",
"Zhang",
"Massagué",
"Seoane",
"Wotton",
"Rhodes",
"Yu",
"Shanker",
"Deshpande",
"Varambally",
"Ghosh",
"Barrette",
"Pandey",
"Chinnaiyan",
"Lukiw",
"Zhao",
"Cui",
"Saba",
"Gushue",
"Huzarewich",
"Manguiat",
"Medina",
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"Booth",
"Parkhurst",
"Yang",
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"Lafaille",
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"Gan",
"Schafer",
"Lehrman",
"Kautzman",
"Koyama",
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"Greenberg",
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] | [
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"Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions. N Richter, S Wendt, P B Georgieva, D Hambardzumyan, C Nolte, H Kettenmann, Neurosci Lett. 583Richter N, Wendt S, Georgieva PB, Hambardzumyan D, Nolte C, Kettenmann H. Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions. Neurosci Lett. 2014; 583:130-35.",
"Epidermal growth factor (EGF)-enhanced vascular cell adhesion molecule-1 (VCAM-1) expression promotes macrophage and glioblastoma cell interaction and tumor cell invasion. Y Zheng, W Yang, K Aldape, J He, Z Lu, J Biol Chem. 288Zheng Y, Yang W, Aldape K, He J, Lu Z. Epidermal growth factor (EGF)-enhanced vascular cell adhesion molecule-1 (VCAM-1) expression promotes macrophage and glioblastoma cell interaction and tumor cell invasion. J Biol Chem. 2013; 288:31488-95.",
"Epidermal growth factor is a motility factor for microglial cells in vitro: evidence for EGF receptor expression. C Nolte, F Kirchhoff, H Kettenmann, Eur J Neurosci. 9Nolte C, Kirchhoff F, Kettenmann H. Epidermal growth factor is a motility factor for microglial cells in vitro: evidence for EGF receptor expression. Eur J Neurosci. 1997; 9:1690-98.",
"NGF promotes microglial migration through the activation of its high affinity receptor: modulation by TGF-β. De Simone, R Ambrosini, E Carnevale, D , Ajmone-Cat , M A Minghetti, L , J Neuroimmunol. 190De Simone R, Ambrosini E, Carnevale D, Ajmone-Cat MA, Minghetti L. NGF promotes microglial migration through the activation of its high affinity receptor: modulation by TGF-β. J Neuroimmunol. 2007; 190:53-60.",
"Transforming growth factor-beta suppresses activation www.oncotarget.com and proliferation of microglia in vitro. A Suzumura, M Sawada, H Yamamoto, T Marunouchi, J Immunol. 151Suzumura A, Sawada M, Yamamoto H, Marunouchi T. Transforming growth factor-beta suppresses activation www.oncotarget.com and proliferation of microglia in vitro. J Immunol. 1993; 151:2150-58.",
"TGFβ produced by IL-10 redirected astrocytes attenuates microglial activation. D M Norden, A M Fenn, A Dugan, J P Godbout, 53. 53Glia. 6253. 53. Norden DM, Fenn AM, Dugan A, Godbout JP. TGFβ produced by IL-10 redirected astrocytes attenuates microglial activation. Glia. 2014; 62:881-95.",
"Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro. B Spittau, L Wullkopf, X Zhou, J Rilka, D Pfeifer, K Krieglstein, Glia. 61Spittau B, Wullkopf L, Zhou X, Rilka J, Pfeifer D, Krieglstein K. Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro. Glia. 2013; 61:287-300.",
"TGF-β1 disrupts endotoxin signaling in microglial cells through Smad3 and MAPK pathways. Y Le, P Iribarren, W Gong, Y Cui, X Zhang, J M Wang, J Immunol. 173Le Y, Iribarren P, Gong W, Cui Y, Zhang X, Wang JM. TGF-β1 disrupts endotoxin signaling in microglial cells through Smad3 and MAPK pathways. J Immunol. 2004; 173:962-68.",
"Transforming growth factor-β stimulates β amyloid uptake by microglia through Smad3-dependent mechanisms. J E Tichauer, Von Bernhardi, R , J Neurosci Res. 90Tichauer JE, von Bernhardi R. Transforming growth factor-β stimulates β amyloid uptake by microglia through Smad3-dependent mechanisms. J Neurosci Res. 2012; 90:1970-80.",
"SMAD4 is involved in the development of endotoxin tolerance in microglia. X Liu, Y Qin, A Dai, Y Zhang, H Xue, H Ni, L Han, L Zhu, D Yuan, T Tao, M Cao, Cell Mol Neurobiol. 36Liu X, Qin Y, Dai A, Zhang Y, Xue H, Ni H, Han L, Zhu L, Yuan D, Tao T, Cao M. SMAD4 is involved in the development of endotoxin tolerance in microglia. Cell Mol Neurobiol. 2016; 36:777-88.",
"Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development. I Costello, C A Biondi, J M Taylor, E K Bikoff, E J Robertson, BMC Dev Biol. 954Costello I, Biondi CA, Taylor JM, Bikoff EK, Robertson EJ. Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development. BMC Dev Biol. 2009; 9:54.",
"Smad4 dependency defines two classes of transforming growth factor {β} (TGF-{β}) target genes and distinguishes TGF-{β}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses. L Levy, C S Hill, Mol Cell Biol. 25Levy L, Hill CS. Smad4 dependency defines two classes of transforming growth factor {β} (TGF-{β}) target genes and distinguishes TGF-{β}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses. Mol Cell Biol. 2005; 25:8108-25.",
"Changes in microglial inflammationrelated and brain-enriched MicroRNAs expressions in response to in vitro oxygen-glucose deprivation. H Kong, A Omran, M U Ashhab, N Gan, J Peng, F He, L Wu, X Deng, F Yin, Neurochem Res. 39Kong H, Omran A, Ashhab MU, Gan N, Peng J, He F, Wu L, Deng X, Yin F. Changes in microglial inflammation- related and brain-enriched MicroRNAs expressions in response to in vitro oxygen-glucose deprivation. Neurochem Res. 2014; 39:233-43.",
"Up-regulation of miRNA-146a in progressive, age-related inflammatory neurodegenerative disorders of the human CNS. P N Alexandrov, P Dua, W J Lukiw, Front Neurol. 5181Alexandrov PN, Dua P, Lukiw WJ. Up-regulation of miRNA-146a in progressive, age-related inflammatory neurodegenerative disorders of the human CNS. Front Neurol. 2014; 5:181.",
"miR-146a negatively regulates the induction of proinflammatory cytokines in response to Japanese encephalitis virus infection in microglial cells. M Deng, G Du, J Zhao, X Du, Arch Virol. 162Deng M, Du G, Zhao J, Du X. miR-146a negatively regulates the induction of proinflammatory cytokines in response to Japanese encephalitis virus infection in microglial cells. Arch Virol. 2017; 162:1495-505.",
"Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. M Katakowski, B Buller, X Zheng, Y Lu, T Rogers, O Osobamiro, W Shu, F Jiang, M Chopp, Cancer Lett. 335Katakowski M, Buller B, Zheng X, Lu Y, Rogers T, Osobamiro O, Shu W, Jiang F, Chopp M. Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett. 2013; 335:201-04.",
"C6 glioma cell insoluble matrix components enhance interferon-γ-stimulated inducible nitric-oxide synthase/ nitric oxide production in BV2 microglial cells. Y J Kim, S Y Hwang, J S Hwang, J W Lee, E S Oh, I O Han, J Biol Chem. 283Kim YJ, Hwang SY, Hwang JS, Lee JW, Oh ES, Han IO. C6 glioma cell insoluble matrix components enhance interferon-γ-stimulated inducible nitric-oxide synthase/ nitric oxide production in BV2 microglial cells. J Biol Chem. 2008; 283:2526-33.",
"Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype. X Shen, M A Burguillos, A M Osman, J Frijhoff, A Carrillo-Jiménez, S Kanatani, M Augsten, D Saidi, J Rodhe, E Kavanagh, A Rongvaux, V Rraklli, U Nyman, Nat Immunol. 17Shen X, Burguillos MA, Osman AM, Frijhoff J, Carrillo- Jiménez A, Kanatani S, Augsten M, Saidi D, Rodhe J, Kavanagh E, Rongvaux A, Rraklli V, Nyman U, et al. Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype. Nat Immunol. 2016; 17:1282-90.",
"MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. K Mallilankaraman, C Cárdenas, P J Doonan, H C Chandramoorthy, K M Irrinki, T Golenár, G Csordás, P Madireddi, J Yang, M Müller, R Miller, J E Kolesar, J Molgó, Nat Cell Biol. 14Mallilankaraman K, Cárdenas C, Doonan PJ, Chandramoorthy HC, Irrinki KM, Golenár T, Csordás G, Madireddi P, Yang J, Müller M, Miller R, Kolesar JE, Molgó J, et al. MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. Nat Cell Biol. 2012; 14:1336-43.",
"Analysis of relative gene expression data using real-time quantitative PCR and the 2. K J Livak, T D Schmittgen, C(T)) Method. Methods. 25Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Δ Δ C(T)) Method. Methods. 2001; 25:402-08."
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"Hostile takeover: glioma stem cells recruit TAMs to support tumor progression",
"Human glioblastoma tumours and neural cancer stem cells express the chemokine CX3CL1 and its receptor CX3CR1",
"Endogenous brain pericytes are widely activated and contribute to mouse glioma microvasculature",
"Microglia in brain tumors",
"Distribution and characterization of microglia/macrophages in human brain tumors",
"Microglial activation and its implications in the brain diseases",
"IL-4 drives microglia and macrophages toward a phenotype conducive for tissue repair and functional recovery after spinal cord injury",
"Ffrench-Constant C. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination",
"Microglial/macrophage polarization dynamics following traumatic brain injury",
"Differential roles of M1 and M2 microglia in neurodegenerative diseases",
"KCa3.1 inhibition switches the phenotype of glioma-infiltrating microglia/macrophages",
"Glioma grade is associated with the accumulation and activity of cells bearing M2 monocyte markers",
"Gliomaassociated microglia/macrophages display an expression profile different from M1 and M2 polarization and highly express Gpnmb and Spp1",
"Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype",
"Molecular definition of the pro-tumorigenic phenotype of glioma-activated microglia",
"The role of microglia and macrophages in glioma maintenance and progression",
"Signal transduction by the TGF-β superfamily",
"Ginsenoside Rh2 induces cell cycle arrest and differentiation in human leukemia cells by upregulating TGF-β expression",
"Abstract 3400: an angiogenesis gene signature points to active TGF-beta/ JAK signaling pathways in a subset of human pancreatic ductal adenocarcinoma cancer patients that are distinct from pathways in pancreatic neuroendocrine tumors",
"A long noncoding RNA activated by TGF-β promotes the invasion-metastasis cascade in hepatocellular carcinoma",
"PRRX2 as a novel TGF-β-induced factor enhances invasion and migration in mammary epithelial cell and correlates with poor prognosis in breast cancer",
"Abstract 1690: IGF-1 and TGF-β promote EMT and angiogenesis in 3D cultures of lung adenocarcinoma cells: a pilot study",
"High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene",
"USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma",
"and Cancer Genome Atlas Research Network. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1",
"Insights into SMAD4 loss in pancreatic cancer from inducible restoration of TGF-β signaling",
"Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer",
"TGF-β as a therapeutic target in high grade gliomas -promises and challenges",
"Microglia-derived TGF-beta as an important regulator of glioblastoma invasion-an inhibition of TGFbeta-dependent effects by shrna against human TGF-beta type II receptor",
"microRNA-200b modulates microglia-mediated neuroinflammation via the cJun/MAPK pathway",
"HDAC inhibitor sodium butyrate-mediated epigenetic regulation enhances neuroprotective function of microglia during ischemic stroke",
"Micrornas: key players in microglia and astrocyte mediated inflammation in cns pathologies",
"NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses",
"MicroRNA-146a: a key regulator of astrocyte-mediated inflammatory response",
"MicroRNA-146a: a dominant, negative regulator of the innate immune response",
"MicroRNA-146a inhibits glioma development by targeting Notch1",
"Smad transcription factors",
"ONCOMINE: a cancer microarray database and integrated data-mining platform",
"An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in Alzheimer disease and in stressed human brain cells",
"MicroRNA 146a (miR-146a) is over-expressed during prion disease and modulates the innate immune response and the microglial activation state",
"Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor",
"Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner",
"Synaptic pruning by microglia is necessary for normal brain development",
"Biology of microglia in the developing brain",
"Microglia/macrophages promote glioma progression",
"Evaluation of TgH(CX3CR1-EGFP) mice implanted with mCherry-GL261 cells as an in vivo model for morphometrical analysis of glioma-microglia interaction",
"Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions",
"Epidermal growth factor (EGF)-enhanced vascular cell adhesion molecule-1 (VCAM-1) expression promotes macrophage and glioblastoma cell interaction and tumor cell invasion",
"Epidermal growth factor is a motility factor for microglial cells in vitro: evidence for EGF receptor expression",
"NGF promotes microglial migration through the activation of its high affinity receptor: modulation by TGF-β",
"Transforming growth factor-beta suppresses activation www.oncotarget.com and proliferation of microglia in vitro",
"TGFβ produced by IL-10 redirected astrocytes attenuates microglial activation",
"Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro",
"TGF-β1 disrupts endotoxin signaling in microglial cells through Smad3 and MAPK pathways",
"Transforming growth factor-β stimulates β amyloid uptake by microglia through Smad3-dependent mechanisms",
"SMAD4 is involved in the development of endotoxin tolerance in microglia",
"Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development",
"Smad4 dependency defines two classes of transforming growth factor {β} (TGF-{β}) target genes and distinguishes TGF-{β}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses",
"Changes in microglial inflammationrelated and brain-enriched MicroRNAs expressions in response to in vitro oxygen-glucose deprivation",
"Up-regulation of miRNA-146a in progressive, age-related inflammatory neurodegenerative disorders of the human CNS",
"miR-146a negatively regulates the induction of proinflammatory cytokines in response to Japanese encephalitis virus infection in microglial cells",
"Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth",
"C6 glioma cell insoluble matrix components enhance interferon-γ-stimulated inducible nitric-oxide synthase/ nitric oxide production in BV2 microglial cells",
"Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype",
"MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism",
"Analysis of relative gene expression data using real-time quantitative PCR and the 2"
] | [
"Cell Stem Cell",
"Eur J Cancer",
"PLoS One",
"Glia",
"Acta Neuropathol",
"Curr Med Chem",
"Glia",
"Nat Neurosci",
"J Neurotrauma",
"Mol Neurobiol",
"Cell Death Dis",
"Clin Cancer Res",
"PLoS One",
"JCI Insight",
"Glia",
"Nat Neurosci",
"Science",
"Carcinogenesis",
"Cancer Res",
"Cancer Cell",
"Mol Carcinog",
"Cancer Res",
"Cancer Cell",
"Nat Med",
"Cancer Cell",
"Mol Endocrinol",
"Genes Dev",
"Biochem Pharmacol",
"Oncogene",
"J Neurochem",
"Mol Neurobiol",
"Curr Med Chem",
"Proc Natl Acad Sci USA",
"PLoS One",
"Front Immunol",
"Mol Cell Biol",
"Genes Dev",
"Neoplasia",
"J Biol Chem",
"PLoS One",
"Cell",
"Neuron",
"Science",
"J Neuropathol Exp Neurol",
"Glia",
"BMC Cancer",
"Neurosci Lett",
"J Biol Chem",
"Eur J Neurosci",
"J Neuroimmunol",
"J Immunol",
"Glia",
"Glia",
"J Immunol",
"J Neurosci Res",
"Cell Mol Neurobiol",
"BMC Dev Biol",
"Mol Cell Biol",
"Neurochem Res",
"Front Neurol",
"Arch Virol",
"Cancer Lett",
"J Biol Chem",
"Nat Immunol",
"Nat Cell Biol",
"C(T)) Method. Methods"
] | [
"\nFigure 1 :\n1GCM induces TGFβ signaling pathway in microglia. ELISA assay quantification revealed that GCM contains ~5ng/ml of TGFβ (A).",
"\nFigure 2 :\n2SMAD4 and tumor supportive genes MMP9 and VEGFa are upregulated in glioma associated microglia.",
"\nFigure 3 :\n3SMAD4 is expressed in microglia associated with human glioblastoma samples. An analysis of the TCGA profiling data reveals a significant increase in SMAD4 mRNA levels in brain glioblastoma tissue as compared to normal non-malignant tissue (A). Panel shows expression of SMAD4 in human glioblastoma tumor tissue (GB 34) (B). Note that there is evident expression of SMAD4 (red) expression in Iba1 positive microglial cells (green). Cell nuclei are labelled with DAPI (blue). Scale=30μm (low magnification), Scale=10μm (high magnification). Table depicts the percentage of Iba1 positive microglial cells per tumor (C). www.oncotarget.com",
"\nFigure 4 :\n4SMAD4 regulates expression of tumor supportive factor MMP9 in microglia. Quantitative RT-PCR and western blot analyses shows a decrease in mRNA and protein expression of SMAD4 in shSMAD4 BV2 cells as compared to negative control (A, C). Data represent Mean±SD, (n=3), Students t-test, *** p<0.001. Quantitative RT-PCR and western blot analyses show that shRNAmediated knockdown of SMAD4 resulted in a decrease in the mRNA and protein expression of MMP9 (B, C). Mean±SD, (n=4), Students t-test, * p<0.05.",
"\nFigure 5 :\n5shRNA-mediated knockdown of SMAD4 suppresses microglial migration. Trans-well membrane image panels",
"\nFigure 6 :\n6Effect of conditioned medium derived from shSMAD4 knockdown microglial cells on glioma cell viability.Histogram depicts MTS assay absorbance values at 24, 48 and 72h after treatment of glioma cells with conditioned medium derived from microglia. A significant increase in glioma viability was observed after treatment with microglia conditioned medium at all time points (A). This was further confirmed using alamar blue viability assay wherein an increase in % of reduction of alamar blue was observed in glioma cells treated with microglia conditioned medium at all time points (B). Histogram further shows the MTS assay absorbance values at 24, 48 and 72h treatment of glioma cells with conditioned medium derived from shSMAD4 microglial cells (C). A decrease in absorbance values indicates a decrease in viability of glioma cells after treatment with conditioned medium from shSMAD4 cells as compared to glioma cells treated with negative control medium (C). In addition, alamar blue assay results (D) show a decrease in percentage of alamar blue reduction in glioma cells treated with medium from shSMAD4 microglia as compared to glioma cells treated with negative control medium. Data represent Mean±SD, (n=4), *** p<0.001, **** p<0.0001. www.oncotarget.com",
"\nFigure 7 :\n7MiR-146a is downregulated in GCM treated microglia and targets SMAD4. TargetScan software predicted that miR-146a putatively binds to 3'UTR of SMAD4 mRNA (A).",
"\nFigure 8 :\n8MiR-146a regulates SMAD4 in GCM-treated microglia. Western blot analyses show that transfection of microglial cells with miR-146a mimics suppressed the GCM induced induction of SMAD4 protein (A, C) Data represent mean±SD, (n=3), * p<0.05. MiR-146a inhibition increases the protein levels of SMAD4 upon GCM treatment of microglia as seen by western blot analyses and densitometry",
"\nFigure 9 :\n9MiR-146a alters expression of tumor supportive gene MMP9 in glioma associated microglia and suppressed microglial migration towards GCM. Overexpression of miR-146a suppressed expression of MMP9 at the protein level and conversely, inhibition of miR-146a in microglia was found to increase levels of MMP9 as depicted by immunoblotting and densitometry analysis (A, B). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Image panel shows a decrease in the number of migrated microglial cells (purple) after overexpression of miR-146a in response to GCM as compared to Neg Cont cells (C-F). Histogram depicts fold change in migrating cells (G). Data represent mean±SD, (n=3), ** p<0.01. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com",
"\nFigure 10 :\n10Effect of conditioned medium derived from miR-146a overexpression in microglia on viability of glioma cells. Histogram depicts MTS assay absorbance values 24, 48 and 72h treatment of glioma cells with conditioned medium derived from miR-146a overexpression in microglia. A decrease in absorbance values at all time points indicates an inhibition of viability of glioma cells after treatment with medium from miR-146a overexpressed cells as compared to glioma cells treated with negative control (scrambled miRNA) transfected medium (A). Further, alamar blue assay results show a decrease in percentage of alamar blue reduction, indicating a suppression of glioma cell viability upon treatment with medium from miR-146a overexpressed microglia as compared to glioma cells treated with scrambled control transfected medium (B). Data represent Mean±SD, (n=4), ** p<0.01, **** p<0.0001. www.oncotarget.com",
"\nFigure 11 :\n11SMAD4, regulated by microRNA-146a, promotes microglial cell migration and tumor progression in glioma environment. Glioma cells secrete a number of factors including TGFβ (A) that activates the TGFβ pathway in microglia associated with glioma. Glioma conditioned medium was found to induce phosphorylation of SMAD2/3 complex and increase the level of SMAD4 (B) in microglia. Concurrently, miR-146a which was found to target SMAD4, was downregulated in glioma associated microglia (C). Downregulation of miR-146a in glioma-associated microglia increases the levels of tumor supportive factors (D), including SMAD4 and MMP9 which promote glioma progression and microglial migration. www.oncotarget.com",
"\n\n(Qiagen, Cat No: 217004) as per the manufacturer's instructions. RNA isolate (10ng) was used for conversion of miRNA to cDNA using Universal cDNA Synthesis Kit II (Exiqon, Cat No: 203301). Quantification of miRNA expression in control and GCM treated groups was carried out using ExiLENT SYBR ® Green master mix (Exiqon, Cat No: 203421). LNA-modified miR-146a-5p primers (Exiqon, Cat No: 204688) and small nuclear U6 RNA primers (Exiqon, Cat No: 203907) were used for miRNA real time PCR. cDNA conversion and quantitative Real-time PCR (mRNA) The total RNA (2000ng) was converted to cDNA for gene expression analysis using a master mix consisting of M-MLV Reverse transcriptase enzyme (Promega, Cat No: M170A), M-MLV Reverse Transcriptase 5X Reaction Buffer (Promega, Cat No: M531A), dNTP (Promega, Cat No: U1511) and RNasin ® Ribonuclease inhibitor (Promega, Cat No: N2111) in a 25μl reaction volume.",
"\nTable 1 :\n1Tumor sample grade and typeSample Name \nTumor Type \nTumor Grade \n\nGB-21 \nGlioblastoma \ngrade IV \n\nGB-22 \nGlioblastoma \ngrade IV \n\nGBO-24 \nGlioblastoma with oligodendroglial component \ngrade IV \n\nGB-25 \nGlioblastoma \ngrade IV \n\nGBO-26 \nGlioblastoma with oligodendroglioma \ngrade IV \n\nGB-28 \nGlioblastoma \ngrade IV \n\nGB-34 \nGlioblastoma \ngrade IV \n\n",
"\nTable 2 :\n2Primer sequencesGene \nForward Primer \nReverse Primer \n\nSMAD4 \nTCCAACACCCGCCAAGTAAT \nGCTGGCTGAGCAGTAAATCC \n\nMMP9 \nGCGTGTCTGGAGATTCGACTT \nTATCCACGCGAATGACGCT \n\nβ-Actin \nGGATTCCATACCCAAGAAGGA \nGGATTCCATACCCAAGAAGGA \n",
"\n\nCat No: 4464066) and miRNA inhibitors (Exiqon, Cat No: 4100679-001). BV2 cells were also transfected with scrambled mimics (Ambion, Cat No: 4464058) or inhibitors (Exiqon, Cat No: 199006-001) as negative controls. The mimic and inhibitors complexes for transfection were prepared using X-tremeGENE siRNA Transfection Reagent (Roche, Cat No: 04476093001) in Opti-MEM medium at concentrations of 20 and 40nM, respectively. RNA and protein analyses were performed at 48h and 72h post transfection, respectively. Protein extracts from BV2 microglial cells were obtained using a cocktail of M-PER ™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Cat No: 78501), Halt ™ Protease Inhibitor (ThermoFisher Scientific, Cat No: 78430) and phosphatase inhibitor (ThermoFisher Scientific, Cat No: 78427). Protein quantification was performed by Bradford assay using the Bio-Rad protein Assay Kit (Bio-Rad, Cat No: 5000001). Total protein lysate (30μg) was denatured at 95ºC for 10min. Proteins were loaded onto a 10% SDS-polyacrylamide gel (PAGE) electrophoresis setup and transferred to membranes (Bio-Rad, Cat No: 162-0177). Blocking of non-specific sites on the membrane was done using 5% milk or bovine serum albumin (BSA). Membranes were incubated overnight at 4ºC with primary antibodies as follows: SMAD4-1:1000 (Cell Signaling Technology, Cat No: 9515), VEGFa-1:500 (SantaCruz, Cat No: sc-152) and MMP9-1:2000 (EMD Millipore, Cat No: AB19016) and subsequently incubated with horseradish peroxidase conjugated secondary antibodies (ThermoFisher Scientific, Cat No: 31430, Cat No: 31460) 1h. Pico Chemiluminescent substrate (ThermoFisher Scientific, Cat No: 37070) was used to develop the blots and the protein expression level was quantified densitometrically (Bio-Rad Quantity One ® 1-D Analysis Software, Cat No: 1709600).Western blotting \n\n"
] | [
"GCM induces TGFβ signaling pathway in microglia. ELISA assay quantification revealed that GCM contains ~5ng/ml of TGFβ (A).",
"SMAD4 and tumor supportive genes MMP9 and VEGFa are upregulated in glioma associated microglia.",
"SMAD4 is expressed in microglia associated with human glioblastoma samples. An analysis of the TCGA profiling data reveals a significant increase in SMAD4 mRNA levels in brain glioblastoma tissue as compared to normal non-malignant tissue (A). Panel shows expression of SMAD4 in human glioblastoma tumor tissue (GB 34) (B). Note that there is evident expression of SMAD4 (red) expression in Iba1 positive microglial cells (green). Cell nuclei are labelled with DAPI (blue). Scale=30μm (low magnification), Scale=10μm (high magnification). Table depicts the percentage of Iba1 positive microglial cells per tumor (C). www.oncotarget.com",
"SMAD4 regulates expression of tumor supportive factor MMP9 in microglia. Quantitative RT-PCR and western blot analyses shows a decrease in mRNA and protein expression of SMAD4 in shSMAD4 BV2 cells as compared to negative control (A, C). Data represent Mean±SD, (n=3), Students t-test, *** p<0.001. Quantitative RT-PCR and western blot analyses show that shRNAmediated knockdown of SMAD4 resulted in a decrease in the mRNA and protein expression of MMP9 (B, C). Mean±SD, (n=4), Students t-test, * p<0.05.",
"shRNA-mediated knockdown of SMAD4 suppresses microglial migration. Trans-well membrane image panels",
"Effect of conditioned medium derived from shSMAD4 knockdown microglial cells on glioma cell viability.Histogram depicts MTS assay absorbance values at 24, 48 and 72h after treatment of glioma cells with conditioned medium derived from microglia. A significant increase in glioma viability was observed after treatment with microglia conditioned medium at all time points (A). This was further confirmed using alamar blue viability assay wherein an increase in % of reduction of alamar blue was observed in glioma cells treated with microglia conditioned medium at all time points (B). Histogram further shows the MTS assay absorbance values at 24, 48 and 72h treatment of glioma cells with conditioned medium derived from shSMAD4 microglial cells (C). A decrease in absorbance values indicates a decrease in viability of glioma cells after treatment with conditioned medium from shSMAD4 cells as compared to glioma cells treated with negative control medium (C). In addition, alamar blue assay results (D) show a decrease in percentage of alamar blue reduction in glioma cells treated with medium from shSMAD4 microglia as compared to glioma cells treated with negative control medium. Data represent Mean±SD, (n=4), *** p<0.001, **** p<0.0001. www.oncotarget.com",
"MiR-146a is downregulated in GCM treated microglia and targets SMAD4. TargetScan software predicted that miR-146a putatively binds to 3'UTR of SMAD4 mRNA (A).",
"MiR-146a regulates SMAD4 in GCM-treated microglia. Western blot analyses show that transfection of microglial cells with miR-146a mimics suppressed the GCM induced induction of SMAD4 protein (A, C) Data represent mean±SD, (n=3), * p<0.05. MiR-146a inhibition increases the protein levels of SMAD4 upon GCM treatment of microglia as seen by western blot analyses and densitometry",
"MiR-146a alters expression of tumor supportive gene MMP9 in glioma associated microglia and suppressed microglial migration towards GCM. Overexpression of miR-146a suppressed expression of MMP9 at the protein level and conversely, inhibition of miR-146a in microglia was found to increase levels of MMP9 as depicted by immunoblotting and densitometry analysis (A, B). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Image panel shows a decrease in the number of migrated microglial cells (purple) after overexpression of miR-146a in response to GCM as compared to Neg Cont cells (C-F). Histogram depicts fold change in migrating cells (G). Data represent mean±SD, (n=3), ** p<0.01. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com",
"Effect of conditioned medium derived from miR-146a overexpression in microglia on viability of glioma cells. Histogram depicts MTS assay absorbance values 24, 48 and 72h treatment of glioma cells with conditioned medium derived from miR-146a overexpression in microglia. A decrease in absorbance values at all time points indicates an inhibition of viability of glioma cells after treatment with medium from miR-146a overexpressed cells as compared to glioma cells treated with negative control (scrambled miRNA) transfected medium (A). Further, alamar blue assay results show a decrease in percentage of alamar blue reduction, indicating a suppression of glioma cell viability upon treatment with medium from miR-146a overexpressed microglia as compared to glioma cells treated with scrambled control transfected medium (B). Data represent Mean±SD, (n=4), ** p<0.01, **** p<0.0001. www.oncotarget.com",
"SMAD4, regulated by microRNA-146a, promotes microglial cell migration and tumor progression in glioma environment. Glioma cells secrete a number of factors including TGFβ (A) that activates the TGFβ pathway in microglia associated with glioma. Glioma conditioned medium was found to induce phosphorylation of SMAD2/3 complex and increase the level of SMAD4 (B) in microglia. Concurrently, miR-146a which was found to target SMAD4, was downregulated in glioma associated microglia (C). Downregulation of miR-146a in glioma-associated microglia increases the levels of tumor supportive factors (D), including SMAD4 and MMP9 which promote glioma progression and microglial migration. www.oncotarget.com",
"(Qiagen, Cat No: 217004) as per the manufacturer's instructions. RNA isolate (10ng) was used for conversion of miRNA to cDNA using Universal cDNA Synthesis Kit II (Exiqon, Cat No: 203301). Quantification of miRNA expression in control and GCM treated groups was carried out using ExiLENT SYBR ® Green master mix (Exiqon, Cat No: 203421). LNA-modified miR-146a-5p primers (Exiqon, Cat No: 204688) and small nuclear U6 RNA primers (Exiqon, Cat No: 203907) were used for miRNA real time PCR. cDNA conversion and quantitative Real-time PCR (mRNA) The total RNA (2000ng) was converted to cDNA for gene expression analysis using a master mix consisting of M-MLV Reverse transcriptase enzyme (Promega, Cat No: M170A), M-MLV Reverse Transcriptase 5X Reaction Buffer (Promega, Cat No: M531A), dNTP (Promega, Cat No: U1511) and RNasin ® Ribonuclease inhibitor (Promega, Cat No: N2111) in a 25μl reaction volume.",
"Tumor sample grade and type",
"Primer sequences",
"Cat No: 4464066) and miRNA inhibitors (Exiqon, Cat No: 4100679-001). BV2 cells were also transfected with scrambled mimics (Ambion, Cat No: 4464058) or inhibitors (Exiqon, Cat No: 199006-001) as negative controls. The mimic and inhibitors complexes for transfection were prepared using X-tremeGENE siRNA Transfection Reagent (Roche, Cat No: 04476093001) in Opti-MEM medium at concentrations of 20 and 40nM, respectively. RNA and protein analyses were performed at 48h and 72h post transfection, respectively. Protein extracts from BV2 microglial cells were obtained using a cocktail of M-PER ™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Cat No: 78501), Halt ™ Protease Inhibitor (ThermoFisher Scientific, Cat No: 78430) and phosphatase inhibitor (ThermoFisher Scientific, Cat No: 78427). Protein quantification was performed by Bradford assay using the Bio-Rad protein Assay Kit (Bio-Rad, Cat No: 5000001). Total protein lysate (30μg) was denatured at 95ºC for 10min. Proteins were loaded onto a 10% SDS-polyacrylamide gel (PAGE) electrophoresis setup and transferred to membranes (Bio-Rad, Cat No: 162-0177). Blocking of non-specific sites on the membrane was done using 5% milk or bovine serum albumin (BSA). Membranes were incubated overnight at 4ºC with primary antibodies as follows: SMAD4-1:1000 (Cell Signaling Technology, Cat No: 9515), VEGFa-1:500 (SantaCruz, Cat No: sc-152) and MMP9-1:2000 (EMD Millipore, Cat No: AB19016) and subsequently incubated with horseradish peroxidase conjugated secondary antibodies (ThermoFisher Scientific, Cat No: 31430, Cat No: 31460) 1h. Pico Chemiluminescent substrate (ThermoFisher Scientific, Cat No: 37070) was used to develop the blots and the protein expression level was quantified densitometrically (Bio-Rad Quantity One ® 1-D Analysis Software, Cat No: 1709600)."
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"Figure 3B",
"Figure 3B",
"Figure 3C",
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"Figure 4A",
"Figure 4B",
"Figure 5A-5D",
"Figure 5B",
"Figure 5A",
"Figure 5C",
"Figure 5F",
"Figure 5H",
"Figure 5J",
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"Gliomas are malignant brain tumors with a wide range of clinical features. Gliomas arise from the glial cells of the brain, progressing through the benign stage (WHO Grade I) to highly malignant (WHO Grades II to IV) stages. Higher grades of glioma are heterogeneous in nature, consisting of neoplastic cells, glioma-like stem cells, extensive vasculature and immune cells [1][2][3]. Microglia, the resident immune cells of the brain, form a significant portion of the infiltrating immune cell population in a tumor, making up to one-third of the tumor mass in some higher grade tumors [4,5]. As the first responders to any injury, insult or infection in the central nervous system (CNS), microglia become activated, secrete a variety of pro-inflammatory cytokines and exhibit phagocytic activity to clear tissue debris [6]. Subsequently, microglia facilitate repair and regeneration of the affected region in the CNS via release of growth factors and anti-inflammatory cytokines [7,8]. Activated microglia exhibit neurotoxic and neuroprotective roles in neuropathology and based on their functions, microglia are categorized as the classical (pro-inflammatory) phenotype and the alternative (anti-inflammatory) phenotype [9,10]. Microglial function in glioma tumors is an alternative form of activation wherein microglia secrete cytokines and chemokines that are gliomagenic and support the growth of the tumor [11,12]. However, recent studies suggest that tumor-associated microglia express genes that are distinct from either activation state [13,14], thus emphasizing the complex nature of tumor-associated microglia and its roles in a glioma microenvironment. This tumorigenic nature of microglia in glioma tumors may be attributed to molecular and epigenetic pathways that are altered by signaling molecules released from cancerous cells in the microenvironment.",
"Neoplastic cells within a tumor secrete a number of soluble cytokines, chemokines and growth factors that affect microglial motility, proliferation and phagocytosis [15,16]. A key signaling molecule that is highly enriched in the glioma microenvironment is the Transforming Growth Factor-beta (TGFβ) which activates the TGFβ pathway that is mediated by SMAD2 and 3, substrates for the TGFβ family of receptors. Upon binding of the TGFβ ligand to its receptor, the SMAD2/3 complex is phosphorylated and coupled with the common mediator SMAD4, translocated to the nucleus where the complex regulates the transcription of TGFβ responsive genes [17]. TGFβ is a known inhibitor of cell cycle progression [18] and thus, functions as a tumor suppressor in the early stages of certain cancers. On the contrary, TGFβ signaling can be pro-tumorigenic by inducing genes that promote tumorigenic aspects of glioma progression such as angiogenesis [19], metastasis [20,21] and epithelialmesenchymal transition [22]. Hyperactive TGFβ signaling is associated with certain subtypes of glioblastoma tumors, such as the mesenchymal subset and contributes to aggressiveness of the tumor and poor prognosis in patients [23][24][25]. In tumors with activated TGFβ signaling such as hepatocellular cancer, elevated SMAD4 has been shown to mediate tumor promoting signaling [26], while in other cancers such as pancreatic cancer, deletion of SMAD4 is associated with tumor progression and metastasis [27,28]. Therapeutic approaches using TGFβ antagonists and oligonucleotides coding anti-sense TGFβ2 have proven successful in reversal of TGFβ-aided immunosuppression in glioma [29,30]. However, systemic inhibition of TGFβ pathway can lead to unfavorable effects as TGFβ is involved in several cellular signaling pathways. This led us to investigate alternate specific mechanisms by which the TGFβ signaling pathways can be disrupted to attenuate the tumor supportive phenotype of microglia. Moreover, the role of SMAD4 in microglial functions in gliomas has been poorly understood and hence, this study is aimed to understand the role of SMAD4 in tumor-associated microglia in mediating tumor progression.",
"In addition to altered signaling pathways, activated microglia in different neuropathologies exhibit dysregulated epigenetic mechanisms such as chromatin modifications, changes in gene-specific histone acetylation and methylation and differential microRNA (miRNA) expression [31,32]. In particular, miRNAs have emerged as a central class of epigenetic mediators that post-transcriptionally regulate gene expression [33]. Dysregulation of miRNAs in activated microglia has been shown to contribute to development and progression of neurodegenerative diseases and brain injuries [33]. A global miRNA microarray analysis of activated primary microglial cells identified several miRNAs that were differentially expressed in activated microglia. The micro RNA 146a (miR-146a) was found to be upregulated in activated microglia as compared to control microglia (unpublished data). MiR-146a, which is enriched in activated macrophages and microglia [34], has been shown to target and suppress mediators of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling pathway in activated microglia and astrocytes, thereby functioning as a negative feedback regulator of microglial activation [35,36]. In addition, miR-146a was reported to target Notch1 in glioma cells www.oncotarget.com and further inhibit the process of gliomagenesis by suppressing migration and proliferation of cancer cells [37]. Further, our bioinformatics analysis predicted miR-146a to target SMAD4. Given the important role of miR-146a in microglia activation and gliomagenesis and its putative effect on SMAD4, this study attempted to understand the role of miR-146a and its putative target SMAD4 in microglia functions in tumor progression in glioma environment.",
"It was hypothesized that altered molecular and epigenetic mechanisms regulate the tumor supportive behavior of glioma-associated microglia. In this study, SMAD4, a mediator of the TGFβ signaling pathway was upregulated in microglia exposed to glioma conditioned medium and was found to be robustly expressed in microglia associated with human glioblastoma tissues. Stable loss of SMAD4 in microglia decreased expression level of a tumor promoter, MMP9, which resulted in decreased migratory potential of microglia in a transwell migration assay. In addition, miR-146a which was predicted to target SMAD4, was downregulated in microglia exposed to glioma conditioned medium treatment and regulated the expression levels of tumor supportive gene MMP9 in microglia. Overall, the present study implicates the role of miR-146a-SMAD4 in regulating microglial functions in glioma tumors.",
"To understand the effect of glioma microenvironment on microglia, BV2 microglial cells were cultured in glioma conditioned medium (GCM) derived from C6 glioma cells. The concentration of TGFβ in the GCM was assessed to be ~5ng/ml as compared to undetectable levels of TGFβ in serum containing medium ( Figure 1A). First, the expression of total and phosphorylated SMAD2/3, which mediates the TGFβ signaling, was determined in GCM-treated microglia. Western blot analysis showed an increase in the phosphorylated levels of SMAD2 and SMAD3 in GCM treated microglia as compared to control microglia ( Figure 1B, 1C). Upon phosphorylation of SMAD2 and SMAD3, the complex has been shown to bind to SMAD4 and translocate to the nucleus [38]. Confocal imaging revealed a co-localization of the pSMAD2/3 with SMAD4 in nuclei of microglial cells treated with GCM ( Figure 1D). Further, immunocytochemistry revealed increased co-localization of the SMAD4 and total SMAD2/3 in microglia treated with GCM as compared to control cells ( Figure 1E), suggesting that the TGFβ pathway is activated in GCMtreated microglia.",
"As SMAD2/3 forms a complex with SMAD4 to regulate TGFβ responsive genes, the effect of glioma microenvironment on SMAD4 expression in microglia was determined by western blot and immunocytochemistry. Upregulation of SMAD4 protein expression was clearly evident in microglia treated with GCM as compared to that of control (Figure 2A, 2B). Concomitantly, GCM treated microglial cells showed an upregulation in proteins involved in TGFβ signaling pathway such as matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGFa) (Figure 2A, 2B), suggesting that glioma-associated microglia exhibit upregulated expression of tumor supportive factors.",
"In light of the above results, we sought to validate the expression of SMAD4 in human glioblastoma tissue to better understand its role in glioma pathogenesis. An analysis of the expression profiling data of glioblastoma samples in The Cancer Genome Atlas database using Oncomine software showed a significant increase (3.271 fold) in the expression of SMAD4 (Reporter ID: 202527_s_at) mRNA in glioblastoma tissues (n=542) when compared with non-malignant brain tissue samples available in the database ( Figure 3A) [39].",
"In the present study, double immunofluorescence analysis was performed to determine SMAD4 protein expression in glioblastoma tissues ( Figure 3B). Confocal imaging showed expression of SMAD4 in Iba1-positive microglia, as well as non-microglial cells in different tissue samples ( Figure 3B). The Iba1-immunoreactive cells appeared to be an amoeboid or rounded phenotype, indicative of activated state of the cell type. High magnification images show evident nuclear expression of SMAD4 in Iba1 positive microglia ( Figure 3B). Quantitative analysis of Iba1-positive cell bodies revealed a high percentage of microglia in the tumor samples ( Figure 3C).",
"In order to ascertain the role of SMAD4 in microglia, stable knockdown of SMAD4 in microglia was carried out by transduction of small-hairpin RNA (shRNA) against the Smad4 gene. ShRNA-mediated silencing of the Smad4 gene resulted in 90% decrease in mRNA levels of Smad4 ( Figure 4A) and 80% decrease in the protein level of SMAD4 ( Figure 4A, 4C). A downregulation of MMP9 at mRNA and protein level was observed in microglia www.oncotarget.com Western Blot (B) shows an increase in phosphorylated form of pSMAD2/3 in microglia treated with GCM when compared with control. Histogram depicts densitometric quantification of pSMAD2/3 normalized against total SMAD2/3 expression (C) Data represent mean±SD, (n=4), Students t-test, * p<0.05, ** p<0.01. Confocal images show pSMAD2/3 (green, D), total SMAD2/3 expression (green, E) and SMAD4 expression (red, D, E) in BV2 microglia (indicated by arrows). Cell nuclei are labelled with DAPI (blue). GCM increases immunofluorescence intensity of pSMAD2/3 as compared to control cells (D). Immunocolocalization of pSMAD2/3, total SMAD2/3 and SMAD4 reveals that pSMAD2/3 and total SMAD2/3 colocalize with SMAD4 in microglia treated with GCM (D, E). Scale bar=30μm. www.oncotarget.com Western blot analysis shows an increase in SMAD4 protein levels and tumor supportive genes, VEGFa and MMP9 in BV2 microglia upon GCM treatment (A). Histogram depicts densitometric quantification of protein levels of SMAD4, VEGFa and MMP9 in microglia exposed to GCM (B). Data represent Mean±SD, (n=3-5), Students t-test, * p<0.05. after knockdown of SMAD4 compared to cells transduced with the empty vector, which served as a negative control ( Figure 4B, 4C). This suggests that SMAD4 regulates the expression of tumor supportive factor, MMP9 in microglia.",
"The transwell migration assay was performed, wherein microglial cells were seeded in serum-free medium in the upper chamber of a transwell insert and allowed to migrate towards the lower chamber containing GCM or chemoattractants such as TGFβ and EGF, to assess the migratory potential of microglia towards GCM ( Figure 5A-5D). There is a significant increase in the number of microglial cells migrating towards GCM ( Figure 5B, 5E) in the lower chamber as compared to the number of microglial cells migrating to serum containing medium which served as a control ( Figure 5A). In addition, microglial cells showed increased migration towards chambers containing soluble TGFβ and EGF ( Figure 5C, 5D, 5E) as compared to control. Further, the role of SMAD4 in the migration of microglia towards GCM was studied. A significant decrease in the number of shSMAD4 cells migrating towards the serum containing medium in the lower chamber, was observed as compared to control cells which were transfected with empty vector ( Figure 5F, 5G, 5J). This indicates that SMAD4 plays a significant role in the migratory potential of microglia. On the other hand, upon exposure to GCM, there was an increase in the number of migrating control and shSMAD4 microglial cells compared to that of control and shSMAD4 microglial cells, respectively. However, the increase in migration of shSMAD4 microglial cells exposed to GCM was significantly less than that of control microglial cells show an increase in the number of migrated microglial cells (purple) after exposure to GCM (B), TGFβ (C) and EGF (D) as compared to cells exposed to serum containing medium (A). Histogram depicts fold change in migrating cells exposed to GCM, TGFβ and EGF as compared with control group (E). Data represent mean±SD, * p<0.05, ** p<0.01, (n=3). There was a decrease in the number of migrated shSMAD4 microglial cells (purple) in response to GCM and serum containing medium (I, G) as compared to negative control (Neg Cont) microglial cells exposed to GCM and serum containing medium (H, F). Histogram depicts fold change in migrating cells (J). Data represent mean±SD, (n=3), * p<0.05, ** p<0.01. www.oncotarget.com exposed to GCM ( Figure 5H, 5I, 5J). In addition, GCMinduced migration index of both control and shSMAD4 microglial cells appear to be comparable ( Figure 5J), suggesting that factors involving TGFβ pathway present in GCM could play a role in migration of microglia.",
"Microglial cells have been shown to promote glioma progression through secretion of factors that aid in tumor growth. In the present study, the viability of glioma cells in response to conditioned medium from microglia was evaluated using an MTS assay and alamar blue assay. The results indicate that there is a significant increase in cell viability ( Figure 6A, 6B) of glioma cells after 24, 48 and 72h of treatment with medium derived from control microglial cells, confirming that microglial cells secrete factors that promote glioma cell growth. In order to determine the effect of knockdown of SMAD4 in microglia on glioma cell viability, glioma cells were treated with conditioned medium derived from shSMAD4 microglial cells. The results indicate that there is a decrease in viability of glioma cells when treated with medium from SMAD4 knockdown microglial cells ( Figure 6C), suggesting that suppression of SMAD4 in microglia decreases the viability of glioma cells. This was further confirmed with an alamar blue assay wherein a significant decrease in reduction of alamar blue was observed in vitro after treatment of glioma cells with medium derived from shSMAD4 microglial cells as compared to the corresponding control ( Figure 6D).",
"In order to delineate the epigenetic mechanisms that regulate SMAD4 expression in glioma-associated microglia, we examined the 3'UTR of the Smad4 mRNA sequence to identify miRNA binding sites. TargetScan algorithms software (http://www.targetscan.org/mmu_71/) revealed putative complimentary binding site for miR-146a in the 3'UTR of Smad4 ( Figure 7A). Given the role of miR-146a in activation of microglia [36,40,41], its role in the tumor supportive phenotype of microglia was determined. The quantitative RT-PCR analysis showed that the expression level of miR-146a-5p was significantly decreased in glioma-associated microglia as compared to control cells ( Figure 7B). This is concurrent with an increase in mRNA and protein levels of SMAD4 ( Figure 7B, 2A), demonstrating an inverse relationship between miR-146a and SMAD4 in microglia.",
"Further, 3'UTR luciferase assay was performed to confirm that SMAD4 is a target of miR-146a. BV2 microglial cells were transfected with a luciferase vector containing the 3'UTR of the mouse Smad4 gene together with miR-146a overexpression (mimics) or scrambled probes. A significant decrease in the luciferase activity in BV2 microglia was observed upon co-transfection of the mimics and the luciferase vector, indicating that the miR-146a binds to the 3'UTR of Smad4 in luciferase vector ( Figure 7C). Transfection efficiency of the miRNA overexpression and inhibition was determined by examining the levels of the miR-146a in microglial cells after transfection of mimics and inhibitors. Overexpression of miR-146a mimics was observed to increase miR-146a levels in microglia by nearly 400-fold while inhibition of the miR-146a resulted in a ~50% decrease in miR-146a levels ( Figure 7D). The mRNA expression of SMAD4 after overexpression and inhibition of the miR-146a was analyzed. MiR-146a mimic transfection resulted in decrease in mRNA and protein expression levels of SMAD4 and conversely, inhibition of miRNA function resulted in an increase in the mRNA and protein levels of SMAD4 ( Figure 7E, 7F, 7G) in microglia, suggesting that miR-146a directly targets SMAD4 in microglia.",
"In order to understand the functional relationship between miR-146a and SMAD4 in GCM-induced microglia, loss-of and gain-of function experiments were carried out. MiR-146a overexpression was found to significantly decrease SMAD4 expression at protein levels in microglia treated with GCM, as determined by western blot (Figure 8A, 8C). In contrast, inhibition of the miR-146a resulted in a marginal increase in the protein levels of SMAD4 in microglia treated with GCM as revealed by western blot analyses ( Figure 8B, 8D). Immunocytochemistry analysis also revealed that miR-146a overexpression with mimics decreased the expression of SMAD4 in microglia treated with or without GCM as compared to that transfected with scrambled probes ( Figure 8E). This suggests that miR-146a regulates SMAD4 expression in glioma-associated microglia.",
"Microglia treated with GCM showed an induction of tumor promoter gene MMP9, concomitant with an increase in SMAD4 level. In order to determine if miR-146a regulates tumor supportive gene expression in microglia, expression of MMP9 upon overexpression and knockdown of miR-146a was analyzed. Overexpression of the miR-146a in microglia resulted in a significant suppression of MMP9 protein expression while inhibition of miR-146a was found to increase MMP9 expression ( Figure 9A, 9B). In addition, overexpression of miR-146a in microglia resulted in a significant decrease in migration of microglia towards GCM in a transwell migration assay ( Figure 9C-9G), indicating that miR-146a suppresses microglial migration through regulation of SMAD4 and its downstream gene MMP9. www.oncotarget.com Histogram depicts a significant decrease in the levels of miR-146a and a significant increase in mRNA levels of SMAD4, indicating an inverse relationship between SMAD4 and miR-146a in microglia treated with GCM (B). Histogram shows a significant decrease in the luciferase activity in cells co-transfected with miR-146a mimic and luciferase vector as compared to cells co-transfected with scrambled control miRNA and luciferase vector, indicating that miR-146a targets SMAD4 (C). Data represent mean±SD, (n=3), Students t-test, * p<0.05. MiRNA mimics and inhibitor transfection efficiency was verified using qRT-PCR. Histogram shows an about 400-fold increase in miR-146a levels in microglia after mimic transfection and about 50% decrease in miR-146a levels after inhibitor transfection (D). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Histogram shows a decrease in the mRNA levels of SMAD4 upon overexpression of miR-146a and conversely an increase in SMAD4 mRNA upon inhibition of miR-146a (E). Data represent mean±SD, (n=3), Students t-test, * p<0.05. Western blot (F) and densitometry analysis (G) confirm that overexpression of miR-146a suppresses SMAD4 protein levels and inhibition of miR-146a increases SMAD4 protein level as compared to scrambled probe transfected cells. Data represent mean±SD, (n=3), Students t-test, * p<0.05, ** p<0.01. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com",
"To determine the effect of miR-146a overexpression in microglia on glioma cell viability, glioma cells were treated with conditioned medium derived from miR-146a overexpressed microglial cells. The MTS results indicate that there is a decreased viability of glioma cells across different time points after treatment with miR-146a overexpressed microglia-derived medium as compared with that of scrambled control transfected cells ( Figure 10A). This was further confirmed with an alamar blue assay wherein a significant decrease in alamar blue reduction was observed in vitro after treatment of glioma cells with medium from miR-146a overexpressed microglial cells ( Figure 10B), indicating that overexpression of miR-146a in microglia decreased the viability of glioma cells.",
"It is well documented that microglia play diverse roles, either detrimental or beneficial, during CNS pathology [6]. In the normal healthy brain, microglia monitor the brain microenvironment for pathogens and injury and are involved in functions such as neuronal synapse formation, maintenance and pruning [42][43][44]. Upon activation, the microglia with processes rapidly transform into amoeboid phenotype and are involved in phagocytosis of debris in brain parenchyma [45]. In the present study, microglia within gliomas exhibit primarily amoeboid phenotype, suggesting that they are activated, probably in response to the glioma secretome which consists of signaling molecules released by neoplastic and non-neoplastic cells such as vascular endothelial cells, astrocytes and cancer stem cells of the tumor that lie in quantification (B, D). Data represent mean±SD, (n=5), * p<0.05. Immunofluorescence labelling shows that miR-146a overexpression attenuated SMAD4 expression (red) in BV2 microglia nuclei with or without GCM treatment as compared to cells transfected with scrambled miRNA (Neg Cont) probes (E). DAPI-nuclei staining, blue. Scale bar=30μm. MiR-146a OE-MiR-146a overexpression; MiR-146a KD-MiR-146a knockdown. www.oncotarget.com spatial proximity to microglial cells. This is supported by recent experimental evidence, which showed that microglia exposed to glioma conditioned medium in vitro and microglia associated with glioma tumors in mice models in vivo exhibit an amoeboid phenotype that is characteristic of a state of activation [46][47][48]. Further, the glioma tumors analyzed in the present study showed a high percentage of Iba1-positive microglial cells, with certain glioblastoma tumors hosting nearly 25%-50% of microglial cells in the tumor mass. A higher frequency of microglial cells in the tumor may be attributed to migration of microglia in the brain parenchyma in response to factors released by the glioma cells as can be seen in the in vitro migration assay. Several studies have shown that soluble factors such as EGF [49,50] and TGFβ [51] serve as potent chemotactic factors in tumors and may promote migration of microglia towards the tumor as observed in the present study.",
"In addition to its role as a chemoattractant, the TGFβ ligand activates an anti-inflammatory signaling pathway in microglia, exerting an opposing effect on pro-inflammatory signaling that is widely known to be neurotoxic to brain tissue [52,53]. TGFβ has been shown to act on microglia in an autocrine manner and maintain microglial quiescence [54]. It has also been shown that microglia-derived TGFβ enhanced the invasiveness and tumorigenicity of the glioma cells and siRNA-mediated knockdown of TGFβ Receptor II in glioma cells disrupted this tumor promoting effect of TGFβ [30]. The present study shows increased levels of total and pSMAD2/3, which mediate TGFβ signaling pathway, in microglia after GCM treatment in vitro. While the roles of SMAD2/3 have been widely studied in microglia [55,56], the role of SMAD4 in microglial activation, specifically in context of the TGFβ signaling has remained unclear. There is evidence showing that SMAD4 is upregulated in LPS- activated microglia and acts as a negative feedback inhibitor of NFκB, a pro-inflammatory signaling response in the activated microglia [57]. In the present study, glioma-associated microglia expressed SMAD4 in human glioblastoma tumors in vivo and microglia exposed to GCM showed increased expression of SMAD4 in vitro. This was further supported by an analysis of the data in The Cancer Genome Atlas which revealed an upregulated SMAD4 level in glioblastoma tumors as compared to normal brain samples. Further, shRNA-mediated silencing of SMAD4 in microglia was associated with a decrease in the expression of MMP9, an extracellular matrix metalloproteinase and suppression of microglial migration. This has been evidenced in hepatocellular carcinoma wherein knockdown of SMAD4 reduced migratory capacity and colony formation ability of the cancer cells [26]. SMAD4 has also been shown to control endodermal cell migration during embryonic development through regulation of extracellular matrix modelling enzymes, MMP9 and MMP14 [58]. In addition, SMAD4 silencing in pancreatic tumor cells and keratinocytes has been shown to abolish TGFβ induced migration [59], therefore highlighting a vital role for SMAD4 in microglial migration.",
"Microglial cells are highly secretory in nature. In the present study, microglia-derived growth factors in the conditioned medium were found to promote glioma cell viability in vitro. This is an important finding as it may lead to a novel therapeutic strategy which may focus on suppressing microglia-mediated tumorigenesis. Moreover, this study demonstrates that glioma-associated microglia promote tumorigenesis through SMAD4 expression, since glioma cells treated with conditioned medium derived from shSMAD4 microglial cells showed decreased viability. This decreased viability of glioma cells is also possible via SMAD4-induced negative regulation of NF-κB pathway in microglia, since recent studies have shown that SMAD4 knockdown in microglia induced pro-inflammatory cytokine, IL-6 in an NF-κB dependent manner [57]. Overall, these results indicate that the possible interaction between SMAD4 and NF-κB in glioma-associated microglia may determine the tumor progression. The epigenetic regulation of SMAD4 was investigated, as these mechanisms provide an additional layer of post-transcriptional control. In this study, SMAD4 was found to be targeted by miR-146a in microglia. MiR-146a, a miRNA enriched in immune cells, is known to be upregulated in activated microglia and macrophages in pathological conditions such as infection [41], ischemic stroke [60] and Alzheimer's disease [40,61]. Several studies have shown that miR-146a modulates the innate immune response of activated microglial cells through regulation of the pro-inflammatory transcription factor, NFκB [41,62]. Recently, the miRNA-146b, which shows sequence similarity with miR-146a has been shown to inhibit glioma growth in vitro through modulation of its target EGFR [63]. Downregulation of miR-146a in gliomaassociated microglia as observed in the present study may favour tumorigenesis through increased expression of SMAD4 and its downstream genes which are involved in tumor progression. This also indicates that the gliomaassociated microglia do not exhibit activated microglial phenotype as observed in other neuropathologies such as Alzheimer's and stroke. Functional studies further confirmed that miR-146a is a negative regulator of tumorigenic gene expression in microglia via its target SMAD4, as its overexpression in microglia resulted in suppression of MMP9, which is a tumorigenic factor that promotes migration of microglia towards GCM and the glioma viability in vitro.",
"As the immune cell of the brain, microglia show complex phenotypes in the brain tumor and did not reject or phagocytose the tumor cells. Instead, they conglomerate within the tumor core and support the tumor progression. It is thus imperative to study microglial function in context of different molecular and genetic subtypes of glioblastoma. In this regard, this study shows robust expression of SMAD4 in glioblastoma tumors and in glioma-associated microglia. It has also been demonstrated that SMAD4 which was found to be post-transcriptionally regulated by miR-146a, regulates the migration of microglial cells in response to glioma conditioned medium. This study further established that miR-146a suppresses tumorigenic gene, MMP9 in gliomaassociated microglia and glioma cell viability through its target SMAD4 (Figure 11). Further in vivo studies are required to evaluate the therapeutic effect of miR-146a and SMAD4 on glioma growth and progression. ",
"Graded brain tumor specimens were obtained with written informed consent, as part of a study protocol approved by the SingHealth Centralised Institutional Review Board A and the National Healthcare Group Domain-Specific Review Board A ( Table 1). All protocols were approved by the Institutional Review Board, National University of Singapore. Use of glioblastoma human tissues was reviewed and approved by the National University of Singapore Institutional Review Board (NUS-IRB Reference Code: B-16-049E).",
"Human tumor sections were fixed, frozen, sectioned (7μm thick) and mounted onto slides. Tissue sections were blocked in 3% bovine serum albumin (BSA) solution in phosphate-buffered-saline (PBS) containing 0.3% Triton-X (TX). Primary antibody against the full length human SMAD4 protein was a kind gift from Dr. Lu Lei from the Nanyang Technological University, Singapore. Sections were incubated with primary antibodies in 1% BSA overnight at 4ºC at the following concentrations: SMAD4-1:300, Iba1-1:50 (Abcam, ab5076). Further, sections were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:5000) and mounted with a fluorescent mounting medium. Fluorescence images were captured using a confocal microscope (Olympus, FV1000 Fluoview). Iba1 positive cell bodies showing a well-defined DAPI stained nuclei were manually counted across 4 fields of the tumor section and plotted as a percentage of total DAPI cell nuclei.",
"BV2 murine microglial cells and C6 rat astrocytoma cells obtained from American Tissue type Culture Collection (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a 5% CO 2 incubator at 37ºC and regularly passaged with Trypsin-EDTA solution to allow healthy growth of cells.",
"To mimic a glioma microenvironment in vitro, microglial BV2 cells were treated with conditioned medium from the C6 cell line [64,65]. Briefly, C6 cells were seeded in 10cm culture dishes at a density of 2X10 6 cells. Cells were allowed to settle overnight and DMEM supplemented with 10% FBS was added to the culture the next day. Culture supernatant was collected after 48h and filtered through a 0.22μm filter to remove cellular debris. GCM was stored at -80ºC and freeze-thaw cycles were minimized.",
"Stable knockdown of SMAD4 was performed by lentiviral mediated transduction of SMAD4 specific shRNA in BV2 microglial cells. Microglial cells were transduced with 4 shRNA individual clones (Dharmacon, GE Healthcare) against the SMAD4 gene (Accession Number: NM_008540). Selection pressure was applied 48h after transduction using puromycin at the concentration of 2μg/ml. Cells were maintained in puromycin containing medium for 6-10 days and expanded [66]. Efficiency of knockdown in microglia was confirmed using western blotting analysis and the shSMAD4 clone that induced maximal knockdown, shSMAD4_2, was selected for further analysis (Supplementary Figure 1).",
"RNA was isolated from the BV2 cells after transfection or treatment, using miRNeasy Mini Kit The qRT-PCR for gene expression was performed using Fast SYBR Green Master Mix (ThermoFisher Scientific, Cat No: 4385614) with 1:10 ratio dilution of cDNA in nuclease-free water. Primer sequences for PCR reactions are given in Table 2. Fold change between control and experimental groups was calculated as per 2 -ΔΔCt method [67]. All PCR reactions were carried out in a real time Applied Biosystems PCR system (Life Technologies, Model No: 7900HT).",
"The Transwell migration assay was carried out to assess the migratory potential of microglia towards GCM, TGFβ and EGF. 40,000 microglial cells were seeded in the upper chamber of the Transwell migration insert (Corning, Cat No:3422) and was placed in the lower chamber containing glioma conditioned medium and medium containing TGFβ (PeproTech, Cat No 100-21C) and human recombinant EGF (Life Technologies, PHG0311).",
"The cells were allowed to migrate for 15h. Inserts were fixed in 100% methanol for 10min and stained for visualization using 0.5% cresyl violet solution. Cells on the upper membrane of the insert were removed using a cotton swab. 5 random fields from 3 biological replicates were quantified and the results are plotted as fold change between treatment and control groups.",
"BV2 cells were plated at a density of 2X10 5 in 6-well plates and gain-of and loss-of-function studies of miR-146a-5p was carried out using miRNA mimics (Ambion, ",
"A luciferase assay was performed to verify if miR-146a targets SMAD4 mRNA. BV2 microglial cells were plated at a density of 20,000 cells in 24 well plates. The luciferase vector containing the 3`UTR of SMAD4 was commercially purchased from GeneCopoeia (Cat No: MmiT027594). Cells were co-transfected with mimics and scrambled probes (30nm) and luciferase vector (1000ng) and the medium containing secreted luciferase was collected at 24h. The Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia Cat No: SPDA-D010) was used to determine luminescence of secreted Gaussia luciferase (GLuc). A secondary reporter, secreted alkaline phosphatase (SEAP), served as an internal control. GLuc/SEAP ratio was determined to measure the luminescence output of the transfected sample.",
"BV2 microglial cells were grown on poly-L-lysine coated coverslips. Following transfection and/or treatment, cells were fixed with 4% paraformaldehyde for 15min at room temperature. Permeabilization of cell membranes was achieved using 0.1% Triton-X containing PBS. Following this, the slides were blocked using 5% normal goat serum and incubated overnight at 4ºC with the following antibodies-SMAD4-1:100 (Santa Cruz, sc-7966), pSMAD2/3-1:200 (Cell signaling technology, Cat No: 8828) and SMAD2/3-1:200 (Cell Signaling Technology, Cat No: 8685). Following this, cells were incubated with fluorophore tagged secondary antibodies: anti-rabbit Cy3 (Sigma, Cat No: c2306), anti-mouse Cy3 (Sigma, Cat No: C2181) and FITC conjugated lectin (Sigma, Cat No: L0401) was used as a marker for microglial cells. Cell nuclei were counterstained with DAPI for visualization. Fluorescence images were captured using a confocal microscope (Olympus, FV1000 Fluoview).",
"TGFβ in the C6 glioma conditioned medium (GCM) was quantified using the Quantikine ELISA Kit (RND Systems, MB100B) as per the manufacturer's instructions. Briefly, latent TGFβ1 was activated to its immunoreactive form by addition of 20μl of 1M HCl to 100μl of the GCM, and subsequently neutralized by adding 20μl of 1.2N NaOH. The TGFβ1 standard was reconstituted and serially diluted using the diluent solution provided in the kit. 50μl of GCM and the TGFβ1 standard samples were added to the TGF-β1 antibody pre-coated ELISA plate and incubated for 2h. Subsequently, the samples were discarded, and the wells were thoroughly washed using wash buffer. Next, 100μl of the TGFβ1 conjugate was added to each well and incubated for 2h. Following washing, the plate was incubated with substrate solution, and reaction was stopped using the stop solution provided in the kit. The optical density was measured at 450nm using a plate reader.",
"In order to evaluate the effect of SMAD4 knockdown in microglia on glioma cell viability, an MTS and alamar blue assay was performed. Conditioned medium was collected from control microglia (microglial CM) and from microglia after knockdown of SMAD4 (shSMAD4-CM) or miR-146a overexpression (MiR-146a OE-CM). Conditioned medium from microglia transduced with the empty vector (Neg Control CM (stables)) and the scrambled probes (Neg Control CM (miRNA)) served as controls. About 10,000 of C6 glioma cells were seeded in 96-well plates and treated with the conditioned medium from different groups. Post 24, 48 and 72h of treatment, 20μl of MTS reagent (Promega, Cat No. G358C) was added to the cells and incubated at 37°C for 2h. Following this, absorbance was read at 490nm in a 96-well plate reader. The alamar blue assay was performed by adding 10μl of the alamar blue reagent (ThermoFisher Scientific, Cat No. DAL1100) to the cells after treatment with conditioned medium. Absorbance was read at 570 nm and normalized against absorbance at 600nm. The results are plotted as a percentage of alamar blue reduction.",
"Data from at least three biological replicates were analyzed using GraphPad Prism and Microsoft Excel software and represented as mean ± S.D. In comparing 2 experimental groups, the Student's t-test was used. Multiple groups were analyzed using one-way or two-way ANOVA tests, followed by post-hoc Tukey's and Sidak's test. Data sets were considered significant at p<0.05."
] | [] | [
"INTRODUCTION",
"RESULTS",
"Glioma conditioned medium induced phosphorylation of SMAD2 and SMAD3 in microglia",
"Microglia treated with GCM show increased expression of SMAD4 and tumor supportive genes",
"SMAD4 is expressed in microglia associated with human glioblastoma samples",
"SMAD4 regulates the expression of tumor supportive factor MMP9",
"ShRNA-mediated silencing of SMAD4 suppresses the migration of microglia towards glioma conditioned medium",
"Microglial conditioned medium from shSMAD4 cells inhibits glioma viability",
"MiR-146a regulates SMAD4 expression in GCM treated microglia",
"MiR-146a regulates the expression of MMP9 in microglia and suppresses migration of microglia towards GCM",
"miR-146a overexpression in microglia suppresses glioma viability and growth",
"DISCUSSION",
"MATERIALS AND METHODS",
"Human tissue samples",
"Immunofluorescence",
"Cell culture",
"Preparation of glioma conditioned medium (GCM)",
"Generation of SMAD4 knockdown stable cells in microglia",
"RNA extraction, cDNA conversion and quantitative real-time PCR (miRNA)",
"Migration assay",
"Transfection of miRNA mimics and inhibitors",
"Luciferase assay",
"Immunocytochemistry",
"ELISA based quantification of TGFβ in GCM",
"MTS and alamar blue cell viability assay",
"Statistical analysis",
"Figure 1 :",
"Figure 2 :",
"Figure 3 :",
"Figure 4 :",
"Figure 5 :",
"Figure 6 :",
"Figure 7 :",
"Figure 8 :",
"Figure 9 :",
"Figure 10 :",
"Figure 11 :",
"Table 1 :",
"Table 2 :"
] | [
"Sample Name \nTumor Type \nTumor Grade \n\nGB-21 \nGlioblastoma \ngrade IV \n\nGB-22 \nGlioblastoma \ngrade IV \n\nGBO-24 \nGlioblastoma with oligodendroglial component \ngrade IV \n\nGB-25 \nGlioblastoma \ngrade IV \n\nGBO-26 \nGlioblastoma with oligodendroglioma \ngrade IV \n\nGB-28 \nGlioblastoma \ngrade IV \n\nGB-34 \nGlioblastoma \ngrade IV \n\n",
"Gene \nForward Primer \nReverse Primer \n\nSMAD4 \nTCCAACACCCGCCAAGTAAT \nGCTGGCTGAGCAGTAAATCC \n\nMMP9 \nGCGTGTCTGGAGATTCGACTT \nTATCCACGCGAATGACGCT \n\nβ-Actin \nGGATTCCATACCCAAGAAGGA \nGGATTCCATACCCAAGAAGGA \n",
"Western blotting \n\n"
] | [
"Table 1",
"Table 2"
] | [
"Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment",
"Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment"
] | [
"Oncotarget"
] |
52,345,307 | 2022-03-01T11:33:18Z | CCBY | https://www.frontiersin.org/articles/10.3389/fbioe.2018.00129/pdf | GOLD | 9960fef033677c702d4cb29f872f36d2ac1e99b3 | null | null | null | null | 10.3389/fbioe.2018.00129 | 2894102246 | 30320079 | 6165858 |
a section of the journal Frontiers in Bioengineering and Biotechnology Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding
September 2018. 2018
May Griffith
Damien Harkin
Hirak Kumar Patra
Peter Ruminski
Imaninezhad M Schober
J
Griggs D
Ruminski P
Kuljanishvili I
Mozhdeh Imaninezhad
Biomedical Engineering
Saint Louis University
Saint LouisMOUnited States
Joseph Schober
Pharmaceutical Sciences
Southern Illinois University
EdwardsvilleILUnited States
David Griggs
Molecular Microbiology & Immunology
Saint Louis University
Saint LouisMOUnited States
Peter Ruminski
Center for World Health and Medicine
Saint Louis University
United States, 5 PhysicsSaint LouisMO
Saint Louis University
Saint LouisMOUnited States
†
Irma Kuljanishvili
Silviya Petrova Zustiak
Biomedical Engineering
Saint Louis University
Saint LouisMOUnited States
Université de Montréal
Canada
Queensland University of Technology
Australia
Department of Medicine
Division of Oncology
University of Cambridge
United Kingdom
Washington University
St. LouisMOUnited States
a section of the journal Frontiers in Bioengineering and Biotechnology Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding
Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding. Front. Bioeng. Biotechnol
6129September 2018. 201810.3389/fbioe.2018.00129Specialty section: This article was submitted to Biomaterials, Received: 14 May 2018 Accepted: 30 August 2018 Published: 24 September 2018 Citation:ORIGINAL RESEARCH Edited by: Reviewed by: *Correspondence: Silviya Petrova Zustiak [email protected] † Present Address:carbon nanotubeshydrogelcell spreadingintegrinsprotein adsorptionfibronectin
Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes.
INTRODUCTION
Carbon nanotubes (CNTs) are cylindrical and have nanometer diameters but typically micrometer lengths, resulting in high aspect ratios. Owing to their exceptional physical, chemical, and mechanical properties, they have been extensively studied for their effect on cellular behaviors (Ryoo et al., 2010;Bosi et al., 2013). Accounts of tissue engineered products including CNTs have proliferated in recent years, which could be coupled to our growing awareness of the nanodimensionality of nature (Harrison and Atala, 2007;Shah et al., 2015). In addition, due to their electro-conductivity, CNTs have been used as components of engineered excitable tissues for neural (Lewitus et al., 2011;Bosi et al., 2013) and cardiac (Shin et al., 2013) tissue engineering applications. Due to their exceptional mechanical stability and low weight-to-strength ratio, they have also been used in orthopedic applications (Sahithi et al., 2010), where CNT-based nanofibers have even been shown to select for osteoblast adhesion (Price et al., 2004).
However, very little is known about the mechanism of cell attachment and spreading on CNTs. In one study of soluble multi-walled (MWCNTs) toxicity toward A549 lung cancer cells, the authors observed that some of the MWCNTs coated the surface of the cells (Kaiser et al., 2013). The authors GRAPHICAL ABSTRACT | Cell attachment and spreading onto individual single-walled carbon nanotubes (SWCNTs) and multi-walled CNT aggregates (MWCNTs) is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive extracellular matrix (ECM) proteins to the nanotubes.
suggested that cells could be adhering directly to the MWCNTs via integrins or to serum proteins which have adsorbed onto the MWCNTs first (Kaiser et al., 2013). In another study of neural cells onto MWCNTs aggregates, it was suggested that cells adhered via physical entanglements of cellular processes with MWCNT aggregates but only when both were of similar dimensions (Sorkin et al., 2009). However, another recent study of mesenchymal stem cells (MSCs) has shown that cells can recognize, adhere to and spread along individual single-walled (SWCNTs) of a diameter <2 nm; non-specific cell adhesion was controlled through PEG passivation (Namgung et al., 2011). While the mechanism of cell attachment and spreading was not studied, the authors showed that cells formed robust focal adhesion complexes on the SWCNT patterned substrates (Namgung et al., 2011). A different group has shown that NIH 3T3 cells not only formed focal adhesion complexes when grown on MWCNTs films, but the complexes were larger in number and smaller in area compared to a glass substrate, suggesting high affinity for the MWCNTs (Ryoo et al., 2010). Lastly, research on a non-adhesive SiO 2 substrate has shown that topography alone can contribute to cell adhesion (Fan et al., 2002) and nanometer surface roughness has been shown to increase osteoblast adhesion to carbon nanofibers (Price et al., 2004).
Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function (Geiger et al., 2001). These interactions play an integral role in the development and maintenance of tissues (Huang et al., 2003). Due to its significance, mechanisms of cell attachment and spreading have been widely explored in various fields such as cellular biology (Kwon et al., 2007) or biomedical applications (Wang et al., 2009). In vitro most mammalian cells are anchorage-dependent and attach firmly to the substrate (Sagvolden et al., 1999). Upon cell adhesion, cells undergo morphologic alteration driven by passive deformation and active reorganization of the cytoskeleton. Integrin receptors and heterodimeric transmembrane proteins play a central role in cell adhesion and spreading. For example, fibroblast cells adhesiveness to fibronectin is reduced by impairing α5β1 integrin (Zou et al., 2002). Specific integrin binding provides not only a mechanical linkage between the intercellular actin cytoskeleton and the extracellular matrix, but also a bidirectional transmembrane signaling pathway (Hynes, 1987;Geiger et al., 2001;Van der Flier and Sonnenberg, 2001). Hence, cell adhesion and spreading on the underlying substrate is an important consideration in biomaterial design and development. Further, the requirements for cell adhesion and spreading will differ for different applications and could also be cell-specific (Huang et al., 2003). Surface properties of materials also influence the composition of the adsorbed protein layers, which subsequently regulate a variety of cell behaviors such as attachment, viability, spreading, migration, and differentiation (Webb et al., 2000).
To date there have been very few and contradicting reports on the mechanism of cell attachment and spreading on CNTs, hence no consensus has been reached. Importantly, the mechanism of cell attachment and spreading to individual SWCNTs has not been studied. Here, we hypothesized that cell attachment and spreading to both individual SWCNTs and MWCNT aggregates is governed by the same process. Specifically, we suggest that cell attachment and spreading onto nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes.
MATERIALS AND METHODS
Materials
Single crystal ST cut quartz wafers (diameter of 76.2 mm, thickness of 500 µm) were purchased from University Wafer (Boston, MA). Poly(ethylene glycol) diacrylate (PEGDA, MW 5 kDa) was purchased from Laysan Bio (Arab, AL). Phosphate buffered saline (PBS, 10X, pH 7.4), Hoechst 33258, Alexa 488 phalloidin, bovine serum albumin (BSA) and bovine fibronectin were purchased from Thermo Scientific (Waltham, MA). Rabbit anti-fibronectin antibody was purchased from Abcam (Cambridge, MA). Goat anti-rabbit IgG conjugated with TRITC was purchased from Jackson ImmunoResearch Inc. (West Grove, PA). Irgacure 2959 was purchased from BASF Corporation (Florham Park, NJ). GelBond (GelBond PAG film for polyacrylamide) was purchased from GE Health Care (Filial Sverige, Sweden). Silicone spacers were purchased from Grace Bio-Labs (Bend, Oregon). RainX were purchased from a general store. Ham's F12K medium with 2 mM glutamine and 1.5 g/L sodium bicarbonate, penicillin/streptomycin (pen/strep), and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Dulbecco's modified eagle's medium (DMEM) and N2 supplement (100X) were purchased from Gibco TM (Logan, UT). NIH 3T3 cells were purchased from ATCC (Manassas, Virginia). PC12 cells were generously provided by Dr. Grant Kolar (Department of Pathology, Saint Louis University). 18 × 18 mm #2 glass coverslips and PBS, without calcium and magnesium were purchased from Corning Life Sciences (Manassas, VA). Triton X-100 and formaldehyde were purchased from Sigma Aldrich (St. Louis, MO). Peptidomimetics CWHM-12 and CWHM-96 were synthesized and generously provided by Drs. Griggs and Ruminski (Saint Louis University) (Henderson et al., 2013). A sample of semiconducting multi-walled carbon nanotube powder (MWCNTs) (20 ± 3 nm in diameter and 3 ± 2 µm in length) produced via catalytic chemical vapor deposition was generously provided from MerCorp (Tucson, AZ).
4-Arm PEG-Ac Modification With RGDS
4-arm PEG-RGDS with 80% modification efficiency was prepared following a previously developed protocol . Briefly, Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) was dissolved in 5% acetic acid in de-ionized (DI) water and 4-arm PEG-Ac was dissolved in 0.3 M TEA buffer. The two solutions were combined at a 1:4 molar ratio of RGDS:Ac and reacted for 30 min at room temperature. The concentrations were chosen such that, assuming an ideal reaction, one of the acrylate groups of each PEG-Ac can be conjugated with RGDS. Upon reaction the solution was placed at −80 • C for 15 min and then lyophilized (VirTis Sentry 2.0 Lyphilizer) overnight. The 4-arm PEG-RGDS product was purged with argon and stored in a desiccated environment at −20 • C until use.
Polyethylene Glycol Hydrogel Preparation
Stock solution of 1% w/v Irgacure 2959 was prepared by dissolving Irgacure 2959 in DI water and sonicating it in a bath sonicator (Fisher Scientific, Waltham, MA) for 1 h. The Irgacure solution was stored shielded from light at 4 • C until use. Stock solution of 30% w/v PEGDA was prepared in 1X PBS, pH 7.4 and stored at 4 • C for up to 1 wk. Then, 10% and 20% w/v PEGDA hydrogel precursor solutions containing 0.1% w/v Irgacure 2959 were prepared by diluting the PEGDA and Irgacure stock solutions with 1X PBS pH 7.4 and vortexing for 30 s. To make PEG-RGDS gels, 1% w/v of functionalized 4-arm PEG-RGDS was added to the PEGDA precursor solution (the total PEG concentration was kept at 10 or 20% w/v for all gels). To prepare hydrogels in slab geometry, 50 µl of a hydrogel precursor solution was deposited on a glass plate, which was hydrophobic-coated with RainX. The hydrogel precursor solution was then sandwiched with a second glass plate, where the two plates were separated by silicon spacers (0.5 mm in height). Hydrogels were formed by exposure to 365 nm ultraviolet (UV) light (4.81 mW/cm 2 ) for 10 min.
Cell Culture and Maintenance
PC12 cells were cultured in Ham's F12K medium with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 10% FBS, and 1% pen/strep in a humidified environment at 37 • C and 5% CO 2 . NIH 3T3 cells were cultured in DMEM medium supplemented with 10% FBS and 1% pen/strep in a humidified environment at 37 • C and 5% CO 2 . For cell studies in "serum-free medium, " cells were pre-conditioned for 24 h with DMEM medium supplemented with 1X N2 supplement and 1% pen/strep. Follow up experiments were conducted in a serum-free medium of the same composition. For cell studies in "conditioned serum-free medium, " cells were pre-conditioned for 24 h with DMEM medium supplemented with 1X N2 supplement and 1% pen/strep. This "conditioned serum-free medium, " which was expected to contain soluble cell-secreted adhesive proteins such as fibronectin, was then collected and used in further experiments. For cell maintenance, medium was replaced every 2-3 days until cell confluency was reached. To subculture, confluent cells were harvested by a 5 min exposure to 0.05% trypsin/0.02% EDTA.
SWCNT Transfer From Quartz Wafers Onto Hydrogels (SWCNTs/Hydrogel) Figure 1A illustrates the developed technique for transferring quartz-grown single walled carbon nanotubes (SWCNTs) onto a hydrogel substrate, which was previously reported (Imaninezhad et al., 2017). Briefly, aligned SWCNTs were grown on a quartz wafer via a catalytic vapor deposition as described by us previously (Imaninezhad et al., 2017). To transfer the SWCNTs from the quartz wafer onto the hydrogel, 20 µl of the hydrogel precursor solution was deposited directly on the wafer. The hydrogel precursor solution was then sandwiched with a GelBond film, hydrophilic side-down. GelBond is a transparent, flexible film that has a hydrophilic and a hydrophobic side, where the hydrophilic side adheres to the hydrogel during gelation and facilitates easy hydrogel removal from the quartz. Upon gelation, the gel sandwich was soaked in DI water for 2 h to allow for hydrogel swelling and to dissolve remaining salts and Irgacure 2959. Finally, the GelBond/hydrogel was peeled off from the quartz wafer with transferred SWCNTs on its surface.
Stamped MWCNTs on PEG Hydrogels (MWCNTs/Hydrogel)
To prepare stamped MWCNTs on PEGDA hydrogels ( Figure 1B), first, PEGDA hydrogels were made as described Representative SEM image shows the actual SWCNTs (scale bar is 10 µm). Then a hydrogel precursor solution is deposited on the quartz-grown SWCNTs, sandwiched with a GelBond flexible plastic support and exposed to 365 nm ultraviolet (UV) light (4.81 mW/cm 2 ) for 10 min. Lastly, the GelBond/hydrogel construct is peeled off from the wafer with SWCNTS transferred onto the hydrogel. This nanocomposite is termed PEG/SWCNTs. (B) A hydrogel slab is made in a first step and then "stamped" onto dispersed multi-walled carbon nanotubes (MWCNTs) to form PEG/MWCNTs nanocomposites.
above. Then MWCNTs powder was dispersed on a glass plate and the PEGDA gel slabs were pressed firmly against the MWCNT powder to embed the nanotubes into the hydrogel. The MWCNTs stamped gels were then gently rinsed with PBS to remove loose MWCNTs.
Scanning Electron Microscopy Imaging of SWCNTs on Quartz Wafers
SWCNTs were imaged by scanning electron microscopy (SEM, Zeiss EVO LS15 SEM, Oberkochen, Germany). Images were acquired with low acceleration voltages of 1-1.5 kV at various magnifications under a high vacuum environment.
Evaluation of Cell Area and Shape
To test the ability of SWCNT/hydrogel and MWCNT/hydrogel composites to support cell adhesion and spreading, hydrogels were prepared as described above with and without CNTs. For the control samples without CNTs, PEG hydrogels containing immobilized RGDS (0.08 mM) were prepared to elicit cell attachment. All hydrogel samples (prepared in the form of slabs) were placed at the bottom of 24 well-plates, one sample per well and were sterilized under UV (302 nm) for 60 min. Then, 50 µl of cell suspension was carefully pipetted on top of each hydrogel to achieve a density of 5 × 10 4 cells/cm 2 and the plates were placed in an incubated environment at 37 • C and 5% CO 2 for 30 min to initiate cell attachment. Then 500 µl of additional medium was added carefully to each sample and the cells were cultured for up to 24 h. At 24 h cells were washed gently with PBS, covered again with fresh medium, and imaged under an inverted fluorescent microscope (Zeiss, Axiovert 200M, Oberkochen, Germany) at 10X zoom. All images were analyzed with ImageJ software available freely from the NIH at http://rsbweb.nih.gov/ij/. Shape factor was calculated as:
f = 4πA P 2 (1)
where, A is cell area and P is cell perimeter. A shape factor close to 1 indicates circularity and a shape factor close to 0 indicates elongation.
Immunofluorescence for Fibronectin Detection
Fibronectin was passively adsorbed to the PEG, MWCNTs/ hydrogel and SWCNTs/hydrogel samples. Fibronectin was diluted to 50 µg/ml in 1X PBS and 10 µl of the diluted solution was deposited on each sample and placed in the incubator for 1 h at 37 • C and 5% CO 2 . The samples were rinsed with 1X PBS and then 50 µl cell suspension of NIH 3T3 cells was seeded onto each sample to give a cell seeding density of 5 × 10 4 cells/cm 2 . Samples were placed in a humidified incubator at 37 • C and 5% CO 2 for 30 min to allow initial cell attachment and then 500 µl of fresh medium was added to each sample for further culturing. At specified time points, the samples were fixed in 4% formaldehyde and 0.1% Triton X-100. After fixation, samples were washed in DI water, and blocked for 20 min at 22 • C with 2% w/v BSA solubilized in PBS. Samples were incubated with Alexa 488 phalloidin, Hoechst 33258, and rabbit anti-fibronectin followed by anti-rabbit TRITC secondary antibody, and then washed, and mounted onto standard glass slides. A drop of Aqua/Poly mount was added onto the hydrogels and then hydrogels were covered with a plain glass coverslip. Images were acquired with a Leica DMi8 inverted epifluorescence microscope fitted with a 12-bit grayscale CCD camera using a 40X dry objective. All images were acquired using Metamorph software.
Peptidomimetic Studies
CWHM-12 is a small molecule peptidomimetic of the amino acid motif Arg-Gly-Asp (RGD) which potently inhibits (IC 50 < 10 nM) the binding of integrins αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and α5β1 to their respective extracellular matrix ligands, as has been described previously (Henderson et al., 2013). CWHM-96 is the R-isomer of CWWM-12 differing only in the orientation of its carboxyl (CO 2 H) group and is 100-1000-fold less potent than CWHM-12 for inhibition of these same integrins. Therefore, it was used as a highly structurally similar negative control compound. Stock solutions of 1 mM of the peptidomimetic compounds were prepared in 50% DMSO (in sterile water). PC12 and NIH 3T3 cells were seeded at 5×10 4 cells/cm 2 on SWCNTs/hydrogel, MWCNTs/hydrogel, and control PEG-RGDS substrates and cultured for 24 h. After 24 h of culture, the peptidomemetics were added at a final concentration of 10 µM directly to the cell medium and the cells were exposed to peptidomemetics for additional 24 h. The cell medium was exchanged with fresh medium and samples were imaged under an inverted fluorescent microscope (Zeiss, Axiovert 200M, Oberkochen, Germany).
Statistical Analysis
Results are reported as averages ± standard deviation. Statistical significance between multiple samples was tested by ANOVA followed by a post-hoc analysis and between two samples by Student's t-test (p < 0.05). A minimum of three samples from three independent experiments were tested per condition. For cell analysis, a minimum of 20 cells per image, from a minimum of six images were analyzed per condition. Figure 2 shows NIH 3T3 cell attachment and spreading in serum, serum-free and conditioned serum-free medium on PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels. Note that PEG only hydrogel was also tested as a control, but cell attachment was not observed (data not shown). Hence, PEG-RGDS was used, where the RGDS ligand was covalently tethered to support integrin-mediated cell adhesion . To produce the conditioned serum-free medium, cells were exposed to serum-free medium for 24 h upon which the conditioned serum-free medium (i.e., containing cell-secreted biomolecules) was collected and used for experiments. Phase contrast images showed that cells adhered on all substrates independently of serum presence but exhibited serum-dependent morphology (Figure 2A). Cells had higher cell spreading area (per individual cell) and an elongated morphology (low shape factor) in the presence of serum in the medium and lower cell spreading area and a more rounded morphology (high shape factor) in the absence of serum. Quantification revealed that cell spreading area in the presence of serum was significantly higher than other media for all hydrogel types ( Figure 2B). The differences were largest between the serum and serumfree media: ∼56% for cells seeded on the PEG-RGDS and the PEG/MWCNTs gels and ∼70% for cells seeded on the PEG/SWCNTs gels. The difference between the serum-free and conditioned serum-free medium was largest for cells seeded on the PEG/MWCNTs gels (∼56%) and not significant for cells seeded on the PEG/SWCNTs gels. Shape factor was inversely proportional to cell spreading area, where lowest shape factor (i.e., highest elongation) was observed in the serum medium, followed by the conditioned serum-free and lastly by the serumfree medium ( Figure 2C). Overall, our results confirmed that serum proteins facilitated cell spreading and elongation for cells seeded on the nanocomposite hydrogels and the control PEG-RGDS gels. Conditioning the serum-free medium with cell-secreted proteins improved cell spreading and elongation over the serum-free condition but was not as effective as serum in the media. Importantly our results indicate that cells were able to adhere and spread onto unmodified SWCNTs and MWCNTs embedded in the inert PEG gels even in the absence of any adhesive ligands on the material. We suggest that the reason for cell adhesion and spreading was the ability of SWCNTs and MWCNTs to adsorb adhesive proteins (e.g., fibronectin) secreted by cells or present in serum (Khang et al., 2007), which then could elicit cell attachment and subsequent spreading.
RESULTS
Cells Adhered and Spread Onto SWCNTs and MWCNTs Due to Adsorption of Adhesive Proteins
Exogenous and Cell-Secreted Fibronectin Adsorbed Onto SWCNTs and MWCNTs; Intracellular Fibronectin Not Affected by CNTs Presence
We studied the effect of fibronectin adsorption, both exogenous and cell-secreted, onto PEG/SWCNTs, and PEG/MWCNTs nanocomposite hydrogels via epifluorescence microscopy. To ascertain anti-fibronectin antibody specificity a negative control was used, where samples stained with normal rabbit IgG fraction were compared to samples stained with the affinity purified, rabbit anti-fibronectin antibody. Only the sample stained with the anti-fibronectin antibody showed intense TRITC fluorescence (indicated with arrows) indicating specificity ( Figure S1).
NIH 3T3 cells were stained for fibronectin, actin and cell nucleus to visualize intracellular and extracellular fibronectin at 2 h post-seeding in a serum-free medium for cells seeded on top of PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels (Figure 3). All cells stained positive for fibronectin, and there appeared to be some extracellular fibronectin at that time point, particularly on the PEG/MWCNTs nanocomposites (yellow arrows in images). At this early time point cells on all hydrogel types appeared rounded and the number of attached cells was low.
To determine whether pre-absorbed fibronectin will facilitate better early cell attachment and spreading, we imaged cells at 2 h of culture in serum-free medium on fibronectin-coated PEG/MWCNTs nanocomposite and the PEG-RGDS control gels (Figure 4). We specifically focused on the MWCNTs/hydrogel composite, as opposed to the SWCNTs/hydrogel one, as a representative substrate due to the easier nanotube visualization. Upon staining, we noted intracellular and some extracellular fibronectin. As expected, extracellular fibronectin was not seen on the inert PEG-RGDS gels but was seen on the PEG/MWCNTs nanocomposite due to fibronectin adsorption onto the MWCNTs. Note that we observed fibronectin only on the smaller MWCNTs clusters and not the larger ones, because we could not image the surface of the black MWCNTs with an inverted microscope. Importantly, we observed cell spreading even at this early time point and a higher number of adhered cells compared to the no-exogenousfibronectin condition (Figure 3), especially for cells seeded on the nanocomposite hydrogel. Our results indicate that passive adsorption of adhesive proteins, such as fibronectin, FIGURE 4 | Fluorescence images of NIH 3T3 cells on fibronectin-coated PEG/MWCNTs and PEG-RGDS hydrogels at 2 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm.
FIGURE 5 | Fluorescence images of NIH 3T3 cells on PEG/MWCNTs hydrogels (−Fn) and fibronectin-coated PEG/MWCNTs hydrogels (+Fn) at 6 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm. Arrows indicate extracellular fibronectin.
facilitate cell adhesion and spreading on CNT/hydrogel nanocomposites.
Additionally, to determine whether the presence of MWCNTs affected cell production of fibronectin (i.e., intracellular fibronectin amount), we quantified the amount of fibronectin per cell. Comparing cells on the PEG/MWCNTs substrate vs. the PEG-RGDS substrate we observed no difference in intracellular fibronectin. The mean integrated pixel intensity ± s.e.m. in the PEG/MWCNT and PEG-RGDS fibronectin images was 2.7 × 10 6 ± 0.2 × 10 6 (n = 27 cells) and 3.3 × 10 6 ± 0.3 × 10 6 (n = 33 cells), respectively, yielding a p-value of 0.11. Hence, we conclude that MWCNT presence did not affect intracellular fibronectin levels.
Lastly, we stained for fibronectin at a longer time point of 6 h for cells seeded onto PEG/MWCNTs nanocomposites expecting to see more cell-secreted extracellular fibronectin and higher number of adhered cells (Figure 5). We tested two conditions: exogenously coated fibronectin (+Fn) and no exogenous fibronectin (-Fn). We observed a similar number of attached cells irrespective of exogenous fibronectin. All cells stained positive for intracellular fibronectin; we did not note qualitative differences between cells on the fibronectin-coated samples (+Fn) and the uncoated ones (−Fn). Similarly to cell images at 2 h post-seeing, at 6 h post-seeding we observed some extracellular fibronectin in the (−Fn) condition (indicated by yellow arrows in the images), which appeared to accumulate around MWCNTs in the gel and was not seen in MWCNTdevoid regions. The fibronectin-coated samples (+Fn) exhibited an overall higher level of fluorescence from the hydrogel surface indicative of protein adsorption onto the MWCNTs. Our results indicate that cells were able to secrete fibronectin, which was subsequently adsorbed onto the MWCNTs in the nanocomposites and elicited cell adhesion and spreading. Lastly, while the addition of exogenous fibronectin led to improved cell adhesion at 2 h post-seeding (Figures 3, 4), it did not seem to affect cell adhesion at 6 h post-seeding; similar number of adhered cells were noted on both (−Fn) and (+Fn) substrates.
Cells Adhered and Spread Onto SWCNTs and MWCNTs on PEG Hydrogels via Integrin Binding
Next, we tested whether cell attachment and spreading onto PEG/MWCNTs and PEG/SWCNTs hydrogels was facilitated FIGURE 6 | (A) Phase contrast images of NIH 3T3 cells in serum, (B) serum-free, (C) conditioned serum-free media on PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS hydrogels at 24 h after exposure to peptidomimetics (scale bar is 100 µm). (D) Cell area and (E) shape factor of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium in the presence of CWHM-12 (active) and CWHM-96 (inactive control) peptidomimetics. *Asterisks designate significant differences (120 cells from n = 3, p < 0.05).
by integrin binding (Figure 6). Note that the SWCNTs in the PEG/SWCNTs hydrogels were 1-2 nm in diameter (Imaninezhad et al., 2017); hence physical entanglement of cell processes on SWCNTs alone could not explain cell attachment and spreading (Sorkin et al., 2009). We tested a highly stable synthetic RGD peptidomimetic, namely CWHM-12, with selective inhibition toward integrins ανβ1, ανβ3, ανβ5, ανβ6, ανβ8, and α5β1, which collectively mediate binding to RGD-containing biomolecules present in serum such as fibronectin and vitronectin. In contrast, the R-enantiomer of this compound, namely CWHM-96, has very low activity toward the same integrins.
We tested the effect of the integrin inhibitor CWHM-12 and the inactive control CWHM-96 on NIH 3T3 cell attachment and spreading onto PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels in serum (Figure 6A), serum-free ( Figure 6B) and conditioned serum-free (Figure 6C) medium. For all conditions tested, the cells rounded up and formed cell aggregates on top of the gels when exposed to the active CWHM-12, but not when exposed to the inactive CWHM-96 compound. When quantifying cell area ( Figure 6D) and shape factor (Figure 6E), we observed that both depended on the media and the substrate in the presence of the inactive compound CWHM-96, but not in the presence of the active compound CWHM-12 where all cells were rounded. As expected, in the presence of CWHM-96 cells were most spread and elongated in the presence of serum in the media, followed by conditioned serum-free, followed by serumfree medium. The differences between the three media conditions were most pronounced for the PEG-RGDS and PEG/MWCNTs gels, but were minimal for cells seeded on the PEG/SWCNTs gels. In the presence of the active compound CWHM-12 all cells had a similar cell area of ∼400 µm 2 and a shape factor close to 1, which is indicative of a circular shape, suggesting an inhibition of integrin binding.
We also tested cells seeded on PEG/SWCNTs gels in serumfree and conditioned serum-free medium after a shorter exposure of 4 h to peptidomimetics CWHM-12 and CWHM-96 (data not shown). We observed cells rounding upon exposure to the active CWHM-12 compound in both medium conditions and not in response to the inactive CWHM-96 one, similarly to data presented for 24 h of exposure. Overall, our results show that cells attached and spread via integrin binding onto all substrates in all media conditions, indicating that serum or cell-secreted adhesive proteins adsorb on the nanotubes and elicit cell attachment and subsequent spreading.
Integrin-Facilitated Cell Adhesion and Spreading to SWCNTs and MWCNTs Was Cell-Type Independent
Lastly, to confirm that cell attachment and spreading onto SWCNTs and MWCNTs was not specific to NIH 3T3 cells, we used PC12 cells in one representative experiment (Figure 7). Specifically, we seeded PC12 cells on PEG/SWCNTS, PEG/MWCNTS, and PEG-RGDS gels in serum-free medium containing inactive CWHM-96 or active CWHM-12 compounds. As with NIH 3T3 cells, PC12 cells were able to attach and spread onto all substrates in all media conditions. When exposed to the inactive CWHM-96 compound cells seemed more spread and elongated but were completely rounded in the presence of the active CWHM-12 compound. Our data indicates that PC12 cells, similarly to NIH 3T3 cells adhered and spread onto SWCNTs and MWCNTs via integrin binding to cell-secreted proteins adsorbed onto the nanotubes.
DISCUSSION
The objective of this study was to understand how cells adhere and spread onto MWCNTs aggregates and individual SWCNTs of diameter less than 2 nm embedded in an inert PEG hydrogel substrate. The significance of embedding the nanotubes in PEG substrate was to isolate cell adhesion and spreading onto the nanotubes, both SWCNTs and MWCNTs, from adhesion and spreading onto the underlying substrate: PEG is inert and does not support cell adhesion (Imaninezhad et al., 2017). We postulated that cells adhere and spread onto individual SWCNTs and MWCNTs aggregates via integrin binding because of serum or cell-secreted proteins adsorbed onto the CNTs' surface. Proteins are known to adsorb tightly onto the CNT surface due to pi-stacking (Salvador-Morales et al., 2006;Kaiser et al., 2013). This study was enabled by our previous work, where we developed a method to transfer individual SWCNTs onto hydrogel substrates and demonstrated robust cell adhesion to these novel nanocomposite materials (Imaninezhad et al., 2017). Because the SWCNTs were of a very small diameter (<2 nm) and dispersed individually, we postulated that physical entanglement between cellular processes and SWCNTs could not explain cell adhesion and spreading, especially because the cellular processes were of greater dimension than the individual SWCNTs; previous work has shown that neural cells can attach to MWCNT aggregates by physical entanglement of cellular processes with the nanotubes if both have similar dimensions (Sorkin et al., 2009).
To test our hypothesis, we cultured cells on PEG/SWCNTs nanocomposites, PEG/MWCNTs nanocomposites and PEG-RGDS hydrogels (no CNTs control) in various media, namely serum, serum-free, and conditioned serum-free. Note that both nanocomposite substrates, PEG/MWCNTs and PEG/SWCNTs, were developed and characterized by us previously, and have shown excellent biocompatibility with >90% cells viability retained at 24 h post-culture (Shah et al., 2015;Imaninezhad et al., 2017). The conditioned serum-free medium was used to control for the effect of cell-secreted adhesive proteins (e.g., fibronectin) as this medium was considered enriched in such proteins. The PEG-RGDS gels were used as a control substrate, since cells could only attach to the RGDS ligand in those gels. We anticipated that cell-secreted proteins will adsorb onto the CNTs and facilitate a more robust cell adhesion and spreading in this condition compared to the serum-free condition (Vogel et al., 1980). At 24 h of culture, we noted some cell adhesion in all media conditions with the highest number of cells observed in the serum, followed by the conditioned serum-free, followed by the serum-free media (Figure 2). It is important to note that cells clustered around nanotube-rich areas, which was most clearly visible on the PEG/MWCNTs substrates. As expected, the highest cell spreading was also seen in the serum medium and the lowest in the serum-free medium. Further, since fibroblasts are known to secrete adhesive proteins shortly after culturing in serum-free medium (Grinnell and Feld, 1979), our 24 h time point meant that even cells in the serum-free medium would be able to secrete proteins to facilitate their adhesion and spreading onto the surface. This result led us to believe that adsorption of adhesive proteins, either from the serum or cell-secreted, was critical and facilitated cell adhesion and spreading onto both SWCNTs and MWCNTs.
To probe for cell adhesion and spreading as well as for adhesive protein secretion at shorter time points, we stained cells cultured in a serum-free medium for 2 and 6 h for intracellular fibronectin (Figures 3-5). Fibronectins are adhesive glycoproteins on the cell surface and in the extracellular matrix and a common soluble protein secreted by cells (Akiyama and Yamada, 1985). Fibronectin has been shown to adsorb tightly and with high affinity onto carbon nanotubes (Khang et al., 2007). On the other hand, studies on the effect of soluble fibronectin binding to the surface of cells have shown that fibronectin almost exclusively binds to the external surface of the plasma membrane (Akiyama and Yamada, 1985). Fibroblast cells in particular have been shown to attach and spread in a serum-free medium because of cell-secreted fibronectin within 60 min of seeding (Grinnell and Feld, 1979). We hypothesized that cells were able to secrete fibronectin, which would then adsorb with high affinity to the SWCNTs and MWCNTs, facilitating cell adhesion and spreading. As anticipated, at 2 h we noted that cells on all substrates stained positive for intracellular fibronectin, but we observed minimal extracellular fibronectin adsorbed on the carbon nanotube substrates (Figure 3). It could be due to the early time point, but it also could be due to the high intensity of intracellular fibronectin obscuring any weaker signal from the extracellular one. Hence, to confirm that fibronectin indeed facilitated cell adhesion, we pre-coated PEG/MWCNTs and PEG-RGDS substrates with exogenous fibronectin and cultured cells for 2 h (Figure 4). We observed a higher number of attached cells on the exogenously coated PEG/MWCNTs nanocomposites compared to the noncoated ones. We then repeated the experiment at a later time point, namely 6 h, at which point we anticipated that cells would secrete sufficient amount of fibronectin to facilitate robust cell adhesion and spreading (Grinnell and Feld, 1979) (Figure 5). At 6 h, we saw a larger number of cells adhering to the PEG/MWCNTs substrate compared to the 2 h time point in serum-free medium (48 ± 21 cells/mm 2 vs. 27 ± 14 cells/mm 2 , respectively), where the cells were also more spread. Importantly, at 6 h there was no difference between numbers of adhered cells on the fibronectin-pre-coated vs. non-coated PEG/MWCNTs nanocomposites. Hence, our results suggested that a soluble cell-secreted adhesive protein such as fibronectin facilitated cell adhesion and spreading onto CNTs.
Finally, we did not see a significant difference in intracellular fibronectin expression for cells seeded on PEG/MWCNTs vs. PEG-RGDS substrates (Figure 4), suggesting that MWCNTs did not stimulate fibronectin expression. The effect of surface tethered MWCNTs on fibronectin expression from fibroblast cells had not been studied before. However, multiple studies have indicated that the microenvironment could affect fibronectin expression and secretion by cells. For example, it has been shown that surface roughness can increase fibronectin expression and secretion by fibroblast cells, and that fibronectin expression is greatly enhanced in high serum conditions and minimal in low serum conditions (Chou et al., 1995). Here, the PEG/MWCNTs substrate should have presented higher roughness than PEG-RGDS due to the presence of MWCNT aggregates, but the cells were cultured in the absence of serum, which could explain the similar expression levels for both conditions. However, another study on the effect of soluble SWCNTs on HEK293 cells has shown that SWCNTs down-regulated the expression of several adhesion proteins such as laminin, fibronectin, cadherin, FAK, and collagen IV (Cui et al., 2005). The difference between this finding and our results could be due to the different presentation of CNTs (soluble vs. tethered) as well as the different cell types and CNTs types used.
While we showed that cell adhesion and spreading was facilitated by adhesive proteins adsorbed onto the nanotubes, the question still remained whether the cell binding and spreading onto SWCNTs and MWCNTs was integrin-dependent. Integrins are heterodimers consisting of an α and a β subunit that together determine ligand specificity and initiate intracellular signaling events (Lowin and Straub, 2011). Fibroblast cells express the β1, β3, β5, αv, and α5 subunits of RGD-binding integrins that mediate cellular processes such as adhesion, spreading and migration (Lowin and Straub, 2011). Within the aminoacid sequences of proteins such as fibronectin and vitronectin, integrins bind to the arginine-glycine-aspartic acid (RGD) motifs (Sarin et al., 2005). Interaction with the RGD sequence is an important switch for integrin activation and subsequent cellular changes. The same ECM molecules can recognize various integrin combinations. For example, fibronectin is recognized by α4β1, α5β1, ανβ1, ανβ3, and α3β1, and weakly by others (Lowin and Straub, 2011).
Here, we used a potent small-molecule inhibitor of α5 (α5β1) and αν integrins (ανβ1, ανβ3, ανβ5, ανβ6, and ανβ8), namely the RGD peptidomimetic CWHM-12, and an inactive peptidomimetic CWHM-96 as control (Henderson et al., 2013). Specifically, we studied the effect of the peptidomimetics on cell attachment and spreading on PEG/SWCNTs nanocomposites, PEG/MWCNTs nanocomposites and PEG-RGDS control hydrogels in serum, serum-free and conditioned serum-free media (Figure 6). Our goal was to determine whether cell attachment and spreading onto the nanotubes in the various media conditions was dependent on RGD-binding integrins. On all substrates and in all media conditions, cells adhered and spread (higher spreading in the serum and conditioned serum-free media) in the presence of the inactive CWHM-96 compound, but balled up and formed clusters in the presence of the active CWHM-12 compound. Similar responses to the peptidomimetics were seen with another cell type, namely PC12 cells (Figure 7), indicating that the response was not cell-specific. Note that cell rounding was not caused by peptidomimetic toxicity but due to its ability to inhibit integrin binding with the substrate (Henderson et al., 2013). Hence, our results indicated that cell adhesion and spreading onto SWCNTs and MWCNTs was similar to the cell adhesion and spreading onto the control PEG-RGD gels and was integrin-dependent. While studies with RGD-like peptidomimetics have not been performed before, other studies have shown that cells are able to form focal adhesion complexes on CNTs (Ryoo et al., 2010), indicative of integrin binding.
One limitation of our study is that we did not specifically study short time points (<30 min) to determine if initial cell attachment is also dominated by integrin binding. For example, some recent work on cell adhesion to charged polymers has suggested that short term cell adhesion in the presence and absence of serum in the media is different and could not be fully explained by either integrin binding or charged interactions (Hoshiba et al., 2018). Hence, it is possible that a different and yet unidentified mechanism is guiding cell adhesion to CNTs initially, but is masked by a predominant integrin-dependent adhesion at the later time points studied here. We were also not able to unequivocally rule out that cells were able to attach directly to the CNTs. It has been suggested previously that SWCNTs can bind directly to integrins as well as to ECM proteins first, where the whole complex subsequently binds to integrins (Kaiser et al., 2013). However, it was only a suggestion based on observations by the researchers and was not proven unequivocally. Studies to answer such follow up questions will be included in our future work.
CONCLUSION
In summary, we observed that NIH 3T3 cells attached and spread onto the PEG/SWCNTs and PEG/MWCNTs nanocomposites due to adhesive proteins from serum in the medium, where proteins adsorbed onto the SWCNTs and MWCNTs. Cells also adhered and spread onto the nanocomposites in serumfree medium due to cell-secreted adhesive proteins, such as fibronectin. Conditioning the serum-free medium with cellsecreted proteins enhanced cell attachment and spreading. All cells stained positive for intracellular fibronectin at 2 and 6 h of culture, where MWCNTs and SWCNTs presence did not affect intracellular fibronectin expression. Inhibition of cell attachment and spreading onto the nanocomposite materials by a broadaction RGD peptidomimetic (active against α ν β 1 , α ν β 3, α ν β 5 , α ν β 6, and α ν β 8 integrins) confirmed that cell attachment and spreading onto the nanotubes was integrin-dependent. Similar results were observed with two different cell types, namely NIH 3T3 fibroblasts and PC12 neural-like cells, indicating that the process of cell attachment and spreading onto nanotubes was cell type-independent. This was the first study to explore why and how cells attach and spread onto both individual SWCNTs (<2 nm in diameter) and MWCNTs aggregates, embedded in an inert PEG substrate (to prevent non-specific cell attachment). Our results are significant because they demonstrated that cells used the same process for adhesion and spreading onto nanotubes and adhesive ligands such as RGDS. Importantly, we were able to show that cells recognized and bound to nanotubes which were significantly smaller than the smallest cell processes.
AUTHOR CONTRIBUTIONS
MI conducted most of the experiments and wrote a first draft of the paper. JS performed the immunostaining work and provided associated figures and some text related to this work. DG and PR provided the peptidomimetics and contributed to experimental design related to peptidomimetics. IK assisted with SWCNT samples grown on quartz. SZ conceptualized the idea, guided experimental design and data analysis and wrote the final paper.
FIGURE 1 |
1Schematic of CNT-hydrogel nanocomposites preparation. (A) Single-walled carbon nanotubes (SWCNTs) are grown on a quartz wafer in a first step.
FIGURE 2 |
2(A) Phase contrast images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium (scale bar is 100 µm). (B) Cell spreading area and (C) cell shape factor (inverse of elongation) for NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium. *Asterisks designate significant differences (120 cells from n = 3, p < 0.05).
FIGURE 3 |
3Fluorescence images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels at 2 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm. Arrows indicate extracellular fibronectin.
FIGURE 7 |
7Phase contrast images of PC12 cells in serum-free medium upon exposure to CWHM-12 (pos.) and CWHM-96 (neg.) peptidomimetics. Scale bar is 100 µm.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org
September 2018 | Volume 6 | Article 129
ACKNOWLEDGMENTSWe acknowledge support from start-up funds provided to SZ by Saint Louis University and a Spark Microgrant awarded to SZ and IK by Saint Louis University. MI acknowledges support from Barta Scholarship and Dissertation Fellowship awarded by Saint Louis University. We thank Isy Okafor for technical assistance with SWCNTs growth on quartz wafers.SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2018.00129/full#supplementary-material
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Enhanced fibronectin adsorption on carbon nanotube/poly (carbonate) urethane: independent role of surface nanoroughness and associated surface energy. D Khang, S Y Kim, P Liu-Snyder, G T Palmore, S M Durbin, T J Webster, 10.1016/j.biomaterials.2007.07.018Biomaterials. 28Khang, D., Kim, S. Y., Liu-Snyder, P., Palmore, G. T., Durbin, S. M., and Webster, T. J. (2007). Enhanced fibronectin adsorption on carbon nanotube/poly (carbonate) urethane: independent role of surface nano- roughness and associated surface energy. Biomaterials 28, 4756-4768. doi: 10.1016/j.biomaterials.2007.07.018
Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference. K W Kwon, S S Choi, S H Lee, B Kim, S N Lee, M C Park, 10.1039/b710054jLab Chip. 7Kwon, K. W., Choi, S. S., Lee, S. H., Kim, B., Lee, S. N., Park, M. C., et al. (2007). Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference. Lab Chip. 7, 1461-1468. doi: 10.1039/b710054j
Biohybrid carbon nanotube/agarose fibers for neural tissue engineering. D Y Lewitus, J Landers, J Branch, K L Smith, G Callegari, J Kohn, 10.1002/adfm.201002429Adv. Funct. Mater. 21Lewitus, D. Y., Landers, J., Branch, J., Smith, K. L., Callegari, G., Kohn, J., et al. (2011). Biohybrid carbon nanotube/agarose fibers for neural tissue engineering. Adv. Funct. Mater. 21, 2624-2632. doi: 10.1002/adfm.201002429
Integrins and their ligands in rheumatoid arthritis. T Lowin, R H Straub, 10.1186/ar3464Arthritis Res. Ther. 13Lowin, T., and Straub, R. H. (2011). Integrins and their ligands in rheumatoid arthritis. Arthritis Res. Ther. 13, 244-244. doi: 10.1186/ar3464
Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes. S Namgung, K Y Baik, J Park, S Hong, 10.1021/nn2023057ACS Nano. 5Namgung, S., Baik, K. Y., Park, J., and Hong, S. (2011). Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes. ACS Nano 5, 7383-7390. doi: 10.1021/nn2023057
Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts. R L Price, K Ellison, K M Haberstroh, T J Webster, 10.1002/jbm.a.30073J. Biomed. Mater. Res. Part A. 70Price, R. L., Ellison, K., Haberstroh, K. M., and Webster, T. J. (2004). Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts. J. Biomed. Mater. Res. Part A. 70, 129-138. doi: 10.1002/jbm.a.30073
Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. S R Ryoo, Y K Kim, M H Kim, Min , D H , 10.1021/nn1018279ACS Nano. 4Ryoo, S. R., Kim, Y. K., Kim, M. H., and Min, D. H. (2010). Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. ACS Nano 4, 6587-6598. doi: 10.1021/nn1018279
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Complement activation and protein adsorption by carbon nanotubes. C Salvador-Morales, E Flahaut, E Sim, J Sloan, M L Green, R B Sim, 10.1016/j.molimm.2005.02.006Mol. Immunol. 43Salvador-Morales, C., Flahaut, E., Sim, E., Sloan, J., Green, M. L., and Sim, R. B. (2006). Complement activation and protein adsorption by carbon nanotubes. Mol. Immunol. 43, 193-201. doi: 10.1016/j.molimm.2005.02.006
Arginineglycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via α(5)β(1) integrin. V Sarin, R D Gaffin, G A Meininger, M Muthuchamy, 10.1113/jphysiol.2005.083238J. Physiol. 564Sarin, V., Gaffin, R. D., Meininger, G. A., and Muthuchamy, M. (2005). Arginine- glycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via α(5)β(1) integrin. J. Physiol. 564, 603-617. doi: 10.1113/jphysiol.2005.083238
Development and characterization of polyethylene glycol-carbon nanotube hydrogel composite. K Shah, D Vasileva, A Karadaghy, S Zustiak, 10.1039/C5TB01047KJ. Mater. Chem. B. 3Shah, K., Vasileva, D., Karadaghy, A., and Zustiak, S. (2015). Development and characterization of polyethylene glycol-carbon nanotube hydrogel composite. J. Mater. Chem. B. 3, 7950-7962. doi: 10.1039/C5TB01047K
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Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces. K Webb, V Hlady, P A Tresco, 10.1002/(SICI)1097-4636(20000305)49:3<362::AID-JBM9>3.0.CO;2-SJ. Biomed. Mater. Res. 493<362::AID-JBM9>3.0.CO;2-SWebb, K., Hlady, V., and Tresco, P. A. (2000). Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces. J. Biomed. Mater. Res. 49, 362-368. doi: 10.1002/(SICI)1097-4636(20000305) 49:3<362::AID-JBM9>3.0.CO;2-S
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Influence of cell-adhesive peptide ligands on poly (ethylene glycol) hydrogel physical, mechanical and transport properties. S P Zustiak, R Durbal, J B Leach, 10.1016/j.actbio.2010.03.040Acta Biomater. 6Zustiak, S. P., Durbal, R., and Leach, J. B. (2010). Influence of cell-adhesive peptide ligands on poly (ethylene glycol) hydrogel physical, mechanical and transport properties. Acta Biomater. 6, 3404-3414. doi: 10.1016/j.actbio.2010. 03.040
Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. S P Zustiak, J B Leach, 10.1021/bm100137qdoi: 10.1021/ bm100137qBiomacromolecules. 11Zustiak, S. P., and Leach, J. B. (2010). Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. Biomacromolecules 11, 1348-1357. doi: 10.1021/ bm100137q
| [
"Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes."
] | [
"May Griffith ",
"Damien Harkin ",
"Hirak Kumar Patra ",
"Peter Ruminski ",
"Imaninezhad M Schober ",
"J ",
"Griggs D ",
"Ruminski P ",
"Kuljanishvili I ",
"Mozhdeh Imaninezhad \nBiomedical Engineering\nSaint Louis University\nSaint LouisMOUnited States\n",
"Joseph Schober \nPharmaceutical Sciences\nSouthern Illinois University\nEdwardsvilleILUnited States\n",
"David Griggs \nMolecular Microbiology & Immunology\nSaint Louis University\nSaint LouisMOUnited States\n",
"Peter Ruminski \nCenter for World Health and Medicine\nSaint Louis University\nUnited States, 5 PhysicsSaint LouisMO\n\nSaint Louis University\nSaint LouisMOUnited States\n",
"† ",
"Irma Kuljanishvili ",
"Silviya Petrova Zustiak \nBiomedical Engineering\nSaint Louis University\nSaint LouisMOUnited States\n",
"\nUniversité de Montréal\nCanada\n",
"\nQueensland University of Technology\nAustralia\n",
"\nDepartment of Medicine\nDivision of Oncology\nUniversity of Cambridge\nUnited Kingdom\n",
"\nWashington University\nSt. LouisMOUnited States\n"
] | [
"Biomedical Engineering\nSaint Louis University\nSaint LouisMOUnited States",
"Pharmaceutical Sciences\nSouthern Illinois University\nEdwardsvilleILUnited States",
"Molecular Microbiology & Immunology\nSaint Louis University\nSaint LouisMOUnited States",
"Center for World Health and Medicine\nSaint Louis University\nUnited States, 5 PhysicsSaint LouisMO",
"Saint Louis University\nSaint LouisMOUnited States",
"Biomedical Engineering\nSaint Louis University\nSaint LouisMOUnited States",
"Université de Montréal\nCanada",
"Queensland University of Technology\nAustralia",
"Department of Medicine\nDivision of Oncology\nUniversity of Cambridge\nUnited Kingdom",
"Washington University\nSt. LouisMOUnited States"
] | [
"May",
"Damien",
"Hirak",
"Kumar",
"Peter",
"Imaninezhad",
"M",
"J",
"Griggs",
"D",
"Ruminski",
"P",
"Kuljanishvili",
"I",
"Mozhdeh",
"Joseph",
"David",
"Peter",
"†",
"Irma",
"Silviya",
"Petrova"
] | [
"Griffith",
"Harkin",
"Patra",
"Ruminski",
"Schober",
"Imaninezhad",
"Schober",
"Griggs",
"Ruminski",
"Kuljanishvili",
"Zustiak"
] | [
"S K Akiyama, ",
"K M Yamada, ",
"S Bosi, ",
"A Fabbro, ",
"L Ballerini, ",
"M Prato, ",
"L Chou, ",
"J D Firth, ",
"J Uitto, ",
"D M Brunette, ",
"D Cui, ",
"F Tian, ",
"C S Ozkan, ",
"M Wang, ",
"H Gao, ",
"Y W Fan, ",
"F Z Cui, ",
"S P Hou, ",
"Q Y Xu, ",
"L N Chen, ",
"I S Lee, ",
"B Geiger, ",
"A Bershadsky, ",
"R Pankov, ",
"K M Yamada, ",
"F Grinnell, ",
"M K Feld, ",
"B S Harrison, ",
"Atala , ",
"A , ",
"N C Henderson, ",
"T D Arnold, ",
"Y Katamura, ",
"M M Giacomini, ",
"J D Rodriguez, ",
"J H Mccarty, ",
"T Hoshiba, ",
"C Yoshikawa, ",
"K Sakakibara, ",
"W Huang, ",
"B Anvari, ",
"J H Torres, ",
"R G Lebaron, ",
"K A Athanasiou, ",
"R O Hynes, ",
"M Imaninezhad, ",
"I Kuljanishvili, ",
"S P Zustiak, ",
"J P Kaiser, ",
"T Buerki-Thurnherr, ",
"P Wick, ",
"D Khang, ",
"S Y Kim, ",
"P Liu-Snyder, ",
"G T Palmore, ",
"S M Durbin, ",
"T J Webster, ",
"K W Kwon, ",
"S S Choi, ",
"S H Lee, ",
"B Kim, ",
"S N Lee, ",
"M C Park, ",
"D Y Lewitus, ",
"J Landers, ",
"J Branch, ",
"K L Smith, ",
"G Callegari, ",
"J Kohn, ",
"T Lowin, ",
"R H Straub, ",
"S Namgung, ",
"K Y Baik, ",
"J Park, ",
"S Hong, ",
"R L Price, ",
"K Ellison, ",
"K M Haberstroh, ",
"T J Webster, ",
"S R Ryoo, ",
"Y K Kim, ",
"M H Kim, ",
"Min , ",
"D H , ",
"G Sagvolden, ",
"I Giaever, ",
"E O Pettersen, ",
"J Feder, ",
"K Sahithi, ",
"M Swetha, ",
"K Ramasamy, ",
"N Srinivasan, ",
"N Selvamurugan, ",
"C Salvador-Morales, ",
"E Flahaut, ",
"E Sim, ",
"J Sloan, ",
"M L Green, ",
"R B Sim, ",
"V Sarin, ",
"R D Gaffin, ",
"G A Meininger, ",
"M Muthuchamy, ",
"K Shah, ",
"D Vasileva, ",
"A Karadaghy, ",
"S Zustiak, ",
"S R Shin, ",
"S M Jung, ",
"M Zalabany, ",
"K Kim, ",
"P Zorlutuna, ",
"N M Kim, ",
"R Sorkin, ",
"A Greenbaum, ",
"M David-Pur, ",
"S Anava, ",
"A Ayali, ",
"E Ben-Jacob, ",
"A Van Der Flier, ",
"A Sonnenberg, ",
"A Vogel, ",
"R Ross, ",
"E Raines, ",
"C C Wang, ",
"Y C Hsu, ",
"F C Su, ",
"S C Lu, ",
"T M Lee, ",
"K Webb, ",
"V Hlady, ",
"P A Tresco, ",
"J X Zou, ",
"Y Liu, ",
"E B Pasquale, ",
"E Ruoslahti, ",
"S P Zustiak, ",
"R Durbal, ",
"J B Leach, ",
"S P Zustiak, ",
"J B Leach, "
] | [
"S",
"K",
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"A",
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"Atala",
"A",
"N",
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] | [
"Akiyama",
"Yamada",
"Bosi",
"Fabbro",
"Ballerini",
"Prato",
"Chou",
"Firth",
"Uitto",
"Brunette",
"Cui",
"Tian",
"Ozkan",
"Wang",
"Gao",
"Fan",
"Cui",
"Hou",
"Xu",
"Chen",
"Lee",
"Geiger",
"Bershadsky",
"Pankov",
"Yamada",
"Grinnell",
"Feld",
"Harrison",
"Henderson",
"Arnold",
"Katamura",
"Giacomini",
"Rodriguez",
"Mccarty",
"Hoshiba",
"Yoshikawa",
"Sakakibara",
"Huang",
"Anvari",
"Torres",
"Lebaron",
"Athanasiou",
"Hynes",
"Imaninezhad",
"Kuljanishvili",
"Zustiak",
"Kaiser",
"Buerki-Thurnherr",
"Wick",
"Khang",
"Kim",
"Liu-Snyder",
"Palmore",
"Durbin",
"Webster",
"Kwon",
"Choi",
"Lee",
"Kim",
"Lee",
"Park",
"Lewitus",
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"Branch",
"Smith",
"Callegari",
"Kohn",
"Lowin",
"Straub",
"Namgung",
"Baik",
"Park",
"Hong",
"Price",
"Ellison",
"Haberstroh",
"Webster",
"Ryoo",
"Kim",
"Kim",
"Sagvolden",
"Giaever",
"Pettersen",
"Feder",
"Sahithi",
"Swetha",
"Ramasamy",
"Srinivasan",
"Selvamurugan",
"Salvador-Morales",
"Flahaut",
"Sim",
"Sloan",
"Green",
"Sim",
"Sarin",
"Gaffin",
"Meininger",
"Muthuchamy",
"Shah",
"Vasileva",
"Karadaghy",
"Zustiak",
"Shin",
"Jung",
"Zalabany",
"Kim",
"Zorlutuna",
"Kim",
"Sorkin",
"Greenbaum",
"David-Pur",
"Anava",
"Ayali",
"Ben-Jacob",
"Van Der Flier",
"Sonnenberg",
"Vogel",
"Ross",
"Raines",
"Wang",
"Hsu",
"Su",
"Lu",
"Lee",
"Webb",
"Hlady",
"Tresco",
"Zou",
"Liu",
"Pasquale",
"Ruoslahti",
"Zustiak",
"Durbal",
"Leach",
"Zustiak",
"Leach"
] | [
"The interaction of plasma fibronectin with fibroblastic cells in suspension. S K Akiyama, K M Yamada, J. Biol. Chem. 260Akiyama, S. K., and Yamada, K. M. (1985). The interaction of plasma fibronectin with fibroblastic cells in suspension. J. Biol. Chem. 260, 4492-4500.",
"Carbon nanotubes: a promise for nerve tissue engineering?. S Bosi, A Fabbro, L Ballerini, M Prato, 10.1515/ntrev-2012-0067Nanotechnol. Rev. 2Bosi, S., Fabbro, A., Ballerini, L., and Prato, M. (2013). Carbon nanotubes: a promise for nerve tissue engineering? Nanotechnol. Rev. 2, 47-57. doi: 10.1515/ntrev-2012-0067",
"Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts. L Chou, J D Firth, J Uitto, D M Brunette, J. Cell Sci. 108Chou, L., Firth, J. D., Uitto, J., and Brunette, D. M. (1995). Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts. J. Cell Sci.. 108, 1563-1573.",
"Effect of single wall carbon nanotubes on human HEK293 cells. D Cui, F Tian, C S Ozkan, M Wang, H Gao, 10.1016/j.toxlet.2004.08.015Toxicol. Lett. 155Cui, D., Tian, F., Ozkan, C. S., Wang, M., and Gao, H. (2005). Effect of single wall carbon nanotubes on human HEK293 cells. Toxicol. Lett. 155, 73-85. doi: 10.1016/j.toxlet.2004.08.015",
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"Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. N C Henderson, T D Arnold, Y Katamura, M M Giacomini, J D Rodriguez, J H Mccarty, 10.1038/nm.3282Nat. Med. 19Henderson, N. C., Arnold, T. D., Katamura, Y., Giacomini, M. M., Rodriguez, J. D., McCarty, J. H., et al. (2013). Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat. Med. 19, 1617-1624. doi: 10.1038/nm.3282",
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"A two-step method for transferring single-walled carbon nanotubes onto a hydrogel substrate. M Imaninezhad, I Kuljanishvili, S P Zustiak, 10.1002/mabi.201600261Macromol. Biosci. 171600261Imaninezhad, M., Kuljanishvili, I., and Zustiak, S. P. (2017). A two-step method for transferring single-walled carbon nanotubes onto a hydrogel substrate. Macromol. Biosci. 17:1600261-n/a. doi: 10.1002/mabi.201600261",
"Influence of single walled carbon nanotubes at subtoxical concentrations on cell adhesion and other cell parameters of human epithelial cells. J P Kaiser, T Buerki-Thurnherr, P Wick, 10.1016/j.jksus.2012.06.003J. King Saud Univ. Sci. 25Kaiser, J. P., Buerki-Thurnherr, T., and Wick, P. (2013). Influence of single walled carbon nanotubes at subtoxical concentrations on cell adhesion and other cell parameters of human epithelial cells. J. King Saud Univ. Sci. 25, 15-27. doi: 10.1016/j.jksus.2012.06.003",
"Enhanced fibronectin adsorption on carbon nanotube/poly (carbonate) urethane: independent role of surface nanoroughness and associated surface energy. D Khang, S Y Kim, P Liu-Snyder, G T Palmore, S M Durbin, T J Webster, 10.1016/j.biomaterials.2007.07.018Biomaterials. 28Khang, D., Kim, S. Y., Liu-Snyder, P., Palmore, G. T., Durbin, S. M., and Webster, T. J. (2007). Enhanced fibronectin adsorption on carbon nanotube/poly (carbonate) urethane: independent role of surface nano- roughness and associated surface energy. Biomaterials 28, 4756-4768. doi: 10.1016/j.biomaterials.2007.07.018",
"Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference. K W Kwon, S S Choi, S H Lee, B Kim, S N Lee, M C Park, 10.1039/b710054jLab Chip. 7Kwon, K. W., Choi, S. S., Lee, S. H., Kim, B., Lee, S. N., Park, M. C., et al. (2007). Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference. Lab Chip. 7, 1461-1468. doi: 10.1039/b710054j",
"Biohybrid carbon nanotube/agarose fibers for neural tissue engineering. D Y Lewitus, J Landers, J Branch, K L Smith, G Callegari, J Kohn, 10.1002/adfm.201002429Adv. Funct. Mater. 21Lewitus, D. Y., Landers, J., Branch, J., Smith, K. L., Callegari, G., Kohn, J., et al. (2011). Biohybrid carbon nanotube/agarose fibers for neural tissue engineering. Adv. Funct. Mater. 21, 2624-2632. doi: 10.1002/adfm.201002429",
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"Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes. S Namgung, K Y Baik, J Park, S Hong, 10.1021/nn2023057ACS Nano. 5Namgung, S., Baik, K. Y., Park, J., and Hong, S. (2011). Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes. ACS Nano 5, 7383-7390. doi: 10.1021/nn2023057",
"Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts. R L Price, K Ellison, K M Haberstroh, T J Webster, 10.1002/jbm.a.30073J. Biomed. Mater. Res. Part A. 70Price, R. L., Ellison, K., Haberstroh, K. M., and Webster, T. J. (2004). Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts. J. Biomed. Mater. Res. Part A. 70, 129-138. doi: 10.1002/jbm.a.30073",
"Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. S R Ryoo, Y K Kim, M H Kim, Min , D H , 10.1021/nn1018279ACS Nano. 4Ryoo, S. R., Kim, Y. K., Kim, M. H., and Min, D. H. (2010). Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. ACS Nano 4, 6587-6598. doi: 10.1021/nn1018279",
"Cell adhesion force microscopy. G Sagvolden, I Giaever, E O Pettersen, J Feder, 10.1073/pnas.96.2.471Proc. Natl. Acad. Sci. U.S.A. 96Sagvolden, G., Giaever, I., Pettersen, E. O., and Feder, J. (1999). Cell adhesion force microscopy. Proc. Natl. Acad. Sci. U.S.A. 96, 471-476. doi: 10.1073/pnas.96.2.471",
"Polymeric composites containing carbon nanotubes for bone tissue engineering. K Sahithi, M Swetha, K Ramasamy, N Srinivasan, N Selvamurugan, 10.1016/j.ijbiomac.2010.01.006Int. J. Biol. Macromol. 46Sahithi, K., Swetha, M., Ramasamy, K., Srinivasan, N., and Selvamurugan, N. (2010). Polymeric composites containing carbon nanotubes for bone tissue engineering. Int. J. Biol. Macromol. 46, 281-283. doi: 10.1016/j.ijbiomac.2010.01.006",
"Complement activation and protein adsorption by carbon nanotubes. C Salvador-Morales, E Flahaut, E Sim, J Sloan, M L Green, R B Sim, 10.1016/j.molimm.2005.02.006Mol. Immunol. 43Salvador-Morales, C., Flahaut, E., Sim, E., Sloan, J., Green, M. L., and Sim, R. B. (2006). Complement activation and protein adsorption by carbon nanotubes. Mol. Immunol. 43, 193-201. doi: 10.1016/j.molimm.2005.02.006",
"Arginineglycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via α(5)β(1) integrin. V Sarin, R D Gaffin, G A Meininger, M Muthuchamy, 10.1113/jphysiol.2005.083238J. Physiol. 564Sarin, V., Gaffin, R. D., Meininger, G. A., and Muthuchamy, M. (2005). Arginine- glycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via α(5)β(1) integrin. J. Physiol. 564, 603-617. doi: 10.1113/jphysiol.2005.083238",
"Development and characterization of polyethylene glycol-carbon nanotube hydrogel composite. K Shah, D Vasileva, A Karadaghy, S Zustiak, 10.1039/C5TB01047KJ. Mater. Chem. B. 3Shah, K., Vasileva, D., Karadaghy, A., and Zustiak, S. (2015). Development and characterization of polyethylene glycol-carbon nanotube hydrogel composite. J. Mater. Chem. B. 3, 7950-7962. doi: 10.1039/C5TB01047K",
"Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators. S R Shin, S M Jung, M Zalabany, K Kim, P Zorlutuna, N M Kim, 10.1021/nn305559jACS Nano. 7Shin, S. R., Jung, S. M., Zalabany, M., Kim, K., Zorlutuna, P., Kim, N. M., et al. (2013). Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators. ACS Nano 7, 2369-2380. doi: 10.1021/nn305559j",
"Process entanglement as a neuronal anchorage mechanism to rough surfaces. R Sorkin, A Greenbaum, M David-Pur, S Anava, A Ayali, E Ben-Jacob, 10.1088/0957-4484/20/1/015101Nanotechnology. 2015101Sorkin, R., Greenbaum, A., David-Pur, M., Anava, S., Ayali, A., Ben-Jacob, E., et al. (2009). Process entanglement as a neuronal anchorage mechanism to rough surfaces. Nanotechnology 20:015101. doi: 10.1088/0957-4484/20/1/015101",
"Function and interactions of integrins. A Van Der Flier, A Sonnenberg, 10.1007/s004410100417Cell Tissue Res. 305Van der Flier, A., and Sonnenberg, A. (2001). Function and interactions of integrins. Cell Tissue Res. 305, 285-298. doi: 10.1007/s004410100417",
"Role of serum components in densitydependent inhibition of growth of cells in culture. Platelet-derived growth factor is the major serum determinant of saturation density. A Vogel, R Ross, E Raines, 10.1083/jcb.85.2.377J. Cell Biol. 85Vogel, A., Ross, R., and Raines, E. (1980). Role of serum components in density- dependent inhibition of growth of cells in culture. Platelet-derived growth factor is the major serum determinant of saturation density. J. Cell Biol.. 85, 377-385. doi: 10.1083/jcb.85.2.377",
"Effects of passivation treatments on titanium alloy with nanometric scale roughness and induced changes in fibroblast initial adhesion evaluated by a cytodetacher. C C Wang, Y C Hsu, F C Su, S C Lu, T M Lee, 10.1002/jbm.a.31604J. Biomed. Mater. Res. A. 88Wang, C. C., Hsu, Y. C., Su, F. C., Lu, S. C., and Lee, T. M. (2009). Effects of passivation treatments on titanium alloy with nanometric scale roughness and induced changes in fibroblast initial adhesion evaluated by a cytodetacher. J. Biomed. Mater. Res. A 88, 370-383. doi: 10.1002/jbm.a.31604",
"Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces. K Webb, V Hlady, P A Tresco, 10.1002/(SICI)1097-4636(20000305)49:3<362::AID-JBM9>3.0.CO;2-SJ. Biomed. Mater. Res. 493<362::AID-JBM9>3.0.CO;2-SWebb, K., Hlady, V., and Tresco, P. A. (2000). Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces. J. Biomed. Mater. Res. 49, 362-368. doi: 10.1002/(SICI)1097-4636(20000305) 49:3<362::AID-JBM9>3.0.CO;2-S",
"Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity. J X Zou, Y Liu, E B Pasquale, E Ruoslahti, 10.1074/jbc.M103133200J. Biol. Chem. 277Zou, J. X., Liu, Y., Pasquale, E. B., and Ruoslahti, E. (2002). Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity. J. Biol. Chem. 277, 1824-1827. doi: 10.1074/jbc.M103133200",
"Influence of cell-adhesive peptide ligands on poly (ethylene glycol) hydrogel physical, mechanical and transport properties. S P Zustiak, R Durbal, J B Leach, 10.1016/j.actbio.2010.03.040Acta Biomater. 6Zustiak, S. P., Durbal, R., and Leach, J. B. (2010). Influence of cell-adhesive peptide ligands on poly (ethylene glycol) hydrogel physical, mechanical and transport properties. Acta Biomater. 6, 3404-3414. doi: 10.1016/j.actbio.2010. 03.040",
"Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. S P Zustiak, J B Leach, 10.1021/bm100137qdoi: 10.1021/ bm100137qBiomacromolecules. 11Zustiak, S. P., and Leach, J. B. (2010). Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. Biomacromolecules 11, 1348-1357. doi: 10.1021/ bm100137q"
] | [
"(Ryoo et al., 2010;",
"Bosi et al., 2013)",
"(Harrison and Atala, 2007;",
"Shah et al., 2015)",
"(Lewitus et al., 2011;",
"Bosi et al., 2013)",
"(Shin et al., 2013)",
"(Sahithi et al., 2010)",
"(Price et al., 2004)",
"(Kaiser et al., 2013)",
"(Kaiser et al., 2013)",
"(Sorkin et al., 2009)",
"(Namgung et al., 2011)",
"(Namgung et al., 2011)",
"(Ryoo et al., 2010)",
"(Fan et al., 2002)",
"(Price et al., 2004)",
"(Geiger et al., 2001)",
"(Huang et al., 2003)",
"(Kwon et al., 2007)",
"(Wang et al., 2009)",
"(Sagvolden et al., 1999)",
"(Zou et al., 2002)",
"(Hynes, 1987;",
"Geiger et al., 2001;",
"Van der Flier and Sonnenberg, 2001)",
"(Huang et al., 2003)",
"(Webb et al., 2000)",
"(Henderson et al., 2013)",
"(Imaninezhad et al., 2017)",
"(Imaninezhad et al., 2017)",
"(Henderson et al., 2013)",
"(Khang et al., 2007)",
"(Imaninezhad et al., 2017)",
"(Sorkin et al., 2009)",
"(Imaninezhad et al., 2017)",
"(Salvador-Morales et al., 2006;",
"Kaiser et al., 2013)",
"(Imaninezhad et al., 2017)",
"(Sorkin et al., 2009)",
"(Shah et al., 2015;",
"Imaninezhad et al., 2017)",
"(Vogel et al., 1980)",
"(Grinnell and Feld, 1979)",
"(Akiyama and Yamada, 1985)",
"(Khang et al., 2007)",
"(Akiyama and Yamada, 1985)",
"(Grinnell and Feld, 1979)",
"(Grinnell and Feld, 1979)",
"(Chou et al., 1995)",
"(Cui et al., 2005)",
"(Lowin and Straub, 2011)",
"(Lowin and Straub, 2011)",
"(Sarin et al., 2005)",
"(Lowin and Straub, 2011)",
"(Henderson et al., 2013)",
"(Henderson et al., 2013)",
"(Ryoo et al., 2010)",
"(Hoshiba et al., 2018)",
"(Kaiser et al., 2013)"
] | [
"The interaction of plasma fibronectin with fibroblastic cells in suspension",
"Carbon nanotubes: a promise for nerve tissue engineering?",
"Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts",
"Effect of single wall carbon nanotubes on human HEK293 cells",
"Culture of neural cells on silicon wafers with nano-scale surface topograph",
"Transmembrane crosstalk between the extracellular matrix-cytoskeleton crosstalk",
"Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin",
"Carbon nanotube applications for tissue engineering",
"Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs",
"Characterization of initial cell adhesion on charged polymer substrates in serum-containing and serum-free media",
"Temporal effects of cell adhesion on mechanical characteristics of the single chondrocyte",
"Integrins: a family of cell surface receptors",
"A two-step method for transferring single-walled carbon nanotubes onto a hydrogel substrate",
"Influence of single walled carbon nanotubes at subtoxical concentrations on cell adhesion and other cell parameters of human epithelial cells",
"Enhanced fibronectin adsorption on carbon nanotube/poly (carbonate) urethane: independent role of surface nanoroughness and associated surface energy",
"Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference",
"Biohybrid carbon nanotube/agarose fibers for neural tissue engineering",
"Integrins and their ligands in rheumatoid arthritis",
"Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes",
"Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts",
"Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies",
"Cell adhesion force microscopy",
"Polymeric composites containing carbon nanotubes for bone tissue engineering",
"Complement activation and protein adsorption by carbon nanotubes",
"Arginineglycine-aspartic acid (RGD)-containing peptides inhibit the force production of mouse papillary muscle bundles via α(5)β(1) integrin",
"Development and characterization of polyethylene glycol-carbon nanotube hydrogel composite",
"Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators",
"Process entanglement as a neuronal anchorage mechanism to rough surfaces",
"Function and interactions of integrins",
"Role of serum components in densitydependent inhibition of growth of cells in culture. Platelet-derived growth factor is the major serum determinant of saturation density",
"Effects of passivation treatments on titanium alloy with nanometric scale roughness and induced changes in fibroblast initial adhesion evaluated by a cytodetacher",
"Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces",
"Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity",
"Influence of cell-adhesive peptide ligands on poly (ethylene glycol) hydrogel physical, mechanical and transport properties",
"Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties"
] | [
"J. Biol. Chem",
"Nanotechnol. Rev",
"J. Cell Sci",
"Toxicol. Lett",
"J. Neurosci. Methods",
"Nat. Rev. Mol. Cell Biol",
"Cell",
"Biomaterials",
"Nat. Med",
"Langmuir",
"J. Ortho. Res",
"Cell",
"Macromol. Biosci",
"J. King Saud Univ. Sci",
"Biomaterials",
"Lab Chip",
"Adv. Funct. Mater",
"Arthritis Res. Ther",
"ACS Nano",
"J. Biomed. Mater. Res. Part A",
"ACS Nano",
"Proc. Natl. Acad. Sci. U.S.A",
"Int. J. Biol. Macromol",
"Mol. Immunol",
"J. Physiol",
"J. Mater. Chem. B",
"ACS Nano",
"Nanotechnology",
"Cell Tissue Res",
"J. Cell Biol",
"J. Biomed. Mater. Res. A",
"J. Biomed. Mater. Res",
"J. Biol. Chem",
"Acta Biomater",
"Biomacromolecules"
] | [
"\nFIGURE 1 |\n1Schematic of CNT-hydrogel nanocomposites preparation. (A) Single-walled carbon nanotubes (SWCNTs) are grown on a quartz wafer in a first step.",
"\nFIGURE 2 |\n2(A) Phase contrast images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium (scale bar is 100 µm). (B) Cell spreading area and (C) cell shape factor (inverse of elongation) for NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium. *Asterisks designate significant differences (120 cells from n = 3, p < 0.05).",
"\nFIGURE 3 |\n3Fluorescence images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels at 2 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm. Arrows indicate extracellular fibronectin.",
"\nFIGURE 7 |\n7Phase contrast images of PC12 cells in serum-free medium upon exposure to CWHM-12 (pos.) and CWHM-96 (neg.) peptidomimetics. Scale bar is 100 µm."
] | [
"Schematic of CNT-hydrogel nanocomposites preparation. (A) Single-walled carbon nanotubes (SWCNTs) are grown on a quartz wafer in a first step.",
"(A) Phase contrast images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium (scale bar is 100 µm). (B) Cell spreading area and (C) cell shape factor (inverse of elongation) for NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium. *Asterisks designate significant differences (120 cells from n = 3, p < 0.05).",
"Fluorescence images of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels at 2 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm. Arrows indicate extracellular fibronectin.",
"Phase contrast images of PC12 cells in serum-free medium upon exposure to CWHM-12 (pos.) and CWHM-96 (neg.) peptidomimetics. Scale bar is 100 µm."
] | [
"Figure 1A",
"Figure 1B)",
"Figure 2",
"(Figure 2A)",
"Figure 2B",
"Figure 2C)",
"Figure S1",
"(Figure 3)",
"(Figure 4)",
"(Figure 3)",
"(Figure 5)",
"(Figures 3, 4)",
"(Figure 6)",
"(Figure 6A)",
"Figure 6B",
"(Figure 6C)",
"Figure 6D",
"(Figure 6E)",
"(Figure 7)",
"(Figure 2)",
"(Figures 3-5)",
"(Figure 3)",
"(Figure 4)",
"(Figure 5)",
"(Figure 4)",
"(Figure 6",
"(Figure 7)"
] | [
"f = 4πA P 2 (1)"
] | [
"Carbon nanotubes (CNTs) are cylindrical and have nanometer diameters but typically micrometer lengths, resulting in high aspect ratios. Owing to their exceptional physical, chemical, and mechanical properties, they have been extensively studied for their effect on cellular behaviors (Ryoo et al., 2010;Bosi et al., 2013). Accounts of tissue engineered products including CNTs have proliferated in recent years, which could be coupled to our growing awareness of the nanodimensionality of nature (Harrison and Atala, 2007;Shah et al., 2015). In addition, due to their electro-conductivity, CNTs have been used as components of engineered excitable tissues for neural (Lewitus et al., 2011;Bosi et al., 2013) and cardiac (Shin et al., 2013) tissue engineering applications. Due to their exceptional mechanical stability and low weight-to-strength ratio, they have also been used in orthopedic applications (Sahithi et al., 2010), where CNT-based nanofibers have even been shown to select for osteoblast adhesion (Price et al., 2004).",
"However, very little is known about the mechanism of cell attachment and spreading on CNTs. In one study of soluble multi-walled (MWCNTs) toxicity toward A549 lung cancer cells, the authors observed that some of the MWCNTs coated the surface of the cells (Kaiser et al., 2013). The authors GRAPHICAL ABSTRACT | Cell attachment and spreading onto individual single-walled carbon nanotubes (SWCNTs) and multi-walled CNT aggregates (MWCNTs) is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive extracellular matrix (ECM) proteins to the nanotubes.",
"suggested that cells could be adhering directly to the MWCNTs via integrins or to serum proteins which have adsorbed onto the MWCNTs first (Kaiser et al., 2013). In another study of neural cells onto MWCNTs aggregates, it was suggested that cells adhered via physical entanglements of cellular processes with MWCNT aggregates but only when both were of similar dimensions (Sorkin et al., 2009). However, another recent study of mesenchymal stem cells (MSCs) has shown that cells can recognize, adhere to and spread along individual single-walled (SWCNTs) of a diameter <2 nm; non-specific cell adhesion was controlled through PEG passivation (Namgung et al., 2011). While the mechanism of cell attachment and spreading was not studied, the authors showed that cells formed robust focal adhesion complexes on the SWCNT patterned substrates (Namgung et al., 2011). A different group has shown that NIH 3T3 cells not only formed focal adhesion complexes when grown on MWCNTs films, but the complexes were larger in number and smaller in area compared to a glass substrate, suggesting high affinity for the MWCNTs (Ryoo et al., 2010). Lastly, research on a non-adhesive SiO 2 substrate has shown that topography alone can contribute to cell adhesion (Fan et al., 2002) and nanometer surface roughness has been shown to increase osteoblast adhesion to carbon nanofibers (Price et al., 2004).",
"Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function (Geiger et al., 2001). These interactions play an integral role in the development and maintenance of tissues (Huang et al., 2003). Due to its significance, mechanisms of cell attachment and spreading have been widely explored in various fields such as cellular biology (Kwon et al., 2007) or biomedical applications (Wang et al., 2009). In vitro most mammalian cells are anchorage-dependent and attach firmly to the substrate (Sagvolden et al., 1999). Upon cell adhesion, cells undergo morphologic alteration driven by passive deformation and active reorganization of the cytoskeleton. Integrin receptors and heterodimeric transmembrane proteins play a central role in cell adhesion and spreading. For example, fibroblast cells adhesiveness to fibronectin is reduced by impairing α5β1 integrin (Zou et al., 2002). Specific integrin binding provides not only a mechanical linkage between the intercellular actin cytoskeleton and the extracellular matrix, but also a bidirectional transmembrane signaling pathway (Hynes, 1987;Geiger et al., 2001;Van der Flier and Sonnenberg, 2001). Hence, cell adhesion and spreading on the underlying substrate is an important consideration in biomaterial design and development. Further, the requirements for cell adhesion and spreading will differ for different applications and could also be cell-specific (Huang et al., 2003). Surface properties of materials also influence the composition of the adsorbed protein layers, which subsequently regulate a variety of cell behaviors such as attachment, viability, spreading, migration, and differentiation (Webb et al., 2000).",
"To date there have been very few and contradicting reports on the mechanism of cell attachment and spreading on CNTs, hence no consensus has been reached. Importantly, the mechanism of cell attachment and spreading to individual SWCNTs has not been studied. Here, we hypothesized that cell attachment and spreading to both individual SWCNTs and MWCNT aggregates is governed by the same process. Specifically, we suggest that cell attachment and spreading onto nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes.",
"Single crystal ST cut quartz wafers (diameter of 76.2 mm, thickness of 500 µm) were purchased from University Wafer (Boston, MA). Poly(ethylene glycol) diacrylate (PEGDA, MW 5 kDa) was purchased from Laysan Bio (Arab, AL). Phosphate buffered saline (PBS, 10X, pH 7.4), Hoechst 33258, Alexa 488 phalloidin, bovine serum albumin (BSA) and bovine fibronectin were purchased from Thermo Scientific (Waltham, MA). Rabbit anti-fibronectin antibody was purchased from Abcam (Cambridge, MA). Goat anti-rabbit IgG conjugated with TRITC was purchased from Jackson ImmunoResearch Inc. (West Grove, PA). Irgacure 2959 was purchased from BASF Corporation (Florham Park, NJ). GelBond (GelBond PAG film for polyacrylamide) was purchased from GE Health Care (Filial Sverige, Sweden). Silicone spacers were purchased from Grace Bio-Labs (Bend, Oregon). RainX were purchased from a general store. Ham's F12K medium with 2 mM glutamine and 1.5 g/L sodium bicarbonate, penicillin/streptomycin (pen/strep), and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Dulbecco's modified eagle's medium (DMEM) and N2 supplement (100X) were purchased from Gibco TM (Logan, UT). NIH 3T3 cells were purchased from ATCC (Manassas, Virginia). PC12 cells were generously provided by Dr. Grant Kolar (Department of Pathology, Saint Louis University). 18 × 18 mm #2 glass coverslips and PBS, without calcium and magnesium were purchased from Corning Life Sciences (Manassas, VA). Triton X-100 and formaldehyde were purchased from Sigma Aldrich (St. Louis, MO). Peptidomimetics CWHM-12 and CWHM-96 were synthesized and generously provided by Drs. Griggs and Ruminski (Saint Louis University) (Henderson et al., 2013). A sample of semiconducting multi-walled carbon nanotube powder (MWCNTs) (20 ± 3 nm in diameter and 3 ± 2 µm in length) produced via catalytic chemical vapor deposition was generously provided from MerCorp (Tucson, AZ).",
"4-arm PEG-RGDS with 80% modification efficiency was prepared following a previously developed protocol . Briefly, Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) was dissolved in 5% acetic acid in de-ionized (DI) water and 4-arm PEG-Ac was dissolved in 0.3 M TEA buffer. The two solutions were combined at a 1:4 molar ratio of RGDS:Ac and reacted for 30 min at room temperature. The concentrations were chosen such that, assuming an ideal reaction, one of the acrylate groups of each PEG-Ac can be conjugated with RGDS. Upon reaction the solution was placed at −80 • C for 15 min and then lyophilized (VirTis Sentry 2.0 Lyphilizer) overnight. The 4-arm PEG-RGDS product was purged with argon and stored in a desiccated environment at −20 • C until use.",
"Stock solution of 1% w/v Irgacure 2959 was prepared by dissolving Irgacure 2959 in DI water and sonicating it in a bath sonicator (Fisher Scientific, Waltham, MA) for 1 h. The Irgacure solution was stored shielded from light at 4 • C until use. Stock solution of 30% w/v PEGDA was prepared in 1X PBS, pH 7.4 and stored at 4 • C for up to 1 wk. Then, 10% and 20% w/v PEGDA hydrogel precursor solutions containing 0.1% w/v Irgacure 2959 were prepared by diluting the PEGDA and Irgacure stock solutions with 1X PBS pH 7.4 and vortexing for 30 s. To make PEG-RGDS gels, 1% w/v of functionalized 4-arm PEG-RGDS was added to the PEGDA precursor solution (the total PEG concentration was kept at 10 or 20% w/v for all gels). To prepare hydrogels in slab geometry, 50 µl of a hydrogel precursor solution was deposited on a glass plate, which was hydrophobic-coated with RainX. The hydrogel precursor solution was then sandwiched with a second glass plate, where the two plates were separated by silicon spacers (0.5 mm in height). Hydrogels were formed by exposure to 365 nm ultraviolet (UV) light (4.81 mW/cm 2 ) for 10 min.",
"PC12 cells were cultured in Ham's F12K medium with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 10% FBS, and 1% pen/strep in a humidified environment at 37 • C and 5% CO 2 . NIH 3T3 cells were cultured in DMEM medium supplemented with 10% FBS and 1% pen/strep in a humidified environment at 37 • C and 5% CO 2 . For cell studies in \"serum-free medium, \" cells were pre-conditioned for 24 h with DMEM medium supplemented with 1X N2 supplement and 1% pen/strep. Follow up experiments were conducted in a serum-free medium of the same composition. For cell studies in \"conditioned serum-free medium, \" cells were pre-conditioned for 24 h with DMEM medium supplemented with 1X N2 supplement and 1% pen/strep. This \"conditioned serum-free medium, \" which was expected to contain soluble cell-secreted adhesive proteins such as fibronectin, was then collected and used in further experiments. For cell maintenance, medium was replaced every 2-3 days until cell confluency was reached. To subculture, confluent cells were harvested by a 5 min exposure to 0.05% trypsin/0.02% EDTA.",
"SWCNT Transfer From Quartz Wafers Onto Hydrogels (SWCNTs/Hydrogel) Figure 1A illustrates the developed technique for transferring quartz-grown single walled carbon nanotubes (SWCNTs) onto a hydrogel substrate, which was previously reported (Imaninezhad et al., 2017). Briefly, aligned SWCNTs were grown on a quartz wafer via a catalytic vapor deposition as described by us previously (Imaninezhad et al., 2017). To transfer the SWCNTs from the quartz wafer onto the hydrogel, 20 µl of the hydrogel precursor solution was deposited directly on the wafer. The hydrogel precursor solution was then sandwiched with a GelBond film, hydrophilic side-down. GelBond is a transparent, flexible film that has a hydrophilic and a hydrophobic side, where the hydrophilic side adheres to the hydrogel during gelation and facilitates easy hydrogel removal from the quartz. Upon gelation, the gel sandwich was soaked in DI water for 2 h to allow for hydrogel swelling and to dissolve remaining salts and Irgacure 2959. Finally, the GelBond/hydrogel was peeled off from the quartz wafer with transferred SWCNTs on its surface.",
"To prepare stamped MWCNTs on PEGDA hydrogels ( Figure 1B), first, PEGDA hydrogels were made as described Representative SEM image shows the actual SWCNTs (scale bar is 10 µm). Then a hydrogel precursor solution is deposited on the quartz-grown SWCNTs, sandwiched with a GelBond flexible plastic support and exposed to 365 nm ultraviolet (UV) light (4.81 mW/cm 2 ) for 10 min. Lastly, the GelBond/hydrogel construct is peeled off from the wafer with SWCNTS transferred onto the hydrogel. This nanocomposite is termed PEG/SWCNTs. (B) A hydrogel slab is made in a first step and then \"stamped\" onto dispersed multi-walled carbon nanotubes (MWCNTs) to form PEG/MWCNTs nanocomposites.",
"above. Then MWCNTs powder was dispersed on a glass plate and the PEGDA gel slabs were pressed firmly against the MWCNT powder to embed the nanotubes into the hydrogel. The MWCNTs stamped gels were then gently rinsed with PBS to remove loose MWCNTs.",
"SWCNTs were imaged by scanning electron microscopy (SEM, Zeiss EVO LS15 SEM, Oberkochen, Germany). Images were acquired with low acceleration voltages of 1-1.5 kV at various magnifications under a high vacuum environment.",
"To test the ability of SWCNT/hydrogel and MWCNT/hydrogel composites to support cell adhesion and spreading, hydrogels were prepared as described above with and without CNTs. For the control samples without CNTs, PEG hydrogels containing immobilized RGDS (0.08 mM) were prepared to elicit cell attachment. All hydrogel samples (prepared in the form of slabs) were placed at the bottom of 24 well-plates, one sample per well and were sterilized under UV (302 nm) for 60 min. Then, 50 µl of cell suspension was carefully pipetted on top of each hydrogel to achieve a density of 5 × 10 4 cells/cm 2 and the plates were placed in an incubated environment at 37 • C and 5% CO 2 for 30 min to initiate cell attachment. Then 500 µl of additional medium was added carefully to each sample and the cells were cultured for up to 24 h. At 24 h cells were washed gently with PBS, covered again with fresh medium, and imaged under an inverted fluorescent microscope (Zeiss, Axiovert 200M, Oberkochen, Germany) at 10X zoom. All images were analyzed with ImageJ software available freely from the NIH at http://rsbweb.nih.gov/ij/. Shape factor was calculated as:",
"where, A is cell area and P is cell perimeter. A shape factor close to 1 indicates circularity and a shape factor close to 0 indicates elongation.",
"Fibronectin was passively adsorbed to the PEG, MWCNTs/ hydrogel and SWCNTs/hydrogel samples. Fibronectin was diluted to 50 µg/ml in 1X PBS and 10 µl of the diluted solution was deposited on each sample and placed in the incubator for 1 h at 37 • C and 5% CO 2 . The samples were rinsed with 1X PBS and then 50 µl cell suspension of NIH 3T3 cells was seeded onto each sample to give a cell seeding density of 5 × 10 4 cells/cm 2 . Samples were placed in a humidified incubator at 37 • C and 5% CO 2 for 30 min to allow initial cell attachment and then 500 µl of fresh medium was added to each sample for further culturing. At specified time points, the samples were fixed in 4% formaldehyde and 0.1% Triton X-100. After fixation, samples were washed in DI water, and blocked for 20 min at 22 • C with 2% w/v BSA solubilized in PBS. Samples were incubated with Alexa 488 phalloidin, Hoechst 33258, and rabbit anti-fibronectin followed by anti-rabbit TRITC secondary antibody, and then washed, and mounted onto standard glass slides. A drop of Aqua/Poly mount was added onto the hydrogels and then hydrogels were covered with a plain glass coverslip. Images were acquired with a Leica DMi8 inverted epifluorescence microscope fitted with a 12-bit grayscale CCD camera using a 40X dry objective. All images were acquired using Metamorph software. ",
"CWHM-12 is a small molecule peptidomimetic of the amino acid motif Arg-Gly-Asp (RGD) which potently inhibits (IC 50 < 10 nM) the binding of integrins αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and α5β1 to their respective extracellular matrix ligands, as has been described previously (Henderson et al., 2013). CWHM-96 is the R-isomer of CWWM-12 differing only in the orientation of its carboxyl (CO 2 H) group and is 100-1000-fold less potent than CWHM-12 for inhibition of these same integrins. Therefore, it was used as a highly structurally similar negative control compound. Stock solutions of 1 mM of the peptidomimetic compounds were prepared in 50% DMSO (in sterile water). PC12 and NIH 3T3 cells were seeded at 5×10 4 cells/cm 2 on SWCNTs/hydrogel, MWCNTs/hydrogel, and control PEG-RGDS substrates and cultured for 24 h. After 24 h of culture, the peptidomemetics were added at a final concentration of 10 µM directly to the cell medium and the cells were exposed to peptidomemetics for additional 24 h. The cell medium was exchanged with fresh medium and samples were imaged under an inverted fluorescent microscope (Zeiss, Axiovert 200M, Oberkochen, Germany).",
"Results are reported as averages ± standard deviation. Statistical significance between multiple samples was tested by ANOVA followed by a post-hoc analysis and between two samples by Student's t-test (p < 0.05). A minimum of three samples from three independent experiments were tested per condition. For cell analysis, a minimum of 20 cells per image, from a minimum of six images were analyzed per condition. Figure 2 shows NIH 3T3 cell attachment and spreading in serum, serum-free and conditioned serum-free medium on PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels. Note that PEG only hydrogel was also tested as a control, but cell attachment was not observed (data not shown). Hence, PEG-RGDS was used, where the RGDS ligand was covalently tethered to support integrin-mediated cell adhesion . To produce the conditioned serum-free medium, cells were exposed to serum-free medium for 24 h upon which the conditioned serum-free medium (i.e., containing cell-secreted biomolecules) was collected and used for experiments. Phase contrast images showed that cells adhered on all substrates independently of serum presence but exhibited serum-dependent morphology (Figure 2A). Cells had higher cell spreading area (per individual cell) and an elongated morphology (low shape factor) in the presence of serum in the medium and lower cell spreading area and a more rounded morphology (high shape factor) in the absence of serum. Quantification revealed that cell spreading area in the presence of serum was significantly higher than other media for all hydrogel types ( Figure 2B). The differences were largest between the serum and serumfree media: ∼56% for cells seeded on the PEG-RGDS and the PEG/MWCNTs gels and ∼70% for cells seeded on the PEG/SWCNTs gels. The difference between the serum-free and conditioned serum-free medium was largest for cells seeded on the PEG/MWCNTs gels (∼56%) and not significant for cells seeded on the PEG/SWCNTs gels. Shape factor was inversely proportional to cell spreading area, where lowest shape factor (i.e., highest elongation) was observed in the serum medium, followed by the conditioned serum-free and lastly by the serumfree medium ( Figure 2C). Overall, our results confirmed that serum proteins facilitated cell spreading and elongation for cells seeded on the nanocomposite hydrogels and the control PEG-RGDS gels. Conditioning the serum-free medium with cell-secreted proteins improved cell spreading and elongation over the serum-free condition but was not as effective as serum in the media. Importantly our results indicate that cells were able to adhere and spread onto unmodified SWCNTs and MWCNTs embedded in the inert PEG gels even in the absence of any adhesive ligands on the material. We suggest that the reason for cell adhesion and spreading was the ability of SWCNTs and MWCNTs to adsorb adhesive proteins (e.g., fibronectin) secreted by cells or present in serum (Khang et al., 2007), which then could elicit cell attachment and subsequent spreading.",
"We studied the effect of fibronectin adsorption, both exogenous and cell-secreted, onto PEG/SWCNTs, and PEG/MWCNTs nanocomposite hydrogels via epifluorescence microscopy. To ascertain anti-fibronectin antibody specificity a negative control was used, where samples stained with normal rabbit IgG fraction were compared to samples stained with the affinity purified, rabbit anti-fibronectin antibody. Only the sample stained with the anti-fibronectin antibody showed intense TRITC fluorescence (indicated with arrows) indicating specificity ( Figure S1).",
"NIH 3T3 cells were stained for fibronectin, actin and cell nucleus to visualize intracellular and extracellular fibronectin at 2 h post-seeding in a serum-free medium for cells seeded on top of PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels (Figure 3). All cells stained positive for fibronectin, and there appeared to be some extracellular fibronectin at that time point, particularly on the PEG/MWCNTs nanocomposites (yellow arrows in images). At this early time point cells on all hydrogel types appeared rounded and the number of attached cells was low.",
"To determine whether pre-absorbed fibronectin will facilitate better early cell attachment and spreading, we imaged cells at 2 h of culture in serum-free medium on fibronectin-coated PEG/MWCNTs nanocomposite and the PEG-RGDS control gels (Figure 4). We specifically focused on the MWCNTs/hydrogel composite, as opposed to the SWCNTs/hydrogel one, as a representative substrate due to the easier nanotube visualization. Upon staining, we noted intracellular and some extracellular fibronectin. As expected, extracellular fibronectin was not seen on the inert PEG-RGDS gels but was seen on the PEG/MWCNTs nanocomposite due to fibronectin adsorption onto the MWCNTs. Note that we observed fibronectin only on the smaller MWCNTs clusters and not the larger ones, because we could not image the surface of the black MWCNTs with an inverted microscope. Importantly, we observed cell spreading even at this early time point and a higher number of adhered cells compared to the no-exogenousfibronectin condition (Figure 3), especially for cells seeded on the nanocomposite hydrogel. Our results indicate that passive adsorption of adhesive proteins, such as fibronectin, FIGURE 4 | Fluorescence images of NIH 3T3 cells on fibronectin-coated PEG/MWCNTs and PEG-RGDS hydrogels at 2 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm.",
"FIGURE 5 | Fluorescence images of NIH 3T3 cells on PEG/MWCNTs hydrogels (−Fn) and fibronectin-coated PEG/MWCNTs hydrogels (+Fn) at 6 h in serum-free medium. Nucleus was stained with Hoechst 33258 (blue), actin was stained with Alexa 488 phalloidin (green) and fibronectin was stained with polyclonal anti-fibronectin (red). Scale bar is 50 µm. Arrows indicate extracellular fibronectin.",
"facilitate cell adhesion and spreading on CNT/hydrogel nanocomposites.",
"Additionally, to determine whether the presence of MWCNTs affected cell production of fibronectin (i.e., intracellular fibronectin amount), we quantified the amount of fibronectin per cell. Comparing cells on the PEG/MWCNTs substrate vs. the PEG-RGDS substrate we observed no difference in intracellular fibronectin. The mean integrated pixel intensity ± s.e.m. in the PEG/MWCNT and PEG-RGDS fibronectin images was 2.7 × 10 6 ± 0.2 × 10 6 (n = 27 cells) and 3.3 × 10 6 ± 0.3 × 10 6 (n = 33 cells), respectively, yielding a p-value of 0.11. Hence, we conclude that MWCNT presence did not affect intracellular fibronectin levels.",
"Lastly, we stained for fibronectin at a longer time point of 6 h for cells seeded onto PEG/MWCNTs nanocomposites expecting to see more cell-secreted extracellular fibronectin and higher number of adhered cells (Figure 5). We tested two conditions: exogenously coated fibronectin (+Fn) and no exogenous fibronectin (-Fn). We observed a similar number of attached cells irrespective of exogenous fibronectin. All cells stained positive for intracellular fibronectin; we did not note qualitative differences between cells on the fibronectin-coated samples (+Fn) and the uncoated ones (−Fn). Similarly to cell images at 2 h post-seeing, at 6 h post-seeding we observed some extracellular fibronectin in the (−Fn) condition (indicated by yellow arrows in the images), which appeared to accumulate around MWCNTs in the gel and was not seen in MWCNTdevoid regions. The fibronectin-coated samples (+Fn) exhibited an overall higher level of fluorescence from the hydrogel surface indicative of protein adsorption onto the MWCNTs. Our results indicate that cells were able to secrete fibronectin, which was subsequently adsorbed onto the MWCNTs in the nanocomposites and elicited cell adhesion and spreading. Lastly, while the addition of exogenous fibronectin led to improved cell adhesion at 2 h post-seeding (Figures 3, 4), it did not seem to affect cell adhesion at 6 h post-seeding; similar number of adhered cells were noted on both (−Fn) and (+Fn) substrates.",
"Next, we tested whether cell attachment and spreading onto PEG/MWCNTs and PEG/SWCNTs hydrogels was facilitated FIGURE 6 | (A) Phase contrast images of NIH 3T3 cells in serum, (B) serum-free, (C) conditioned serum-free media on PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS hydrogels at 24 h after exposure to peptidomimetics (scale bar is 100 µm). (D) Cell area and (E) shape factor of NIH 3T3 cells on PEG/SWCNTs, PEG/MWCNTs and PEG-RGDS hydrogels in serum, serum-free and conditioned serum-free medium in the presence of CWHM-12 (active) and CWHM-96 (inactive control) peptidomimetics. *Asterisks designate significant differences (120 cells from n = 3, p < 0.05).",
"by integrin binding (Figure 6). Note that the SWCNTs in the PEG/SWCNTs hydrogels were 1-2 nm in diameter (Imaninezhad et al., 2017); hence physical entanglement of cell processes on SWCNTs alone could not explain cell attachment and spreading (Sorkin et al., 2009). We tested a highly stable synthetic RGD peptidomimetic, namely CWHM-12, with selective inhibition toward integrins ανβ1, ανβ3, ανβ5, ανβ6, ανβ8, and α5β1, which collectively mediate binding to RGD-containing biomolecules present in serum such as fibronectin and vitronectin. In contrast, the R-enantiomer of this compound, namely CWHM-96, has very low activity toward the same integrins.",
"We tested the effect of the integrin inhibitor CWHM-12 and the inactive control CWHM-96 on NIH 3T3 cell attachment and spreading onto PEG/SWCNTs, PEG/MWCNTs, and PEG-RGDS gels in serum (Figure 6A), serum-free ( Figure 6B) and conditioned serum-free (Figure 6C) medium. For all conditions tested, the cells rounded up and formed cell aggregates on top of the gels when exposed to the active CWHM-12, but not when exposed to the inactive CWHM-96 compound. When quantifying cell area ( Figure 6D) and shape factor (Figure 6E), we observed that both depended on the media and the substrate in the presence of the inactive compound CWHM-96, but not in the presence of the active compound CWHM-12 where all cells were rounded. As expected, in the presence of CWHM-96 cells were most spread and elongated in the presence of serum in the media, followed by conditioned serum-free, followed by serumfree medium. The differences between the three media conditions were most pronounced for the PEG-RGDS and PEG/MWCNTs gels, but were minimal for cells seeded on the PEG/SWCNTs gels. In the presence of the active compound CWHM-12 all cells had a similar cell area of ∼400 µm 2 and a shape factor close to 1, which is indicative of a circular shape, suggesting an inhibition of integrin binding.",
"We also tested cells seeded on PEG/SWCNTs gels in serumfree and conditioned serum-free medium after a shorter exposure of 4 h to peptidomimetics CWHM-12 and CWHM-96 (data not shown). We observed cells rounding upon exposure to the active CWHM-12 compound in both medium conditions and not in response to the inactive CWHM-96 one, similarly to data presented for 24 h of exposure. Overall, our results show that cells attached and spread via integrin binding onto all substrates in all media conditions, indicating that serum or cell-secreted adhesive proteins adsorb on the nanotubes and elicit cell attachment and subsequent spreading.",
"Lastly, to confirm that cell attachment and spreading onto SWCNTs and MWCNTs was not specific to NIH 3T3 cells, we used PC12 cells in one representative experiment (Figure 7). Specifically, we seeded PC12 cells on PEG/SWCNTS, PEG/MWCNTS, and PEG-RGDS gels in serum-free medium containing inactive CWHM-96 or active CWHM-12 compounds. As with NIH 3T3 cells, PC12 cells were able to attach and spread onto all substrates in all media conditions. When exposed to the inactive CWHM-96 compound cells seemed more spread and elongated but were completely rounded in the presence of the active CWHM-12 compound. Our data indicates that PC12 cells, similarly to NIH 3T3 cells adhered and spread onto SWCNTs and MWCNTs via integrin binding to cell-secreted proteins adsorbed onto the nanotubes.",
"The objective of this study was to understand how cells adhere and spread onto MWCNTs aggregates and individual SWCNTs of diameter less than 2 nm embedded in an inert PEG hydrogel substrate. The significance of embedding the nanotubes in PEG substrate was to isolate cell adhesion and spreading onto the nanotubes, both SWCNTs and MWCNTs, from adhesion and spreading onto the underlying substrate: PEG is inert and does not support cell adhesion (Imaninezhad et al., 2017). We postulated that cells adhere and spread onto individual SWCNTs and MWCNTs aggregates via integrin binding because of serum or cell-secreted proteins adsorbed onto the CNTs' surface. Proteins are known to adsorb tightly onto the CNT surface due to pi-stacking (Salvador-Morales et al., 2006;Kaiser et al., 2013). This study was enabled by our previous work, where we developed a method to transfer individual SWCNTs onto hydrogel substrates and demonstrated robust cell adhesion to these novel nanocomposite materials (Imaninezhad et al., 2017). Because the SWCNTs were of a very small diameter (<2 nm) and dispersed individually, we postulated that physical entanglement between cellular processes and SWCNTs could not explain cell adhesion and spreading, especially because the cellular processes were of greater dimension than the individual SWCNTs; previous work has shown that neural cells can attach to MWCNT aggregates by physical entanglement of cellular processes with the nanotubes if both have similar dimensions (Sorkin et al., 2009).",
"To test our hypothesis, we cultured cells on PEG/SWCNTs nanocomposites, PEG/MWCNTs nanocomposites and PEG-RGDS hydrogels (no CNTs control) in various media, namely serum, serum-free, and conditioned serum-free. Note that both nanocomposite substrates, PEG/MWCNTs and PEG/SWCNTs, were developed and characterized by us previously, and have shown excellent biocompatibility with >90% cells viability retained at 24 h post-culture (Shah et al., 2015;Imaninezhad et al., 2017). The conditioned serum-free medium was used to control for the effect of cell-secreted adhesive proteins (e.g., fibronectin) as this medium was considered enriched in such proteins. The PEG-RGDS gels were used as a control substrate, since cells could only attach to the RGDS ligand in those gels. We anticipated that cell-secreted proteins will adsorb onto the CNTs and facilitate a more robust cell adhesion and spreading in this condition compared to the serum-free condition (Vogel et al., 1980). At 24 h of culture, we noted some cell adhesion in all media conditions with the highest number of cells observed in the serum, followed by the conditioned serum-free, followed by the serum-free media (Figure 2). It is important to note that cells clustered around nanotube-rich areas, which was most clearly visible on the PEG/MWCNTs substrates. As expected, the highest cell spreading was also seen in the serum medium and the lowest in the serum-free medium. Further, since fibroblasts are known to secrete adhesive proteins shortly after culturing in serum-free medium (Grinnell and Feld, 1979), our 24 h time point meant that even cells in the serum-free medium would be able to secrete proteins to facilitate their adhesion and spreading onto the surface. This result led us to believe that adsorption of adhesive proteins, either from the serum or cell-secreted, was critical and facilitated cell adhesion and spreading onto both SWCNTs and MWCNTs.",
"To probe for cell adhesion and spreading as well as for adhesive protein secretion at shorter time points, we stained cells cultured in a serum-free medium for 2 and 6 h for intracellular fibronectin (Figures 3-5). Fibronectins are adhesive glycoproteins on the cell surface and in the extracellular matrix and a common soluble protein secreted by cells (Akiyama and Yamada, 1985). Fibronectin has been shown to adsorb tightly and with high affinity onto carbon nanotubes (Khang et al., 2007). On the other hand, studies on the effect of soluble fibronectin binding to the surface of cells have shown that fibronectin almost exclusively binds to the external surface of the plasma membrane (Akiyama and Yamada, 1985). Fibroblast cells in particular have been shown to attach and spread in a serum-free medium because of cell-secreted fibronectin within 60 min of seeding (Grinnell and Feld, 1979). We hypothesized that cells were able to secrete fibronectin, which would then adsorb with high affinity to the SWCNTs and MWCNTs, facilitating cell adhesion and spreading. As anticipated, at 2 h we noted that cells on all substrates stained positive for intracellular fibronectin, but we observed minimal extracellular fibronectin adsorbed on the carbon nanotube substrates (Figure 3). It could be due to the early time point, but it also could be due to the high intensity of intracellular fibronectin obscuring any weaker signal from the extracellular one. Hence, to confirm that fibronectin indeed facilitated cell adhesion, we pre-coated PEG/MWCNTs and PEG-RGDS substrates with exogenous fibronectin and cultured cells for 2 h (Figure 4). We observed a higher number of attached cells on the exogenously coated PEG/MWCNTs nanocomposites compared to the noncoated ones. We then repeated the experiment at a later time point, namely 6 h, at which point we anticipated that cells would secrete sufficient amount of fibronectin to facilitate robust cell adhesion and spreading (Grinnell and Feld, 1979) (Figure 5). At 6 h, we saw a larger number of cells adhering to the PEG/MWCNTs substrate compared to the 2 h time point in serum-free medium (48 ± 21 cells/mm 2 vs. 27 ± 14 cells/mm 2 , respectively), where the cells were also more spread. Importantly, at 6 h there was no difference between numbers of adhered cells on the fibronectin-pre-coated vs. non-coated PEG/MWCNTs nanocomposites. Hence, our results suggested that a soluble cell-secreted adhesive protein such as fibronectin facilitated cell adhesion and spreading onto CNTs.",
"Finally, we did not see a significant difference in intracellular fibronectin expression for cells seeded on PEG/MWCNTs vs. PEG-RGDS substrates (Figure 4), suggesting that MWCNTs did not stimulate fibronectin expression. The effect of surface tethered MWCNTs on fibronectin expression from fibroblast cells had not been studied before. However, multiple studies have indicated that the microenvironment could affect fibronectin expression and secretion by cells. For example, it has been shown that surface roughness can increase fibronectin expression and secretion by fibroblast cells, and that fibronectin expression is greatly enhanced in high serum conditions and minimal in low serum conditions (Chou et al., 1995). Here, the PEG/MWCNTs substrate should have presented higher roughness than PEG-RGDS due to the presence of MWCNT aggregates, but the cells were cultured in the absence of serum, which could explain the similar expression levels for both conditions. However, another study on the effect of soluble SWCNTs on HEK293 cells has shown that SWCNTs down-regulated the expression of several adhesion proteins such as laminin, fibronectin, cadherin, FAK, and collagen IV (Cui et al., 2005). The difference between this finding and our results could be due to the different presentation of CNTs (soluble vs. tethered) as well as the different cell types and CNTs types used.",
"While we showed that cell adhesion and spreading was facilitated by adhesive proteins adsorbed onto the nanotubes, the question still remained whether the cell binding and spreading onto SWCNTs and MWCNTs was integrin-dependent. Integrins are heterodimers consisting of an α and a β subunit that together determine ligand specificity and initiate intracellular signaling events (Lowin and Straub, 2011). Fibroblast cells express the β1, β3, β5, αv, and α5 subunits of RGD-binding integrins that mediate cellular processes such as adhesion, spreading and migration (Lowin and Straub, 2011). Within the aminoacid sequences of proteins such as fibronectin and vitronectin, integrins bind to the arginine-glycine-aspartic acid (RGD) motifs (Sarin et al., 2005). Interaction with the RGD sequence is an important switch for integrin activation and subsequent cellular changes. The same ECM molecules can recognize various integrin combinations. For example, fibronectin is recognized by α4β1, α5β1, ανβ1, ανβ3, and α3β1, and weakly by others (Lowin and Straub, 2011).",
"Here, we used a potent small-molecule inhibitor of α5 (α5β1) and αν integrins (ανβ1, ανβ3, ανβ5, ανβ6, and ανβ8), namely the RGD peptidomimetic CWHM-12, and an inactive peptidomimetic CWHM-96 as control (Henderson et al., 2013). Specifically, we studied the effect of the peptidomimetics on cell attachment and spreading on PEG/SWCNTs nanocomposites, PEG/MWCNTs nanocomposites and PEG-RGDS control hydrogels in serum, serum-free and conditioned serum-free media (Figure 6). Our goal was to determine whether cell attachment and spreading onto the nanotubes in the various media conditions was dependent on RGD-binding integrins. On all substrates and in all media conditions, cells adhered and spread (higher spreading in the serum and conditioned serum-free media) in the presence of the inactive CWHM-96 compound, but balled up and formed clusters in the presence of the active CWHM-12 compound. Similar responses to the peptidomimetics were seen with another cell type, namely PC12 cells (Figure 7), indicating that the response was not cell-specific. Note that cell rounding was not caused by peptidomimetic toxicity but due to its ability to inhibit integrin binding with the substrate (Henderson et al., 2013). Hence, our results indicated that cell adhesion and spreading onto SWCNTs and MWCNTs was similar to the cell adhesion and spreading onto the control PEG-RGD gels and was integrin-dependent. While studies with RGD-like peptidomimetics have not been performed before, other studies have shown that cells are able to form focal adhesion complexes on CNTs (Ryoo et al., 2010), indicative of integrin binding.",
"One limitation of our study is that we did not specifically study short time points (<30 min) to determine if initial cell attachment is also dominated by integrin binding. For example, some recent work on cell adhesion to charged polymers has suggested that short term cell adhesion in the presence and absence of serum in the media is different and could not be fully explained by either integrin binding or charged interactions (Hoshiba et al., 2018). Hence, it is possible that a different and yet unidentified mechanism is guiding cell adhesion to CNTs initially, but is masked by a predominant integrin-dependent adhesion at the later time points studied here. We were also not able to unequivocally rule out that cells were able to attach directly to the CNTs. It has been suggested previously that SWCNTs can bind directly to integrins as well as to ECM proteins first, where the whole complex subsequently binds to integrins (Kaiser et al., 2013). However, it was only a suggestion based on observations by the researchers and was not proven unequivocally. Studies to answer such follow up questions will be included in our future work.",
"In summary, we observed that NIH 3T3 cells attached and spread onto the PEG/SWCNTs and PEG/MWCNTs nanocomposites due to adhesive proteins from serum in the medium, where proteins adsorbed onto the SWCNTs and MWCNTs. Cells also adhered and spread onto the nanocomposites in serumfree medium due to cell-secreted adhesive proteins, such as fibronectin. Conditioning the serum-free medium with cellsecreted proteins enhanced cell attachment and spreading. All cells stained positive for intracellular fibronectin at 2 and 6 h of culture, where MWCNTs and SWCNTs presence did not affect intracellular fibronectin expression. Inhibition of cell attachment and spreading onto the nanocomposite materials by a broadaction RGD peptidomimetic (active against α ν β 1 , α ν β 3, α ν β 5 , α ν β 6, and α ν β 8 integrins) confirmed that cell attachment and spreading onto the nanotubes was integrin-dependent. Similar results were observed with two different cell types, namely NIH 3T3 fibroblasts and PC12 neural-like cells, indicating that the process of cell attachment and spreading onto nanotubes was cell type-independent. This was the first study to explore why and how cells attach and spread onto both individual SWCNTs (<2 nm in diameter) and MWCNTs aggregates, embedded in an inert PEG substrate (to prevent non-specific cell attachment). Our results are significant because they demonstrated that cells used the same process for adhesion and spreading onto nanotubes and adhesive ligands such as RGDS. Importantly, we were able to show that cells recognized and bound to nanotubes which were significantly smaller than the smallest cell processes.",
"MI conducted most of the experiments and wrote a first draft of the paper. JS performed the immunostaining work and provided associated figures and some text related to this work. DG and PR provided the peptidomimetics and contributed to experimental design related to peptidomimetics. IK assisted with SWCNT samples grown on quartz. SZ conceptualized the idea, guided experimental design and data analysis and wrote the final paper."
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Materials",
"4-Arm PEG-Ac Modification With RGDS",
"Polyethylene Glycol Hydrogel Preparation",
"Cell Culture and Maintenance",
"Stamped MWCNTs on PEG Hydrogels (MWCNTs/Hydrogel)",
"Scanning Electron Microscopy Imaging of SWCNTs on Quartz Wafers",
"Evaluation of Cell Area and Shape",
"Immunofluorescence for Fibronectin Detection",
"Peptidomimetic Studies",
"Statistical Analysis",
"RESULTS",
"Cells Adhered and Spread Onto SWCNTs and MWCNTs Due to Adsorption of Adhesive Proteins",
"Exogenous and Cell-Secreted Fibronectin Adsorbed Onto SWCNTs and MWCNTs; Intracellular Fibronectin Not Affected by CNTs Presence",
"Cells Adhered and Spread Onto SWCNTs and MWCNTs on PEG Hydrogels via Integrin Binding",
"Integrin-Facilitated Cell Adhesion and Spreading to SWCNTs and MWCNTs Was Cell-Type Independent",
"DISCUSSION",
"CONCLUSION",
"AUTHOR CONTRIBUTIONS",
"FIGURE 1 |",
"FIGURE 2 |",
"FIGURE 3 |",
"FIGURE 7 |"
] | [] | [] | [
"a section of the journal Frontiers in Bioengineering and Biotechnology Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding",
"a section of the journal Frontiers in Bioengineering and Biotechnology Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding"
] | [
"Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding. Front. Bioeng. Biotechnol"
] |
235,650,634 | 2022-01-11T12:01:59Z | CCBY | https://jeccr.biomedcentral.com/track/pdf/10.1186/s13046-021-01946-2 | GOLD | edbeca39b8b453a70ec5510c0bdbe4d8888ac159 | null | null | null | null | 10.1186/s13046-021-01946-2 | null | 34174926 | 8235815 |
Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression
Marius Kemper
Alina Schiecke
Hanna Maar
Sergey Nikulin
Andrey Poloznikov
Vladimir Galatenko
Michael Tachezy
Florian Gebauer
Tobias Lange
Kristoffer Riecken
Alexander Tonevitsky
Achim Aigner
Jakob Izbicki
Udo Schumacher
Daniel Wicklein
Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression
10.1186/s13046-021-01946-2R E S E A R C H Open Access
Background: Mesothelial E-and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis.Methods: Knockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated. Results: The knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-β and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E-and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients.Conclusion: Combined depletion of ITGAV in PDA cells and E-and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E-and P-selectins and ITGAV in PDA.
Background
Pancreatic ductal adenocarcinoma (PDA) belongs to the most lethal malignancies in industrialized countries. By 2040 an increase to 777,423 death per year worldwide is expected [1]. Despite advances in diagnostics and therapy, the five-year survival rate is only 9% [1]. These data indicate that the vast majority of patients relapse within 5 years after surgery. Besides locoregional relapse or distant metastasis to lungs or liver, recurrence manifests as peritoneal carcinomatosis in many cases [2], making this process an important target for therapeutic intervention. In fact, the median overall survival for patients diagnosed with intraperitoneal carcinomatosis amounts 14.1 months only [2].
In a previous study, we demonstrated that tumor cell adherence to the peritoneal mesothelium is highly dependent on the binding of carbohydrate structures present of the cancer cells' outer cell membrane to E-and P-selectins expressed on the peritoneal mesothelial cells using a xenograft model with selectin-deficient mice [3]. In the absence of selectins, only a minority of cells could adhere to the peritoneum, which considerably inhibited intraperitoneal tumor growth [3]. In the present study, we investigated the underlying mechanism for the remaining metastatic deposit in selectin-deficient mice in which we found Integrin αV (ITGAV) to be functionally involved. ITGAV has recently been reported to be an important driver of cancer progression in prostate [4,5] and colon carcinoma [6]. In PDA, recent studies demonstrate that the dimer of Integrins αVβ6 (ITGAV / ITGB6) is overexpressed in most tumors (with overexpression being retained in the corresponding metastases) and is a feasible target for novel therapeutic approaches [7,8].
Methods
Cell lines and RNA interference-mediated ITGAV knockdown
Authenticated PDA cell lines PaCa 5061 [9] and BxPC3 [10] were used. ITGAV knockdown was achieved using a short hairpin RNA (shRNA) mediated approach: A 65 bp hairpin DNA oligomer containing a 19 bp anti-ITGAV sequence (GGATGGTGTCCACTTCAAA) was inserted into the pLVX vector (Clontech). Potential offtarget effects were checked (NCBI BLAST). An shRNA sequence against firefly luciferase was used for the control cell line [11]. Knockdown and control cells were seeded at three cells per well. Sublines with normal ITGAV and low ITGAV expression were pooled respectively and tested for Mycoplasma. The passage number of used cell lines never exceeded 20.
Flow cytometry
Flow cytometry was performed as described [12]. Following antibodies were used: ITGAV (327,907, BioLegend), ITGB1 (11-0299, Thermo Fisher Scientific), ITGB3 (336, 403, BioLegend), ITGB5 (11-0497-42, Thermo Fisher Scientific), ITGB6 (FAB4155P, R&D), HLA-DR (347,401, BD). Stained cells were subjected to FACS Calibur Flow Cytometry System (BD).
Enzyme-linked immunosorbent assay (ELISA)
Cells were seeded in a T25 cell culture flask. After 48 h, they were changed to 1.5 mL serum-free medium. After another 24 h of incubation, the medium was harvested and centrifuged. Supernatants were collected. The measurement of TGF-β1 was made using the Free Active TGF-β1 ELISA Kit (cat. 437,707, BioLegend), respectively Total TGF-β1 ELISA Kit (cat. 436,707, BioLegend).
In vitro characterization of ITGAV knockdown cells
Cell proliferation was assessed using the XTT assay (Roche Diagnostics). 3 × 10 3 cells were plated per well (96-well plate) and incubated for 72 h. Fifty microliter XTT labeling mixture was added per well. After 5 h incubation, spectrophotometrical absorbance (450 nm) was measured using a microplate reader (MR5000 Multiplate Reader, Dynatech).
Static cell adhesion assays were performed in fibronectin-coated μ-Slides (ibidi). Cells were expanded to a concentration of 1 × 10 5 cells and added to a suspension volume of 60 μL. After 1 h of incubation, visual fields were documented (Axio Cam MRm, Carl Zeiss). To calculate the fraction of adherent cells, slides were washed with PBS and the same predefined visual fields were documented.
Differences in cell migration were assessed using the FluoroBlok Migration Assay with a 24-well plate with 8.0-μm pore size inserts (BD Bioscience). Cells were trypsinized and resuspended in serum-free medium in a concentration of 3 × 10 5 cells/ml. Four hundred microliter cell suspension was added to the apical chamber and 1200 μl medium with 10% FCS (Gibco) as chemoattractant was added to the bottom chamber. The assay was incubated for 24 h. After removing the chemoattractant from the bottom chamber, visualization of migrated cells was performed by adding 500 μl/well HBSS buffer with Calcein AM (Invitrogen) 4 μg/mL in the bottom well and incubating for 1 h. The readout was conducted at 485/530 nm (Ex/Em) on a Genios bottomreading fluorescence plate reader (Tecan). To assess the invasive potential, cells were seeded on a 24-multiwell insert plate uniformly coated with basement membrane equivalent Matrigel (FluoroBlok Invasion System, BD Biosciences). After rehydration according to the manufacturer's guidelines for use, all further steps were identical to those described for the migration assay.
For the colony-forming assay, 1500 μl single-cell suspension (1200 cells/ml) was mixed with 1500 μl growth factor reduced Matrigel (cat.: 356230, BD Biosciences). Fifty microliter of this suspension was seeded per well (96-well plate). After 30 min incubation, 200 μl medium was added per well. The plates were kept in an incubator for 14 d.
Animal experiments: xenograft mouse model C57BL/6 pfp −/− /rag2 −/− mice and E-and P-selectin double deficient pfp −/− /rag2 −/− [3] at the age of 8-12 weeks and bodyweight of 21-29 g were used for the study. For the intraperitoneal and the subcutaneous xenograft model, 1 × 10 6 viable, mycoplasma-free tumor cells suspended in 200 μl RPMI were injected intraperitoneally or subcutaneously. Peritoneal carcinomatoses were quantified by an adapted peritoneal carcinomatosis index (PCI). Briefly, the murine peritoneum was divided into 9 sections (upper left to lower right) and every section was attributed with a carcinoma score from 0 to 3, resulting in a total PCI score of 0 to 27 for each animal [3,13]. For the subcutaneous experiment, mice were injected subcutaneously directly under the right scapula with 1 × 10 6 mycoplasma-free cells. Animals were sacrificed when primary tumors exceeded 1.5 cm 3 or ulcerated the mouse skin. All animal experiments were performed following the United Kingdom Coordinating Committee of Cancer Research Guidelines [14]. The experiment was approved by the local licensing authority (project no. G09/88 and G10/55).
Quantitative real-time polymerase chain reaction (qRT-PCR)
After sacrifice, the left lungs were homogenized (Tissue-Lyser II, Qiagen) and subjected to DNA isolation (QIAamp DNA Mini Kit, Qiagen). Two hundred microliter blood was subjected to DNA isolation. DNA concentrations were quantified (NanoDrop, Peqlab). All lung DNA samples were normalized to 30 ng/μL. The concentrations of blood DNA were similar in all samples (approx. 10 ng/μL) and were therefore not normalized. qRT-PCR was performed with established humanspecific Alu primers [15]. Numerical data were determined against a standard curve as described [16].
Western blot
Protein concentration was determined by bicinchoninic acid assay. Western blots were performed as described [17]. Briefly, 30 μg of protein of each sample was added per well. Electrophoresis was performed in 10% polyacrylamide separating gel with a 5% stacking gel. Proteins were transferred to nitrocellulose membranes (0.45 μm, GE Healthcare). After overnight incubation in blocking solution (5% milk powder), membranes were incubated with the respective antibody: ITGAV (cat. sc-376,156, Santa Cruz), ITGB1 (cat sc-374,429, Santa Cruz), ITGB6 (cat. c-293,194, Santa Cruz) and a cterminal SMAD 4 antibody (cat. ab217267, Abcam). HSC70 (cat. Sc-7298, Santa Cruz) was used as intrinsic control. Goat anti-Mouse IgG (cat. P0447, Dako) or, respectively, Goat anti-rabbit IgG (cat. Sc-2054, Santa Cruz) were used as a secondary antibodies.
Affymetrix gene arrays
RNA was extracted (miRNeasy Mini Kit, Qiagen) and concentrations were determined (NanoDrop). The RNA integrity number was higher than 7 for all samples (Agilent Bioanalyzer 2100 system). Expression analysis was performed using the GeneChip Human Transcriptome Array 2.0 (Thermo Fisher Scientific). For cDNA synthesis, labeling and hybridization, the GeneChip WT PLUS Reagent Kit and GeneChip Hybridization, Wash, and Stain Kit (Thermo Fisher Scientific) were used.
Immunohistochemical analysis
Sections of formalin-fixed, paraffin-embedded tumors were used. Briefly, after antigen retrieval, the following primary antibodies were used: ITGAV (sc-376,156, Santa Cruz,); ITGB1 (AB3167, Abcam), ITGB6 (HPA023626, Atlas), CEACAM7 (LS-B13068, LSBio), TGFBR1 (PA5-98192, Thermo Scientific), phospho-SMAD2 (AB3849, Millipore), TGFBI (MA526731, Thermo Fisher Scientific), Twist (ab50581, Abcam), Ki 67 (M7240, Agilent); human-specific fibronectin (NCL-FIB, Leica Biosystems); fibronectin, cross-reacting with the fibronectinequivalent protein in mouse (A0245, Dako); STAT1 (HPA000931, Sigma-Aldrich), HLA-DR (M0746, Agilent). Vectastain ABC kit (Vector) was used. Slides were scanned by Axio Scan Z1 (Zeiss). For quantification with Image J (NIH), images of four randomly chosen vision fields (not containing necrotic areas) were analyzed. If artifacts interfered with automated quantification by Image J, the staining was quantified by two experienced investigators: grade 0: no reaction or weak focal reaction; grade 1 intense focal or diffuse weak reaction; grade 2 moderate diffuse reaction; grade 3 for an intense diffuse reaction.
Study population
Two hundred nine patients with PDA who underwent pancreaticoduodenectomy at the University Medical Centre Hamburg-Eppendorf between 2000 and 2012 were included. Written informed consent was obtained from all participants. The study protocol was approved by the Hamburg Medical Chamber's ethics committee (Approval number: PV3548).
Tissue microarray (TMA) construction and analysis TMA construction was performed as previously described [18]. Only punches with clearly detectable tumor tissue were included. The staining intensity and quantity of the tumor cells of each tissue spot were scored. To analyze the phospho-SMAD2 staining, all cell nuclei in a visual field were counted and the percentage of phospho-SMAD2 -positive cell nuclei was determined. If this was more than 75%, the tissue sample was classified as high. Of the initial 209 samples, ITGAV could be analyzed in 183, pSMAD2 in 180, STAT1 in 160, and HLA-DR in 192 samples. Immunohistochemical analysis of the sections was performed without knowledge of the patients' identity or clinical status.
Data analyses
GraphPad Prism was used for in vitro and in vivo statistical calculations (Student's t-test, Kaplan-Meier method, Log-rank test). To analyze the TMA, SPSS was used: Relationships between categorical variables were calculated using chi-square tests. Survival curves were plotted using the Kaplan-Meier method and analyzed by the log-rank test. For data obtained by gene expression array, statistical analysis was performed using Transcriptome Analysis Console (4.0.0.25) with summation method Gene + Exon -SST-RMA (version 1) and empirical Bayes method. Calculated p-values were corrected for multiple testing using the Benjamini-Hochberg (FDR) procedure. The sample sets with expression fold changes of at least +/− 1.5 and with an FDR p-value < 0.05 were considered significantly differentially expressed. Further analysis was performed with R 3.5.1 programming language with IDE RStudio 1.1. The heat maps were constructed with the "heatmap.2" function from "gplots" package.
Results
Integrin expression was upregulated in intraperitoneal carcinomatosis grown in selectin-deficient mice
The gene expression profiles of tumors that had developed in the host animals despite selectin deficiency were compared with the gene expression profiles in tumors grown in wild-type Pfp −/− /Rag2 −/− mice: Integrins β1 (1.56-fold), α2 (1.67-fold), β6 (1.69-fold) and αV (1.70-fold) were found to be upregulated in the tumors of the selectin deficient animals. We selected integrin alpha-V (ITGAV) -which displayed the highest upregulation -to further study integrin influence in PDA. We confirmed the upregulation of ITGAV in xenograft carcinomatoses using immunohistochemistry (Fig. 1). We then generated a subline of the PaCa 5061 cell line with stable knockdown of ITGAV (shRNA), termed PaCa 5061 ITGAV KD and a respective control subline with unchanged ITGAV designated as PaCa 5061 control. The expression of ITGAV was reduced by > 65% in PaCa 5061 as determined by flow cytometry and western blot (Fig. 2a).
ITGAV KD massively suppressed intraperitoneal carcinomatosis and reduced primary tumor development and distant metastasis in SMAD4-intact PaCa 5061 cells
To answer the question of whether ITGAV on the tumor cells alone or in combination with E-and Pselectins in the tumor environment has a functional effect on intraperitoneal carcinomatosis formation, PaCa 5061 ITGAV KD and PaCa 5061 control cells were intraperitoneally injected into selectin-deficient and wildtype Pfp −/− /Rag2 −/− mice (12 animals each group for the selectin-deficient mice, 14 and 15 selectin-competent animals for the control PaCa 5061 and the ITGAV KD group, respectively). After a growth period of 62 days, the ITGAV KD alone almost completely abolished intraperitoneal carcinomatosis in wild-type mice. Only 7 of 15 mice (47%) showed any sign of tumor development (small tumors at the injection site with only one animal displaying macroscopically visible intraperitoneal carcinomatosis (with a minimal PCI of 1.) In the experimental group with control PaCa 5061 cells injected into selectin-deficient mice, 11 of 12 mice showed a tumor take (92%), with all of them displaying intraperitoneal carcinomatoses (mean PCI of 12.92; P < 0.001, Fig. 3a). After 62 d, only 2 of 12 selectin k.o. mice (17%) injected with ITGAV KD cells displayed any small tumors at the injection site and no carcinomatosis was observed. Due to this, the presumed synergistic effect of the selectin Of the latter, half of the mice developed no peritoneal carcinomatosis, while in the remaining half, only minimal peritoneal carcinomatosis was observed, resulting in a mean PCI of 0.67. In contrast, injection of ITGAV KD cells into wild-type mice led to a mean PCI of 3.13 after 77 days, with at least small macroscopically visible tumor masses being present in all eight animals with tumor take. As mentioned above, PaCa 5061 cells formed subcutaneous primary tumors in and around the injection channel after intraperitoneal injection: tumor weight in the group of wild-type mice with ITGAV KD cells was significantly reduced (mean of 116 mg, P = 0.003) and a synergistic effect could be observed for the selectindeficient animals with ITGAV KD cells. Here, tumor weights of the injection channel tumors (mean of 22 mg, P = 0.017) were again significantly reduced compared with their wild-type counterparts (Fig. 3b).
Due to the different sizes of injection-site tumors of ITGAV KD and control cells in selectin-deficient and wild-type mice, we expected a functional effect of ITGAV on primary tumor development. To verify this observation, we performed an additional subcutaneous xenograft experiment with ITGAV KD and control cells in selectin wild-type mice. In other cancer entities, it could be shown that ITGAV activates latent TGF-β in the extracellular matrix (ECM) [19]. As SMAD4 is part of the transforming growth factor-β (TGF-β) signaling pathway and is inactivated in 50% of PDA patients [20] resulting in dysfunctional canonical TGF-β signaling in the corresponding tumorswe additionally included the cell line BxPC3 with SMAD4 deletion as a model cell line for this patient subset. SMAD4 deletion was confirmed by absent SMAD4 protein levels in the case of BxPC3 (Fig. 3d). Again, sublines with and without stable knockdown of ITGAV, designated BxPC3 ITGAV KD and BxPC3 control, were generated. The expression of ITGAV was reduced by > 65% in the KD cell line compared to the expression levels of the control cells (Fig. 2).
After subcutaneous tumor cell injection (10 pfp − − /rag2 −− mice each s.c. injected with control and ITGAV KD PaCa 5061, respectively), 9 of 10 animals (90%) injected with control cells and 7 of 10 (70%) mice injected with ITGAV KD cells developed s.c. tumors until the experiment was terminated after 199 days. Mice inoculated with PaCa 5061 ITGAV KD cells showed a significantly prolonged overall survival until reaching the termination criteria (median survival 198.5 vs. 115 d for the control cells, P = 0.01, Fig. 3e). Similar results were found for BxPC3 (median survival 74 vs. 99 d, P < 0.001, Fig. 3h). All animals injected with BxPC3 cells (11 with control and 10 with ITGAV KD) developed s.c. tumors, except one animal from the ITGAV KD group, found dead 1 week after injection. It showed no sign of tumor development and was thus excluded from all further analyses. As tumor size and weight at the time of death did not differ significantly between the ITGAV and control groups for both cell lines, a significantly reduced growth of the ITGAV KD tumors can be assumed for PaCa 5061 and BxPC3 cells.
To analyze the influence of ITGAV knockdown on hematogenous metastasis, tumor cells in the animals' lungs and blood were quantified for all mice by qRT-PCR. The PaCa 5061 ITGAV KD (P = 0.045, Fig. 3f) and BxPC3 ITGAV KD (P = 0.026, Fig. 3i) groups showed significantly less human DNA in the lungs, reflecting significantly fewer PDA cells than the control group. The numbers of circulating tumor cells in the animals' peripheral blood decreased due to the ITGAV KD, albeit not reaching statistical significance ( Fig. 3g and j).
(See figure on previous page.) Fig. 3 Knockdown of ITGAV in PaCa 5061 led to an effective reduction of intraperitoneal carcinomatosis after 62 d compared with control PaCa 5061 in E−/P-selectin deficient mice: Only 1/15 animals of the PaCa 5061 ITGAV KD group in wild-type mice showed carcinomatosis formation vs. 11/12 mice with carcinomatoses in the control group (P < 0.0001, a). ITGAV knockdown also reduced tumor growth at the injection site (P < 0.0001, B). 7/15 (ITGAV KD cells in E−/P-selectin k.o. mice) displayed tumors at the injection site vs. 11/12 in the control group (control PaCa 5061 in E−/P-selectin deficient mice). Combination of ITGAV KD and selectin knockout led to further reduction of injection site tumors (P = 0.012; only 2/14 animals with tumors, b). After 77 days, this synergistic effect of ITGAV and E−/P-selectins could also be demonstrated for intraperitoneal carcinomatosis formation (P = 0.017, C; 8/13 mice with tumor take with all eight displaying carcinomatosis with ITGAV KD in wild-type mice vs. 6/ 14 mice with tumor take and 3 of them displaying carcinomatosis with ITGAV KD in E−/P-selectin deficient animals). Using western blot, the Cterminal deletion of SMAD 4 in BxPC3 cells was confirmed (d). Wild-type mice inoculated subcutaneously with PaCa 5061 ITGAV KD cells showed significantly prolonged survival (tumor take rate 7/10) compared with control cells in wild-type animals (take rate 9/10; P = 0.002, e). Similar results were found for BxPC3 (100% take rate, both groups; P < 0.001, 2H). The number of human cells in the animals' lungs was significantly reduced in mice injected with PaCa 5061 (P = 0.045, f) and BxPC3 ITGAV KD cells (P = 0.045, I). There was no significant difference for the PaCa5061 (g) or BxPC3 tumor cells circulating in the animals' blood (j) The possible integrin-beta subunits corresponding with ITGAV (ITGB1, ITGB3, ITGB5, ITGB6 and ITGB8) were determined in vitro using flow cytometry. Of these, only ITGB1 and ITGB6 were relevantly expressed in PaCa5061 and BxPC3 cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells in vitro (Fig. 2b). In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchanged (Fig. 2b). These changes were confirmed for the xenograft tumors by western blot (Fig. 2c) and immunohistochemistry (Fig. 6).
Effects of ITGAV knockdown in PaCa 5061 und BxPC3 cells in vitro
To elucidate whether ITGAV activates TGF-β1 in vitro, an ELISA was used to determine the concentration of active and latent TGF-β1 in supernatants from cell culture medium. The concentration of active TGF-β1 decreased by 57% to 5.11 pg/mL (P = 0.005, Fig. 4a), whereas the concentration of total TGF-β1 did not differ significantly. Obviously, ITGAV activates latent TGF-β1 for PaCa 5061 cells in vitro.
To characterize the functional effects of ITGAV KD in vitro, proliferation, adhesion, migration, invasion and colony-forming assays were performed. No significant differences were detected in cell proliferation between knockdown and control cells in vitro as determined by XTT assays (Fig. 4b). In line with this finding, in vivo proliferation rates analyzed by determining the Ki-67 staining index of vital xenograft tumor tissue revealed no significant differences between the groups (Fig. 6).
The basal membrane / extracellular Matrix (ECM) lies open between the peritoneal cells [21], which might enable direct contact between ITGAV on the intraperitoneal tumor cells and its ligands such as fibronectin. Adhesion of ITGAV KD cells on fibronectin was analyzed under static conditions: PaCa 5061 cells with ITGAV KD showed a distinctly reduced number of adhering cells, with 11% compared with 39% for the controls (P = 0.006). Likewise, a reduction in the adhesive potential of BxPC3 cells could be demonstrated (P = 0.025, Fig. 4c).
The proportion of migrating tumor cells was reduced for both cell lines upon ITGAV KD (P < 0.001, Fig. 4d). Moreover, BxPC3 ITGAV knockdown cells showed a reduced invasive potential compared with the control cells (P = 0.048). PaCa 5061 cells were not able to pass the Matrigel layer at all (Fig. 4e). In line with the invasion assay finding, BxPC3 ITGAV KD cells formed fewer spheroid colonies per well than the control cells (P = 0.0134, Fig. 4f). Representative images of colonies formed by BxPC3 control and BxPC3 ITGAV KD cells are shown in Supplementary Fig. 1. PaCa 5061 cells were not able to form colonies in Matrigel after 14 days.
Gene expression and immunohistochemical analysis of the primary tumors
To gain insight into how the ITGAV knockdown might cause the reduction in tumor growth and metastasis/carcinomatosis formation observed in vivo, we performed whole human genome expression analyses. Genes of interest were defined and categorized into groups according to their functional context (Table 1, Fig. 5). Differences in selected proteins were validated by immunohistochemistry (Fig. 6).
Starting with the SMAD4-intact PaCa 5061 cells, changes regarding the TGF-β signaling are compiled first: The non-signaling reservoir TGF-β receptor 3 (TGFBR3) was found to be downregulated in the ITGAV KD tumors. In contrast, the expression of TGF-β2 (activation not dependent on ITGAV) and TGF-βR1 (receptor for TGF-β1, 2 and 3) were upregulated. Expression of the fibronectin-anchored, latent-transforming growth factor beta-binding protein 1 (LTBP1), which is essential for activating TGF-β3 with integrin αvβ6 [22], was downregulated in PaCa 5061 ITGAV KD tumors. Regarding the regulation of the SMAD2/3:SMAD4 complex, expression levels of several involved genes were significantly regulated, including E2F4, PPM1A, SERP INE1, SKIL and STUB1. To confirm the changed activity of TGF-β activation, staining for phospho-SMAD2 was performed (Fig. 6): The proportion of phospho-SMAD2positive tumor cell nuclei was significantly higher in the PaCa 5061 control group compared with the ITGAV KD group (P = 0.03, Fig. 6b). Besides, transforming growth factor beta-induced (TGFBI), located in the ECM and associated with poor survival [23], is downregulated as a result of the ITGAV knockdown, which was again immunohistochemically confirmed (Fig. 6b).
Secondly, changes regarding epithelial-mesenchymal transition (EMT) were as follows: Weaker intranuclear staining signals for Twist, a master regulator of EMT, which is controlled by TGF-β1 [24], was observed in PaCa 5061 ITGAV KD tumors (Fig. 6b). In line with the KD cells' more epithelial phenotype, Occludin was found to be 17.35-fold upregulated in the PaCa 5061 ITGAV KD tumors; accordingly, E-cadherin staining signals were enhanced in the ITGAV KD tumors (Fig. 6b). CD44, which correlated with EMT and poor survival in a clinical study of PDA [25], was downregulated 8.94fold upon knockdown of ITGAV. The secretion of matrix proteins and proteases was also found to be modulated to favor tumor progression in PDA [19]. Exemplarily, MMP-10 was decreased upon knockdown of ITGAV accompanied by increased stromal (murine) fibronectin: Staining for both human and murine fibronectin using a cross-reactive antibody revealed a moderate signal in the ITGAV knockdown groups, whereas the control groups were predominantly only weakly stained for fibronectin (Fig. 6). In contrast, specific staining for human fibronectin revealed no differences between the two groups, indicating that the stromal fibronectin was deposited by murine stromal cells and not by human cancer cells. Thirdly, changes regarding the immune status were as follows: MHC class II molecules, part of the human leukocyte antigen (HLA) system, were upregulated in the ITGAV KD xenograft tumors (i.e. HLA-DRA, HLA-DRB1, HLA-DRB4, HLA-DRB5, HLA-DPA1 and HLA-DQB2). Exemplarily, we verified the upregulation of HLA-DR by immunohistochemistry (Fig. 6). Additionally, the upregulation of HLA-DR on PaCa 5061 ITGAV KD cells was also confirmed in vitro using flow Fig. 2). Increased JAK-STAT signaling leads to increased expression of MHC class II genes [26] and, indeed, both JAK1 and STAT1 were found to be increased in the ITGAV KD xenografts. Using immunohistochemistry, an increased intranuclear STAT1 signal could also be confirmed in the corresponding ITGAV KD xenograft tumors at the protein level (Fig. 6).
When gene expression profiles of the SMAD4deficient BxPC3 xenografts with knockdown of ITGAV were analyzed, no genes associated with TGF-β signaling, EMT or immune status were found to be significantly regulated. We attribute this gene expression pattern to the malfunction of the canonical TGF-β signaling pathway caused by the confirmed deletion of SMAD4 (Fig. 3d). Table 1). E.g., the adhesion molecule CEAC AM7 was significantly upregulated in both models, as demonstrated by immunohistochemistry (Fig. 6).
Clinical proof of concept using a tissue microarray
A high level of ITGAV expression in the PDA cells was found in 129 tissue samples (71%). These patients showed a significantly poorer outcome with a median survival of 11 (95% CI 8.6-13.3) months instead of 21 (95% CI 15.5-26.5) months in the group of patients with low ITGAV expression (P = 0.004, Fig. 7a). For 125 tissue samples (69%), a high percentage of pSMAD2positive tumor cell nuclei was identified and ITGAV and phospho-SMAD2 staining intensities were positively correlated (Supplementary Table 2, P = 0.014). However, there was no significant correlation for phospho-SMAD2 with survival (P = 0.127). Ninety three tissue samples (58%) displayed a high expression of STAT1. No significant association existed between STAT1 and survival (P = 0.310). High expression of HLA-DR was found in 22 tumor samples. Of these 22 tumors, 17 were also STAT1 positive. Hence, a positive correlation between STAT1 and HLA-DR expression was observed (Supplementary Table 2, P = 0.019). This observation supports the connection between JAK-STAT signaling and the expression of HLA-DR. In our cohort, a better survival could be demonstrated for patients with high expression of HLA-DR by tumor cells with a median survival of 35 (95% CI 0.6-69.4) months compared with 15 (95% CI 12.9-17.1) months for patients with low HLA-DR expression (Fig. 7b). None of the staining results showed any correlation with gender, grading or tumor stage (Supplementary Table 2).
Discussion
In the present study, we could show that ITGAV expression was upregulated in intraperitoneal pancreatic ductal adenocarcinoma xenograft tumor cells that grew under selectin-deficient conditions, suggesting a compensatory increase in integrin expression. We could demonstrate that the knockdown of ITGAV led to a massive reduction in intraperitoneal carcinomatosis, primary tumor growth and pulmonary metastasis. Even more importantly, the effects of the absence of host selectins and ITGAV were synergistic, as clearly demonstrated in our intraperitoneal tumor model. This is the first model to describe such a synergism experimentally. These findings validate the hypothesis that cancer cell adhesion to mesothelial cells follows similar mechanisms as the leukocyte adhesion cascade, which is initiated by selectins followed by integrins [27]. Integrin-mediated cell adhesion also seems to be essential in compensating for the absence of selectin expression [28]. The observed increased expression in other cell adhesion molecules in the ITGAV KD tumors (e.g. the adhesion molecule CEACAM-7) might partly compensate for the reduction of ITGAV. Our findings indicate that the adhesion to the peritoneal mesothelium or its underlying basal lamina is the rate-limiting step of peritoneal carcinomatosis formation in PDA. Fortunately, this step is particularly amenable to therapeutic invention as the molecules involved are located on the cell surface. Therefore, blockage of both selectins and integrins is an attractive option to inhibit peritoneal metastasis in PDA. A body of literature suggests that ITGAV activates latent TGF-β in the ECM [19,29]. We could demonstrate this for the SMAD4-intact PaCa 5061 cells, for which the concentration of active TGF-β1 is reduced due to the knockdown of ITGAV in vitro. Furthermore, the knockdown of ITGAV reduced the activity of phospho-SMAD2 in the xenograft tumors of the SMAD4-intact PaCa 5061 cells in vivo. Our gene expression analyses confirmed altered TGF-β-signaling due to the knockdown of ITGAV in PaCa 5061 tumors, as described above. Moreover, there is an association between ITGAV and pSMAD2-positive tumors in the analyzed patient samples: high expression of ITGAV is connected with poorer survival. This observation corroborates data in the GEPIA2 webserver ( Supplementary Fig. 3, [30]) and Human Protein Atlas [31], which show a correlation between ITGAV mRNA expression level and poor patient survival. Moreover, a recent study demonstrated that elevated serum soluble TGF-β predicted poor survival in PDA [32].
At the beginning of the tumorigenic process, TGF-β1 functions as a tumor suppressor due to its ability to suppress cell division in epithelial cells [33]. Hezel et al. used a genetically engineered mouse model with Kras G12D -initiated, SMAD4-deficient murine PDAs reflecting an early disease state and identified increased tumor cell proliferation through the blockade of integrin αvβ6 [34]. The cell lines used in our study originate from locally advanced human tumors, as commonly present at the time of diagnosis. Our proliferation assay in vitro and the determination of the Ki-67 Labeling Index in the xenograft tumors show no changes in cell proliferation (Fig. 5b), suggesting that the examined cell lines are already resistant to the anti-proliferative effects of TGF-β.
Bates et al. reported that a high level of integrin αvβ6 is accompanied by a poorer overall survival for colon carcinoma. They assumed the reason for this to be the integrin αvβ6-dependent activation of TGF-β1 and the resulting stimulation of EMT [35]. In PDA, high integrin αvβ6 mRNA levels were associated with shortened patient survival and antibody therapy directed against this dimer suppressed the pro-tumorogenic microenvironment (e.g. by suppression of TGF-β signaling) in mouse models [7]. Besides the critical alterations induced by ITGAV in TGF-β signaling for SMAD4 intact PDA (represented by PaCa 5061 in this study), the xenograft model with the SMAD4 dysfunctional BxPC3 cells demonstrates that there are also TGF-β independent effects of ITGAV in PDA. The TMA data further corroborate this observation: Although Tumors were not stratified for functional SMAD4, ITGAV expression was still prognostic in the overall cohort. Together with the fact that half of all PDA display deletions / inactivating mutations of SMAD4 [20], this indicates that ITGAV is prognostic for SMAD4 functional as well as dysfunctional tumors. In prostate cancer, for example, AKT activation has been described as an additional mechanism for ITGAV involvement in a recent study [4]. Our study shows that the proportion of Twist-positive cells decreases through the knockdown of ITGAV in the SMAD4-intact PaCa 5061 cells. Twist controls the expression of epithelial gene signatures such as Occludin and E-cadherin [36] and indeed, expression of these epithelial genes increased upon ITGAV KD. In pharyngeal carcinoma, Van Aarsen et al. demonstrated that blocking with an integrin αvβ6 antibody reduced TGF-β-induced SMAD2 phosphorylation, which resulted in diminished tumor growth and reduced invasive potential while an impact of the treatment on cell proliferation was not observed [37]. These effects were confirmed for PDA in our study.
In vivo, TFG-β1 is a potent activator of the transdifferentiation of fibroblasts into myofibroblasts, which are referred to as cancer-associated fibroblasts (CAFs) [38]. The increase in the ECM contraction caused by CAFs increases the probability of TGF-β activation [38]. Interestingly, it has been shown that the heterogeneity of CAFs in PDA is mediated by JAK/STAT signaling antagonized by TGF-β [39]. CAFs themselves interact with integrins and secrete a large number of proteins into the ECM matrix, proteases and cytokines, which can further increase cancer progression [40]. For example, CAFs deposit fibronectin in the ECM, thereby creating promigratory tracks [41]. It is assumed that CAFs themselves express ITGAV and hence can also activate latent TGF-β [42]. Consistently, we found an upregulation of murine fibronectin in the xenograft tumors with ITGAV KD (see Fig. 5a and e).
Highly interesting is the immunomodulatory effect of the ITGAV knockdown found in our study, especially since results for immunotherapy of PDA [43] must be considered disappointing so far. To our knowledge, the observed changes in MHC-II expression by PDA have not been described before. However, to fully investigate the implications of these alterations, a suitable new in vivo model with an intact adaptive immune system would be needed, e.g. a syngenic model. As MHC-II is required for CD4+ T-cell activation, which also plays essential roles in antitumor immunity [44], further investigations in this field could prove highly rewarding.
Conclusions
The ITGAV knockdown of PDA massively suppressed the intraperitoneal carcinomatosis of these cells. Moreover, the effects of the absence of selectins and reduced expression of ITGAV on intraperitoneal carcinomatosis are synergistic, confirming the hypothesis of a multistep and partly redundant leukocyte adhesion cascade as the rate-limiting step within the metastatic cascade. Mechanistically, ITGAV activates TGF-β and drives epithelialmesenchymal transition PDA cells. Specific inhibition of ITGAV may have the potential to impede intraperitoneal carcinomatosis, tumor growth and distant metastasis.
Fig. 1
1In a xenograft model of human PDAC, Integrin αV is upregulated in intraperitoneal carcinomatoses grown in E-and P-selectin knockout mice. Immunohistochemical staining for human Integrin αV: Carcinomatosis from wild-type (a) compared with those grown in E−/P-selectindeficient pfp −− /rag2 −− mice (b). Scale bar: 50 μm PaCa5061 ITGAV KD BxPC3 ITGAV KD
Fig. 3 (
3See legend on next page.) not be determined. Therefore, we extended the experimental time to 77 days and demonstrated a synergistic effect of ITGAV knockdown and selectin knockout (13 and 14 selectin-deficient Pfp −/− / Rag2 −/− for the control cells and the ITGAV KD cells, respectively, P = 0.017,Fig. 3c). Tumor take rates were as follows: selectin k.o. mice injected with control cells: 8 of 13 (62%) and k.o. mice injected with ITGAV KD cells: 6 of 14 (43%).
Fig. 4
4Concentration of active TGF-β1 and total TFG-β1 measured in the cell culture supernatant of PaCa 5061 ITGAV KD cells and control cells using ELISA (a). The proliferation assay showed no significant differences between the ITGAV knockdown groups and the corresponding control groups (b). The static adhesion on fibronectin was reduced by ITGAV knockdown (c). The migratory potential of tumor cells was decreased by the ITGAV KD (d), while the invasive potential was only impaired in the case of BxPC3 cells (e). In the colony-forming assay, BxPC3 ITGAV KD cells formed fewer spheroid colonies than the control cells. PaCa 5061 cells were not able to form colonies in Matrigel after 14 days (f)
Fig. 5
5Heat map and clustering of gene expression data for PaCa 5061 and BxPC3 xenograft tumors: TGF-β signaling (a), epithelial-mesenchymal transition (b) and immune status (c). A significantly higher expression is shown in red and a lower expression in green. In contrast to the gene expression changes in BxPC3 xenograft tumors (no canonical signaling due to the deletion of SMAD4), changes in PaCa 5061 (SMAD4 intact) xenograft tumors appeared organized Finally, ITGAV targets concordantly altered in both PaCa5061 and BxPC3 xenograft tumors could be assigned to the group of cell surface interactions (s. Supplementary
Fig. 7 (
7See legend on next page.)
(
See figure on previous page.) Fig. 7 Patients with high expression of ITGAV (N = 129) had a significantly poorer survival with a mean survival of 11 months than patients with low expression of ITGAV (N = 54) with a mean survival of 21 months (a). Patients with high expression of HLA-DR (N = 22) showed a significantly better survival with a mean survival of 35 months than patients with low expression of HLA-DR (N = 170) with a mean survival of 15 months (b). Representative tissue samples are given (c). Scale bar: 50 μm
Abbreviations PDA: Pancreatic ductal adenocarcinoma; shRNA: Short hairpin RNA; ELISA: Enzyme-linked immunosorbent assay; PCI: Peritoneal carcinomatosis index; qRT-PCR: Quantitative real-time polymerase chain reaction; TMA: Tissue microarray; KD: Knockdown; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; HLA: Human leucocyte antigen; CAFs: Cancer-associated fibroblasts
Fig. 2Changes of ITGAV and corresponding beta-subunits ITGB1 und ITGB6 (a) using flow cytometry (b) and Western blot (c). The expression of ITGAV was reduced by > 65% in PaCa 5061 and BxPC3 ITGAV KD cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells. In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchangedFlow cytometry
Western blot
Flow cytometry
Western blot
ITGAV
ITGB1
5%
ITGB6
B
PaCa5061
BxPC3
C
PaCa5061
BxPC3
Control ITGAV KD Control ITGAV KD
Signal
ITGAV KD: 98.02
Control:
229.52
ITGAV
ITGB1
ITGB6
Signal
ITGAV KD: 93.17
Control:
151.66
Signal
ITGAV KD: 831.15
Control:
1152.14
Signal
ITGAV KD: 985.50
Control:
1000.50
Signal
ITGAV KD: 104.82
Control:
130.17
Signal
ITGAV KD: 56.15
Control:
61.90
ITGAV
ITGB1
ITGB6
HSC-70
A
70%
90%
66%
87 %
31%
19%
16%
73%
51%
69%
21
0.0
0.2
0.4
0.6
0.8
1.0
P = 0.012
P < 0.0001
Paca 5061
control in
selectin deficient
Paca 5061
ITGAV KD in
selectin deficient
Paca 5061
ITGAV KD in
w ildtype
tumor weight injection site (g)
HSC70
control
SMAD4
C-Terminus
C
terminated after 77 d
D
G
PaCa 5061 BxPC3
B
terminated after 62 d
A
terminated after 62 d
H
I
J
E
F
Table 1
1Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font)Gene Symbol
Fold Change
P
FDR
adj. P
TGF-β signaling
CDK8
1.8
0.007
0.108
E2F4
−2.3
< 0.001
0.007
LTBP1
− 2.61
0.0003
0.0201
NEDD4L
−1.3
0.096
0.462
PARD3
− 2.43
< 0.001
0.001
PPM1A
2.42
0.002
0.051
PPP1CA
−1.55
< 0.001
0.013
PPP1CB
2.59
< 0.001
0.003
RAP1B
1.5
0.0064
0.1065
SERPINE1
−2.83
0.004
0.077
SKIL
2.92
0.002
0.051
SMAD1
−1.18
0.310
0.733
SMAD2
1.09
0.809
0.958
SMAD3
1.79
0.273
0.704
SMAD4
−1.08
0.917
0.982
SMAD6
−1.13
0.289
0.717
SMAD7
1.06
0.505
0.851
SMAD9
−1.01
0.595
0.887
STUB1
−1.84
< 0.001
0.019
TGFB1
−1.59
0.014
0.170
TGFB2
3.61
0.003
0.071
TGFBI
−0.11
0.065
0.384
TGFBR1
1.68
< 0.001
0.015
TGFBR2
1.22
0.971
0.994
TGFBR3
−2.75
0.005
0.090
Epithelial-mesenchymal transition
ACTA2
−1.04
0.6051
0.8912
ADAM10
2.26
0.003
0.063
ADAM12
1.01
0.897
0.979
BSG
−2.65
< 0.001
0.002
CDH1
−1.21
0.3769
0.7813
CDH2
−5.04
< 0.001
< 0.001
FGFBP1
−1.86
0.007
0.107
FLG
265.8
< 0.001
0.002
FN1
−2.57
0.002
0.048
HSPG2
−1.78
0.001
0.039
JAG1
−3.05
< 0.001
0.005
KLK13
2.06
0.001
0.043
KLK7
3.18
0.008
0.125
LAMA2
−1.61
0.001
0.028
LAMA3
−6.57
< 0.001
0.003
LAMA5
−1.74
0.072
0.404
Table 1
1Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font) (Continued)Gene Symbol
Fold Change
P
FDR
adj. P
HLA-DMB
1.41
0.009
0.129
HLA-DOA
1.26
0.009
0.129
HLA-DOB
1.09
0.437
0.817
HLA-DPA1
3.32
< 0.001
0.005
HLA-DPB1
1.4
0.019
0.197
HLA-DPB2
1.21
0.031
0.257
HLA-DQA1
−1.14
0.294
0.721
HLA-DQA2
1.77
0.008
0.123
HLA-DQB1
−1.14
0.492
0.845
HLA-DQB2
1.41
0.003
0.065
HLA-DRA
14.81
< 0.001
0.004
HLA-DRB1
19.95
0.001
0.025
HLA-DRB4
12.38
< 0.001
0.005
HLA-DRB5
3.45
< 0.001
0.002
HLA-E
1.17
0.199
0.628
HLA-G
1.64
0.048
0.328
HLA-H
1.13
0.298
0.724
ICAM1
1.71
0.065
0.385
IFNG
−1.07
0.179
0.603
IRF1
1.37
0.005
0.094
IRF2
− 1.56
0.014
0.170
JAK1
1.82
< 0.001
0.015
KIF3B
−1.73
0.004
0.076
LMO7
5.19
< 0.001
0.005
MALT1
1.72
0.003
0.069
MID1
−3.3
< 0.001
0.007
MT2A
−2.36
0.002
0.049
OAS1
1.78
0.014
0.169
OSBPL1A
−2.52
< 0.001
0.010
PAK1
−2.66
< 0.001
0.004
PTAFR
−2.09
0.006
0.102
RIPK2
2.56
0.020
0.205
SLCO4C1
−2.93
0.001
0.040
STAT1
2.58
< 0.001
0.019
TIMP2
4.83
< 0.001
0.008
TRIM22
2.28
< 0.001
0.004
AcknowledgementsWe thank Prof. Dr. Guido Sauter for providing the tissue microarray and Christine Knies, Tobias Gosau, Jenniffer Schröder-Schwarz, Maike Märker, Lisa Staffeldt and Ursula Makowski for excellent technical and organizational support.Availability of data and materialsThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.2Institute of Anatomy and Experimental Morphology, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany.3Supplementary InformationThe online version contains supplementary material available at https://doi. org/10.1186/s13046-021-01946-2. Additional file 3:Supplementary Figure 3. For an in silico analysis of the association between gene expression and patient survival in the GEPI A2 webserver, we used the heterodimer ITGAV and ITGB6 as a signature. High expression of ITGAV and ITGB6 is associated with poor survival (P < 0.001).
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| [
"Background: Mesothelial E-and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis.Methods: Knockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated. Results: The knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-β and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E-and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients.Conclusion: Combined depletion of ITGAV in PDA cells and E-and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E-and P-selectins and ITGAV in PDA."
] | [
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"Hanna Maar ",
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"Andrey Poloznikov ",
"Vladimir Galatenko ",
"Michael Tachezy ",
"Florian Gebauer ",
"Tobias Lange ",
"Kristoffer Riecken ",
"Alexander Tonevitsky ",
"Achim Aigner ",
"Jakob Izbicki ",
"Udo Schumacher ",
"Daniel Wicklein "
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"Marius",
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] | [
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"Immunotherapy for pancreatic cancer: present and future. F Aroldi, A Zaniboni, 10.2217/imt-2016-0142Immunotherapy. 97Aroldi F, Zaniboni A. Immunotherapy for pancreatic cancer: present and future. Immunotherapy. 2017;9(7):607-16. https://doi.org/10.2217/imt-201 6-0142.",
"How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules?. O Haabeth, A Tveita, M Fauskanger, F Schjesvold, K Lorvik, P Hofgaard, Front Immunol. 5174Haabeth O, Tveita A, Fauskanger M, Schjesvold F, Lorvik K, Hofgaard P, et al. How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules? Front Immunol. 2014;5:174.",
"Publisher's Note. Publisher's Note",
"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."
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"Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods",
"Metaanalysis of recurrence pattern after resection for pancreatic cancer",
"Selectin binding is essential for peritoneal carcinomatosis in a xenograft model of human pancreatic adenocarcinoma in pfp−−/rag2--mice",
"Large oncosomes overexpressing integrin alpha-V promote prostate cancer adhesion and invasion via AKT activation",
"Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype",
"Identification of biomarkers associated with extracellular vesicles based on an integrative pan-cancer bioinformatics analysis",
"The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy",
"Integrin αvβ6-specific therapy for pancreatic cancer developed from footand-mouth-disease virus",
"Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung",
"Characterization of a new primary human pancreatic tumor line",
"Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis",
"RNAi technology to block the expression of molecules relevant to metastasis: the cell adhesion molecule CEACAM1 as an instructive example",
"Surgical responsibilities in the management of peritoneal carcinomatosis",
"Guidelines for the welfare and use of animals in cancer research",
"Comparison of two techniques for the screening of human tumor cells in mouse blood: quantitative real-time polymerase chain reaction (qRT-PCR) versus laser scanning cytometry (LSC)",
"Human prostate cancer in a clinically relevant Xenograft mouse model: identification of β(1,6)-branched oligosaccharides as a marker of tumor progression",
"Expression pattern of the AP-1 family in breast cancer: association of fosB expression with a well-differentiated, receptor-positive tumor phenotype",
"Methods in molecular biology",
"The role of integrins in TGFβ activation in the tumour stroma",
"Whole genomes redefine the mutational landscape of pancreatic cancer",
"An electron microscopic study of the invasion of ascites tumor cells into the abdominal wall",
"Transforming growth factor beta-induced, an extracellular matrix interacting protein, enhances glycolysis and promotes pancreatic cancer cell migration",
"Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis",
"Expression of CD44 in pancreatic cancer and its significance",
"MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation",
"The functional role of integrins during intraand extravasation within the metastatic cascade",
"Cell adhesion molecules in metastatic neuroblastoma models",
"Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs",
"GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses",
"A pathology atlas of the human cancer transcriptome",
"The prognostic role of soluble TGF-beta and its dynamics in unresectable pancreatic cancer treated with chemotherapy",
"The role of TGF-β/SMAD4 signaling in cancer",
"TGF-β and αvβ6 integrin act in a common pathway to suppress pancreatic cancer progression",
"Transcriptional activation of integrin β6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma",
"TGF-beta-induced epithelial to mesenchymal transition",
"Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism",
"Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix",
"IL1-induced JAK/ STAT signaling is antagonized by TGFβ to shape CAF heterogeneity in pancreatic ductal adenocarcinoma",
"Insidious changes in stromal matrix fuel cancer progression",
"Cancerassociated fibroblasts lead tumor invasion through integrin-β3-dependent fibronectin assembly",
"It has to be the αv: myofibroblast integrins activate latent TGF-β1",
"Immunotherapy for pancreatic cancer: present and future",
"How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules?"
] | [
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"Oncotarget",
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"J Pathol",
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"Publisher's Note",
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] | [
"\nFig. 1\n1In a xenograft model of human PDAC, Integrin αV is upregulated in intraperitoneal carcinomatoses grown in E-and P-selectin knockout mice. Immunohistochemical staining for human Integrin αV: Carcinomatosis from wild-type (a) compared with those grown in E−/P-selectindeficient pfp −− /rag2 −− mice (b). Scale bar: 50 μm PaCa5061 ITGAV KD BxPC3 ITGAV KD",
"\nFig. 3 (\n3See legend on next page.) not be determined. Therefore, we extended the experimental time to 77 days and demonstrated a synergistic effect of ITGAV knockdown and selectin knockout (13 and 14 selectin-deficient Pfp −/− / Rag2 −/− for the control cells and the ITGAV KD cells, respectively, P = 0.017,Fig. 3c). Tumor take rates were as follows: selectin k.o. mice injected with control cells: 8 of 13 (62%) and k.o. mice injected with ITGAV KD cells: 6 of 14 (43%).",
"\nFig. 4\n4Concentration of active TGF-β1 and total TFG-β1 measured in the cell culture supernatant of PaCa 5061 ITGAV KD cells and control cells using ELISA (a). The proliferation assay showed no significant differences between the ITGAV knockdown groups and the corresponding control groups (b). The static adhesion on fibronectin was reduced by ITGAV knockdown (c). The migratory potential of tumor cells was decreased by the ITGAV KD (d), while the invasive potential was only impaired in the case of BxPC3 cells (e). In the colony-forming assay, BxPC3 ITGAV KD cells formed fewer spheroid colonies than the control cells. PaCa 5061 cells were not able to form colonies in Matrigel after 14 days (f)",
"\nFig. 5\n5Heat map and clustering of gene expression data for PaCa 5061 and BxPC3 xenograft tumors: TGF-β signaling (a), epithelial-mesenchymal transition (b) and immune status (c). A significantly higher expression is shown in red and a lower expression in green. In contrast to the gene expression changes in BxPC3 xenograft tumors (no canonical signaling due to the deletion of SMAD4), changes in PaCa 5061 (SMAD4 intact) xenograft tumors appeared organized Finally, ITGAV targets concordantly altered in both PaCa5061 and BxPC3 xenograft tumors could be assigned to the group of cell surface interactions (s. Supplementary",
"\nFig. 7 (\n7See legend on next page.)",
"\n(\nSee figure on previous page.) Fig. 7 Patients with high expression of ITGAV (N = 129) had a significantly poorer survival with a mean survival of 11 months than patients with low expression of ITGAV (N = 54) with a mean survival of 21 months (a). Patients with high expression of HLA-DR (N = 22) showed a significantly better survival with a mean survival of 35 months than patients with low expression of HLA-DR (N = 170) with a mean survival of 15 months (b). Representative tissue samples are given (c). Scale bar: 50 μm",
"\n\nAbbreviations PDA: Pancreatic ductal adenocarcinoma; shRNA: Short hairpin RNA; ELISA: Enzyme-linked immunosorbent assay; PCI: Peritoneal carcinomatosis index; qRT-PCR: Quantitative real-time polymerase chain reaction; TMA: Tissue microarray; KD: Knockdown; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; HLA: Human leucocyte antigen; CAFs: Cancer-associated fibroblasts",
"\n\nFig. 2Changes of ITGAV and corresponding beta-subunits ITGB1 und ITGB6 (a) using flow cytometry (b) and Western blot (c). The expression of ITGAV was reduced by > 65% in PaCa 5061 and BxPC3 ITGAV KD cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells. In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchangedFlow cytometry \nWestern blot \nFlow cytometry \nWestern blot \n\nITGAV \n\nITGB1 \n5% \n\nITGB6 \n\nB \n\nPaCa5061 \nBxPC3 \n\nC \n\nPaCa5061 \nBxPC3 \n\nControl ITGAV KD Control ITGAV KD \n\nSignal \nITGAV KD: 98.02 \nControl: \n229.52 \n\nITGAV \n\nITGB1 \n\nITGB6 \n\nSignal \nITGAV KD: 93.17 \nControl: \n151.66 \n\nSignal \nITGAV KD: 831.15 \nControl: \n1152.14 \n\nSignal \nITGAV KD: 985.50 \nControl: \n1000.50 \n\nSignal \nITGAV KD: 104.82 \nControl: \n130.17 \n\nSignal \nITGAV KD: 56.15 \nControl: \n61.90 \n\nITGAV \n\nITGB1 \n\nITGB6 \n\nHSC-70 \n\nA \n\n70% \n90% \n66% \n87 % \n\n31% \n19% \n16% \n\n73% \n51% \n69% \n21 \n\n0.0 \n\n0.2 \n\n0.4 \n\n0.6 \n\n0.8 \n\n1.0 \n\nP = 0.012 \n\nP < 0.0001 \n\nPaca 5061 \ncontrol in \nselectin deficient \n\nPaca 5061 \nITGAV KD in \nselectin deficient \n\nPaca 5061 \nITGAV KD in \nw ildtype \n\ntumor weight injection site (g) \n\nHSC70 \ncontrol \n\nSMAD4 \nC-Terminus \n\nC \n\nterminated after 77 d \n\nD \n\nG \n\nPaCa 5061 BxPC3 \n\nB \n\nterminated after 62 d \n\nA \n\nterminated after 62 d \n\nH \nI \nJ \n\nE \nF \n\n",
"\nTable 1\n1Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font)Gene Symbol \nFold Change \nP \nFDR \nadj. P \n\nTGF-β signaling \n\nCDK8 \n1.8 \n0.007 \n0.108 \n\nE2F4 \n−2.3 \n< 0.001 \n0.007 \n\nLTBP1 \n− 2.61 \n0.0003 \n0.0201 \n\nNEDD4L \n−1.3 \n0.096 \n0.462 \n\nPARD3 \n− 2.43 \n< 0.001 \n0.001 \n\nPPM1A \n2.42 \n0.002 \n0.051 \n\nPPP1CA \n−1.55 \n< 0.001 \n0.013 \n\nPPP1CB \n2.59 \n< 0.001 \n0.003 \n\nRAP1B \n1.5 \n0.0064 \n0.1065 \n\nSERPINE1 \n−2.83 \n0.004 \n0.077 \n\nSKIL \n2.92 \n0.002 \n0.051 \n\nSMAD1 \n−1.18 \n0.310 \n0.733 \n\nSMAD2 \n1.09 \n0.809 \n0.958 \n\nSMAD3 \n1.79 \n0.273 \n0.704 \n\nSMAD4 \n−1.08 \n0.917 \n0.982 \n\nSMAD6 \n−1.13 \n0.289 \n0.717 \n\nSMAD7 \n1.06 \n0.505 \n0.851 \n\nSMAD9 \n−1.01 \n0.595 \n0.887 \n\nSTUB1 \n−1.84 \n< 0.001 \n0.019 \n\nTGFB1 \n−1.59 \n0.014 \n0.170 \n\nTGFB2 \n3.61 \n0.003 \n0.071 \n\nTGFBI \n−0.11 \n0.065 \n0.384 \n\nTGFBR1 \n1.68 \n< 0.001 \n0.015 \n\nTGFBR2 \n1.22 \n0.971 \n0.994 \n\nTGFBR3 \n−2.75 \n0.005 \n0.090 \n\nEpithelial-mesenchymal transition \n\nACTA2 \n−1.04 \n0.6051 \n0.8912 \n\nADAM10 \n2.26 \n0.003 \n0.063 \n\nADAM12 \n1.01 \n0.897 \n0.979 \n\nBSG \n−2.65 \n< 0.001 \n0.002 \n\nCDH1 \n−1.21 \n0.3769 \n0.7813 \n\nCDH2 \n−5.04 \n< 0.001 \n< 0.001 \n\nFGFBP1 \n−1.86 \n0.007 \n0.107 \n\nFLG \n265.8 \n< 0.001 \n0.002 \n\nFN1 \n−2.57 \n0.002 \n0.048 \n\nHSPG2 \n−1.78 \n0.001 \n0.039 \n\nJAG1 \n−3.05 \n< 0.001 \n0.005 \n\nKLK13 \n2.06 \n0.001 \n0.043 \n\nKLK7 \n3.18 \n0.008 \n0.125 \n\nLAMA2 \n−1.61 \n0.001 \n0.028 \n\nLAMA3 \n−6.57 \n< 0.001 \n0.003 \n\nLAMA5 \n−1.74 \n0.072 \n0.404 \n",
"\nTable 1\n1Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font) (Continued)Gene Symbol \nFold Change \nP \nFDR \nadj. P \n\nHLA-DMB \n1.41 \n0.009 \n0.129 \n\nHLA-DOA \n1.26 \n0.009 \n0.129 \n\nHLA-DOB \n1.09 \n0.437 \n0.817 \n\nHLA-DPA1 \n3.32 \n< 0.001 \n0.005 \n\nHLA-DPB1 \n1.4 \n0.019 \n0.197 \n\nHLA-DPB2 \n1.21 \n0.031 \n0.257 \n\nHLA-DQA1 \n−1.14 \n0.294 \n0.721 \n\nHLA-DQA2 \n1.77 \n0.008 \n0.123 \n\nHLA-DQB1 \n−1.14 \n0.492 \n0.845 \n\nHLA-DQB2 \n1.41 \n0.003 \n0.065 \n\nHLA-DRA \n14.81 \n< 0.001 \n0.004 \n\nHLA-DRB1 \n19.95 \n0.001 \n0.025 \n\nHLA-DRB4 \n12.38 \n< 0.001 \n0.005 \n\nHLA-DRB5 \n3.45 \n< 0.001 \n0.002 \n\nHLA-E \n1.17 \n0.199 \n0.628 \n\nHLA-G \n1.64 \n0.048 \n0.328 \n\nHLA-H \n1.13 \n0.298 \n0.724 \n\nICAM1 \n1.71 \n0.065 \n0.385 \n\nIFNG \n−1.07 \n0.179 \n0.603 \n\nIRF1 \n1.37 \n0.005 \n0.094 \n\nIRF2 \n− 1.56 \n0.014 \n0.170 \n\nJAK1 \n1.82 \n< 0.001 \n0.015 \n\nKIF3B \n−1.73 \n0.004 \n0.076 \n\nLMO7 \n5.19 \n< 0.001 \n0.005 \n\nMALT1 \n1.72 \n0.003 \n0.069 \n\nMID1 \n−3.3 \n< 0.001 \n0.007 \n\nMT2A \n−2.36 \n0.002 \n0.049 \n\nOAS1 \n1.78 \n0.014 \n0.169 \n\nOSBPL1A \n−2.52 \n< 0.001 \n0.010 \n\nPAK1 \n−2.66 \n< 0.001 \n0.004 \n\nPTAFR \n−2.09 \n0.006 \n0.102 \n\nRIPK2 \n2.56 \n0.020 \n0.205 \n\nSLCO4C1 \n−2.93 \n0.001 \n0.040 \n\nSTAT1 \n2.58 \n< 0.001 \n0.019 \n\nTIMP2 \n4.83 \n< 0.001 \n0.008 \n\nTRIM22 \n2.28 \n< 0.001 \n0.004 \n"
] | [
"In a xenograft model of human PDAC, Integrin αV is upregulated in intraperitoneal carcinomatoses grown in E-and P-selectin knockout mice. Immunohistochemical staining for human Integrin αV: Carcinomatosis from wild-type (a) compared with those grown in E−/P-selectindeficient pfp −− /rag2 −− mice (b). Scale bar: 50 μm PaCa5061 ITGAV KD BxPC3 ITGAV KD",
"See legend on next page.) not be determined. Therefore, we extended the experimental time to 77 days and demonstrated a synergistic effect of ITGAV knockdown and selectin knockout (13 and 14 selectin-deficient Pfp −/− / Rag2 −/− for the control cells and the ITGAV KD cells, respectively, P = 0.017,Fig. 3c). Tumor take rates were as follows: selectin k.o. mice injected with control cells: 8 of 13 (62%) and k.o. mice injected with ITGAV KD cells: 6 of 14 (43%).",
"Concentration of active TGF-β1 and total TFG-β1 measured in the cell culture supernatant of PaCa 5061 ITGAV KD cells and control cells using ELISA (a). The proliferation assay showed no significant differences between the ITGAV knockdown groups and the corresponding control groups (b). The static adhesion on fibronectin was reduced by ITGAV knockdown (c). The migratory potential of tumor cells was decreased by the ITGAV KD (d), while the invasive potential was only impaired in the case of BxPC3 cells (e). In the colony-forming assay, BxPC3 ITGAV KD cells formed fewer spheroid colonies than the control cells. PaCa 5061 cells were not able to form colonies in Matrigel after 14 days (f)",
"Heat map and clustering of gene expression data for PaCa 5061 and BxPC3 xenograft tumors: TGF-β signaling (a), epithelial-mesenchymal transition (b) and immune status (c). A significantly higher expression is shown in red and a lower expression in green. In contrast to the gene expression changes in BxPC3 xenograft tumors (no canonical signaling due to the deletion of SMAD4), changes in PaCa 5061 (SMAD4 intact) xenograft tumors appeared organized Finally, ITGAV targets concordantly altered in both PaCa5061 and BxPC3 xenograft tumors could be assigned to the group of cell surface interactions (s. Supplementary",
"See legend on next page.)",
"See figure on previous page.) Fig. 7 Patients with high expression of ITGAV (N = 129) had a significantly poorer survival with a mean survival of 11 months than patients with low expression of ITGAV (N = 54) with a mean survival of 21 months (a). Patients with high expression of HLA-DR (N = 22) showed a significantly better survival with a mean survival of 35 months than patients with low expression of HLA-DR (N = 170) with a mean survival of 15 months (b). Representative tissue samples are given (c). Scale bar: 50 μm",
"Abbreviations PDA: Pancreatic ductal adenocarcinoma; shRNA: Short hairpin RNA; ELISA: Enzyme-linked immunosorbent assay; PCI: Peritoneal carcinomatosis index; qRT-PCR: Quantitative real-time polymerase chain reaction; TMA: Tissue microarray; KD: Knockdown; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; HLA: Human leucocyte antigen; CAFs: Cancer-associated fibroblasts",
"Fig. 2Changes of ITGAV and corresponding beta-subunits ITGB1 und ITGB6 (a) using flow cytometry (b) and Western blot (c). The expression of ITGAV was reduced by > 65% in PaCa 5061 and BxPC3 ITGAV KD cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells. In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchanged",
"Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font)",
"Selected gene expression changes in PaCa 5061 xenograft tumors upon ITGAV KD. FDR adjusted P-values < 0.10 are highlighted (bold font) (Continued)"
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"Pancreatic ductal adenocarcinoma (PDA) belongs to the most lethal malignancies in industrialized countries. By 2040 an increase to 777,423 death per year worldwide is expected [1]. Despite advances in diagnostics and therapy, the five-year survival rate is only 9% [1]. These data indicate that the vast majority of patients relapse within 5 years after surgery. Besides locoregional relapse or distant metastasis to lungs or liver, recurrence manifests as peritoneal carcinomatosis in many cases [2], making this process an important target for therapeutic intervention. In fact, the median overall survival for patients diagnosed with intraperitoneal carcinomatosis amounts 14.1 months only [2].",
"In a previous study, we demonstrated that tumor cell adherence to the peritoneal mesothelium is highly dependent on the binding of carbohydrate structures present of the cancer cells' outer cell membrane to E-and P-selectins expressed on the peritoneal mesothelial cells using a xenograft model with selectin-deficient mice [3]. In the absence of selectins, only a minority of cells could adhere to the peritoneum, which considerably inhibited intraperitoneal tumor growth [3]. In the present study, we investigated the underlying mechanism for the remaining metastatic deposit in selectin-deficient mice in which we found Integrin αV (ITGAV) to be functionally involved. ITGAV has recently been reported to be an important driver of cancer progression in prostate [4,5] and colon carcinoma [6]. In PDA, recent studies demonstrate that the dimer of Integrins αVβ6 (ITGAV / ITGB6) is overexpressed in most tumors (with overexpression being retained in the corresponding metastases) and is a feasible target for novel therapeutic approaches [7,8].",
"Authenticated PDA cell lines PaCa 5061 [9] and BxPC3 [10] were used. ITGAV knockdown was achieved using a short hairpin RNA (shRNA) mediated approach: A 65 bp hairpin DNA oligomer containing a 19 bp anti-ITGAV sequence (GGATGGTGTCCACTTCAAA) was inserted into the pLVX vector (Clontech). Potential offtarget effects were checked (NCBI BLAST). An shRNA sequence against firefly luciferase was used for the control cell line [11]. Knockdown and control cells were seeded at three cells per well. Sublines with normal ITGAV and low ITGAV expression were pooled respectively and tested for Mycoplasma. The passage number of used cell lines never exceeded 20.",
"Flow cytometry was performed as described [12]. Following antibodies were used: ITGAV (327,907, BioLegend), ITGB1 (11-0299, Thermo Fisher Scientific), ITGB3 (336, 403, BioLegend), ITGB5 (11-0497-42, Thermo Fisher Scientific), ITGB6 (FAB4155P, R&D), HLA-DR (347,401, BD). Stained cells were subjected to FACS Calibur Flow Cytometry System (BD).",
"Cells were seeded in a T25 cell culture flask. After 48 h, they were changed to 1.5 mL serum-free medium. After another 24 h of incubation, the medium was harvested and centrifuged. Supernatants were collected. The measurement of TGF-β1 was made using the Free Active TGF-β1 ELISA Kit (cat. 437,707, BioLegend), respectively Total TGF-β1 ELISA Kit (cat. 436,707, BioLegend).",
"Cell proliferation was assessed using the XTT assay (Roche Diagnostics). 3 × 10 3 cells were plated per well (96-well plate) and incubated for 72 h. Fifty microliter XTT labeling mixture was added per well. After 5 h incubation, spectrophotometrical absorbance (450 nm) was measured using a microplate reader (MR5000 Multiplate Reader, Dynatech).",
"Static cell adhesion assays were performed in fibronectin-coated μ-Slides (ibidi). Cells were expanded to a concentration of 1 × 10 5 cells and added to a suspension volume of 60 μL. After 1 h of incubation, visual fields were documented (Axio Cam MRm, Carl Zeiss). To calculate the fraction of adherent cells, slides were washed with PBS and the same predefined visual fields were documented.",
"Differences in cell migration were assessed using the FluoroBlok Migration Assay with a 24-well plate with 8.0-μm pore size inserts (BD Bioscience). Cells were trypsinized and resuspended in serum-free medium in a concentration of 3 × 10 5 cells/ml. Four hundred microliter cell suspension was added to the apical chamber and 1200 μl medium with 10% FCS (Gibco) as chemoattractant was added to the bottom chamber. The assay was incubated for 24 h. After removing the chemoattractant from the bottom chamber, visualization of migrated cells was performed by adding 500 μl/well HBSS buffer with Calcein AM (Invitrogen) 4 μg/mL in the bottom well and incubating for 1 h. The readout was conducted at 485/530 nm (Ex/Em) on a Genios bottomreading fluorescence plate reader (Tecan). To assess the invasive potential, cells were seeded on a 24-multiwell insert plate uniformly coated with basement membrane equivalent Matrigel (FluoroBlok Invasion System, BD Biosciences). After rehydration according to the manufacturer's guidelines for use, all further steps were identical to those described for the migration assay.",
"For the colony-forming assay, 1500 μl single-cell suspension (1200 cells/ml) was mixed with 1500 μl growth factor reduced Matrigel (cat.: 356230, BD Biosciences). Fifty microliter of this suspension was seeded per well (96-well plate). After 30 min incubation, 200 μl medium was added per well. The plates were kept in an incubator for 14 d.",
"Animal experiments: xenograft mouse model C57BL/6 pfp −/− /rag2 −/− mice and E-and P-selectin double deficient pfp −/− /rag2 −/− [3] at the age of 8-12 weeks and bodyweight of 21-29 g were used for the study. For the intraperitoneal and the subcutaneous xenograft model, 1 × 10 6 viable, mycoplasma-free tumor cells suspended in 200 μl RPMI were injected intraperitoneally or subcutaneously. Peritoneal carcinomatoses were quantified by an adapted peritoneal carcinomatosis index (PCI). Briefly, the murine peritoneum was divided into 9 sections (upper left to lower right) and every section was attributed with a carcinoma score from 0 to 3, resulting in a total PCI score of 0 to 27 for each animal [3,13]. For the subcutaneous experiment, mice were injected subcutaneously directly under the right scapula with 1 × 10 6 mycoplasma-free cells. Animals were sacrificed when primary tumors exceeded 1.5 cm 3 or ulcerated the mouse skin. All animal experiments were performed following the United Kingdom Coordinating Committee of Cancer Research Guidelines [14]. The experiment was approved by the local licensing authority (project no. G09/88 and G10/55).",
"After sacrifice, the left lungs were homogenized (Tissue-Lyser II, Qiagen) and subjected to DNA isolation (QIAamp DNA Mini Kit, Qiagen). Two hundred microliter blood was subjected to DNA isolation. DNA concentrations were quantified (NanoDrop, Peqlab). All lung DNA samples were normalized to 30 ng/μL. The concentrations of blood DNA were similar in all samples (approx. 10 ng/μL) and were therefore not normalized. qRT-PCR was performed with established humanspecific Alu primers [15]. Numerical data were determined against a standard curve as described [16].",
"Protein concentration was determined by bicinchoninic acid assay. Western blots were performed as described [17]. Briefly, 30 μg of protein of each sample was added per well. Electrophoresis was performed in 10% polyacrylamide separating gel with a 5% stacking gel. Proteins were transferred to nitrocellulose membranes (0.45 μm, GE Healthcare). After overnight incubation in blocking solution (5% milk powder), membranes were incubated with the respective antibody: ITGAV (cat. sc-376,156, Santa Cruz), ITGB1 (cat sc-374,429, Santa Cruz), ITGB6 (cat. c-293,194, Santa Cruz) and a cterminal SMAD 4 antibody (cat. ab217267, Abcam). HSC70 (cat. Sc-7298, Santa Cruz) was used as intrinsic control. Goat anti-Mouse IgG (cat. P0447, Dako) or, respectively, Goat anti-rabbit IgG (cat. Sc-2054, Santa Cruz) were used as a secondary antibodies.",
"RNA was extracted (miRNeasy Mini Kit, Qiagen) and concentrations were determined (NanoDrop). The RNA integrity number was higher than 7 for all samples (Agilent Bioanalyzer 2100 system). Expression analysis was performed using the GeneChip Human Transcriptome Array 2.0 (Thermo Fisher Scientific). For cDNA synthesis, labeling and hybridization, the GeneChip WT PLUS Reagent Kit and GeneChip Hybridization, Wash, and Stain Kit (Thermo Fisher Scientific) were used.",
"Sections of formalin-fixed, paraffin-embedded tumors were used. Briefly, after antigen retrieval, the following primary antibodies were used: ITGAV (sc-376,156, Santa Cruz,); ITGB1 (AB3167, Abcam), ITGB6 (HPA023626, Atlas), CEACAM7 (LS-B13068, LSBio), TGFBR1 (PA5-98192, Thermo Scientific), phospho-SMAD2 (AB3849, Millipore), TGFBI (MA526731, Thermo Fisher Scientific), Twist (ab50581, Abcam), Ki 67 (M7240, Agilent); human-specific fibronectin (NCL-FIB, Leica Biosystems); fibronectin, cross-reacting with the fibronectinequivalent protein in mouse (A0245, Dako); STAT1 (HPA000931, Sigma-Aldrich), HLA-DR (M0746, Agilent). Vectastain ABC kit (Vector) was used. Slides were scanned by Axio Scan Z1 (Zeiss). For quantification with Image J (NIH), images of four randomly chosen vision fields (not containing necrotic areas) were analyzed. If artifacts interfered with automated quantification by Image J, the staining was quantified by two experienced investigators: grade 0: no reaction or weak focal reaction; grade 1 intense focal or diffuse weak reaction; grade 2 moderate diffuse reaction; grade 3 for an intense diffuse reaction.",
"Two hundred nine patients with PDA who underwent pancreaticoduodenectomy at the University Medical Centre Hamburg-Eppendorf between 2000 and 2012 were included. Written informed consent was obtained from all participants. The study protocol was approved by the Hamburg Medical Chamber's ethics committee (Approval number: PV3548).",
"Tissue microarray (TMA) construction and analysis TMA construction was performed as previously described [18]. Only punches with clearly detectable tumor tissue were included. The staining intensity and quantity of the tumor cells of each tissue spot were scored. To analyze the phospho-SMAD2 staining, all cell nuclei in a visual field were counted and the percentage of phospho-SMAD2 -positive cell nuclei was determined. If this was more than 75%, the tissue sample was classified as high. Of the initial 209 samples, ITGAV could be analyzed in 183, pSMAD2 in 180, STAT1 in 160, and HLA-DR in 192 samples. Immunohistochemical analysis of the sections was performed without knowledge of the patients' identity or clinical status.",
"GraphPad Prism was used for in vitro and in vivo statistical calculations (Student's t-test, Kaplan-Meier method, Log-rank test). To analyze the TMA, SPSS was used: Relationships between categorical variables were calculated using chi-square tests. Survival curves were plotted using the Kaplan-Meier method and analyzed by the log-rank test. For data obtained by gene expression array, statistical analysis was performed using Transcriptome Analysis Console (4.0.0.25) with summation method Gene + Exon -SST-RMA (version 1) and empirical Bayes method. Calculated p-values were corrected for multiple testing using the Benjamini-Hochberg (FDR) procedure. The sample sets with expression fold changes of at least +/− 1.5 and with an FDR p-value < 0.05 were considered significantly differentially expressed. Further analysis was performed with R 3.5.1 programming language with IDE RStudio 1.1. The heat maps were constructed with the \"heatmap.2\" function from \"gplots\" package.",
"The gene expression profiles of tumors that had developed in the host animals despite selectin deficiency were compared with the gene expression profiles in tumors grown in wild-type Pfp −/− /Rag2 −/− mice: Integrins β1 (1.56-fold), α2 (1.67-fold), β6 (1.69-fold) and αV (1.70-fold) were found to be upregulated in the tumors of the selectin deficient animals. We selected integrin alpha-V (ITGAV) -which displayed the highest upregulation -to further study integrin influence in PDA. We confirmed the upregulation of ITGAV in xenograft carcinomatoses using immunohistochemistry (Fig. 1). We then generated a subline of the PaCa 5061 cell line with stable knockdown of ITGAV (shRNA), termed PaCa 5061 ITGAV KD and a respective control subline with unchanged ITGAV designated as PaCa 5061 control. The expression of ITGAV was reduced by > 65% in PaCa 5061 as determined by flow cytometry and western blot (Fig. 2a).",
"To answer the question of whether ITGAV on the tumor cells alone or in combination with E-and Pselectins in the tumor environment has a functional effect on intraperitoneal carcinomatosis formation, PaCa 5061 ITGAV KD and PaCa 5061 control cells were intraperitoneally injected into selectin-deficient and wildtype Pfp −/− /Rag2 −/− mice (12 animals each group for the selectin-deficient mice, 14 and 15 selectin-competent animals for the control PaCa 5061 and the ITGAV KD group, respectively). After a growth period of 62 days, the ITGAV KD alone almost completely abolished intraperitoneal carcinomatosis in wild-type mice. Only 7 of 15 mice (47%) showed any sign of tumor development (small tumors at the injection site with only one animal displaying macroscopically visible intraperitoneal carcinomatosis (with a minimal PCI of 1.) In the experimental group with control PaCa 5061 cells injected into selectin-deficient mice, 11 of 12 mice showed a tumor take (92%), with all of them displaying intraperitoneal carcinomatoses (mean PCI of 12.92; P < 0.001, Fig. 3a). After 62 d, only 2 of 12 selectin k.o. mice (17%) injected with ITGAV KD cells displayed any small tumors at the injection site and no carcinomatosis was observed. Due to this, the presumed synergistic effect of the selectin Of the latter, half of the mice developed no peritoneal carcinomatosis, while in the remaining half, only minimal peritoneal carcinomatosis was observed, resulting in a mean PCI of 0.67. In contrast, injection of ITGAV KD cells into wild-type mice led to a mean PCI of 3.13 after 77 days, with at least small macroscopically visible tumor masses being present in all eight animals with tumor take. As mentioned above, PaCa 5061 cells formed subcutaneous primary tumors in and around the injection channel after intraperitoneal injection: tumor weight in the group of wild-type mice with ITGAV KD cells was significantly reduced (mean of 116 mg, P = 0.003) and a synergistic effect could be observed for the selectindeficient animals with ITGAV KD cells. Here, tumor weights of the injection channel tumors (mean of 22 mg, P = 0.017) were again significantly reduced compared with their wild-type counterparts (Fig. 3b).",
"Due to the different sizes of injection-site tumors of ITGAV KD and control cells in selectin-deficient and wild-type mice, we expected a functional effect of ITGAV on primary tumor development. To verify this observation, we performed an additional subcutaneous xenograft experiment with ITGAV KD and control cells in selectin wild-type mice. In other cancer entities, it could be shown that ITGAV activates latent TGF-β in the extracellular matrix (ECM) [19]. As SMAD4 is part of the transforming growth factor-β (TGF-β) signaling pathway and is inactivated in 50% of PDA patients [20] resulting in dysfunctional canonical TGF-β signaling in the corresponding tumorswe additionally included the cell line BxPC3 with SMAD4 deletion as a model cell line for this patient subset. SMAD4 deletion was confirmed by absent SMAD4 protein levels in the case of BxPC3 (Fig. 3d). Again, sublines with and without stable knockdown of ITGAV, designated BxPC3 ITGAV KD and BxPC3 control, were generated. The expression of ITGAV was reduced by > 65% in the KD cell line compared to the expression levels of the control cells (Fig. 2).",
"After subcutaneous tumor cell injection (10 pfp − − /rag2 −− mice each s.c. injected with control and ITGAV KD PaCa 5061, respectively), 9 of 10 animals (90%) injected with control cells and 7 of 10 (70%) mice injected with ITGAV KD cells developed s.c. tumors until the experiment was terminated after 199 days. Mice inoculated with PaCa 5061 ITGAV KD cells showed a significantly prolonged overall survival until reaching the termination criteria (median survival 198.5 vs. 115 d for the control cells, P = 0.01, Fig. 3e). Similar results were found for BxPC3 (median survival 74 vs. 99 d, P < 0.001, Fig. 3h). All animals injected with BxPC3 cells (11 with control and 10 with ITGAV KD) developed s.c. tumors, except one animal from the ITGAV KD group, found dead 1 week after injection. It showed no sign of tumor development and was thus excluded from all further analyses. As tumor size and weight at the time of death did not differ significantly between the ITGAV and control groups for both cell lines, a significantly reduced growth of the ITGAV KD tumors can be assumed for PaCa 5061 and BxPC3 cells.",
"To analyze the influence of ITGAV knockdown on hematogenous metastasis, tumor cells in the animals' lungs and blood were quantified for all mice by qRT-PCR. The PaCa 5061 ITGAV KD (P = 0.045, Fig. 3f) and BxPC3 ITGAV KD (P = 0.026, Fig. 3i) groups showed significantly less human DNA in the lungs, reflecting significantly fewer PDA cells than the control group. The numbers of circulating tumor cells in the animals' peripheral blood decreased due to the ITGAV KD, albeit not reaching statistical significance ( Fig. 3g and j).",
"(See figure on previous page.) Fig. 3 Knockdown of ITGAV in PaCa 5061 led to an effective reduction of intraperitoneal carcinomatosis after 62 d compared with control PaCa 5061 in E−/P-selectin deficient mice: Only 1/15 animals of the PaCa 5061 ITGAV KD group in wild-type mice showed carcinomatosis formation vs. 11/12 mice with carcinomatoses in the control group (P < 0.0001, a). ITGAV knockdown also reduced tumor growth at the injection site (P < 0.0001, B). 7/15 (ITGAV KD cells in E−/P-selectin k.o. mice) displayed tumors at the injection site vs. 11/12 in the control group (control PaCa 5061 in E−/P-selectin deficient mice). Combination of ITGAV KD and selectin knockout led to further reduction of injection site tumors (P = 0.012; only 2/14 animals with tumors, b). After 77 days, this synergistic effect of ITGAV and E−/P-selectins could also be demonstrated for intraperitoneal carcinomatosis formation (P = 0.017, C; 8/13 mice with tumor take with all eight displaying carcinomatosis with ITGAV KD in wild-type mice vs. 6/ 14 mice with tumor take and 3 of them displaying carcinomatosis with ITGAV KD in E−/P-selectin deficient animals). Using western blot, the Cterminal deletion of SMAD 4 in BxPC3 cells was confirmed (d). Wild-type mice inoculated subcutaneously with PaCa 5061 ITGAV KD cells showed significantly prolonged survival (tumor take rate 7/10) compared with control cells in wild-type animals (take rate 9/10; P = 0.002, e). Similar results were found for BxPC3 (100% take rate, both groups; P < 0.001, 2H). The number of human cells in the animals' lungs was significantly reduced in mice injected with PaCa 5061 (P = 0.045, f) and BxPC3 ITGAV KD cells (P = 0.045, I). There was no significant difference for the PaCa5061 (g) or BxPC3 tumor cells circulating in the animals' blood (j) The possible integrin-beta subunits corresponding with ITGAV (ITGB1, ITGB3, ITGB5, ITGB6 and ITGB8) were determined in vitro using flow cytometry. Of these, only ITGB1 and ITGB6 were relevantly expressed in PaCa5061 and BxPC3 cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells in vitro (Fig. 2b). In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchanged (Fig. 2b). These changes were confirmed for the xenograft tumors by western blot (Fig. 2c) and immunohistochemistry (Fig. 6).",
"To elucidate whether ITGAV activates TGF-β1 in vitro, an ELISA was used to determine the concentration of active and latent TGF-β1 in supernatants from cell culture medium. The concentration of active TGF-β1 decreased by 57% to 5.11 pg/mL (P = 0.005, Fig. 4a), whereas the concentration of total TGF-β1 did not differ significantly. Obviously, ITGAV activates latent TGF-β1 for PaCa 5061 cells in vitro.",
"To characterize the functional effects of ITGAV KD in vitro, proliferation, adhesion, migration, invasion and colony-forming assays were performed. No significant differences were detected in cell proliferation between knockdown and control cells in vitro as determined by XTT assays (Fig. 4b). In line with this finding, in vivo proliferation rates analyzed by determining the Ki-67 staining index of vital xenograft tumor tissue revealed no significant differences between the groups (Fig. 6).",
"The basal membrane / extracellular Matrix (ECM) lies open between the peritoneal cells [21], which might enable direct contact between ITGAV on the intraperitoneal tumor cells and its ligands such as fibronectin. Adhesion of ITGAV KD cells on fibronectin was analyzed under static conditions: PaCa 5061 cells with ITGAV KD showed a distinctly reduced number of adhering cells, with 11% compared with 39% for the controls (P = 0.006). Likewise, a reduction in the adhesive potential of BxPC3 cells could be demonstrated (P = 0.025, Fig. 4c).",
"The proportion of migrating tumor cells was reduced for both cell lines upon ITGAV KD (P < 0.001, Fig. 4d). Moreover, BxPC3 ITGAV knockdown cells showed a reduced invasive potential compared with the control cells (P = 0.048). PaCa 5061 cells were not able to pass the Matrigel layer at all (Fig. 4e). In line with the invasion assay finding, BxPC3 ITGAV KD cells formed fewer spheroid colonies per well than the control cells (P = 0.0134, Fig. 4f). Representative images of colonies formed by BxPC3 control and BxPC3 ITGAV KD cells are shown in Supplementary Fig. 1. PaCa 5061 cells were not able to form colonies in Matrigel after 14 days.",
"To gain insight into how the ITGAV knockdown might cause the reduction in tumor growth and metastasis/carcinomatosis formation observed in vivo, we performed whole human genome expression analyses. Genes of interest were defined and categorized into groups according to their functional context (Table 1, Fig. 5). Differences in selected proteins were validated by immunohistochemistry (Fig. 6).",
"Starting with the SMAD4-intact PaCa 5061 cells, changes regarding the TGF-β signaling are compiled first: The non-signaling reservoir TGF-β receptor 3 (TGFBR3) was found to be downregulated in the ITGAV KD tumors. In contrast, the expression of TGF-β2 (activation not dependent on ITGAV) and TGF-βR1 (receptor for TGF-β1, 2 and 3) were upregulated. Expression of the fibronectin-anchored, latent-transforming growth factor beta-binding protein 1 (LTBP1), which is essential for activating TGF-β3 with integrin αvβ6 [22], was downregulated in PaCa 5061 ITGAV KD tumors. Regarding the regulation of the SMAD2/3:SMAD4 complex, expression levels of several involved genes were significantly regulated, including E2F4, PPM1A, SERP INE1, SKIL and STUB1. To confirm the changed activity of TGF-β activation, staining for phospho-SMAD2 was performed (Fig. 6): The proportion of phospho-SMAD2positive tumor cell nuclei was significantly higher in the PaCa 5061 control group compared with the ITGAV KD group (P = 0.03, Fig. 6b). Besides, transforming growth factor beta-induced (TGFBI), located in the ECM and associated with poor survival [23], is downregulated as a result of the ITGAV knockdown, which was again immunohistochemically confirmed (Fig. 6b).",
"Secondly, changes regarding epithelial-mesenchymal transition (EMT) were as follows: Weaker intranuclear staining signals for Twist, a master regulator of EMT, which is controlled by TGF-β1 [24], was observed in PaCa 5061 ITGAV KD tumors (Fig. 6b). In line with the KD cells' more epithelial phenotype, Occludin was found to be 17.35-fold upregulated in the PaCa 5061 ITGAV KD tumors; accordingly, E-cadherin staining signals were enhanced in the ITGAV KD tumors (Fig. 6b). CD44, which correlated with EMT and poor survival in a clinical study of PDA [25], was downregulated 8.94fold upon knockdown of ITGAV. The secretion of matrix proteins and proteases was also found to be modulated to favor tumor progression in PDA [19]. Exemplarily, MMP-10 was decreased upon knockdown of ITGAV accompanied by increased stromal (murine) fibronectin: Staining for both human and murine fibronectin using a cross-reactive antibody revealed a moderate signal in the ITGAV knockdown groups, whereas the control groups were predominantly only weakly stained for fibronectin (Fig. 6). In contrast, specific staining for human fibronectin revealed no differences between the two groups, indicating that the stromal fibronectin was deposited by murine stromal cells and not by human cancer cells. Thirdly, changes regarding the immune status were as follows: MHC class II molecules, part of the human leukocyte antigen (HLA) system, were upregulated in the ITGAV KD xenograft tumors (i.e. HLA-DRA, HLA-DRB1, HLA-DRB4, HLA-DRB5, HLA-DPA1 and HLA-DQB2). Exemplarily, we verified the upregulation of HLA-DR by immunohistochemistry (Fig. 6). Additionally, the upregulation of HLA-DR on PaCa 5061 ITGAV KD cells was also confirmed in vitro using flow Fig. 2). Increased JAK-STAT signaling leads to increased expression of MHC class II genes [26] and, indeed, both JAK1 and STAT1 were found to be increased in the ITGAV KD xenografts. Using immunohistochemistry, an increased intranuclear STAT1 signal could also be confirmed in the corresponding ITGAV KD xenograft tumors at the protein level (Fig. 6).",
"When gene expression profiles of the SMAD4deficient BxPC3 xenografts with knockdown of ITGAV were analyzed, no genes associated with TGF-β signaling, EMT or immune status were found to be significantly regulated. We attribute this gene expression pattern to the malfunction of the canonical TGF-β signaling pathway caused by the confirmed deletion of SMAD4 (Fig. 3d). Table 1). E.g., the adhesion molecule CEAC AM7 was significantly upregulated in both models, as demonstrated by immunohistochemistry (Fig. 6).",
"A high level of ITGAV expression in the PDA cells was found in 129 tissue samples (71%). These patients showed a significantly poorer outcome with a median survival of 11 (95% CI 8.6-13.3) months instead of 21 (95% CI 15.5-26.5) months in the group of patients with low ITGAV expression (P = 0.004, Fig. 7a). For 125 tissue samples (69%), a high percentage of pSMAD2positive tumor cell nuclei was identified and ITGAV and phospho-SMAD2 staining intensities were positively correlated (Supplementary Table 2, P = 0.014). However, there was no significant correlation for phospho-SMAD2 with survival (P = 0.127). Ninety three tissue samples (58%) displayed a high expression of STAT1. No significant association existed between STAT1 and survival (P = 0.310). High expression of HLA-DR was found in 22 tumor samples. Of these 22 tumors, 17 were also STAT1 positive. Hence, a positive correlation between STAT1 and HLA-DR expression was observed (Supplementary Table 2, P = 0.019). This observation supports the connection between JAK-STAT signaling and the expression of HLA-DR. In our cohort, a better survival could be demonstrated for patients with high expression of HLA-DR by tumor cells with a median survival of 35 (95% CI 0.6-69.4) months compared with 15 (95% CI 12.9-17.1) months for patients with low HLA-DR expression (Fig. 7b). None of the staining results showed any correlation with gender, grading or tumor stage (Supplementary Table 2).",
"In the present study, we could show that ITGAV expression was upregulated in intraperitoneal pancreatic ductal adenocarcinoma xenograft tumor cells that grew under selectin-deficient conditions, suggesting a compensatory increase in integrin expression. We could demonstrate that the knockdown of ITGAV led to a massive reduction in intraperitoneal carcinomatosis, primary tumor growth and pulmonary metastasis. Even more importantly, the effects of the absence of host selectins and ITGAV were synergistic, as clearly demonstrated in our intraperitoneal tumor model. This is the first model to describe such a synergism experimentally. These findings validate the hypothesis that cancer cell adhesion to mesothelial cells follows similar mechanisms as the leukocyte adhesion cascade, which is initiated by selectins followed by integrins [27]. Integrin-mediated cell adhesion also seems to be essential in compensating for the absence of selectin expression [28]. The observed increased expression in other cell adhesion molecules in the ITGAV KD tumors (e.g. the adhesion molecule CEACAM-7) might partly compensate for the reduction of ITGAV. Our findings indicate that the adhesion to the peritoneal mesothelium or its underlying basal lamina is the rate-limiting step of peritoneal carcinomatosis formation in PDA. Fortunately, this step is particularly amenable to therapeutic invention as the molecules involved are located on the cell surface. Therefore, blockage of both selectins and integrins is an attractive option to inhibit peritoneal metastasis in PDA. A body of literature suggests that ITGAV activates latent TGF-β in the ECM [19,29]. We could demonstrate this for the SMAD4-intact PaCa 5061 cells, for which the concentration of active TGF-β1 is reduced due to the knockdown of ITGAV in vitro. Furthermore, the knockdown of ITGAV reduced the activity of phospho-SMAD2 in the xenograft tumors of the SMAD4-intact PaCa 5061 cells in vivo. Our gene expression analyses confirmed altered TGF-β-signaling due to the knockdown of ITGAV in PaCa 5061 tumors, as described above. Moreover, there is an association between ITGAV and pSMAD2-positive tumors in the analyzed patient samples: high expression of ITGAV is connected with poorer survival. This observation corroborates data in the GEPIA2 webserver ( Supplementary Fig. 3, [30]) and Human Protein Atlas [31], which show a correlation between ITGAV mRNA expression level and poor patient survival. Moreover, a recent study demonstrated that elevated serum soluble TGF-β predicted poor survival in PDA [32].",
"At the beginning of the tumorigenic process, TGF-β1 functions as a tumor suppressor due to its ability to suppress cell division in epithelial cells [33]. Hezel et al. used a genetically engineered mouse model with Kras G12D -initiated, SMAD4-deficient murine PDAs reflecting an early disease state and identified increased tumor cell proliferation through the blockade of integrin αvβ6 [34]. The cell lines used in our study originate from locally advanced human tumors, as commonly present at the time of diagnosis. Our proliferation assay in vitro and the determination of the Ki-67 Labeling Index in the xenograft tumors show no changes in cell proliferation (Fig. 5b), suggesting that the examined cell lines are already resistant to the anti-proliferative effects of TGF-β.",
"Bates et al. reported that a high level of integrin αvβ6 is accompanied by a poorer overall survival for colon carcinoma. They assumed the reason for this to be the integrin αvβ6-dependent activation of TGF-β1 and the resulting stimulation of EMT [35]. In PDA, high integrin αvβ6 mRNA levels were associated with shortened patient survival and antibody therapy directed against this dimer suppressed the pro-tumorogenic microenvironment (e.g. by suppression of TGF-β signaling) in mouse models [7]. Besides the critical alterations induced by ITGAV in TGF-β signaling for SMAD4 intact PDA (represented by PaCa 5061 in this study), the xenograft model with the SMAD4 dysfunctional BxPC3 cells demonstrates that there are also TGF-β independent effects of ITGAV in PDA. The TMA data further corroborate this observation: Although Tumors were not stratified for functional SMAD4, ITGAV expression was still prognostic in the overall cohort. Together with the fact that half of all PDA display deletions / inactivating mutations of SMAD4 [20], this indicates that ITGAV is prognostic for SMAD4 functional as well as dysfunctional tumors. In prostate cancer, for example, AKT activation has been described as an additional mechanism for ITGAV involvement in a recent study [4]. Our study shows that the proportion of Twist-positive cells decreases through the knockdown of ITGAV in the SMAD4-intact PaCa 5061 cells. Twist controls the expression of epithelial gene signatures such as Occludin and E-cadherin [36] and indeed, expression of these epithelial genes increased upon ITGAV KD. In pharyngeal carcinoma, Van Aarsen et al. demonstrated that blocking with an integrin αvβ6 antibody reduced TGF-β-induced SMAD2 phosphorylation, which resulted in diminished tumor growth and reduced invasive potential while an impact of the treatment on cell proliferation was not observed [37]. These effects were confirmed for PDA in our study.",
"In vivo, TFG-β1 is a potent activator of the transdifferentiation of fibroblasts into myofibroblasts, which are referred to as cancer-associated fibroblasts (CAFs) [38]. The increase in the ECM contraction caused by CAFs increases the probability of TGF-β activation [38]. Interestingly, it has been shown that the heterogeneity of CAFs in PDA is mediated by JAK/STAT signaling antagonized by TGF-β [39]. CAFs themselves interact with integrins and secrete a large number of proteins into the ECM matrix, proteases and cytokines, which can further increase cancer progression [40]. For example, CAFs deposit fibronectin in the ECM, thereby creating promigratory tracks [41]. It is assumed that CAFs themselves express ITGAV and hence can also activate latent TGF-β [42]. Consistently, we found an upregulation of murine fibronectin in the xenograft tumors with ITGAV KD (see Fig. 5a and e).",
"Highly interesting is the immunomodulatory effect of the ITGAV knockdown found in our study, especially since results for immunotherapy of PDA [43] must be considered disappointing so far. To our knowledge, the observed changes in MHC-II expression by PDA have not been described before. However, to fully investigate the implications of these alterations, a suitable new in vivo model with an intact adaptive immune system would be needed, e.g. a syngenic model. As MHC-II is required for CD4+ T-cell activation, which also plays essential roles in antitumor immunity [44], further investigations in this field could prove highly rewarding.",
"The ITGAV knockdown of PDA massively suppressed the intraperitoneal carcinomatosis of these cells. Moreover, the effects of the absence of selectins and reduced expression of ITGAV on intraperitoneal carcinomatosis are synergistic, confirming the hypothesis of a multistep and partly redundant leukocyte adhesion cascade as the rate-limiting step within the metastatic cascade. Mechanistically, ITGAV activates TGF-β and drives epithelialmesenchymal transition PDA cells. Specific inhibition of ITGAV may have the potential to impede intraperitoneal carcinomatosis, tumor growth and distant metastasis. "
] | [] | [
"Background",
"Methods",
"Cell lines and RNA interference-mediated ITGAV knockdown",
"Flow cytometry",
"Enzyme-linked immunosorbent assay (ELISA)",
"In vitro characterization of ITGAV knockdown cells",
"Quantitative real-time polymerase chain reaction (qRT-PCR)",
"Western blot",
"Affymetrix gene arrays",
"Immunohistochemical analysis",
"Study population",
"Data analyses",
"Results",
"Integrin expression was upregulated in intraperitoneal carcinomatosis grown in selectin-deficient mice",
"ITGAV KD massively suppressed intraperitoneal carcinomatosis and reduced primary tumor development and distant metastasis in SMAD4-intact PaCa 5061 cells",
"Effects of ITGAV knockdown in PaCa 5061 und BxPC3 cells in vitro",
"Gene expression and immunohistochemical analysis of the primary tumors",
"Clinical proof of concept using a tissue microarray",
"Discussion",
"Conclusions",
"Fig. 1",
"Fig. 3 (",
"Fig. 4",
"Fig. 5",
"Fig. 7 (",
"(",
"Table 1",
"Table 1"
] | [
"Flow cytometry \nWestern blot \nFlow cytometry \nWestern blot \n\nITGAV \n\nITGB1 \n5% \n\nITGB6 \n\nB \n\nPaCa5061 \nBxPC3 \n\nC \n\nPaCa5061 \nBxPC3 \n\nControl ITGAV KD Control ITGAV KD \n\nSignal \nITGAV KD: 98.02 \nControl: \n229.52 \n\nITGAV \n\nITGB1 \n\nITGB6 \n\nSignal \nITGAV KD: 93.17 \nControl: \n151.66 \n\nSignal \nITGAV KD: 831.15 \nControl: \n1152.14 \n\nSignal \nITGAV KD: 985.50 \nControl: \n1000.50 \n\nSignal \nITGAV KD: 104.82 \nControl: \n130.17 \n\nSignal \nITGAV KD: 56.15 \nControl: \n61.90 \n\nITGAV \n\nITGB1 \n\nITGB6 \n\nHSC-70 \n\nA \n\n70% \n90% \n66% \n87 % \n\n31% \n19% \n16% \n\n73% \n51% \n69% \n21 \n\n0.0 \n\n0.2 \n\n0.4 \n\n0.6 \n\n0.8 \n\n1.0 \n\nP = 0.012 \n\nP < 0.0001 \n\nPaca 5061 \ncontrol in \nselectin deficient \n\nPaca 5061 \nITGAV KD in \nselectin deficient \n\nPaca 5061 \nITGAV KD in \nw ildtype \n\ntumor weight injection site (g) \n\nHSC70 \ncontrol \n\nSMAD4 \nC-Terminus \n\nC \n\nterminated after 77 d \n\nD \n\nG \n\nPaCa 5061 BxPC3 \n\nB \n\nterminated after 62 d \n\nA \n\nterminated after 62 d \n\nH \nI \nJ \n\nE \nF \n\n",
"Gene Symbol \nFold Change \nP \nFDR \nadj. P \n\nTGF-β signaling \n\nCDK8 \n1.8 \n0.007 \n0.108 \n\nE2F4 \n−2.3 \n< 0.001 \n0.007 \n\nLTBP1 \n− 2.61 \n0.0003 \n0.0201 \n\nNEDD4L \n−1.3 \n0.096 \n0.462 \n\nPARD3 \n− 2.43 \n< 0.001 \n0.001 \n\nPPM1A \n2.42 \n0.002 \n0.051 \n\nPPP1CA \n−1.55 \n< 0.001 \n0.013 \n\nPPP1CB \n2.59 \n< 0.001 \n0.003 \n\nRAP1B \n1.5 \n0.0064 \n0.1065 \n\nSERPINE1 \n−2.83 \n0.004 \n0.077 \n\nSKIL \n2.92 \n0.002 \n0.051 \n\nSMAD1 \n−1.18 \n0.310 \n0.733 \n\nSMAD2 \n1.09 \n0.809 \n0.958 \n\nSMAD3 \n1.79 \n0.273 \n0.704 \n\nSMAD4 \n−1.08 \n0.917 \n0.982 \n\nSMAD6 \n−1.13 \n0.289 \n0.717 \n\nSMAD7 \n1.06 \n0.505 \n0.851 \n\nSMAD9 \n−1.01 \n0.595 \n0.887 \n\nSTUB1 \n−1.84 \n< 0.001 \n0.019 \n\nTGFB1 \n−1.59 \n0.014 \n0.170 \n\nTGFB2 \n3.61 \n0.003 \n0.071 \n\nTGFBI \n−0.11 \n0.065 \n0.384 \n\nTGFBR1 \n1.68 \n< 0.001 \n0.015 \n\nTGFBR2 \n1.22 \n0.971 \n0.994 \n\nTGFBR3 \n−2.75 \n0.005 \n0.090 \n\nEpithelial-mesenchymal transition \n\nACTA2 \n−1.04 \n0.6051 \n0.8912 \n\nADAM10 \n2.26 \n0.003 \n0.063 \n\nADAM12 \n1.01 \n0.897 \n0.979 \n\nBSG \n−2.65 \n< 0.001 \n0.002 \n\nCDH1 \n−1.21 \n0.3769 \n0.7813 \n\nCDH2 \n−5.04 \n< 0.001 \n< 0.001 \n\nFGFBP1 \n−1.86 \n0.007 \n0.107 \n\nFLG \n265.8 \n< 0.001 \n0.002 \n\nFN1 \n−2.57 \n0.002 \n0.048 \n\nHSPG2 \n−1.78 \n0.001 \n0.039 \n\nJAG1 \n−3.05 \n< 0.001 \n0.005 \n\nKLK13 \n2.06 \n0.001 \n0.043 \n\nKLK7 \n3.18 \n0.008 \n0.125 \n\nLAMA2 \n−1.61 \n0.001 \n0.028 \n\nLAMA3 \n−6.57 \n< 0.001 \n0.003 \n\nLAMA5 \n−1.74 \n0.072 \n0.404 \n",
"Gene Symbol \nFold Change \nP \nFDR \nadj. P \n\nHLA-DMB \n1.41 \n0.009 \n0.129 \n\nHLA-DOA \n1.26 \n0.009 \n0.129 \n\nHLA-DOB \n1.09 \n0.437 \n0.817 \n\nHLA-DPA1 \n3.32 \n< 0.001 \n0.005 \n\nHLA-DPB1 \n1.4 \n0.019 \n0.197 \n\nHLA-DPB2 \n1.21 \n0.031 \n0.257 \n\nHLA-DQA1 \n−1.14 \n0.294 \n0.721 \n\nHLA-DQA2 \n1.77 \n0.008 \n0.123 \n\nHLA-DQB1 \n−1.14 \n0.492 \n0.845 \n\nHLA-DQB2 \n1.41 \n0.003 \n0.065 \n\nHLA-DRA \n14.81 \n< 0.001 \n0.004 \n\nHLA-DRB1 \n19.95 \n0.001 \n0.025 \n\nHLA-DRB4 \n12.38 \n< 0.001 \n0.005 \n\nHLA-DRB5 \n3.45 \n< 0.001 \n0.002 \n\nHLA-E \n1.17 \n0.199 \n0.628 \n\nHLA-G \n1.64 \n0.048 \n0.328 \n\nHLA-H \n1.13 \n0.298 \n0.724 \n\nICAM1 \n1.71 \n0.065 \n0.385 \n\nIFNG \n−1.07 \n0.179 \n0.603 \n\nIRF1 \n1.37 \n0.005 \n0.094 \n\nIRF2 \n− 1.56 \n0.014 \n0.170 \n\nJAK1 \n1.82 \n< 0.001 \n0.015 \n\nKIF3B \n−1.73 \n0.004 \n0.076 \n\nLMO7 \n5.19 \n< 0.001 \n0.005 \n\nMALT1 \n1.72 \n0.003 \n0.069 \n\nMID1 \n−3.3 \n< 0.001 \n0.007 \n\nMT2A \n−2.36 \n0.002 \n0.049 \n\nOAS1 \n1.78 \n0.014 \n0.169 \n\nOSBPL1A \n−2.52 \n< 0.001 \n0.010 \n\nPAK1 \n−2.66 \n< 0.001 \n0.004 \n\nPTAFR \n−2.09 \n0.006 \n0.102 \n\nRIPK2 \n2.56 \n0.020 \n0.205 \n\nSLCO4C1 \n−2.93 \n0.001 \n0.040 \n\nSTAT1 \n2.58 \n< 0.001 \n0.019 \n\nTIMP2 \n4.83 \n< 0.001 \n0.008 \n\nTRIM22 \n2.28 \n< 0.001 \n0.004 \n"
] | [
"The possible integrin-beta subunits corresponding with ITGAV (ITGB1, ITGB3, ITGB5, ITGB6 and ITGB8)",
"Table 1",
"Table 2",
"Table 2",
"Table 2"
] | [
"Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression",
"Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression"
] | [] |
25,168,281 | 2022-03-12T00:44:17Z | CCBYNCSA | null | null | 24de1f9dc9b5993cc0588f752f1aaff43f177df3 | null | null | null | null | null | 2410324918 | 26636277 | 5628591 |
Differential effects of low-magnitude high-frequency vibration on reloading hind-limb soleus and gastrocnemius medialis muscles in 28-day tail-suspended rats
K-T Sun
Department of Orthopaedics and Traumatology
The Chinese University of Hong Kong
Shatin
New Territories
Hong Kong SAR
K-S Leung
Department of Orthopaedics and Traumatology
The Chinese University of Hong Kong
Shatin
New Territories
Hong Kong SAR
P
M-F Siu
Department of Health Technology and Informatics
The Hong Kong Polytechnic University
Hung HomKowloon, Hong KongChina
L Qin
Department of Orthopaedics and Traumatology
The Chinese University of Hong Kong
Shatin
New Territories
Hong Kong SAR
The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System
The Chinese University of Hong Kong Shenzhen Research Institute
ShenzhenThe People's Republic of China
Wing-Hoi Cheung
Department of Orthopaedics and Traumatology
Clinical Science Building
Hong Kong SAR
5/FChina
The Chinese University of Hong Kong
Shatin, Hong KongNew TerritoriesChina
W-H Cheung
Department of Orthopaedics and Traumatology
The Chinese University of Hong Kong
Shatin
New Territories
Hong Kong SAR
The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System
The Chinese University of Hong Kong Shenzhen Research Institute
ShenzhenThe People's Republic of China
Wing-Hoi Cheung
Department of Orthopaedics and Traumatology
Clinical Science Building
Hong Kong SAR
5/FChina
The Chinese University of Hong Kong
Shatin, Hong KongNew TerritoriesChina
Differential effects of low-magnitude high-frequency vibration on reloading hind-limb soleus and gastrocnemius medialis muscles in 28-day tail-suspended rats
Edited by: J. Rittweger Accepted 10 July 2015Original Article Hylonome This study was funded by the General Research Fund (Ref: 469911) from the University Grants Committee, Corresponding author:VibrationSkeletal MuscleReloadingContractile FunctionFiber Type
Skeletal muscles are highly mechanical sensitive and show high plasticity to altered activity levels 1-4 . Muscle disuse (or unloading), as in astronauts and bed-rest patients, leads to muscle atrophy and functional loss 5-6 . When designing treatment options for disuse atrophy, one must keep in mind that disused muscles are more susceptible to damage than normal muscles and hence over-exerting a disuse muscle may cause further deterioration, rather than improvement of muscle function 7-8 .Despite the numerous investigations on muscle disuse, less information about reloading from disuse and the suitable intervention is available. Moreover, most existing animal studies were conducted in a relatively short, 14-day tail suspension (TS) model and soleus muscle was the major muscle of interest. The duration of unloading and muscles of interests in fact are critical factors that lead to different degree of muscle-dependent, progressive physiological changes. For instance, myofibrillar protein loss was more prominent and became stable after 28 days of unloading than 7 and 14 days protocol whereas soleus was more affected than plantaris and gastrocnemius muscle (Details see Review byThomason, 1990)[9][10][11]. A better understanding of the reloading effects on unloaded limb muscles will provide valuable information for determining safe and even customized intervention for the susceptible disused muscles during reloading.Low-magnitude high-frequency vibration (LMHFV) is a J Musculoskelet Neuronal Interact 2015; 15(4):316-324AbstractObjectives: Low-magnitude high-frequency vibration (LMHFV) was reported beneficial to muscle contractile functions in clinical and preclinical studies. This study aims to investigate the effects of LMHFV on myofibers, myogenic cells and functional properties of disused soleus (Sol) and gastrocnemius medialis (GM) during reloading. Methods: Sprague Dawley rats were hind-limb unloaded for 28 days and assigned to reloading control (Ctrl) or LMHFV group (Vib). Sol and GM of both groups were harvested for fiber typing, proliferating myogenic cell counting and in vitro functional assessment. Results: Myogenic cells proliferation was promoted by LMHFV in both Sol and GM (p<0.001 and p<0.05 respectively). Force generating capacity was not much affected (Vib=Ctrl, p>0.05) but fast-fiber favorable changes in fiber type switching (more type IIA but lower type I in Vib; p<0.05 and 0.01 respectively) and fiber hypertrophy (type I, Vib<Ctrl; p<0.01) were observed mainly in GM. Conclusion: LMHFV was not detrimental to reloading muscles but the outcomes were muscle dependent. The unique fiber type composition and anatomical differences between Sol and GM might render the differential muscle responses to LMHFV. Further investigations on myofibers type specific responses to different LMHFV regimes and myogenic cell interaction with associated myofiber were proposed.
Introduction non-invasive biophysical modality, and its beneficial effects on musculoskeletal system are widely reported in various preclinical and clinical studies. Clinical trials showed that LMHFV prevented muscle atrophy in bed-rest patients and improved muscle performance with high compliance in community elderly [12][13] . Preclinical work from Xie and his colleague suggested vibration stimulated mice lower limbs muscle to hypertrophy 14 . Promoting proliferation and differentiation of myogenic cells as well as down-regulating genes involved in atrophy pathway could be the possible mechanisms of the muscle improvement by vibration treatment [15][16] .
With the evidences of LMHFV effectiveness in muscle improvement, it was hypothesized that LMHFV improves the outcomes of reloading disused muscles. The objectives of the current study were to investigate the effects of LMFHV treatment on: 1) fiber morphology, 2) myogenic cells proliferation and, 3) contractile function of soleus (Sol) and gastrocnemius medialis (GM), two weight-bearing limb synergist muscles, during 21 days of reloading period. Given the lack of information about reloading from prolonged disuse, the current work would also demonstrate any differential outcomes of reloading on Sol and GM.
Materials and methods
Animal care and experimental design
A total of 48 6-month-old male Sprague-Dawley adult rats were obtained from the Laboratory Animal Service Centre of the Chinese University of Hong Kong. All animals were housed in temperature-controlled rooms with 12:12 hour darklight cycle. All procedures performed in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Ref: 10/093/MIS5).
Animals were hind-limb unloaded for 28 days individually based on Morey's tail suspension (TS) protocol 17 . Briefly, zincoxide plaster with a harness was wrapped around the tail and secured by surgical tapes. Animals were then suspended in head-down position at torso-to-ground angle of ≤30°, while hind-limbs were dangled down without any solid support from the tail-suspension cage. Free-cage movement, access to water and standard rat chow ad libitum with their forelimbs were allowed. The health status of the animals was monitored daily. Age-matched weight bearing rats (WB, n=6) were euthanized at the same time for TS model verification.
After 28 days of TS, part of the unloading rats were sacrificed immediately (without reloading) and served as control of unloading (TS, or referred as Day 0 baseline data, n=6). The remaining rats were reloaded by allowing free-cage movement by four limbs in standard rat cage independently. The reloading rats were randomly assigned to either reloading control (Ctrl) or reloading plus vibration (Vib). Animals in Vib received LMHFV (0.6g, 35Hz; g=gravitational acceleration) 20 min/day and 5 days/week. Animals were euthanized by overdosed pentobarbital 7, 14 and 21 days after reloading (n=6/treatment/timepoint) 18 . Left Sol and GM were freshly harvested, weighted and subjected to in vitro functional assess-ment; the contralateral muscles were snap-frozen in melting isopentane, embedded in OCT compound and stored at -80°C until cryosectioning.
Proliferative cell labeling
To label proliferative cells in reloading muscles, a time-released pellet of 5-bromo-2'-deoxyuridine (BrdU, nucleotide analog to thymidine) (Innovative Research of America, FL, USA) was implanted subcutaneously 14 days before each endpoint 19 . Briefly, the animal was first anesthetized by isoflurane and according to manufacturer's instructions, the neck was shaved and disinfected by alcohol before a 5 mm longitudinal incision was made. A BrdU pellet was then put into a pocket 20 mm beyond the incision site subcutaneously. For the rats euthanized at Day 7 post-TS, BrdU pellet was implanted when the rats were still tail-suspended (i.e. day 21 of TS).
Histology
Consecutive 7μm cross-sections of right Sol and GM muscles were cut using cryostat. ATPase staining conditioned at pH 4.6 at room temperature was performed to distinguish the three muscle fibers: type I (darkest), IIA (lightest) and IIB (intermediate), based on Hintz's protocol and images of section were captured under the light microscope (Leica DFC490, Leica Microsystems) 20 . The whole section of Sol and the core region in the proximal head of GM (with mixed fibers profile) were analyzed 21 . Three random fields were captured to analyze the effects of LMHFV on different fiber types. The fiber crosssectional area (FCSA) and the proportion (%) of fiber types I, IIA and IIB were measured with ImagePro Plus analysis software (v5.1.0.20, Media Cybernetics, MD, USA).
Immunohistochemistry
To identify proliferative myogenic cells and the associated fiber types in both Sol and GM, a BrdU/laminin double-staining protocol was performed on the ATPase stained cryosections as modified from Siu's protocol 19 . Primary antibodies included mouse anti-BrdU (1:100, Abcam) and rabbit anti-rat laminin (1:200, Abcam). Secondary antibodies included Alexa Fluor555-conjugated goat anti-mouse IgG (γ2a) secondary antibody (Zymed) and Alexa Fluor488-conjugated donkey antirabbit IgG(H+L) antibody working concentration at 4 μg/ml. BrdU-positive nuclei lying on the laminin-stained basement membrane were counted and considered as proliferative myogenic cells. Immunofluorescent images obtained were co-localized with ATPase staining to identify the fiber types that the proliferative myogenic cells associated with.
In vitro muscle functional assessment
The protocols for muscle functional assessment were modified from Plant and Segal's studies [22][23] . The distal tendon of muscle was sutured and hung onto the transducer (305C-LR, Aurora Scientific Inc.) with proximal end anchored to the in vitro functional test apparatus (805A, Aurora Scientific Inc., Ontario, Canada). Muscles were stimulated to contract by square-wave pulses (0.2 ms width) at supramaximal voltage (80V). The detected forces were recorded by Dynamic Muscle Control (DMC v5.1) and analyzed with Dynamic Muscle Analysis (DMA v3.2) software (Aurora Scientific Inc.). All muscles were isometrically stimulated at optimal length (Lo) at room temperature (around 25℃ in air-conditioned laboratory) which was determined by eliciting isometric twitch with increasing muscle length until maximal force was generated 22 .
For Sol, force-frequency relationship was determined by stimulating the muscle for 1s at 10, 20, 40, 60, 80 and 100 Hz with 5-minute resting intervals. Maximal tetanic force of Sol was defined as the largest force obtained from the force-frequency relationship. For GM, continuous tetanic stimulation was unfavorable due to potential core anoxia induced by poor oxygen/nutrient perfusion to the bulky GM that deteriorated contractile function and would lead to severe force underestimation. Hence, isometric tetanic force of GM was achieved by delivering supramaximal stimulation, 500 ms, 150Hz at optimal length once only. All force measurement was normalized by physiological cross-sectional area (pCSA). It was estimated by pCSA= mass (g)/[muscle length (cm) x muscle density (gcm -3 )], where muscle density is assumed as 1.056 (gcm -3 ) 24 .
Statistical analysis
All data were expressed in mean ± standard deviation. Twoway analysis of variance (ANOVA) test was applied to analyze the main effects amongst treatment and reloading period. Posthoc multiple comparison corrected by Bonferroni adjustment was performed when significant main effects were detected. When significant interaction was detected, independent Student's t-test were used for further comparisons between Vib and Ctrl groups. All statistical analyses were performed with SPSS 20.0 (IBM, NY, USA). Statistical significance was set at p<0.05.
Results
TS model verification
The Sol muscle mass (Mm), pCSA and FCSA were significantly decreased by 33%, 31% and 40% respectively in TS group whereas in GM, these were decreased by 26%, 23% and 43% respectively (all p< 0.001; Table 1). Muscle fibers atrophy and slow-to-fast fiber type transition could be observed in both Sol and GM. Besides, increase in interstitial space in TS was observed ( Figure 1).
Muscle morphology
The morphological data of Sol and GM muscles in Ctrl and Vib groups at different reloading period were summarized in Tables 2 and 3 respectively. Reloading induced an increase in most of the morphological outcomes during reloading but differential responses to reloading and LMHFV treatment could be observed between Sol and GM. Particularly, decrease of Lo (~3%, p<0.05) during reloading compared with TS and smaller overall FCSA (Vib<Ctrl; p<0.01) in LMHFV group could only be observed in GM. Instead, Lo of Sol increased with reloading compared with TS (~7%, p<0.05) and LMHFV treatment did not demonstrate any effects on Sol overall FCSA (Vib=Ctrl; p>0.05). Other morphological parameters including muscle mass (~35%, p<0.001), pCSA (~28%, p<0.001) and FCSA (~27%, p<0.01) increased in Sol during reloading and
Fiber typing -FCSA and fiber proportion
The changes of FCSA of different fiber types (type I, IIA and IIB) in Sol and GM were illustrated in Figure 2a and 2b respectively. Fiber-type specific FCSA of Sol were similar and no difference (p>0.05) was detected during reloading (Figure 2a), whereas GM showed significant increase at Day 21 compared with Day 7 and Day 14 in type I (both p<0.01) and type IIB fibers (both p<0.001) (Figure 2b). Besides, Ctrl type I FCSA, but not type IIA or IIB, of GM was larger than that of Vib (p<0.01) and echoed with the overall FCSA decrease in Vib.
For the fiber type proportion, the percentage of all fiber types in Sol and GM were illustrated in Figures 3a and 3b respectively. Sol was made up of >90% type I fibers and increasing trend of type I percentage was observed during reloading (Day 21>Day 7, p=0.052). On the contrary, Sol type IIB percentage decreased from Day 14 to Day 21 significantly (p<0.05). Vibration treatment showed no effects on Sol fiber types switching.
Regarding GM fiber percentage, GM was composed of a mix of three fiber types with type IIB constituting ~50% of the total fibers in the core region of GM. Different from Sol, fiber type transition during reloading was absent in GM. Comparing Ctrl and Vib, there was a higher type I proportion in Ctrl (p<0.01; interaction with p<0.05) whereas type IIA was higher in Vib (p<0.05; treatment*timepoints interaction with p=0.05). Significant interactions were found in these two main effects i.e. reloading time and treatment. Therefore post-hoc test was carried out to compare the differences between Ctrl and Vib across timepoints. The post-hoc independent t-test analysis showed a higher type I proportion in Ctrl at Day 7 (Ctrl>Vib, p<0.01) whereas that for type IIA proportion was higher in Vib at Day 7 (Vib>Ctrl, p<0.05) and Day 21 (Vib>Ctrl, p=0.05).
Proliferating myogenic cell counting
The counting results were illustrated in Figure 4 (a-c). The Vib group generally showed higher counting in both Sol and GM. Overall counting, and fiber type specific counts in type I and IIA in Sol were higher in Vib than Ctrl (all p<0.001, Fig-ures 4a and 4b). Although overall counting showed no difference between Vib and Ctrl in GM, fiber type specific counting revealed higher counts in type IIB fiber of Vib group (p<0.05, Figure 4c). Significant treatment*timepoints interaction (p<0.001) was detected in this observable increase of myogenic cells proliferation in Vib during reloading. The post-hoc independent t-tests showed that the increase of type IIB count in Vib was at Day 14 (Day 14-Vib>Day 14-Ctrl, p<0.001) while the other two timepoints had no differences.
Moreover, the peak counting during reloading period was at Day 14 in all three fiber types. The counts actually covered all the proliferative activities happened from day 0 to day 14 of reloading (See Method for details). The time of peak activity was apparently independent of the fiber types, treatment and muscles of interest (except type IIA in Sol which the counts maintained high in Vib, see Figure 4b).
Contractile properties -specific force (sP o ) and time to peak twitch (TpT)
The contractile properties of Sol and GM were illustrated in Figure 5 (a-d). Increase in sP o during reloading was observed in Sol (Figure 5a; Day 21>TS and Day 7, both p<0.001 and Day 14, p<0.01) while force deficit, compared to TS level, was evident in GM throughout the reloading period in spite of the increasing trend from Day 14 to Day 21 (Figure 5b; p<0.05). LMHFV treatment appeared promoting force generating capacity in Sol as indicated from higher sP o in Vib compared with TS (Vib>TS, p<0.05) which was, however, absent in Ctrl (Figure 5a). GM sP o in Vib was not different from the Ctrl nor the TS. The contraction time, TpT of Sol appeared longer at Day 21 as compared with Day 14. Nevertheless, given a significant interaction detected (p<0.01), further post-hoc comparison showed that TpT of Ctrl-Day 21 was significantly longer than Vib-Day21 (p<0.05) and TS (p<0.01) (Figure 5c). As for GM, TpT decreased and was significantly shorter than TS during the reloading period. Otherwise, the changes of TpT amongst all comparisons between Vib and Ctrl or among timepoints were not different significantly (p>0.05) (Figure 5d).
Discussion
The current study aims to examine the effects of LMHFV intervention on the regrowth process of reloading muscles from prolonged disuse at functional, histo-morphological and cellular levels. The results showed LMHFV intervention was not injurious to reloading muscles, both Sol and GM, while myogenic cells proliferative activities were enhanced. Only in the GM demonstrated type I fiber hypertrophy and a type I to type IIA fiber transition was observed upon LMHFV treatment during reloading. Further investigations on the fiber type specific responses of various muscles toward LMHFV and interaction of myogenic cells with the associated myofiber were recommended.
The 28-day tail suspension prolonged muscle disuse model (TS) in rat was applied to investigate the effects of LMHFV treatment on two reloading lower limbs muscles, Sol and GM. It was well known slow-twitch muscle fibers were more susceptible to disuse atrophy compared with the fast counterparts 9-10 . Hence differential influences of 28-day TS on Sol (slow-dominant) and GM (fast-dominant except the mixed core region) should be expected. Our results were consistent with the previous findings that the gross morphology of Sol was reduced more than that of GM in response to TS [25][26] . For FCSA, the changes appeared similar between Sol and the core region of GM. The peripheral region of GM, in fact composed mainly of type IIB fibers, showed no changes in FCSA within the region (data not shown). Hence, if whole GM was considered, the effects of TS on GM were not as prominent as in Sol. This intrinsic difference should be taken into consideration in interpreting the differential responses of Sol and GM to reloading.
In Sol, reloading regrowth could be observed from morphological (gain of Mm, Lo, and pCSA) and fiber typing data (increased FCSA and fast-to-slow type transition). Coupled with functional data, the increasing specific sP o and longer TpT, reloading induced regrowth of Sol was evident and consistent with previous report 8 . Furthermore, it was apparent that Vib promoted gain of force generating capacity as indicated from the significantly higher sP o compared with TS. This increasing trend, nevertheless, was not significant in Ctrl. Longer timepoints of Vib treatment may allow a clearer picture of this beneficial effect. Comparing with Sol, the case in GM was rather more complicated. Although Mm, pCSA and FCSA increased during reloading suggesting apparent muscle regrowth, Lo decreased at the early timepoints and specific force deficit in GM was observed during the whole reloading period. This deficit was neither be improved nor deteriorated by LMHFV. To explain the deficit, reloading induced injury in GM was suggested 8 .
Reloading injury is caused by myofiber disruption due to stretching or increased muscle strain when the shortened disused myofibers were reloaded and lengthened. It was known that TS unloaded muscle fibers were shortened to adapt to the prolonged plantar flexion of the foot, which was typically observed in prolonged TS model 27 . In addition, the anatomy of gastrocnemius muscle which connects two joints (ankle and knee) and is composed of fast-fibers in majority may render its higher susceptibility toward eccentric/ stretching injury during reloading than Sol 8,28 . It might explain our current findings that GM showed a prolonged functional deficit during reloading even it was less affected by unloading than Sol 28 . Given Sol also experienced fiber shortening, it was expected to be affected by reloading injury and demonstrated force deficit. Previous studies showed the onset of force decrement in Sol was around 1-2 days from reloading and the weakness may last no longer than 9 days 8,29 . Our results did not contradict with these findings that force generating capacity remained similar to TS (no increase) at Day 7 (the earliest timepoint of this study) and an increase could be observed since Day 14. In summary, LMHFV have mild effects on Sol functional recovery and is non-injurious to mechanical injury-prone GM during reloading. Optimization of LMHFV treatment period might enhance the beneficial outcomes and lead to significant difference from Ctrl in Sol without adverse effects on GM.
Myogenic cell proliferative activities were found elevated by counting overall myogenic cells regardless of the associated fiber types in Sol only. In addition to overall counting, the current investigation adopted the novel fiber type specific myogenic cell analysis to evaluate the effects of LMHFV on myogenic cell activities. The concept of fiber associated myogenic cell progeny was suggested from Verdijk's group including reduced satellite cells in aging type II skeletal muscle fibers and increased SC counts in type II fibers after eccentric exercises [30][31] . The effects of LMHFV on fiber-type specific counts were found in both Sol (type I and IIA) and GM (type IIB). Stimulatory effects of LMHFV on myogenic cells proliferation were therefore evident in both Sol and GM. This indicated that the analysis of overall myogenic cell activities might underestimate or even misinterpret the effects of particular intervention.
Referring to Verdijk's studies, the fiber type specific changes could be primary to the vulnerable type II fibers in eccentric and aging models [30][31] . Hence it was questionable if the fiber type specific effects observed in this study were related to fiber susceptibility to injury. Our functional results showed vibration was not injurious to Sol and even in the mechanical sensitive GM. Instead, trend of faster force regain in Sol was found. Similarly, administration of vibration of similar regime (45 Hz, 0.6 g) on dystrophic mice model, with skeletal muscles well-known susceptible to mechanical damages, showed no detrimental effects as well 32 . Hence damages to muscle cells by vibration treatment should be absent or negligible, if any. In the same study, 7 days of treatment could upregulate muscle paired box gene-7 (Pax7) gene expression, a myogenic cell marker particularly in quiescent muscle satellite cell, which suggested promotion of myogenic cells activities in vivo 32 . In vitro vibration treatment on myogenic cell line, C2C12, was also reported to increase cell proliferation and myogenic differentiation without any injury or challenge to the cultured cells 15 . Therefore, it was conceived to regard the current findings primarily as direct promotion of myogenic cell proliferation and less likely related to induced myofiber injury. However, it remained questionable why myogenic cells associated with particular fiber types appeared more responsive to LMHFV. Recruitment of different kinds of motor units during vibration treatment, according to tonic vibration reflex (TVR) was evident and suggested only fibers under the activated motor units responded to contract [33][34][35] . Besides, communication mechanisms between the associated myofibers and adjacent myogenic cells have been reported [36][37] . It implied the possibility that LMHFV might trigger specific types of fiber to contract and stimulate the associated myogenic cell activities leading to the fiber type specific myogenic cell responses. More speculations on interaction of myogenic cells and the associated fibers upon mechanical stimuli are suggested.
In addition to stimulation of type IIB myogenic cells activities, LMHFV induced fast fiber type specific effects on other outcomes of GM, for instance, suppression of type I fiber hypertrophy and shift of type IIA fibers from type I counterparts. A trend of shorter TpT at Day 21 could be observed in GM as well. LMHFV hence apparently favored GM fast fiber changes during reloading. It was interesting that Sol showed increases in both type I and IIA associated myogenic cell activities. Also, Xie's study showed vibration stimulated both type I and type IIA fiber hypertrophy in growing mice 14 . Although fast-to-slow fiber type transition in Sol was not affected by LMHFV, significantly shorter TpT was detected. It was unclear if other factors like calcium handling and cross-bridge kinetics attributed to the difference. In general, the fiber type specific responses appeared muscle-dependent. It was worthy for further investigations on the differential responses of the two muscles to various vibration regimes (e.g. changes in frequency and magnitude) and to examine the fiber type specific changes.
In conclusion, LMHFV intervention did no harm on the contractile functions of reloading muscles during 21 days of reloading while myogenic cell activities could be promoted in both Sol and GM. However, a limitation of the current study was that the cell fate of the increased number of myogenic cells was uncertain. It remains to be seen whether the fiber type specific changes due to LMHFV were dependent on the elevated myogenic cell activity. The reloading effects on Sol and GM were different and the concerns of customizing intervention for particular muscles should be well-considered so that therapeutic effects were maximized without compromis-ing other muscles toward injury. The use of LMHFV in treating prolonged disused muscles should be further investigated to depict the regulatory mechanisms of the myofibers on the associated myogenic cells as well as the fiber type specific responses in different LMHFV regimes.
Figure 2 .
2Fiber cross-sectional area (FCSA) (10 3 μm 2 ) of three fiber types: type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): No significant difference detected amongst all comparisons; b) gastrocnemius medialis (GM): a Reloading control (Ctrl) larger than reloading plus vibration (Vib) in type I FCSA, p<0.01; b,c Day 21 larger than Day 7, p<0.01 and p<0.001 respectively; d,e Day 21 larger than Day 14, p<0.01 and p<0.001 respectively; main effects detected from Two-way ANOVA coupled with post-hoc Bonferroni adjustment. No significant interaction detected.similarly in GM with muscle mass (~23%, p<0.001), pCSA (~17%, p<0.001) and FCSA (~26%, p<0.001), all compared with the corresponding value in TS.
Figure 3 .
3The proportion (%) of three fiber types, type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): a higher % type I in Day 21 than Day 7, p = 0.05; b lower % type IIB in Day 21 than Day 14, p<0.05; b) gastrocnemius medialis (GM): a higher % type I in reloading control (Ctrl) than reloading plus vibration (Vib), p<0.01 (interaction, p<0.05); b higher % type IIA in Vib than Ctrl, p<0.05 (interaction, p=0.05); main effects and interaction detected from Twoway ANOVA coupled with post-hoc Bonferroni test.
Figure 4 .
4Proliferating myogenic cell counts (count / 100 fibers) from soleus (Sol) and gastrocnemius medialis (GM) muscles. a) overall bromodeoxyuridine (BrdU) + /myogenic cell in Sol (left panel) and GM (right panel
Figure 5 .
5Contractile properties -specific force (sP o , gcm -2 ) and contraction time to peak twitch (TpT, ms) of soleus (Sol) and gastrocnemius medialis (GM) during reloading. The dotted line in each graph was the corresponding mean in unloading (TS) group. a) sP o -Sol: a reloading plus vibration (Vib) larger than TS, p<0.05; b Day 21 larger than TS, p<0.001; c Day 21 larger than Day 7, p<0.001; d Day 21 larger than Day 14, p<0.01; b) sP o -GM: a Day 21 larger than Day 14, p<0.05; c) TpT -Sol: a Day 21 slower than Day 14, p<0.05 (interaction, p<0.01); d) TpT -GM: a reloading control (Ctrl) faster than TS, p<0.001; b Vib faster than TS, p<0.001; c Day 7 faster than TS, p<0.05; d Day 14 faster than TS, p<0.001; e Day 21 faster than TS, p<0.05; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test.
Figure 1. Typical ATPase (pH4.6) staining images of weight bearing (WB, left column) and unloading (TS, right column) from soleus (Sol, upper panel) and gastrocnemius medialis (GM, lower panel). Type I (I, darkest), type IIA (IIA, lightest) and type IIB (IIB, intermediate) were indicated. *Note that no type IIB in Sol WB (upper left). Magnification: 200X.Table 2. Morphological data summary of Sol from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.001 respectively; e,f Day21 larger than TS, p<0.05 and p<0.001 respectively; g,h Day 14 larger than TS, p<0.01 and p<0.001; main effects detected from Two-way ANOVA. No significant interaction detected.Table 3. Morphological data summary of GM from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.01 respectively; e Day21 larger than TS, p<0.001; f TS larger than Day 14, p<0.05; g Day 14 larger than TS, p<0.001; h Ctrl larger than Vib, p<0.01; main effects detected from Two-way ANOVA. No significant interaction detected.TS (n=6)
0.200 ± 0.017 a
6.20 ± 0.62 a
2.87± 0.12 a
1.164 ± 0.090 a
25.68 ± 2.55 a
2.59 ± 0.20 a
WB (n=6)
0.300 ± 0.024
8.85 ± 0.94
4.74 ± 0.31
1.556 ± 0.100
33.14 ± 2.07
4.57 ± 0.63
Table 1. The Sol (Left column) and GM (Right column) muscle mass (Mm), physiological cross-sectional area (pCSA) and fiber cross-sectional
area (FCSA) between weight-bearing group (WB) and hind-limb unloading group (TS) (n=6, mean ± SD). a WB larger than TS, p<0.001 by in-
dependent samples t-test.
Group/
Sol
Timepoint
Mm (g)
Lo (cm)
pCSA (10 -2 cm 2 )
FCSA (10 3 μm 2 )
TS
0.200 ± 0.017 b,d,f,h
3.06 ± 0.11 e
6.20 ± 0.62 b,d,f,h
2.87 ± 0.12 a,c,g
Day 7
Ctrl
0.221 ± 0.034
2.89 ± 0.10
7.21 ± 1.00
3.51 ± 0.58
Vib
0.226 ± 0.021
3.01 ± 0.08
7.09 ± 0.68
3.37 ± 0.17
Day 14
Ctrl
0.268 ± 0.017
3.10 ± 0.16
8.40 ± 0.87
3.86 ± 0.27
Vib
0.268 ± 0.007
3.09 ± 0.10
8.20 ± 0.16
3.44 ± 0.23
Day 21
Ctrl
0.273 ± 0.025
3.34 ± 0.02
7.84 ± 0.71
3.25 ± 0.31
Vib
0.271 ± 0.015
3.21 ± 0.16
7.97 ± 0.56
3.49 ± 0.41
Group/
GM
Timepoint
Mm (g)
Lo (cm)
pCSA (10 -2 cm 2 )
FCSA (10 3 μm 2 )
TS
1.164 ± 0.100 a,d,e,g
4.30 ± 0.08 f
25.68 ± 2.55 b,d,e,f
2.59 ± 0.20 b,c,e
Day 7
Ctrl
1.195 ± 0.091
4.12 ± 0.10
27.44 ± 2.05
3.05 ± 0.21
Vib
1.213 ± 0.063
4.24 ± 0.08
27.13 ± 1.28
3.09 ± 0.32
Day 14
Ctrl
1.386 ±0.107
4.09 ± 0.16
32.10 ± 2.12
3.06 ± 0.16
Vib
1.376 ±0.132
4.21 ± 0.10
30.07 ± 1.58
2.62 ± 0.21
Day 21
Ctrl
1.403 ± 0.049
4.38 ± 0.03
30.43 ± 1.20
3.79 ± 0.35 h
Vib
1.426 ± 0.112
4.34 ± 0.05
30.34 ± 1.48
3.27 ± 0.33
). a Reloading plus vibration (Vib) more than reloading control (Ctrl) in Sol, p<0.001; b Day 14 more than Day 7 in Sol, p<0.05; c Day 14 more than Day 21 in Sol, p=0.052; d Day 14 more than Day 7 in GM; e Day 14 more than Day 21 in GM, p<0.001; b) Sol: fiber type specific BrdU + /myogenic cell associated with type I, IIA and IIB fibers. a Vib more than Ctrl in type I, p<0.05; b Vib more than Ctrl in type IIA, p< 0.05; c Day 14 > Day 7 in type IIB, p<0.001; d Day 14 > Day 21 in type IIB, p<0.001; c) GM: fiber type specific BrdU + /myogenic cell associaed with type I, IIA and IIB fibers. a Vib more than Ctrl in type IIB, p<0.05 (interaction, p<0.001); b Day 14 > Day 7 in type IIA, p<0.01; c Day 14 > Day 21 in type IIA, p<0.01; d Day 14 > Day 7 in type IIB, p<0.01; e Day 14 > Day 21 in type IIB, p<0.001; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test.
AcknowledgementsWe would like to thank Prof. Anthony Bakker, The University of Western Australia, for his technical experience and support in in vitro functional assessment.
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| [
"Skeletal muscles are highly mechanical sensitive and show high plasticity to altered activity levels 1-4 . Muscle disuse (or unloading), as in astronauts and bed-rest patients, leads to muscle atrophy and functional loss 5-6 . When designing treatment options for disuse atrophy, one must keep in mind that disused muscles are more susceptible to damage than normal muscles and hence over-exerting a disuse muscle may cause further deterioration, rather than improvement of muscle function 7-8 .Despite the numerous investigations on muscle disuse, less information about reloading from disuse and the suitable intervention is available. Moreover, most existing animal studies were conducted in a relatively short, 14-day tail suspension (TS) model and soleus muscle was the major muscle of interest. The duration of unloading and muscles of interests in fact are critical factors that lead to different degree of muscle-dependent, progressive physiological changes. For instance, myofibrillar protein loss was more prominent and became stable after 28 days of unloading than 7 and 14 days protocol whereas soleus was more affected than plantaris and gastrocnemius muscle (Details see Review byThomason, 1990)[9][10][11]. A better understanding of the reloading effects on unloaded limb muscles will provide valuable information for determining safe and even customized intervention for the susceptible disused muscles during reloading.Low-magnitude high-frequency vibration (LMHFV) is a J Musculoskelet Neuronal Interact 2015; 15(4):316-324AbstractObjectives: Low-magnitude high-frequency vibration (LMHFV) was reported beneficial to muscle contractile functions in clinical and preclinical studies. This study aims to investigate the effects of LMHFV on myofibers, myogenic cells and functional properties of disused soleus (Sol) and gastrocnemius medialis (GM) during reloading. Methods: Sprague Dawley rats were hind-limb unloaded for 28 days and assigned to reloading control (Ctrl) or LMHFV group (Vib). Sol and GM of both groups were harvested for fiber typing, proliferating myogenic cell counting and in vitro functional assessment. Results: Myogenic cells proliferation was promoted by LMHFV in both Sol and GM (p<0.001 and p<0.05 respectively). Force generating capacity was not much affected (Vib=Ctrl, p>0.05) but fast-fiber favorable changes in fiber type switching (more type IIA but lower type I in Vib; p<0.05 and 0.01 respectively) and fiber hypertrophy (type I, Vib<Ctrl; p<0.01) were observed mainly in GM. Conclusion: LMHFV was not detrimental to reloading muscles but the outcomes were muscle dependent. The unique fiber type composition and anatomical differences between Sol and GM might render the differential muscle responses to LMHFV. Further investigations on myofibers type specific responses to different LMHFV regimes and myogenic cell interaction with associated myofiber were proposed."
] | [
"K-T Sun \nDepartment of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin\n\nNew Territories\nHong Kong SAR\n\n",
"K-S Leung \nDepartment of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin\n\nNew Territories\nHong Kong SAR\n\n",
"P ",
"M-F Siu \nDepartment of Health Technology and Informatics\nThe Hong Kong Polytechnic University\nHung HomKowloon, Hong KongChina\n",
"L Qin \nDepartment of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin\n\nNew Territories\nHong Kong SAR\n\n\nThe CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System\nThe Chinese University of Hong Kong Shenzhen Research Institute\nShenzhenThe People's Republic of China\n\nWing-Hoi Cheung\nDepartment of Orthopaedics and Traumatology\nClinical Science Building\nHong Kong SAR\n5/FChina\n\nThe Chinese University of Hong Kong\nShatin, Hong KongNew TerritoriesChina\n",
"W-H Cheung \nDepartment of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin\n\nNew Territories\nHong Kong SAR\n\n\nThe CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System\nThe Chinese University of Hong Kong Shenzhen Research Institute\nShenzhenThe People's Republic of China\n\nWing-Hoi Cheung\nDepartment of Orthopaedics and Traumatology\nClinical Science Building\nHong Kong SAR\n5/FChina\n\nThe Chinese University of Hong Kong\nShatin, Hong KongNew TerritoriesChina\n"
] | [
"Department of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin",
"New Territories\nHong Kong SAR\n",
"Department of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin",
"New Territories\nHong Kong SAR\n",
"Department of Health Technology and Informatics\nThe Hong Kong Polytechnic University\nHung HomKowloon, Hong KongChina",
"Department of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin",
"New Territories\nHong Kong SAR\n",
"The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System\nThe Chinese University of Hong Kong Shenzhen Research Institute\nShenzhenThe People's Republic of China",
"Wing-Hoi Cheung\nDepartment of Orthopaedics and Traumatology\nClinical Science Building\nHong Kong SAR\n5/FChina",
"The Chinese University of Hong Kong\nShatin, Hong KongNew TerritoriesChina",
"Department of Orthopaedics and Traumatology\nThe Chinese University of Hong Kong\nShatin",
"New Territories\nHong Kong SAR\n",
"The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System\nThe Chinese University of Hong Kong Shenzhen Research Institute\nShenzhenThe People's Republic of China",
"Wing-Hoi Cheung\nDepartment of Orthopaedics and Traumatology\nClinical Science Building\nHong Kong SAR\n5/FChina",
"The Chinese University of Hong Kong\nShatin, Hong KongNew TerritoriesChina"
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"A J Blazevich, ",
"M Fluck, ",
"H Hoppeler, ",
"M Fluck, ",
"H Hoppeler, ",
"M Fluck, ",
"P M Droppert, ",
"C Mercier, ",
"J Jobin, ",
"C Lepine, ",
"C Simard, ",
"J Frenette, ",
"M St-Pierre, ",
"C H Cote, ",
"E Mylona, ",
"F X Pizza, ",
"J J Widrick, ",
"G F Maddalozzo, ",
"H Hu, ",
"J C Herron, ",
"U T Iwaniec, ",
"R T Turner, ",
"D B Thomason, ",
"R E Herrick, ",
"D Surdyka, ",
"K M Baldwin, ",
"D B Thomason, ",
"F W Booth, ",
"A M Hanson, ",
"B C Harrison, ",
"M H Young, ",
"L S Stodieck, ",
"M Moriggi, ",
"M Vasso, ",
"C Fania, ",
"D Capitanio, ",
"G Bonifacio, ",
"M Salanova, ",
"D Blottner, ",
"J Rittweger, ",
"D Felsenberg, ",
"P Cerretelli, ",
"C Gelfi, ",
"K S Leung, ",
"C Y Li, ",
"Y K Tse, ",
"T K Choy, ",
"P C Leung, ",
"V W Hung, ",
"S Y Chan, ",
"A H Leung, ",
"W H Cheung, ",
"L Xie, ",
"C Rubin, ",
"S Judex, ",
"C Z Wang, ",
"G J Wang, ",
"M L Ho, ",
"Y H Wang, ",
"M L Yeh, ",
"C H Chen, ",
"G Ceccarelli, ",
"L Benedetti, ",
"D Galli, ",
"D Pre, ",
"G Silvani, ",
"N Crosetto, ",
"G Magenes, ",
"Cusella De Angelis, ",
"M G , ",
"E R Morey-Holton, ",
"R K Globus, ",
"D H Chow, ",
"K S Leung, ",
"L Qin, ",
"A H Leung, ",
"W H Cheung, ",
"P M Siu, ",
"E E Pistilli, ",
"D C Butler, ",
"S E Alway, ",
"C S Hintz, ",
"E F Coyle, ",
"K K Kaiser, ",
"M M Chi, ",
"O H Lowry, ",
"L C Wang, ",
"D Kernell, ",
"D R Plant, ",
"F Beitzel, ",
"G S Lynch, ",
"S S Segal, ",
"J A Faulkner, ",
"A M Payne, ",
"S L Dodd, ",
"C Leeuwenburgh, ",
"P K Jain, ",
"P K Banerjee, ",
"N S Baboo, ",
"E M Iyer, ",
"M E Tischler, ",
"E J Henriksen, ",
"K A Munoz, ",
"C S Stump, ",
"C R Woodman, ",
"C R Kirby, ",
"D A Riley, ",
"G R Slocum, ",
"J L Bain, ",
"F R Sedlak, ",
"T E Sowa, ",
"J W Mellender, ",
"Bryan Dixon, ",
"J , ",
"D Pottle, ",
"L E Gosselin, ",
"L B Verdijk, ",
"R Koopman, ",
"G Schaart, ",
"K Meijer, ",
"H H Savelberg, ",
"L J Van Loon, ",
"N M Cermak, ",
"T Snijders, ",
"B R Mckay, ",
"G Parise, ",
"L B Verdijk, ",
"M A Tarnopolsky, ",
"M J Gibala, ",
"L J Loon, ",
"S A Novotny, ",
"M D Eckhoff, ",
"B C Eby, ",
"J A Call, ",
"D Nuckley, ",
"D A Lowe, ",
"R D Pollock, ",
"R C Woledge, ",
"F C Martin, ",
"D J Newham, ",
"C Delecluse, ",
"M Roelants, ",
"S Verschueren, ",
"B J Martin, ",
"H S Park, ",
"R Bischoff, ",
"P Tavi, ",
"T Korhonen, ",
"S L Hanninen, ",
"J D Bruton, ",
"S Loof, ",
"A Simon, ",
"H Westerblad, "
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] | [
"Effects of physical training and detraining, immobilisation, growth and aging on human fascicle geometry. A J Blazevich, Sports Med. 36Blazevich AJ. Effects of physical training and detraining, immobilisation, growth and aging on human fascicle geometry. Sports Med 2006;36:1003-1017.",
"Molecular basis of skeletal muscle plasticity -from gene to form and function. M Fluck, H Hoppeler, Rev Physiol Biochem Pharmacol. 146Fluck M, Hoppeler H. Molecular basis of skeletal muscle plasticity -from gene to form and function. Rev Physiol Biochem Pharmacol 2003;146:159-216.",
"Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli. M Fluck, J Exp Biol. 209Fluck M. Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli. J Exp Biol 2006;209:2239-2248.",
"Normal mammalian skeletal muscle and its phenotypic plasticity. H Hoppeler, M Fluck, J Exp Biol. 205Hoppeler H, Fluck M. Normal mammalian skeletal mus- cle and its phenotypic plasticity. J Exp Biol 2002; 205:2143-2152.",
"A review of muscle atrophy in microgravity and during prolonged bed rest. P M Droppert, J Br Interplanet Soc. 46Droppert PM. A review of muscle atrophy in microgravity and during prolonged bed rest. J Br Interplanet Soc 1993; 46:83-86.",
"Effects of hindlimb suspension on contractile properties of young and old rat muscles and the impact of electrical stimulation on the recovery process. C Mercier, J Jobin, C Lepine, C Simard, Mech Ageing Dev. 106Mercier C, Jobin J, Lepine C, Simard C. Effects of hindlimb suspension on contractile properties of young and old rat muscles and the impact of electrical stimulation on the re- covery process. Mech Ageing Dev 1999; 106:305-320.",
"Muscle impairment occurs rapidly and precedes inflammatory cell accumulation after mechanical loading. J Frenette, M St-Pierre, C H Cote, E Mylona, F X Pizza, Am J Physiol Regul Integr Comp Physiol. 282Frenette J, St-Pierre M, Cote CH, Mylona E, Pizza FX. Muscle impairment occurs rapidly and precedes inflam- matory cell accumulation after mechanical loading. Am J Physiol Regul Integr Comp Physiol 2002;282:R351-357.",
"Detrimental effects of reloading recovery on force, shortening velocity, and power of soleus muscles from hindlimb-unloaded rats. J J Widrick, G F Maddalozzo, H Hu, J C Herron, U T Iwaniec, R T Turner, Am J Physiol Regul Integr Comp Physiol. 295Widrick JJ, Maddalozzo GF, Hu H, Herron JC, Iwaniec UT, Turner RT. Detrimental effects of reloading recovery on force, shortening velocity, and power of soleus mus- cles from hindlimb-unloaded rats. Am J Physiol Regul In- tegr Comp Physiol 2008;295:R1585-1592.",
"Time course of soleus muscle myosin expression during hindlimb suspension and recovery. D B Thomason, R E Herrick, D Surdyka, K M Baldwin, J Appl Physiol. 63Thomason DB, Herrick RE, Surdyka D, Baldwin KM. Time course of soleus muscle myosin expression during hindlimb suspension and recovery. J Appl Physiol 1987;63:130-137.",
"Atrophy of the soleus muscle by hindlimb unweighting. D B Thomason, F W Booth, J Appl Physiol. 68Thomason DB, Booth FW. Atrophy of the soleus muscle by hindlimb unweighting. J Appl Physiol (1985) 1990; 68:1-12.",
"Ferguson VL. Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension. A M Hanson, B C Harrison, M H Young, L S Stodieck, Muscle Nerve. 48Hanson AM, Harrison BC, Young MH, Stodieck LS, Fer- guson VL. Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension. Muscle Nerve 2013;48:393-402.",
"Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation. M Moriggi, M Vasso, C Fania, D Capitanio, G Bonifacio, M Salanova, D Blottner, J Rittweger, D Felsenberg, P Cerretelli, C Gelfi, Proteomics. 10Moriggi M, Vasso M, Fania C, Capitanio D, Bonifacio G, Salanova M, Blottner D, Rittweger J, Felsenberg D, Cer- retelli P, Gelfi C. Long term bed rest with and without vi- bration exercise countermeasures: effects on human muscle protein dysregulation. Proteomics 2010;10:3756-3774.",
"Effects of 18-month low-magnitude high-frequency vibration on fall rate and fracture risks in 710 community elderly -a cluster-randomized controlled trial. K S Leung, C Y Li, Y K Tse, T K Choy, P C Leung, V W Hung, S Y Chan, A H Leung, W H Cheung, Osteoporos Int. 25Leung KS, Li CY, Tse YK, Choy TK, Leung PC, Hung VW, Chan SY, Leung AH, Cheung WH. Effects of 18- month low-magnitude high-frequency vibration on fall rate and fracture risks in 710 community elderly -a clus- ter-randomized controlled trial. Osteoporos Int 2014; 25:1785-1795.",
"Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations. L Xie, C Rubin, S Judex, J Appl Physiol. 104Xie L, Rubin C, Judex S. Enhancement of the adolescent murine musculoskeletal system using low-level mechan- ical vibrations. J Appl Physiol 2008;104:1056-1062.",
"Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts. C Z Wang, G J Wang, M L Ho, Y H Wang, M L Yeh, C H Chen, J Appl Physiol. 109Wang CZ, Wang GJ, Ho ML, Wang YH, Yeh ML, Chen CH. Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts. J Appl Physiol 2010; 109:840-848.",
"Lowamplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells. G Ceccarelli, L Benedetti, D Galli, D Pre, G Silvani, N Crosetto, G Magenes, Cusella De Angelis, M G , J Tissue Eng Regen Med. 8Ceccarelli G, Benedetti L, Galli D, Pre D, Silvani G, Crosetto N, Magenes G, Cusella De Angelis MG. Low- amplitude high frequency vibration down-regulates myo- statin and atrogin-1 expression, two components of the atrophy pathway in muscle cells. J Tissue Eng Regen Med 2014;8:396-406.",
"Hindlimb unloading rodent model: technical aspects. E R Morey-Holton, R K Globus, J Appl Physiol. 92Morey-Holton ER, Globus RK. Hindlimb unloading ro- dent model: technical aspects. J Appl Physiol 2002; 92:1367-1377.",
"Low-magnitude high-frequency vibration (LMHFV) enhances bone remodeling in osteoporotic rat femoral fracture healing. D H Chow, K S Leung, L Qin, A H Leung, W H Cheung, J Orthop Res. 29Chow DH, Leung KS, Qin L, Leung AH, Cheung WH. Low-magnitude high-frequency vibration (LMHFV) en- hances bone remodeling in osteoporotic rat femoral frac- ture healing. J Orthop Res 2011;29:746-752.",
"Aging influences cellular and molecular responses of apoptosis to skeletal muscle unloading. P M Siu, E E Pistilli, D C Butler, S E Alway, Am J Physiol Cell Physiol. 288Siu PM, Pistilli EE, Butler DC, Alway SE. Aging influ- ences cellular and molecular responses of apoptosis to skeletal muscle unloading. Am J Physiol Cell Physiol 2005;288:C338-349.",
"Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining. C S Hintz, E F Coyle, K K Kaiser, M M Chi, O H Lowry, J Histochem Cytochem. 32Hintz CS, Coyle EF, Kaiser KK, Chi MM, Lowry OH. Comparison of muscle fiber typing by quantitative en- zyme assays and by myosin ATPase staining. J Histochem Cytochem 1984;32:655-660.",
"Proximo-distal organization and fibre type regionalization in rat hindlimb muscles. L C Wang, D Kernell, J Muscle Res Cell Motil. 21Wang LC, Kernell D. Proximo-distal organization and fibre type regionalization in rat hindlimb muscles. J Mus- cle Res Cell Motil 2000;21:587-598.",
"Length-tension relationships are altered in regenerating muscles of the rat after bupivacaine injection. D R Plant, F Beitzel, G S Lynch, J Appl Physiol. 98Plant DR, Beitzel F, Lynch GS. Length-tension relation- ships are altered in regenerating muscles of the rat after bupivacaine injection. J Appl Physiol 2005;98:1998-2003.",
"Temperature-dependent physiological stability of rat skeletal muscle in vitro. S S Segal, J A Faulkner, Am J Physiol. 248Segal SS, Faulkner JA. Temperature-dependent physio- logical stability of rat skeletal muscle in vitro. Am J Phys- iol 1985;248:C265-270.",
"Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space. A M Payne, S L Dodd, C Leeuwenburgh, J Appl Physiol. 95Payne AM, Dodd SL, Leeuwenburgh C. Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space. J Appl Physiol (1985) 2003;95:2554-2562.",
"Physiological properties of rat hind limb muscles after 15 days of simulated weightless environment. P K Jain, P K Banerjee, N S Baboo, E M Iyer, Indian J Physiol Pharmacol. 41Jain PK, Banerjee PK, Baboo NS, Iyer EM. Physiological properties of rat hind limb muscles after 15 days of sim- ulated weightless environment. Indian J Physiol Pharma- col 1997;41:23-28.",
"Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats. M E Tischler, E J Henriksen, K A Munoz, C S Stump, C R Woodman, C R Kirby, J Appl Physiol. 74Tischler ME, Henriksen EJ, Munoz KA, Stump CS, Wood- man CR, Kirby CR. Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats. J Appl Physiol (1985) 1993;74:2161-2165.",
"Rat hindlimb unloading: soleus histochemistry, ultrastructure, and electromyography. D A Riley, G R Slocum, J L Bain, F R Sedlak, T E Sowa, J W Mellender, J Appl Physiol. 69Riley DA, Slocum GR, Bain JL, Sedlak FR, Sowa TE, Mellender JW. Rat hindlimb unloading: soleus histo- chemistry, ultrastructure, and electromyography. J Appl Physiol (1985) 1990;69:58-66.",
"Gastrocnemius vs. soleus strain: how to differentiate and deal with calf muscle injuries. Bryan Dixon, J , Curr Rev Musculoskelet Med. 2Bryan Dixon J. Gastrocnemius vs. soleus strain: how to differentiate and deal with calf muscle injuries. Curr Rev Musculoskelet Med 2009;2:74-77.",
"Impact of mechanical load on functional recovery after muscle reloading. D Pottle, L E Gosselin, Med Sci Sports Exerc. 32Pottle D, Gosselin LE. Impact of mechanical load on functional recovery after muscle reloading. Med Sci Sports Exerc 2000;32:2012-2017.",
"Satellite cell content is specifically reduced in type II skeletal muscle fibers in the elderly. L B Verdijk, R Koopman, G Schaart, K Meijer, H H Savelberg, L J Van Loon, Am J Physiol Endocrinol Metab. 292Verdijk LB, Koopman R, Schaart G, Meijer K, Savelberg HH, van Loon LJ. Satellite cell content is specifically re- duced in type II skeletal muscle fibers in the elderly. Am J Physiol Endocrinol Metab 2007;292:E151-157.",
"Eccentric Exercise Increases Satellite Cell Content in Type II Muscle Fibers. N M Cermak, T Snijders, B R Mckay, G Parise, L B Verdijk, M A Tarnopolsky, M J Gibala, L J Loon, Med Sci Sports Exerc. 45Cermak NM, Snijders T, McKay BR, Parise G, Verdijk LB, Tarnopolsky MA, Gibala MJ, Loon LJ. Eccentric Ex- ercise Increases Satellite Cell Content in Type II Muscle Fibers. Med Sci Sports Exerc 2013;45:230-237.",
"Musculoskeletal response of dystrophic mice to short term, low intensity, high frequency vibration. S A Novotny, M D Eckhoff, B C Eby, J A Call, D Nuckley, D A Lowe, J Musculoskelet Neuronal Interact. 13Novotny SA, Eckhoff MD, Eby BC, Call JA, Nuckley D, Lowe DA. Musculoskeletal response of dystrophic mice to short term, low intensity, high frequency vibration. J Musculoskelet Neuronal Interact 2013;13:418-429.",
"Effects of whole body vibration on motor unit recruitment and threshold. R D Pollock, R C Woledge, F C Martin, D J Newham, J Appl Physiol. 112Pollock RD, Woledge RC, Martin FC, Newham DJ. Ef- fects of whole body vibration on motor unit recruitment and threshold. J Appl Physiol (1985) 2012;112:388-395.",
"Strength increase after whole-body vibration compared with resistance training. C Delecluse, M Roelants, S Verschueren, Med Sci Sports Exerc. 35Delecluse C, Roelants M, Verschueren S. Strength in- crease after whole-body vibration compared with resist- ance training. Med Sci Sports Exerc 2003;35:1033-1041.",
"Analysis of the tonic vibration reflex: influence of vibration variables on motor unit synchronization and fatigue. B J Martin, H S Park, Eur J Appl Physiol Occup Physiol. 75Martin BJ, Park HS. Analysis of the tonic vibration reflex: influence of vibration variables on motor unit synchro- nization and fatigue. Eur J Appl Physiol Occup Physiol 1997;75:504-511.",
"Interaction between satellite cells and skeletal muscle fibers. R Bischoff, Development. 109Bischoff R. Interaction between satellite cells and skeletal muscle fibers. Development 1990;109:943-952.",
"Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes. P Tavi, T Korhonen, S L Hanninen, J D Bruton, S Loof, A Simon, H Westerblad, J Cell Physiol. 223Tavi P, Korhonen T, Hanninen SL, Bruton JD, Loof S, Simon A, Westerblad H. Myogenic skeletal muscle satel- lite cells communicate by tunnelling nanotubes. J Cell Physiol 2010;223:376-383."
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"Effects of physical training and detraining, immobilisation, growth and aging on human fascicle geometry",
"Molecular basis of skeletal muscle plasticity -from gene to form and function",
"Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli",
"Normal mammalian skeletal muscle and its phenotypic plasticity",
"A review of muscle atrophy in microgravity and during prolonged bed rest",
"Effects of hindlimb suspension on contractile properties of young and old rat muscles and the impact of electrical stimulation on the recovery process",
"Muscle impairment occurs rapidly and precedes inflammatory cell accumulation after mechanical loading",
"Detrimental effects of reloading recovery on force, shortening velocity, and power of soleus muscles from hindlimb-unloaded rats",
"Time course of soleus muscle myosin expression during hindlimb suspension and recovery",
"Atrophy of the soleus muscle by hindlimb unweighting",
"Ferguson VL. Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension",
"Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation",
"Effects of 18-month low-magnitude high-frequency vibration on fall rate and fracture risks in 710 community elderly -a cluster-randomized controlled trial",
"Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations",
"Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts",
"Lowamplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells",
"Hindlimb unloading rodent model: technical aspects",
"Low-magnitude high-frequency vibration (LMHFV) enhances bone remodeling in osteoporotic rat femoral fracture healing",
"Aging influences cellular and molecular responses of apoptosis to skeletal muscle unloading",
"Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining",
"Proximo-distal organization and fibre type regionalization in rat hindlimb muscles",
"Length-tension relationships are altered in regenerating muscles of the rat after bupivacaine injection",
"Temperature-dependent physiological stability of rat skeletal muscle in vitro",
"Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space",
"Physiological properties of rat hind limb muscles after 15 days of simulated weightless environment",
"Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats",
"Rat hindlimb unloading: soleus histochemistry, ultrastructure, and electromyography",
"Gastrocnemius vs. soleus strain: how to differentiate and deal with calf muscle injuries",
"Impact of mechanical load on functional recovery after muscle reloading",
"Satellite cell content is specifically reduced in type II skeletal muscle fibers in the elderly",
"Eccentric Exercise Increases Satellite Cell Content in Type II Muscle Fibers",
"Musculoskeletal response of dystrophic mice to short term, low intensity, high frequency vibration",
"Effects of whole body vibration on motor unit recruitment and threshold",
"Strength increase after whole-body vibration compared with resistance training",
"Analysis of the tonic vibration reflex: influence of vibration variables on motor unit synchronization and fatigue",
"Interaction between satellite cells and skeletal muscle fibers",
"Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes"
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] | [
"\nFigure 2 .\n2Fiber cross-sectional area (FCSA) (10 3 μm 2 ) of three fiber types: type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): No significant difference detected amongst all comparisons; b) gastrocnemius medialis (GM): a Reloading control (Ctrl) larger than reloading plus vibration (Vib) in type I FCSA, p<0.01; b,c Day 21 larger than Day 7, p<0.01 and p<0.001 respectively; d,e Day 21 larger than Day 14, p<0.01 and p<0.001 respectively; main effects detected from Two-way ANOVA coupled with post-hoc Bonferroni adjustment. No significant interaction detected.similarly in GM with muscle mass (~23%, p<0.001), pCSA (~17%, p<0.001) and FCSA (~26%, p<0.001), all compared with the corresponding value in TS.",
"\nFigure 3 .\n3The proportion (%) of three fiber types, type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): a higher % type I in Day 21 than Day 7, p = 0.05; b lower % type IIB in Day 21 than Day 14, p<0.05; b) gastrocnemius medialis (GM): a higher % type I in reloading control (Ctrl) than reloading plus vibration (Vib), p<0.01 (interaction, p<0.05); b higher % type IIA in Vib than Ctrl, p<0.05 (interaction, p=0.05); main effects and interaction detected from Twoway ANOVA coupled with post-hoc Bonferroni test.",
"\nFigure 4 .\n4Proliferating myogenic cell counts (count / 100 fibers) from soleus (Sol) and gastrocnemius medialis (GM) muscles. a) overall bromodeoxyuridine (BrdU) + /myogenic cell in Sol (left panel) and GM (right panel",
"\nFigure 5 .\n5Contractile properties -specific force (sP o , gcm -2 ) and contraction time to peak twitch (TpT, ms) of soleus (Sol) and gastrocnemius medialis (GM) during reloading. The dotted line in each graph was the corresponding mean in unloading (TS) group. a) sP o -Sol: a reloading plus vibration (Vib) larger than TS, p<0.05; b Day 21 larger than TS, p<0.001; c Day 21 larger than Day 7, p<0.001; d Day 21 larger than Day 14, p<0.01; b) sP o -GM: a Day 21 larger than Day 14, p<0.05; c) TpT -Sol: a Day 21 slower than Day 14, p<0.05 (interaction, p<0.01); d) TpT -GM: a reloading control (Ctrl) faster than TS, p<0.001; b Vib faster than TS, p<0.001; c Day 7 faster than TS, p<0.05; d Day 14 faster than TS, p<0.001; e Day 21 faster than TS, p<0.05; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test.",
"\n\nFigure 1. Typical ATPase (pH4.6) staining images of weight bearing (WB, left column) and unloading (TS, right column) from soleus (Sol, upper panel) and gastrocnemius medialis (GM, lower panel). Type I (I, darkest), type IIA (IIA, lightest) and type IIB (IIB, intermediate) were indicated. *Note that no type IIB in Sol WB (upper left). Magnification: 200X.Table 2. Morphological data summary of Sol from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.001 respectively; e,f Day21 larger than TS, p<0.05 and p<0.001 respectively; g,h Day 14 larger than TS, p<0.01 and p<0.001; main effects detected from Two-way ANOVA. No significant interaction detected.Table 3. Morphological data summary of GM from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.01 respectively; e Day21 larger than TS, p<0.001; f TS larger than Day 14, p<0.05; g Day 14 larger than TS, p<0.001; h Ctrl larger than Vib, p<0.01; main effects detected from Two-way ANOVA. No significant interaction detected.TS (n=6) \n0.200 ± 0.017 a \n6.20 ± 0.62 a \n2.87± 0.12 a \n1.164 ± 0.090 a \n25.68 ± 2.55 a \n2.59 ± 0.20 a \nWB (n=6) \n0.300 ± 0.024 \n8.85 ± 0.94 \n4.74 ± 0.31 \n1.556 ± 0.100 \n33.14 ± 2.07 \n4.57 ± 0.63 \n\nTable 1. The Sol (Left column) and GM (Right column) muscle mass (Mm), physiological cross-sectional area (pCSA) and fiber cross-sectional \narea (FCSA) between weight-bearing group (WB) and hind-limb unloading group (TS) (n=6, mean ± SD). a WB larger than TS, p<0.001 by in-\ndependent samples t-test. \n\nGroup/ \nSol \n\nTimepoint \nMm (g) \nLo (cm) \npCSA (10 -2 cm 2 ) \nFCSA (10 3 μm 2 ) \n\nTS \n0.200 ± 0.017 b,d,f,h \n3.06 ± 0.11 e \n6.20 ± 0.62 b,d,f,h \n2.87 ± 0.12 a,c,g \n\nDay 7 \nCtrl \n0.221 ± 0.034 \n2.89 ± 0.10 \n7.21 ± 1.00 \n3.51 ± 0.58 \nVib \n0.226 ± 0.021 \n3.01 ± 0.08 \n7.09 ± 0.68 \n3.37 ± 0.17 \n\nDay 14 \nCtrl \n0.268 ± 0.017 \n3.10 ± 0.16 \n8.40 ± 0.87 \n3.86 ± 0.27 \nVib \n0.268 ± 0.007 \n3.09 ± 0.10 \n8.20 ± 0.16 \n3.44 ± 0.23 \n\nDay 21 \nCtrl \n0.273 ± 0.025 \n3.34 ± 0.02 \n7.84 ± 0.71 \n3.25 ± 0.31 \nVib \n0.271 ± 0.015 \n3.21 ± 0.16 \n7.97 ± 0.56 \n3.49 ± 0.41 \n\nGroup/ \nGM \n\nTimepoint \nMm (g) \nLo (cm) \npCSA (10 -2 cm 2 ) \nFCSA (10 3 μm 2 ) \n\nTS \n1.164 ± 0.100 a,d,e,g \n4.30 ± 0.08 f \n25.68 ± 2.55 b,d,e,f \n2.59 ± 0.20 b,c,e \n\nDay 7 \nCtrl \n1.195 ± 0.091 \n4.12 ± 0.10 \n27.44 ± 2.05 \n3.05 ± 0.21 \nVib \n1.213 ± 0.063 \n4.24 ± 0.08 \n27.13 ± 1.28 \n3.09 ± 0.32 \nDay 14 \nCtrl \n1.386 ±0.107 \n4.09 ± 0.16 \n32.10 ± 2.12 \n3.06 ± 0.16 \nVib \n1.376 ±0.132 \n4.21 ± 0.10 \n30.07 ± 1.58 \n2.62 ± 0.21 \nDay 21 \nCtrl \n1.403 ± 0.049 \n4.38 ± 0.03 \n30.43 ± 1.20 \n3.79 ± 0.35 h \nVib \n1.426 ± 0.112 \n4.34 ± 0.05 \n30.34 ± 1.48 \n3.27 ± 0.33 \n\n",
"\n\n). a Reloading plus vibration (Vib) more than reloading control (Ctrl) in Sol, p<0.001; b Day 14 more than Day 7 in Sol, p<0.05; c Day 14 more than Day 21 in Sol, p=0.052; d Day 14 more than Day 7 in GM; e Day 14 more than Day 21 in GM, p<0.001; b) Sol: fiber type specific BrdU + /myogenic cell associated with type I, IIA and IIB fibers. a Vib more than Ctrl in type I, p<0.05; b Vib more than Ctrl in type IIA, p< 0.05; c Day 14 > Day 7 in type IIB, p<0.001; d Day 14 > Day 21 in type IIB, p<0.001; c) GM: fiber type specific BrdU + /myogenic cell associaed with type I, IIA and IIB fibers. a Vib more than Ctrl in type IIB, p<0.05 (interaction, p<0.001); b Day 14 > Day 7 in type IIA, p<0.01; c Day 14 > Day 21 in type IIA, p<0.01; d Day 14 > Day 7 in type IIB, p<0.01; e Day 14 > Day 21 in type IIB, p<0.001; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test."
] | [
"Fiber cross-sectional area (FCSA) (10 3 μm 2 ) of three fiber types: type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): No significant difference detected amongst all comparisons; b) gastrocnemius medialis (GM): a Reloading control (Ctrl) larger than reloading plus vibration (Vib) in type I FCSA, p<0.01; b,c Day 21 larger than Day 7, p<0.01 and p<0.001 respectively; d,e Day 21 larger than Day 14, p<0.01 and p<0.001 respectively; main effects detected from Two-way ANOVA coupled with post-hoc Bonferroni adjustment. No significant interaction detected.similarly in GM with muscle mass (~23%, p<0.001), pCSA (~17%, p<0.001) and FCSA (~26%, p<0.001), all compared with the corresponding value in TS.",
"The proportion (%) of three fiber types, type I, IIA and IIB from pH4.6 ATPase staining. a) soleus (Sol): a higher % type I in Day 21 than Day 7, p = 0.05; b lower % type IIB in Day 21 than Day 14, p<0.05; b) gastrocnemius medialis (GM): a higher % type I in reloading control (Ctrl) than reloading plus vibration (Vib), p<0.01 (interaction, p<0.05); b higher % type IIA in Vib than Ctrl, p<0.05 (interaction, p=0.05); main effects and interaction detected from Twoway ANOVA coupled with post-hoc Bonferroni test.",
"Proliferating myogenic cell counts (count / 100 fibers) from soleus (Sol) and gastrocnemius medialis (GM) muscles. a) overall bromodeoxyuridine (BrdU) + /myogenic cell in Sol (left panel) and GM (right panel",
"Contractile properties -specific force (sP o , gcm -2 ) and contraction time to peak twitch (TpT, ms) of soleus (Sol) and gastrocnemius medialis (GM) during reloading. The dotted line in each graph was the corresponding mean in unloading (TS) group. a) sP o -Sol: a reloading plus vibration (Vib) larger than TS, p<0.05; b Day 21 larger than TS, p<0.001; c Day 21 larger than Day 7, p<0.001; d Day 21 larger than Day 14, p<0.01; b) sP o -GM: a Day 21 larger than Day 14, p<0.05; c) TpT -Sol: a Day 21 slower than Day 14, p<0.05 (interaction, p<0.01); d) TpT -GM: a reloading control (Ctrl) faster than TS, p<0.001; b Vib faster than TS, p<0.001; c Day 7 faster than TS, p<0.05; d Day 14 faster than TS, p<0.001; e Day 21 faster than TS, p<0.05; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test.",
"Figure 1. Typical ATPase (pH4.6) staining images of weight bearing (WB, left column) and unloading (TS, right column) from soleus (Sol, upper panel) and gastrocnemius medialis (GM, lower panel). Type I (I, darkest), type IIA (IIA, lightest) and type IIB (IIB, intermediate) were indicated. *Note that no type IIB in Sol WB (upper left). Magnification: 200X.Table 2. Morphological data summary of Sol from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.001 respectively; e,f Day21 larger than TS, p<0.05 and p<0.001 respectively; g,h Day 14 larger than TS, p<0.01 and p<0.001; main effects detected from Two-way ANOVA. No significant interaction detected.Table 3. Morphological data summary of GM from TS, Ctrl and Vib at different timepoints including muscle mass (Mm), optimal length (Lo), physiological cross-sectional area (pCSA) and fiber cross-sectional area (FCSA). a,b Ctrl larger than TS, p<0.01 and p<0.001 respectively; c,d Vib larger than TS, p<0.05 and p<0.01 respectively; e Day21 larger than TS, p<0.001; f TS larger than Day 14, p<0.05; g Day 14 larger than TS, p<0.001; h Ctrl larger than Vib, p<0.01; main effects detected from Two-way ANOVA. No significant interaction detected.",
"). a Reloading plus vibration (Vib) more than reloading control (Ctrl) in Sol, p<0.001; b Day 14 more than Day 7 in Sol, p<0.05; c Day 14 more than Day 21 in Sol, p=0.052; d Day 14 more than Day 7 in GM; e Day 14 more than Day 21 in GM, p<0.001; b) Sol: fiber type specific BrdU + /myogenic cell associated with type I, IIA and IIB fibers. a Vib more than Ctrl in type I, p<0.05; b Vib more than Ctrl in type IIA, p< 0.05; c Day 14 > Day 7 in type IIB, p<0.001; d Day 14 > Day 21 in type IIB, p<0.001; c) GM: fiber type specific BrdU + /myogenic cell associaed with type I, IIA and IIB fibers. a Vib more than Ctrl in type IIB, p<0.05 (interaction, p<0.001); b Day 14 > Day 7 in type IIA, p<0.01; c Day 14 > Day 21 in type IIA, p<0.01; d Day 14 > Day 7 in type IIB, p<0.01; e Day 14 > Day 21 in type IIB, p<0.001; main effects and interaction detected from Two-way ANOVA coupled with post-hoc Bonferroni test."
] | [
"Figure 1",
"Figure 2a",
"(Figure 2a)",
"(Figure 2b)",
"Figures 3a and 3b",
"Figure 4 (a-c)",
"Fig-ures 4a and 4b)",
"Figure 4c",
"Figure 4b",
"Figure 5 (a-d)",
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"(Figure 5b; p<0.05",
"(Figure 5a)",
"(Figure 5c",
"(Figure 5d"
] | [] | [
"Introduction non-invasive biophysical modality, and its beneficial effects on musculoskeletal system are widely reported in various preclinical and clinical studies. Clinical trials showed that LMHFV prevented muscle atrophy in bed-rest patients and improved muscle performance with high compliance in community elderly [12][13] . Preclinical work from Xie and his colleague suggested vibration stimulated mice lower limbs muscle to hypertrophy 14 . Promoting proliferation and differentiation of myogenic cells as well as down-regulating genes involved in atrophy pathway could be the possible mechanisms of the muscle improvement by vibration treatment [15][16] .",
"With the evidences of LMHFV effectiveness in muscle improvement, it was hypothesized that LMHFV improves the outcomes of reloading disused muscles. The objectives of the current study were to investigate the effects of LMFHV treatment on: 1) fiber morphology, 2) myogenic cells proliferation and, 3) contractile function of soleus (Sol) and gastrocnemius medialis (GM), two weight-bearing limb synergist muscles, during 21 days of reloading period. Given the lack of information about reloading from prolonged disuse, the current work would also demonstrate any differential outcomes of reloading on Sol and GM.",
"A total of 48 6-month-old male Sprague-Dawley adult rats were obtained from the Laboratory Animal Service Centre of the Chinese University of Hong Kong. All animals were housed in temperature-controlled rooms with 12:12 hour darklight cycle. All procedures performed in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Ref: 10/093/MIS5).",
"Animals were hind-limb unloaded for 28 days individually based on Morey's tail suspension (TS) protocol 17 . Briefly, zincoxide plaster with a harness was wrapped around the tail and secured by surgical tapes. Animals were then suspended in head-down position at torso-to-ground angle of ≤30°, while hind-limbs were dangled down without any solid support from the tail-suspension cage. Free-cage movement, access to water and standard rat chow ad libitum with their forelimbs were allowed. The health status of the animals was monitored daily. Age-matched weight bearing rats (WB, n=6) were euthanized at the same time for TS model verification.",
"After 28 days of TS, part of the unloading rats were sacrificed immediately (without reloading) and served as control of unloading (TS, or referred as Day 0 baseline data, n=6). The remaining rats were reloaded by allowing free-cage movement by four limbs in standard rat cage independently. The reloading rats were randomly assigned to either reloading control (Ctrl) or reloading plus vibration (Vib). Animals in Vib received LMHFV (0.6g, 35Hz; g=gravitational acceleration) 20 min/day and 5 days/week. Animals were euthanized by overdosed pentobarbital 7, 14 and 21 days after reloading (n=6/treatment/timepoint) 18 . Left Sol and GM were freshly harvested, weighted and subjected to in vitro functional assess-ment; the contralateral muscles were snap-frozen in melting isopentane, embedded in OCT compound and stored at -80°C until cryosectioning.",
"To label proliferative cells in reloading muscles, a time-released pellet of 5-bromo-2'-deoxyuridine (BrdU, nucleotide analog to thymidine) (Innovative Research of America, FL, USA) was implanted subcutaneously 14 days before each endpoint 19 . Briefly, the animal was first anesthetized by isoflurane and according to manufacturer's instructions, the neck was shaved and disinfected by alcohol before a 5 mm longitudinal incision was made. A BrdU pellet was then put into a pocket 20 mm beyond the incision site subcutaneously. For the rats euthanized at Day 7 post-TS, BrdU pellet was implanted when the rats were still tail-suspended (i.e. day 21 of TS).",
"Consecutive 7μm cross-sections of right Sol and GM muscles were cut using cryostat. ATPase staining conditioned at pH 4.6 at room temperature was performed to distinguish the three muscle fibers: type I (darkest), IIA (lightest) and IIB (intermediate), based on Hintz's protocol and images of section were captured under the light microscope (Leica DFC490, Leica Microsystems) 20 . The whole section of Sol and the core region in the proximal head of GM (with mixed fibers profile) were analyzed 21 . Three random fields were captured to analyze the effects of LMHFV on different fiber types. The fiber crosssectional area (FCSA) and the proportion (%) of fiber types I, IIA and IIB were measured with ImagePro Plus analysis software (v5.1.0.20, Media Cybernetics, MD, USA).",
"To identify proliferative myogenic cells and the associated fiber types in both Sol and GM, a BrdU/laminin double-staining protocol was performed on the ATPase stained cryosections as modified from Siu's protocol 19 . Primary antibodies included mouse anti-BrdU (1:100, Abcam) and rabbit anti-rat laminin (1:200, Abcam). Secondary antibodies included Alexa Fluor555-conjugated goat anti-mouse IgG (γ2a) secondary antibody (Zymed) and Alexa Fluor488-conjugated donkey antirabbit IgG(H+L) antibody working concentration at 4 μg/ml. BrdU-positive nuclei lying on the laminin-stained basement membrane were counted and considered as proliferative myogenic cells. Immunofluorescent images obtained were co-localized with ATPase staining to identify the fiber types that the proliferative myogenic cells associated with.",
"The protocols for muscle functional assessment were modified from Plant and Segal's studies [22][23] . The distal tendon of muscle was sutured and hung onto the transducer (305C-LR, Aurora Scientific Inc.) with proximal end anchored to the in vitro functional test apparatus (805A, Aurora Scientific Inc., Ontario, Canada). Muscles were stimulated to contract by square-wave pulses (0.2 ms width) at supramaximal voltage (80V). The detected forces were recorded by Dynamic Muscle Control (DMC v5.1) and analyzed with Dynamic Muscle Analysis (DMA v3.2) software (Aurora Scientific Inc.). All muscles were isometrically stimulated at optimal length (Lo) at room temperature (around 25℃ in air-conditioned laboratory) which was determined by eliciting isometric twitch with increasing muscle length until maximal force was generated 22 .",
"For Sol, force-frequency relationship was determined by stimulating the muscle for 1s at 10, 20, 40, 60, 80 and 100 Hz with 5-minute resting intervals. Maximal tetanic force of Sol was defined as the largest force obtained from the force-frequency relationship. For GM, continuous tetanic stimulation was unfavorable due to potential core anoxia induced by poor oxygen/nutrient perfusion to the bulky GM that deteriorated contractile function and would lead to severe force underestimation. Hence, isometric tetanic force of GM was achieved by delivering supramaximal stimulation, 500 ms, 150Hz at optimal length once only. All force measurement was normalized by physiological cross-sectional area (pCSA). It was estimated by pCSA= mass (g)/[muscle length (cm) x muscle density (gcm -3 )], where muscle density is assumed as 1.056 (gcm -3 ) 24 .",
"All data were expressed in mean ± standard deviation. Twoway analysis of variance (ANOVA) test was applied to analyze the main effects amongst treatment and reloading period. Posthoc multiple comparison corrected by Bonferroni adjustment was performed when significant main effects were detected. When significant interaction was detected, independent Student's t-test were used for further comparisons between Vib and Ctrl groups. All statistical analyses were performed with SPSS 20.0 (IBM, NY, USA). Statistical significance was set at p<0.05.",
"The Sol muscle mass (Mm), pCSA and FCSA were significantly decreased by 33%, 31% and 40% respectively in TS group whereas in GM, these were decreased by 26%, 23% and 43% respectively (all p< 0.001; Table 1). Muscle fibers atrophy and slow-to-fast fiber type transition could be observed in both Sol and GM. Besides, increase in interstitial space in TS was observed ( Figure 1).",
"The morphological data of Sol and GM muscles in Ctrl and Vib groups at different reloading period were summarized in Tables 2 and 3 respectively. Reloading induced an increase in most of the morphological outcomes during reloading but differential responses to reloading and LMHFV treatment could be observed between Sol and GM. Particularly, decrease of Lo (~3%, p<0.05) during reloading compared with TS and smaller overall FCSA (Vib<Ctrl; p<0.01) in LMHFV group could only be observed in GM. Instead, Lo of Sol increased with reloading compared with TS (~7%, p<0.05) and LMHFV treatment did not demonstrate any effects on Sol overall FCSA (Vib=Ctrl; p>0.05). Other morphological parameters including muscle mass (~35%, p<0.001), pCSA (~28%, p<0.001) and FCSA (~27%, p<0.01) increased in Sol during reloading and ",
"The changes of FCSA of different fiber types (type I, IIA and IIB) in Sol and GM were illustrated in Figure 2a and 2b respectively. Fiber-type specific FCSA of Sol were similar and no difference (p>0.05) was detected during reloading (Figure 2a), whereas GM showed significant increase at Day 21 compared with Day 7 and Day 14 in type I (both p<0.01) and type IIB fibers (both p<0.001) (Figure 2b). Besides, Ctrl type I FCSA, but not type IIA or IIB, of GM was larger than that of Vib (p<0.01) and echoed with the overall FCSA decrease in Vib.",
"For the fiber type proportion, the percentage of all fiber types in Sol and GM were illustrated in Figures 3a and 3b respectively. Sol was made up of >90% type I fibers and increasing trend of type I percentage was observed during reloading (Day 21>Day 7, p=0.052). On the contrary, Sol type IIB percentage decreased from Day 14 to Day 21 significantly (p<0.05). Vibration treatment showed no effects on Sol fiber types switching.",
"Regarding GM fiber percentage, GM was composed of a mix of three fiber types with type IIB constituting ~50% of the total fibers in the core region of GM. Different from Sol, fiber type transition during reloading was absent in GM. Comparing Ctrl and Vib, there was a higher type I proportion in Ctrl (p<0.01; interaction with p<0.05) whereas type IIA was higher in Vib (p<0.05; treatment*timepoints interaction with p=0.05). Significant interactions were found in these two main effects i.e. reloading time and treatment. Therefore post-hoc test was carried out to compare the differences between Ctrl and Vib across timepoints. The post-hoc independent t-test analysis showed a higher type I proportion in Ctrl at Day 7 (Ctrl>Vib, p<0.01) whereas that for type IIA proportion was higher in Vib at Day 7 (Vib>Ctrl, p<0.05) and Day 21 (Vib>Ctrl, p=0.05).",
"The counting results were illustrated in Figure 4 (a-c). The Vib group generally showed higher counting in both Sol and GM. Overall counting, and fiber type specific counts in type I and IIA in Sol were higher in Vib than Ctrl (all p<0.001, Fig-ures 4a and 4b). Although overall counting showed no difference between Vib and Ctrl in GM, fiber type specific counting revealed higher counts in type IIB fiber of Vib group (p<0.05, Figure 4c). Significant treatment*timepoints interaction (p<0.001) was detected in this observable increase of myogenic cells proliferation in Vib during reloading. The post-hoc independent t-tests showed that the increase of type IIB count in Vib was at Day 14 (Day 14-Vib>Day 14-Ctrl, p<0.001) while the other two timepoints had no differences.",
"Moreover, the peak counting during reloading period was at Day 14 in all three fiber types. The counts actually covered all the proliferative activities happened from day 0 to day 14 of reloading (See Method for details). The time of peak activity was apparently independent of the fiber types, treatment and muscles of interest (except type IIA in Sol which the counts maintained high in Vib, see Figure 4b).",
"The contractile properties of Sol and GM were illustrated in Figure 5 (a-d). Increase in sP o during reloading was observed in Sol (Figure 5a; Day 21>TS and Day 7, both p<0.001 and Day 14, p<0.01) while force deficit, compared to TS level, was evident in GM throughout the reloading period in spite of the increasing trend from Day 14 to Day 21 (Figure 5b; p<0.05). LMHFV treatment appeared promoting force generating capacity in Sol as indicated from higher sP o in Vib compared with TS (Vib>TS, p<0.05) which was, however, absent in Ctrl (Figure 5a). GM sP o in Vib was not different from the Ctrl nor the TS. The contraction time, TpT of Sol appeared longer at Day 21 as compared with Day 14. Nevertheless, given a significant interaction detected (p<0.01), further post-hoc comparison showed that TpT of Ctrl-Day 21 was significantly longer than Vib-Day21 (p<0.05) and TS (p<0.01) (Figure 5c). As for GM, TpT decreased and was significantly shorter than TS during the reloading period. Otherwise, the changes of TpT amongst all comparisons between Vib and Ctrl or among timepoints were not different significantly (p>0.05) (Figure 5d).",
"The current study aims to examine the effects of LMHFV intervention on the regrowth process of reloading muscles from prolonged disuse at functional, histo-morphological and cellular levels. The results showed LMHFV intervention was not injurious to reloading muscles, both Sol and GM, while myogenic cells proliferative activities were enhanced. Only in the GM demonstrated type I fiber hypertrophy and a type I to type IIA fiber transition was observed upon LMHFV treatment during reloading. Further investigations on the fiber type specific responses of various muscles toward LMHFV and interaction of myogenic cells with the associated myofiber were recommended.",
"The 28-day tail suspension prolonged muscle disuse model (TS) in rat was applied to investigate the effects of LMHFV treatment on two reloading lower limbs muscles, Sol and GM. It was well known slow-twitch muscle fibers were more susceptible to disuse atrophy compared with the fast counterparts 9-10 . Hence differential influences of 28-day TS on Sol (slow-dominant) and GM (fast-dominant except the mixed core region) should be expected. Our results were consistent with the previous findings that the gross morphology of Sol was reduced more than that of GM in response to TS [25][26] . For FCSA, the changes appeared similar between Sol and the core region of GM. The peripheral region of GM, in fact composed mainly of type IIB fibers, showed no changes in FCSA within the region (data not shown). Hence, if whole GM was considered, the effects of TS on GM were not as prominent as in Sol. This intrinsic difference should be taken into consideration in interpreting the differential responses of Sol and GM to reloading.",
"In Sol, reloading regrowth could be observed from morphological (gain of Mm, Lo, and pCSA) and fiber typing data (increased FCSA and fast-to-slow type transition). Coupled with functional data, the increasing specific sP o and longer TpT, reloading induced regrowth of Sol was evident and consistent with previous report 8 . Furthermore, it was apparent that Vib promoted gain of force generating capacity as indicated from the significantly higher sP o compared with TS. This increasing trend, nevertheless, was not significant in Ctrl. Longer timepoints of Vib treatment may allow a clearer picture of this beneficial effect. Comparing with Sol, the case in GM was rather more complicated. Although Mm, pCSA and FCSA increased during reloading suggesting apparent muscle regrowth, Lo decreased at the early timepoints and specific force deficit in GM was observed during the whole reloading period. This deficit was neither be improved nor deteriorated by LMHFV. To explain the deficit, reloading induced injury in GM was suggested 8 .",
"Reloading injury is caused by myofiber disruption due to stretching or increased muscle strain when the shortened disused myofibers were reloaded and lengthened. It was known that TS unloaded muscle fibers were shortened to adapt to the prolonged plantar flexion of the foot, which was typically observed in prolonged TS model 27 . In addition, the anatomy of gastrocnemius muscle which connects two joints (ankle and knee) and is composed of fast-fibers in majority may render its higher susceptibility toward eccentric/ stretching injury during reloading than Sol 8,28 . It might explain our current findings that GM showed a prolonged functional deficit during reloading even it was less affected by unloading than Sol 28 . Given Sol also experienced fiber shortening, it was expected to be affected by reloading injury and demonstrated force deficit. Previous studies showed the onset of force decrement in Sol was around 1-2 days from reloading and the weakness may last no longer than 9 days 8,29 . Our results did not contradict with these findings that force generating capacity remained similar to TS (no increase) at Day 7 (the earliest timepoint of this study) and an increase could be observed since Day 14. In summary, LMHFV have mild effects on Sol functional recovery and is non-injurious to mechanical injury-prone GM during reloading. Optimization of LMHFV treatment period might enhance the beneficial outcomes and lead to significant difference from Ctrl in Sol without adverse effects on GM.",
"Myogenic cell proliferative activities were found elevated by counting overall myogenic cells regardless of the associated fiber types in Sol only. In addition to overall counting, the current investigation adopted the novel fiber type specific myogenic cell analysis to evaluate the effects of LMHFV on myogenic cell activities. The concept of fiber associated myogenic cell progeny was suggested from Verdijk's group including reduced satellite cells in aging type II skeletal muscle fibers and increased SC counts in type II fibers after eccentric exercises [30][31] . The effects of LMHFV on fiber-type specific counts were found in both Sol (type I and IIA) and GM (type IIB). Stimulatory effects of LMHFV on myogenic cells proliferation were therefore evident in both Sol and GM. This indicated that the analysis of overall myogenic cell activities might underestimate or even misinterpret the effects of particular intervention.",
"Referring to Verdijk's studies, the fiber type specific changes could be primary to the vulnerable type II fibers in eccentric and aging models [30][31] . Hence it was questionable if the fiber type specific effects observed in this study were related to fiber susceptibility to injury. Our functional results showed vibration was not injurious to Sol and even in the mechanical sensitive GM. Instead, trend of faster force regain in Sol was found. Similarly, administration of vibration of similar regime (45 Hz, 0.6 g) on dystrophic mice model, with skeletal muscles well-known susceptible to mechanical damages, showed no detrimental effects as well 32 . Hence damages to muscle cells by vibration treatment should be absent or negligible, if any. In the same study, 7 days of treatment could upregulate muscle paired box gene-7 (Pax7) gene expression, a myogenic cell marker particularly in quiescent muscle satellite cell, which suggested promotion of myogenic cells activities in vivo 32 . In vitro vibration treatment on myogenic cell line, C2C12, was also reported to increase cell proliferation and myogenic differentiation without any injury or challenge to the cultured cells 15 . Therefore, it was conceived to regard the current findings primarily as direct promotion of myogenic cell proliferation and less likely related to induced myofiber injury. However, it remained questionable why myogenic cells associated with particular fiber types appeared more responsive to LMHFV. Recruitment of different kinds of motor units during vibration treatment, according to tonic vibration reflex (TVR) was evident and suggested only fibers under the activated motor units responded to contract [33][34][35] . Besides, communication mechanisms between the associated myofibers and adjacent myogenic cells have been reported [36][37] . It implied the possibility that LMHFV might trigger specific types of fiber to contract and stimulate the associated myogenic cell activities leading to the fiber type specific myogenic cell responses. More speculations on interaction of myogenic cells and the associated fibers upon mechanical stimuli are suggested.",
"In addition to stimulation of type IIB myogenic cells activities, LMHFV induced fast fiber type specific effects on other outcomes of GM, for instance, suppression of type I fiber hypertrophy and shift of type IIA fibers from type I counterparts. A trend of shorter TpT at Day 21 could be observed in GM as well. LMHFV hence apparently favored GM fast fiber changes during reloading. It was interesting that Sol showed increases in both type I and IIA associated myogenic cell activities. Also, Xie's study showed vibration stimulated both type I and type IIA fiber hypertrophy in growing mice 14 . Although fast-to-slow fiber type transition in Sol was not affected by LMHFV, significantly shorter TpT was detected. It was unclear if other factors like calcium handling and cross-bridge kinetics attributed to the difference. In general, the fiber type specific responses appeared muscle-dependent. It was worthy for further investigations on the differential responses of the two muscles to various vibration regimes (e.g. changes in frequency and magnitude) and to examine the fiber type specific changes.",
"In conclusion, LMHFV intervention did no harm on the contractile functions of reloading muscles during 21 days of reloading while myogenic cell activities could be promoted in both Sol and GM. However, a limitation of the current study was that the cell fate of the increased number of myogenic cells was uncertain. It remains to be seen whether the fiber type specific changes due to LMHFV were dependent on the elevated myogenic cell activity. The reloading effects on Sol and GM were different and the concerns of customizing intervention for particular muscles should be well-considered so that therapeutic effects were maximized without compromis-ing other muscles toward injury. The use of LMHFV in treating prolonged disused muscles should be further investigated to depict the regulatory mechanisms of the myofibers on the associated myogenic cells as well as the fiber type specific responses in different LMHFV regimes."
] | [] | [
"Materials and methods",
"Animal care and experimental design",
"Proliferative cell labeling",
"Histology",
"Immunohistochemistry",
"In vitro muscle functional assessment",
"Statistical analysis",
"Results",
"TS model verification",
"Muscle morphology",
"Fiber typing -FCSA and fiber proportion",
"Proliferating myogenic cell counting",
"Contractile properties -specific force (sP o ) and time to peak twitch (TpT)",
"Discussion",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 ."
] | [
"TS (n=6) \n0.200 ± 0.017 a \n6.20 ± 0.62 a \n2.87± 0.12 a \n1.164 ± 0.090 a \n25.68 ± 2.55 a \n2.59 ± 0.20 a \nWB (n=6) \n0.300 ± 0.024 \n8.85 ± 0.94 \n4.74 ± 0.31 \n1.556 ± 0.100 \n33.14 ± 2.07 \n4.57 ± 0.63 \n\nTable 1. The Sol (Left column) and GM (Right column) muscle mass (Mm), physiological cross-sectional area (pCSA) and fiber cross-sectional \narea (FCSA) between weight-bearing group (WB) and hind-limb unloading group (TS) (n=6, mean ± SD). a WB larger than TS, p<0.001 by in-\ndependent samples t-test. \n\nGroup/ \nSol \n\nTimepoint \nMm (g) \nLo (cm) \npCSA (10 -2 cm 2 ) \nFCSA (10 3 μm 2 ) \n\nTS \n0.200 ± 0.017 b,d,f,h \n3.06 ± 0.11 e \n6.20 ± 0.62 b,d,f,h \n2.87 ± 0.12 a,c,g \n\nDay 7 \nCtrl \n0.221 ± 0.034 \n2.89 ± 0.10 \n7.21 ± 1.00 \n3.51 ± 0.58 \nVib \n0.226 ± 0.021 \n3.01 ± 0.08 \n7.09 ± 0.68 \n3.37 ± 0.17 \n\nDay 14 \nCtrl \n0.268 ± 0.017 \n3.10 ± 0.16 \n8.40 ± 0.87 \n3.86 ± 0.27 \nVib \n0.268 ± 0.007 \n3.09 ± 0.10 \n8.20 ± 0.16 \n3.44 ± 0.23 \n\nDay 21 \nCtrl \n0.273 ± 0.025 \n3.34 ± 0.02 \n7.84 ± 0.71 \n3.25 ± 0.31 \nVib \n0.271 ± 0.015 \n3.21 ± 0.16 \n7.97 ± 0.56 \n3.49 ± 0.41 \n\nGroup/ \nGM \n\nTimepoint \nMm (g) \nLo (cm) \npCSA (10 -2 cm 2 ) \nFCSA (10 3 μm 2 ) \n\nTS \n1.164 ± 0.100 a,d,e,g \n4.30 ± 0.08 f \n25.68 ± 2.55 b,d,e,f \n2.59 ± 0.20 b,c,e \n\nDay 7 \nCtrl \n1.195 ± 0.091 \n4.12 ± 0.10 \n27.44 ± 2.05 \n3.05 ± 0.21 \nVib \n1.213 ± 0.063 \n4.24 ± 0.08 \n27.13 ± 1.28 \n3.09 ± 0.32 \nDay 14 \nCtrl \n1.386 ±0.107 \n4.09 ± 0.16 \n32.10 ± 2.12 \n3.06 ± 0.16 \nVib \n1.376 ±0.132 \n4.21 ± 0.10 \n30.07 ± 1.58 \n2.62 ± 0.21 \nDay 21 \nCtrl \n1.403 ± 0.049 \n4.38 ± 0.03 \n30.43 ± 1.20 \n3.79 ± 0.35 h \nVib \n1.426 ± 0.112 \n4.34 ± 0.05 \n30.34 ± 1.48 \n3.27 ± 0.33 \n\n"
] | [
"Table 1",
"Tables 2 and 3"
] | [
"Differential effects of low-magnitude high-frequency vibration on reloading hind-limb soleus and gastrocnemius medialis muscles in 28-day tail-suspended rats",
"Differential effects of low-magnitude high-frequency vibration on reloading hind-limb soleus and gastrocnemius medialis muscles in 28-day tail-suspended rats"
] | [] |
237,533,192 | 2022-01-10T04:10:36Z | CCBY | https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/fsn3.2466 | GOLD | 34ef968bf1b60d5eefce5f2a00dc213dc82fde3a | null | null | null | null | 10.1002/fsn3.2466 | 3182000383 | 34532017 | 8441376 |
Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts
2021
Sang-Soo Lee
Department of Biochemistry
Chungnam National University
DaejeonKorea
Yong-An Kim
Department of Biochemistry
Chungnam National University
DaejeonKorea
| Bokkee
Core Laboratory for Convergent Translational Research
Korea University College of Medicine
SeoulKorea
Eun
Jayeon Yoo
National Institute of Animal Science
RDA
WanjugunKorea
Eun-Mi Kim
Department of Predictive Toxicology
Korea Institute of Toxicology
DaejeonSouth Korea
| Myoung
Soo Nam [email protected]
Division of Animal Resource Science
Department of Biochemistry
Chungnam National University
DaejeonKorea
Division of Animal Resource Science
Chungnam National University
34134DaejeonKorea
Chungnam National University
34134DaejeonKorea
| Kee
K Kim [email protected]
Department of Biochemistry
Chungnam National University
DaejeonKorea
Sang-Soo Lee
Yong-An Kim
Soo Myoung
Kee K Nam
Kim
Kee K Correspondence
Kim
Myoung Soo Nam
Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts
Food Sci Nutr
9202110.1002/fsn3.2466Received: 5 May 2021 | Revised: 13 June 2021 | Accepted: 29 June 2021O R I G I N A L R E S E A R C H Funding information Rural Development Administration, Grant/ Award Number: PJ01354401
Sarcopenia is a disease characterized by the loss of muscle mass and function that occurs mainly in older adults. The present study was designed to investigate the hypothesis that water extract of Lycium chinense (WELC) would improve muscle function and promote myogenesis for sarcopenia. We investigated the effect of water extracts of L. chinense on muscular endurance function and myogenesis to examine its efficacy in sarcopenia.Intake of WELC-containing cheese enhanced the muscular endurance function of mice in treadmill endurance tests. In addition, the crosssectional areas of muscle fibers in the gastrocnemius muscle of L. chinense-fed mice were greater than that of control mice. Furthermore, WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. Taken together, our results suggest that L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions.
| INTRODUC TI ON
Muscle formation occurs during development, and during regeneration of muscle tissue following damage by exercise or disease (Menko & Boettiger, 1987;Yu et al., 2014).
During the process of muscle regeneration, myocardial cells exit the stationary/quiescence state and differentiate to form myofibers through cell fusion. This differentiation process is called myogenesis and is controlled by various signaling cascades.
Various myogenic proteins, such as paired Box Protein Pax-7 (Pax7), myoblast determination protein 1 (MyoD), myogenin (MyoG), and myosin heavy chain 3 (Myh3) represent the different myogenic stages (Rudnicki et al., 1992;Weintraub et al., 1989;Wright et al., 1989). Myoblasts form thick and long myotube cells through the process of myogenesis and the expression of myogenic proteins and the length and thickness of myotubes are very important factors in assessing myogenesis. Suppression of myogenesis due to various factors, such as aging, inhibits myotube formation that prevents the normal formation of muscle tissues, including skeletal muscle, resulting in muscle loss that leads to reduction in the total amount of muscles (Bodine et al., 2001;Janssen et al., 2004;Suetta et al., 2013).
Muscle loss is a typical muscle and nervous system disease and is one of the symptoms of aging. The total amount of skeletal muscle decreases gradually due to the natural process of aging and is replaced by adipose tissue resulting in a condition called sarcopenia (Roche, 1994). Sarcopenia mainly occurs in adults over the age of 65 years, and the number of older adults is increasing (Bae & Kim, 2017;von Haehling et al., 2010). Sarcopenia decreases the total amount of skeletal muscles and consequently increases the risk of fractures due to insufficient protection of the bones by skeletal muscles leading to decrease in the amount of activity and metabolic diseases such as diabetes, and thus has a tremendous impact on human health (Butcher et al., 2018;Leslie et al., 2020). Although sarcopenia is influenced by several factors, such as genetics or drugs, it is also affected by diet. Recent studies have confirmed that muscle function is improved by eating cheese with high protein and saturated fatty acid content (Alemán-Mateo et al., 2012;Devries & Phillips, 2015).
In addition, the effect of various natural products on myogenesis are being actively investigated (Chen et al., 2019;Cho et al., 2020;Lee et al., 2020).
Lycium chinense is a medicinal herb cultivated throughout Asia.
It protects the liver, has beneficial effects in osteoporosis, and has been used as a herbal medicine for its anticancer and antioxidant properties (Mocan et al., 2014). The efficacy of L. chinense is due to its high content of polyphenols, carotenoids, and amino acids (Kruczek et al., 2020;Thiruvengadam et al., 2020). L. chinense also contains betaine, a natural amino acid and a precursor of methionine.
Although betaine has been studied for its role in improving muscle function, the effect of L. chinense with a high content of betaine on myogenesis and muscle function has not yet been studied (Cholewa et al., 2018;Senesi et al., 2013).
In this study, the effect of water extract of L. chinense (WELC) on muscle endurance and muscle formation was investigated. In addition, treatment with WELC and betaine was shown to influence myogenesis in C2C12 and primary myoblasts.
| MATERIAL S AND ME THODS
| Animals and experimental diets
BALB/c mice (8-week-old male; Daehan Biolink) were housed at 22 ± 2 ℃, with 50% ± 5% relative humidity under 12 hr light/dark cycle with ad libitum access to standard chow diet and water. All mice were acclimated for 7 days before use in experiments. Mice were divided into three groups (n = 4 mice/group) as follows: standard chow diet (Cont.), standard chow diet containing 40% cheese (Che.), and Che. with 3% L. chinense (Che. + LC). During the 6-week feeding period, body weight was recorded every 7 days, and food intake was recorded every 3 days.
2.
2 | Extracts, asiago cheese, experimental diets, and chemical preparation Lycium chinense was gifted by the Gogi Berry Research Institute, Cheongyang, Republic of Korea. Dried L. chinense (25 g) was extracted with distilled water (1 L) at boiling temperature for 2 hr.
After the water evaporated, the extracts were freeze-dried.
Six month-fermented asiago cheese, and 6 month-fermented asiago cheese containing 3% L. chinense extract were used. Betaine (Sigma Aldrich) was used as a marker for L. chinense. The Asiago
Cheese was prepared by the Segato et al. method (Segato et al., 2007). The amount of added L. chinense extract was 0% and 3.0%. Ripening of the control of Asiago Cheese was performed for 6 months in the ripening room (10 ± 2 °C and 85 ± 5% of relative humidity).
| Exercise treadmill test
The exercise treadmill test was performed at a rate of 10 cm/s for 3 min with a 0% slope. The speed was gradually increased to 15 cm/s and then maintained at 45 cm/s until the mouse was unable to run for more than 10 s.
| Serum biochemical analysis
The effects of the experimental diets on serum glutamic-oxaloacetic transaminase (GOT), glucose, blood urea nitrogen (BUN), total cholesterol, triglyceride, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured using an auto-analyzer (TBA-40FR; Toshiba).
| Cell culture
C2C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% streptomycin/penicillin at 37 °C in a humidified 5% CO 2 atmosphere. When the cells reached 90% confluence, the culture medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% horse serum (Gibco) and 1% streptomycin/penicillin. While inducing myogenesis, the medium was replaced with fresh medium every day.
| PLO and collagen coating
To coat the myoblast culture dishes or plates with poly-L-ornithine (PLO, Sigma) and collagen (Santa Cruz Biotechnology), the dishes and plates were incubated 6 hr at 25 ℃ with 0.001% solution of PLO in sterile water at 5 µg/cm 2 or 0.3 mg/ml solution of collagen in sterile water at 5 µg/cm 2 . The extra solution was aspirated and dishes or plates were dried in UV lamp exposure and washed two times with phosphate-buffered saline (PBS).
| Isolation of mouse primary myoblasts
Mouse primary myoblasts were isolated from 3-day-after-birth mouse hind limb skeletal muscles. Muscles were separated from the bones and fat and subjected treatment with 1.5 U/ml collagenase D and 2.4 U/ml dispase solution (1:1) for 1 hr to isolate myoblasts.
Isolated myoblasts were centrifuged at 300 g for 5 min and resuspended in growth medium consisting of Ham's F-10 medium with 10% calf serum (GIBCO), 1% penicillin-streptomycin, and 2.5 μg/ml fibroblast growth factor. Mouse primary myoblasts were cultured in poly-L-ornithine (PLO, Sigma) coated dish for culture, or collagen (Santa Cruz Biotechnology) coated dish to induce myogenesis at 37 ℃ in a humidified 5% CO 2 atmosphere. When the mouse primary myoblasts reached 90% confluence, the culture medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% horse serum (Gibco) and 1% streptomycin/penicillin. When inducing myogenesis, the medium was replaced with fresh medium every day.
| Immunoblot analysis
After at least 3 days of differentiation, the C2C12 cells or mouse primary myoblasts were lysed with cold tris-triton lysis buffer.
Identical amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk in PBS with 0.1% tween 20 (PBST) for 1 hr and incubated with primary anti-Myh3 (1:500, SC-53091, Santa Cruz), anti-MyoG (1:500, SC-52903, Santa Cruz), and anti-GAPDH (1:2,000, SC-47724, Santa Cruz) antibodies for 12 hr. After washing the membranes in PBST, immunoreactive signals were obtained using horseradish peroxidase-conjugated secondary antibodies (Abcam) and Super Signal system (Pierce Chemical).
| Immunofluorescence
Mouse primary myoblasts were plated in a 4-well collagen-coated chamber slide, and myogenesis was induced for 5 days. The myoblasts were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, blocked with 5% goat serum for 1 hr, and incubated overnight with primary antibodies against Myh3 (1:250, SC-53091, Santa Cruz) at 4 °C.
Alexa Fluor 488-conjugated goat antibody (1:500, Thermo Fisher Scientific) against mouse IgG was used as the secondary antibody. Nuclei were co-stained with DAPI (1:1,000; Thermo Fisher Scientific).
| Statistical analysis
All results are expressed as the mean ± standard error of the mean of at least three separate experiments for each group. Comparisons between experimental groups were performed using two-tailed Student's t test (SPSS software, version 12.0; BMI). Significance was set at p < 0.05.
| RE SULTS
| Physical characteristics and food supplements
To compare the protein and fat contents of cheese-containing diets, we quantified the nutrients in the experimental diets (Table 1). The carbohydrate content of Cont. group (49.1 g/100 g) was higher than that of the Che. (30.5 g/100 g) and Che. + LC (30.4 g/100 g) experimental diet groups. Protein content of the Che. (25.4 g/100 g) and Che. + LC (24.8 g/100 g) experimental diets groups was higher than that of the Cont. group (20.1 g/100 g). Fat content of Che. (20.5 g/100 g) and Che. + LC (20.9 g/100 g) experimental diets groups was higher than that of the Cont. group (4.2 g/100 g). Calorie from the fat of Che. (45.2% of total calories), and Che. + LC (46.0% of total calories) groups was approximately fourfold higher than that of the Cont. (11.9% of total calories) group. As the experimental groups were fed highfat diets, we investigated whether experimental high-fat diets induced weight gain or metabolic change in each mouse group by measuring bodyweight after 6-week ad libitum feeding. We found that the mouse body weights increased by 8.9 and 8.5 g in the Che. and Che. + LC groups, respectively, whereas it increased by 5.9 g in the Cont. group (Figure 1a). Although the food intake did not differ among the groups, the calorie intake of Che. and Che. + LC groups was higher than that of the Cont. group (Figure 1b). As the calorie intake of the experimental groups was higher than that of the Cont. group, we measured the weight of the heart, liver, FAT, gastrocnemius (GAS), and soleus (SOL) muscles to examine metabolic changes in the three groups. Although the heart, liver, and GAS muscle mass did not differ among the groups, fat mass in the Che. group (0.98 ± 0.22 g) was higher than that in Cont.
(0.49 ± 0.18 g) and Che. + LC groups (0.75 ± 0.31 g) (Figure 1c).
Moreover, the soleus muscle mass was slightly increased in the Che. + LC group (8.15 ± 0.52 mg) compared to that in the Cont.
(7.16 ± 0.76 mg) and Che. groups (7.46 ± 0.39 mg). These results suggest that L. chinense extracts decreased fat mass and increased SOL muscle mass, whereas the other organs and tissue masses remained unaffected.
| Serum levels of lipids, GOT, BUN, and blood glucose
As the fat mass in the L. chinense-fed group decreased and SOL muscle mass increased, we examined the metabolic changes in the groups by serum analysis. GOT, BUN, blood glucose, cholesterol, triglyceride, HDL, and LDL levels were compared among the groups ( Figure 2). GOT, BUN, and blood glucose levels were nor-
| Muscle fiber composition and muscle endurance function
As the SOL muscle mass was increased in L. chinense-fed mice ( Figure 1c), we measured the distribution of muscle fiber size and thickness of GAS muscles by H&E staining (Figure 3a). The fiber size distribution showed a shift toward larger fiber diameter ( Figure 3b). Composition of the hypertrophic muscle fibers (˃1,750 μm 2 ) in Che. + LC group (37.5%) was also higher than that of the Cont. (20.9%), and Che. groups (14.3%). Further, the average muscle fiber area of Che. + LC group (1,665.1 ± 141.9 μm 2 ) was larger than that of the Cont. (1,478.0 ± 23.9 μm 2 ) and Che. groups Che. groups (481.7 ± 47.1 m). These results suggest that L. chinense extract improves the fiber size and endurance function of the GAS muscles.
| Activity of betaine on myogenesis
Because betaine is a marker of L. chinense extracts, we investigated whether betaine is a key factor in muscle improvement.
We analyzed the betaine content in WELC by HPLC and found F I G U R E 1 Changes in body weight, food intake, and relative organ weights of mice fed experimental diets. Mice were provided normal diet, cheese (40% of feed), or cheese containing L. chinense (40% of feed) for 6 weeks. (a) Body weights and weight gain were measured every week. (b) Intake of food and calorie were measured every 3 days. (c) Tissue/organ weights were measured as representative organ.
All experimental data are presented as means ± standard deviation (n = 4/group). N.S., not significant; *, p < 0.05 (paired t test)
that 1 mg of WELC contained 14.72 μg of betaine ( Figure S1).
Betaine showed no cytotoxicity in C2C12 cells at concentrations of up to 5,000 μM ( Figure S2). C2C12 cells were differentiated in the presence of betaine for the indicated number of days, and
Myh3 and MyoG protein levels were examined by immunoblot analysis. MyoG expression increased 2.23-fold on day 5 after treatment with 50 μM betaine (Figure 4a). Myh3 expression was also increased by betaine treatment in a dose-dependent manner. In addition, betaine treatment increased myotube length in a dose-dependent manner (Figure 4b). These results suggest that betaine is an effective substance in L. chinense extracts that is involved in myogenesis. As myogenesis of C2C12 cells was promoted by betaine treatment, we examined the effect of betaine and WELC on myogenesis of mouse primary myoblasts. Mouse primary myoblasts were differentiated in the presence of 50 μM betaine. Myh3 expression was significantly increased by betaine treatment (Figure 5a). In addition, the function of betaine in promoting myotube formation in C2C12 cells was confirmed in mouse primary myoblasts (Figure 5b). Taken together, L. chinense and its marker betaine enhance myogenesis and muscle endurance in mice.
| D ISCUSS I ON
Skeletal muscle is one of the main organs in the body that performs various functions, such as protecting bones, providing muscle strength, and is important for maintaining health (Danielewicz et al., 2019). Nutrients, including proteins, are among the most important factors in maintaining skeletal muscle (Chanet et al., 2017;Hamarsland et al., 2019). Several recent studies have focused on the effect of continuous intake of foods rich in nutrients, including protein and saturated fatty acids, such as cheese, on improvement in muscle function. L. chinense, a herb cultivated all over Asia, has been used as a herbal supplement for a long time and contains several physiologically active substances including a high content of betaine, which is well known to have a positive effect on muscle formation.
The purpose of this study was to determine the effect of L. chinense on muscle function under protein-rich dietary conditions via intake of cheese.
The skeletal muscle is comprised of slow and fast muscles, which affect endurance and quickness, respectively (Sciote et al., 1994). Therefore, to analyze muscle function, appropriate experimental methods need to be selected and analyzed. Methods for evaluating the muscle function of skeletal muscles include a treadmill test or a wheel running test to evaluate muscle endurance, and a grab test to evaluate grip strength .
In this study, we evaluated muscle function through a treadmill test and confirmed that the group that consumed L. chinense had the most outstanding muscle function. Given that among the various methods of evaluating muscle functions, the treadmill test is particularly suitable for evaluating muscle endurance, it is significant to note that muscle endurance was significantly increased in rats that consumed L. chinense. Once it was confirmed that muscle endurance, a function of the slow muscles, was improved, the gastrocnemius muscle, which has a high proportion of slow muscle among the leg muscles of the mouse, was analyzed. Although there was no significant difference in its weight compared to that of the other muscles, cross-sectional analysis revealed that the average thickness of the muscle fibers of the L. chinense-fed group increased, and the amount of muscle fibers with a thickness of 1,750 μm 2 or more was also increased. Since the function of the muscle tissue is strongly influenced by the thickness of each muscle fiber than the amount of muscle fiber, this result shows that the average thickness of the slow muscle fibers of the L. chinense-fed group increased, resulting in an increase in muscle endurance. In addition, analysis of the weights of the major organ and various F I G U R E 2 Blood profile of mice fed experimental diets. Changes in GOT, BUN, blood glucose, cholesterol, triglyceride, HDL, and LDL levels were measured. BUN, blood urea nitrogen; GOT, glutamic-oxaloacetic transaminase; HDL, high-density lipoprotein; LDL, low-density lipoprotein. All experimental data are presented as means ± standard deviation (n = 4/group). N.S., not significant; *, p < 0.05 (paired t test)
blood indices did not reveal any significant changes, indicating that WELC increased muscle endurance without any toxicity to mouse metabolism. F I G U R E 4 Effect of betaine on myogenesis of C2C12 cells. C2C12 cells were cultured for the indicated periods in differentiation medium containing betaine. (a) Immunoblot analysis was performed with anti-myosin heavy chain (Myh3) and anti-myogenin (MyoG) antibodies. Anti-Gapdh served as an internal control. Relative band intensities of Myh3 and MyoG were quantified using ImageJ software. (b) Representative images of myotubes were obtained at the indicated time points. Length of myotubes from each condition was quantified. All experimental data are presented as means ± standard deviation. *, p < 0.05 (paired t test) protein and therefore it is possible to evaluate myogenesis based on these changes (Venuti et al., 1995;Whalen et al., 1981). In this study, MyoG and Myh3 were selected as myogenesis indicators.
Myogenesis is a process in which myoblasts
The expression of MyoG and Myh3 were confirmed to be increased in C2C12 cells and primary myoblasts when treated with WELC and its constituent, betaine. In addition to the overall increase in the expression of Myh3, the expression level of MyoG was slightly increased on the 3rd day and decreased again on the 5th day, confirming that myogenesis was significantly promoted.
MyoG expression was increased in the early stage of myogenesis and decreased after the myotube was formed (Li, 2014;Yablonka-Reuveni & Paterson, 2001). Immunofluorescence analysis con- Currently, studies on substances that improve sarcopenia and muscle loss are limited. The efficacy of L. chinense extract and its indicator substance, betaine, in promoting myogenesis, that was confirmed through the results of this study adds significant value by contributing to basic knowledge that may be used for further research on the prevention and treatment of sarcopenia. In addition, considering that L. chinense is cultivated in a wide area, it can be potentially used to improve sarcopenia more effectively through the development of functional foods that strengthen muscle function.
ACK N OWLED G M ENTS
This work was carried out with the support of "Cooperative Research
Program for Agriculture Science and Technology Development (Project No. PJ01354401)," Rural Development Administration, Republic of Korea.
CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interest. were performed in accordance with the institutional guidelines.
AUTH O R CO NTR I B UTI
F I G U R E 5
Effect of betaine and L. chinense on myogenesis of primary mouse myoblasts. Primary mouse myoblasts were differentiated under the indicated condition for 5 days. (a) Immunoblot analysis was performed with anti-Myh3 antibody. Anti-Gapdh served as an internal control. Relative band intensities of Myh3 were quantified using ImageJ software. (b) Primary mouse myoblasts were observed by immunofluorescence microscopy. Myh3 (green) and DAPI (blue) were analyzed as representative markers for myotube and nuclear staining, respectively. Length of myotubes and fusion index were quantified. All experimental data are presented as means ± standard deviation. *, p < 0.05 (paired t test)
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
O RCI D
Myoung Soo Nam https://orcid.org/0000-0003-0866-1041
Kee K. Kim https://orcid.org/0000-0002-1088-3383
( 1 ,
1456.4 ± 105.4 μm 2 ). The observation of these histological changes in skeletal muscle in the L. chinense-fed mice prompted us to analyze the muscle endurance function via a treadmill test (Figure 3c). We measured running distance and time until exhaustion. The results of running distance before exhaustion showed that the Che. + LC (636.7 ± 45.0 m) group ran more than the Cont. (305.0 ± 90.2 m) and
form myotube cells through cell fusion and eventually form myofibers. Progression of myogenesis is marked by changes in the expression of myogenic F I G U R E 3 Changes in muscle fiber composition and exercise function in mice fed experimental diets. (a) Representative histological image of hematoxylin and eosin-stained cross-section of gastrocnemius muscle in experimental mice. (b) Cross-sectional area of myofiber in gastrocnemius muscle from each group. Frequency distributions of fibers (left), and measurement and comparison of average fiber area (right). (c) Treadmill endurance tests were performed with progressively increasing speed and time. All experimental data are presented as means ± standard deviation (n = 4/group). *, p < 0.05 (paired t test)
firmed that the cells with higher expression of myh3 formed larger myotubes compared to other cells treated with L. chinense and betaine, and it was confirmed that the number of nuclei contained in each myotube also increased. During myogenesis, myoblasts form long and large myotubes through cell fusion composed of myosin heavy chains. Treatment with L. chinense extracts and betaine promoted cell fusion and myotube formation in myoblasts, thereby facilitating myogenesis.
O N Sang-Soo Lee: Data curation (equal); Formal analysis (equal); Methodology (equal); Visualization (equal); Writing-original draft (equal). Yong-An Kim: Data curation (equal); Methodology (equal); Visualization (equal). Bokkee Eun: Resources (equal). Jayeon Yoo: Resources (equal). Eun-mi Kim: Resources (equal). Kee K. Kim: Funding acquisition (equal); Project administration (equal); Supervision (equal). Myoung Soo Nam: Resources (equal).E TH I C A L A PPROVA LResearch and animal care protocols were approved by the Animal Experimental Ethics Committee of the Chungnam National University (Daejeon, Korea, approval no. 202009-CNU-158) and
TA B L E 1 Composition of experimental dietsCont.
Che.
Che. + LC
Nutrients
Carbohydrate
g/100 g
49.1
30.5
30.4
% of total calorie
62.5
29.9
29.8
Protein
g/100 g
20.1
25.4
24.8
% of total calorie
25.6
24.9
24.3
Fat
g/100 g
4.2
20.5
20.9
% of total calorie
11.9
45.2
46.0
Total calorie (kcal/100 g)
314.2
408.4
409.3
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Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. W E Wright, D A Sassoon, V K Lin, 10.1016/0092-8674(89)90583-7Cell. 564Wright, W. E., Sassoon, D. A., & Lin, V. K. (1989). Myogenin, a factor reg- ulating myogenesis, has a domain homologous to MyoD. Cell, 56(4), 607-617. https://doi.org/10.1016/0092-8674(89)90583 -7
MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts. Z Yablonka-Reuveni, B M Paterson, 10.1177/002215540104900405Journal of Histochemistry & Cytochemistry. 4944900405Yablonka-Reuveni, Z., & Paterson, B. M. (2001). MyoD and myogenin ex- pression patterns in cultures of fetal and adult chicken myoblasts. Journal of Histochemistry & Cytochemistry, 49(4), 455-462. https:// doi.org/10.1177/00221 55401 04900405
Decreased eccentric exercise-induced macrophage infiltration in skeletal muscle after supplementation with a class of ginseng-derived steroids. S H Yu, C Y Huang, S D Lee, M F Hsu, R Y Wang, C L Kao, C H Kuo, 10.1371/journal.pone.0114649PLoS One. 912Yu, S. H., Huang, C. Y., Lee, S. D., Hsu, M. F., Wang, R. Y., Kao, C. L., & Kuo, C. H. (2014). Decreased eccentric exercise-induced macro- phage infiltration in skeletal muscle after supplementation with a class of ginseng-derived steroids. PLoS One, 9(12), e114649. https:// doi.org/10.1371/journ al.pone.0114649
| [
"Sarcopenia is a disease characterized by the loss of muscle mass and function that occurs mainly in older adults. The present study was designed to investigate the hypothesis that water extract of Lycium chinense (WELC) would improve muscle function and promote myogenesis for sarcopenia. We investigated the effect of water extracts of L. chinense on muscular endurance function and myogenesis to examine its efficacy in sarcopenia.Intake of WELC-containing cheese enhanced the muscular endurance function of mice in treadmill endurance tests. In addition, the crosssectional areas of muscle fibers in the gastrocnemius muscle of L. chinense-fed mice were greater than that of control mice. Furthermore, WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. Taken together, our results suggest that L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions."
] | [
"Sang-Soo Lee \nDepartment of Biochemistry\nChungnam National University\nDaejeonKorea\n",
"Yong-An Kim \nDepartment of Biochemistry\nChungnam National University\nDaejeonKorea\n",
"| Bokkee \nCore Laboratory for Convergent Translational Research\nKorea University College of Medicine\nSeoulKorea\n",
"Eun ",
"Jayeon Yoo \nNational Institute of Animal Science\nRDA\nWanjugunKorea\n",
"Eun-Mi Kim \nDepartment of Predictive Toxicology\nKorea Institute of Toxicology\nDaejeonSouth Korea\n",
"| Myoung ",
"Soo Nam [email protected] \nDivision of Animal Resource Science\nDepartment of Biochemistry\nChungnam National University\nDaejeonKorea\n\nDivision of Animal Resource Science\nChungnam National University\n34134DaejeonKorea\n\nChungnam National University\n34134DaejeonKorea\n",
"| Kee ",
"K Kim [email protected] \nDepartment of Biochemistry\nChungnam National University\nDaejeonKorea\n",
"Sang-Soo Lee ",
"Yong-An Kim ",
"Soo Myoung ",
"Kee K Nam ",
"Kim ",
"Kee K Correspondence ",
"Kim ",
"Myoung Soo Nam "
] | [
"Department of Biochemistry\nChungnam National University\nDaejeonKorea",
"Department of Biochemistry\nChungnam National University\nDaejeonKorea",
"Core Laboratory for Convergent Translational Research\nKorea University College of Medicine\nSeoulKorea",
"National Institute of Animal Science\nRDA\nWanjugunKorea",
"Department of Predictive Toxicology\nKorea Institute of Toxicology\nDaejeonSouth Korea",
"Division of Animal Resource Science\nDepartment of Biochemistry\nChungnam National University\nDaejeonKorea",
"Division of Animal Resource Science\nChungnam National University\n34134DaejeonKorea",
"Chungnam National University\n34134DaejeonKorea",
"Department of Biochemistry\nChungnam National University\nDaejeonKorea"
] | [
"Sang-Soo",
"Yong-An",
"|",
"Eun",
"Jayeon",
"Eun-Mi",
"|",
"Soo",
"|",
"K",
"Sang-Soo",
"Yong-An",
"Soo",
"Kee",
"K",
"Kee",
"K",
"Myoung",
"Soo"
] | [
"Lee",
"Kim",
"Bokkee",
"Yoo",
"Kim",
"Myoung",
"Nam",
"Kee",
"Kim",
"Lee",
"Kim",
"Myoung",
"Nam",
"Kim",
"Correspondence",
"Kim",
"Nam"
] | [
"H Alemán-Mateo, ",
"L Macías, ",
"J Esparza-Romero, ",
"H Astiazaran-García, ",
"A L Blancas, ",
"E.-J Bae, ",
"Y.-H Kim, ",
"S C Bodine, ",
"E Latres, ",
"S Baumhueter, ",
"V K Lai, ",
"L Nunez, ",
"B A Clarke, ",
"W T Poueymirou, ",
"F J Panaro, ",
"E Na, ",
"K Dharmarajan, ",
"Z Q Pan, ",
"D M Valenzuela, ",
"T M Dechiara, ",
"T N Stitt, ",
"G D Yancopoulos, ",
"D J Glass, ",
"J T Butcher, ",
"J D Mintz, ",
"S Larion, ",
"S Qiu, ",
"L Ruan, ",
"D J Fulton, ",
"D W Stepp, ",
"A Chanet, ",
"J Salles, ",
"C Guillet, ",
"C Giraudet, ",
"A Berry, ",
"V Patrac, ",
"C Domingues-Faria, ",
"C Tagliaferri, ",
"K Bouton, ",
"J Bertrand-Michel, ",
"M Van Dijk, ",
"M Jourdan, ",
"Y Luiking, ",
"S Verlaan, ",
"C Pouyet, ",
"P Denis, ",
"Y Boirie, ",
"S Walrand, ",
"S Chen, ",
"H S Lee, ",
"H S Han, ",
"K K Kim, ",
"Y S Cho, ",
"E Kim, ",
"S K Choi, ",
"W H Lee, ",
"H S Han, ",
"K K Kim, ",
"J M Cholewa, ",
"A Hudson, ",
"T Cicholski, ",
"A Cervenka, ",
"K Barreno, ",
"K Broom, ",
"M Barch, ",
"S A S Craig, ",
"A Danielewicz, ",
"J Morze, ",
"M Obara-Gołębiowska, ",
"M Przybyłowicz, ",
"K E Przybyłowicz, ",
"M C Devries, ",
"S M Phillips, ",
"H Hamarsland, ",
"M K Johansen, ",
"F Seeberg, ",
"M Brochmann, ",
"I Garthe, ",
"H B Benestad, ",
"T Raastad, ",
"I Janssen, ",
"R N Baumgartner, ",
"R Ross, ",
"I H Rosenberg, ",
"R Roubenoff, ",
"Y J Kim, ",
"H J Kim, ",
"W J Lee, ",
"J K Seong, ",
"A Kruczek, ",
"M Krupa-Malkiewicz, ",
"S Lachowicz, ",
"J Oszmianski, ",
"I Ochmian, ",
"S S Lee, ",
"E Kim, ",
"N J Cho, ",
"H S Han, ",
"K K Kim, ",
"W D Leslie, ",
"J T Schousboe, ",
"S N Morin, ",
"P Martineau, ",
"L M Lix, ",
"H Johansson, ",
"E V Mccloskey, ",
"N C Harvey, ",
"J A Kanis, ",
"W Li, ",
"A S Menko, ",
"D Boettiger, ",
"A Mocan, ",
"L Vlase, ",
"D C Vodnar, ",
"C Bischin, ",
"D Hanganu, ",
"A M Gheldiu, ",
"R Oprean, ",
"R Silaghi-Dumitrescu, ",
"G Crisan, ",
"A F Roche, ",
"M A Rudnicki, ",
"T Braun, ",
"S Hinuma, ",
"R Jaenisch, ",
"J J Sciote, ",
"A M Rowlerson, ",
"C Hopper, ",
"N P Hunt, ",
"S Segato, ",
"S Balzan, ",
"C A Elia, ",
"L Lignitto, ",
"A Granata, ",
"L Magro, ",
"B Contiero, ",
"I Andrighetto, ",
"E Novelli, ",
"P Senesi, ",
"L Luzi, ",
"A Montesano, ",
"N Mazzocchi, ",
"I Terruzzi, ",
"C Suetta, ",
"U Frandsen, ",
"A L Mackey, ",
"L Jensen, ",
"L G Hvid, ",
"M L Bayer, ",
"S J Petersson, ",
"H D Schrøder, ",
"J L Andersen, ",
"P Aagaard, ",
"P Schjerling, ",
"M Kjaer, ",
"M Thiruvengadam, ",
"B K Ghimire, ",
"S H Kim, ",
"C Y Yu, ",
"D H Oh, ",
"R Chelliah, ",
"C Kwon, ",
"Y J Kim, ",
"I M Chung, ",
"J M Venuti, ",
"J H Morris, ",
"J L Vivian, ",
"E N Olson, ",
"W H Klein, ",
"S Von Haehling, ",
"J E Morley, ",
"S D Anker, ",
"H Weintraub, ",
"S J Tapscott, ",
"R L Davis, ",
"M J Thayer, ",
"M A Adam, ",
"A B Lassar, ",
"A D Miller, ",
"R G Whalen, ",
"S M Sell, ",
"G S Butler-Browne, ",
"K Schwartz, ",
"P Bouveret, ",
"I Pinset-Harstom, ",
"W E Wright, ",
"D A Sassoon, ",
"V K Lin, ",
"Z Yablonka-Reuveni, ",
"B M Paterson, ",
"S H Yu, ",
"C Y Huang, ",
"S D Lee, ",
"M F Hsu, ",
"R Y Wang, ",
"C L Kao, ",
"C H Kuo, "
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] | [
"Alemán-Mateo",
"Macías",
"Esparza-Romero",
"Astiazaran-García",
"Blancas",
"Bae",
"Kim",
"Bodine",
"Latres",
"Baumhueter",
"Lai",
"Nunez",
"Clarke",
"Poueymirou",
"Panaro",
"Na",
"Dharmarajan",
"Pan",
"Valenzuela",
"Dechiara",
"Stitt",
"Yancopoulos",
"Glass",
"Butcher",
"Mintz",
"Larion",
"Qiu",
"Ruan",
"Fulton",
"Stepp",
"Chanet",
"Salles",
"Guillet",
"Giraudet",
"Berry",
"Patrac",
"Domingues-Faria",
"Tagliaferri",
"Bouton",
"Bertrand-Michel",
"Van Dijk",
"Jourdan",
"Luiking",
"Verlaan",
"Pouyet",
"Denis",
"Boirie",
"Walrand",
"Chen",
"Lee",
"Han",
"Kim",
"Cho",
"Kim",
"Choi",
"Lee",
"Han",
"Kim",
"Cholewa",
"Hudson",
"Cicholski",
"Cervenka",
"Barreno",
"Broom",
"Barch",
"Craig",
"Danielewicz",
"Morze",
"Obara-Gołębiowska",
"Przybyłowicz",
"Przybyłowicz",
"Devries",
"Phillips",
"Hamarsland",
"Johansen",
"Seeberg",
"Brochmann",
"Garthe",
"Benestad",
"Raastad",
"Janssen",
"Baumgartner",
"Ross",
"Rosenberg",
"Roubenoff",
"Kim",
"Kim",
"Lee",
"Seong",
"Kruczek",
"Krupa-Malkiewicz",
"Lachowicz",
"Oszmianski",
"Ochmian",
"Lee",
"Kim",
"Cho",
"Han",
"Kim",
"Leslie",
"Schousboe",
"Morin",
"Martineau",
"Lix",
"Johansson",
"Mccloskey",
"Harvey",
"Kanis",
"Li",
"Menko",
"Boettiger",
"Mocan",
"Vlase",
"Vodnar",
"Bischin",
"Hanganu",
"Gheldiu",
"Oprean",
"Silaghi-Dumitrescu",
"Crisan",
"Roche",
"Rudnicki",
"Braun",
"Hinuma",
"Jaenisch",
"Sciote",
"Rowlerson",
"Hopper",
"Hunt",
"Segato",
"Balzan",
"Elia",
"Lignitto",
"Granata",
"Magro",
"Contiero",
"Andrighetto",
"Novelli",
"Senesi",
"Luzi",
"Montesano",
"Mazzocchi",
"Terruzzi",
"Suetta",
"Frandsen",
"Mackey",
"Jensen",
"Hvid",
"Bayer",
"Petersson",
"Schrøder",
"Andersen",
"Aagaard",
"Schjerling",
"Kjaer",
"Thiruvengadam",
"Ghimire",
"Kim",
"Yu",
"Oh",
"Chelliah",
"Kwon",
"Kim",
"Chung",
"Venuti",
"Morris",
"Vivian",
"Olson",
"Klein",
"Von Haehling",
"Morley",
"Anker",
"Weintraub",
"Tapscott",
"Davis",
"Thayer",
"Adam",
"Lassar",
"Miller",
"Whalen",
"Sell",
"Butler-Browne",
"Schwartz",
"Bouveret",
"Pinset-Harstom",
"Wright",
"Sassoon",
"Lin",
"Yablonka-Reuveni",
"Paterson",
"Yu",
"Huang",
"Lee",
"Hsu",
"Wang",
"Kao",
"Kuo"
] | [
"Physiological effects beyond the significant gain in muscle mass in sarcopenic elderly men: Evidence from a randomized clinical trial using a protein-rich food. H Alemán-Mateo, L Macías, J Esparza-Romero, H Astiazaran-García, A L Blancas, 10.2147/CIA.S32356Clinical Interventions in Aging. 7Alemán-Mateo, H., Macías, L., Esparza-Romero, J., Astiazaran-García, H., & Blancas, A. L. (2012). Physiological effects beyond the sig- nificant gain in muscle mass in sarcopenic elderly men: Evidence from a randomized clinical trial using a protein-rich food. Clinical Interventions in Aging, 7, 225-234. https://doi.org/10.2147/CIA. S32356",
"Factors affecting sarcopenia in Korean adults by age groups. Osong Public Health and Research Perspectives. E.-J Bae, Y.-H Kim, 10.24171/j.phrp.2017.8.3.038Bae, E.-J., & Kim, Y.-H. (2017). Factors affecting sarcopenia in Korean adults by age groups. Osong Public Health and Research Perspectives, 8(3), 169-178. https://doi.org/10.24171/ j.phrp.2017.8.3.03",
"Identification of ubiquitin ligases required for skeletal muscle atrophy. S C Bodine, E Latres, S Baumhueter, V K Lai, L Nunez, B A Clarke, W T Poueymirou, F J Panaro, E Na, K Dharmarajan, Z Q Pan, D M Valenzuela, T M Dechiara, T N Stitt, G D Yancopoulos, D J Glass, 10.1126/science.1065874Science. 2945547Bodine, S. C., Latres, E., Baumhueter, S., Lai, V. K., Nunez, L., Clarke, B. A., Poueymirou, W. T., Panaro, F. J., Na, E., Dharmarajan, K., Pan, Z. Q., Valenzuela, D. M., DeChiara, T. M., Stitt, T. N., Yancopoulos, G. D., & Glass, D. J. (2001). Identification of ubiquitin ligases required for skeletal muscle atrophy. Science, 294(5547), 1704-1708. https://doi. org/10.1126/scien ce.1065874",
"Increased muscle mass protects against hypertension and renal injury in obesity. J T Butcher, J D Mintz, S Larion, S Qiu, L Ruan, D J Fulton, D W Stepp, 10.1161/JAHA.118.009358Journal of the American Heart Association. 716Butcher, J. T., Mintz, J. D., Larion, S., Qiu, S., Ruan, L., Fulton, D. J., & Stepp, D. W. (2018). Increased muscle mass protects against hypertension and renal injury in obesity. Journal of the American Heart Association, 7(16), e009358. https://doi.org/10.1161/JAHA.118.009358",
"Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition. A Chanet, J Salles, C Guillet, C Giraudet, A Berry, V Patrac, C Domingues-Faria, C Tagliaferri, K Bouton, J Bertrand-Michel, M Van Dijk, M Jourdan, Y Luiking, S Verlaan, C Pouyet, P Denis, Y Boirie, S Walrand, 10.1016/j.jnutbio.2017.02.024The Journal of Nutritional Biochemistry. 46Chanet, A., Salles, J., Guillet, C., Giraudet, C., Berry, A., Patrac, V., Domingues-Faria, C., Tagliaferri, C., Bouton, K., Bertrand-Michel, J., Van Dijk, M., Jourdan, M., Luiking, Y., Verlaan, S., Pouyet, C., Denis, P., Boirie, Y., & Walrand, S. (2017). Vitamin D supplementation re- stores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition. The Journal of Nutritional Biochemistry, 46, 30-38. https://doi.org/10.1016/j.jnutb io.2017.02.024",
"Investigation of the effect of Terminalia chebula fruit extract and its active ingredient, gallic aicd on muscle differentiation. S Chen, H S Lee, H S Han, K K Kim, ?g=kissm eta&m=exp&enc=BCBA2 A9705 FFBF8 FC923 DE941 3C53B65. 34Chen, S., Lee, H. S., Han, H. S., & Kim, K. K. (2019). Investigation of the ef- fect of Terminalia chebula fruit extract and its active ingredient, gallic aicd on muscle differentiation. The Korea Journal of Herbology, 34(2), 59-66. http://kiss.kstudy.com/thesi s/thesi s-view.asp?g=kissm eta&m=exp&enc=BCBA2 A9705 FFBF8 FC923 DE941 3C53B65",
"Investigation of the effect of Blueberry hydrothermal extracts on myoblast differentiation. Y S Cho, E Kim, S K Choi, W H Lee, H S Han, K K Kim, The Korea Journal of Herbology. 353Cho, Y. S., Kim, E., Choi, S. K., Lee, W. H., Han, H. S., & Kim, K. K. (2020). Investigation of the effect of Blueberry hydrothermal extracts on myoblast differentiation. The Korea Journal of Herbology, 35(3), 25- 32. http://kiss.kstudy.com/thesi s/thesi s-view.asp?g=kissm eta&m=- exp&enc=A1EA3 1E583 54786 EA8F4 90B58 69CA0AB",
"The effects of chronic betaine supplementation on body composition and performance in collegiate females: A double-blind, randomized, placebo controlled trial. J M Cholewa, A Hudson, T Cicholski, A Cervenka, K Barreno, K Broom, M Barch, S A S Craig, 10.1186/s12970-018-0243-xJournal of the International Society of Sports Nutrition. 151Cholewa, J. M., Hudson, A., Cicholski, T., Cervenka, A., Barreno, K., Broom, K., Barch, M., & Craig, S. A. S. (2018). The effects of chronic betaine supplementation on body composition and performance in collegiate females: A double-blind, randomized, placebo controlled trial. Journal of the International Society of Sports Nutrition, 15(1), 37. https://doi.org/10.1186/s1297 0-018-0243-x",
"Nutrient patterns and the skeletal muscle mass index among Polish women: A cross-sectional study. A Danielewicz, J Morze, M Obara-Gołębiowska, M Przybyłowicz, K E Przybyłowicz, 10.1038/s41598-019-55367-5Scientific Reports. 9118930Danielewicz, A., Morze, J., Obara-Gołębiowska, M., Przybyłowicz, M., & Przybyłowicz, K. E. (2019). Nutrient patterns and the skeletal muscle mass index among Polish women: A cross-sectional study. Scientific Reports, 9(1), 18930. https://doi.org/10.1038/s4159 8-019-55367 -5",
"Supplemental protein in support of muscle mass and health: Advantage whey. M C Devries, S M Phillips, 10.1111/1750-3841.12802Journal of Food Science. 801SupplDevries, M. C., & Phillips, S. M. (2015). Supplemental protein in support of muscle mass and health: Advantage whey. Journal of Food Science, 80(Suppl 1), A8-A15. https://doi.org/10.1111/1750-3841.12802",
"Native whey induces similar adaptation to strength training as milk, despite higher levels of leucine, in elderly individuals. H Hamarsland, M K Johansen, F Seeberg, M Brochmann, I Garthe, H B Benestad, T Raastad, 10.3390/nu11092094Nutrients. 119Hamarsland, H., Johansen, M. K., Seeberg, F., Brochmann, M., Garthe, I., Benestad, H. B., & Raastad, T. (2019). Native whey induces sim- ilar adaptation to strength training as milk, despite higher levels of leucine, in elderly individuals. Nutrients, 11(9), 2094. https://doi. org/10.3390/nu110 92094",
"Skeletal muscle cutpoints associated with elevated physical disability risk in older men and women. I Janssen, R N Baumgartner, R Ross, I H Rosenberg, R Roubenoff, 10.1093/aje/kwh058American Journal of Epidemiology. 1594Janssen, I., Baumgartner, R. N., Ross, R., Rosenberg, I. H., & Roubenoff, R. (2004). Skeletal muscle cutpoints associated with elevated phys- ical disability risk in older men and women. American Journal of Epidemiology, 159(4), 413-421. https://doi.org/10.1093/aje/kwh058",
"A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model. Y J Kim, H J Kim, W J Lee, J K Seong, 10.1186/s42826-019-0035-8Laboratory Animal Research. 361Kim, Y. J., Kim, H. J., Lee, W. J., & Seong, J. K. (2020). A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model. Laboratory Animal Research, 36(1), 3. https://doi.org/10.1186/ s4282 6-019-0035-8",
"Health-promoting capacities of in vitro and cultivated goji (Lycium chinense Mill.) fruit and leaves; Polyphenols, antimicrobial activity, macro-and microelements and heavy metals. A Kruczek, M Krupa-Malkiewicz, S Lachowicz, J Oszmianski, I Ochmian, 10.3390/molecules25225314Molecules. 2522Kruczek, A., Krupa-Malkiewicz, M., Lachowicz, S., Oszmianski, J., & Ochmian, I. (2020). Health-promoting capacities of in vitro and cul- tivated goji (Lycium chinense Mill.) fruit and leaves; Polyphenols, an- timicrobial activity, macro-and microelements and heavy metals. Molecules, 25(22), 5314. https://doi.org/10.3390/molec ules2 5225314",
"Investigation of the effect of Lacca Sinica Exsiccata water extract on myoblast differentiation. S S Lee, E Kim, N J Cho, H S Han, K K Kim, The Korea Journal of Herbology. 353Lee, S. S., Kim, E., Cho, N. J., Han, H. S., & Kim, K. K. (2020). Investigation of the effect of Lacca Sinica Exsiccata water extract on myo- blast differentiation. The Korea Journal of Herbology, 35(3), 9-16. http://kiss.kstudy.com/thesi s/thesi s-view.asp?g=kissm eta&m=ex- p&enc=17DCF C1E96 88488 8F64F EDE45 1265514",
"Loss in DXA-estimated total body lean mass but not fat mass predicts incident major osteoporotic fracture and hip fracture independently from FRAX: A registry-based cohort study. W D Leslie, J T Schousboe, S N Morin, P Martineau, L M Lix, H Johansson, E V Mccloskey, N C Harvey, J A Kanis, 10.1007/s11657-020-00773-wArchives of Osteoporosis. 15196Leslie, W. D., Schousboe, J. T., Morin, S. N., Martineau, P., Lix, L. M., Johansson, H., McCloskey, E. V., Harvey, N. C., & Kanis, J. A. (2020). Loss in DXA-estimated total body lean mass but not fat mass predicts incident major osteoporotic fracture and hip fracture independently from FRAX: A registry-based cohort study. Archives of Osteoporosis, 15(1), 96. https://doi.org/10.1007/s1165 7-020-00773 -w",
"Characterization and expression analysis of myogenin gene in white muscle of chinese mandarin fish, Siniperca chuatsi. W Li, 10.4172/jpb.1000304Journal of Proteomics & Bioinformatics. 7Li, W. (2014). Characterization and expression analysis of myogenin gene in white muscle of chinese mandarin fish, Siniperca chuatsi. Journal of Proteomics & Bioinformatics, 7, 71-76. https://doi.org/10.4172/ jpb.1000304",
"Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation. A S Menko, D Boettiger, 10.1016/0092-8674(87)90009-2Cell. 511Menko, A. S., & Boettiger, D. (1987). Occupation of the extracellular ma- trix receptor, integrin, is a control point for myogenic differentiation. Cell, 51(1), 51-57. https://doi.org/10.1016/0092-8674(87)90009 -2",
"Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves. A Mocan, L Vlase, D C Vodnar, C Bischin, D Hanganu, A M Gheldiu, R Oprean, R Silaghi-Dumitrescu, G Crisan, 10.3390/molecules190710056Molecules. 197Mocan, A., Vlase, L., Vodnar, D. C., Bischin, C., Hanganu, D., Gheldiu, A. M., Oprean, R., Silaghi-Dumitrescu, R., & Crisan, G. (2014). Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves. Molecules, 19(7), 10056-10073. https://doi.org/10.3390/molec ules1 90710056",
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"Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. M A Rudnicki, T Braun, S Hinuma, R Jaenisch, 10.1016/0092-8674(92)90508-ACell. 713Rudnicki, M. A., Braun, T., Hinuma, S., & Jaenisch, R. (1992). Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell, 71(3), 383-390. https://doi.org/10.1016/0092-8674(92)90508 -A",
"Fibre type classification and myosin isoforms in the human masseter muscle. J J Sciote, A M Rowlerson, C Hopper, N P Hunt, 10.1016/0022-510x(94)90089-2Journal of the Neurological Sciences. 1261Sciote, J. J., Rowlerson, A. M., Hopper, C., & Hunt, N. P. (1994). Fibre type classification and myosin isoforms in the human masseter mus- cle. Journal of the Neurological Sciences, 126(1), 15-24. https://doi. org/10.1016/0022-510x(94)90089 -2",
"Effect of period of milk production and ripening on quality traits of Asiago cheese. S Segato, S Balzan, C A Elia, L Lignitto, A Granata, L Magro, B Contiero, I Andrighetto, E Novelli, 10.4081/ijas.2007.1s.469Italian Journal of Animal Science. 6sup1Segato, S., Balzan, S., Elia, C. A., Lignitto, L., Granata, A., Magro, L., Contiero, B., Andrighetto, I., & Novelli, E. (2007). Effect of period of milk production and ripening on quality traits of Asiago cheese. Italian Journal of Animal Science, 6(sup1), 469-471. https://doi. org/10.4081/ijas.2007.1s.469",
"Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation. P Senesi, L Luzi, A Montesano, N Mazzocchi, I Terruzzi, 10.1186/1479-5876-11-174Journal of Translational Medicine. 11Senesi, P., Luzi, L., Montesano, A., Mazzocchi, N., & Terruzzi, I. (2013). Betaine supplement enhances skeletal muscle differentiation in mu- rine myoblasts via IGF-1 signaling activation. Journal of Translational Medicine, 11, 174. https://doi.org/10.1186/1479-5876-11-174",
"Ageing is associated with diminished muscle regrowth and myogenic precursor cell expansion early after immobilityinduced atrophy in human skeletal muscle. C Suetta, U Frandsen, A L Mackey, L Jensen, L G Hvid, M L Bayer, S J Petersson, H D Schrøder, J L Andersen, P Aagaard, P Schjerling, M Kjaer, 10.1113/jphysiol.2013.257121The Journal of Physiology. 59115Suetta, C., Frandsen, U., Mackey, A. L., Jensen, L., Hvid, L. G., Bayer, M. L., Petersson, S. J., Schrøder, H. D., Andersen, J. L., Aagaard, P., Schjerling, P., & Kjaer, M. (2013). Ageing is associated with diminished muscle re- growth and myogenic precursor cell expansion early after immobility- induced atrophy in human skeletal muscle. The Journal of Physiology, 591(15), 3789-3804. https://doi.org/10.1113/jphys iol.2013.257121",
"Assessment of mineral and phenolic profiles and their association with the antioxidant, cytotoxic effect, and antimicrobial potential of Lycium chinense Miller. M Thiruvengadam, B K Ghimire, S H Kim, C Y Yu, D H Oh, R Chelliah, C Kwon, Y J Kim, I M Chung, 10.3390/plants9081023Plants-Basel. 981023Thiruvengadam, M., Ghimire, B. K., Kim, S. H., Yu, C. Y., Oh, D. H., Chelliah, R., Kwon, C., Kim, Y. J., & Chung, I. M. (2020). Assessment of mineral and phenolic profiles and their association with the antioxidant, cy- totoxic effect, and antimicrobial potential of Lycium chinense Miller. Plants-Basel, 9(8), 1023. https://doi.org/10.3390/plant s9081023",
"Myogenin is required for late but not early aspects of myogenesis during mouse development. J M Venuti, J H Morris, J L Vivian, E N Olson, W H Klein, 10.1083/jcb.128.4.563Journal of Cell Biology. 1284Venuti, J. M., Morris, J. H., Vivian, J. L., Olson, E. N., & Klein, W. H. (1995). Myogenin is required for late but not early aspects of myogenesis during mouse development. Journal of Cell Biology, 128(4), 563-576. https://doi.org/10.1083/jcb.128.4.563",
"An overview of sarcopenia: Facts and numbers on prevalence and clinical impact. S Von Haehling, J E Morley, S D Anker, 10.1007/s13539-010-0014-2Journal of Cachexia, Sarcopenia and Muscle. 12von Haehling, S., Morley, J. E., & Anker, S. D. (2010). An overview of sarcopenia: Facts and numbers on prevalence and clinical impact. Journal of Cachexia, Sarcopenia and Muscle, 1(2), 129-133. https://doi. org/10.1007/s1353 9-010-0014-2",
"Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. H Weintraub, S J Tapscott, R L Davis, M J Thayer, M A Adam, A B Lassar, A D Miller, 10.1073/pnas.86.14.5434Proceedings of the National Academy of Sciences of the United States of America. the National Academy of Sciences of the United States of America86Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adam, M. A., Lassar, A. B., & Miller, A. D. (1989). Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proceedings of the National Academy of Sciences of the United States of America, 86(14), 5434-5438. https://doi. org/10.1073/pnas.86.14.5434",
"Three myosin heavy-chain isozymes appear sequentially in rat muscle development. R G Whalen, S M Sell, G S Butler-Browne, K Schwartz, P Bouveret, I Pinset-Harstom, 10.1038/292805a0Nature. 2925826Whalen, R. G., Sell, S. M., Butler-Browne, G. S., Schwartz, K., Bouveret, P., & Pinset-Harstom, I. (1981). Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature, 292(5826), 805-809. https://doi.org/10.1038/292805a0",
"Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. W E Wright, D A Sassoon, V K Lin, 10.1016/0092-8674(89)90583-7Cell. 564Wright, W. E., Sassoon, D. A., & Lin, V. K. (1989). Myogenin, a factor reg- ulating myogenesis, has a domain homologous to MyoD. Cell, 56(4), 607-617. https://doi.org/10.1016/0092-8674(89)90583 -7",
"MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts. Z Yablonka-Reuveni, B M Paterson, 10.1177/002215540104900405Journal of Histochemistry & Cytochemistry. 4944900405Yablonka-Reuveni, Z., & Paterson, B. M. (2001). MyoD and myogenin ex- pression patterns in cultures of fetal and adult chicken myoblasts. Journal of Histochemistry & Cytochemistry, 49(4), 455-462. https:// doi.org/10.1177/00221 55401 04900405",
"Decreased eccentric exercise-induced macrophage infiltration in skeletal muscle after supplementation with a class of ginseng-derived steroids. S H Yu, C Y Huang, S D Lee, M F Hsu, R Y Wang, C L Kao, C H Kuo, 10.1371/journal.pone.0114649PLoS One. 912Yu, S. H., Huang, C. Y., Lee, S. D., Hsu, M. F., Wang, R. Y., Kao, C. L., & Kuo, C. H. (2014). Decreased eccentric exercise-induced macro- phage infiltration in skeletal muscle after supplementation with a class of ginseng-derived steroids. PLoS One, 9(12), e114649. https:// doi.org/10.1371/journ al.pone.0114649"
] | [
"(Menko & Boettiger, 1987;",
"Yu et al., 2014)",
"(Rudnicki et al., 1992;",
"Weintraub et al., 1989;",
"Wright et al., 1989)",
"(Bodine et al., 2001;",
"Janssen et al., 2004;",
"Suetta et al., 2013)",
"(Roche, 1994)",
"(Bae & Kim, 2017;",
"von Haehling et al., 2010)",
"(Butcher et al., 2018;",
"Leslie et al., 2020)",
"(Alemán-Mateo et al., 2012;",
"Devries & Phillips, 2015)",
"(Chen et al., 2019;",
"Cho et al., 2020;",
"Lee et al., 2020)",
"(Mocan et al., 2014)",
"(Kruczek et al., 2020;",
"Thiruvengadam et al., 2020)",
"(Cholewa et al., 2018;",
"Senesi et al., 2013)",
"(Segato et al., 2007)",
"(Danielewicz et al., 2019)",
"(Chanet et al., 2017;",
"Hamarsland et al., 2019)",
"(Sciote et al., 1994)",
"(Venuti et al., 1995;",
"Whalen et al., 1981)",
"(Li, 2014;",
"Yablonka-Reuveni & Paterson, 2001)"
] | [
"Physiological effects beyond the significant gain in muscle mass in sarcopenic elderly men: Evidence from a randomized clinical trial using a protein-rich food",
"Identification of ubiquitin ligases required for skeletal muscle atrophy",
"Increased muscle mass protects against hypertension and renal injury in obesity",
"Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition",
"Investigation of the effect of Terminalia chebula fruit extract and its active ingredient, gallic aicd on muscle differentiation",
"Investigation of the effect of Blueberry hydrothermal extracts on myoblast differentiation",
"The effects of chronic betaine supplementation on body composition and performance in collegiate females: A double-blind, randomized, placebo controlled trial",
"Nutrient patterns and the skeletal muscle mass index among Polish women: A cross-sectional study",
"Supplemental protein in support of muscle mass and health: Advantage whey",
"Native whey induces similar adaptation to strength training as milk, despite higher levels of leucine, in elderly individuals",
"Skeletal muscle cutpoints associated with elevated physical disability risk in older men and women",
"A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model",
"Health-promoting capacities of in vitro and cultivated goji (Lycium chinense Mill.) fruit and leaves; Polyphenols, antimicrobial activity, macro-and microelements and heavy metals",
"Investigation of the effect of Lacca Sinica Exsiccata water extract on myoblast differentiation",
"Loss in DXA-estimated total body lean mass but not fat mass predicts incident major osteoporotic fracture and hip fracture independently from FRAX: A registry-based cohort study",
"Characterization and expression analysis of myogenin gene in white muscle of chinese mandarin fish, Siniperca chuatsi",
"Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation",
"Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves",
"Sarcopenia: A critical review of its measurements and health-related significance in the middle-aged and elderly",
"Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development",
"Fibre type classification and myosin isoforms in the human masseter muscle",
"Effect of period of milk production and ripening on quality traits of Asiago cheese",
"Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation",
"Ageing is associated with diminished muscle regrowth and myogenic precursor cell expansion early after immobilityinduced atrophy in human skeletal muscle",
"Assessment of mineral and phenolic profiles and their association with the antioxidant, cytotoxic effect, and antimicrobial potential of Lycium chinense Miller",
"Myogenin is required for late but not early aspects of myogenesis during mouse development",
"An overview of sarcopenia: Facts and numbers on prevalence and clinical impact",
"Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD",
"Three myosin heavy-chain isozymes appear sequentially in rat muscle development",
"Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD",
"MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts",
"Decreased eccentric exercise-induced macrophage infiltration in skeletal muscle after supplementation with a class of ginseng-derived steroids"
] | [
"the National Academy of Sciences of the United States of America",
"Clinical Interventions in Aging",
"Factors affecting sarcopenia in Korean adults by age groups. Osong Public Health and Research Perspectives",
"Science",
"Journal of the American Heart Association",
"The Journal of Nutritional Biochemistry",
"?g=kissm eta&m=exp&enc=BCBA2 A9705 FFBF8 FC923 DE941 3C53B65",
"The Korea Journal of Herbology",
"Journal of the International Society of Sports Nutrition",
"Scientific Reports",
"Journal of Food Science",
"Nutrients",
"American Journal of Epidemiology",
"Laboratory Animal Research",
"Molecules",
"The Korea Journal of Herbology",
"Archives of Osteoporosis",
"Journal of Proteomics & Bioinformatics",
"Cell",
"Molecules",
"American Journal of Human Biology",
"Cell",
"Journal of the Neurological Sciences",
"Italian Journal of Animal Science",
"Journal of Translational Medicine",
"The Journal of Physiology",
"Plants-Basel",
"Journal of Cell Biology",
"Journal of Cachexia, Sarcopenia and Muscle",
"Proceedings of the National Academy of Sciences of the United States of America",
"Nature",
"Cell",
"Journal of Histochemistry & Cytochemistry",
"PLoS One"
] | [
"\n( 1 ,\n1456.4 ± 105.4 μm 2 ). The observation of these histological changes in skeletal muscle in the L. chinense-fed mice prompted us to analyze the muscle endurance function via a treadmill test (Figure 3c). We measured running distance and time until exhaustion. The results of running distance before exhaustion showed that the Che. + LC (636.7 ± 45.0 m) group ran more than the Cont. (305.0 ± 90.2 m) and",
"\n\nform myotube cells through cell fusion and eventually form myofibers. Progression of myogenesis is marked by changes in the expression of myogenic F I G U R E 3 Changes in muscle fiber composition and exercise function in mice fed experimental diets. (a) Representative histological image of hematoxylin and eosin-stained cross-section of gastrocnemius muscle in experimental mice. (b) Cross-sectional area of myofiber in gastrocnemius muscle from each group. Frequency distributions of fibers (left), and measurement and comparison of average fiber area (right). (c) Treadmill endurance tests were performed with progressively increasing speed and time. All experimental data are presented as means ± standard deviation (n = 4/group). *, p < 0.05 (paired t test)",
"\n\nfirmed that the cells with higher expression of myh3 formed larger myotubes compared to other cells treated with L. chinense and betaine, and it was confirmed that the number of nuclei contained in each myotube also increased. During myogenesis, myoblasts form long and large myotubes through cell fusion composed of myosin heavy chains. Treatment with L. chinense extracts and betaine promoted cell fusion and myotube formation in myoblasts, thereby facilitating myogenesis.",
"\n\nO N Sang-Soo Lee: Data curation (equal); Formal analysis (equal); Methodology (equal); Visualization (equal); Writing-original draft (equal). Yong-An Kim: Data curation (equal); Methodology (equal); Visualization (equal). Bokkee Eun: Resources (equal). Jayeon Yoo: Resources (equal). Eun-mi Kim: Resources (equal). Kee K. Kim: Funding acquisition (equal); Project administration (equal); Supervision (equal). Myoung Soo Nam: Resources (equal).E TH I C A L A PPROVA LResearch and animal care protocols were approved by the Animal Experimental Ethics Committee of the Chungnam National University (Daejeon, Korea, approval no. 202009-CNU-158) and",
"\n\nTA B L E 1 Composition of experimental dietsCont. \nChe. \nChe. + LC \n\nNutrients \n\nCarbohydrate \n\ng/100 g \n49.1 \n30.5 \n30.4 \n\n% of total calorie \n62.5 \n29.9 \n29.8 \n\nProtein \n\ng/100 g \n20.1 \n25.4 \n24.8 \n\n% of total calorie \n25.6 \n24.9 \n24.3 \n\nFat \n\ng/100 g \n4.2 \n20.5 \n20.9 \n\n% of total calorie \n11.9 \n45.2 \n46.0 \n\nTotal calorie (kcal/100 g) \n314.2 \n408.4 \n409.3 \n"
] | [
"456.4 ± 105.4 μm 2 ). The observation of these histological changes in skeletal muscle in the L. chinense-fed mice prompted us to analyze the muscle endurance function via a treadmill test (Figure 3c). We measured running distance and time until exhaustion. The results of running distance before exhaustion showed that the Che. + LC (636.7 ± 45.0 m) group ran more than the Cont. (305.0 ± 90.2 m) and",
"form myotube cells through cell fusion and eventually form myofibers. Progression of myogenesis is marked by changes in the expression of myogenic F I G U R E 3 Changes in muscle fiber composition and exercise function in mice fed experimental diets. (a) Representative histological image of hematoxylin and eosin-stained cross-section of gastrocnemius muscle in experimental mice. (b) Cross-sectional area of myofiber in gastrocnemius muscle from each group. Frequency distributions of fibers (left), and measurement and comparison of average fiber area (right). (c) Treadmill endurance tests were performed with progressively increasing speed and time. All experimental data are presented as means ± standard deviation (n = 4/group). *, p < 0.05 (paired t test)",
"firmed that the cells with higher expression of myh3 formed larger myotubes compared to other cells treated with L. chinense and betaine, and it was confirmed that the number of nuclei contained in each myotube also increased. During myogenesis, myoblasts form long and large myotubes through cell fusion composed of myosin heavy chains. Treatment with L. chinense extracts and betaine promoted cell fusion and myotube formation in myoblasts, thereby facilitating myogenesis.",
"O N Sang-Soo Lee: Data curation (equal); Formal analysis (equal); Methodology (equal); Visualization (equal); Writing-original draft (equal). Yong-An Kim: Data curation (equal); Methodology (equal); Visualization (equal). Bokkee Eun: Resources (equal). Jayeon Yoo: Resources (equal). Eun-mi Kim: Resources (equal). Kee K. Kim: Funding acquisition (equal); Project administration (equal); Supervision (equal). Myoung Soo Nam: Resources (equal).E TH I C A L A PPROVA LResearch and animal care protocols were approved by the Animal Experimental Ethics Committee of the Chungnam National University (Daejeon, Korea, approval no. 202009-CNU-158) and",
"TA B L E 1 Composition of experimental diets"
] | [
"(Figure 1a)",
"(Figure 1b)",
"(Figure 1c)",
"Figure 2)",
"Figure 1c",
"(Figure 3a",
"Figure 3b",
"Figure S1",
"Figure S2",
"(Figure 4a",
"(Figure 4b)",
"(Figure 5a",
"(Figure 5b"
] | [] | [
"Muscle formation occurs during development, and during regeneration of muscle tissue following damage by exercise or disease (Menko & Boettiger, 1987;Yu et al., 2014).",
"During the process of muscle regeneration, myocardial cells exit the stationary/quiescence state and differentiate to form myofibers through cell fusion. This differentiation process is called myogenesis and is controlled by various signaling cascades.",
"Various myogenic proteins, such as paired Box Protein Pax-7 (Pax7), myoblast determination protein 1 (MyoD), myogenin (MyoG), and myosin heavy chain 3 (Myh3) represent the different myogenic stages (Rudnicki et al., 1992;Weintraub et al., 1989;Wright et al., 1989). Myoblasts form thick and long myotube cells through the process of myogenesis and the expression of myogenic proteins and the length and thickness of myotubes are very important factors in assessing myogenesis. Suppression of myogenesis due to various factors, such as aging, inhibits myotube formation that prevents the normal formation of muscle tissues, including skeletal muscle, resulting in muscle loss that leads to reduction in the total amount of muscles (Bodine et al., 2001;Janssen et al., 2004;Suetta et al., 2013).",
"Muscle loss is a typical muscle and nervous system disease and is one of the symptoms of aging. The total amount of skeletal muscle decreases gradually due to the natural process of aging and is replaced by adipose tissue resulting in a condition called sarcopenia (Roche, 1994). Sarcopenia mainly occurs in adults over the age of 65 years, and the number of older adults is increasing (Bae & Kim, 2017;von Haehling et al., 2010). Sarcopenia decreases the total amount of skeletal muscles and consequently increases the risk of fractures due to insufficient protection of the bones by skeletal muscles leading to decrease in the amount of activity and metabolic diseases such as diabetes, and thus has a tremendous impact on human health (Butcher et al., 2018;Leslie et al., 2020). Although sarcopenia is influenced by several factors, such as genetics or drugs, it is also affected by diet. Recent studies have confirmed that muscle function is improved by eating cheese with high protein and saturated fatty acid content (Alemán-Mateo et al., 2012;Devries & Phillips, 2015).",
"In addition, the effect of various natural products on myogenesis are being actively investigated (Chen et al., 2019;Cho et al., 2020;Lee et al., 2020).",
"Lycium chinense is a medicinal herb cultivated throughout Asia.",
"It protects the liver, has beneficial effects in osteoporosis, and has been used as a herbal medicine for its anticancer and antioxidant properties (Mocan et al., 2014). The efficacy of L. chinense is due to its high content of polyphenols, carotenoids, and amino acids (Kruczek et al., 2020;Thiruvengadam et al., 2020). L. chinense also contains betaine, a natural amino acid and a precursor of methionine.",
"Although betaine has been studied for its role in improving muscle function, the effect of L. chinense with a high content of betaine on myogenesis and muscle function has not yet been studied (Cholewa et al., 2018;Senesi et al., 2013).",
"In this study, the effect of water extract of L. chinense (WELC) on muscle endurance and muscle formation was investigated. In addition, treatment with WELC and betaine was shown to influence myogenesis in C2C12 and primary myoblasts.",
"BALB/c mice (8-week-old male; Daehan Biolink) were housed at 22 ± 2 ℃, with 50% ± 5% relative humidity under 12 hr light/dark cycle with ad libitum access to standard chow diet and water. All mice were acclimated for 7 days before use in experiments. Mice were divided into three groups (n = 4 mice/group) as follows: standard chow diet (Cont.), standard chow diet containing 40% cheese (Che.), and Che. with 3% L. chinense (Che. + LC). During the 6-week feeding period, body weight was recorded every 7 days, and food intake was recorded every 3 days.",
"2 | Extracts, asiago cheese, experimental diets, and chemical preparation Lycium chinense was gifted by the Gogi Berry Research Institute, Cheongyang, Republic of Korea. Dried L. chinense (25 g) was extracted with distilled water (1 L) at boiling temperature for 2 hr.",
"After the water evaporated, the extracts were freeze-dried.",
"Six month-fermented asiago cheese, and 6 month-fermented asiago cheese containing 3% L. chinense extract were used. Betaine (Sigma Aldrich) was used as a marker for L. chinense. The Asiago",
"Cheese was prepared by the Segato et al. method (Segato et al., 2007). The amount of added L. chinense extract was 0% and 3.0%. Ripening of the control of Asiago Cheese was performed for 6 months in the ripening room (10 ± 2 °C and 85 ± 5% of relative humidity).",
"The exercise treadmill test was performed at a rate of 10 cm/s for 3 min with a 0% slope. The speed was gradually increased to 15 cm/s and then maintained at 45 cm/s until the mouse was unable to run for more than 10 s.",
"The effects of the experimental diets on serum glutamic-oxaloacetic transaminase (GOT), glucose, blood urea nitrogen (BUN), total cholesterol, triglyceride, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured using an auto-analyzer (TBA-40FR; Toshiba).",
"C2C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% streptomycin/penicillin at 37 °C in a humidified 5% CO 2 atmosphere. When the cells reached 90% confluence, the culture medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% horse serum (Gibco) and 1% streptomycin/penicillin. While inducing myogenesis, the medium was replaced with fresh medium every day.",
"To coat the myoblast culture dishes or plates with poly-L-ornithine (PLO, Sigma) and collagen (Santa Cruz Biotechnology), the dishes and plates were incubated 6 hr at 25 ℃ with 0.001% solution of PLO in sterile water at 5 µg/cm 2 or 0.3 mg/ml solution of collagen in sterile water at 5 µg/cm 2 . The extra solution was aspirated and dishes or plates were dried in UV lamp exposure and washed two times with phosphate-buffered saline (PBS).",
"Mouse primary myoblasts were isolated from 3-day-after-birth mouse hind limb skeletal muscles. Muscles were separated from the bones and fat and subjected treatment with 1.5 U/ml collagenase D and 2.4 U/ml dispase solution (1:1) for 1 hr to isolate myoblasts.",
"Isolated myoblasts were centrifuged at 300 g for 5 min and resuspended in growth medium consisting of Ham's F-10 medium with 10% calf serum (GIBCO), 1% penicillin-streptomycin, and 2.5 μg/ml fibroblast growth factor. Mouse primary myoblasts were cultured in poly-L-ornithine (PLO, Sigma) coated dish for culture, or collagen (Santa Cruz Biotechnology) coated dish to induce myogenesis at 37 ℃ in a humidified 5% CO 2 atmosphere. When the mouse primary myoblasts reached 90% confluence, the culture medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% horse serum (Gibco) and 1% streptomycin/penicillin. When inducing myogenesis, the medium was replaced with fresh medium every day.",
"After at least 3 days of differentiation, the C2C12 cells or mouse primary myoblasts were lysed with cold tris-triton lysis buffer.",
"Identical amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and",
"transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk in PBS with 0.1% tween 20 (PBST) for 1 hr and incubated with primary anti-Myh3 (1:500, SC-53091, Santa Cruz), anti-MyoG (1:500, SC-52903, Santa Cruz), and anti-GAPDH (1:2,000, SC-47724, Santa Cruz) antibodies for 12 hr. After washing the membranes in PBST, immunoreactive signals were obtained using horseradish peroxidase-conjugated secondary antibodies (Abcam) and Super Signal system (Pierce Chemical).",
"Mouse primary myoblasts were plated in a 4-well collagen-coated chamber slide, and myogenesis was induced for 5 days. The myoblasts were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, blocked with 5% goat serum for 1 hr, and incubated overnight with primary antibodies against Myh3 (1:250, SC-53091, Santa Cruz) at 4 °C.",
"Alexa Fluor 488-conjugated goat antibody (1:500, Thermo Fisher Scientific) against mouse IgG was used as the secondary antibody. Nuclei were co-stained with DAPI (1:1,000; Thermo Fisher Scientific).",
"All results are expressed as the mean ± standard error of the mean of at least three separate experiments for each group. Comparisons between experimental groups were performed using two-tailed Student's t test (SPSS software, version 12.0; BMI). Significance was set at p < 0.05.",
"To compare the protein and fat contents of cheese-containing diets, we quantified the nutrients in the experimental diets (Table 1). The carbohydrate content of Cont. group (49.1 g/100 g) was higher than that of the Che. (30.5 g/100 g) and Che. + LC (30.4 g/100 g) experimental diet groups. Protein content of the Che. (25.4 g/100 g) and Che. + LC (24.8 g/100 g) experimental diets groups was higher than that of the Cont. group (20.1 g/100 g). Fat content of Che. (20.5 g/100 g) and Che. + LC (20.9 g/100 g) experimental diets groups was higher than that of the Cont. group (4.2 g/100 g). Calorie from the fat of Che. (45.2% of total calories), and Che. + LC (46.0% of total calories) groups was approximately fourfold higher than that of the Cont. (11.9% of total calories) group. As the experimental groups were fed highfat diets, we investigated whether experimental high-fat diets induced weight gain or metabolic change in each mouse group by measuring bodyweight after 6-week ad libitum feeding. We found that the mouse body weights increased by 8.9 and 8.5 g in the Che. and Che. + LC groups, respectively, whereas it increased by 5.9 g in the Cont. group (Figure 1a). Although the food intake did not differ among the groups, the calorie intake of Che. and Che. + LC groups was higher than that of the Cont. group (Figure 1b). As the calorie intake of the experimental groups was higher than that of the Cont. group, we measured the weight of the heart, liver, FAT, gastrocnemius (GAS), and soleus (SOL) muscles to examine metabolic changes in the three groups. Although the heart, liver, and GAS muscle mass did not differ among the groups, fat mass in the Che. group (0.98 ± 0.22 g) was higher than that in Cont.",
"(0.49 ± 0.18 g) and Che. + LC groups (0.75 ± 0.31 g) (Figure 1c).",
"Moreover, the soleus muscle mass was slightly increased in the Che. + LC group (8.15 ± 0.52 mg) compared to that in the Cont.",
"(7.16 ± 0.76 mg) and Che. groups (7.46 ± 0.39 mg). These results suggest that L. chinense extracts decreased fat mass and increased SOL muscle mass, whereas the other organs and tissue masses remained unaffected.",
"As the fat mass in the L. chinense-fed group decreased and SOL muscle mass increased, we examined the metabolic changes in the groups by serum analysis. GOT, BUN, blood glucose, cholesterol, triglyceride, HDL, and LDL levels were compared among the groups ( Figure 2). GOT, BUN, and blood glucose levels were nor- ",
"As the SOL muscle mass was increased in L. chinense-fed mice ( Figure 1c), we measured the distribution of muscle fiber size and thickness of GAS muscles by H&E staining (Figure 3a). The fiber size distribution showed a shift toward larger fiber diameter ( Figure 3b). Composition of the hypertrophic muscle fibers (˃1,750 μm 2 ) in Che. + LC group (37.5%) was also higher than that of the Cont. (20.9%), and Che. groups (14.3%). Further, the average muscle fiber area of Che. + LC group (1,665.1 ± 141.9 μm 2 ) was larger than that of the Cont. (1,478.0 ± 23.9 μm 2 ) and Che. groups Che. groups (481.7 ± 47.1 m). These results suggest that L. chinense extract improves the fiber size and endurance function of the GAS muscles.",
"Because betaine is a marker of L. chinense extracts, we investigated whether betaine is a key factor in muscle improvement.",
"We analyzed the betaine content in WELC by HPLC and found F I G U R E 1 Changes in body weight, food intake, and relative organ weights of mice fed experimental diets. Mice were provided normal diet, cheese (40% of feed), or cheese containing L. chinense (40% of feed) for 6 weeks. (a) Body weights and weight gain were measured every week. (b) Intake of food and calorie were measured every 3 days. (c) Tissue/organ weights were measured as representative organ.",
"All experimental data are presented as means ± standard deviation (n = 4/group). N.S., not significant; *, p < 0.05 (paired t test)",
"that 1 mg of WELC contained 14.72 μg of betaine ( Figure S1).",
"Betaine showed no cytotoxicity in C2C12 cells at concentrations of up to 5,000 μM ( Figure S2). C2C12 cells were differentiated in the presence of betaine for the indicated number of days, and",
"Myh3 and MyoG protein levels were examined by immunoblot analysis. MyoG expression increased 2.23-fold on day 5 after treatment with 50 μM betaine (Figure 4a). Myh3 expression was also increased by betaine treatment in a dose-dependent manner. In addition, betaine treatment increased myotube length in a dose-dependent manner (Figure 4b). These results suggest that betaine is an effective substance in L. chinense extracts that is involved in myogenesis. As myogenesis of C2C12 cells was promoted by betaine treatment, we examined the effect of betaine and WELC on myogenesis of mouse primary myoblasts. Mouse primary myoblasts were differentiated in the presence of 50 μM betaine. Myh3 expression was significantly increased by betaine treatment (Figure 5a). In addition, the function of betaine in promoting myotube formation in C2C12 cells was confirmed in mouse primary myoblasts (Figure 5b). Taken together, L. chinense and its marker betaine enhance myogenesis and muscle endurance in mice.",
"Skeletal muscle is one of the main organs in the body that performs various functions, such as protecting bones, providing muscle strength, and is important for maintaining health (Danielewicz et al., 2019). Nutrients, including proteins, are among the most important factors in maintaining skeletal muscle (Chanet et al., 2017;Hamarsland et al., 2019). Several recent studies have focused on the effect of continuous intake of foods rich in nutrients, including protein and saturated fatty acids, such as cheese, on improvement in muscle function. L. chinense, a herb cultivated all over Asia, has been used as a herbal supplement for a long time and contains several physiologically active substances including a high content of betaine, which is well known to have a positive effect on muscle formation.",
"The purpose of this study was to determine the effect of L. chinense on muscle function under protein-rich dietary conditions via intake of cheese.",
"The skeletal muscle is comprised of slow and fast muscles, which affect endurance and quickness, respectively (Sciote et al., 1994). Therefore, to analyze muscle function, appropriate experimental methods need to be selected and analyzed. Methods for evaluating the muscle function of skeletal muscles include a treadmill test or a wheel running test to evaluate muscle endurance, and a grab test to evaluate grip strength .",
"In this study, we evaluated muscle function through a treadmill test and confirmed that the group that consumed L. chinense had the most outstanding muscle function. Given that among the various methods of evaluating muscle functions, the treadmill test is particularly suitable for evaluating muscle endurance, it is significant to note that muscle endurance was significantly increased in rats that consumed L. chinense. Once it was confirmed that muscle endurance, a function of the slow muscles, was improved, the gastrocnemius muscle, which has a high proportion of slow muscle among the leg muscles of the mouse, was analyzed. Although there was no significant difference in its weight compared to that of the other muscles, cross-sectional analysis revealed that the average thickness of the muscle fibers of the L. chinense-fed group increased, and the amount of muscle fibers with a thickness of 1,750 μm 2 or more was also increased. Since the function of the muscle tissue is strongly influenced by the thickness of each muscle fiber than the amount of muscle fiber, this result shows that the average thickness of the slow muscle fibers of the L. chinense-fed group increased, resulting in an increase in muscle endurance. In addition, analysis of the weights of the major organ and various F I G U R E 2 Blood profile of mice fed experimental diets. Changes in GOT, BUN, blood glucose, cholesterol, triglyceride, HDL, and LDL levels were measured. BUN, blood urea nitrogen; GOT, glutamic-oxaloacetic transaminase; HDL, high-density lipoprotein; LDL, low-density lipoprotein. All experimental data are presented as means ± standard deviation (n = 4/group). N.S., not significant; *, p < 0.05 (paired t test)",
"blood indices did not reveal any significant changes, indicating that WELC increased muscle endurance without any toxicity to mouse metabolism. F I G U R E 4 Effect of betaine on myogenesis of C2C12 cells. C2C12 cells were cultured for the indicated periods in differentiation medium containing betaine. (a) Immunoblot analysis was performed with anti-myosin heavy chain (Myh3) and anti-myogenin (MyoG) antibodies. Anti-Gapdh served as an internal control. Relative band intensities of Myh3 and MyoG were quantified using ImageJ software. (b) Representative images of myotubes were obtained at the indicated time points. Length of myotubes from each condition was quantified. All experimental data are presented as means ± standard deviation. *, p < 0.05 (paired t test) protein and therefore it is possible to evaluate myogenesis based on these changes (Venuti et al., 1995;Whalen et al., 1981). In this study, MyoG and Myh3 were selected as myogenesis indicators.",
"The expression of MyoG and Myh3 were confirmed to be increased in C2C12 cells and primary myoblasts when treated with WELC and its constituent, betaine. In addition to the overall increase in the expression of Myh3, the expression level of MyoG was slightly increased on the 3rd day and decreased again on the 5th day, confirming that myogenesis was significantly promoted.",
"MyoG expression was increased in the early stage of myogenesis and decreased after the myotube was formed (Li, 2014;Yablonka-Reuveni & Paterson, 2001). Immunofluorescence analysis con- Currently, studies on substances that improve sarcopenia and muscle loss are limited. The efficacy of L. chinense extract and its indicator substance, betaine, in promoting myogenesis, that was confirmed through the results of this study adds significant value by contributing to basic knowledge that may be used for further research on the prevention and treatment of sarcopenia. In addition, considering that L. chinense is cultivated in a wide area, it can be potentially used to improve sarcopenia more effectively through the development of functional foods that strengthen muscle function.",
"This work was carried out with the support of \"Cooperative Research",
"Program for Agriculture Science and Technology Development (Project No. PJ01354401),\" Rural Development Administration, Republic of Korea.",
"The authors have declared no conflicts of interest. were performed in accordance with the institutional guidelines.",
"Effect of betaine and L. chinense on myogenesis of primary mouse myoblasts. Primary mouse myoblasts were differentiated under the indicated condition for 5 days. (a) Immunoblot analysis was performed with anti-Myh3 antibody. Anti-Gapdh served as an internal control. Relative band intensities of Myh3 were quantified using ImageJ software. (b) Primary mouse myoblasts were observed by immunofluorescence microscopy. Myh3 (green) and DAPI (blue) were analyzed as representative markers for myotube and nuclear staining, respectively. Length of myotubes and fusion index were quantified. All experimental data are presented as means ± standard deviation. *, p < 0.05 (paired t test)",
"The data that support the findings of this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.",
"Myoung Soo Nam https://orcid.org/0000-0003-0866-1041",
"Kee K. Kim https://orcid.org/0000-0002-1088-3383"
] | [] | [
"| INTRODUC TI ON",
"| MATERIAL S AND ME THODS",
"| Animals and experimental diets",
"2.",
"| Exercise treadmill test",
"| Serum biochemical analysis",
"| Cell culture",
"| PLO and collagen coating",
"| Isolation of mouse primary myoblasts",
"| Immunoblot analysis",
"| Immunofluorescence",
"| Statistical analysis",
"| RE SULTS",
"| Physical characteristics and food supplements",
"| Serum levels of lipids, GOT, BUN, and blood glucose",
"| Muscle fiber composition and muscle endurance function",
"| Activity of betaine on myogenesis",
"| D ISCUSS I ON",
"Myogenesis is a process in which myoblasts",
"ACK N OWLED G M ENTS",
"CO N FLI C T O F I NTE R E S T",
"AUTH O R CO NTR I B UTI",
"F I G U R E 5",
"DATA AVA I L A B I L I T Y S TAT E M E N T",
"O RCI D",
"( 1 ,"
] | [
"Cont. \nChe. \nChe. + LC \n\nNutrients \n\nCarbohydrate \n\ng/100 g \n49.1 \n30.5 \n30.4 \n\n% of total calorie \n62.5 \n29.9 \n29.8 \n\nProtein \n\ng/100 g \n20.1 \n25.4 \n24.8 \n\n% of total calorie \n25.6 \n24.9 \n24.3 \n\nFat \n\ng/100 g \n4.2 \n20.5 \n20.9 \n\n% of total calorie \n11.9 \n45.2 \n46.0 \n\nTotal calorie (kcal/100 g) \n314.2 \n408.4 \n409.3 \n"
] | [
"(Table 1"
] | [
"Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts",
"Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts"
] | [
"Food Sci Nutr"
] |
44,512,168 | 2022-03-02T21:09:58Z | CCBY | http://www.jbc.org/content/284/5/2867.full.pdf | HYBRID | 06b518538228068205d2dc74068dd7828e13b96e | null | null | null | null | 10.1074/jbc.m807526200 | 1973032259 | 19008232 | null |
Ski Regulates Muscle Terminal Differentiation by Transcriptional Activation of Myog in a Complex with Six1 and Eya3 * □ S
JBC Papers in PressCopyright JBC Papers in PressNovember 12, 2008
Hong Zhang
Department of Biochemistry
Case Western Reserve University
44106ClevelandOhio
Ed Stavnezer
Department of Biochemistry
Case Western Reserve University
44106ClevelandOhio
Ski Regulates Muscle Terminal Differentiation by Transcriptional Activation of Myog in a Complex with Six1 and Eya3 * □ S
JBC Papers in PressNovember 12, 200810.1074/jbc.M807526200Received for publication, September 29, 2008, and in revised form, November 12, 2008
Overexpression of the Ski pro-oncogene has been shown to induce myogenesis in non-muscle cells, to promote muscle hypertrophy in postnatal mice, and to activate transcription of muscle-specific genes. However, the precise role of Ski in muscle cell differentiation and its underlying molecular mechanism are not fully understood. To elucidate the involvement of Ski in muscle terminal differentiation, two retroviral systems were used to achieve conditional overexpression or knockdown of Ski in satellite cell-derived C2C12 myoblasts. We found that enforced expression of Ski promoted differentiation, whereas loss of Ski severely impaired it. Compromised terminal differentiation in the absence of Ski was likely because of the failure to induce myogenin (Myog) and p21 despite normal expression of MyoD. Chromatin immunoprecipitation and transcriptional reporter experiments showed that Ski occupied the endogenous Myog regulatory region and activated transcription from the Myog regulatory region upon differentiation. Transactivation of Myog was largely dependent on a MEF3 site bound by Six1, not on the binding site of MyoD or MEF2. Activation of the MEF3 site required direct interaction of Ski with Six1 and Eya3 mediated by the evolutionarily conserved Dachshund homology domain of Ski. Our results indicate that Ski is necessary for muscle terminal differentiation and that it exerts this role, at least in part, through its association with Six1 and Eya3 to regulate the Myog transcription.
tion from the 184-bp Myog regulatory region (36). However, no direct interactions between Ski and these muscle-specific transcription factors have been reported.
In vitro studies have revealed that terminal differentiation of myoblasts proceeds through a highly ordered sequence of events. These cells express MyoD while proliferating, but when growth stimuli are removed, they initiate expression of Myog followed by the induction of the cyclin-dependent kinase inhibitor p21 and irreversible withdrawal from the cell cycle. Subsequently these postmitotic myocytes express muscle-specific contractile proteins such as myosin heavy chain (MHC) and finally fuse into multinucleated myotubes (43). This process is governed mainly by two families of transcription factors, the MRFs and MEF2 (44 -46). The MRF gene family includes MyoD, Myf5, Myog, and MRF4 (44,47,48). All MRF family members share a highly conserved basic region and adjacent helix-loop-helix motif (bHLH) that mediates binding to a consensus DNA sequence, CANNTG, known as the E box, that is present in the regulatory regions of many muscle-specific genes. Forced expression of any MRF gene is capable of inducing expression of muscle-specific genes and activation of myogenic differentiation even in non-muscle cells. The MEF2 proteins belong to the superfamily of MADS (MCM1-agamous-deficient serum response factor) box transcription factors and directly bind an AϩT-rich element found in the promoters and enhancers of many muscle specific genes (49). Genetic analysis reveals that members of the MEF2 family are also essential for terminal muscle differentiation (50).
Another cis element, the MEF3 site (consensus sequence, TCAGGTT), is also present in the 133-bp Myog regulatory region. Studies of transgenic mice demonstrated that mutation of this MEF3 site abolishes correct expression of a Myog-LacZ transgene during embryogenesis (51). Two skeletal musclespecific members of the Six family (sine oculis homeodomaincontaining transcription factors), Six1 and Six4, bind to the MEF3 element and transactivate Myog transcription (51). Drosophila sine oculis (so) has been shown to act synergistically with eyes absent (eya) and dachshund (dac) by direct proteinprotein interactions. Similar interactions underlie the synergism of their mammalian homologues Six, Eya, and Dach (10,(52)(53)(54)(55)(56)(57)(58)(59). This evolutionarily conserved regulatory network of Eya/Six/Dach has been shown to regulate myogenesis in chicken somite culture and in the chick limb and to activate transcription of reporters containing the Myog MEF3 site (52,53). Interaction of mammalian Dach with Six protein is mediated by the evolutionarily conserved DHD motif (60). This has led to the suggestion that by virtue of its possession of these conserved domains (7,10,12) Ski might also interact with Six and Eya proteins to regulate Myog expression and thereby control commitment of myogenic cells to terminal differentiation (52).
In this study, we addressed this possibility by exploiting the well characterized mouse muscle satellite cell line, C2C12, as a model system. Using retroviral vectors to achieve tetracyclineregulated overexpression or knockdown of Ski, we asked whether Ski might not only stimulate but also be required for terminal differentiation of C2C12 cells. To probe the mechanism underlying the transcriptional regulation of Myog by Ski, the E box, MEF2, and MEF3 sites in the Myog regulatory region were investigated as the possible cis response elements. Using co-immunoprecipitation, we asked whether direct binding of Ski to MyoD, MEF2c, Six1, and/or Eya3 mediates its transcriptional activity. Finally chromatin immunoprecipitation (ChIP) assays were performed to determine whether Ski resides at the endogenous Myog regulatory region prior to or concomitant with the initiation of terminal differentiation.
EXPERIMENTAL PROCEDURES
Construction of Retroviral Vectors-The replication-defective retroviral vector LNITX was kindly provided by Dr. Fage and was described earlier (61). A DNA fragment containing the entire coding region of the human SKI cDNA was excised from the plasmid pSHHSKIN1D using BamHI and ClaI, blunt-ended with T4 DNA polymerase, and inserted into the LINTX vector at the PmeI site to produce LNIT-huSKI.
The replication-defective retroviral vector TMP-tTA was modified from the SIN-TREmiR30-PIG (TMP) vector (kindly provided by Dr. Scott Lowe) (62)(63)(64) by replacing its GFP gene with the tetracycline transactivator (65) gene (66). The sequences of shRNAs targeting mouse Ski were chosen using RNAi Codex and are designated by their positions in the mouse Ski cDNA sequence (GenBank TM accession number AF435852): mSki1145 (bases 1145-1163), mSki1819 (bases 1819 -1837), and mSki977 (bases 977-995). DNA forms of these shRNA inserts were generated by PCR amplification of 97-base synthetic oligonucleotides using Pfu DNA polymerase (Invitrogen) and a common set of primers (5Ј-cagaggctcgagaaggtatattgctgttgacagtgagcg-3Ј and 5Ј-cgcggcgaattccgaggcagtaggca-3Ј). The PCR products were subsequently digested with XhoI and EcoRI and inserted between these sites within TMP-tTA vector to generate TMP-tTA-mSki1145, TMP-tTA-mSki1819, and TMP-tTA-mSki977. Clones containing these shRNA-encoding inserts were sequence-verified.
Tissue Culture and Transfection-Proliferating mouse C2C12 myoblasts were maintained in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (Atlanta Biologicals), 100 g/ml penicillin, 100 units/ml streptomycin, and 0.002% Fungizone (Invitrogen). To avoid spontaneous differentiation, cells were always kept in subconfluent (60 -70%) conditions. Terminal differentiation was induced by switching subconfluent cells (80%) to differentiation medium (DM) consisting of Dulbecco's modified Eagle's medium, 2% heatinactivated horse serum, and antibiotics as in GM. Morphological differentiation, judged by myotube formation, was documented by digital photography of phase-contrast microscopic images.
Retroviral Packaging and Infection-The retrovirus packaging cell line PA317 (ATCC number SD3443) was cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 g/ml penicillin, and 100 units/ml streptomycin. They were transfected with retroviral constructs by using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. 24 h after transfection, the medium was harvested, and live cells were removed by centrifugation at 1000 ϫ g for 10 min. The retrovirus-containing supernatant was used to infect exponentially growing C2C12 cells at 40% confluence in the presence of 10 ng/ml Polybrene (Sigma). The infection was repeated with freshly harvested virus 12 h later. After an additional 12 h of culture, C2C12 cells were switched to GM plus 2 g/ml doxycycline (Dox; an analog of tetracycline) and antibiotics (G418 at 750 g/ml for LNITX-based constructs and puromycin at 2 ng/ml for TMP-tTA-based constructs). Two weeks later the resulting individual colonies were isolated using cloning discs according to the manufacturer's protocol (PGC Scientifics, 62-6151-14) and expanded in GM with Dox and G418 or puromycin. After culture in GM minus Dox, clones were screened by Western blot analysis for tetracycline-regulated Ski overexpression or knockdown.
Western Blotting-Whole-cell extracts were prepared from confluent C2C12 cells on 100-mm culture dishes as follows: cells were scraped into 400 l of lysis buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 0.1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 1 mM NaF, and Complete protease inhibitor mixture (Roche Applied Science)). After 10-min incubation on ice, the suspension was subjected to three freeze-thaw cycles, and cell debris were removed by centrifugation at 12,500 rpm for 15 min at 4°C. Protein concentrations were determined by Bradford assay (Bio-Rad), and equal amounts of proteins were boiled in protein loading buffer (100 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 0.2% bromphenol blue, and 2% -mercaptoethanol), separated by 6% SDS-PAGE, and transferred to Immobilon-P membranes (0.45 m, Millipore).
For most antibodies, blots were preblocked for 1 h at room temperature in blocking buffer (20 mM Tris, pH 7.6, 125 mM NaCl, 0.1% Tween 20, and 5% (w/v) nonfat dry milk) and incubated with primary antibodies in the same solution overnight at 4°C. The membranes were then washed with TBST (20 mM Tris, pH 7.6, 125 mM NaCl, and 0.1% Tween 20) and incubated for 1 h at room temperature with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase followed by washes with TBST. Signals were detected using either enhanced chemiluminescent horseradish peroxidase substrate (Pierce) or CDP-Star alkaline phosphatase chemiluminescent substrate (1:50; Tropix) and exposure to HyBlot CL autoradiography film (Denville Scientific).
When using anti-Ski G8 monoclonal antibody, the blots were preblocked overnight at 4°C in blocking buffer (PBS (7.7 mM Na 2 HPO 4 , 2.7 mM NaH 2 PO 4 , and 150 mM NaCl, pH 7.2), 0.4% casein, 1% polyvinylpyrrolidone (40,000), 10 mM EDTA, and 0.2% Tween, pH 7.2) and then incubated with primary antibody in the same solution for 30 min at room temperature. The membranes were washed with blocking buffer and further incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma) for 30 min at room temperature. Subsequently the blots were washed with blocking buffer and then with assay buffer (0.1 M EDTA, 1 mM MgCl 2 , and 0.02% azide, pH 10.0), and the signal was detected with CDP-Star alkaline phosphatase chemiluminescent substrate as above.
The following primary antibodies were used for immunoblotting: Ski (monoclonal antibody (1:2000; G8, Learner Research Institute Hybridoma Core Facility) or rabbit polyclonal antibody (1:1000; H329, Santa Cruz Biotechnology, Inc.)), Myog (1:1000; F5D, Santa Cruz Biotechnology, Inc.), TFIIE-␣ (1:3000; C-17, Santa Cruz Biotechnology, Inc.), MHC (1:1000; MF-20, Developmental Studies Hybridoma Bank), MyoD (1:1000; 5.8A, Santa Cruz Biotechnology, Inc.), and anti-FLAG (1:5000; M2, Sigma). The secondary antibodies used were: alkaline phosphatase-conjugated anti-mouse IgG (Fcspecific) and anti-rabbit IgG (whole molecule) (1:30,000; Sigma) and horseradish peroxidase-conjugated Mouse IgG TrueBlot TM and Rabbit IgG TrueBlot (1:1000; eBioscience).
Immunofluorescence-C2C12 myoblasts (2 ϫ 10 4 ) were seeded into each well of LAB-TEK 8-well chamber slides (Nunc) coated with 1 g/cm 2 laminin (Invitrogen) in GM and switched to DM for 3 days. Cells were fixed in 3.7% paraformaldehyde, PBS for 30 min at room temperature; permeabilized with 10% goat serum, 1% Triton X-100, and PBS for 10 min; and incubated with blocking buffer (PBS, 10% goat serum, and 0.1% Tween 20) for 1 h at room temperature and then with primary antibodies overnight at 4°C. Chambers were washed with PBST (PBS and 0.1% Tween 20), incubated with secondary antibodies for 1 h at room temperature, washed with PBS, and mounted in Vectashield aqueous mounting medium with DAPI (Vector Laboratories). Images were obtained using an Olympus BX50 upright fluorescence microscope equipped with Polaroid digital camera PDMC2, Polaroid PDMC2 software, and fluorescent illumination. Images were assembled using Photoshop CS (Adobe).
The following primary antibodies were used for immunofluorescence: MyoD (1:100; 5.8A, Santa Cruz Biotechnology, Inc.), Myog (1:100; F5D, Santa Cruz Biotechnology, Inc.), MHC (1:200; MF20, Developmental Studies Hybridoma Bank), and p21 (1:100; SX118, BD Pharmingen). Secondary antibodies were Alexa 488-or Alexa594-conjugated goat anti-mouse IgG antibody (1:300; Molecular Probes). Control experiments performed with normal IgG as the primary antibody yielded no signal above the background.
Quantification was performed by counting at least 1000 DAPI-stained nuclei in more than 10 random fields per culture plate. For MHC, the differentiation index ϭ nuclei within MHC-stained multinucleate myotubes/total number of DAPIstained nuclei, and the fusion index ϭ the average number of nuclei per MHC-stained myotube. For nuclear proteins, the differentiation index ϭ number of antibody-stained nuclei/total number of DAPI-stained nuclei. All experiments were performed in triplicate on three independent cultures, and the standard deviation was calculated.
Real Time PCR-RNA was isolated using the RNeasy kit (Qiagen) with DNase I treatment according to the manufacturer's protocol, and cDNA was generated using reverse transcriptase SuperScript TM III (Invitrogen) with random hexamer primers according to the manufacturer's instructions. Quantitative real time PCR (iCycler iQ TM , Bio-Rad) was performed using SYBR Green PCR Core Reagents (Applied Bioscience) according to the manufacturer's protocols. PCR was performed for 40 cycles of 94°C for 15 s, 60°C for 20 s, and 72°C for 20 s followed by a single 72°C extension step for 5 min. Primer sequences used for real time PCR will be provided upon request. Analyses were performed in triplicate on RNA samples from three independent experiments. Threshold cycles (Ct) of target genes were normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and relative transcript levels were calculated from the Ct values as Y ϭ 2 Ϫ⌬Ct where Y is -fold difference in amount of target gene versus Gapdh and ⌬Ct ϭ Ct x Ϫ Ct Gapdh .
ChIP-Chromatin immunoprecipitation experiments were performed essentially as described before (67). Briefly C2C12 cells cultured in GM or DM for 2 days were cross-linked with 1% formaldehyde, PBS for 10 min at room temperature. Fixed cells were scraped and resuspended at 2 ϫ 10 7 cells/ml in lysis buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% deoxycholate, and 0.1% SDS). Suspensions were sonicated to yield chromatin with an average DNA length of 200 -500 bp. Equal amounts of chromatin from each sample (2 ϫ 10 6 cells/assay) were preabsorbed at 4°C for 1 h with 40 l of a 50% slurry of preblocked protein A beads (Repligen; previously incubated with 1 mg/ml salmon testes DNA, 10 mg/ml bovine serum albumin, and 0.05% sodium azide in TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA)). After pelleting the beads, supernates were incubated overnight at 4°C with either 2 g of rabbit polyclonal Ski antibody (H329, Santa Cruz Biotechnology, Inc.) or normal rabbit IgG. Antibody-chromatin complexes were then captured by incubation with 40 l of a 50% slurry of preblocked protein A beads at 4°C for 1 h. The beads were washed sequentially in lysis buffer, high salt buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton-100, 0.1% deoxycholate, and 0.1% SDS), lithium salt buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% Nonidet P-40, and 0.5% deoxycholate), and TE buffer. Complexes were eluted from the beads in 150 l of elution buffer (10 mM Tris, pH 8.0, 5 mM EDTA, and 1% SDS), and formaldehyde cross-linking was reversed by overnight incubation at 65°C. After treatment with RNase A and proteinase K, DNA was isolated by phenol extraction and ethanol precipitation. The optimal PCR cycle numbers were determined by real time PCR, and 5% of purified DNA was analyzed by regular PCR using HotStart-IT Taq Master Mix (USB). 25% of each reaction mixture was resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. The following PCR primer sets were used: the mouse Myog regulatory region (Ϫ169 to ϩ39, GenBank accession number M95800), 5Ј-gggcaaaaggagagggaag-3Ј and 5Ј-agtggcaggaacaagcctt-3Ј; the non-promoter region downstream of Myog gene (ϩ1943 to ϩ2185 downstream of the Myog gene, GenBank accession number NW_001030662.1) serving as a negative control, 5Ј-gtcaagaactgacttaaggcc-3Ј and 5Ј-gacactaggagaagggtggag-3Ј; and the mouse Smad7 regulatory region (Ϫ274 to Ϫ142, GenBank accession number NW_001030635.1), 5-tagaaacccgatctgttgtttgcg-3 and 5-cctctgctcggctggttccactgc-3. For input control, 10% of cross-linked chromatin was purified as described above and assessed for PCR by using the same sets of primers.
Reporter Assays-A 202-bp DNA fragment (Ϫ184 to ϩ18, GenBank accession number M95800) containing the Myog regulatory region was amplified by PCR using C57BL/6 genomic DNA as template. Myog184-luciferase reporter carrying the luciferase gene downstream of this Myog regulatory region was generated by inserting HindIII-StuI-digested PCR product into the HindIII-SmaI site of the pGL3-Basic vector (Promega).
Myog-luciferase constructs with E box, MEF2, and MEF3 mutations (Myog184-E1E2m, Myog184-MEF2m, and Myog184-MEF3m) were generated from Myog184-luciferase using the QuikChange site-directed mutagenesis kit (Stratagene). SKI expression vector pCDNA-huSKI was described previously (23).
Cells (1 ϫ 10 5 ) were seeded in 12-well plates and transfected at 80% confluence using Lipofectamine 2000 (Roche Applied Science) with a combined total of 4 g of expression vector and reporter plasmid DNAs. 18 h after transfection, cells were switched to DM for 48 h prior to harvest and lysis in 1ϫ Reporter lysis buffer (Promega) with one round of freeze-thaw followed by incubation at room temperature for 20 min. Cell debris were removed by centrifugation, and luciferase activity in the supernatant was determined by a Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol using a MAXline microplate luminometer (Molecular Devices). The relative light units were generated by normalizing firefly luciferase units to Renilla luciferase units of the co-transfected pTK-Renilla-luc vector. The experiments were done in duplicate, and the reported results represent at least three independent experiments.
Co-immunoprecipitation-C2C12 cells (ϳ50% confluent, 100-mm dishes) were transfected with expression plasmids for FLAG-tagged Six1, FLAG-tagged Eya3, FLAG-tagged Mef2c, MyoD, and full-length SKI or its mutants using Lipofectamine 2000. 18 h after transfection, cells were refed GM or switched to DM and harvested 2 days later in 1 ml of NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, and Complete protease inhibitors mixtures). After brief sonication, cell debris were removed by centrifuge at 10,000 rpm for 15 min at 4°C. The lysates were preabsorbed with protein A beads, and protein concentrations were determined by the Bradford protein assay. Immunoprecipitations using equal amounts of proteins with either rabbit polyclonal anti-Ski or normal rabbit IgG were collected by overnight incubation with protein A beads (Repligen) at 4°C. Precipitates were washed five times in NETN buffer, resuspended and boiled in protein loading buffer, separated by 6% SDS-PAGE, and analyzed by Western blotting. If precipitating and primary Western blotting antibodies were from the same species, either horseradish peroxidase-conjugated Mouse IgG TrueBlot or Rabbit IgG TrueBlot was used as the secondary antibody accordingly.
RESULTS
Forced Expression of SKI Stimulates Myogenic Differentiation in C2C12
Cells-Overexpression of Ski has been reported to induce non-muscle fibroblasts to differentiate into myotubes (30,31), suggesting a role of Ski in myogenic lineage determination and terminal differentiation. To assess the role of Ski in terminal differentiation independent of its possible role in myogenic lineage determination, C2C12 cells that have already committed to myogenic fate and require only serum deprivation to undergo terminal differentiation were used in this study. To regulate Ski expression, C2C12 cells were infected with LNIT-huSKI, a retroviral vector that allowed both G418 selection and Dox regulation of SKI expression (Fig. 1A). Several G418-resistant clones were isolated and propagated in GM containing Dox to suppress SKI overexpression. They were then subdivided and tested for SKI expression after 3-day cultures in either the presence or absence of Dox. Western blot analysis of cell extracts revealed obvious induction of SKI expression in several LNIT-huSKI clones after Dox withdrawal (Fig. 1B), whereas a clone infected with the LNITX empty vec-tor did not overexpress SKI regardless of Dox treatment (Fig. 1D, right panel).
Using these clones, we examined the effect of SKI expression on myotube formation. LNIT-huSKI cells were cultured in GM with or without Dox for 4 days. Upon reaching 80% confluence, the cells were induced to differentiate by switching to DM while maintaining the presence or absence of Dox. 1 day after initiating differentiation, LNIT-huSKI cells that expressed exogenous SKI had prematurely formed myotubes, but no myotubes were observed in the same clone cultured in the presence of Dox to suppress SKI overexpression (Fig. 1C). Western analysis of these cells revealed expression of Myog only in cells that expressed exogenous SKI (Fig. 1D, left panel). These results were not due to Dox treatment because the vector-only controls showed no obvious difference in Myog or endogenous Ski expression in the presence or absence of Dox (Fig. 1D, right panel). These data indicate that the increased SKI expression stimulates the commitment of C2C12 myoblasts to terminal muscle differentiation.
Generation of C2C12 Clones with Inducible Knockdown of Ski Expression-The gain-of-function study above indicated that, as in avian cells, overexpression of Ski induces muscle terminal differentiation of C2C12 cells. However, those results do not necessarily implicate endogenous Ski in this process. To address this issue we asked whether knocking down endogenous mouse Ski expression would affect the differentiation of C2C12 cells. To accomplish this and avoid potential deleterious effects on the long term cell viability due to the loss of endogenous Ski, we used tetracycline-regulated expression of shRNAs to knock down Ski in C2C12 cells. The TMP-tTA vector used for this purpose was modified from the TMP retroviral vector (62-64) by substituting the tTA gene for the GFP gene so that this single retrovirus carries both the tetracycline-controlled transactivator (tTA) and its tetracycline response element (TRE) ( Fig. 2A). DNA sequences that encoded shRNAs targeting mouse Ski were inserted within the framework of micoRNA-30 (miR30) downstream of the TRE-CMV promoter ( Fig. 2A). Transcripts of this cassette resembling natural microRNA-30 could be generated upon removal of Dox, resulting in the knockdown of Ski. Three different shRNA inserts targeting mouse Ski gene were designed, and retroviral constructs, designated as TMP-tTA-mSki977, -1145, and -1819, were generated.
PA317 packaging cells were transfected with each of the three TMP-tTA-mSki vectors (or the empty vector), and viruses were harvested to infect C2C12 myoblasts. Puromycinresistant clones were isolated and propagated in GM plus Dox to prevent shRNA expression. These clones were then subdivided and cultured with or without Dox for 6 days prior to testing for conditional knockdown of Ski. Western analysis of several TMP-tTA-mSki1819 clones revealed a highly efficient Dox-dependent knockdown of Ski without significant leakiness (Fig. 2B). TMP-tTA-mSki cells grown in the absence of Dox produced nearly undetectable Ski levels, whereas the same clones grown in the presence of Dox expressed Ski at levels similar to that of the vector-only control. Of ϳ40 clones tested with each of the three TMP-tTA-mSki shRNAs, 70 -80% (65), the minimal CMV promoter regulated by tetracycline response elements (TRE-CMV), and human SKI coding region (SKI). B, Doxregulated SKI expression in LNIT-huSKI clones. Western analysis of SKI expression in representative C2C12 LNIT-huSKI clones cultured under induced condition (ϪDox) or suppressed condition (ϩDox) for 3 days. A vector-only clone (LNITX) was used as a negative control (Ctrl). TFIIE-␣ was a loading control. C, a representative C2C12 LNIT-huSKI clone (F8) was cultured in GM or DM for 1 day with or without Dox, and phase-contrast microscopy shows myotube formation only in the absence of Dox. D, enhanced Myog expression by overexpression of SKI. LNIT-huSKI F8 clone or a vector-only clone (LNITX) was cultured in GM or DM for 1 day with or without Dox, and Western blot analysis revealed the expression of Myog and SKI. TFIIE-␣ was a loading control. Note that endogenous Ski expression in the right panel was revealed only after much longer exposure than the left panel where overexpression of SKI was detected.
Regulation of Muscle Differentiation by Ski-Six1-Eya3
exhibited Dox-dependent knockdown of Ski, indicating the high efficiency of this system (data not shown).
Ski Knockdown Is Reversible and Dox Dose-dependent-To determine the kinetics of Ski knockdown, a C2C12 TMP-tTA-mSki1145 clone was propagated in Dox-containing medium, transferred to Dox-free medium, and monitored for Ski expression over an 8-day period. Western analysis showed that knockdown of Ski was apparent within 4 days after Dox removal and was virtually complete after 6 days (Fig. 2C). This knockdown was completely reversible; readdition of Dox to these cells restored normal Ski expression within 4 days (Fig. 2D). The extent of Ski knockdown was also doxycycline dose-dependent; expression of Ski was barely detectable at 2 ng/ml Dox or less and was comparable to the vector-only control at 20 ng/ml Dox or more (Fig. 2E). Taken together, these data demonstrated that this single vector system allows tightly regulated and reversible knockdown of endogenous Ski.
Impaired Myotube Formation in the Absence of Ski-Using this Dox-regulated knockdown system, we next evaluated the consequences of the loss of Ski on terminal differentiation. A C2C12 TMP-tTA-mSki1145 clone was either kept in GM plus Dox to maintain Ski expression or switched into GM minus Dox for 7 days to achieve the maximal knockdown of Ski. Subsequently upon reaching 80% confluence, these cells were switched from GM to DM while continuing the maintenance or suppression of Ski expression. Phase microscopy of the cultures prior to switching to DM revealed that the morphology of TMP-tTA-mSki and TMP-tTA cells in GM was similar and not affected by the loss of Ski expression (Fig. 3, A and B, and sup- plemental Fig. S1, A and B). This was not true for cells that were switched into DM to induce differentiation. The TMP-tTA-mSki cells in which Ski expression was maintained exhibited extensive myotube formation (Fig. 3, C and E). However, when Ski expression was knocked down in the TMP-tTA-mSki clone, no obvious myotube formation was visible within 2 days (Fig. 3D), and only a few small myotubes had formed after 4 days (Fig. 3F). The lack of myotube formation was not due to delayed differentiation kinetics because in the absence of Dox TMP-tTA-mSki cells did not show significant myotube formation even after 6 days in DM (data not shown). Furthermore the differentiation defect of TMP-tTA-mSki cells in the absence of Dox was not due to the withdrawal of Dox because myotube formation of the TMP-tTA control was independent of Dox (supplemental Fig. S1, C and D). Thus the results indicate that Ski expression is required for terminal muscle differentiation of C2C12 cells.
Decreased myotube formation in the absence of Ski could be due to a block at any stage in the differentiation pathway or a failure of fully differentiated myocytes to fuse. To distinguish between these possibilities, we performed immunofluorescence for MHC, which is a terminal differentiation marker expressed in both unfused myocytes and multinucleated myotubes. Consistent with the morphological observations above, TMP-tTA-mSki cells exhibited extensive formation of MHCpositive multinucleated myotubes when expressing Ski, whereas the vast majority of cells from the same clonal line were MHC-negative when Ski expression was knocked down (Fig. 3, G and H). The effects of loss of Ski on terminal differentiation and fusion were further quantified by measuring both the percentage of nuclei in MHC-positive cells (differentiation index) and the average number of nuclei per MHC-positive myotube (fusion index). We found that the loss of Ski caused significant (p Ͻ 0.05) drops in both the differentiation index (from 46.4 Ϯ 6.2 to 14.6 Ϯ 9.8%) and fusion index (20.7 Ϯ 7.7 to 3.4 Ϯ 1.7 nuclei per myotube) (Fig. 3, I and J), respectively. The results indicate that in the absence of Ski, both differentiation (ϳ3-fold fewer MHC-positive cells) and myotube formation (ϳ6-fold fewer nuclei per myotube) were severely impaired (Fig. 3, I and J). Thus the loss of Ski resulted in a differentiation block upstream of myocyte fusion.
Loss of Ski Inhibits Commitment to Terminal Muscle Differentiation-To determine the stage at which terminal differentiation was blocked by the absence of Ski, we further examined the expression of an early differentiation marker, Myog, and a late differentiation marker, MHC. As shown in Fig. 4A, when maintained in GM, neither the C2C12 TMP-tTA-mSki1145 cells nor the TMP-tTA cells expressed Myog or MHC at detectable levels regardless of Dox treatment. However, after 3 days of culture in DM, TMP-tTA-mSki cells in which Ski expression was maintained by Dox treatment expressed both Myog and MHC at high levels. This was also the case when TMP-tTA cells were cultured in DM regardless of Dox treatment. In contrast, when the same TMP-tTA-mSki clone with Ski expression knocked down were cultured in DM, Myog and MHC were expressed at very low levels (Fig. 4A). Similar results were obtained using TMP-tTA-mSki clones expressing the other two shRNAs, indicating that the differen-tiation block was not due to an off-target effect of the shRNA (supplemental Fig. S2). In addition, a Dox dose-response experiment with a C2C12 TMP-tTA-mSki clone expressing a different shRNA (mSki1189) revealed that the expression of Myog and MHC dropped in parallel with the decrease in endogenous Ski expression (Fig. 4B). The loss of Myog expression indicated that differentiation was blocked at a very early stage in the absence of Ski expression.
The early stages of muscle differentiation are marked by a well characterized progression of protein expression including the myogenic regulatory factors (MyoD and Myog) and the cell cycle regulator (p21) (43,44,47,48). To obtain a quantitative assessment of the early disruption of the myogenic differentia- tion program due to the loss of Ski expression, we investigated the percentage of cells expressing these proteins during the differentiation of C2C12 TMP-tTA-mSki clones in the absence or presence of Dox. Myog and p21 are markers of commitment to terminal differentiation and withdrawal from the cell cycle, respectively. Their increased expression can be detected in both differentiating myocytes and fully differentiated multinucleated myotubes and is widely used to assess the early stage of differentiation. Within 3 days of switching to DM, 35.5% of TMP-tTA-mSki cells were Myog-positive when cultured in the presence of Dox (Fig. 4, C and I), and 27.9% were p21-positive (Fig. 4, E and J). In sharp contrast, in the same TMP-tTA-mSki clone with Ski expression knocked down, the percentages of Myog-and p21-positive cells decreased to 8.8% (Fig. 4, D and I) and 14.9% (Fig. 4, F and J), respectively. On the other hand, the expression of MyoD, a constitutive myogenic lineage marker of C2C12 cells, was comparable in the presence and absence of Ski expression (Fig. 4, G, H, and K). These results indicate that the loss of Ski blocked a step in the differentiation pathway downstream of MyoD. Results obtained with a representative TMP-tTA-mSki1145 clone are shown in Fig. 4, and similar results were obtained with two other clonal lines (data not shown).
To determine whether the observed Ski-dependent changes in protein expression were due to reductions in the expression of their mRNAs, we performed real time PCR on RNA isolated from TMP-tTA-mSki cells cultured for 3 days in DM plus or minus Dox. Concomitant with the 5.4-fold lower expression of Ski mRNA in TMP-tTA-mSki cells in the absence of Dox, Myog and p21 mRNA levels were reduced by 12-and 5.5-fold, respectively. On the other hand, the level of MyoD mRNA was not significantly affected by the loss of Ski (Fig. 4L). These results mirror those obtained in the analyses of protein expression and indicate that Ski is necessary for the transcription or accumulation of mRNAs that are important for initiating muscle terminal differentiation.
Ski Occupies Myog Regulatory Region in Differentiating Myoblasts-Because Ski is known to be a co-regulator of transcription it seemed likely that the changes we detected in the expression of muscle-specific mRNA might be due to direct effects of Ski on transcription. This possibility was especially appealing for Myog not only because its expression is required for the initiation of terminal differentiation (43,44,47,48) but also because published reporter gene assays have demonstrated that transient overexpression of Ski can transcriptionally activate the Myog regulatory region (36,37).
To investigate whether endogenous Ski might directly regulate Myog transcription, we performed ChIP assays on C2C12 cells cultured in either GM or DM. DNA was isolated from the chromatin immunoprecipitated with anti-Ski antibodies and was analyzed by PCR using primers that amplify the Myog regulatory region including the E1 box, a MEF2 site, and a MEF3 element (40 -42, 51, 68) (Fig. 5A). The ChIP assays revealed that Ski was not bound to this endogenous Myog regulatory region in proliferating C2C12 cells cultured in GM. However, Ski became associated with this region when the cells were stimulated to differentiate by switching them into DM (Fig. 5B, upper panel). The specificity of this interaction was verified by negative results in control ChIP assays using either a nonspe-cific antibody (normal IgG) and the same primers amplifying the Myog regulatory region or the Ski antibody and primers amplifying a non-promoter region downstream of Myog gene (Fig. 5B, top and middle panels). Furthermore the observation that Ski was expressed at similar levels in cells cultured in GM and DM indicated that the increased interaction between Ski and the endogenous Myog regulatory region upon differentiation was not due to a parallel increase in Ski expression (Fig. 5C). Likewise the fact that the Smad7 regulatory region, which is known to be bound by Ski (69), was occupied by Ski in both proliferating and differentiating cells (Fig. 5B, bottom panel) indicated that the chromatin binding ability of Ski did not depend merely on the change from GM to DM. Thus Ski selectively binds the Myog regulatory region in concert with a signal that initiates differentiation. These results suggest that the requirement for endogenous Ski in the initiation of terminal muscle differentiation might be due to its direct role in activating of Myog transcription.
MEF3 Binding Site Is Required for the Activation of Myog Regulatory Region by Ski-In light of the above results we sought to define the Ski-response cis element in the Myog regulatory region. A 184-bp Myog regulatory region sequence has been defined as a muscle-specific regulatory region and contains a number of transcriptional factor binding sites that are critical for activation of Myog transcription during differentiation (38, 40 -42, 51, 68). Among others, they include two consensus MyoD-binding E boxes (proximal E1 box and distal E2 box), a MEF2 binding site, and a MEF3 site (Fig. 6A, upper panel). Ski does not bind DNA directly, but it has previously been shown to synergize with MyoD and MEF2 in activating transcription of Myog reporters containing this 184-bp sequence (36). This synergy requires the binding of these transcription factors to their response elements in the Myog regulatory region. To address whether Ski activation of Myog regulatory region was mediated only through these binding sites, we mutated the MEF2 binding site or both E boxes (E1 and E2) to eliminate binding by their corresponding transcriptional factors (Fig. 6A). In addition, we mutated the MEF3 site, although it had not previously been implicated in activation by Ski. C2C12 myoblasts were transfected with the wild-type Myog reporter (Myog184) or its mutated derivatives (Myog184-MEF3m, Myog184-MEF2m, and Myog184-E1E2m; see Fig. 6A) along with a SKI expression vector or an empty vector, and luciferase activity was measured 2 days after switching the cells to DM. SKI expression potentiated the activity of the wild-type Myog reporter (Fig. 6B) and surprisingly activated the transcription of the reporters carrying mutations of either the MEF2 site or both E boxes to ϳ80% of the level observed with the wild-type reporter (Fig. 6C). These results suggested that activation of Myog regulatory region by SKI was not mediated exclusively by these two regulatory elements. However, activation of the reporter bearing a mutated MEF3 site by SKI was only 50% of that of the wild type (Fig. 6C), indicating that this site may be the major SKI-responsive cis element within the Myog regulatory region.
SKI Associates with Eya3 and Six1 in Differentiating Muscle
Cells-We have shown that activation of Myog transcription by SKI is mediated mainly through the MEF3 site in the regulatory region. Because Ski does not bind DNA directly, it seemed likely that its association with the endogenous Myog regulatory region and its activation of Myog transcription are mediated by its association with transcription factors that bind to the MEF3 element. Six1 has been shown to bind to the MEF3 site of the Myog regulatory region (51) and to synergize with Eya to positively regulate the transcription driven by this cis element (53). In addition, because Dach, a Ski family member, forms a trimeric complex with Six1 and Eya3 to regulate muscle-specific gene expression (53), it seemed possible that Ski may be tethered to Myog regulatory region via an association with Six1 and Eya3 in a similar manner. We therefore investigated whether Ski interacts with Six1 and Eya3 in muscle cells undergoing terminal differentiation. C2C12 cells were co-transfected with SKI and FLAG-tagged Six1 and Eya3 expression vectors, and cells were either cultured in GM or induced to differentiate in DM for 48 h. Extracts of these cells were immunoprecipitated with either rabbit anti-Ski or normal rabbit IgG and analyzed for co-precipitation of Six1 and Eya3 by Western blotting with anti-FLAG. As seen in Fig. 7A, neither Six1 nor Eya3 was precipitated by normal rabbit IgG, whereas Six1 was co-precipitated with SKI at comparable levels in proliferating (GM) and differentiating (DM) cells. In contrast, co-precipitation of Eya3 with SKI was barely above background in proliferating cells but clearly detectable in differentiating cells. Thus in muscle cells Ski is constitutively associated with Six1 but interacts with Eya3 only upon differentiation.
Because a previous study suggested that Ski activated transcription of Myog regulatory region in cooperation with MyoD and MEF2 (36), we performed similar co-precipitation assays to examine the possible interactions of Ski with these proteins. Surprisingly neither MyoD nor MEF2C co-precipitated with Lysates were immunoprecipitated with a rabbit antibody against Ski (␣-Ski lanes), and precipitates were analyzed by Western blotting using anti-Ski, anti-FLAG, or anti-MyoD antibodies. 10% of the input for immunoprecipitation (Input lanes) confirms that comparable amounts of these proteins were used in the immunoprecipitation assays. Immunoprecipitation (IP) with a normal rabbit IgG (IgG lanes) was used as a negative control. SKI in either proliferative or differentiating C2C12 cells (Fig. 7, B and C). Considering that MyoD and MEF2 activate the transcription of some muscle-specific genes in a cooperative manner (44), it seemed possible that their interactions with SKI might require the presence of both proteins. To test this possibility, similar co-immunoprecipitation experiments were performed with extracts of C2C12 cells that were co-transfected with SKI and both MyoD and MEF2 expression vectors. Once again both proteins were expressed at high levels, but neither of them co-precipitated with SKI in C2C12 cells cultured in GM or DM (Fig. 7D). Although negative results are inconclusive, these observations and the results of the reporter assays suggest that transcriptional cooperation of Ski with MyoD and MEF2 may be mediated by an indirect interaction with a DNA-bound Ski-Six1-Eya3 complex.
The DHD of Ski Is Required for Its Association with Six1 and Its Activation of Myog Transcription-It has been shown that Dach interacts with Six1 through its DHD (52,53). It was therefore of interest to determine whether this conserved domain in Ski also mediates its interaction with Six1. The ability of a SKI mutant lacking the DHD to interact with Six1 was examined by coimmunoprecipitation (Fig. 8, A and B). Deletion of the DHD from SKI (SKI⌬DHD) greatly reduced its ability to interact with Six1, although it did not affect interaction with Eya3.
Having identified the DHD as the Six1 binding domain in SKI, we sought to determine the effect of its deletion on transcriptional activation of Myog. Cotransfection experiments showed that SKI⌬DHD failed to activate transcription of the wild-type Myog reporter (Myog184) significantly compared with the wild-type SKI (Fig. 8C). Western blots of the transfected cells demonstrated that the lack of reporter activation by SKI⌬DHD was not due to a failure to express this protein at a level similar to that of the wild-type SKI (Fig. 8D). To confirm these results, we next asked whether the DHD of Ski was also required for activation of endogenous Myog expression upon differentiation. To answer this question we assessed whether reintroducing SKI or SKI⌬DHD into C2C12 TMP-tTA-mSki cells could overcome the loss of Myog and MHC expression due to the knockdown of Ski in these cells. C2C12 TMP-tTA-mSki cells were grown in GM minus Dox to achieve the maximal knockdown of endogenous mouse Ski and transfected with vectors expressing human SKI or human SKI⌬DHD, which are not subject to knockdown by the mouse Ski-targeted shRNA. These cells were analyzed for Myog and MHC expression after 2 days of culture in DM minus Dox. We found that wild-type SKI restored Myog and MHC expression. However, SKI⌬DHD failed to do so, and this inability was not attributable to poor expression of this mutant (Fig. 8E), indicating that the DHD is needed for the activation of Myog transcription.
DISCUSSION
The role of Ski in terminal differentiation has been evidenced by its ability to induce myogenesis in non-muscle cells in vitro and hypertrophy of type II fast muscle of adult mice when it was overexpressed (29 -32). It remained of great interest to determine whether Ski also regulates terminal differentiation of muscle satellite cells, which are the committed myogenic cells and responsible for the skeletal muscle regeneration (70,71). However, because Ski Ϫ/Ϫ mice die at birth, we were not able to address this issue using this mouse genetic model (34). We therefore used a well established in vitro model, satellite cellderived C2C12 myoblasts, to investigate the role of Ski in terminal differentiation and its underlying molecular mechanism. Our findings that differentiation was enhanced by overexpression of SKI and severely impaired in the absence of endogenous Ski indicate that Ski not only induces but also is necessary for differentiation of determined myoblasts. We further established a tight linkage between the myogenic activity of Ski and the expression of Myog at both mRNA and protein levels. Additional results suggest that direct regulation of Myog underlies the myogenic activity of Ski by demonstrating transcriptional activation of Myog by SKI in reporter assays and the association of Ski with the endogenous Myog regulatory region upon differentiation. Surprisingly transcriptional activation of Myog by SKI was largely mediated by its cooperation with Six1 and Eya3 through a MEF3 binding site, not with MyoD or MEF2 through the E boxes or the MEF2 binding site. The necessity of an evolutionarily conserved DHD for the interaction of SKI with Six1 is consistent with published data on the interaction between Dach and Six proteins (52). Our findings support a model in FIGURE 8. The DHD of SKI is required for its association with Six1 and its activation of Myog transcription. A, schematic diagrams represent SKI and the DHD deletion mutant (SKI⌬DHD). B, interaction of SKI with Six1 was mediated by its DHD. C2C12 cells were co-transfected with expression plasmids for FLAG-tagged Six1 and Eya3 and full-length SKI (wild type (WT)) or SKI⌬DHD (described in A) and cultured in DM for 2 days. Immunoprecipitation (IP) assays were performed as described in the legend to Fig. 7. C, C2C12 cells were co-transfected with the wild-type Myog reporter (Myog184) and expression vectors for wild-type SKI or SKI⌬DHD and cultured in DM for 2 days. Luciferase activity of each sample was calculated as described under "Experimental Procedures," and the -fold activation of Myog reporter by wild-type SKI and SKI⌬DHD were calculated as described in the legend to Fig. 6B. Data are expressed as the mean values from three independent experiments performed in triplicate. Error bars represent the S.D. D, Western blotting of the same lysates used for the luciferase assays in C revealed similar expression of wild-type SKI and SKI⌬DHD. TFIIE-␣ was used as a loading control. E, cells were cultured in GM in the absence of Dox for 6 days to achieve maximal knockdown of Ski and then transfected with expression vectors for wild-type human SKI or SKI⌬DHD. Cells were then switched to DM and cultured for 2 day prior to harvest. Western blotting revealed expression of SKI, MHC, and Myog. TFIIE-␣ was used as a loading control (Ctrl).
which Ski substitutes for Dach in the standard Dach-Six1-Eya3 complex to regulate myogenesis.
Our earlier attempts to perform these studies using C2C12 myoblasts were frustrated by our inability to maintain cells in which Ski is constitutively overexpressed or knocked down. Here we surmounted this problem using inducible vectors, which allowed cloning and propagation of transduced cells expressing endogenous Ski at normal levels prior to acute induction or knockdown of Ski expression. The retroviral vector we constructed for regulated knockdown (TMP-tTA) was modified from the TMP retroviral vector of Lowe and co-workers (62) and others (63,64). Because the TMP vector only carries the TRE-driven microRNA cassette whereas a second vector provides the tTA gene, two rounds of infection and antibiotic selection are required. This approach works well for cells whose activity is not compromised by prolonged passage, but it is not optimal for C2C12 myoblasts, which gradually lose their differentiation potential. Our new vector, TMP-tTA, carries the TRE-driven microRNA cassette and the tTA gene in a single retroviral vector so only one round of infection/antibiotic selection is needed. This vector has been proven as effective as the original TMP vector with regard to the efficiency, reversibility, dose dependence, and insignificant leakiness of knockdown. Given its simplicity and effectiveness, this knockdown vector is suitable to study any gene required for cell survival and is especially useful in cells whose activities of interest are sensitive to prolonged cell culture passage.
Earlier work showed that Ski stimulated myogenesis in nonmuscle primary avian cells by inducing both MyoD and Myog, two genes controlling myogenic lineage determination and terminal differentiation, respectively (30). We therefore assumed that loss of Ski would lead to down-regulation of both of these genes and result not only in impaired differentiation but also in loss of myogenic identity. However, in the present report, we observed that loss of Ski only affected the expression level of Myog but not MyoD in determined C2C12 myoblasts, indicating that Ski is not necessary for maintaining myogenic identity. Given the pivotal role of Myog in the initiation of differentiation (43,44,47,48), it is likely that regulation of Myog expression is the key mediator of the effect of Ski on terminal differentiation. Furthermore our data revealed that the regulation of Myog expression by Ski was not only at the protein level but also at the transcript level. This result along with the observed transactivation of Myog reporter by SKI and the occupancy of Ski on the endogenous Myog regulatory region places Myog as a direct transcriptional target of Ski.
As a non-DNA-binding transcription factor, Ski has to be brought into contact with promoters/enhancers by interaction with transcription factors that bind to specific DNA cis regulatory elements (28). Cis elements essential for transcriptional regulation of Myog include two E boxes and a MEF2 site that are bound by MyoD and MEF2, respectively (40 -42, 51, 68). Previous studies have shown that MyoD and MEF2 cooperatively activate the Myog transcription through binding to these elements at the onset of myogenesis (44). This cooperative interaction has also been seen in the upstream regulatory sequences of other muscle-specific genes, including MLC1/3 and MCK (35,(72)(73)(74). Interestingly Ski has been implicated in activation of these promoters in cooperation with MyoD, and a previous report based solely on reporter assays suggested the same mechanism for the transactivation of Myog regulatory region by Ski (36). However, our results, using the same Myog regulatory region, appear to conflict with this report by showing that the destruction of E boxes or the MEF2 site had only a marginal effect on the ability of SKI to activate the transcription of Myog reporters. We cannot explain this discrepancy, but we believe that although reporter assays can be useful for defining cis response elements their biological significance requires confirmation by additional experiments. Our inability to detect direct interactions of SKI with MyoD or MEF2 and the absence of any previous data showing these interactions suggest an indirect mechanism for the cooperation between Ski and MyoD on Myog activation.
Recent studies demonstrating the presence of functional Pbx1 and MEF3 binding sites have revealed that the Myog regulatory region is more complex than previously believed (51,53,68,75,76). Our data underscore this complexity by showing that the MEF3 site instead of the previously implicated E boxes and MEF2 site mediated transactivation of Myog by SKI. These sites reside in close proximity within the Myog regulatory region suggesting that their combined occupancy by Six1, MyoD, and MEF2 may be the basis for the cooperation between Ski and these proteins. Our finding that activation of Myog transcription by Ski requires interaction with the MEF3-binding Six1 protein provides the mechanism for recruitment of Ski to the Myog regulatory region. This interaction and the observation that Ski Ϫ/Ϫ mice exhibited a muscle defect similar to that of the Six1 Ϫ/Ϫ mice suggest a common mechanism by which both Ski and Six1 regulate myogenesis (34,51,59,(77)(78)(79).
The differentiation-dependent interaction between Ski, Six1, and Eya3 correlates well with the observation that the association of Ski with the endogenous Myog regulatory region occurs only in differentiating cells. Because growth factor signaling can block the ability of Eya to interact with Six proteins (80,81), it is possible that the withdrawal of serum growth factors initiates terminal differentiation by freeing Eya to form the Ski-Six-Eya trimeric complex on the Myog regulatory region to activate its transcription. Dachshund has also been reported to transactivate the Myog reporter through the interaction with Six and Eya (53,54). However, because activation of Myog expression and subsequent differentiation was almost abolished in the absence of Ski, we believe that in C2C12 myoblasts it is Ski, not Dach, that is the essential member of this trimeric complex. Our findings shed new light on the mechanism of the myogenic activity of Ski and the importance of the Ski-Six-Eya trimeric complex in muscle terminal differentiation.
FIGURE 1 .
1SKI stimulates differentiation of C2C12 myoblasts. A, schematic representation of retroviral vector LNIT-huSKI showing the 5Ј and 3Ј long terminal repeats (LTR), neomycin resistance gene (neo r ), internal ribosomal entry site (IRES), tTA
FIGURE 2 .
2Dox-regulated knockdown of Ski in C2C12 cells via shRNAs in the context of miR30. A, schematic representation of TMP-tTA-mSki retroviral vector showing the 5Ј long terminal repeat (LTR) and 3Ј self-inactivating long terminal repeat (SIN-LTR), puromycin resistance gene (Puro r ) driven by the phosphoglycerate kinase (PGK) promoter, internal ribosomal entry site (IRES), tTA, and a microRNA cassette downstream of TRE-CMV promoter. The shRNAs targeting mouse Ski were inserted into the microRNA cassette between the 5Ј and 3Ј flanking sequences derived from the miR30 primary transcript (5ЈmiR30 and 3ЈmiR30). B, Dox-regulated knockdown of Ski in TMP-tTA-mSki1819 clones. Western analysis of Ski expression was performed on representative C2C12 TMP-tTA-mSki1819 clones or the vector-only clone (Ctrl) cultured with (ϩDox) or without Dox (ϪDox) for 1 week. C, Western blot analysis revealed decreased Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to removal of Dox. Cells were cultured in the presence of 200 ng/ml Dox for 8 days, switched into Dox-free medium, and analyzed after culture for the indicated number of days. D, Western blot analysis revealed restoration of Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to addition of Dox. Cells were cultured in the absence of Dox for 8 days to achieve the maximal knockdown of Ski and then switched into medium containing 200 ng/ml Dox for the indicated number of days. E, Western blot analysis revealed dose response of Ski knockdown to Dox in a C2C12 TMP-tTA-mSki1145 clone. Cells were cultured in the absence of Dox for 6 days and switched into medium containing the indicated concentration of Dox for another 6 days. TFIIE-␣ was used as a loading control, and a vector-only clone (Ctrl) shows the endogenous Ski level.
FIGURE 3 .
3Loss of Ski prevents terminal differentiation of C2C12 myoblasts.Prior to the following assays, cells were cultured for 7 days in GM either with Dox as a control or without Dox to achieve maximal knockdown of Ski. A-F, a C2C12 TMP-tTA-mSki1145 clone was analyzed for the effect of Ski knockdown on myotube formation by phase-contrast microscopy (left panel). Cells were cultured to 80% confluence in GM with (ϩDox) or without Dox (ϪDox), switched to DM with continued presence or absence of Dox, and cultured for 2 and 4 days. Representative fields are shown at 25ϫ magnification. G and H, TMP-tTA-mSki1145 cells were cultured as described above and kept in DM for 3 days. Myotubes were labeled by indirect immunofluorescence with antibody against MHC (green), and nuclei were counterstained with DAPI (blue). Representative fields are shown at 100ϫ magnification. I and J, quantitative analysis of the immunofluorescence assays in G and H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The percentage of DAPI-stained nuclei in MHC-positive cells (I) and the average number of nuclei per MHC-positive myotube (J) were calculated as described under "Experimental Procedures." Data above the bar represent means of three independent experiments. Error bars show S.D.
FIGURE 4 .
4Loss of Ski reduces the expression of muscle-specific genes at both mRNA and protein levels. A, a representative C2C12 TMP-tTA-mSki1145 clone was cultured as described in the legend to Fig. 3 and kept in DM for 3 days prior to Western analysis for Ski, MHC, and Myog expression. TFIIE-␣ was used as a loading control. B, Western blotting revealed expression of Ski, MHC, and Myog in a C2C12 TMP-tTA-mSki1819 clone cultured in DM for 3 days with the indicated concentrations of Dox (0 -200 ng/ml). C-H, indirect immunofluorescence was performed on a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. Differentiating cells were detected with antibodies against Myog, p21, or MyoD (green), and total nuclei were counterstained with DAPI (blue). Representative fields are shown at 400ϫ magnification. I-K, quantitative analysis of the immunofluorescence assays in C-H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The fraction of nuclei positively stained for Myog (I), p21 (J), and MyoD (K) were calculated as described under "Experimental Procedures." Data above the bar represent means of three independent experiments. Error bars show S.D. L, quantitative real time PCR analysis of Ski, Myog, p21, and MyoD mRNA levels in a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. -Fold change of the transcript level in culture in the presence of Dox over that in the absence of Dox was calculated as described under "Experimental Procedures." Data above the bars represent means from three independent experiments performed in triplicate. Error bars show S.D.
FIGURE 5 .
5Ski binds the endogenous Myog regulatory region upon differentiation. A, schematic representation of the Myog regulatory region. E boxes (E1 and E2), the MEF2 binding site, and the MEF3 binding site are indicated as open boxes relative to the transcriptional start site (ϩ1). Primer sets used to amplify sequences spanning the Myog regulatory region or a nonpromoter region downstream of the Myog gene are represented as arrows. B, ChIP analysis of Ski occupancy of the endogenous Myog regulatory region in proliferating and differentiating cells. Equivalent amounts of cross-linked chromatin from C2C12 cells cultured in GM or in DM for 2 days were precipitated with an antibody against Ski (␣-Ski) or a normal rabbit IgG. The DNA isolated from precipitated chromatin was analyzed by PCR using primers spanning Myog regulatory region (upper), non-promoter region (middle), or Smad7 regulatory region (lower), and following electrophoresis, PCR products were visualized by ethidium bromide staining. C, Western analysis of Ski, MHC, and Myog expression in C2C12 cells cultured in GM or in DM for the indicated periods of time. TFIIE-␣ was used as a loading control.
FIGURE 6 .
6The MEF3 binding site is required for maximal activation of Myog promoter by SKI. A, schematic representation of the 184-bp proximal Myog regulatory region and its mutants. The intact E1 and E2 boxes, MEF2 site, and MEF3 site are indicated as open boxes; the mutated elements are marked by "ϫ." The sequences of wild-type binding sites are indicated in uppercase, and the mutated bases in these binding sites are indicated in lowercase. B and C, C2C12 cells were co-transfected with the wild-type or mutated reporters described above along with a SKI expression vector or an empty vector (Mock) and cultured in DM for 2 days. Luciferase activity (relative light units) of each sample was calculated as described under "Experimental Procedures." B, activation of wild-type Myog regulatory region (Myog184) by SKI was calculated as the -fold change of relative light units in SKI-transfected cells over that in the empty vector-transfected cells, which was set to an arbitrary unit of 1. Data are expressed as the mean values from three independent experiments performed in triplicate. Error bars represent the S.D. C, the activation of wild-type and mutated Myog regulatory region by SKI were calculated as described in B. The activation of the mutated Myog regulatory regions (Myog184-E1E2m, Myog184-MEF2m, and Myog184-MEF3m) by SKI is expressed as the percentage of that with the wild type, which was set at 100%. Data are expressed as the mean data of each reporter from three independent experiments performed in triplicates. Error bars represent the S.D.
FIGURE 7 .
7SKI interacts with Six1 and Eya3 but not with MyoD or MEF2. A-D, C2C12 cells were co-transfected with expression plasmids for SKI and FLAG-tagged Six1 and Eya3 (A), MEF2-FLAG (B), MyoD (C), or MEF2-FLAG and MyoD (D) and cultured in GM or DM for 2 days.
Acknowledgments-We thank Gregory Hannon and Ross
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| [
"Overexpression of the Ski pro-oncogene has been shown to induce myogenesis in non-muscle cells, to promote muscle hypertrophy in postnatal mice, and to activate transcription of muscle-specific genes. However, the precise role of Ski in muscle cell differentiation and its underlying molecular mechanism are not fully understood. To elucidate the involvement of Ski in muscle terminal differentiation, two retroviral systems were used to achieve conditional overexpression or knockdown of Ski in satellite cell-derived C2C12 myoblasts. We found that enforced expression of Ski promoted differentiation, whereas loss of Ski severely impaired it. Compromised terminal differentiation in the absence of Ski was likely because of the failure to induce myogenin (Myog) and p21 despite normal expression of MyoD. Chromatin immunoprecipitation and transcriptional reporter experiments showed that Ski occupied the endogenous Myog regulatory region and activated transcription from the Myog regulatory region upon differentiation. Transactivation of Myog was largely dependent on a MEF3 site bound by Six1, not on the binding site of MyoD or MEF2. Activation of the MEF3 site required direct interaction of Ski with Six1 and Eya3 mediated by the evolutionarily conserved Dachshund homology domain of Ski. Our results indicate that Ski is necessary for muscle terminal differentiation and that it exerts this role, at least in part, through its association with Six1 and Eya3 to regulate the Myog transcription."
] | [
"Hong Zhang \nDepartment of Biochemistry\nCase Western Reserve University\n44106ClevelandOhio\n",
"Ed Stavnezer \nDepartment of Biochemistry\nCase Western Reserve University\n44106ClevelandOhio\n"
] | [
"Department of Biochemistry\nCase Western Reserve University\n44106ClevelandOhio",
"Department of Biochemistry\nCase Western Reserve University\n44106ClevelandOhio"
] | [
"Hong",
"Ed"
] | [
"Zhang",
"Stavnezer"
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"C Sime, ",
"E Graham, ",
"R Baldock, ",
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"D Samols, ",
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"S Kuang, ",
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"Le Grand, ",
"F Rudnicki, ",
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". K Ikeda, Y Watanabe, H Ohto, K Kawakami, Mol. Cell. Biol. 22Ikeda, K., Watanabe, Y., Ohto, H., and Kawakami, K. (2002) Mol. Cell. Biol. 22, 6759 -6766",
". K Kawakami, S Sato, H Ozaki, K Ikeda, BioEssays. 22Kawakami, K., Sato, S., Ozaki, H., and Ikeda, K. (2000) BioEssays 22, 616 -626",
". X Li, V Perissi, F Liu, D W Rose, M G Rosenfeld, Science. 297Li, X., Perissi, V., Liu, F., Rose, D. W., and Rosenfeld, M. G. (2002) Science 297, 1180 -1183",
". S J Odelberg, A Kollhoff, M T Keating, Cell. 103Odelberg, S. J., Kollhoff, A., and Keating, M. T. (2000) Cell 103, 1099 -1109",
". R A Dickins, M T Hemann, J T Zilfou, D R Simpson, I Ibarra, G J Hannon, S W Lowe, Nat. Genet. 37Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J., and Lowe, S. W. (2005) Nat. Genet. 37, 1289 -1295",
". J M Silva, M Z Li, K Chang, W Ge, M C Golding, R J Rickles, D Siolas, G Hu, P J Paddison, M R Schlabach, N Sheth, J Bradshaw, J Burchard, A Kulkarni, G Cavet, R Sachidanandam, W R Mccombie, M A Cleary, S J Elledge, G J Hannon, Nat. Genet. 37Silva, J. M., Li, M. Z., Chang, K., Ge, W., Golding, M. C., Rickles, R. J., Siolas, D., Hu, G., Paddison, P. J., Schlabach, M. R., Sheth, N., Bradshaw, J., Burchard, J., Kulkarni, A., Cavet, G., Sachidanandam, R., McCombie, W. R., Cleary, M. A., Elledge, S. J., and Hannon, G. J. (2005) Nat. Genet. 37, 1281-1288",
". F Stegmeier, G Hu, R J Rickles, G J Hannon, S J Elledge, Proc. Natl. Acad. Sci. U. S. A. 102Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J., and Elledge, S. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 13212-13217",
". A P Monaghan, D R Davidson, C Sime, E Graham, R Baldock, S S Bhattacharya, R E Hill, Development. 112Monaghan, A. P., Davidson, D. R., Sime, C., Graham, E., Baldock, R., Bhat- tacharya, S. S., and Hill, R. E. (1991) Development 112, 1053-1061",
". M Gossen, H Bujard, Proc. Natl. Acad. Sci. U. S. A. 89Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551",
". D P Young, I Kushner, D Samols, J. Immunol. 181Young, D. P., Kushner, I., and Samols, D. (2008) J. Immunol. 181, 2420 -2427",
". A B Heidt, A Rojas, I S Harris, B L Black, Mol. Cell. Biol. 27Heidt, A. B., Rojas, A., Harris, I. S., and Black, B. L. (2007) Mol. Cell. Biol. 27, 5910 -5920",
". N G Denissova, F Liu, J. Biol. Chem. 279Denissova, N. G., and Liu, F. (2004) J. Biol. Chem. 279, 28143-28148",
". S Kuang, M A Rudnicki, Trends Mol. Med. 14Kuang, S., and Rudnicki, M. A. (2008) Trends Mol. Med. 14, 82-91",
". Le Grand, F Rudnicki, M A , Curr. Opin. Cell Biol. 19Le Grand, F., and Rudnicki, M. A. (2007) Curr. Opin. Cell Biol. 19, 628 -633",
". L A Gossett, D J Kelvin, E A Sternberg, E N Olson, Mol. Cell. Biol. 9Gossett, L. A., Kelvin, D. J., Sternberg, E. A., and Olson, E. N. (1989) Mol. Cell. Biol. 9, 5022-5033",
". N Rosenthal, E B Berglund, B M Wentworth, M Donoghue, B Winter, E Bober, T Braun, Arnold , H H , Nucleic Acids Res. 18Rosenthal, N., Berglund, E. B., Wentworth, B. M., Donoghue, M., Winter, B., Bober, E., Braun, T., and Arnold, H. H. (1990) Nucleic Acids Res. 18, 6239 -6246",
". B M Wentworth, M Donoghue, J C Engert, E B Berglund, N Rosenthal, Proc. Natl. Acad. Sci. U. S. A. 88Wentworth, B. M., Donoghue, M., Engert, J. C., Berglund, E. B., and Rosenthal, N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1242-1246",
". L Zhang, Wang , C , Oncogene. 26Zhang, L., and Wang, C. (2007) Oncogene 26, 1595-1605",
". C A Berkes, D A Bergstrom, B H Penn, K J Seaver, P S Knoepfler, S J Tapscott, Mol. Cell. 14Berkes, C. A., Bergstrom, D. A., Penn, B. H., Seaver, K. J., Knoepfler, P. S., and Tapscott, S. J. (2004) Mol. Cell 14, 465-477",
". C Laclef, G Hamard, J Demignon, E Souil, C Houbron, Maire , P , Development. 130Laclef, C., Hamard, G., Demignon, J., Souil, E., Houbron, C., and Maire, P. (2003) Development 130, 2239 -2252",
". C Laclef, E Souil, J Demignon, Maire , P , Mech. Dev. 120Laclef, C., Souil, E., Demignon, J., and Maire, P. (2003) Mech. Dev. 120, 669 -679",
". C Colmenares, H A Heilstedt, L G Shaffer, S Schwartz, M Berk, J C Murray, E Stavnezer, Nat. Genet. 30Colmenares, C., Heilstedt, H. A., Shaffer, L. G., Schwartz, S., Berk, M., Murray, J. C., and Stavnezer, E. (2002) Nat. Genet. 30, 106 -109",
". H Ohto, S Kamada, K Tago, S I Tominaga, H Ozaki, S Sato, K Kawakami, Mol. Cell. Biol. 19Ohto, H., Kamada, S., Tago, K., Tominaga, S. I., Ozaki, H., Sato, S., and Kawakami, K. (1999) Mol. Cell. Biol. 19, 6815-6824",
". H Ozaki, Y Watanabe, K Ikeda, K Kawakami, J. Hum. Genet. 47Ozaki, H., Watanabe, Y., Ikeda, K., and Kawakami, K. (2002) J. Hum. Genet. 47, 107-116"
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"J. Virol",
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] | [
"\nFIGURE 1 .\n1SKI stimulates differentiation of C2C12 myoblasts. A, schematic representation of retroviral vector LNIT-huSKI showing the 5Ј and 3Ј long terminal repeats (LTR), neomycin resistance gene (neo r ), internal ribosomal entry site (IRES), tTA",
"\nFIGURE 2 .\n2Dox-regulated knockdown of Ski in C2C12 cells via shRNAs in the context of miR30. A, schematic representation of TMP-tTA-mSki retroviral vector showing the 5Ј long terminal repeat (LTR) and 3Ј self-inactivating long terminal repeat (SIN-LTR), puromycin resistance gene (Puro r ) driven by the phosphoglycerate kinase (PGK) promoter, internal ribosomal entry site (IRES), tTA, and a microRNA cassette downstream of TRE-CMV promoter. The shRNAs targeting mouse Ski were inserted into the microRNA cassette between the 5Ј and 3Ј flanking sequences derived from the miR30 primary transcript (5ЈmiR30 and 3ЈmiR30). B, Dox-regulated knockdown of Ski in TMP-tTA-mSki1819 clones. Western analysis of Ski expression was performed on representative C2C12 TMP-tTA-mSki1819 clones or the vector-only clone (Ctrl) cultured with (ϩDox) or without Dox (ϪDox) for 1 week. C, Western blot analysis revealed decreased Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to removal of Dox. Cells were cultured in the presence of 200 ng/ml Dox for 8 days, switched into Dox-free medium, and analyzed after culture for the indicated number of days. D, Western blot analysis revealed restoration of Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to addition of Dox. Cells were cultured in the absence of Dox for 8 days to achieve the maximal knockdown of Ski and then switched into medium containing 200 ng/ml Dox for the indicated number of days. E, Western blot analysis revealed dose response of Ski knockdown to Dox in a C2C12 TMP-tTA-mSki1145 clone. Cells were cultured in the absence of Dox for 6 days and switched into medium containing the indicated concentration of Dox for another 6 days. TFIIE-␣ was used as a loading control, and a vector-only clone (Ctrl) shows the endogenous Ski level.",
"\nFIGURE 3 .\n3Loss of Ski prevents terminal differentiation of C2C12 myoblasts.Prior to the following assays, cells were cultured for 7 days in GM either with Dox as a control or without Dox to achieve maximal knockdown of Ski. A-F, a C2C12 TMP-tTA-mSki1145 clone was analyzed for the effect of Ski knockdown on myotube formation by phase-contrast microscopy (left panel). Cells were cultured to 80% confluence in GM with (ϩDox) or without Dox (ϪDox), switched to DM with continued presence or absence of Dox, and cultured for 2 and 4 days. Representative fields are shown at 25ϫ magnification. G and H, TMP-tTA-mSki1145 cells were cultured as described above and kept in DM for 3 days. Myotubes were labeled by indirect immunofluorescence with antibody against MHC (green), and nuclei were counterstained with DAPI (blue). Representative fields are shown at 100ϫ magnification. I and J, quantitative analysis of the immunofluorescence assays in G and H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The percentage of DAPI-stained nuclei in MHC-positive cells (I) and the average number of nuclei per MHC-positive myotube (J) were calculated as described under \"Experimental Procedures.\" Data above the bar represent means of three independent experiments. Error bars show S.D.",
"\nFIGURE 4 .\n4Loss of Ski reduces the expression of muscle-specific genes at both mRNA and protein levels. A, a representative C2C12 TMP-tTA-mSki1145 clone was cultured as described in the legend to Fig. 3 and kept in DM for 3 days prior to Western analysis for Ski, MHC, and Myog expression. TFIIE-␣ was used as a loading control. B, Western blotting revealed expression of Ski, MHC, and Myog in a C2C12 TMP-tTA-mSki1819 clone cultured in DM for 3 days with the indicated concentrations of Dox (0 -200 ng/ml). C-H, indirect immunofluorescence was performed on a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. Differentiating cells were detected with antibodies against Myog, p21, or MyoD (green), and total nuclei were counterstained with DAPI (blue). Representative fields are shown at 400ϫ magnification. I-K, quantitative analysis of the immunofluorescence assays in C-H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The fraction of nuclei positively stained for Myog (I), p21 (J), and MyoD (K) were calculated as described under \"Experimental Procedures.\" Data above the bar represent means of three independent experiments. Error bars show S.D. L, quantitative real time PCR analysis of Ski, Myog, p21, and MyoD mRNA levels in a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. -Fold change of the transcript level in culture in the presence of Dox over that in the absence of Dox was calculated as described under \"Experimental Procedures.\" Data above the bars represent means from three independent experiments performed in triplicate. Error bars show S.D.",
"\nFIGURE 5 .\n5Ski binds the endogenous Myog regulatory region upon differentiation. A, schematic representation of the Myog regulatory region. E boxes (E1 and E2), the MEF2 binding site, and the MEF3 binding site are indicated as open boxes relative to the transcriptional start site (ϩ1). Primer sets used to amplify sequences spanning the Myog regulatory region or a nonpromoter region downstream of the Myog gene are represented as arrows. B, ChIP analysis of Ski occupancy of the endogenous Myog regulatory region in proliferating and differentiating cells. Equivalent amounts of cross-linked chromatin from C2C12 cells cultured in GM or in DM for 2 days were precipitated with an antibody against Ski (␣-Ski) or a normal rabbit IgG. The DNA isolated from precipitated chromatin was analyzed by PCR using primers spanning Myog regulatory region (upper), non-promoter region (middle), or Smad7 regulatory region (lower), and following electrophoresis, PCR products were visualized by ethidium bromide staining. C, Western analysis of Ski, MHC, and Myog expression in C2C12 cells cultured in GM or in DM for the indicated periods of time. TFIIE-␣ was used as a loading control.",
"\nFIGURE 6 .\n6The MEF3 binding site is required for maximal activation of Myog promoter by SKI. A, schematic representation of the 184-bp proximal Myog regulatory region and its mutants. The intact E1 and E2 boxes, MEF2 site, and MEF3 site are indicated as open boxes; the mutated elements are marked by \"ϫ.\" The sequences of wild-type binding sites are indicated in uppercase, and the mutated bases in these binding sites are indicated in lowercase. B and C, C2C12 cells were co-transfected with the wild-type or mutated reporters described above along with a SKI expression vector or an empty vector (Mock) and cultured in DM for 2 days. Luciferase activity (relative light units) of each sample was calculated as described under \"Experimental Procedures.\" B, activation of wild-type Myog regulatory region (Myog184) by SKI was calculated as the -fold change of relative light units in SKI-transfected cells over that in the empty vector-transfected cells, which was set to an arbitrary unit of 1. Data are expressed as the mean values from three independent experiments performed in triplicate. Error bars represent the S.D. C, the activation of wild-type and mutated Myog regulatory region by SKI were calculated as described in B. The activation of the mutated Myog regulatory regions (Myog184-E1E2m, Myog184-MEF2m, and Myog184-MEF3m) by SKI is expressed as the percentage of that with the wild type, which was set at 100%. Data are expressed as the mean data of each reporter from three independent experiments performed in triplicates. Error bars represent the S.D.",
"\nFIGURE 7 .\n7SKI interacts with Six1 and Eya3 but not with MyoD or MEF2. A-D, C2C12 cells were co-transfected with expression plasmids for SKI and FLAG-tagged Six1 and Eya3 (A), MEF2-FLAG (B), MyoD (C), or MEF2-FLAG and MyoD (D) and cultured in GM or DM for 2 days."
] | [
"SKI stimulates differentiation of C2C12 myoblasts. A, schematic representation of retroviral vector LNIT-huSKI showing the 5Ј and 3Ј long terminal repeats (LTR), neomycin resistance gene (neo r ), internal ribosomal entry site (IRES), tTA",
"Dox-regulated knockdown of Ski in C2C12 cells via shRNAs in the context of miR30. A, schematic representation of TMP-tTA-mSki retroviral vector showing the 5Ј long terminal repeat (LTR) and 3Ј self-inactivating long terminal repeat (SIN-LTR), puromycin resistance gene (Puro r ) driven by the phosphoglycerate kinase (PGK) promoter, internal ribosomal entry site (IRES), tTA, and a microRNA cassette downstream of TRE-CMV promoter. The shRNAs targeting mouse Ski were inserted into the microRNA cassette between the 5Ј and 3Ј flanking sequences derived from the miR30 primary transcript (5ЈmiR30 and 3ЈmiR30). B, Dox-regulated knockdown of Ski in TMP-tTA-mSki1819 clones. Western analysis of Ski expression was performed on representative C2C12 TMP-tTA-mSki1819 clones or the vector-only clone (Ctrl) cultured with (ϩDox) or without Dox (ϪDox) for 1 week. C, Western blot analysis revealed decreased Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to removal of Dox. Cells were cultured in the presence of 200 ng/ml Dox for 8 days, switched into Dox-free medium, and analyzed after culture for the indicated number of days. D, Western blot analysis revealed restoration of Ski expression in C2C12 TMP-tTA-mSki1145 clone in response to addition of Dox. Cells were cultured in the absence of Dox for 8 days to achieve the maximal knockdown of Ski and then switched into medium containing 200 ng/ml Dox for the indicated number of days. E, Western blot analysis revealed dose response of Ski knockdown to Dox in a C2C12 TMP-tTA-mSki1145 clone. Cells were cultured in the absence of Dox for 6 days and switched into medium containing the indicated concentration of Dox for another 6 days. TFIIE-␣ was used as a loading control, and a vector-only clone (Ctrl) shows the endogenous Ski level.",
"Loss of Ski prevents terminal differentiation of C2C12 myoblasts.Prior to the following assays, cells were cultured for 7 days in GM either with Dox as a control or without Dox to achieve maximal knockdown of Ski. A-F, a C2C12 TMP-tTA-mSki1145 clone was analyzed for the effect of Ski knockdown on myotube formation by phase-contrast microscopy (left panel). Cells were cultured to 80% confluence in GM with (ϩDox) or without Dox (ϪDox), switched to DM with continued presence or absence of Dox, and cultured for 2 and 4 days. Representative fields are shown at 25ϫ magnification. G and H, TMP-tTA-mSki1145 cells were cultured as described above and kept in DM for 3 days. Myotubes were labeled by indirect immunofluorescence with antibody against MHC (green), and nuclei were counterstained with DAPI (blue). Representative fields are shown at 100ϫ magnification. I and J, quantitative analysis of the immunofluorescence assays in G and H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The percentage of DAPI-stained nuclei in MHC-positive cells (I) and the average number of nuclei per MHC-positive myotube (J) were calculated as described under \"Experimental Procedures.\" Data above the bar represent means of three independent experiments. Error bars show S.D.",
"Loss of Ski reduces the expression of muscle-specific genes at both mRNA and protein levels. A, a representative C2C12 TMP-tTA-mSki1145 clone was cultured as described in the legend to Fig. 3 and kept in DM for 3 days prior to Western analysis for Ski, MHC, and Myog expression. TFIIE-␣ was used as a loading control. B, Western blotting revealed expression of Ski, MHC, and Myog in a C2C12 TMP-tTA-mSki1819 clone cultured in DM for 3 days with the indicated concentrations of Dox (0 -200 ng/ml). C-H, indirect immunofluorescence was performed on a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. Differentiating cells were detected with antibodies against Myog, p21, or MyoD (green), and total nuclei were counterstained with DAPI (blue). Representative fields are shown at 400ϫ magnification. I-K, quantitative analysis of the immunofluorescence assays in C-H. Red bars represent culture in the presence of Dox (ϩDox), and blue bars represent culture in the absence of Dox (ϪDox). The fraction of nuclei positively stained for Myog (I), p21 (J), and MyoD (K) were calculated as described under \"Experimental Procedures.\" Data above the bar represent means of three independent experiments. Error bars show S.D. L, quantitative real time PCR analysis of Ski, Myog, p21, and MyoD mRNA levels in a C2C12 TMP-tTA-mSki1145 clone cultured in DM with or without Dox for 3 days. -Fold change of the transcript level in culture in the presence of Dox over that in the absence of Dox was calculated as described under \"Experimental Procedures.\" Data above the bars represent means from three independent experiments performed in triplicate. Error bars show S.D.",
"Ski binds the endogenous Myog regulatory region upon differentiation. A, schematic representation of the Myog regulatory region. E boxes (E1 and E2), the MEF2 binding site, and the MEF3 binding site are indicated as open boxes relative to the transcriptional start site (ϩ1). Primer sets used to amplify sequences spanning the Myog regulatory region or a nonpromoter region downstream of the Myog gene are represented as arrows. B, ChIP analysis of Ski occupancy of the endogenous Myog regulatory region in proliferating and differentiating cells. Equivalent amounts of cross-linked chromatin from C2C12 cells cultured in GM or in DM for 2 days were precipitated with an antibody against Ski (␣-Ski) or a normal rabbit IgG. The DNA isolated from precipitated chromatin was analyzed by PCR using primers spanning Myog regulatory region (upper), non-promoter region (middle), or Smad7 regulatory region (lower), and following electrophoresis, PCR products were visualized by ethidium bromide staining. C, Western analysis of Ski, MHC, and Myog expression in C2C12 cells cultured in GM or in DM for the indicated periods of time. TFIIE-␣ was used as a loading control.",
"The MEF3 binding site is required for maximal activation of Myog promoter by SKI. A, schematic representation of the 184-bp proximal Myog regulatory region and its mutants. The intact E1 and E2 boxes, MEF2 site, and MEF3 site are indicated as open boxes; the mutated elements are marked by \"ϫ.\" The sequences of wild-type binding sites are indicated in uppercase, and the mutated bases in these binding sites are indicated in lowercase. B and C, C2C12 cells were co-transfected with the wild-type or mutated reporters described above along with a SKI expression vector or an empty vector (Mock) and cultured in DM for 2 days. Luciferase activity (relative light units) of each sample was calculated as described under \"Experimental Procedures.\" B, activation of wild-type Myog regulatory region (Myog184) by SKI was calculated as the -fold change of relative light units in SKI-transfected cells over that in the empty vector-transfected cells, which was set to an arbitrary unit of 1. Data are expressed as the mean values from three independent experiments performed in triplicate. Error bars represent the S.D. C, the activation of wild-type and mutated Myog regulatory region by SKI were calculated as described in B. The activation of the mutated Myog regulatory regions (Myog184-E1E2m, Myog184-MEF2m, and Myog184-MEF3m) by SKI is expressed as the percentage of that with the wild type, which was set at 100%. Data are expressed as the mean data of each reporter from three independent experiments performed in triplicates. Error bars represent the S.D.",
"SKI interacts with Six1 and Eya3 but not with MyoD or MEF2. A-D, C2C12 cells were co-transfected with expression plasmids for SKI and FLAG-tagged Six1 and Eya3 (A), MEF2-FLAG (B), MyoD (C), or MEF2-FLAG and MyoD (D) and cultured in GM or DM for 2 days."
] | [
"(Fig. 1A)",
"(Fig. 1B)",
"(Fig. 1D, right panel)",
"(Fig. 1C)",
"(Fig. 1D, left panel)",
"(Fig. 1D, right panel)",
"Fig. 2A)",
"Fig. 2A)",
"(Fig. 2B)",
"(Fig. 2C",
"(Fig. 2D)",
"(Fig. 2E)",
"(Fig. 3, A and B",
"Fig. S1, A and B)",
"(Fig. 3, C and E)",
"(Fig. 3D)",
"(Fig. 3F)",
"Fig. S1, C and D)",
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"(Fig. 3",
"Fig. 4A",
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"Fig. S2",
"(Fig. 4B)",
"(Fig. 4, C and I)",
"(Fig. 4, E and J)",
"(Fig. 4, D and I)",
"(Fig. 4, F and J)",
"(Fig. 4, G, H, and K)",
"Fig. 4",
"(Fig. 4L)",
"(Fig. 5A)",
"(Fig. 5B, upper panel)",
"(Fig. 5B, top and middle panels)",
"(Fig. 5C",
"(Fig. 5B, bottom panel)",
"(Fig. 6A, upper panel)",
"(Fig. 6A",
"Fig. 6A",
"(Fig. 6B)",
"(Fig. 6C)",
"(Fig. 6C)",
"Fig. 7A",
"(Fig. 7",
"(Fig. 7D)",
"(Fig. 8, A and B)",
"(Fig. 8C)",
"(Fig. 8D)",
"(Fig. 8E)",
"Fig. 7",
"Fig. 6B"
] | [] | [
"tion from the 184-bp Myog regulatory region (36). However, no direct interactions between Ski and these muscle-specific transcription factors have been reported.",
"In vitro studies have revealed that terminal differentiation of myoblasts proceeds through a highly ordered sequence of events. These cells express MyoD while proliferating, but when growth stimuli are removed, they initiate expression of Myog followed by the induction of the cyclin-dependent kinase inhibitor p21 and irreversible withdrawal from the cell cycle. Subsequently these postmitotic myocytes express muscle-specific contractile proteins such as myosin heavy chain (MHC) and finally fuse into multinucleated myotubes (43). This process is governed mainly by two families of transcription factors, the MRFs and MEF2 (44 -46). The MRF gene family includes MyoD, Myf5, Myog, and MRF4 (44,47,48). All MRF family members share a highly conserved basic region and adjacent helix-loop-helix motif (bHLH) that mediates binding to a consensus DNA sequence, CANNTG, known as the E box, that is present in the regulatory regions of many muscle-specific genes. Forced expression of any MRF gene is capable of inducing expression of muscle-specific genes and activation of myogenic differentiation even in non-muscle cells. The MEF2 proteins belong to the superfamily of MADS (MCM1-agamous-deficient serum response factor) box transcription factors and directly bind an AϩT-rich element found in the promoters and enhancers of many muscle specific genes (49). Genetic analysis reveals that members of the MEF2 family are also essential for terminal muscle differentiation (50).",
"Another cis element, the MEF3 site (consensus sequence, TCAGGTT), is also present in the 133-bp Myog regulatory region. Studies of transgenic mice demonstrated that mutation of this MEF3 site abolishes correct expression of a Myog-LacZ transgene during embryogenesis (51). Two skeletal musclespecific members of the Six family (sine oculis homeodomaincontaining transcription factors), Six1 and Six4, bind to the MEF3 element and transactivate Myog transcription (51). Drosophila sine oculis (so) has been shown to act synergistically with eyes absent (eya) and dachshund (dac) by direct proteinprotein interactions. Similar interactions underlie the synergism of their mammalian homologues Six, Eya, and Dach (10,(52)(53)(54)(55)(56)(57)(58)(59). This evolutionarily conserved regulatory network of Eya/Six/Dach has been shown to regulate myogenesis in chicken somite culture and in the chick limb and to activate transcription of reporters containing the Myog MEF3 site (52,53). Interaction of mammalian Dach with Six protein is mediated by the evolutionarily conserved DHD motif (60). This has led to the suggestion that by virtue of its possession of these conserved domains (7,10,12) Ski might also interact with Six and Eya proteins to regulate Myog expression and thereby control commitment of myogenic cells to terminal differentiation (52).",
"In this study, we addressed this possibility by exploiting the well characterized mouse muscle satellite cell line, C2C12, as a model system. Using retroviral vectors to achieve tetracyclineregulated overexpression or knockdown of Ski, we asked whether Ski might not only stimulate but also be required for terminal differentiation of C2C12 cells. To probe the mechanism underlying the transcriptional regulation of Myog by Ski, the E box, MEF2, and MEF3 sites in the Myog regulatory region were investigated as the possible cis response elements. Using co-immunoprecipitation, we asked whether direct binding of Ski to MyoD, MEF2c, Six1, and/or Eya3 mediates its transcriptional activity. Finally chromatin immunoprecipitation (ChIP) assays were performed to determine whether Ski resides at the endogenous Myog regulatory region prior to or concomitant with the initiation of terminal differentiation.",
"Construction of Retroviral Vectors-The replication-defective retroviral vector LNITX was kindly provided by Dr. Fage and was described earlier (61). A DNA fragment containing the entire coding region of the human SKI cDNA was excised from the plasmid pSHHSKIN1D using BamHI and ClaI, blunt-ended with T4 DNA polymerase, and inserted into the LINTX vector at the PmeI site to produce LNIT-huSKI.",
"The replication-defective retroviral vector TMP-tTA was modified from the SIN-TREmiR30-PIG (TMP) vector (kindly provided by Dr. Scott Lowe) (62)(63)(64) by replacing its GFP gene with the tetracycline transactivator (65) gene (66). The sequences of shRNAs targeting mouse Ski were chosen using RNAi Codex and are designated by their positions in the mouse Ski cDNA sequence (GenBank TM accession number AF435852): mSki1145 (bases 1145-1163), mSki1819 (bases 1819 -1837), and mSki977 (bases 977-995). DNA forms of these shRNA inserts were generated by PCR amplification of 97-base synthetic oligonucleotides using Pfu DNA polymerase (Invitrogen) and a common set of primers (5Ј-cagaggctcgagaaggtatattgctgttgacagtgagcg-3Ј and 5Ј-cgcggcgaattccgaggcagtaggca-3Ј). The PCR products were subsequently digested with XhoI and EcoRI and inserted between these sites within TMP-tTA vector to generate TMP-tTA-mSki1145, TMP-tTA-mSki1819, and TMP-tTA-mSki977. Clones containing these shRNA-encoding inserts were sequence-verified.",
"Tissue Culture and Transfection-Proliferating mouse C2C12 myoblasts were maintained in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (Atlanta Biologicals), 100 g/ml penicillin, 100 units/ml streptomycin, and 0.002% Fungizone (Invitrogen). To avoid spontaneous differentiation, cells were always kept in subconfluent (60 -70%) conditions. Terminal differentiation was induced by switching subconfluent cells (80%) to differentiation medium (DM) consisting of Dulbecco's modified Eagle's medium, 2% heatinactivated horse serum, and antibiotics as in GM. Morphological differentiation, judged by myotube formation, was documented by digital photography of phase-contrast microscopic images.",
"Retroviral Packaging and Infection-The retrovirus packaging cell line PA317 (ATCC number SD3443) was cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 g/ml penicillin, and 100 units/ml streptomycin. They were transfected with retroviral constructs by using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. 24 h after transfection, the medium was harvested, and live cells were removed by centrifugation at 1000 ϫ g for 10 min. The retrovirus-containing supernatant was used to infect exponentially growing C2C12 cells at 40% confluence in the presence of 10 ng/ml Polybrene (Sigma). The infection was repeated with freshly harvested virus 12 h later. After an additional 12 h of culture, C2C12 cells were switched to GM plus 2 g/ml doxycycline (Dox; an analog of tetracycline) and antibiotics (G418 at 750 g/ml for LNITX-based constructs and puromycin at 2 ng/ml for TMP-tTA-based constructs). Two weeks later the resulting individual colonies were isolated using cloning discs according to the manufacturer's protocol (PGC Scientifics, 62-6151-14) and expanded in GM with Dox and G418 or puromycin. After culture in GM minus Dox, clones were screened by Western blot analysis for tetracycline-regulated Ski overexpression or knockdown.",
"Western Blotting-Whole-cell extracts were prepared from confluent C2C12 cells on 100-mm culture dishes as follows: cells were scraped into 400 l of lysis buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 0.1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 1 mM NaF, and Complete protease inhibitor mixture (Roche Applied Science)). After 10-min incubation on ice, the suspension was subjected to three freeze-thaw cycles, and cell debris were removed by centrifugation at 12,500 rpm for 15 min at 4°C. Protein concentrations were determined by Bradford assay (Bio-Rad), and equal amounts of proteins were boiled in protein loading buffer (100 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 0.2% bromphenol blue, and 2% -mercaptoethanol), separated by 6% SDS-PAGE, and transferred to Immobilon-P membranes (0.45 m, Millipore).",
"For most antibodies, blots were preblocked for 1 h at room temperature in blocking buffer (20 mM Tris, pH 7.6, 125 mM NaCl, 0.1% Tween 20, and 5% (w/v) nonfat dry milk) and incubated with primary antibodies in the same solution overnight at 4°C. The membranes were then washed with TBST (20 mM Tris, pH 7.6, 125 mM NaCl, and 0.1% Tween 20) and incubated for 1 h at room temperature with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase followed by washes with TBST. Signals were detected using either enhanced chemiluminescent horseradish peroxidase substrate (Pierce) or CDP-Star alkaline phosphatase chemiluminescent substrate (1:50; Tropix) and exposure to HyBlot CL autoradiography film (Denville Scientific).",
"When using anti-Ski G8 monoclonal antibody, the blots were preblocked overnight at 4°C in blocking buffer (PBS (7.7 mM Na 2 HPO 4 , 2.7 mM NaH 2 PO 4 , and 150 mM NaCl, pH 7.2), 0.4% casein, 1% polyvinylpyrrolidone (40,000), 10 mM EDTA, and 0.2% Tween, pH 7.2) and then incubated with primary antibody in the same solution for 30 min at room temperature. The membranes were washed with blocking buffer and further incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma) for 30 min at room temperature. Subsequently the blots were washed with blocking buffer and then with assay buffer (0.1 M EDTA, 1 mM MgCl 2 , and 0.02% azide, pH 10.0), and the signal was detected with CDP-Star alkaline phosphatase chemiluminescent substrate as above.",
"The following primary antibodies were used for immunoblotting: Ski (monoclonal antibody (1:2000; G8, Learner Research Institute Hybridoma Core Facility) or rabbit polyclonal antibody (1:1000; H329, Santa Cruz Biotechnology, Inc.)), Myog (1:1000; F5D, Santa Cruz Biotechnology, Inc.), TFIIE-␣ (1:3000; C-17, Santa Cruz Biotechnology, Inc.), MHC (1:1000; MF-20, Developmental Studies Hybridoma Bank), MyoD (1:1000; 5.8A, Santa Cruz Biotechnology, Inc.), and anti-FLAG (1:5000; M2, Sigma). The secondary antibodies used were: alkaline phosphatase-conjugated anti-mouse IgG (Fcspecific) and anti-rabbit IgG (whole molecule) (1:30,000; Sigma) and horseradish peroxidase-conjugated Mouse IgG TrueBlot TM and Rabbit IgG TrueBlot (1:1000; eBioscience).",
"Immunofluorescence-C2C12 myoblasts (2 ϫ 10 4 ) were seeded into each well of LAB-TEK 8-well chamber slides (Nunc) coated with 1 g/cm 2 laminin (Invitrogen) in GM and switched to DM for 3 days. Cells were fixed in 3.7% paraformaldehyde, PBS for 30 min at room temperature; permeabilized with 10% goat serum, 1% Triton X-100, and PBS for 10 min; and incubated with blocking buffer (PBS, 10% goat serum, and 0.1% Tween 20) for 1 h at room temperature and then with primary antibodies overnight at 4°C. Chambers were washed with PBST (PBS and 0.1% Tween 20), incubated with secondary antibodies for 1 h at room temperature, washed with PBS, and mounted in Vectashield aqueous mounting medium with DAPI (Vector Laboratories). Images were obtained using an Olympus BX50 upright fluorescence microscope equipped with Polaroid digital camera PDMC2, Polaroid PDMC2 software, and fluorescent illumination. Images were assembled using Photoshop CS (Adobe).",
"The following primary antibodies were used for immunofluorescence: MyoD (1:100; 5.8A, Santa Cruz Biotechnology, Inc.), Myog (1:100; F5D, Santa Cruz Biotechnology, Inc.), MHC (1:200; MF20, Developmental Studies Hybridoma Bank), and p21 (1:100; SX118, BD Pharmingen). Secondary antibodies were Alexa 488-or Alexa594-conjugated goat anti-mouse IgG antibody (1:300; Molecular Probes). Control experiments performed with normal IgG as the primary antibody yielded no signal above the background.",
"Quantification was performed by counting at least 1000 DAPI-stained nuclei in more than 10 random fields per culture plate. For MHC, the differentiation index ϭ nuclei within MHC-stained multinucleate myotubes/total number of DAPIstained nuclei, and the fusion index ϭ the average number of nuclei per MHC-stained myotube. For nuclear proteins, the differentiation index ϭ number of antibody-stained nuclei/total number of DAPI-stained nuclei. All experiments were performed in triplicate on three independent cultures, and the standard deviation was calculated.",
"Real Time PCR-RNA was isolated using the RNeasy kit (Qiagen) with DNase I treatment according to the manufacturer's protocol, and cDNA was generated using reverse transcriptase SuperScript TM III (Invitrogen) with random hexamer primers according to the manufacturer's instructions. Quantitative real time PCR (iCycler iQ TM , Bio-Rad) was performed using SYBR Green PCR Core Reagents (Applied Bioscience) according to the manufacturer's protocols. PCR was performed for 40 cycles of 94°C for 15 s, 60°C for 20 s, and 72°C for 20 s followed by a single 72°C extension step for 5 min. Primer sequences used for real time PCR will be provided upon request. Analyses were performed in triplicate on RNA samples from three independent experiments. Threshold cycles (Ct) of target genes were normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and relative transcript levels were calculated from the Ct values as Y ϭ 2 Ϫ⌬Ct where Y is -fold difference in amount of target gene versus Gapdh and ⌬Ct ϭ Ct x Ϫ Ct Gapdh .",
"ChIP-Chromatin immunoprecipitation experiments were performed essentially as described before (67). Briefly C2C12 cells cultured in GM or DM for 2 days were cross-linked with 1% formaldehyde, PBS for 10 min at room temperature. Fixed cells were scraped and resuspended at 2 ϫ 10 7 cells/ml in lysis buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% deoxycholate, and 0.1% SDS). Suspensions were sonicated to yield chromatin with an average DNA length of 200 -500 bp. Equal amounts of chromatin from each sample (2 ϫ 10 6 cells/assay) were preabsorbed at 4°C for 1 h with 40 l of a 50% slurry of preblocked protein A beads (Repligen; previously incubated with 1 mg/ml salmon testes DNA, 10 mg/ml bovine serum albumin, and 0.05% sodium azide in TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA)). After pelleting the beads, supernates were incubated overnight at 4°C with either 2 g of rabbit polyclonal Ski antibody (H329, Santa Cruz Biotechnology, Inc.) or normal rabbit IgG. Antibody-chromatin complexes were then captured by incubation with 40 l of a 50% slurry of preblocked protein A beads at 4°C for 1 h. The beads were washed sequentially in lysis buffer, high salt buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton-100, 0.1% deoxycholate, and 0.1% SDS), lithium salt buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% Nonidet P-40, and 0.5% deoxycholate), and TE buffer. Complexes were eluted from the beads in 150 l of elution buffer (10 mM Tris, pH 8.0, 5 mM EDTA, and 1% SDS), and formaldehyde cross-linking was reversed by overnight incubation at 65°C. After treatment with RNase A and proteinase K, DNA was isolated by phenol extraction and ethanol precipitation. The optimal PCR cycle numbers were determined by real time PCR, and 5% of purified DNA was analyzed by regular PCR using HotStart-IT Taq Master Mix (USB). 25% of each reaction mixture was resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. The following PCR primer sets were used: the mouse Myog regulatory region (Ϫ169 to ϩ39, GenBank accession number M95800), 5Ј-gggcaaaaggagagggaag-3Ј and 5Ј-agtggcaggaacaagcctt-3Ј; the non-promoter region downstream of Myog gene (ϩ1943 to ϩ2185 downstream of the Myog gene, GenBank accession number NW_001030662.1) serving as a negative control, 5Ј-gtcaagaactgacttaaggcc-3Ј and 5Ј-gacactaggagaagggtggag-3Ј; and the mouse Smad7 regulatory region (Ϫ274 to Ϫ142, GenBank accession number NW_001030635.1), 5-tagaaacccgatctgttgtttgcg-3 and 5-cctctgctcggctggttccactgc-3. For input control, 10% of cross-linked chromatin was purified as described above and assessed for PCR by using the same sets of primers.",
"Reporter Assays-A 202-bp DNA fragment (Ϫ184 to ϩ18, GenBank accession number M95800) containing the Myog regulatory region was amplified by PCR using C57BL/6 genomic DNA as template. Myog184-luciferase reporter carrying the luciferase gene downstream of this Myog regulatory region was generated by inserting HindIII-StuI-digested PCR product into the HindIII-SmaI site of the pGL3-Basic vector (Promega).",
"Myog-luciferase constructs with E box, MEF2, and MEF3 mutations (Myog184-E1E2m, Myog184-MEF2m, and Myog184-MEF3m) were generated from Myog184-luciferase using the QuikChange site-directed mutagenesis kit (Stratagene). SKI expression vector pCDNA-huSKI was described previously (23).",
"Cells (1 ϫ 10 5 ) were seeded in 12-well plates and transfected at 80% confluence using Lipofectamine 2000 (Roche Applied Science) with a combined total of 4 g of expression vector and reporter plasmid DNAs. 18 h after transfection, cells were switched to DM for 48 h prior to harvest and lysis in 1ϫ Reporter lysis buffer (Promega) with one round of freeze-thaw followed by incubation at room temperature for 20 min. Cell debris were removed by centrifugation, and luciferase activity in the supernatant was determined by a Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol using a MAXline microplate luminometer (Molecular Devices). The relative light units were generated by normalizing firefly luciferase units to Renilla luciferase units of the co-transfected pTK-Renilla-luc vector. The experiments were done in duplicate, and the reported results represent at least three independent experiments.",
"Co-immunoprecipitation-C2C12 cells (ϳ50% confluent, 100-mm dishes) were transfected with expression plasmids for FLAG-tagged Six1, FLAG-tagged Eya3, FLAG-tagged Mef2c, MyoD, and full-length SKI or its mutants using Lipofectamine 2000. 18 h after transfection, cells were refed GM or switched to DM and harvested 2 days later in 1 ml of NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, and Complete protease inhibitors mixtures). After brief sonication, cell debris were removed by centrifuge at 10,000 rpm for 15 min at 4°C. The lysates were preabsorbed with protein A beads, and protein concentrations were determined by the Bradford protein assay. Immunoprecipitations using equal amounts of proteins with either rabbit polyclonal anti-Ski or normal rabbit IgG were collected by overnight incubation with protein A beads (Repligen) at 4°C. Precipitates were washed five times in NETN buffer, resuspended and boiled in protein loading buffer, separated by 6% SDS-PAGE, and analyzed by Western blotting. If precipitating and primary Western blotting antibodies were from the same species, either horseradish peroxidase-conjugated Mouse IgG TrueBlot or Rabbit IgG TrueBlot was used as the secondary antibody accordingly.",
"Cells-Overexpression of Ski has been reported to induce non-muscle fibroblasts to differentiate into myotubes (30,31), suggesting a role of Ski in myogenic lineage determination and terminal differentiation. To assess the role of Ski in terminal differentiation independent of its possible role in myogenic lineage determination, C2C12 cells that have already committed to myogenic fate and require only serum deprivation to undergo terminal differentiation were used in this study. To regulate Ski expression, C2C12 cells were infected with LNIT-huSKI, a retroviral vector that allowed both G418 selection and Dox regulation of SKI expression (Fig. 1A). Several G418-resistant clones were isolated and propagated in GM containing Dox to suppress SKI overexpression. They were then subdivided and tested for SKI expression after 3-day cultures in either the presence or absence of Dox. Western blot analysis of cell extracts revealed obvious induction of SKI expression in several LNIT-huSKI clones after Dox withdrawal (Fig. 1B), whereas a clone infected with the LNITX empty vec-tor did not overexpress SKI regardless of Dox treatment (Fig. 1D, right panel).",
"Using these clones, we examined the effect of SKI expression on myotube formation. LNIT-huSKI cells were cultured in GM with or without Dox for 4 days. Upon reaching 80% confluence, the cells were induced to differentiate by switching to DM while maintaining the presence or absence of Dox. 1 day after initiating differentiation, LNIT-huSKI cells that expressed exogenous SKI had prematurely formed myotubes, but no myotubes were observed in the same clone cultured in the presence of Dox to suppress SKI overexpression (Fig. 1C). Western analysis of these cells revealed expression of Myog only in cells that expressed exogenous SKI (Fig. 1D, left panel). These results were not due to Dox treatment because the vector-only controls showed no obvious difference in Myog or endogenous Ski expression in the presence or absence of Dox (Fig. 1D, right panel). These data indicate that the increased SKI expression stimulates the commitment of C2C12 myoblasts to terminal muscle differentiation.",
"Generation of C2C12 Clones with Inducible Knockdown of Ski Expression-The gain-of-function study above indicated that, as in avian cells, overexpression of Ski induces muscle terminal differentiation of C2C12 cells. However, those results do not necessarily implicate endogenous Ski in this process. To address this issue we asked whether knocking down endogenous mouse Ski expression would affect the differentiation of C2C12 cells. To accomplish this and avoid potential deleterious effects on the long term cell viability due to the loss of endogenous Ski, we used tetracycline-regulated expression of shRNAs to knock down Ski in C2C12 cells. The TMP-tTA vector used for this purpose was modified from the TMP retroviral vector (62-64) by substituting the tTA gene for the GFP gene so that this single retrovirus carries both the tetracycline-controlled transactivator (tTA) and its tetracycline response element (TRE) ( Fig. 2A). DNA sequences that encoded shRNAs targeting mouse Ski were inserted within the framework of micoRNA-30 (miR30) downstream of the TRE-CMV promoter ( Fig. 2A). Transcripts of this cassette resembling natural microRNA-30 could be generated upon removal of Dox, resulting in the knockdown of Ski. Three different shRNA inserts targeting mouse Ski gene were designed, and retroviral constructs, designated as TMP-tTA-mSki977, -1145, and -1819, were generated.",
"PA317 packaging cells were transfected with each of the three TMP-tTA-mSki vectors (or the empty vector), and viruses were harvested to infect C2C12 myoblasts. Puromycinresistant clones were isolated and propagated in GM plus Dox to prevent shRNA expression. These clones were then subdivided and cultured with or without Dox for 6 days prior to testing for conditional knockdown of Ski. Western analysis of several TMP-tTA-mSki1819 clones revealed a highly efficient Dox-dependent knockdown of Ski without significant leakiness (Fig. 2B). TMP-tTA-mSki cells grown in the absence of Dox produced nearly undetectable Ski levels, whereas the same clones grown in the presence of Dox expressed Ski at levels similar to that of the vector-only control. Of ϳ40 clones tested with each of the three TMP-tTA-mSki shRNAs, 70 -80% (65), the minimal CMV promoter regulated by tetracycline response elements (TRE-CMV), and human SKI coding region (SKI). B, Doxregulated SKI expression in LNIT-huSKI clones. Western analysis of SKI expression in representative C2C12 LNIT-huSKI clones cultured under induced condition (ϪDox) or suppressed condition (ϩDox) for 3 days. A vector-only clone (LNITX) was used as a negative control (Ctrl). TFIIE-␣ was a loading control. C, a representative C2C12 LNIT-huSKI clone (F8) was cultured in GM or DM for 1 day with or without Dox, and phase-contrast microscopy shows myotube formation only in the absence of Dox. D, enhanced Myog expression by overexpression of SKI. LNIT-huSKI F8 clone or a vector-only clone (LNITX) was cultured in GM or DM for 1 day with or without Dox, and Western blot analysis revealed the expression of Myog and SKI. TFIIE-␣ was a loading control. Note that endogenous Ski expression in the right panel was revealed only after much longer exposure than the left panel where overexpression of SKI was detected.",
"exhibited Dox-dependent knockdown of Ski, indicating the high efficiency of this system (data not shown).",
"Ski Knockdown Is Reversible and Dox Dose-dependent-To determine the kinetics of Ski knockdown, a C2C12 TMP-tTA-mSki1145 clone was propagated in Dox-containing medium, transferred to Dox-free medium, and monitored for Ski expression over an 8-day period. Western analysis showed that knockdown of Ski was apparent within 4 days after Dox removal and was virtually complete after 6 days (Fig. 2C). This knockdown was completely reversible; readdition of Dox to these cells restored normal Ski expression within 4 days (Fig. 2D). The extent of Ski knockdown was also doxycycline dose-dependent; expression of Ski was barely detectable at 2 ng/ml Dox or less and was comparable to the vector-only control at 20 ng/ml Dox or more (Fig. 2E). Taken together, these data demonstrated that this single vector system allows tightly regulated and reversible knockdown of endogenous Ski.",
"Impaired Myotube Formation in the Absence of Ski-Using this Dox-regulated knockdown system, we next evaluated the consequences of the loss of Ski on terminal differentiation. A C2C12 TMP-tTA-mSki1145 clone was either kept in GM plus Dox to maintain Ski expression or switched into GM minus Dox for 7 days to achieve the maximal knockdown of Ski. Subsequently upon reaching 80% confluence, these cells were switched from GM to DM while continuing the maintenance or suppression of Ski expression. Phase microscopy of the cultures prior to switching to DM revealed that the morphology of TMP-tTA-mSki and TMP-tTA cells in GM was similar and not affected by the loss of Ski expression (Fig. 3, A and B, and sup- plemental Fig. S1, A and B). This was not true for cells that were switched into DM to induce differentiation. The TMP-tTA-mSki cells in which Ski expression was maintained exhibited extensive myotube formation (Fig. 3, C and E). However, when Ski expression was knocked down in the TMP-tTA-mSki clone, no obvious myotube formation was visible within 2 days (Fig. 3D), and only a few small myotubes had formed after 4 days (Fig. 3F). The lack of myotube formation was not due to delayed differentiation kinetics because in the absence of Dox TMP-tTA-mSki cells did not show significant myotube formation even after 6 days in DM (data not shown). Furthermore the differentiation defect of TMP-tTA-mSki cells in the absence of Dox was not due to the withdrawal of Dox because myotube formation of the TMP-tTA control was independent of Dox (supplemental Fig. S1, C and D). Thus the results indicate that Ski expression is required for terminal muscle differentiation of C2C12 cells.",
"Decreased myotube formation in the absence of Ski could be due to a block at any stage in the differentiation pathway or a failure of fully differentiated myocytes to fuse. To distinguish between these possibilities, we performed immunofluorescence for MHC, which is a terminal differentiation marker expressed in both unfused myocytes and multinucleated myotubes. Consistent with the morphological observations above, TMP-tTA-mSki cells exhibited extensive formation of MHCpositive multinucleated myotubes when expressing Ski, whereas the vast majority of cells from the same clonal line were MHC-negative when Ski expression was knocked down (Fig. 3, G and H). The effects of loss of Ski on terminal differentiation and fusion were further quantified by measuring both the percentage of nuclei in MHC-positive cells (differentiation index) and the average number of nuclei per MHC-positive myotube (fusion index). We found that the loss of Ski caused significant (p Ͻ 0.05) drops in both the differentiation index (from 46.4 Ϯ 6.2 to 14.6 Ϯ 9.8%) and fusion index (20.7 Ϯ 7.7 to 3.4 Ϯ 1.7 nuclei per myotube) (Fig. 3, I and J), respectively. The results indicate that in the absence of Ski, both differentiation (ϳ3-fold fewer MHC-positive cells) and myotube formation (ϳ6-fold fewer nuclei per myotube) were severely impaired (Fig. 3, I and J). Thus the loss of Ski resulted in a differentiation block upstream of myocyte fusion.",
"Loss of Ski Inhibits Commitment to Terminal Muscle Differentiation-To determine the stage at which terminal differentiation was blocked by the absence of Ski, we further examined the expression of an early differentiation marker, Myog, and a late differentiation marker, MHC. As shown in Fig. 4A, when maintained in GM, neither the C2C12 TMP-tTA-mSki1145 cells nor the TMP-tTA cells expressed Myog or MHC at detectable levels regardless of Dox treatment. However, after 3 days of culture in DM, TMP-tTA-mSki cells in which Ski expression was maintained by Dox treatment expressed both Myog and MHC at high levels. This was also the case when TMP-tTA cells were cultured in DM regardless of Dox treatment. In contrast, when the same TMP-tTA-mSki clone with Ski expression knocked down were cultured in DM, Myog and MHC were expressed at very low levels (Fig. 4A). Similar results were obtained using TMP-tTA-mSki clones expressing the other two shRNAs, indicating that the differen-tiation block was not due to an off-target effect of the shRNA (supplemental Fig. S2). In addition, a Dox dose-response experiment with a C2C12 TMP-tTA-mSki clone expressing a different shRNA (mSki1189) revealed that the expression of Myog and MHC dropped in parallel with the decrease in endogenous Ski expression (Fig. 4B). The loss of Myog expression indicated that differentiation was blocked at a very early stage in the absence of Ski expression.",
"The early stages of muscle differentiation are marked by a well characterized progression of protein expression including the myogenic regulatory factors (MyoD and Myog) and the cell cycle regulator (p21) (43,44,47,48). To obtain a quantitative assessment of the early disruption of the myogenic differentia- tion program due to the loss of Ski expression, we investigated the percentage of cells expressing these proteins during the differentiation of C2C12 TMP-tTA-mSki clones in the absence or presence of Dox. Myog and p21 are markers of commitment to terminal differentiation and withdrawal from the cell cycle, respectively. Their increased expression can be detected in both differentiating myocytes and fully differentiated multinucleated myotubes and is widely used to assess the early stage of differentiation. Within 3 days of switching to DM, 35.5% of TMP-tTA-mSki cells were Myog-positive when cultured in the presence of Dox (Fig. 4, C and I), and 27.9% were p21-positive (Fig. 4, E and J). In sharp contrast, in the same TMP-tTA-mSki clone with Ski expression knocked down, the percentages of Myog-and p21-positive cells decreased to 8.8% (Fig. 4, D and I) and 14.9% (Fig. 4, F and J), respectively. On the other hand, the expression of MyoD, a constitutive myogenic lineage marker of C2C12 cells, was comparable in the presence and absence of Ski expression (Fig. 4, G, H, and K). These results indicate that the loss of Ski blocked a step in the differentiation pathway downstream of MyoD. Results obtained with a representative TMP-tTA-mSki1145 clone are shown in Fig. 4, and similar results were obtained with two other clonal lines (data not shown).",
"To determine whether the observed Ski-dependent changes in protein expression were due to reductions in the expression of their mRNAs, we performed real time PCR on RNA isolated from TMP-tTA-mSki cells cultured for 3 days in DM plus or minus Dox. Concomitant with the 5.4-fold lower expression of Ski mRNA in TMP-tTA-mSki cells in the absence of Dox, Myog and p21 mRNA levels were reduced by 12-and 5.5-fold, respectively. On the other hand, the level of MyoD mRNA was not significantly affected by the loss of Ski (Fig. 4L). These results mirror those obtained in the analyses of protein expression and indicate that Ski is necessary for the transcription or accumulation of mRNAs that are important for initiating muscle terminal differentiation.",
"Ski Occupies Myog Regulatory Region in Differentiating Myoblasts-Because Ski is known to be a co-regulator of transcription it seemed likely that the changes we detected in the expression of muscle-specific mRNA might be due to direct effects of Ski on transcription. This possibility was especially appealing for Myog not only because its expression is required for the initiation of terminal differentiation (43,44,47,48) but also because published reporter gene assays have demonstrated that transient overexpression of Ski can transcriptionally activate the Myog regulatory region (36,37).",
"To investigate whether endogenous Ski might directly regulate Myog transcription, we performed ChIP assays on C2C12 cells cultured in either GM or DM. DNA was isolated from the chromatin immunoprecipitated with anti-Ski antibodies and was analyzed by PCR using primers that amplify the Myog regulatory region including the E1 box, a MEF2 site, and a MEF3 element (40 -42, 51, 68) (Fig. 5A). The ChIP assays revealed that Ski was not bound to this endogenous Myog regulatory region in proliferating C2C12 cells cultured in GM. However, Ski became associated with this region when the cells were stimulated to differentiate by switching them into DM (Fig. 5B, upper panel). The specificity of this interaction was verified by negative results in control ChIP assays using either a nonspe-cific antibody (normal IgG) and the same primers amplifying the Myog regulatory region or the Ski antibody and primers amplifying a non-promoter region downstream of Myog gene (Fig. 5B, top and middle panels). Furthermore the observation that Ski was expressed at similar levels in cells cultured in GM and DM indicated that the increased interaction between Ski and the endogenous Myog regulatory region upon differentiation was not due to a parallel increase in Ski expression (Fig. 5C). Likewise the fact that the Smad7 regulatory region, which is known to be bound by Ski (69), was occupied by Ski in both proliferating and differentiating cells (Fig. 5B, bottom panel) indicated that the chromatin binding ability of Ski did not depend merely on the change from GM to DM. Thus Ski selectively binds the Myog regulatory region in concert with a signal that initiates differentiation. These results suggest that the requirement for endogenous Ski in the initiation of terminal muscle differentiation might be due to its direct role in activating of Myog transcription.",
"MEF3 Binding Site Is Required for the Activation of Myog Regulatory Region by Ski-In light of the above results we sought to define the Ski-response cis element in the Myog regulatory region. A 184-bp Myog regulatory region sequence has been defined as a muscle-specific regulatory region and contains a number of transcriptional factor binding sites that are critical for activation of Myog transcription during differentiation (38, 40 -42, 51, 68). Among others, they include two consensus MyoD-binding E boxes (proximal E1 box and distal E2 box), a MEF2 binding site, and a MEF3 site (Fig. 6A, upper panel). Ski does not bind DNA directly, but it has previously been shown to synergize with MyoD and MEF2 in activating transcription of Myog reporters containing this 184-bp sequence (36). This synergy requires the binding of these transcription factors to their response elements in the Myog regulatory region. To address whether Ski activation of Myog regulatory region was mediated only through these binding sites, we mutated the MEF2 binding site or both E boxes (E1 and E2) to eliminate binding by their corresponding transcriptional factors (Fig. 6A). In addition, we mutated the MEF3 site, although it had not previously been implicated in activation by Ski. C2C12 myoblasts were transfected with the wild-type Myog reporter (Myog184) or its mutated derivatives (Myog184-MEF3m, Myog184-MEF2m, and Myog184-E1E2m; see Fig. 6A) along with a SKI expression vector or an empty vector, and luciferase activity was measured 2 days after switching the cells to DM. SKI expression potentiated the activity of the wild-type Myog reporter (Fig. 6B) and surprisingly activated the transcription of the reporters carrying mutations of either the MEF2 site or both E boxes to ϳ80% of the level observed with the wild-type reporter (Fig. 6C). These results suggested that activation of Myog regulatory region by SKI was not mediated exclusively by these two regulatory elements. However, activation of the reporter bearing a mutated MEF3 site by SKI was only 50% of that of the wild type (Fig. 6C), indicating that this site may be the major SKI-responsive cis element within the Myog regulatory region.",
"Cells-We have shown that activation of Myog transcription by SKI is mediated mainly through the MEF3 site in the regulatory region. Because Ski does not bind DNA directly, it seemed likely that its association with the endogenous Myog regulatory region and its activation of Myog transcription are mediated by its association with transcription factors that bind to the MEF3 element. Six1 has been shown to bind to the MEF3 site of the Myog regulatory region (51) and to synergize with Eya to positively regulate the transcription driven by this cis element (53). In addition, because Dach, a Ski family member, forms a trimeric complex with Six1 and Eya3 to regulate muscle-specific gene expression (53), it seemed possible that Ski may be tethered to Myog regulatory region via an association with Six1 and Eya3 in a similar manner. We therefore investigated whether Ski interacts with Six1 and Eya3 in muscle cells undergoing terminal differentiation. C2C12 cells were co-transfected with SKI and FLAG-tagged Six1 and Eya3 expression vectors, and cells were either cultured in GM or induced to differentiate in DM for 48 h. Extracts of these cells were immunoprecipitated with either rabbit anti-Ski or normal rabbit IgG and analyzed for co-precipitation of Six1 and Eya3 by Western blotting with anti-FLAG. As seen in Fig. 7A, neither Six1 nor Eya3 was precipitated by normal rabbit IgG, whereas Six1 was co-precipitated with SKI at comparable levels in proliferating (GM) and differentiating (DM) cells. In contrast, co-precipitation of Eya3 with SKI was barely above background in proliferating cells but clearly detectable in differentiating cells. Thus in muscle cells Ski is constitutively associated with Six1 but interacts with Eya3 only upon differentiation.",
"Because a previous study suggested that Ski activated transcription of Myog regulatory region in cooperation with MyoD and MEF2 (36), we performed similar co-precipitation assays to examine the possible interactions of Ski with these proteins. Surprisingly neither MyoD nor MEF2C co-precipitated with Lysates were immunoprecipitated with a rabbit antibody against Ski (␣-Ski lanes), and precipitates were analyzed by Western blotting using anti-Ski, anti-FLAG, or anti-MyoD antibodies. 10% of the input for immunoprecipitation (Input lanes) confirms that comparable amounts of these proteins were used in the immunoprecipitation assays. Immunoprecipitation (IP) with a normal rabbit IgG (IgG lanes) was used as a negative control. SKI in either proliferative or differentiating C2C12 cells (Fig. 7, B and C). Considering that MyoD and MEF2 activate the transcription of some muscle-specific genes in a cooperative manner (44), it seemed possible that their interactions with SKI might require the presence of both proteins. To test this possibility, similar co-immunoprecipitation experiments were performed with extracts of C2C12 cells that were co-transfected with SKI and both MyoD and MEF2 expression vectors. Once again both proteins were expressed at high levels, but neither of them co-precipitated with SKI in C2C12 cells cultured in GM or DM (Fig. 7D). Although negative results are inconclusive, these observations and the results of the reporter assays suggest that transcriptional cooperation of Ski with MyoD and MEF2 may be mediated by an indirect interaction with a DNA-bound Ski-Six1-Eya3 complex.",
"The DHD of Ski Is Required for Its Association with Six1 and Its Activation of Myog Transcription-It has been shown that Dach interacts with Six1 through its DHD (52,53). It was therefore of interest to determine whether this conserved domain in Ski also mediates its interaction with Six1. The ability of a SKI mutant lacking the DHD to interact with Six1 was examined by coimmunoprecipitation (Fig. 8, A and B). Deletion of the DHD from SKI (SKI⌬DHD) greatly reduced its ability to interact with Six1, although it did not affect interaction with Eya3.",
"Having identified the DHD as the Six1 binding domain in SKI, we sought to determine the effect of its deletion on transcriptional activation of Myog. Cotransfection experiments showed that SKI⌬DHD failed to activate transcription of the wild-type Myog reporter (Myog184) significantly compared with the wild-type SKI (Fig. 8C). Western blots of the transfected cells demonstrated that the lack of reporter activation by SKI⌬DHD was not due to a failure to express this protein at a level similar to that of the wild-type SKI (Fig. 8D). To confirm these results, we next asked whether the DHD of Ski was also required for activation of endogenous Myog expression upon differentiation. To answer this question we assessed whether reintroducing SKI or SKI⌬DHD into C2C12 TMP-tTA-mSki cells could overcome the loss of Myog and MHC expression due to the knockdown of Ski in these cells. C2C12 TMP-tTA-mSki cells were grown in GM minus Dox to achieve the maximal knockdown of endogenous mouse Ski and transfected with vectors expressing human SKI or human SKI⌬DHD, which are not subject to knockdown by the mouse Ski-targeted shRNA. These cells were analyzed for Myog and MHC expression after 2 days of culture in DM minus Dox. We found that wild-type SKI restored Myog and MHC expression. However, SKI⌬DHD failed to do so, and this inability was not attributable to poor expression of this mutant (Fig. 8E), indicating that the DHD is needed for the activation of Myog transcription.",
"The role of Ski in terminal differentiation has been evidenced by its ability to induce myogenesis in non-muscle cells in vitro and hypertrophy of type II fast muscle of adult mice when it was overexpressed (29 -32). It remained of great interest to determine whether Ski also regulates terminal differentiation of muscle satellite cells, which are the committed myogenic cells and responsible for the skeletal muscle regeneration (70,71). However, because Ski Ϫ/Ϫ mice die at birth, we were not able to address this issue using this mouse genetic model (34). We therefore used a well established in vitro model, satellite cellderived C2C12 myoblasts, to investigate the role of Ski in terminal differentiation and its underlying molecular mechanism. Our findings that differentiation was enhanced by overexpression of SKI and severely impaired in the absence of endogenous Ski indicate that Ski not only induces but also is necessary for differentiation of determined myoblasts. We further established a tight linkage between the myogenic activity of Ski and the expression of Myog at both mRNA and protein levels. Additional results suggest that direct regulation of Myog underlies the myogenic activity of Ski by demonstrating transcriptional activation of Myog by SKI in reporter assays and the association of Ski with the endogenous Myog regulatory region upon differentiation. Surprisingly transcriptional activation of Myog by SKI was largely mediated by its cooperation with Six1 and Eya3 through a MEF3 binding site, not with MyoD or MEF2 through the E boxes or the MEF2 binding site. The necessity of an evolutionarily conserved DHD for the interaction of SKI with Six1 is consistent with published data on the interaction between Dach and Six proteins (52). Our findings support a model in FIGURE 8. The DHD of SKI is required for its association with Six1 and its activation of Myog transcription. A, schematic diagrams represent SKI and the DHD deletion mutant (SKI⌬DHD). B, interaction of SKI with Six1 was mediated by its DHD. C2C12 cells were co-transfected with expression plasmids for FLAG-tagged Six1 and Eya3 and full-length SKI (wild type (WT)) or SKI⌬DHD (described in A) and cultured in DM for 2 days. Immunoprecipitation (IP) assays were performed as described in the legend to Fig. 7. C, C2C12 cells were co-transfected with the wild-type Myog reporter (Myog184) and expression vectors for wild-type SKI or SKI⌬DHD and cultured in DM for 2 days. Luciferase activity of each sample was calculated as described under \"Experimental Procedures,\" and the -fold activation of Myog reporter by wild-type SKI and SKI⌬DHD were calculated as described in the legend to Fig. 6B. Data are expressed as the mean values from three independent experiments performed in triplicate. Error bars represent the S.D. D, Western blotting of the same lysates used for the luciferase assays in C revealed similar expression of wild-type SKI and SKI⌬DHD. TFIIE-␣ was used as a loading control. E, cells were cultured in GM in the absence of Dox for 6 days to achieve maximal knockdown of Ski and then transfected with expression vectors for wild-type human SKI or SKI⌬DHD. Cells were then switched to DM and cultured for 2 day prior to harvest. Western blotting revealed expression of SKI, MHC, and Myog. TFIIE-␣ was used as a loading control (Ctrl).",
"which Ski substitutes for Dach in the standard Dach-Six1-Eya3 complex to regulate myogenesis.",
"Our earlier attempts to perform these studies using C2C12 myoblasts were frustrated by our inability to maintain cells in which Ski is constitutively overexpressed or knocked down. Here we surmounted this problem using inducible vectors, which allowed cloning and propagation of transduced cells expressing endogenous Ski at normal levels prior to acute induction or knockdown of Ski expression. The retroviral vector we constructed for regulated knockdown (TMP-tTA) was modified from the TMP retroviral vector of Lowe and co-workers (62) and others (63,64). Because the TMP vector only carries the TRE-driven microRNA cassette whereas a second vector provides the tTA gene, two rounds of infection and antibiotic selection are required. This approach works well for cells whose activity is not compromised by prolonged passage, but it is not optimal for C2C12 myoblasts, which gradually lose their differentiation potential. Our new vector, TMP-tTA, carries the TRE-driven microRNA cassette and the tTA gene in a single retroviral vector so only one round of infection/antibiotic selection is needed. This vector has been proven as effective as the original TMP vector with regard to the efficiency, reversibility, dose dependence, and insignificant leakiness of knockdown. Given its simplicity and effectiveness, this knockdown vector is suitable to study any gene required for cell survival and is especially useful in cells whose activities of interest are sensitive to prolonged cell culture passage.",
"Earlier work showed that Ski stimulated myogenesis in nonmuscle primary avian cells by inducing both MyoD and Myog, two genes controlling myogenic lineage determination and terminal differentiation, respectively (30). We therefore assumed that loss of Ski would lead to down-regulation of both of these genes and result not only in impaired differentiation but also in loss of myogenic identity. However, in the present report, we observed that loss of Ski only affected the expression level of Myog but not MyoD in determined C2C12 myoblasts, indicating that Ski is not necessary for maintaining myogenic identity. Given the pivotal role of Myog in the initiation of differentiation (43,44,47,48), it is likely that regulation of Myog expression is the key mediator of the effect of Ski on terminal differentiation. Furthermore our data revealed that the regulation of Myog expression by Ski was not only at the protein level but also at the transcript level. This result along with the observed transactivation of Myog reporter by SKI and the occupancy of Ski on the endogenous Myog regulatory region places Myog as a direct transcriptional target of Ski.",
"As a non-DNA-binding transcription factor, Ski has to be brought into contact with promoters/enhancers by interaction with transcription factors that bind to specific DNA cis regulatory elements (28). Cis elements essential for transcriptional regulation of Myog include two E boxes and a MEF2 site that are bound by MyoD and MEF2, respectively (40 -42, 51, 68). Previous studies have shown that MyoD and MEF2 cooperatively activate the Myog transcription through binding to these elements at the onset of myogenesis (44). This cooperative interaction has also been seen in the upstream regulatory sequences of other muscle-specific genes, including MLC1/3 and MCK (35,(72)(73)(74). Interestingly Ski has been implicated in activation of these promoters in cooperation with MyoD, and a previous report based solely on reporter assays suggested the same mechanism for the transactivation of Myog regulatory region by Ski (36). However, our results, using the same Myog regulatory region, appear to conflict with this report by showing that the destruction of E boxes or the MEF2 site had only a marginal effect on the ability of SKI to activate the transcription of Myog reporters. We cannot explain this discrepancy, but we believe that although reporter assays can be useful for defining cis response elements their biological significance requires confirmation by additional experiments. Our inability to detect direct interactions of SKI with MyoD or MEF2 and the absence of any previous data showing these interactions suggest an indirect mechanism for the cooperation between Ski and MyoD on Myog activation.",
"Recent studies demonstrating the presence of functional Pbx1 and MEF3 binding sites have revealed that the Myog regulatory region is more complex than previously believed (51,53,68,75,76). Our data underscore this complexity by showing that the MEF3 site instead of the previously implicated E boxes and MEF2 site mediated transactivation of Myog by SKI. These sites reside in close proximity within the Myog regulatory region suggesting that their combined occupancy by Six1, MyoD, and MEF2 may be the basis for the cooperation between Ski and these proteins. Our finding that activation of Myog transcription by Ski requires interaction with the MEF3-binding Six1 protein provides the mechanism for recruitment of Ski to the Myog regulatory region. This interaction and the observation that Ski Ϫ/Ϫ mice exhibited a muscle defect similar to that of the Six1 Ϫ/Ϫ mice suggest a common mechanism by which both Ski and Six1 regulate myogenesis (34,51,59,(77)(78)(79).",
"The differentiation-dependent interaction between Ski, Six1, and Eya3 correlates well with the observation that the association of Ski with the endogenous Myog regulatory region occurs only in differentiating cells. Because growth factor signaling can block the ability of Eya to interact with Six proteins (80,81), it is possible that the withdrawal of serum growth factors initiates terminal differentiation by freeing Eya to form the Ski-Six-Eya trimeric complex on the Myog regulatory region to activate its transcription. Dachshund has also been reported to transactivate the Myog reporter through the interaction with Six and Eya (53,54). However, because activation of Myog expression and subsequent differentiation was almost abolished in the absence of Ski, we believe that in C2C12 myoblasts it is Ski, not Dach, that is the essential member of this trimeric complex. Our findings shed new light on the mechanism of the myogenic activity of Ski and the importance of the Ski-Six-Eya trimeric complex in muscle terminal differentiation."
] | [
"JBC Papers in Press",
"JBC Papers in Press"
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"EXPERIMENTAL PROCEDURES",
"RESULTS",
"Forced Expression of SKI Stimulates Myogenic Differentiation in C2C12",
"Regulation of Muscle Differentiation by Ski-Six1-Eya3",
"SKI Associates with Eya3 and Six1 in Differentiating Muscle",
"DISCUSSION",
"FIGURE 1 .",
"FIGURE 2 .",
"FIGURE 3 .",
"FIGURE 4 .",
"FIGURE 5 .",
"FIGURE 6 .",
"FIGURE 7 ."
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"Ski Regulates Muscle Terminal Differentiation by Transcriptional Activation of Myog in a Complex with Six1 and Eya3 * □ S",
"Ski Regulates Muscle Terminal Differentiation by Transcriptional Activation of Myog in a Complex with Six1 and Eya3 * □ S"
] | [] |
247,047,030 | 2022-03-03T06:16:05Z | CCBY | null | null | 95794b37505b88470053792e5588759d0f578b78 | null | null | null | null | 10.3390/metabo12020193 | null | 35208266 | 8879184 |
Published: 19 February 2022
Teruo Miyazaki
Takashi Ito
Alessia Baseggio Conrado
Shigeru Murakami
Department of Neurology
Ghent University Hospital
9000GhentBelgium
Neuromuscular Reference Center
Ghent University Hospital
9000GhentBelgium
Published: 19 February 202210.3390/metabo12020193Received: 21 January 2022 Accepted: 16 February 2022Citation: Merckx, C.; De Paepe, B. The Role of Taurine in Skeletal Muscle Functioning and Its Potential as a Supportive Treatment for Duchenne Muscular Dystrophy. Metabolites 2022, 12, 193. https:// Academic Editors: Cholsoon Jang,
Introduction
Taurine or 2-aminoethane-sulfonic acid is primarily a free occurring sulfur-containing amino acid. Unlike most other amino acids, it is not a building block for proteins, yet classifies as a conditionally essential amino acid that is abundant in excitable tissues such as brain, retina, heart, and skeletal muscle, where intracellular concentrations range from 20 to 70 mmol/kg. Taurine is either taken up from diet, for example from fish and meat, or can be synthesized from other amino acids such as cysteine or methionine. Taurine has versatile functions: it plays an important role in osmoregulation, acts as a stabilizer of the cell membrane and of proteins, has anti-oxidant and anti-inflammatory functions, regulates mitochondrial tRNA activities, is involved in calcium homeostasis, etc. [1][2][3].
In this review, we focused on the role of taurine in muscle disease, especially in Duchenne Muscular Dystrophy (DMD), a progressive muscle wasting disorder affecting approximately 1 per 5000 male births [4]. Muscle weakness is conspicuous in the hip-and pelvic area first, and later spreads to distal regions. Patients become wheelchair-dependent in their early teens and eventually require cardiac and respiratory care since the muscles of the heart and respiratory system are affected in a life-threatening manner. While awaiting curative treatments to enter the clinic, glucocorticoids are the standard of care, and can prolong life-expectancy of DMD patients [5,6]. Although the precise mechanism by which glucocorticoids slow down disease progression in DMD is not completely understood, its anti-inflammatory action might play a crucial role. However, the use of glucocorticoids negatively influences bone health, which is already impaired in DMD patients [7]. A comprehensive overview of emerging genetic therapies in DMD is provided in the review of Sun et al. [8] The genetic cause of DMD is mutations in the dystrophin gene, located on chromosome X, which hampers the production of functional dystrophin protein. The latter is a keycomponent of the dystrophin-associated protein complex (DAPC) that provides stability to muscle fibers during contraction and relaxation by connecting the intracellular actin cytoskeleton to the basal lamina [7]. Besides membrane stabilization, DAPC also fulfils a role in signal transduction. Dystrophic muscles encounter chronic inflammation, oxidative stress, and ischemia. Eventually these detrimental processes lead to loss of muscle mass and muscle fibrosis [7].
This review explores the physiological role of taurine in skeletal muscle and focuses on the consequences of its disturbed balance in DMD. The therapeutic potential of taurine as a dietary supplement for DMD will be scrutinized.
Involvement of Taurine in Physiological Skeletal Muscle Functioning
Knowledge Gained from Knockout Models
A first clue towards an important role for taurine in the muscle was its relatively high abundance. Proper insights regarding the function of taurine in physiological muscle function were acquired upon the generation of taurine transporter (TauT) knock-out (KO) mouse models and ablation of muscle taurine content. Lack of taurine impaired the conductance velocity of the muscle without affecting nerve conductance speed [2]. In addition, exercise performance was seriously hampered in TauT KO mice as shown by a significantly lower running speed. In a different experimental set-up, the total running distance was only 20% of the distance travelled by age-matched wild type (WT) mice [2,9,10]. Besides running tests, the reduced exercise capacity of TauT KO mice also became apparent during a weight-loaded swimming test that showed an 80% decrease in swimming time compared to WT [11]. This study reported structural changes in morphology of TauT KO muscle; however, Warskulat et al. hypothesized that hampered exercise performance was likely attributed to muscle dysfunction, resulting from taurine deficiency. In evidence, serum creatine kinase levels are increased in TauT KO and serum lactate levels were raised after exercise [2]. Some of the pathological characteristics of TauT KO models such as necrotic myofibers and reduced exercise capacity resemble the features of the mdx mouse model [12,13]. Another approach to evaluate the effect of taurine depletion is the use of guanodinoethane sulfonate (GES). GES, a taurine transporter antagonist, reduces muscle taurine content by 60% [14]. Previously taurine was shown to enhance calcium uptake and release in myofibers concurrently with an increase in force production, whereas myofibers derived from GES-treated mice showed reduced force production at relevant stimulation frequencies. Interestingly, fatigue was reportedly attenuated upon GES-treatment [14].
Furthermore, the lifespan of TauT KO male mice is significantly lower than those of WT mice, 511 and 686 days, respectively. Reduced life expectancy together with increased expression of p16INK4a, an indicator of senescence, in TauT KO mice allowed to hypothesize that taurine might be involved in aging [15,16]. Furthermore, it was suggested that taurine delays muscle-specific senescence including sarcopenia in tumor necrosis factor (TNF)-α stimulated L6 myogenic rat cells. In evidence, differences in the regulation of inflammation, autophagy, and apoptosis have been reported upon taurine treatment [17]. In addition, TNF-α stimulation of L6 myogenic rat cells hampered muscle differentiation which could be restored by taurine. Presumably this effect was mediated through the PI3/AKT signaling pathway since myocyte enhancer factor-2 (MEF-2), a transcription factor involved in myogenic differentiation, was markedly decreased after knockdown of AKT [17]. Furthermore, expression of TauT increased during muscle differentiation and was even further enhanced upon binding of MEF-2 to the promotor region of TauT [18].
In conclusion, depletion of muscle taurine levels either through TauT KO or via pharmacological inhibition of the taurine transporter by GES alters force output and exercise performance. Therefore, taurine seems essential for the preservation of physiological muscle function. The sections below provide an overview of the main cellular processes in which taurine plays a role in relation to the muscle.
Taurine and Its Role in Osmotic Homeostasis
Exposure to hyperosmolar conditions can induce several detrimental cellular effects including interference with transcriptional and translational activity, induction of oxidative stress, DNA damage, and can even elicit apoptosis under certain conditions [19,20]. Thus, safeguarding the osmotic equilibrium is essential for ensuring cellular health. When cells are exposed to an environment high on NaCl, fluid is retracted from the intracellular compartment causing cellular shrinkage, molecular crowding, and increased ionic forces. In order to counteract these deleterious processes, the cell's response is a regulatory volume increase (RVI) that includes activation of inorganic ion transporters (e.g., Na + /K + /2Cl − cotransporter, the Na + /H +/− , and the Cl − /HCO 3 − exchanger), allowing an influx of ions accompanied by osmotic uptake of water [19][20][21][22][23]. However, this condition is unfavorable over time due to increased intracellular ionic forces that could interact with macromolecules. Thus secondarily, accumulation of organic osmolytes (e.g., taurine) that replace inorganic electrolytes results in normalization of ionic strength whilst preserving cellular volume and protein stability [19,20]. Rather than stimulation of de novo synthesis, osmotic stress is most likely to enhance cellular import of taurine [20]. Both the designated TauT as well as the proton-coupled amino-acid transporter (PAT) 1 are capable of accumulating taurine in the cell [24]. However, TauT is considered as the principal transporter of taurine in muscle cells as evidenced by a 98% reduction of taurine content in a TauT KO mouse [10]. Transcription of TauT mRNA is upregulated under hypertonic conditions due to binding of Nuclear Factor of Activated T-cells 5 (NFAT-5) to the 5 flank region of the TauT gene. NFAT-5, also known as tonicity responsive element binding protein (TonEBP) acts as a transcription factor of SLC6A6, the gene encoding TauT, and thus allows cellular accumulation of taurine [25,26].
Exercise can affect the osmotic balance in muscle fibers. Muscle subjected to an intensive exercise protocol resulted in increased myofiber volume, cross-sectional area, and water concentration by more than 15%, indicative for muscle fiber swelling [27,28]. The rise in myofiber water content can be partially explained by water production in cellular metabolic processes that take place during exercise [27][28][29]. Additionally, intracellular solute concentrations might be elevated during exercise as a result of phosphocreatine splitting and increased lactate and H + , ensuing water influx in order to retain osmotic balance [28] and could contribute to myofiber swelling as well. This volume increase is followed by a compensatory mechanism named the regulatory volume decrease (RVD) that releases electrolytes (e.g., K + , HCO 3 − , Cl − ) and osmolytes such as taurine concurrently with water in order to normalize cellular volume [19,20,[26][27][28][29][30][31]. Thus, taurine is released in order to counteract myofiber swelling, a phenomenon which occurs during exercise [28,31].
Taurine and Its Role in Protein and Membrane Stabilization
The stabilizing effect of taurine is mentioned in many papers. However, the mechanism by which taurine is able to exert stabilization is poorly described. In order to comprehend this characteristic, it is important to understand its interaction with water molecules, considering the chemical properties related to its molecular structure. One of the most popular hypotheses that could explain protein stabilization by osmolytes is based on preferential exclusion [32][33][34][35]. This principle builds on unfavorable interactions between proteins and osmolytes in terms of Gibbs adsorption isotherm [32]. In a denatured state, the area of the peptide backbone by which osmolytes can interact is larger and results in increased Gibbs energy (unfavorable). In order to reduce these interactions, the thermodynamic component drives the folding equilibrium towards its native state, also referred to as the osmophobic effect, which is associated with a much lower Gibbs energy. This simplified explanation implies that in the presence of stabilizing osmolytes, the Gibbs energy of the denatured state is much higher compared to Gibbs energy of the folded state [32]. Therefore, the folded protein conformation is favored and osmolytes act as protein stabilizers [32,33]. In general, the presence of osmolytes results in a specific distribution of water molecules around the proteins in a preferential hydrated state and osmolyte exclusion from the protein backbone [32][33][34][35].
Furthermore, stabilizing actions have been attributed to taurine, as well as direct interaction with protein side chains [33][34][35]. The amino group of taurine orients itself preferentially to the protein side chain. This strengthens the hydrogen bonded network of water surrounding the protein and stabilizes its native form. The latter appears to contradict the preferential exclusion theory; however, such interactions between osmolytes and side chains have also been discussed by Bolen et al. [32]. It should be noted that protein side chains are associated with other characteristics than the protein backbone and favorable interactions between osmolyte and side chains might occur. Supposedly, the latter does not substantially alter the protein folding state [32]. In the article by Brudziak et al., the protein was hydrated in the presence of taurine [35]. This might suggest that besides limited interactions between osmolytes and protein side chains, the protein is still preferentially surrounded by water molecules. Taurine was able to increase the thermal stability of both lysozyme and ubiquitin protein [35][36][37][38], although the extent of stabilization was protein specific [35].
In addition to protein stabilization, membrane stabilizing properties of taurine were hypothesized by Huxtable and Bressler [39]. Taurine inhibits the activity of phospholipid methyltransferase, which catalyzes the methylation of phosphatidylethanolamine to form phosphatidylcholine and thus taurine could alter the composition and consequently the properties and stability of phospholipid membranes [40][41][42]. In evidence, the presence of taurine decreased the viscosity of erythrocytic membranes, suggesting taurine might increase membrane fluidity [43].
Eccentric muscle contraction might induce denaturation of myofibrillar proteins as hypothesized by Paulsen et al. [44]. In addition, the unfolded protein response (UPR) is activated during exercise [45,46] which might indicate that proteins struggle to maintain native folding conformations. Interestingly, prolonged exercise increased the denaturation temperature of albumin, pointing to enhanced thermal stability [47]. The importance of taurine in protein stabilization is illustrated in the TauT KO mouse model [15]. It is assumed that a lack of taurine allows accumulation of unfolded and/or misfolded proteins in skeletal muscle which activates expression of genes involved in the UPR. Thus, taurine plays a key role in protein homeostasis of skeletal muscle [15,48].
Taurine and Its Role in Oxidative Stress
Under physiological conditions, reactive oxygen species (ROS) are balanced by antioxidant mechanisms that detoxify reactive species. A limited amount of ROS is produced during exercise and exerts advantageous effects on force generation. In addition, low levels of ROS might protect against injury through adaptations in cellular signaling upon regular training exercise [27,47,49], whereas high levels of ROS are associated with muscle dysfunction [50]. Although direct scavenging of the main ROS (e.g., superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH)) by taurine is considered unlikely [51,52], taurine is believed to relieve oxidative stress through neutralization of hypochlorous acid (HOCl) and upregulation of antioxidant enzymes [53][54][55]. Upon inflammation, neutrophils become activated and release myeloperoxidase (MPO). MPO catalyzes the reaction of chloride and H 2 O 2 , a classic ROS molecule, resulting in the formation of HOCl. HOCl has toxic effects and interferes with cellular processes including molecular transport and pump capacity [56]. Recently, it has been hypothesized that HOCl could alter excitation-contraction (E-C) coupling and impairs muscle force production [57]. HOCl is converted to TauCl after interaction with taurine. TauCl possesses anti-inflammatory properties and increases antioxidant enzymes including heme oxygenase 1 in murine microglial cells and muscle cells [58,59]. Taurine supplementation increased activity of antioxidant enzymes such as superoxide dismutase and catalase, measured in the blood of patients with type II diabetes [60]. Similar results were obtained in the liver and kidney of an ethanol-induced oxidative stress mouse model [61].
Furthermore, antioxidant effects of taurine on superoxide production and lipid peroxidation were observed in the muscle of eccentric exercised rats [62]. Similarly oxidative lipid damage was reduced by taurine treatment in a mouse model of muscle overuse [63].
The Role of Taurine in Mitochondrial Protein Synthesis
Taurine participates in the synthesis of mitochondrial proteins, more specifically proteins that require tRNA (Leu) and tRNA (Lys) with uridine on a Wobble position [64]. If the first base of the tRNA anticodon is uridine, then the classic Watson-Crick rules are substituted by Wobble base pairing rules. According to this hypothesis, H-bonds are formed between the first base of the anticodon (tRNA) and the third position of the mRNA codon. Contrastingly to Watson-Crick base pairing, Wobble pairing suggests that uridine on a Wobble position at the anticodon can form H-bonds with A, U, G, and C on the third position of the mRNA codon mRNA. Whereas, taurine-conjugated uridine tRNA will promote the formation of H-bonds between uridine and A or G on the third codon position and is required for appropriate translation to leucine (UUA/UUG) [64]. Taurine modification of uridine is required in some mitochondrial tRNAs to ensure a more specific codon-anticodon interaction and proper mitochondrial protein synthesis. Cytochrome b, ND5, and ND6 are mitochondrial proteins containing UUG codons, and synthesis of these proteins is potentially hampered if taurine conjugation of mitochondrial tRNA Leu(UUR) is deficient. ND5 and ND6 are functional subunits of oxidative phosphorylation complex I, that catalyzes electron transport from NADH to coenzyme Q [64,65]. As the process of oxidative phosphorylation is one of the main sources of ROS [50] in myofibers, preservation of mitochondrial function is considered essential in the safeguarding of oxidative stress.
Specific mutations in tRNA (Lys) and tRNA (Leu) are associated with respectively myoclonic epilepsy with ragged red fibers (MERRF) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). MERRF and MELAS are mitochondrial diseases that show disturbed protein synthesis and pathological characteristics such as exercise intolerance, thus of which some aspects resemble the TauT KO phenotype [9,64,66]. Of note, taurine supplementation in MELAS patients improved the occurrence of stroke-like episodes [67].
Taurine and Its Role in Calcium Homeostasis
Calcium is an essential cellular building block and a universal carrier of biological information. As a diffusible intracellular second messenger, calcium is involved in signaling transduction pathways and can regulate many different processes ranging from neurotransmitter release, bone formation, and blood coagulation to muscle contraction [68]. More specifically, calcium is essential during the E-C coupling mechanism that transforms the electrical input (action potential) to a mechanical output (contraction). Upon adequate stimulation, an action potential is generated and propagates over the sarcolemma to the transverse tubules that contain L-type calcium channels e.g., dihydropyridine receptors (DHPR). In the skeletal muscle, DHPR are in close contact with the ryanodine receptors (RyR) of the sarcoplasmic reticulum (SR), which releases calcium into the cytosol. Binding of calcium to troponin C induces the translocation of tropomyosin, thereby allowing the formation of cross-bridges between actin and myosin filaments and subsequent contraction [69].
Temporal and spatial changes of calcium concentration in cytoplasm or organelles are monitored by a multitude of calcium sensing proteins that determine the character and duration of the cellular response. The SR is an organelle involved in storage of calcium which is released into the cytoplasmic environment upon muscle stimulation. Taurine partially preserved SR function upon exposure to phospholipase C, which is known to hamper calcium transport, in SR derived from rat skeletal muscle. Furthermore, in the presence of 15 mM taurine, uptake of calcium oxalate by the SR was increased by more than 20% [39].
Similarly, taurine significantly increased the accumulation of calcium in SR of skinned EDL rat myofibers [70]. In this experiment, the membrane of the myofibers was mechanically removed which allowed to control intracellular taurine concentrations. The enhanced calcium SR load and subsequent release upon stimulation might explain the increment in force response upon depolarization. The authors hypothesized that taurine modulates SR calcium pump activity, allowing increased calcium accumulation [70]. These results are consistent with observations in human skeletal muscle fibers type I and II; interestingly, these different muscle fibers contain a different type of SR calcium transport ATPase (SERCA)-isoform, thus taurine might modulate either activity and/or calcium affinity [71]. Of note, a small decline in calcium sensitivity was observed in the presence of taurine in SR of skinned EDL myofibers [70].
Furthermore, voltage clamp experiments carried out in cardiomyocyte derived from guinea pig showed that the effect of taurine on calcium influx is highly dependent of the extracellular calcium concentration. In the presence of taurine (20 mM), calcium influx was slightly increased upon low extracellular calcium concentration (0.8 mM) and vice versa, the calcium influx was slightly yet significant decreased when extracellular calcium concentrations were high (3.6 mM). No effect of taurine on inward calcium current could be observed at an extracellular calcium level of 1.8 mM, suggesting that taurine aims at maintaining intracellular calcium homeostasis [72]. However, taurine modulated resting membrane potential regardless of extracellular calcium channels.
Pathophysiological Characteristics of Taurine in DMD
Regulation in Dystrophin Deficiency
Previously, it has been suggested that alterations in taurine and/or its regulation associate with dystrophinopathology [73][74][75]. Lack of dystrophin results in destabilization of DAPC and thereby rendering the sarcolemma more susceptible to contraction-induced damage. Membrane fragility is observed in dystrophin deficient muscle as evidenced by increased permeability to dyes such as Evans Blue and Procion Orange, accumulation of serum proteins such as albumin and IgG, increased levels of creatine kinase in the blood, and sarcolemma rupture in myofibers [7,76,77].
Taurine regulation is altered in dystrophic tissues of mouse models. Some studies reported a significant downregulation of the TauT in muscles of the mdx mouse model [73], whereas others reported no significant differences between mdx mice and age-matched control mice [78,79]. Seemingly, TauT is significantly downregulated in young mdx mouse and normalizes to control levels over time.
Similar to the expression of TauT, differences in muscle taurine content with age were reported as well, yet with considerable differences between studies. Taurine content of mdx muscle was comparable to controls at age 3-4 weeks, but increased by age 10 weeks [78,80]. A significant downregulation of taurine in mdx mice aged 4 and 6 weeks compared to age-matched control mice was reported [59,73], whereas other studies found no significant difference in taurine content between control and mdx mice at age 6 weeks [73,81]. A decline in taurine content was on the other hand observed in the plantaris muscle of 6-month-old mdx mice compared to wet weight yet did not hold up when compared to dry weight [79]. Contrasting results have been reported regarding the taurine content in mdx mice, however most of these studies conclude that taurine is differentially regulated in mdx mice compared to control mice at a certain time point. The expression of TauT and taurine content in the muscle of mdx mice compared to age-matched control mice is summarized in Table 1. Interestingly, muscle taurine levels increased in glucocorticoid treated mdx mice, pointing to its reestablishment by immunosuppressive therapeutic interventions [75]. As opposed to the observations in the mdx mouse model a significant increase in muscle taurine and its transporter were discerned in 8-month-old Golden Retriever Muscular dystrophy (GRMD) canine model [74]. Similarly, taurine regulation in DMD patients differs from healthy control patients, as shown by increased levels of muscle TauT protein in DMD patients [82,83]. Thus, expression of TauT is differently regulated in the mdx mouse than in the GRMD-model and DMD patients. Interestingly, the phenotype in the mdx mouse model is milder than in GRMD dogs and DMD patients. Myofiber necrosis and muscle weakness become conspicuous approximately 3 weeks after mdx mice are born. Myofiber necrosis in the mdx mouse is most explicit in the young to juvenile period, followed by necrosis at a slower pace in adult mice [74,80]. The course of disease in mdx mice thereby differs from the more progressive pathology in GRMD dogs and DMD patients, that is characterized by fatty replacement and fibrosis at an early age [74]. These differences in pathological features might be linked to regulation of TauT.
Role in Oxidative Stress Management and Mitochondrial Protein Synthesis
Mitochondria participate in cellular energy production by synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation [84]. This process includes transfer of electrons, required for ATP-synthesis, and is inevitably linked to production of ROS. Under physiological conditions, basal levels of ROS are generated as a by-product of oxidative phosphorylation. However, ROS production is enhanced upon mitochondrial dysfunction [84]. Upon oxidative phosphorylation, electron transfer is facilitated by mitochondrial protein complexes that resides within the inner mitochondrial membrane [84]. However, Onopiuk found significantly reduced protein levels of complex III, cytochrome-c reductase, and complex V, the ATP synthase, in immortalized myoblasts of mdx mice. Of note, myoblasts of mdx mice (SC-5), and control myoblasts (IMO) were used [85]. Neither of these myoblasts expressed dystrophin protein, only dystrophin mRNA was present in control myoblasts. In addition, Onopiuk et al. showed an increase in mitochondrial membrane potential [85]. Taken together, these results suggest mitochondrial dysfunction and inevitably, increased ROS production in dystrophin deficient cells. Taurine reduces ROS by upregulation of antioxidant enzymes and might preserve electron transport chain activity by safeguarding mitochondrial protein synthesis of subunits involved in the respiratory chain. In evidence, reduced taurine levels hamper expression of ND6, subunit of complex I in the mitochondria of cardiomyocytes derived from rat [64,86].
Dysregulation of Calcium Homeostasis
Excessive calcium levels are observed in dystrophic myofibers; however, calcium entry through sarcolemmal tears is not considered as the main contributor to calcium overload in dystrophic myofibers. Apparently, the open probability of calcium leak channels in the proximity of micro-tears are increased, thereby allowing an increased calcium influx [87].
Additionally, increased expression and activation of store-operated calcium channels, presumably induced by calcium-independent phospholipase A2, is observed in dystrophin deficient myofibers and could contribute to calcium overload as well [84,[88][89][90]. Moreover, a correlation between the dystrophic phenotype and expression of a stretch-activated channel, transient receptor potential canonical (TRPC) channel 1, was discovered in different muscles of the mdx mouse. The diaphragm of mdx mice was affected the most, as shown by increased Evans Blue permeability, and showed a significant upregulation of TRPC1 expression compared to controls [88]. Thus, the involvement of the TRPC channels might also contribute to the calcium overload as evidenced by increased expression of various TRPC channels in mdx mice. The cytoplasmic calcium concentration is not only determined by calcium channels/exchangers but also by the SR uptake and release of calcium, which plays an essential role during E-C coupling. RyR, responsible for the release of calcium from SR upon depolarization, is hyper nitrosylated in dystrophic muscle and consecutive depletion of calstabin-1 upon RyR-nitrosylation results in calcium leakage [84,[89][90][91][92]. During relaxation, cytoplasmic calcium ions are sequestered by SERCA, thereby lowering its cytoplasmic concentration. In the mdx mouse model, the removal of calcium ions by the SR is hampered, suggesting reduced SERCA functioning. In conclusion, calcium homeostasis is disturbed in dystrophic muscle on many levels. Chronic cytoplasmic calcium overload will induce activation of degrading pathways mediated by phospholipase 2 and proteases which eventually can lead to myofiber death [7,90].
In addition, cytoplasmic calcium overload can induce accumulation of calcium in the mitochondria and mitochondrial dysfunction. Multiple pathways have been proposed by which mitochondrial calcium overload can induce ROS production, such as mitochondrial permeability transition (MPT)-mediated release of anti-oxidative enzymes, dislocation of mitochondrial proteins involved in electron transports including cytochrome C, induction of NO, etc. [84,93]. Mitochondrial calcium overload can elicit MPT pore formation. A permeable pore is formed that spans inner and outer mitochondrial membranes and results in mitochondrial swelling [84]. Furthermore, dystrophic muscle cells show an increased susceptibility to calcium and therefore are more prone to MPT pore formation, that eventually cause mitochondrial swelling and death [87,91]. In evidence, mitochondrial swelling, indicative for MPT pore formation, was induced at a lower calcium load in mitochondria compared to WT [84,90,[92][93][94]. Interestingly, taurine is able to attenuate calcium-induced swelling of mitochondria derived from skeletal muscle [95].
Taurine Supplementation as a Therapeutic Strategy for DMD
Effect of Taurine on Muscle Force
A beneficial effect of taurine on muscle force remains controversial: supported by some studies yet disproved by others. A significant increase in peak twitch force was obtained in taurine supplemented (2.5% w/v in drinking water) mdx mice compared to untreated mdx mice at 4 weeks. However, this effect was abrogated in juvenile and adult mdx mice, respectively aged 10 weeks (2.5% w/v) and 6 months (3% w/v) [78,79]. Similarly, a >50% increase in maximum isometric tetanic specific force (sPo) was obtained by taurine supplementation in mdx mice aged 4 and 6 weeks [78,81]. However, no treatment effect (1 g/kg/day) on specific tetanic force was detected in muscle of mice aged 5-7 weeks, whereas fore limb force, assessed by means of a grip strength meter, was ameliorated [96]. Furthermore, a study performed by Barker reported no effects of taurine treatment (2.5% w/v) on peak twitch force, maximum specific force, nor fatigue or recovery in mdx mice treated from week 2 until week 4 [97]. Similarly, no effect on maximum specific force in 6-week-old mdx mice was observed when high doses of taurine (16 g/kg/day) were administered [98].
In 6-month-old mdx mice, taurine could not ameliorate specific maximum isometric force production. However, after a fatigue protocol consisting of recurrent electrical stimulation, the EDL of taurine treated mdx animals was more resistant to fatigue, as shown by a significant higher force production at the end of stimulation relative to force production at the beginning. Furthermore, EDL muscle of taurine treated mdx animals showed a significantly better recovery capacity at 10-60 min after stimulation than untreated mdx animals [79]. Taurine treatment in exercised mdx mice significantly increased in vivo forelimb muscle strength normalized to body weight [96,99], but did not alter locomotor activity of mdx mice. A similar finding was reported in a study that supplemented unexercised mdx mice. Taurine supplementation (±4 g/kg/day) in 6-week-old unexercised mdx mice significantly increased grip strength [81]. Whereas no effect of high taurine treatment (16 g/kg/day) was observed on normalized grip strength [98].
In summary, benefit of taurine supplementation on muscle force differed considerably between published studies. Although multiple studies have been executed, these used different concentrations, ingestion methods, and treatment protocols, which might explain the obtained conflicting results, and which hampers interpretation.
Effect of Taurine on Oxidative Stress and Inflammation
Although different treatment conditions and read-outs were used, multiple studies have shown anti-inflammatory and anti-oxidative effects of taurine treatment in mdx mice [59,80,81,95]. The anti-oxidant and anti-inflammatory actions of taurine have been proposed as a mechanism by which taurine protects dystrophic tissue from damage. In evidence, muscle of taurine-treated mdx mice showed significantly less NF-κβ positive fibers, TNF-α levels, neutrophil elastase, and MPO activity [59,80,81,96]. Accordingly, taurine-treated mdx mice showed lower levels of disulfide and protein thiol oxidation in muscle compared to untreated mdx mice, which could point towards protection against oxidative stress [59,81,98,100]. Similarly, ROS levels were significantly reduced upon taurine treatment in muscles of exercised dystrophic mice, as shown by dihydroethidium (DHE) staining, which reacts with O 2 − [96]. In addition, taurine normalized the resting macroscopic ionic conductance (gm), a measure for ROS production, to WT levels [96].
Effects of Taurine on E-C Coupling
In mdx mice, the threshold of the membrane potential at which a contraction is elicited is shifted towards a more negative value than in control mice. Thus, contraction is induced upon a lower depolarization state than in WT animals [96,101]. This mechanical threshold is indicative for E-C functioning and suggests alterations in E-C-coupling and/or calcium homeostasis [96,101]. Interestingly, taurine treatment in mdx mice has been shown to partially restore the mechanical threshold [96,99,101]. Taurine treatment in mdx did not alter expression of proteins involved in E-C coupling such as calcium sensitive receptors (RyR), calcium channels (DHPR channels), calcium ATP-ase pump (SERCA) and calcium binding proteins (calsequestrin) [78]. Contrastingly, taurine supplementation (2.5% w/v) in rats significantly increased expression of calsequestrin 1 in the muscle [102]. Although in the mdx mouse and rat study the same dose of taurine was used (2.5% w/v), this discrepancy in outcome might be explained by age-dependent calsequestrin regulation, changes in calsequestrin regulation upon dystrophin deficiency, species-dependent regulation, or differences in treatment protocol. However, a specific explanation cannot be pinpointed with the current literature studies available.
Effect of Taurine on Histopathological Characteristics of the Mdx Mouse
Histopathological characteristics such as necrotic myofibers and fibers with centralized nuclei, indicative for regeneration ensuing myofiber damage, are conspicuous in Hematoxylin-Eosin stained sections of mdx muscles and are used to evaluate therapeutic effectiveness [59]. In the study of Barker, taurine (2.5% w/v) reduced the amount of noncontractile area in young mdx mice, whereas no effect on the percentage regenerative fibers could be observed [78]. Other studies have reported a significant increase in healthy myofibers with peripheral nuclei upon taurine treatment [59], a decrease of histopathological features and myofiber necrosis [80,96]. Thus, taurine seems to alleviate histological features related to dystrophinopathy.
Combinatory Use of Taurine and Glucocorticoids
Combined use of taurine (1 g/kg) and α-methylprednisolone (PDN) (1 mg/kg), a synthetic adrenocortical hormone, in the exercised mdx mouse model significantly improved muscle strength in such a way that a synergistic effect was proposed by Cozzoli et al. [103]. The increase in fore limb muscle strength after 4 weeks of treatment was significantly higher in mice that received combined therapy compared to mice treated with PDN. Similarly, the increment in muscle force normalized to body weight was remarkably elevated in mice that received taurine + PDN compared to single-drug treatments. Furthermore, combined treatment normalized the rheobase potential to WT values, however similar results were observed in taurine-treated mice. No synergistic effect of combination treatment compared to PDN-treatment was detected regarding histopathological markers that included the amount of centronucleated fibers, necrosis and non-muscle tissue [103].
Contrastingly, the study of Barker et al. reported no effects of combined treatment on muscle strength. These opposing findings might be explained by differences in experimental set-up since in the latter study therapeutic intervention was initiated more closely to onset of damage, higher taurine concentrations were used for a shorter period of time and analysis occurred at the peak of damage [97]. Besides possible synergistic effects of combined treatment, taurine could also counteract side-effects related to corticosteroids. Dexamethasone causes muscle atrophy and significantly lowers the myotube diameter, which is restored upon taurine treatment [18]. Furthermore, taurine protects against glucocorticoid-induced mitochondrial dysfunction of the bone and might attenuate corticoid-induced osteoporosis and or osteonecrosis, pathological features that are conspicuous in glucocorticoid-treated DMD patients [104][105][106].
Potential Caveats of Taurine Treatment
In some rat studies, different test regimes of taurine supplementation (3% w/v for 4 weeks [107], 100 mg/kg for 2 weeks [108]; 500 mg/kg for 2 weeks [109]) resulted in increased muscle taurine content, whilst other long-term studies (1% w/v ≈ 50 mg/kg for 22 weeks [110]) could not report increased muscle taurine levels. Similarly, in mouse studies muscle taurine levels were elevated upon taurine supplementation (4% w/v for 3 weeks [59]; 3% w/v for 4 weeks [79]). Interestingly, one study showed that continuous taurine supplementation (2.5% w/v) in mdx mice increased muscle taurine concentration up to the age of 4 weeks, but when treatment was prolonged up to the age of 10 weeks, muscle taurine concentration was significantly decreased in taurine supplemented mdx mice compared to untreated mdx mice [78]. Similarly, high doses of taurine (8% w/v ≈ 16 g/kg/day) added to the drinking water up until the age of 6 weeks, did not increase muscle taurine content [98]. Therefore, it might be hypothesized that the muscle taurine content is strictly regulated. Since chronic treatment might not be able to increase intramuscular taurine levels it is not known if long-term taurine treatment could effectively attenuate muscle damage. Furthermore, taurine intake (≈5 g/day) for a period of 7 days was reported not to alter muscle taurine levels in humans [111].
In general, taurine is well tolerated and safe if used in appropriate concentrations [112]. One study has reported gastro-intestinal complaints; however, taurine was used in combination with other nutritional supplements [112,113]. Another study aimed to investigate taurine supplementation in patients with end-stage renal failure. In healthy subjects, excessive taurine is excreted; however, due to kidney failure the taurine overload was not immediately cleared and these patients suffered from dizziness and vertigo [114] which was the reason to discontinue the study.
High taurine treatment (16 g/kg) in 6 week-old animals significantly reduced the body weight of mdx mice by more than 20% and shortened tibia length by 10%. Of note, the body weight and tibia length of untreated mdx mice were comparable to those of WT animals in this experiment [98]. Similarly, taurine treatment (3% w/v) for 4 weeks in adult mdx mice (5 months old) reduced body weight and muscle mass of mdx mice; however, the weight of these animals still exceeded that of WT animals [79]. Interestingly, no effect of taurine on body weight was observed in WT animals. Obesity is commonly observed in DMD patients, especially in glucocorticoid-treated patients [7,115,116]. Therefore, it remains unclear if taurine-induced decreased body weight would be disadvantageous in these patients. Obviously if growth is hampered, this should be avoided. A study conducted in piglets reported a reduced gain to feed ratio upon higher taurine supplementation (3% taurine diet) compared to untreated piglets whilst supplementation with 0.3% taurine might improve growth [117]. Terrill et al. have hypothesized that high taurine supplementation in young animals interferes with the taurine synthesis pathway, shifting the equilibrium to the left inducing increased cysteine levels in the plasma which could hamper growth [98].
Other Nutritional Supplements Used in Duchenne Muscular Dystrophy
Approximately 50-65% of DMD patients are using vitamins or nutritional supplements [115,116], which indicates that the quest for supportive treatment, including but not limited to taurine, in DMD is relevant. A detailed overview of nutritional supplements that are under investigation for the treatment of DMD is provided in the review of Boccanegra et al. [118].
The current nutritional guidelines for patients are very similar to those used for the general population. If serum 25-hydroxy-vitamin D drops below 30 ng/mL or calcium intake is low, the use of vitamin D and, respectively, calcium intake under the form of calcium citrate or calcium carbonate is advised in order to stimulate bone health, which could be impaired in DMD patients [116,117,119]. In addition, other nutrients are currently under investigation. Coenzyme Q10, a naturally occurring anti-oxidant, significantly improved muscle strength in steroid-treated DMD patients [118,120]. Similarly, beneficial effects of L-arginine, creatine, omega-3 fatty acids have been observed in clinical trials [118].
Conclusions
Taurine is involved in numerous processes such as protein stabilization and osmotic homeostasis and appears to be indispensable for physiological muscle function as evidenced in the TauT KO mouse. We further zoomed in on DMD, a severe muscle disorder that showed altered regulation of taurine and its transporter. Multiple facets of dystrophinopathology and potential mechanisms on how taurine might act on these pathological features have been proposed and discussed in view of the mdx mouse model. Promising results have been obtained in the mdx mouse model in terms of inflammation, muscle strength, oxidative stress, etc. and led to the conclusion that taurine supplementation is relevant for DMD pathology. Former clinical trials conducted to evaluate the anti-aging or mood stabilizing effects of taurine have shown that taurine is well tolerated and considered safe upon appropriate use. However, up until now, no clinical trials have been conducted that evaluated taurine as a treatment for DMD patients. We propose that taurine has the potential to act as supportive therapy in combination with glucocorticoids for the treatment of DMD patients and further studies should be conducted to evaluate the effectiveness of chronic taurine supplementation.
Table 1 .
1Summary of TauT and taurine expression in the mdx mouse.Timepoint of Analysis
Muscle TauT Content
Muscle Taurine Content
Muscle Type
Reference
18 days
↓ in mdx mice
≈ controls
quadriceps
73
22 days
/
≈ controls
quadriceps
80
28 days
28 days
≈ controls
≈ controls
↓ in mdx mice
≈ controls
quadriceps
tibialis anterior
73
78
6 weeks
6 weeks
6 weeks
↓ in mdx mice
/
/
≈ controls
↓ in mdx mice
≈ controls
quadriceps
quad/gas
tibialis anterior
73
59
81
10 weeks
≈ controls
↑ in mdx
tibialis anterior
78
6 months
≈ controls
↓ in mdx mice (wet weight)
≈ controls
(dry weight)
EDL (TauT); plantaris
(taurine)
79
Funding: This research received no external funding.Conflicts of Interest:The authors declare no conflict of interest.
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Taurine and methylprednisolone administration at close proximity to the onset of muscle degeneration is ineffective at attenuating force loss in the hind-limb of 28 days mdx mice. R G Barker, C Van Der Poel, D Horvath, R M Murphy, 10.3390/sports60401096Barker, R.G.; Van der Poel, C.; Horvath, D.; Murphy, R.M. Taurine and methylprednisolone administration at close proximity to the onset of muscle degeneration is ineffective at attenuating force loss in the hind-limb of 28 days mdx mice. Sports 2018, 6, 109. [CrossRef]
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"Teruo Miyazaki ",
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"\nNeuromuscular Reference Center\nGhent University Hospital\n9000GhentBelgium\n"
] | [
"Department of Neurology\nGhent University Hospital\n9000GhentBelgium",
"Neuromuscular Reference Center\nGhent University Hospital\n9000GhentBelgium"
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"Teruo",
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] | [
"Wen",
"Li",
"Zhang",
"Duan",
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] | [
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"Oxidative stress caused by mitochondrial calcium overload. T I Peng, M J Jou, 10.1111/j.1749-6632.2010.05634.xAnn. N. Y. Acad. Sci. 1201PubMedPeng, T.I.; Jou, M.J. Oxidative stress caused by mitochondrial calcium overload. Ann. N. Y. Acad. Sci. 2010, 1201, 183-188. [CrossRef] [PubMed]",
"Duchenne Muscular Dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. M V Dubinin, E Y Talanov, K S Tenkov, V S Starinets, I B Mikheeva, M G Sharapov, K N Belosludtsev, 10.1016/j.bbadis.2020.165674Biochim. Biophys. Acta Mol. Basis Dis. 2020, 1866, 165674. [CrossRefDubinin, M.V.; Talanov, E.Y.; Tenkov, K.S.; Starinets, V.S.; Mikheeva, I.B.; Sharapov, M.G.; Belosludtsev, K.N. Duchenne Muscular Dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. Biochim. Biophys. Acta Mol. Basis Dis. 2020, 1866, 165674. [CrossRef]",
"Taurine enhances skeletal muscle mitochondrial function in a rat model of resistance training. M M Ommati, O Farshad, A Jamshidzadeh, R Heidari, 10.1016/j.phanu.2019.1001619100161Ommati, M.M.; Farshad, O.; Jamshidzadeh, A.; Heidari, R. Taurine enhances skeletal muscle mitochondrial function in a rat model of resistance training. PharmaNutrition 2019, 9, 100161. [CrossRef]",
"Assessment of resveratrol, apocynin and taurine on mechanical-metabolic uncoupling and oxidative stress in a mouse model of Duchenne Muscular Dystrophy: A comparison with the gold standard, α-methyl prednisolone. R F Capogrosso, A Cozzoli, P Mantuano, G M Camerino, A M Massari, V T Sblendorio, A De Luca, 10.1016/j.phrs.2016.02.016Pharmacol. Res. 106Capogrosso, R.F.; Cozzoli, A.; Mantuano, P.; Camerino, G.M.; Massari, A.M.; Sblendorio, V.T.; De Luca, A. Assessment of resveratrol, apocynin and taurine on mechanical-metabolic uncoupling and oxidative stress in a mouse model of Duchenne Muscular Dystrophy: A comparison with the gold standard, α-methyl prednisolone. Pharmacol. Res. 2016, 106, 101-113. [CrossRef]",
"Taurine and methylprednisolone administration at close proximity to the onset of muscle degeneration is ineffective at attenuating force loss in the hind-limb of 28 days mdx mice. R G Barker, C Van Der Poel, D Horvath, R M Murphy, 10.3390/sports60401096Barker, R.G.; Van der Poel, C.; Horvath, D.; Murphy, R.M. Taurine and methylprednisolone administration at close proximity to the onset of muscle degeneration is ineffective at attenuating force loss in the hind-limb of 28 days mdx mice. Sports 2018, 6, 109. [CrossRef]",
"Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction. J R Terrill, G J Pinniger, K V Nair, M D Grounds, P G Arthur, 10.1371/journal.pone.0187317PLoS ONE. 12PubMedTerrill, J.R.; Pinniger, G.J.; Nair, K.V.; Grounds, M.D.; Arthur, P.G. Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction. PLoS ONE 2017, 12, e0187317. [CrossRef] [PubMed]",
"Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1. A De Luca, S Pierno, A Liantonio, M Cetrone, C Camerino, B Fraysse, D C Camerino, 10.1124/jpet.102.041343J. Pharmacol. Exp. Ther. 304PubMedDe Luca, A.; Pierno, S.; Liantonio, A.; Cetrone, M.; Camerino, C.; Fraysse, B.; Camerino, D.C. Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1. J. Pharmacol. Exp. Ther. 2003, 304, 453-463. [CrossRef] [PubMed]",
"Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: Influence of sample manipulation, oxidative stress and ageing. D Giustarini, I Dalle-Donne, A Milzani, R Rossi, 10.1016/j.mad.2011.02.001Mech. Ageing Dev. 132PubMedGiustarini, D.; Dalle-Donne, I.; Milzani, A.; Rossi, R. Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: Influence of sample manipulation, oxidative stress and ageing. Mech. Ageing Dev. 2011, 132, 141-148. [CrossRef] [PubMed]",
"Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine. A De Luca, S Pierno, A Liantonio, M Cetrone, C Camerino, S Simonetti, D C Camerino, 10.1038/sj.bjp.0703907Br. J. Pharmacol. 132De Luca, A.; Pierno, S.; Liantonio, A.; Cetrone, M.; Camerino, C.; Simonetti, S.; Camerino, D.C. Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine. Br. J. Pharmacol. 2001, 132, 1047-1054. [CrossRef]",
"Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation. C A Goodman, D Horvath, C Stathis, T Mori, K Croft, R M Murphy, A Hayes, 10.1152/japplphysiol.00040.2009J. Appl. Physiol. 107PubMedGoodman, C.A.; Horvath, D.; Stathis, C.; Mori, T.; Croft, K.; Murphy, R.M.; Hayes, A. Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation. J. Appl. Physiol. 2009, 107, 144-154. [CrossRef] [PubMed]",
"Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne Muscular Dystrophy. A Cozzoli, J F Rolland, R F Capogrosso, V T Sblendorio, V Longo, S Simonetti, A De Luca, 10.1111/j.1365-2990.2010.01106.xNeuropathol. Appl. Neurobiol. 37PubMedCozzoli, A.; Rolland, J.F.; Capogrosso, R.F.; Sblendorio, V.T.; Longo, V.; Simonetti, S.; De Luca, A. Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne Muscular Dystrophy. Neuropathol. Appl. Neurobiol. 2011, 37, 243-256. [CrossRef] [PubMed]",
"Taurine inhibits glucocorticoid-induced bone mitochondrial injury, preventing osteonecrosis in rabbits and cultured osteocytes. H Hirata, S Ueda, T Ichiseki, M Shimasaki, Y Ueda, A Kaneuji, N Kawahara, 10.3390/ijms21186892Int. J. Mol. Sci. 2020Hirata, H.; Ueda, S.; Ichiseki, T.; Shimasaki, M.; Ueda, Y.; Kaneuji, A.; Kawahara, N. Taurine inhibits glucocorticoid-induced bone mitochondrial injury, preventing osteonecrosis in rabbits and cultured osteocytes. Int. J. Mol. Sci. 2020, 21, 6892. [CrossRef]",
"Potential role of arginine, glutamine and taurine in ameliorating osteoporotic biomarkers in ovariectomized rats. H Hanaa, A H Hamza, Rep. Opin. 1Hanaa, H.; Hamza, A.H. Potential role of arginine, glutamine and taurine in ameliorating osteoporotic biomarkers in ovariec- tomized rats. Rep. Opin 2009, 1, 24-35.",
"Medication-related osteonecrosis of the jaw in a Duchenne Muscular Dystrophy patient. L Campos, L N B Miziara, M Gallottini, K Ortega, F Martins, 10.1016/j.pdpdt.2020.101826Photodiagnosis Photodyn. Ther. 31PubMedCampos, L.; Miziara, L.N.B.; Gallottini, M.; Ortega, K.; Martins, F. Medication-related osteonecrosis of the jaw in a Duchenne Muscular Dystrophy patient. Photodiagnosis Photodyn. Ther. 2020, 31, 101826. [CrossRef] [PubMed]",
"The cytoprotective role of taurine in exercise-induced muscle injury. R Dawson, Jr, M Biasetti, S Messina, J Dominy, 10.1007/s007260200017Amino Acids. 22PubMedDawson, R., Jr.; Biasetti, M.; Messina, S.; Dominy, J. The cytoprotective role of taurine in exercise-induced muscle injury. Amino Acids 2002, 22, 309-324. [CrossRef] [PubMed]",
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"Effects of taurine administration in rat skeletal muscles on exercise. Y Yatabe, S Miyakawa, T Miyazaki, Y Matsuzaki, N Ochiai, 10.1007/s10776-002-0636-1J. Orthop. Sci. 8Yatabe, Y.; Miyakawa, S.; Miyazaki, T.; Matsuzaki, Y.; Ochiai, N. Effects of taurine administration in rat skeletal muscles on exercise. J. Orthop. Sci. 2003, 8, 415-419. [CrossRef]",
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"Seven days of oral taurine supplementation does not increase muscle taurine content or alter substrate metabolism during prolonged exercise in humans. S D Galloway, J L Talanian, A K Shoveller, G J Heigenhauser, L L Spriet, J. Appl. Physiol. 105PubMedGalloway, S.D.; Talanian, J.L.; Shoveller, A.K.; Heigenhauser, G.J.; Spriet, L.L. Seven days of oral taurine supplementation does not increase muscle taurine content or alter substrate metabolism during prolonged exercise in humans. J. Appl. Physiol. 2008, 105, 643-651. [PubMed]",
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] | [
"Taurine is involved in energy metabolism in muscles, adipose tissue, and the liver",
"Taurine transporter knockout depletes muscle taurine levels and results in severe skeletal muscle impairment but leaves cardiac function uncompromised",
"The appeal of a safe amino acid for skeletal muscle disorders",
"Global epidemiology of Duchenne Muscular Dystrophy: An updated systematic review and meta-analysis",
"Practice guideline update summary: Corticosteroid treatment of Duchenne Muscular Dystrophy: Report of the guideline development subcommittee of the American academy of neurology",
"Mechanisms and clinical applications of glucocorticoid steroids in muscular dystrophy",
"Aartsma-Rus, A. Duchenne Muscular Dystrophy",
"Therapeutic strategies for Duchenne Muscular Dystrophy: An update",
"Tissue taurine depletion alters metabolic response to exercise and reduces running capacity in mice",
"Phenotype of the taurine transporter knockout mouse",
"Taurine depletion caused by knocking out the taurine transporter gene leads to cardiomyopathy with cardiac atrophy",
"Osmolytes as mediators of the muscle tissue's responses to inflammation: Emerging regulators of myositis with therapeutic potential",
"Cardiac and skeletal muscle abnormality in taurine transporter-knockout mice",
"The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse",
"Tissue depletion of taurine accelerates skeletal muscle senescence and leads to early death in mice",
"Potential anti-aging role of taurine via proper protein folding: A study from taurine transporter knockout mouse",
"Taurine attenuates catabolic processes related to the onset of sarcopenia",
"Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles",
"The role of hyperosmotic stress in inflammation and disease",
"Cellular response to hyperosmotic stresses",
"Dynamic regulation of barrier integrity of the corneal endothelium",
"Activation of ion transport systems during cell volume regulation",
"K+ transport and volume regulatory response by NKCC in resting rat hindlimb skeletal muscle",
"Significance of taurine transporter (TauT) in homeostasis and its layers of regulation",
"Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells",
"TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc",
"Dehydration and exercise-induced muscle damage: Implications for recovery",
"Hyperthermia, dehydration, and osmotic stress: Unconventional sources of exercise-induced reactive oxygen species",
"Effects of endurance exercise on metabolic water production and plasma volume",
"Physiology of cell volume regulation in vertebrates",
"Reactive oxygen species are important mediators of taurine release from skeletal muscle cells",
"The osmophobic effect: Natural selection of a thermodynamic force in protein folding",
"Are stabilizing osmolytes preferentially excluded from the protein surface? FTIR and MD studies",
"Influence of osmolytes on protein and water structure: A step to understanding the mechanism of protein stabilization",
"Taurine as a water structure breaker and protein stabilizer",
"The stabilization of proteins by osmolytes",
"Role of the osmolyte taurine on the folding of a model protein, hen egg white lysozyme, under a crowding condition",
"Thermodynamic analysis of osmolyte effect on thermal stability of ribonuclease A in terms of water activity",
"Effect of taurine on a muscle intracellular membrane",
"Effect of taurine and methionine on sarcoplasmic reticular Ca 2+ transport and phospholipid methyltransferase activity",
"Interaction of taurine with methionine: Inhibition of myocardial phospholipid methyltransferase",
"Physiological role of taurine-from organism to organelle",
"Membrane-modifying effect of taurine",
"Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans",
"The unfolded protein response mediates adaptation to exercise in skeletal muscle through a PGC-1α/ATF6α complex",
"Endoplasmic reticulum unfolded protein response, aging and exercise: An update",
"The effect of prolonged intense physical exercise of special forces volunteers on their plasma protein denaturation profile examined by differential scanning calorimetry",
"Expedition into taurine biology: Structural insights and therapeutic perspective of taurine in neurodegenerative diseases",
"Reactive oxygen species: Impact on skeletal muscle",
"Role of oxidative stress as key regulator of muscle wasting during cachexia",
"The antioxidant action of taurine, hypotaurine and their metabolic precursors",
"Antioxidant effect of taurine against lead-induced oxidative stress",
"Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model",
"The protective effect of taurine against cyclosporine A-induced oxidative stress and hepatotoxicity in rats",
"Effects of the reactive oxygen species hydrogen peroxide and hypochlorite on endothelial nitric oxide production",
"Effects of HOCl oxidation on excitation-contraction coupling: Implications for the pathophysiology of Duchenne Muscular Dystrophy: Calcium signaling and excitation-contraction in cardiac, skeletal and smooth muscle",
"Investigation of the effect of taurine supplementation on muscle taurine content in the mdx mouse model of Duchenne Muscular Dystrophy using chemically specific synchrotron imaging",
"The effects of taurine supplementation on oxidative stress indices and inflammation biomarkers in patients with type 2 diabetes: A randomized, double-blind, placebo-controlled trial",
"Effect of taurine on ethanol-induced oxidative stress in mouse liver and kidney",
"Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise",
"Modulatory effects of taurine on metabolic and oxidative stress parameters in a mice model of muscle overuse",
"The role of taurine in mitochondria health: More than just an antioxidant",
"Mitochondrial respiratory chain disorders I: Mitochondrial DNA defects",
"Role of taurine in the pathologies of MELAS and MERRF",
"Taurine supplementation for prevention of stroke-like episodes in MELAS: A multicentre, open-label, 52-week phase III trial",
"Calcium homeostasis",
"Skeletal muscle fatigue-regulation of excitation-contraction coupling to avoid metabolic catastrophe",
"Effect of taurine on sarcoplasmic reticulum function and force in skinned fast-twitch skeletal muscle fibres of the rat",
"Acute effects of taurine on sarcoplasmic reticulum Ca 2+ accumulation and contractility in human type I and type II skeletal muscle fibers",
"Taurine modulates ion influx through cardiac Ca 2+ channels",
"Taurine deficiency, synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy",
"Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever muscular dystrophy dog model for Duchenne Muscular Dystrophy",
"Nuclear magnetic resonance spectroscopy study of muscle growth, mdx dystrophy and glucocorticoid treatments: Correlation with repair",
"Pathophysiology of Duchenne Muscular Dystrophy: Current hypotheses",
"Benefits of prenatal taurine supplementation in preventing the onset of acute damage in the Mdx mouse",
"The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice",
"Increased taurine in pre-weaned juvenile mdx mice greatly reduces the acute onset of myofibre necrosis and dystropathology and prevents inflammation",
"Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne Muscular Dystrophy",
"Amino acids of plasma and urine in diseases of muscle",
"Activation of osmolyte pathways in inflammatory myopathy and Duchenne Muscular Dystrophy points to osmoregulation as a contributing pathogenic mechanism",
"Mitochondrial dysfunction in Duchenne Muscular Dystrophy",
"Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts",
"Effect of β-alanine treatment on mitochondrial taurine level and 5-taurinomethyluridine content",
"Increased activity of calcium leak channels caused by proteolysis near sarcolemmal ruptures",
"Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice",
"Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle",
"Abnormal calcium handling in Duchenne Muscular Dystrophy: Mechanisms and potential therapies",
"Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases",
"Mitochondrial dysfunctions during progression of dystrophic cardiomyopathy",
"Oxidative stress caused by mitochondrial calcium overload",
"Duchenne Muscular Dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition",
"Assessment of resveratrol, apocynin and taurine on mechanical-metabolic uncoupling and oxidative stress in a mouse model of Duchenne Muscular Dystrophy: A comparison with the gold standard, α-methyl prednisolone",
"Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction",
"Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1",
"Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: Influence of sample manipulation, oxidative stress and ageing",
"Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine",
"Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation",
"Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne Muscular Dystrophy",
"Taurine inhibits glucocorticoid-induced bone mitochondrial injury, preventing osteonecrosis in rabbits and cultured osteocytes",
"Potential role of arginine, glutamine and taurine in ameliorating osteoporotic biomarkers in ovariectomized rats",
"Medication-related osteonecrosis of the jaw in a Duchenne Muscular Dystrophy patient",
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"\nTable 1 .\n1Summary of TauT and taurine expression in the mdx mouse.Timepoint of Analysis \nMuscle TauT Content \nMuscle Taurine Content \nMuscle Type \nReference \n\n18 days \n↓ in mdx mice \n≈ controls \nquadriceps \n73 \n\n22 days \n/ \n≈ controls \nquadriceps \n80 \n\n28 days \n28 days \n\n≈ controls \n≈ controls \n\n↓ in mdx mice \n≈ controls \n\nquadriceps \ntibialis anterior \n\n73 \n78 \n\n6 weeks \n6 weeks \n6 weeks \n\n↓ in mdx mice \n/ \n/ \n\n≈ controls \n↓ in mdx mice \n≈ controls \n\nquadriceps \nquad/gas \ntibialis anterior \n\n73 \n59 \n81 \n\n10 weeks \n≈ controls \n↑ in mdx \ntibialis anterior \n78 \n\n6 months \n≈ controls \n\n↓ in mdx mice (wet weight) \n≈ controls \n(dry weight) \n\nEDL (TauT); plantaris \n(taurine) \n79 \n\n"
] | [
"Summary of TauT and taurine expression in the mdx mouse."
] | [] | [] | [
"Taurine or 2-aminoethane-sulfonic acid is primarily a free occurring sulfur-containing amino acid. Unlike most other amino acids, it is not a building block for proteins, yet classifies as a conditionally essential amino acid that is abundant in excitable tissues such as brain, retina, heart, and skeletal muscle, where intracellular concentrations range from 20 to 70 mmol/kg. Taurine is either taken up from diet, for example from fish and meat, or can be synthesized from other amino acids such as cysteine or methionine. Taurine has versatile functions: it plays an important role in osmoregulation, acts as a stabilizer of the cell membrane and of proteins, has anti-oxidant and anti-inflammatory functions, regulates mitochondrial tRNA activities, is involved in calcium homeostasis, etc. [1][2][3].",
"In this review, we focused on the role of taurine in muscle disease, especially in Duchenne Muscular Dystrophy (DMD), a progressive muscle wasting disorder affecting approximately 1 per 5000 male births [4]. Muscle weakness is conspicuous in the hip-and pelvic area first, and later spreads to distal regions. Patients become wheelchair-dependent in their early teens and eventually require cardiac and respiratory care since the muscles of the heart and respiratory system are affected in a life-threatening manner. While awaiting curative treatments to enter the clinic, glucocorticoids are the standard of care, and can prolong life-expectancy of DMD patients [5,6]. Although the precise mechanism by which glucocorticoids slow down disease progression in DMD is not completely understood, its anti-inflammatory action might play a crucial role. However, the use of glucocorticoids negatively influences bone health, which is already impaired in DMD patients [7]. A comprehensive overview of emerging genetic therapies in DMD is provided in the review of Sun et al. [8] The genetic cause of DMD is mutations in the dystrophin gene, located on chromosome X, which hampers the production of functional dystrophin protein. The latter is a keycomponent of the dystrophin-associated protein complex (DAPC) that provides stability to muscle fibers during contraction and relaxation by connecting the intracellular actin cytoskeleton to the basal lamina [7]. Besides membrane stabilization, DAPC also fulfils a role in signal transduction. Dystrophic muscles encounter chronic inflammation, oxidative stress, and ischemia. Eventually these detrimental processes lead to loss of muscle mass and muscle fibrosis [7].",
"This review explores the physiological role of taurine in skeletal muscle and focuses on the consequences of its disturbed balance in DMD. The therapeutic potential of taurine as a dietary supplement for DMD will be scrutinized.",
"A first clue towards an important role for taurine in the muscle was its relatively high abundance. Proper insights regarding the function of taurine in physiological muscle function were acquired upon the generation of taurine transporter (TauT) knock-out (KO) mouse models and ablation of muscle taurine content. Lack of taurine impaired the conductance velocity of the muscle without affecting nerve conductance speed [2]. In addition, exercise performance was seriously hampered in TauT KO mice as shown by a significantly lower running speed. In a different experimental set-up, the total running distance was only 20% of the distance travelled by age-matched wild type (WT) mice [2,9,10]. Besides running tests, the reduced exercise capacity of TauT KO mice also became apparent during a weight-loaded swimming test that showed an 80% decrease in swimming time compared to WT [11]. This study reported structural changes in morphology of TauT KO muscle; however, Warskulat et al. hypothesized that hampered exercise performance was likely attributed to muscle dysfunction, resulting from taurine deficiency. In evidence, serum creatine kinase levels are increased in TauT KO and serum lactate levels were raised after exercise [2]. Some of the pathological characteristics of TauT KO models such as necrotic myofibers and reduced exercise capacity resemble the features of the mdx mouse model [12,13]. Another approach to evaluate the effect of taurine depletion is the use of guanodinoethane sulfonate (GES). GES, a taurine transporter antagonist, reduces muscle taurine content by 60% [14]. Previously taurine was shown to enhance calcium uptake and release in myofibers concurrently with an increase in force production, whereas myofibers derived from GES-treated mice showed reduced force production at relevant stimulation frequencies. Interestingly, fatigue was reportedly attenuated upon GES-treatment [14].",
"Furthermore, the lifespan of TauT KO male mice is significantly lower than those of WT mice, 511 and 686 days, respectively. Reduced life expectancy together with increased expression of p16INK4a, an indicator of senescence, in TauT KO mice allowed to hypothesize that taurine might be involved in aging [15,16]. Furthermore, it was suggested that taurine delays muscle-specific senescence including sarcopenia in tumor necrosis factor (TNF)-α stimulated L6 myogenic rat cells. In evidence, differences in the regulation of inflammation, autophagy, and apoptosis have been reported upon taurine treatment [17]. In addition, TNF-α stimulation of L6 myogenic rat cells hampered muscle differentiation which could be restored by taurine. Presumably this effect was mediated through the PI3/AKT signaling pathway since myocyte enhancer factor-2 (MEF-2), a transcription factor involved in myogenic differentiation, was markedly decreased after knockdown of AKT [17]. Furthermore, expression of TauT increased during muscle differentiation and was even further enhanced upon binding of MEF-2 to the promotor region of TauT [18].",
"In conclusion, depletion of muscle taurine levels either through TauT KO or via pharmacological inhibition of the taurine transporter by GES alters force output and exercise performance. Therefore, taurine seems essential for the preservation of physiological muscle function. The sections below provide an overview of the main cellular processes in which taurine plays a role in relation to the muscle.",
"Exposure to hyperosmolar conditions can induce several detrimental cellular effects including interference with transcriptional and translational activity, induction of oxidative stress, DNA damage, and can even elicit apoptosis under certain conditions [19,20]. Thus, safeguarding the osmotic equilibrium is essential for ensuring cellular health. When cells are exposed to an environment high on NaCl, fluid is retracted from the intracellular compartment causing cellular shrinkage, molecular crowding, and increased ionic forces. In order to counteract these deleterious processes, the cell's response is a regulatory volume increase (RVI) that includes activation of inorganic ion transporters (e.g., Na + /K + /2Cl − cotransporter, the Na + /H +/− , and the Cl − /HCO 3 − exchanger), allowing an influx of ions accompanied by osmotic uptake of water [19][20][21][22][23]. However, this condition is unfavorable over time due to increased intracellular ionic forces that could interact with macromolecules. Thus secondarily, accumulation of organic osmolytes (e.g., taurine) that replace inorganic electrolytes results in normalization of ionic strength whilst preserving cellular volume and protein stability [19,20]. Rather than stimulation of de novo synthesis, osmotic stress is most likely to enhance cellular import of taurine [20]. Both the designated TauT as well as the proton-coupled amino-acid transporter (PAT) 1 are capable of accumulating taurine in the cell [24]. However, TauT is considered as the principal transporter of taurine in muscle cells as evidenced by a 98% reduction of taurine content in a TauT KO mouse [10]. Transcription of TauT mRNA is upregulated under hypertonic conditions due to binding of Nuclear Factor of Activated T-cells 5 (NFAT-5) to the 5 flank region of the TauT gene. NFAT-5, also known as tonicity responsive element binding protein (TonEBP) acts as a transcription factor of SLC6A6, the gene encoding TauT, and thus allows cellular accumulation of taurine [25,26].",
"Exercise can affect the osmotic balance in muscle fibers. Muscle subjected to an intensive exercise protocol resulted in increased myofiber volume, cross-sectional area, and water concentration by more than 15%, indicative for muscle fiber swelling [27,28]. The rise in myofiber water content can be partially explained by water production in cellular metabolic processes that take place during exercise [27][28][29]. Additionally, intracellular solute concentrations might be elevated during exercise as a result of phosphocreatine splitting and increased lactate and H + , ensuing water influx in order to retain osmotic balance [28] and could contribute to myofiber swelling as well. This volume increase is followed by a compensatory mechanism named the regulatory volume decrease (RVD) that releases electrolytes (e.g., K + , HCO 3 − , Cl − ) and osmolytes such as taurine concurrently with water in order to normalize cellular volume [19,20,[26][27][28][29][30][31]. Thus, taurine is released in order to counteract myofiber swelling, a phenomenon which occurs during exercise [28,31].",
"The stabilizing effect of taurine is mentioned in many papers. However, the mechanism by which taurine is able to exert stabilization is poorly described. In order to comprehend this characteristic, it is important to understand its interaction with water molecules, considering the chemical properties related to its molecular structure. One of the most popular hypotheses that could explain protein stabilization by osmolytes is based on preferential exclusion [32][33][34][35]. This principle builds on unfavorable interactions between proteins and osmolytes in terms of Gibbs adsorption isotherm [32]. In a denatured state, the area of the peptide backbone by which osmolytes can interact is larger and results in increased Gibbs energy (unfavorable). In order to reduce these interactions, the thermodynamic component drives the folding equilibrium towards its native state, also referred to as the osmophobic effect, which is associated with a much lower Gibbs energy. This simplified explanation implies that in the presence of stabilizing osmolytes, the Gibbs energy of the denatured state is much higher compared to Gibbs energy of the folded state [32]. Therefore, the folded protein conformation is favored and osmolytes act as protein stabilizers [32,33]. In general, the presence of osmolytes results in a specific distribution of water molecules around the proteins in a preferential hydrated state and osmolyte exclusion from the protein backbone [32][33][34][35].",
"Furthermore, stabilizing actions have been attributed to taurine, as well as direct interaction with protein side chains [33][34][35]. The amino group of taurine orients itself preferentially to the protein side chain. This strengthens the hydrogen bonded network of water surrounding the protein and stabilizes its native form. The latter appears to contradict the preferential exclusion theory; however, such interactions between osmolytes and side chains have also been discussed by Bolen et al. [32]. It should be noted that protein side chains are associated with other characteristics than the protein backbone and favorable interactions between osmolyte and side chains might occur. Supposedly, the latter does not substantially alter the protein folding state [32]. In the article by Brudziak et al., the protein was hydrated in the presence of taurine [35]. This might suggest that besides limited interactions between osmolytes and protein side chains, the protein is still preferentially surrounded by water molecules. Taurine was able to increase the thermal stability of both lysozyme and ubiquitin protein [35][36][37][38], although the extent of stabilization was protein specific [35].",
"In addition to protein stabilization, membrane stabilizing properties of taurine were hypothesized by Huxtable and Bressler [39]. Taurine inhibits the activity of phospholipid methyltransferase, which catalyzes the methylation of phosphatidylethanolamine to form phosphatidylcholine and thus taurine could alter the composition and consequently the properties and stability of phospholipid membranes [40][41][42]. In evidence, the presence of taurine decreased the viscosity of erythrocytic membranes, suggesting taurine might increase membrane fluidity [43].",
"Eccentric muscle contraction might induce denaturation of myofibrillar proteins as hypothesized by Paulsen et al. [44]. In addition, the unfolded protein response (UPR) is activated during exercise [45,46] which might indicate that proteins struggle to maintain native folding conformations. Interestingly, prolonged exercise increased the denaturation temperature of albumin, pointing to enhanced thermal stability [47]. The importance of taurine in protein stabilization is illustrated in the TauT KO mouse model [15]. It is assumed that a lack of taurine allows accumulation of unfolded and/or misfolded proteins in skeletal muscle which activates expression of genes involved in the UPR. Thus, taurine plays a key role in protein homeostasis of skeletal muscle [15,48].",
"Under physiological conditions, reactive oxygen species (ROS) are balanced by antioxidant mechanisms that detoxify reactive species. A limited amount of ROS is produced during exercise and exerts advantageous effects on force generation. In addition, low levels of ROS might protect against injury through adaptations in cellular signaling upon regular training exercise [27,47,49], whereas high levels of ROS are associated with muscle dysfunction [50]. Although direct scavenging of the main ROS (e.g., superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH)) by taurine is considered unlikely [51,52], taurine is believed to relieve oxidative stress through neutralization of hypochlorous acid (HOCl) and upregulation of antioxidant enzymes [53][54][55]. Upon inflammation, neutrophils become activated and release myeloperoxidase (MPO). MPO catalyzes the reaction of chloride and H 2 O 2 , a classic ROS molecule, resulting in the formation of HOCl. HOCl has toxic effects and interferes with cellular processes including molecular transport and pump capacity [56]. Recently, it has been hypothesized that HOCl could alter excitation-contraction (E-C) coupling and impairs muscle force production [57]. HOCl is converted to TauCl after interaction with taurine. TauCl possesses anti-inflammatory properties and increases antioxidant enzymes including heme oxygenase 1 in murine microglial cells and muscle cells [58,59]. Taurine supplementation increased activity of antioxidant enzymes such as superoxide dismutase and catalase, measured in the blood of patients with type II diabetes [60]. Similar results were obtained in the liver and kidney of an ethanol-induced oxidative stress mouse model [61].",
"Furthermore, antioxidant effects of taurine on superoxide production and lipid peroxidation were observed in the muscle of eccentric exercised rats [62]. Similarly oxidative lipid damage was reduced by taurine treatment in a mouse model of muscle overuse [63].",
"Taurine participates in the synthesis of mitochondrial proteins, more specifically proteins that require tRNA (Leu) and tRNA (Lys) with uridine on a Wobble position [64]. If the first base of the tRNA anticodon is uridine, then the classic Watson-Crick rules are substituted by Wobble base pairing rules. According to this hypothesis, H-bonds are formed between the first base of the anticodon (tRNA) and the third position of the mRNA codon. Contrastingly to Watson-Crick base pairing, Wobble pairing suggests that uridine on a Wobble position at the anticodon can form H-bonds with A, U, G, and C on the third position of the mRNA codon mRNA. Whereas, taurine-conjugated uridine tRNA will promote the formation of H-bonds between uridine and A or G on the third codon position and is required for appropriate translation to leucine (UUA/UUG) [64]. Taurine modification of uridine is required in some mitochondrial tRNAs to ensure a more specific codon-anticodon interaction and proper mitochondrial protein synthesis. Cytochrome b, ND5, and ND6 are mitochondrial proteins containing UUG codons, and synthesis of these proteins is potentially hampered if taurine conjugation of mitochondrial tRNA Leu(UUR) is deficient. ND5 and ND6 are functional subunits of oxidative phosphorylation complex I, that catalyzes electron transport from NADH to coenzyme Q [64,65]. As the process of oxidative phosphorylation is one of the main sources of ROS [50] in myofibers, preservation of mitochondrial function is considered essential in the safeguarding of oxidative stress.",
"Specific mutations in tRNA (Lys) and tRNA (Leu) are associated with respectively myoclonic epilepsy with ragged red fibers (MERRF) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). MERRF and MELAS are mitochondrial diseases that show disturbed protein synthesis and pathological characteristics such as exercise intolerance, thus of which some aspects resemble the TauT KO phenotype [9,64,66]. Of note, taurine supplementation in MELAS patients improved the occurrence of stroke-like episodes [67].",
"Calcium is an essential cellular building block and a universal carrier of biological information. As a diffusible intracellular second messenger, calcium is involved in signaling transduction pathways and can regulate many different processes ranging from neurotransmitter release, bone formation, and blood coagulation to muscle contraction [68]. More specifically, calcium is essential during the E-C coupling mechanism that transforms the electrical input (action potential) to a mechanical output (contraction). Upon adequate stimulation, an action potential is generated and propagates over the sarcolemma to the transverse tubules that contain L-type calcium channels e.g., dihydropyridine receptors (DHPR). In the skeletal muscle, DHPR are in close contact with the ryanodine receptors (RyR) of the sarcoplasmic reticulum (SR), which releases calcium into the cytosol. Binding of calcium to troponin C induces the translocation of tropomyosin, thereby allowing the formation of cross-bridges between actin and myosin filaments and subsequent contraction [69].",
"Temporal and spatial changes of calcium concentration in cytoplasm or organelles are monitored by a multitude of calcium sensing proteins that determine the character and duration of the cellular response. The SR is an organelle involved in storage of calcium which is released into the cytoplasmic environment upon muscle stimulation. Taurine partially preserved SR function upon exposure to phospholipase C, which is known to hamper calcium transport, in SR derived from rat skeletal muscle. Furthermore, in the presence of 15 mM taurine, uptake of calcium oxalate by the SR was increased by more than 20% [39].",
"Similarly, taurine significantly increased the accumulation of calcium in SR of skinned EDL rat myofibers [70]. In this experiment, the membrane of the myofibers was mechanically removed which allowed to control intracellular taurine concentrations. The enhanced calcium SR load and subsequent release upon stimulation might explain the increment in force response upon depolarization. The authors hypothesized that taurine modulates SR calcium pump activity, allowing increased calcium accumulation [70]. These results are consistent with observations in human skeletal muscle fibers type I and II; interestingly, these different muscle fibers contain a different type of SR calcium transport ATPase (SERCA)-isoform, thus taurine might modulate either activity and/or calcium affinity [71]. Of note, a small decline in calcium sensitivity was observed in the presence of taurine in SR of skinned EDL myofibers [70].",
"Furthermore, voltage clamp experiments carried out in cardiomyocyte derived from guinea pig showed that the effect of taurine on calcium influx is highly dependent of the extracellular calcium concentration. In the presence of taurine (20 mM), calcium influx was slightly increased upon low extracellular calcium concentration (0.8 mM) and vice versa, the calcium influx was slightly yet significant decreased when extracellular calcium concentrations were high (3.6 mM). No effect of taurine on inward calcium current could be observed at an extracellular calcium level of 1.8 mM, suggesting that taurine aims at maintaining intracellular calcium homeostasis [72]. However, taurine modulated resting membrane potential regardless of extracellular calcium channels.",
"Previously, it has been suggested that alterations in taurine and/or its regulation associate with dystrophinopathology [73][74][75]. Lack of dystrophin results in destabilization of DAPC and thereby rendering the sarcolemma more susceptible to contraction-induced damage. Membrane fragility is observed in dystrophin deficient muscle as evidenced by increased permeability to dyes such as Evans Blue and Procion Orange, accumulation of serum proteins such as albumin and IgG, increased levels of creatine kinase in the blood, and sarcolemma rupture in myofibers [7,76,77].",
"Taurine regulation is altered in dystrophic tissues of mouse models. Some studies reported a significant downregulation of the TauT in muscles of the mdx mouse model [73], whereas others reported no significant differences between mdx mice and age-matched control mice [78,79]. Seemingly, TauT is significantly downregulated in young mdx mouse and normalizes to control levels over time.",
"Similar to the expression of TauT, differences in muscle taurine content with age were reported as well, yet with considerable differences between studies. Taurine content of mdx muscle was comparable to controls at age 3-4 weeks, but increased by age 10 weeks [78,80]. A significant downregulation of taurine in mdx mice aged 4 and 6 weeks compared to age-matched control mice was reported [59,73], whereas other studies found no significant difference in taurine content between control and mdx mice at age 6 weeks [73,81]. A decline in taurine content was on the other hand observed in the plantaris muscle of 6-month-old mdx mice compared to wet weight yet did not hold up when compared to dry weight [79]. Contrasting results have been reported regarding the taurine content in mdx mice, however most of these studies conclude that taurine is differentially regulated in mdx mice compared to control mice at a certain time point. The expression of TauT and taurine content in the muscle of mdx mice compared to age-matched control mice is summarized in Table 1. Interestingly, muscle taurine levels increased in glucocorticoid treated mdx mice, pointing to its reestablishment by immunosuppressive therapeutic interventions [75]. As opposed to the observations in the mdx mouse model a significant increase in muscle taurine and its transporter were discerned in 8-month-old Golden Retriever Muscular dystrophy (GRMD) canine model [74]. Similarly, taurine regulation in DMD patients differs from healthy control patients, as shown by increased levels of muscle TauT protein in DMD patients [82,83]. Thus, expression of TauT is differently regulated in the mdx mouse than in the GRMD-model and DMD patients. Interestingly, the phenotype in the mdx mouse model is milder than in GRMD dogs and DMD patients. Myofiber necrosis and muscle weakness become conspicuous approximately 3 weeks after mdx mice are born. Myofiber necrosis in the mdx mouse is most explicit in the young to juvenile period, followed by necrosis at a slower pace in adult mice [74,80]. The course of disease in mdx mice thereby differs from the more progressive pathology in GRMD dogs and DMD patients, that is characterized by fatty replacement and fibrosis at an early age [74]. These differences in pathological features might be linked to regulation of TauT.",
"Mitochondria participate in cellular energy production by synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation [84]. This process includes transfer of electrons, required for ATP-synthesis, and is inevitably linked to production of ROS. Under physiological conditions, basal levels of ROS are generated as a by-product of oxidative phosphorylation. However, ROS production is enhanced upon mitochondrial dysfunction [84]. Upon oxidative phosphorylation, electron transfer is facilitated by mitochondrial protein complexes that resides within the inner mitochondrial membrane [84]. However, Onopiuk found significantly reduced protein levels of complex III, cytochrome-c reductase, and complex V, the ATP synthase, in immortalized myoblasts of mdx mice. Of note, myoblasts of mdx mice (SC-5), and control myoblasts (IMO) were used [85]. Neither of these myoblasts expressed dystrophin protein, only dystrophin mRNA was present in control myoblasts. In addition, Onopiuk et al. showed an increase in mitochondrial membrane potential [85]. Taken together, these results suggest mitochondrial dysfunction and inevitably, increased ROS production in dystrophin deficient cells. Taurine reduces ROS by upregulation of antioxidant enzymes and might preserve electron transport chain activity by safeguarding mitochondrial protein synthesis of subunits involved in the respiratory chain. In evidence, reduced taurine levels hamper expression of ND6, subunit of complex I in the mitochondria of cardiomyocytes derived from rat [64,86].",
"Excessive calcium levels are observed in dystrophic myofibers; however, calcium entry through sarcolemmal tears is not considered as the main contributor to calcium overload in dystrophic myofibers. Apparently, the open probability of calcium leak channels in the proximity of micro-tears are increased, thereby allowing an increased calcium influx [87].",
"Additionally, increased expression and activation of store-operated calcium channels, presumably induced by calcium-independent phospholipase A2, is observed in dystrophin deficient myofibers and could contribute to calcium overload as well [84,[88][89][90]. Moreover, a correlation between the dystrophic phenotype and expression of a stretch-activated channel, transient receptor potential canonical (TRPC) channel 1, was discovered in different muscles of the mdx mouse. The diaphragm of mdx mice was affected the most, as shown by increased Evans Blue permeability, and showed a significant upregulation of TRPC1 expression compared to controls [88]. Thus, the involvement of the TRPC channels might also contribute to the calcium overload as evidenced by increased expression of various TRPC channels in mdx mice. The cytoplasmic calcium concentration is not only determined by calcium channels/exchangers but also by the SR uptake and release of calcium, which plays an essential role during E-C coupling. RyR, responsible for the release of calcium from SR upon depolarization, is hyper nitrosylated in dystrophic muscle and consecutive depletion of calstabin-1 upon RyR-nitrosylation results in calcium leakage [84,[89][90][91][92]. During relaxation, cytoplasmic calcium ions are sequestered by SERCA, thereby lowering its cytoplasmic concentration. In the mdx mouse model, the removal of calcium ions by the SR is hampered, suggesting reduced SERCA functioning. In conclusion, calcium homeostasis is disturbed in dystrophic muscle on many levels. Chronic cytoplasmic calcium overload will induce activation of degrading pathways mediated by phospholipase 2 and proteases which eventually can lead to myofiber death [7,90].",
"In addition, cytoplasmic calcium overload can induce accumulation of calcium in the mitochondria and mitochondrial dysfunction. Multiple pathways have been proposed by which mitochondrial calcium overload can induce ROS production, such as mitochondrial permeability transition (MPT)-mediated release of anti-oxidative enzymes, dislocation of mitochondrial proteins involved in electron transports including cytochrome C, induction of NO, etc. [84,93]. Mitochondrial calcium overload can elicit MPT pore formation. A permeable pore is formed that spans inner and outer mitochondrial membranes and results in mitochondrial swelling [84]. Furthermore, dystrophic muscle cells show an increased susceptibility to calcium and therefore are more prone to MPT pore formation, that eventually cause mitochondrial swelling and death [87,91]. In evidence, mitochondrial swelling, indicative for MPT pore formation, was induced at a lower calcium load in mitochondria compared to WT [84,90,[92][93][94]. Interestingly, taurine is able to attenuate calcium-induced swelling of mitochondria derived from skeletal muscle [95].",
"A beneficial effect of taurine on muscle force remains controversial: supported by some studies yet disproved by others. A significant increase in peak twitch force was obtained in taurine supplemented (2.5% w/v in drinking water) mdx mice compared to untreated mdx mice at 4 weeks. However, this effect was abrogated in juvenile and adult mdx mice, respectively aged 10 weeks (2.5% w/v) and 6 months (3% w/v) [78,79]. Similarly, a >50% increase in maximum isometric tetanic specific force (sPo) was obtained by taurine supplementation in mdx mice aged 4 and 6 weeks [78,81]. However, no treatment effect (1 g/kg/day) on specific tetanic force was detected in muscle of mice aged 5-7 weeks, whereas fore limb force, assessed by means of a grip strength meter, was ameliorated [96]. Furthermore, a study performed by Barker reported no effects of taurine treatment (2.5% w/v) on peak twitch force, maximum specific force, nor fatigue or recovery in mdx mice treated from week 2 until week 4 [97]. Similarly, no effect on maximum specific force in 6-week-old mdx mice was observed when high doses of taurine (16 g/kg/day) were administered [98].",
"In 6-month-old mdx mice, taurine could not ameliorate specific maximum isometric force production. However, after a fatigue protocol consisting of recurrent electrical stimulation, the EDL of taurine treated mdx animals was more resistant to fatigue, as shown by a significant higher force production at the end of stimulation relative to force production at the beginning. Furthermore, EDL muscle of taurine treated mdx animals showed a significantly better recovery capacity at 10-60 min after stimulation than untreated mdx animals [79]. Taurine treatment in exercised mdx mice significantly increased in vivo forelimb muscle strength normalized to body weight [96,99], but did not alter locomotor activity of mdx mice. A similar finding was reported in a study that supplemented unexercised mdx mice. Taurine supplementation (±4 g/kg/day) in 6-week-old unexercised mdx mice significantly increased grip strength [81]. Whereas no effect of high taurine treatment (16 g/kg/day) was observed on normalized grip strength [98].",
"In summary, benefit of taurine supplementation on muscle force differed considerably between published studies. Although multiple studies have been executed, these used different concentrations, ingestion methods, and treatment protocols, which might explain the obtained conflicting results, and which hampers interpretation.",
"Although different treatment conditions and read-outs were used, multiple studies have shown anti-inflammatory and anti-oxidative effects of taurine treatment in mdx mice [59,80,81,95]. The anti-oxidant and anti-inflammatory actions of taurine have been proposed as a mechanism by which taurine protects dystrophic tissue from damage. In evidence, muscle of taurine-treated mdx mice showed significantly less NF-κβ positive fibers, TNF-α levels, neutrophil elastase, and MPO activity [59,80,81,96]. Accordingly, taurine-treated mdx mice showed lower levels of disulfide and protein thiol oxidation in muscle compared to untreated mdx mice, which could point towards protection against oxidative stress [59,81,98,100]. Similarly, ROS levels were significantly reduced upon taurine treatment in muscles of exercised dystrophic mice, as shown by dihydroethidium (DHE) staining, which reacts with O 2 − [96]. In addition, taurine normalized the resting macroscopic ionic conductance (gm), a measure for ROS production, to WT levels [96].",
"In mdx mice, the threshold of the membrane potential at which a contraction is elicited is shifted towards a more negative value than in control mice. Thus, contraction is induced upon a lower depolarization state than in WT animals [96,101]. This mechanical threshold is indicative for E-C functioning and suggests alterations in E-C-coupling and/or calcium homeostasis [96,101]. Interestingly, taurine treatment in mdx mice has been shown to partially restore the mechanical threshold [96,99,101]. Taurine treatment in mdx did not alter expression of proteins involved in E-C coupling such as calcium sensitive receptors (RyR), calcium channels (DHPR channels), calcium ATP-ase pump (SERCA) and calcium binding proteins (calsequestrin) [78]. Contrastingly, taurine supplementation (2.5% w/v) in rats significantly increased expression of calsequestrin 1 in the muscle [102]. Although in the mdx mouse and rat study the same dose of taurine was used (2.5% w/v), this discrepancy in outcome might be explained by age-dependent calsequestrin regulation, changes in calsequestrin regulation upon dystrophin deficiency, species-dependent regulation, or differences in treatment protocol. However, a specific explanation cannot be pinpointed with the current literature studies available.",
"Histopathological characteristics such as necrotic myofibers and fibers with centralized nuclei, indicative for regeneration ensuing myofiber damage, are conspicuous in Hematoxylin-Eosin stained sections of mdx muscles and are used to evaluate therapeutic effectiveness [59]. In the study of Barker, taurine (2.5% w/v) reduced the amount of noncontractile area in young mdx mice, whereas no effect on the percentage regenerative fibers could be observed [78]. Other studies have reported a significant increase in healthy myofibers with peripheral nuclei upon taurine treatment [59], a decrease of histopathological features and myofiber necrosis [80,96]. Thus, taurine seems to alleviate histological features related to dystrophinopathy.",
"Combined use of taurine (1 g/kg) and α-methylprednisolone (PDN) (1 mg/kg), a synthetic adrenocortical hormone, in the exercised mdx mouse model significantly improved muscle strength in such a way that a synergistic effect was proposed by Cozzoli et al. [103]. The increase in fore limb muscle strength after 4 weeks of treatment was significantly higher in mice that received combined therapy compared to mice treated with PDN. Similarly, the increment in muscle force normalized to body weight was remarkably elevated in mice that received taurine + PDN compared to single-drug treatments. Furthermore, combined treatment normalized the rheobase potential to WT values, however similar results were observed in taurine-treated mice. No synergistic effect of combination treatment compared to PDN-treatment was detected regarding histopathological markers that included the amount of centronucleated fibers, necrosis and non-muscle tissue [103].",
"Contrastingly, the study of Barker et al. reported no effects of combined treatment on muscle strength. These opposing findings might be explained by differences in experimental set-up since in the latter study therapeutic intervention was initiated more closely to onset of damage, higher taurine concentrations were used for a shorter period of time and analysis occurred at the peak of damage [97]. Besides possible synergistic effects of combined treatment, taurine could also counteract side-effects related to corticosteroids. Dexamethasone causes muscle atrophy and significantly lowers the myotube diameter, which is restored upon taurine treatment [18]. Furthermore, taurine protects against glucocorticoid-induced mitochondrial dysfunction of the bone and might attenuate corticoid-induced osteoporosis and or osteonecrosis, pathological features that are conspicuous in glucocorticoid-treated DMD patients [104][105][106].",
"In some rat studies, different test regimes of taurine supplementation (3% w/v for 4 weeks [107], 100 mg/kg for 2 weeks [108]; 500 mg/kg for 2 weeks [109]) resulted in increased muscle taurine content, whilst other long-term studies (1% w/v ≈ 50 mg/kg for 22 weeks [110]) could not report increased muscle taurine levels. Similarly, in mouse studies muscle taurine levels were elevated upon taurine supplementation (4% w/v for 3 weeks [59]; 3% w/v for 4 weeks [79]). Interestingly, one study showed that continuous taurine supplementation (2.5% w/v) in mdx mice increased muscle taurine concentration up to the age of 4 weeks, but when treatment was prolonged up to the age of 10 weeks, muscle taurine concentration was significantly decreased in taurine supplemented mdx mice compared to untreated mdx mice [78]. Similarly, high doses of taurine (8% w/v ≈ 16 g/kg/day) added to the drinking water up until the age of 6 weeks, did not increase muscle taurine content [98]. Therefore, it might be hypothesized that the muscle taurine content is strictly regulated. Since chronic treatment might not be able to increase intramuscular taurine levels it is not known if long-term taurine treatment could effectively attenuate muscle damage. Furthermore, taurine intake (≈5 g/day) for a period of 7 days was reported not to alter muscle taurine levels in humans [111].",
"In general, taurine is well tolerated and safe if used in appropriate concentrations [112]. One study has reported gastro-intestinal complaints; however, taurine was used in combination with other nutritional supplements [112,113]. Another study aimed to investigate taurine supplementation in patients with end-stage renal failure. In healthy subjects, excessive taurine is excreted; however, due to kidney failure the taurine overload was not immediately cleared and these patients suffered from dizziness and vertigo [114] which was the reason to discontinue the study.",
"High taurine treatment (16 g/kg) in 6 week-old animals significantly reduced the body weight of mdx mice by more than 20% and shortened tibia length by 10%. Of note, the body weight and tibia length of untreated mdx mice were comparable to those of WT animals in this experiment [98]. Similarly, taurine treatment (3% w/v) for 4 weeks in adult mdx mice (5 months old) reduced body weight and muscle mass of mdx mice; however, the weight of these animals still exceeded that of WT animals [79]. Interestingly, no effect of taurine on body weight was observed in WT animals. Obesity is commonly observed in DMD patients, especially in glucocorticoid-treated patients [7,115,116]. Therefore, it remains unclear if taurine-induced decreased body weight would be disadvantageous in these patients. Obviously if growth is hampered, this should be avoided. A study conducted in piglets reported a reduced gain to feed ratio upon higher taurine supplementation (3% taurine diet) compared to untreated piglets whilst supplementation with 0.3% taurine might improve growth [117]. Terrill et al. have hypothesized that high taurine supplementation in young animals interferes with the taurine synthesis pathway, shifting the equilibrium to the left inducing increased cysteine levels in the plasma which could hamper growth [98].",
"Approximately 50-65% of DMD patients are using vitamins or nutritional supplements [115,116], which indicates that the quest for supportive treatment, including but not limited to taurine, in DMD is relevant. A detailed overview of nutritional supplements that are under investigation for the treatment of DMD is provided in the review of Boccanegra et al. [118].",
"The current nutritional guidelines for patients are very similar to those used for the general population. If serum 25-hydroxy-vitamin D drops below 30 ng/mL or calcium intake is low, the use of vitamin D and, respectively, calcium intake under the form of calcium citrate or calcium carbonate is advised in order to stimulate bone health, which could be impaired in DMD patients [116,117,119]. In addition, other nutrients are currently under investigation. Coenzyme Q10, a naturally occurring anti-oxidant, significantly improved muscle strength in steroid-treated DMD patients [118,120]. Similarly, beneficial effects of L-arginine, creatine, omega-3 fatty acids have been observed in clinical trials [118].",
"Taurine is involved in numerous processes such as protein stabilization and osmotic homeostasis and appears to be indispensable for physiological muscle function as evidenced in the TauT KO mouse. We further zoomed in on DMD, a severe muscle disorder that showed altered regulation of taurine and its transporter. Multiple facets of dystrophinopathology and potential mechanisms on how taurine might act on these pathological features have been proposed and discussed in view of the mdx mouse model. Promising results have been obtained in the mdx mouse model in terms of inflammation, muscle strength, oxidative stress, etc. and led to the conclusion that taurine supplementation is relevant for DMD pathology. Former clinical trials conducted to evaluate the anti-aging or mood stabilizing effects of taurine have shown that taurine is well tolerated and considered safe upon appropriate use. However, up until now, no clinical trials have been conducted that evaluated taurine as a treatment for DMD patients. We propose that taurine has the potential to act as supportive therapy in combination with glucocorticoids for the treatment of DMD patients and further studies should be conducted to evaluate the effectiveness of chronic taurine supplementation."
] | [] | [
"Introduction",
"Involvement of Taurine in Physiological Skeletal Muscle Functioning",
"Knowledge Gained from Knockout Models",
"Taurine and Its Role in Osmotic Homeostasis",
"Taurine and Its Role in Protein and Membrane Stabilization",
"Taurine and Its Role in Oxidative Stress",
"The Role of Taurine in Mitochondrial Protein Synthesis",
"Taurine and Its Role in Calcium Homeostasis",
"Pathophysiological Characteristics of Taurine in DMD",
"Regulation in Dystrophin Deficiency",
"Role in Oxidative Stress Management and Mitochondrial Protein Synthesis",
"Dysregulation of Calcium Homeostasis",
"Taurine Supplementation as a Therapeutic Strategy for DMD",
"Effect of Taurine on Muscle Force",
"Effect of Taurine on Oxidative Stress and Inflammation",
"Effects of Taurine on E-C Coupling",
"Effect of Taurine on Histopathological Characteristics of the Mdx Mouse",
"Combinatory Use of Taurine and Glucocorticoids",
"Potential Caveats of Taurine Treatment",
"Other Nutritional Supplements Used in Duchenne Muscular Dystrophy",
"Conclusions",
"Table 1 ."
] | [
"Timepoint of Analysis \nMuscle TauT Content \nMuscle Taurine Content \nMuscle Type \nReference \n\n18 days \n↓ in mdx mice \n≈ controls \nquadriceps \n73 \n\n22 days \n/ \n≈ controls \nquadriceps \n80 \n\n28 days \n28 days \n\n≈ controls \n≈ controls \n\n↓ in mdx mice \n≈ controls \n\nquadriceps \ntibialis anterior \n\n73 \n78 \n\n6 weeks \n6 weeks \n6 weeks \n\n↓ in mdx mice \n/ \n/ \n\n≈ controls \n↓ in mdx mice \n≈ controls \n\nquadriceps \nquad/gas \ntibialis anterior \n\n73 \n59 \n81 \n\n10 weeks \n≈ controls \n↑ in mdx \ntibialis anterior \n78 \n\n6 months \n≈ controls \n\n↓ in mdx mice (wet weight) \n≈ controls \n(dry weight) \n\nEDL (TauT); plantaris \n(taurine) \n79 \n\n"
] | [
"Table 1"
] | [] | [] |
204,943,882 | 2022-02-03T17:39:25Z | CCBY | https://www.frontiersin.org/articles/10.3389/fendo.2019.00741/pdf | GOLD | 81bd6c904a1445c00b06b088979b75c39c749582 | null | null | null | null | 10.3389/fendo.2019.00741 | 2982633354 | 31736878 | 6828820 |
A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice
1 October 2019
Vaclav Kasicka
Martin Hubalek
Yu Zeng [email protected]
Wu Y
Han M Wang
Y
Gao Y
Cui X
Xu P
Ji C
Zhong T
You L
Zeng Y
Yanting Wu
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Affiliated Maternity and Child Health Care Hospital of Nantong University
NanTongChina
Mei Han
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Yan Wang
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Yao Gao
Department of Endocrinology, Children's Hospital
Nanjing Medical University
NanjingChina
Xianwei Cui
Pengfei Xu
Chenbo Ji
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Tianying Zhong
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Lianghui You [email protected]
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Yu Zeng
Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)
NanjingChina
Institute of Organic Chemistry and Biochemistry (ASCR)
Lianghui You
Institute of Organic Chemistry and Biochemistry (ASCR)
Czechia, Czechia
A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice
Frontiers in Endocrinology | www.frontiersin.org
107411 October 201910.3389/fendo.2019.00741Received: 22 July 2019 Accepted: 14 October 2019ORIGINAL RESEARCH Edited by: Honoo Satake, Suntory Foundation for Life Sciences, Japan Reviewed by: *Correspondence: † These authors have contributed equally to this work as joint first authors Specialty section: This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology Citation: (2019) A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice. Front. Endocrinol. 10:741.mass spectrometrysecreted peptideskeletal muscleinsulin resistanceIrs1-Akt signaling pathwayPgc1α
As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.
INTRODUCTION
Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis (1). Accordingly, the dysregulation of skeletal muscle metabolism also arise a number of metabolic disorders such as hyperinsulinaemia, excessive hepatic gluconeogenesis (2), abnormal lipid accumulation (3), impaired glucose uptake and metabolic inflexibility (4). Furthermore, disorders in skeletal muscle play a central role in the development of type 2 diabetic mellitus (T2DM), obesity and lead to other related complications (5). Thus, it is of great interest to deeply characterize the pathogenesis of dysregulation on skeletal muscle glucose/lipid homeostasis to whole-body endocrine and metabolic functions.
As an important secretory organ, skeletal muscle has drawn attention to be a potential target tissue for treating metabolic disorders (6). Thus, analysis of skeletal muscle secretome opens up a novel route for comprehending the communication of this tissue with other tissues such as adipose tissue (7), bone (8), liver (9) and pancreas (10,11). In light of recently reported experimental evidence, a variety of proteins generated by muscles fibers and released into the circulation are classified as myokines (12), most of which have autocrine, paracrine, and endocrine effects not only in muscle fiber growth (13) but also systemic metabolism (14). The first identified myokine IL-6 presented a vital locally muscular effects such as skeletal muscle growth and glucose/lipid metabolism (15, 16). Furthermore, IL-6 also could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues (17)(18)(19). Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM (20). These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. However, knowledge of the skeletal muscle secretome is scarcely reported under pathophysiology of metabolic diseases such as T2DM and obesity. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established.
Peptides in length of 3-50 amino acids residues, which are widely characterized in mouse and human, are termed as a sort of compounds produced or secreted by endocrine gland tissues as well as certain types of cells (21,22). And Abbreviations: T2DM, type 2 diabetic mellitus; GO, gene ontology; KCRM, creatine kinase m-type; Aldo A, fructose-bisphosphate aldolase A; Mr, relative molecular mass; pI, isoelectric point; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FA, formic acid; DDA, data dependent acquisition; FDR, false discovery rate; PSPEP, Proteomics System Performance Evaluation Pipeline; RIPA, Radio Immunoprecipitation Assay; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; TPIS, triosephosphate isomerase; ENOB, beta-enolase; PYGM, glycogen phosphorylase, muscle form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; MLRS, myosin regulatory light chain 2, skeletal muscle isoform; MYH6, myosin heavy chain 6; MYH7, myosin heavy chain 7; MYBPH, myosin-binding protein H; MYL3, myosin light chain 3; MYL1; LDH, myosin light chain 1; lactate dehydrogenase. these endogenous peptides have important physiological action, including neuroregulation (23), cell differentiation (24) and energy metabolism (25) and dysregulation of peptide hormone signaling have been implicated in a wide range of diseases (26,27). In view of that, further insight into identification of novel peptides is of major importance. Benefited from the progresses in peptide extraction method and application of modern analytical methods, (U)HPLC, nano-LC and CE, hyphenated with tandem mass spectrometry (MS/MS) technology (28,29), various techniques used for quantitative peptidomics have been applied to address the challenging question of identifying peptides with potential bioactive under the physiological or disease condition (30). More importantly, the peptidomics is widely used to identify biological markers (31), discover new drug (32) and therapeutic targets (33). Recently, quantative peptidomics has also been conducted in endocrine studies (22,34,35), however, the secreted peptidomics from skeletal muscle under the insulin-resistant condition was not fully characterized.
Herein, we performed liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to help characterize the secretome from cultured skeletal muscle tissues of db/db mice at peptides level and identify putative bioactive peptides. A global secreted peptides were established and bioinformatics analysis of precursor proteins provided a possible relationship of differential peptides with T2DM or insulin resistance. Additionally, the biological effects of these secreted peptides on C2C12 myotubes elucidated a possible regulatory role in insulin signaling-and mitochondrial-related genes expression. Taken together, these observations will encourage us to investigate function of these secreted peptides from cultured skeletal muscle tissues with other tissues under the diabetic state, thus representing a promising strategy for prevention and treatment of insulin resistance as well as the associated metabolic disorders.
MATERIALS AND METHODS
Ethics Statement
All the studies involving mice acquired approval from the Ethical Committee of Nanjing Medical University. All procedure involving mice were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval Number: IACUC-1812053).
Animal Experiments and Sample Preparation
Twelve-week-old male C57BLKS/J db/db mice (n = 8, db/db group) and age-matched WT controls (n = 8, NC group) were purchased from the Model Animal Research Center of Nanjing Medical University. After adaptive raising for one week, mice were sacrificed by cervical dislocation and skeletal muscle tissues were isolated from the left hind leg (each mice of 100 ∼ 150 mg). Subsequent operations were carried out under a laminar airflow hood to decrease contamination. The visible blood vessels and connective tissue were removed from the tissue. After rinsed with PBS, the skeletal muscle tissues were cut into small pieces (3-4 mm 3 ) with scissors as described by an established protocol (36,37). Tissue cutting will lead to release of damaged cells slowly into the medium. Additionally, a small amount of serum proteins in the tissue pieces will diffuse out during culture period. Therefore, necessary washing procedures during culture were adopted to obtain medium samples (referred to as secretome) containing mainly skeletal muscle tissue-derived secreted components as previously reported (38,39). Tissue fragments were placed in a 10 cm plate (200∼300 mg from two mice as one sample) containing 10 mL serum/phenol red free DMEM/F12 medium (Gibco, Grand Island, CA, USA). After incubation in a humidified incubator at 37 • C under 95% O2 and 5% CO2 for 48 h, the medium was immediately supplemented with protease inhibitors and centrifuged (845 g, 10 min, 4 • C) to wipe off cell debris and dead cell. Supernatant samples from each group (NC or db/db group) were harvested from four individual tissue culture dishes independently (n = 4 per group). Lactate dehydrogenase (LDH) (36,40,41) and IL-6 (42) expression levels were detected to evaluate skeletal muscle vitality and capability during the in vitro culture. Then the supernatant samples obtained were stored at −80 • C until further processing.
Peptide Extraction and Desalting
First, both supernatant samples were concentrated to 1-2 mL by centrifuges for speed vacuum (LaboGene, Allerød, Denmark). Then equal volume of U2 solution containing 8 M urea and 100 mM tetraethyl-ammonium bromide in pH 8.0 was added to the concentrated supernatant for denaturation. Followed by centrifugation for 30 min (13,000 g, 30 • C), the medium supernatant was transferred to a new centrifuge tube. Subsequently, proteins were reduced by 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide successively. The protein concentrations of supernatant from cultured skeletal muscle samples were detected by Bradford method (43) and integrity of these samples were evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) combined with silver staining. Afterwards the peptides were separated from samples by treatment with Amicon R Ultra Centrifugal Filters in 10-kDa (Merck Millipore, Billerica, MA, USA) according to the manufacturer's instruction as previously described (37). The "peptidome" present in the filtrates was desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and the desalted peptide solution was vacuum-dried with centrifuges for speed vacuum (SCAN SPEED 40, LaboGene) as previously described (44) and immediately frozen at −80 • C until the following processing.
Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)
The LC-MS/MS analysis was conducted similarly to the previous protocols (45). The peptide samples were redissolved in 2% (v/v) acetonitrile/0.1% formic acid (FA) (v/v) and 5 µL solution was injected into an A Triple TOFTM 5600 mass spectrometer (AB Sciex, Redwood City, CA, USA) coupled to a ekspert TM nanoLC400 liquid chromatography (AB Sciex) via a nanosource electrospray interface equipped with distal coated SilicaTip emitters (New Objective, Woburn, MA, USA). First, load peptide onto a C18 trap column (5 µm, 100 µm × 20 mm, AB Sciex) and elute at 300 nL/min onto a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Shanghai, China) in the gradient as long as 120 min. These two mobile phases included buffer A containing 2% acetonitrile/ 0.1% FA/ 98% H2O (v/v) and buffer B containing 98% acetonitrile/0.1% FA/2% H2O (v/v). Then peptide mixture was eluted at a flow rate of 0.3 µL/min in a gradient generated with Solvent A containing 98% water and 2% acetonitrile containing 0.1% FA (v/v) and Solvent B containing 2% water and 98% acetonitrile containing 0.1% FA (v/v) according to the previously described reports (45,46). The mass spectrometer was operated in positive mode with a spray voltage of 2500 V, 206.84kPa for the curtain gas, 41.37kPa for the nebulizer gas and 150 • C as temperature. A data dependent acquisition (DDA) method was applied and a full scan MS spectrum (300-1500 m/z) with accumulation time of 0.25 s was adopted. Top 30 precursor ions for fragmentation based on the highest intensity were selected. The collections of MS1 spectra were in the range 350-1500 m/z, and MS2 spectra were in the range of 100-1500 m/z. A total of 47,709 MS/MS were collected from all LC-MS/MS analyses.
Peptide Identification and Quantitative Analysis
Protein Pilot Software (https://sciex.com.cn/products/software/ proteinpilot-software, version4.5,AB SCIEX) was adopted to analyze the original MS/MS file data (47). For peptides identification, the Paragon algorithm was employed against the Mus_musculus SwissProt sequence database (a total of 85210 items, updated in January 2019). The following parameters were installed: The parameters were set as follows: 1) cysteine modified with iodoacetamide; 2) biological modifications were selected as ID focus. The strategy of the automatic decoy database search was employed to estimate false discovery rate (FDR) calculation using the Proteomics System Performance Evaluation Pipeline (PSPEP) Software integrated in protein pilot-software. Only unique peptides (global FDR values < 1%) were considered for further analysis. Skyline v4.2 software was employed for MS1 filtering and ion chromatogram extractions for peptides label-free quantification (48,49). And the parameters setting for skyline MS1 filtering were according to previous studies (48). Using the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.
Bioinformatics and Annotations
The relative molecular mass and isoelectric point of each peptide were calculated by the online tool (http://web.expasy. org/compute.pi/). All the precursors protein of differentially expressed peptides as one group were imported into for GO (http://www.geneontology.org/) (50) and pathway analysis (https://www.kegg.jp/) for predicting potential functions. The online tools UniProt Database (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/) were adopted to explore if the peptides' sequences were positioned in the conserved structural domains or regions of their precursors. The Open Targets Platform database (www.targetvalidation.org/) was adopted to investigate precursors associated with diabetes and obesity as previously reported (24). For visualization, clustergram and volcano plot graphs in this study were drawn with R language (http://www.r-project.org/). For determination of differentially expressed peptides, fold change was computed as the average values of biological duplication (n = 4).
Synthetic Peptides
All the peptides used in this study were custom-synthesized and HPLC-purified by Science Peptide Biological Technology Co., Ltd. (Shanghai, China) through the solid-phase method as described reported (51). The purity in 95% for each peptide was confirmed by HPLC-MS method. All the used peptides were stored in lyophilization at−20 • C until dissolved with sterile water immediately for treatment with cells in vitro.
Cell Culture and Peptide Treatment
C2C12 cells, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China) at 37 • C with 5% CO2. On the fourth day of cell differentiation, the C2C12 cells were pre-treated with synthesized peptides or solvent for 48 h at the same concentration of 50 µM, and then starved for 24 h with serumfree L-DMEM (Gibco). Subsequently, myotubes were incubated in L-DMEM in the presence or absence of 100 nM of insulin for 30 min. At the indicated time, the cells were collected for the following analysis.
Western Blot Analysis and Antibodies
Reverse Transcription and Real-Time Quantitative PCR
Total RNA was isolated using trizol reagent (Life Technologies, Carlsbad, CA, USA). And the cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was determined by real-time quantitative PCR (ABI ViiA7, Applied Biosystems, Foster City, California, USA) using the SYBR Green array. The relative gene expression was analyzed based on the 2 − CT method with normalization of the data to PPIA. The primers used for the real-time quantitative PCR were listed in Table S1.
Statistical Analysis
Data were analyzed with GraphPad Prism 7 (San Diego, CA, USA), and the results were shown as the mean ± standard deviation (SD). Peptides with a fold change larger than 1.5 or <0.67 with a Student's t-test p-value <0.05 were selected as differently expressed peptides.
RESULTS
Identification of Secreted Peptides From Cultured db/db Skeletal Muscle Tissues
Considering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity. LDH and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period. LDH content of culture medium was assessed as an indicator of cell lysis at 0 h∼24 h, 24 h∼48 h and 48 h∼72 h; no significant increased in LDH occurred from 48 h to 72 h (not shown in the manuscript). Secretion of IL-6 remained stable from 24 h to 48 h in culture by ELISA (not shown in the manuscript). Therefore, we chose the 24 h∼48 h culture as an optimal time point. The protein composition and integrity of the secreted samples were evaluated by SDS-PAGE with silver staining in Figure S1. After validation of skeletal muscle culture system and samples assessment, peptides from four different culture dishes per group (control and db/db mice) were individually extracted from supernatants and analyzed via label-free mass spectrometry based quantification. To gain more insights into biological differences between control and db/db mice skeletal muscle, we employed LC-MS/MS technology and identified 3384 peptides, of which 2664 peptides showed valid quantitative values in the detection. A total of 63 peptides were identified differentially secreted, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 peptides were down-regulated (fold change < 0.67, P < 0.05) in medium supernatants from insulin resistant-skeletal muscle tissues. Visualization methods such as hierarchical clustering and volcano plot showed distinct peptides secretion profiles as shown in Figures 1A,B. The differentially secreted peptides were summarized in Table 1.
Charactrization of the Basic Feature of Differentially Secreted Peptides
To characterize the general feature of differentially secreted peptides, we further analyzed the relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs. pI. The results indicated that the Mr varied from 1011.1 Da to 2670.9 Da with 70% between 1,300 and 2,100 Da (Figure 2A), and pI varied from 3.7 to 9.7, with 51% between 4 and 7 ( Figure 2B). Additionally, distribution of pI vs. Mr could divide these peptides into three groups around pI4, pI6 and pI10 ( Figure 2C).
Comprehensive Functional Assessment and Intercellular Location of Precursor Proteins of These Differential Peptides
To make comprehensive functional assessment of these differential peptides, we consider all the precursor proteins as one group and performed GO and Pathways analysis of them. First of all, the precursor proteins of these differential peptides identified from the cultured control and db/db skeletal muscle were classified using Gene Ontology categories, which revealed the majority of proteins were associated with striated muscle contraction and glucose metabolic process ( Figure 3A). The top 20 GO terms were listed in Figure 3A. The precursor proteins annotated involved in these GO terms were presented in Table S2. Subsequent pathway analysis in the KEGG database revealed a significant enrichment in metabolic pathway and glycolysis/gluconeogenesis process ( Figure 3B). The top 14 pathway terms were listed in Figure 3B. The precursor proteins annotated involved in these pathway terms were presented in Table S3. We further discriminated the up-and down-regulated peptides and performed GO and Pathways analysis of these two groups of precursor proteins. This analysis may bring overlapping terms such as phosphorylation, phosphagen metabolic process and phosphorus metabolic process in up-or down-regulated peptides, and a smaller number of Search tool for the retrieval of interacting genes/proteins (STRING) was used to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides. The size of node represents the number of interacting proteins and the color of node represents the bitscore of matching results from STRING database. The thicker the line (edge), the higher the reliability (evaluated by combined score >0.4).
terms in Pathway analysis (deriving up-regulated peptides). These analysis were presented in Figure S2. Additionally, the major intracellular locations of the precursor proteins were considered from literature sources, illustrating that 64% proteins were cytoplasmic and 15% proteins were cytoskeletal seen in Figure 3C. Based on above analysis, we found that a vast of precursor proteins were assigned to glucose metabolic process include ALDO A, triosephosphate isomerase (TPIS), beta-enolase (ENOB), glycogen phosphorylase, muscle form (PYGM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1(PGK1), specifically assigned to glycolysis/gluconeogenesis process. Another kind of proteins enriched in regulation of striated muscle contraction terms (GO) and regulation of actin cytoskeleton (Pathway) were myosin regulatory light chain 2, skeletal muscle isoform(MLRS), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin-binding protein H (MYBPH) and myosin light chain 3 (MYL3),both belonging to sarcomeric proteins. Subsequently, we used search tool for the retrieval of interacting genes/proteins (STRING) to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides in Figure 3D. Based on protein-protein interaction and co-occurrence in KEGG pathways and literature mining, this network was constructed from 25 proteins that matched to the STRING database and the matching results were listed in Table S4. As shown from this network, 22 nodes representing precursor protein constituted the interaction diagram, which could form 93 reliable a-to-b interaction relationship by combined score (more than 0.4) as shown in Table S5.
Cleavage Pattern of Differentially Secreted Peptides
It is widely postulated that most peptides can be attributed mainly to the proteolytic enzymes as well as their type and level (cleavage specificity and activity), which can differ during disease (52)(53)(54). Briefly, the N-terminal pre-cleavage site, N terminus, C terminus, and C-terminal post-cleavage site of the identified peptides were commonly used to investigate the nature of proteolytic enzymes within serum (55) or tissues (56). Thus, we analyzed the distribution of peptide cleavage site and found that Lysine (K) was the most frequent cleavage site of N-terminal amino acid of identified peptides and Alanine (A) was the most frequent cleavage site of C-terminal amino acid of identified peptides. Lysine (K) was the most frequent cleavage site of Nterminal amino acid of preceding peptides, while Leucine (L) was the commonest C-terminal amino acid of preceding peptides ( Figure 4A). These data indicate that the pattern of cleavage sites may represent the specificity of cleavage and activity of proteolytic enzymes under the diabetic condition. Additionally, we also tried to align peptide sequences on the same precursor sequence to construct a "peptide alignment map". Searched from our samples, the largest number of identified peptides (n = 1 7) (Figure 4B) originated from KCRM and the second largest number of identified peptides (n = 6) came from ALDO A (Figure 4B), elucidating that these peptides were easily cleaved by certain kind of endogenous enzymes.
Putative Bioactive Peptides Associated With Diabetes and Obesity
To check for specific domain structures or patterns in the identified peptides comparison with its precursor proteins, we retrieved domain information from the UniProt and Pfam database. In order to validate that if the peptides exerted important roles in the related metabolic diseases, we adopted the Open Targets Platform database to investigate protein precursors. In our searching results, most peptides (n = 44) from the identified pepetides (n = 63) were located in the functional domains ( Table 2). Additionally, 27 out of 44 peptides located in the functional domain were predicted closely related with both obesity and diabetes ( Table 3). These observations will encourage us to further investigate properties of the putative secreted peptides in skeletal muscle under the diabetic state.
The Effect of Candidated Peptides (P1-P7) on Insulin and Mitochondrial-Related Pathways in Skeletal Muscle Cells
To explicit the putative function of differentially secreted peptides, 7 differentially secreted peptides, which had already been annotated derived from authentic protein from Uniprot database, located in the functional motifs and showed relatively high abundance from MS detection, were chosen for further analysis. Table S6. Sequentially, we termed these candidated peptides P1-P7 in order. To use C2C12 myotubes as cell models in vitro, the results of changes in inuslin signaling revealed that up-regulated peptides P2-P4 addition could both significantly decrease the phosphorylation of Irs1 (Ser-307) and Akt (Ser-473) under insulin stimulation (100 nM, 30 min) as shown in Figures 5A,B, in spite of bringing a modestly up-regulation of p-Irs1 (Ser-307) at the basal status. Moreover, mitochondrial-related marker protein Pgc1α was significantly attenuated in the insulin-stimulated C2C12 cells by up-regulated peptides P1-P4 as shown in Figures 5E,F. Among these downregulated peptides, only peptide 7 presented a promotion on p-Akt (Ser-473) expression level both under the basal and insulin stimulation status as shown in Figures 5C,D. And these up-regulated peptide 1-4 could decrease Pgc1α protein level and down-regulated peptide 6 and 7 administration enhanced Pgc1α protein level by the stimulation of insulin presented in Figures 5E,F. We further evaluated Gut 4 (a key gene for glucose transport) and Pgc1α mRNA levels by peptides treatment upon insulin stimulation. The results also indicated that Peptide 3 and 4 modestly decreased Pgc1αexpression at mRNA levels seen in Figure 5G, while Peptide 6 and 7 significantly up-regulated both Glut4 and Pgc1α expression at mRNA levels seen in Figure 5H. These observations revealed that these candidated peptides (P1-P7) may affect the expressions of insulin signaling or mitochondrial-related genes in skeletal muscle cells.
DISCUSSION
Emerging as a widely known secrete organ, skeletal muscle tissues have been extensively discussed using a wide range of comparative and quantitative proteomic methods (57). Proteomic scannings of the medium supernatant from skeletal muscle cells have already afford approaches to identify a large number of secreted proteins (58)(59)(60)(61)(62)(63). Particularly, one such research reported the alteration of insulin effect on the secretome profile of skeletal muscle cells, revealing the changes of protein levels secreted from skeletal muscle during activation of insulin signaling pathway (58). A recent study also generated a comprehensive secretome analysis of skeletal muscle cells under palmitic acid-induced insulin resistance (61). In addition to these proteins, many other muscle-secreted compounds have been also come to light, including exosome (64,65), metabolites (66), and miRNAs (67). These studies have greatly expanded our knowledge of skeletal muscle secretome and might afford possibilities for exploring novel molecular targets in the maintenance of skeletal muscle physiology and even whole-body metabolism. However, to date few studies has focused on the peptides present in either skeletal muscle tissues or cells. Therefore, we attempted to comprehensively profile peptides that may play roles in regulating insulin sensitivity and offer enormous promise for exploring molecular mechanisms underlying insulin resistance.
In present study, a total of 63 peptides were differentially secreted in medium supernatants from cultured skeletal muscle tissues of db/db mice, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 were down-regulated (fold change < 0.67, P < 0.05) shown in Figures 1A,B. The differences of dysregulation peptides may indeed reflect the changes between control and insulin-resistant mice in some extent. However, db/db mouse is a well-established leptin receptordeficient animal model (68,69). Despite no studies revealed the association between leptin receptor deficiency and peptide dysregulation/protein degradation so far, the possible effect by this mutation deserves to be taken into account and other nongenetic mice models could be adopted in the future studies. Based on our previous study (70), 10-kDa filters method was used to extract peptides in order to intercept proteins secreted by skeletal muscle to the conditional media. Characterization of the basic feature (Mr and pI) of these identified peptides (Figures 2A,B) only reflected the differences of distribution in the diabetic skeletal muscle tissues, but also proved the reliability of the used peptide extraction method.
Subsequently, GO and Pathway analysis revealed that the precursors protein of these peptides were mostly involved in muscle contraction and metabolism processes (Figures 3A,B). An interesting finding from our study was that most of the differentially identified peptides derived from cytosolic, cytoskeletal and mitochondrial proteins (Figures 3A,B), which differed from the secretory pathway peptides (71). This (34). Similarly, another peptidomic anaylsis of brain slices cultures and media also pointed that vast majority of secreted peptides arose from intracellular proteins (72). There does also exist some evidence that these identified N-or C-terminus protein yielding peptides, rather than internal fragments, raised the possibility that they are produced by selective processing rather than protein degradation (73). Taken together with previous researches, the current results show us meaningful hints that these intracellular peptides may be secreted via nonclassical mechanisms. Actually, another important origin of peptides is proteolytic degradation processes in body fluid under the physiological or pathologically processes. Most regulatory peptides were efficiently degraded by plasma enzymes once secreted into the bloodstream, which exerted anti-diabetic therapeutic function (74,75) or were identified as disease markers (76). Recently, Federico Aletti et al. (77) used protease activity detection and specific enzymes analysis to explain a large presence of circulating peptides under hemorrhagic shock, which gave us a possible way to evaluate peptides origin. Thus, a fundamental validation is whether the proteins-derived peptides are actually secreted from skeletal muscle cells per se or proteolytic degradation and are of biologically active. Among the differentially secreted peptides from cultured skeletal muscle tissues of db/db mice, we found that three peptides derived from GAPDH, four peptides derived from ALDO A and one peptides derived from PYGM were upregulated in the conditional medium from db/db groups as shown in Table 1. As described by Dustin S. Hittel et.al, a global proteomic survey of skeletal muscle revealed a statistically significant up-regulation in glycolytic enzymes GAPDH and ALDO A protein levels in obese/overweight patients (78). Another quantitative protein profile also identified a more abundant levels of GAPDH and PYGM in skeletal muscle from T2DM groups compared with the control groups (79). In addition to pointing the importance of the mitochondrial numbers and impairments under the insulin-resistant states (80)(81)(82), several studies noted that glycolytic capacity is higher in skeletal muscle of patients with T2DM or obesity (83). Notably, stronger changes of peptides derived from sarcomeric proteins such as myosin light chain 1 (MYL1) and MYL3 were also observed in the conditional medium from diabetic muscles as shown in Table 1. MYL1 and MYL3 are representive markers of fast-muscle and slow-muscle respectively, which were both regulated by insulin stimulation (79). And previous studies observed that property of T2DM individuals muscle was shifted to a fast-twitch glycolytic phenotype (84). In fact, deficiency in sarcomeric proteins in skeletal muscle also suggested their importance in skeletal muscle physiologic and pathological processes. On the whole, our results of increased glycolytic enzyme-or sarcomeric proteins-derived peptides secreted from insulin resistant skeletal muscle may support the hypothesis that altered glycolytic capacities or fiber types under the diabetic status contribute to this difference.
Till now, the putative function of these peptides are not clear. Therefore, we evaluated whether these peptides originated from functional domains of the corresponding precursor protein using the UniProt and Pfam database. In our searching results, most peptides (n = 44) from the identified peptides (n = 63) were located in the functional domains as listed in Table 2. Specifically, most peptides derived from functional enzymes were located in the enzymatic activity region, including TPI- TCTP-derived peptide (119-AEQIKHILANF-129) and CAH3-derived peptide . We also found another kind of S-Nitrosylation modifying sites for affecting GAPDH enzymatic activity (90). Therefore, future efforts need to be established for investigating the potential role of peptides on insulin sensitive cells in vitro or whole-body metabolism in vivo.
The above analyses provided a possibility to evaluate the biological effects of these differentially secreted peptides. As widely reported, insulin resistance in skeletal muscle is tightly connected with the deficit in insulin signaling (91). Consequently, the role of phosphorylation of Irs1 and Akt in signaling pathways is very crucial in anti-hyperglycemia and insulin sensitivity (92). In this result, we found these up-regulated peptides 1-4 both exerted a significantly attenuated insulin action in C2C12 cells, evaluated by a decreased level of p-Irs1 (Ser-307) and p-Akt (Ser-473) seen in Figures 5A,B, whereas only one down-regulated peptide P7 could remarkably promote insulin signaling only via Irs1 signaling pathway seen in Figures 5C,D. Peroxisome proliferator-activated receptor γ coactiva-tor-1(Pgc-1), which displays a dominant role through tight modulation of mitochondrial biogenesis and respiration, has also been demonstrated to participate in skeletal muscle insulin signaling and metabolic homeostasis (93). The up-regulated peptides 1-4 also brought out a decreased protein level of Pgc-1α in C2C12 cells as shown in Figures 5E,F, and down-regulated peptide 6 and 7 administration also gave rise to the increased Pgc1α mRNA and protein level in Figures 5E,F,H. Taken together, these selected peptides secreted from db/db mice skeletal muscle presented a promotive or inhibitory effect on insulin and mitochondrialrelated pathways in skeletal muscle cells by an autocrine manner. Notably, peptide 4 (231-RVPTPNVSVVDLTCRLEKPAK−252) derived from GAPDH displayed a most significant inhibitory effect toward these candidate peptides. However, the relationship between GAPDH-derived peptide and its precursor protein is to be determined. On the other hand, more methods need to be employed for the wider cell signaling screen and further research is required to explore the biologic function of skeletal muscle-secreted peptides on adipocytes or liver cells.
To our knowledge, no large-scale quantitative peptidomic analysis has been performed on skeletal muscle to elucidate secreted peptides profiles under the diabetic status. The present study identified and quantified changes with a label-free discovery using LC-MS/MS technology to construct a global secreted peptides picture. Further bioinformatics analysis of precursors comprehensively provided an atlas of peptides that may exist roles in regulating insulin sensitivity. This represented a new perspective toward exploring insulin resistance pathogenesis. Additionally, the detailed biological effects of these secreted peptides on skeletal muscle insulin resistance or cross-talk with other tissues remained to be elucidated in the future study.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
ETHICS STATEMENT
The protocol has been approved by the Institutional Animal Care and Use Committee of Nanjing medical university (Approval Number: IACUC-1812053).
AUTHOR CONTRIBUTIONS
YWu and MH performed experiments and interpreted results of experiments. YWa and YG prepared the figures. XC and PX analyzed the data. CJ and TZ participated discussion. YZ helped write the manuscript with providing assistance. LY conceived and designed experiments, provided funding to regents, and approved final version of manuscript. All authors read and approved the final manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2019.00741/full#supplementary-material Figure S1 | SDS-PAGE associated with silver staining of the medium supernatants from cultured skeletal muscle tissues (Con vs. db groups). Table S1 | Primers used in this study. Table S2 | GO terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. Table S3 | Pathway terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice.
At the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.
FIGURE 1 |
1Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.
FIGURE 2 |
2Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.
FIGURE 3 |
3Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)
FIGURE 4 |
4Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.
FIGURE 5 |
5Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
The up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in
413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),
FUNDING
This study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science
Figure S2 |
S2Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.
TABLE 1 |
1Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.Peptide sequence
Protein ID
Protein name
Mr(KDa)
Fold change
P-value
Up-regulated peptides
AVGAVFDISNADRLGSSEVEQ
P07310
KCRM
2163
9.728
0.005
TNGAFTGEISPGMIKDLGATWV
P17751
TPIS
2264
8.019
0.035
IADLVVGLCTGQIK
P21550
ENOB
1486
6.77
0.000
NYKPQEEYPDLSKHNNHMA
P07310
KCRM
2315
4.843
0.024
EEIEEDAGLGNGGLGRLAAC
Q9WUB3
PYGM
2030
4.411
0.004
GQCGDVLRALGQNPTQAEV
P09542
MYL3
2013
4.077
0.033
AGPLCLQEVDEPPQHAL
P70296
PEBP1
1873
4.038
0.004
EAFTVIDQNRDGIID
P97457
MLRS
1705
3.947
0.026
DLFDPIIQDRHGGY
P07310
KCRM
1645
3.668
0.022
KDLFDPIIQD
P07310
KCRM
1203
3.282
0.000
LDDVIQTGVDNPGHP
P07310
KCRM
1576
3.265
0.001
SEIIDVSSKAEEVK
A2ASS6
TITIN
1533
3.134
0.002
VIACIGEKLDE
P17751
TPIS
1246
2.976
0.003
GTNPTNAEVKKVLGNPSNEEM
Q545G5
MYL1
2180
2.838
0.006
ADRLGSSEVEQVQLV
P07310
KCRM
1629
2.804
0.001
IGQGTPIPDLPEVKRV
A2AQA9
NEB
1719
2.739
0.002
STTAPPIQSPLPVIPHQK
E9PYJ9
LDB3
1910
2.685
0.023
KADDGRPFPQVI
P05064
ALDOA
1342
2.644
0.021
KSMTEQEQQQLIDD
P07310
KCRM
1692
2.442
0.001
GGGASLELLEGKVLPGVDA
P09411
PGK1
1781
2.264
0.028
LEGTLLKPNMVTPGHA
P05064
ALDOA
1677
2.208
0.010
VMVGMGQKDSYVGDEAQSK
P68134
ACTS
2028
2.168
0.021
DLEEATLQHEATAAALR
Q02566
MYH6
1838
2.07
0.038
AELQEVQITEEKPLLPG
Q9QYG0
NDRG2
1935
2.043
0.048
SEGKCAELEEELKTVTNNL
P58771
TPM1
2163
2.038
0.049
RSIKGYTLPPHC
P07310
KCRM
1428
1.967
0.001
NSNSHSSTFDAGAGIALNDNFVK
P16858
G3P
2365
1.935
0.038
TGKATSEASVSTPEETAPEPAKVPT
P70402
MYBPH
2484
1.919
0.003
DNEYGYSNRVVDL
P16858
G3P
1543
1.898
0.004
EELDAMMKEASGPINF
P97457
MLRS
1781
1.853
0.012
SNNKDQGGYEDFVEGLRV
Q545G5
MYL1
2026
1.816
0.016
AEQIKHILANF
P63028
TCTP
1283
1.787
0.044
GADPEDVITGAFK
P97457
MLRS
1319
1.782
0.031
TSIEDAPITVQSKINQ
A2AQA9
NEB
1743
1.739
0.028
LKGGDDLDPNYVLS
P07310
KCRM
1505
1.732
0.040
IQTGVDNPGHPF
Q6P8J7
KCRS
1281
1.600
0.045
AEVKKVLGNPSNEEMNAK
Q545G5
MYL1
1957
1.595
0.009
GQEQWEEGDLYDKEKQQKPF
E9Q8K5
TITIN
2481
1.586
0.036
AGTNGETTTQGLDGLSERCA
P05064
ALDOA
2037
1.582
0.033
RVPTPNVSVVDLTCRLEKPAK
P16858
G3P
2378
1.531
0.022
KVPEKPEVVEKVEPAPLK
F7CR78
F7CR78
2015
1.527
0.026
GETTTQGLDGLSERCAQ
P05064
ALDOA
1822
1.526
0.042
Down-regulated peptides
KIEFTPEQIEEF
P09542
MYL3
1509
0.612
0.021
DLSAIKIEFSKEQQEDF
P05977
MYL1
2026
0.600
0.019
HELYPIAKGDNQSPI
P16015
CAH3
1681
0.559
0.038
NSLTGEFKGKY
P07310
KCRM
1243
0.545
0.027
DEPKPYPYPNLDDF
Q3V1D3
AMPD1
1709
0.543
0.034
ASHHPADFTPAVHA
Q91VB8
HBA-A1
1457
0.537
0.016
(Continued)
Frontiers in Endocrinology | www.frontiersin.org
TABLE 1 |
1ContinuedPeptide sequence
Protein ID
Protein name
Mr(KDa)
Fold change
P-value
SQVGDVLRALGTNPTNAE
Q545G5
MYL1
1841
0.516
0.047
ADEIAKAQVAQPGGDTI
P70349
HINT1
1725
0.515
0.038
DKETPSGFTLDDV
P07310
KCRM
1423
0.478
0.037
LGTNPTNAEVKKVLGNPSNEEMNAK
Q545G5
MYL1
2654
0.467
0.038
PHPYPALTPEQKK
P05064
ALDOA
1505
0.407
0.028
QLVVDGVKLM
P07310
KCRM
1101
0.400
0.001
KDLFDPIIQDRHG
P07310
KCRM
1553
0.395
0.022
KGVVPLAGTNGETTTQGLDGLSER
P05064
ALDOA
2399
0.367
0.015
RVFDKEGNGTVMGAELR
Q545G5
MYL1
1878
0.341
0.043
NADEVGGEALGRL
A8DUK4
HBB-BS
1300
0.339
0.037
NEHLGYVLTCPS
P07310
KCRM
1389
0.328
0.031
ASHTEEEVSVSVPEVQKKTVTEEK
E9Q8K5
TITIN
2669
0.253
0.023
NEEDHLRV
P07310
KCRM
1010
0.212
0.027
VLTCPSNLGTGLRG
P07310
KCRM
1444
0.201
0.018
GGYKPTDKHKTDL
P07310
KCRM
1459
0.127
0.006
observation was also demonstrated by other peptidomic studies.
From Steven W. Taylor group' results, several identified peptides
in the human islet cultures were derived from intracellular and
cytoskeletal proteins such as microtubule-associated protein 4
and ubiquitin, which may result from a greater level of cellular
stress
TABLE 2 |
2Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported asPeptide sequence
Protein
Location
Domain
Description
Up-regualted peptides
AVGAVFDISNADRLGSSEVEQ
KCRM
329-349
125-367
Phosphagen kinase C-terminal
TNGAFTGEISPGMIKDLGATWV
TPIS
121-142
57-295
TIM
IADLVVGLCTGQIK
ENOB
381-394
142-432
Enolase C
NYKPQEEYPDLSKHNNHMA
KCRM
13-31
11-98
Phosphagen kinase N-terminal
EEIEEDAGLGNGGLGRLAAC
PYGM
124-143
113-828
Phosphorylase
GQCGDVLRALGQNPTQAEV
MYL3
83-101
58-95
EF-hand 1
EAFTVIDQNRDGIID
MLRS
32-46
25-60
EF-hand 1
DLFDPIIQDRHGGY
KCRM
87-100
11-98
Phosphagen kinase N-terminal
KDLFDPIIQD
KCRM
86-95
11-98
Phosphagen kinase N-terminal
LDDVIQTGVDNPGHP
KCRM
53-67
11-98
Phosphagen kinase N-terminal
VIACIGEKLDE
TPIS
174-184
57-295
TIM
GTNPTNAEVKKVLGNPSNEEM
MYL1
39-59
6-41
EF-hand
ADRLGSSEVEQVQLV
KCRM
339-353
125-367
Phosphagen kinase C-terminal
KADDGRPFPQVI
ALDOA
87-98
15-364
Glycolytic
KSMTEQEQQQLIDD
KCRM
177-190
125-367
Phosphagen kinase C-terminal
GGGASLELLEGKVLPGVDA
PGK1
395-413
9-406
PGK
LEGTLLKPNMVTPGHA
ALDOA
224-239
15-364
Glycolytic
VMVGMGQKDSYVGDEAQSK
ACTS
45-63
4-377
Actin
DLEEATLQHEATAAALR
MYH6
1,179-1,195
845-1,926
Myosin_tail_1
SEGKCAELEEELKTVTNNL
TPM1
186-204
1-284
Coiled coili
RSIKGYTLPPHC
KCRM
135-146
125-367
Phosphagen kinase C-terminal
SNNKDQGGYEDFVEGLRV
MYL1
77-94
83-118
EF-hand
AEQIKHILANF
TCTP
119-129
1-172
TCTP
GADPEDVITGAFK
MLRS
93-105
95-130
EF-hand 2
LKGGDDLDPNYVLS
KCRM
115-128
125-367
Phosphagen kinase C-terminal
GQEQWEEGDLYDKEKQQKPF
TITIN
1,691-1,710
1,709-1,799
Ig-like
AGTNGETTTQGLDGLSERCA
ALDOA
117-136
15-364
Glycolytic
RVPTPNVSVVDLTCRLEKPAK
GAPDH
232-252
243-248
[IL]-x-C-x-x-[DE] motif
GETTTQGLDGLSERCAQ
ALDOA
121-137
15-364
Glycolytic
Down-regualted peptides
KIEFTPEQIEEF
MYL3
52-63
58-95
EF-hand 1
DLSAIKIEFSKEQQEDF
MYL1
33-49
44-79
EF-hand 1
HELYPIAKGDNQSPI
CAH3
17-31
3-259
Alpha-carbonic anhydrase
NSLTGEFKGKY
KCRM
163-173
125-367
Phosphagen kinase C-terminal
ASHHPADFTPAVHA
HBA-A1
111-124
3-142
GLOBIN
DKETPSGFTLDDV
KCRM
44-56
11-98
Phosphagen kinase N-terminal
LGTNPTNAEVKKVLGNPSNEEMNAK
MYL1
76-100
83-118
EF-hand
QLVVDGVKLM
KCRM
351-360
125-367
Phosphagen kinase C-terminal
KDLFDPIIQDRHG
KCRM
86-98
11-98
Phosphagen kinase N-terminal
KGVVPLAGTNGETTTQGLDGLSER
ALDOA
111-134
15-364
Glycolytic
RVFDKEGNGTVMGAELR
MYL1
147-163
133-150
EF-hand
NADEVGGEALGRL
HBB-BS
20-32
2-147
GLOBIN
NEHLGYVLTCPS
KCRM
274-285
125-367
Phosphagen kinase C-terminal
NEEDHLRV
KCRM
230-237
125-367
Phosphagen kinase C-terminal
VLTCPSNLGTGLRG
KCRM
280-293
125-367
Phosphagen kinase C-terminal
TABLE 3 |
3Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.Protein name
Description
Peptide number
Association score
with obesity #
Association score with
diabetes mellitus #
TPI1
Triosephosphate isomerase
2
0.012
0.012
KCRM
Creatine kinase M-type
17
0.022
0.017
TTN
Titin
1
0.026
0.063
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
1
0.071
0.117
HBA-A1
Alpha globin 1
1
0.014
0.038
ENOB
Beta-enolase
1
0.040
0.048
CAH3
Carbonic anhydrase 3
1
0.074
0.094
TCTP
Translationally-controlled tumor protein
1
0.028
0.040
HBB-BS
Beta-globin
1
0.008
0.020
ACTS
Actin, alpha skeletal muscle
1
0.006
0.188
#
Table S4 |
S4The matching results of the precursor proteins from STRING database.
Table S5 |
S5Precursor proteins-to-precursor proteins interaction from STRING database.
Table S6 |
S6The information of candidate peptides for functional evaluation.
Frontiers in Endocrinology | www.frontiersin.org
October 2019 | Volume 10 | Article 741
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| [
"As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders."
] | [
"Vaclav Kasicka ",
"Martin Hubalek ",
"Yu Zeng [email protected] ",
"Wu Y ",
"Han M Wang ",
"Y ",
"Gao Y ",
"Cui X ",
"Xu P ",
"Ji C ",
"Zhong T ",
"You L ",
"Zeng Y ",
"Yanting Wu \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nAffiliated Maternity and Child Health Care Hospital of Nantong University\nNanTongChina\n",
"Mei Han \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"Yan Wang \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"Yao Gao \nDepartment of Endocrinology, Children's Hospital\nNanjing Medical University\nNanjingChina\n",
"Xianwei Cui ",
"Pengfei Xu ",
"Chenbo Ji \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"Tianying Zhong \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"Lianghui You [email protected] \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"Yu Zeng \nNanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n\nDepartment of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina\n",
"\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nLianghui You\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nCzechia, Czechia\n"
] | [
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Affiliated Maternity and Child Health Care Hospital of Nantong University\nNanTongChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Department of Endocrinology, Children's Hospital\nNanjing Medical University\nNanjingChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Nanjing Maternity and Child Health Care Institute, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Department of Clinical Laboratory, Women's Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital)\nNanjingChina",
"Institute of Organic Chemistry and Biochemistry (ASCR)\nLianghui You\nInstitute of Organic Chemistry and Biochemistry (ASCR)\nCzechia, Czechia"
] | [
"Vaclav",
"Martin",
"Yu",
"Wu",
"Y",
"Han",
"M",
"Y",
"Gao",
"Y",
"Cui",
"X",
"Xu",
"P",
"Ji",
"C",
"Zhong",
"T",
"You",
"L",
"Zeng",
"Y",
"Yanting",
"Mei",
"Yan",
"Yao",
"Xianwei",
"Pengfei",
"Chenbo",
"Tianying",
"Lianghui",
"Yu"
] | [
"Kasicka",
"Hubalek",
"Zeng",
"Wang",
"Wu",
"Han",
"Wang",
"Gao",
"Cui",
"Xu",
"Ji",
"Zhong",
"You",
"Zeng"
] | [
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"E Jacot, ",
"R A Defronzo, ",
"E Maeder, ",
"E Jequier, ",
"J P Felber, ",
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"D S Paul, ",
"O Ilkayeva, ",
"T R Koves, ",
"F Pascual, ",
"W Wu, ",
"Z Xu, ",
"L Zhang, ",
"J Liu, ",
"J Feng, ",
"X Wang, ",
"L A Consitt, ",
"G Saxena, ",
"A Saneda, ",
"J A Houmard, ",
"K F Petersen, ",
"S Dufour, ",
"D B Savage, ",
"S Bilz, ",
"G Solomon, ",
"S Yonemitsu, ",
"J Giudice, ",
"J M Taylor, ",
"P Boström, ",
"J Wu, ",
"M P Jedrychowski, ",
"A Korde, ",
"L Ye, ",
"J C Lo, ",
"C Tagliaferri, ",
"Y Wittrant, ",
"M J Davicco, ",
"S Walrand, ",
"V Coxam, ",
"M K Montgomery, ",
"R Mokhtar, ",
"J Bayliss, ",
"H C Parkington, ",
"V M Suturin, ",
"C R Bruce, ",
"C S Christensen, ",
"D P Christensen, ",
"M Lundh, ",
"M S Dahllöf, ",
"T N Haase, ",
"J M Velasquez, ",
"K Kozinski, ",
"M Jazurek, ",
"P Dobrzyn, ",
"J Janikiewicz, ",
"K Kolczynska, ",
"A Gajda, ",
"L Pedersen, ",
"P Hojman, ",
"A P Lightfoot, ",
"R G Cooper, ",
"K Eckardt, ",
"S W Görgens, ",
"S Raschke, ",
"J Eckel, ",
"I Nieto-Vazquez, ",
"S Fernández-Veledo, ",
"C De Alvaro, ",
"M Lorenzo, ",
"L Al-Khalili, ",
"K Bouzakri, ",
"S Glund, ",
"F Lönnqvist, ",
"H A Koistinen, ",
"A Krook, ",
"G Van Hall, ",
"A Steensberg, ",
"M Sacchetti, ",
"C Fischer, ",
"C Keller, ",
"P Schjerling, ",
"H J Kim, ",
"T Higashimori, ",
"S Y Park, ",
"H Choi, ",
"J Dong, ",
"Y J Kim, ",
"J P Barlow, ",
"T P Solomon, ",
"B P Carson, ",
"K Sasaki, ",
"N Takahashi, ",
"M Satoh, ",
"M Yamasaki, ",
"N Minamino, ",
"G P Roberts, ",
"P Larraufie, ",
"P Richards, ",
"R G Kay, ",
"S G Galvin, ",
"E L Miedzybrodzka, ",
"Y C Liu, ",
"H J Patel, ",
"A M Khawaja, ",
"M G Belvisi, ",
"D F Rogers, ",
"X Wang, ",
"S Xu, ",
"L Chen, ",
"D Shen, ",
"Y Cao, ",
"R Tang, ",
"A Bartolomucci, ",
"La Corte, ",
"G Possenti, ",
"R Locatelli, ",
"V Rigamonti, ",
"A E Torsello, ",
"A , ",
"N E Zois, ",
"E D Bartels, ",
"I Hunter, ",
"B S Kousholt, ",
"L H Olsen, ",
"J P Goetze, ",
"J O Dasilva, ",
"G P Amorino, ",
"E V Casarez, ",
"B Pemberton, ",
"S J Parsons, ",
"M Schrader, ",
"S Štěpánová, ",
"V Kašička, ",
"L Fricker, ",
"J Klein, ",
"A Ramirez-Torres, ",
"A Ericsson, ",
"Y Huang, ",
"B Breuil, ",
"J Siwy, ",
"M Krochmal, ",
"J P Schanstra, ",
"H Mischak, ",
"S Wang, ",
"A Blois, ",
"T El Rayes, ",
"J F Liu, ",
"M S Hirsch, ",
"K Gravdal, ",
"S W Taylor, ",
"S E Nikoulina, ",
"N L Andon, ",
"C Lowe, ",
"H Ye, ",
"J Wang, ",
"Z Tian, ",
"F Ma, ",
"J Dowell, ",
"Q Bremer, ",
"L Zhu, ",
"M Hou, ",
"B Sun, ",
"J Burén, ",
"L Zhang, ",
"J Yi, ",
"G Jia, ",
"H Tao, ",
"Y Xue, ",
"S Xu, ",
"K Xue, ",
"Q Zhu, ",
"G Alvarez-Llamas, ",
"E Szalowska, ",
"M P De Vries, ",
"D Weening, ",
"K Landman, ",
"A Hoek, ",
"A Roca-Rivada, ",
"Alonso J Al-Massadi, ",
"O Castelao, ",
"C Peinado, ",
"J R Seoane, ",
"L M , ",
"X Casabiell, ",
"V Pineiro, ",
"R Peino, ",
"M Lage, ",
"J Camiña, ",
"R Gallego, ",
"S A Phillips, ",
"T P Ciaraldi, ",
"D K Oh, ",
"M K Savu, ",
"R R Henry, ",
"A Roca-Rivada, ",
"O Al-Massadi, ",
"C Castelao, ",
"L L Senín, ",
"J Alonso, ",
"L M Seoane, ",
"H K Maehre, ",
"L Dalheim, ",
"G K Edvinsen, ",
"E O Elvevoll, ",
"I J Jensen, ",
"M M Robinson, ",
"S Dasari, ",
"H Karakelides, ",
"H R Bergen, ",
"K S Nair, ",
"X Zhang, ",
"Q Liu, ",
"W Zhou, ",
"P Li, ",
"R N Alolga, ",
"L W Qi, ",
"P Yin, ",
"A M Knolhoff, ",
"H J Rosenberg, ",
"L J Millet, ",
"M U Gillette, ",
"J V Sweedler, ",
"I V Shilov, ",
"S L Seymour, ",
"A A Patel, ",
"A Loboda, ",
"W H Tang, ",
"S P Keating, ",
"B Schilling, ",
"M J Rardin, ",
"B X Maclean, ",
"A M Zawadzka, ",
"B E Frewen, ",
"M P Cusack, ",
"M J Rardin, ",
"J C Newman, ",
"J M Held, ",
"M P Cusack, ",
"D J Sorensen, ",
"B Li, ",
"M Ashburner, ",
"C A Ball, ",
"J A Blake, ",
"D Botstein, ",
"H Butler, ",
"J M Cherry, ",
"D Shen, ",
"Y Li, ",
"X Wang, ",
"F Wang, ",
"F Huang, ",
"Y Cao, ",
"J Villanueva, ",
"J Philip, ",
"C A Chaparro, ",
"Y Li, ",
"R Toledo-Crow, ",
"L Denoyer, ",
"J M Koomen, ",
"D Li, ",
"L C Xiao, ",
"T C Liu, ",
"K R Coombes, ",
"J Abbruzzese, ",
"J Villanueva, ",
"D R Shaffer, ",
"J Philip, ",
"C A Chaparro, ",
"H Erdjument-Bromage, ",
"A B Olshen, ",
"F Wang, ",
"J Zhu, ",
"L Hu, ",
"H Qin, ",
"M Ye, ",
"H Zou, ",
"J Zhang, ",
"D Liang, ",
"Q Cheng, ",
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"Y Wu, ",
"Y Wang, ",
"A S Deshmukh, ",
"J H Yoon, ",
"K Yea, ",
"J Kim, ",
"Y S Choi, ",
"S Park, ",
"H Lee, ",
"F Norheim, ",
"T Raastad, ",
"B Thiede, ",
"A C Rustan, ",
"C A Drevon, ",
"F Haugen, ",
"S Raschke, ",
"K Eckardt, ",
"K Bjørklund Holven, ",
"J Jensen, ",
"J Eckel, ",
"A S Deshmukh, ",
"J Cox, ",
"L J Jensen, ",
"F Meissner, ",
"M Mann, ",
"J H Yoon, ",
"D Kim, ",
"J H Jang, ",
"J Ghim, ",
"S Park, ",
"P Song, ",
"Y Furuichi, ",
"Y Manabe, ",
"M Takagi, ",
"M Aoki, ",
"N L Fujii, ",
"A Forterre, ",
"A Jalabert, ",
"K Chikh, ",
"S Pesenti, ",
"V Euthine, ",
"A Granjon, ",
"A Jalabert, ",
"G Vial, ",
"C Guay, ",
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"J Z Nordin, ",
"H Aswad, ",
"A Ibrahim, ",
"M Neinast, ",
"Z P Arany, ",
"S Nielsen, ",
"C Scheele, ",
"C Yfanti, ",
"T Akerström, ",
"A R Nielsen, ",
"B K Pedersen, ",
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"X Wang, ",
"F Huang, ",
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"Y Li, ",
"X Wang, ",
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"T Takao, ",
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"I Berezniuk, ",
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"M Okon, ",
"R E Reid, ",
"L P Mcintosh, ",
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"K A Hucker, ",
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"D R Park, ",
"K H Park, ",
"B J Kim, ",
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"U H Kim, ",
"J Jia, ",
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"X , "
] | [
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"\n\nAt the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.",
"\nFIGURE 1 |\n1Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.",
"\nFIGURE 2 |\n2Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.",
"\nFIGURE 3 |\n3Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)",
"\nFIGURE 4 |\n4Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.",
"\nFIGURE 5 |\n5Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.",
"\n\nThe up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in",
"\n\n413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),",
"\nFUNDING\nThis study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science",
"\nFigure S2 |\nS2Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.",
"\nTABLE 1 |\n1Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nUp-regulated peptides \n\nAVGAVFDISNADRLGSSEVEQ \nP07310 \nKCRM \n2163 \n9.728 \n0.005 \n\nTNGAFTGEISPGMIKDLGATWV \nP17751 \nTPIS \n2264 \n8.019 \n0.035 \n\nIADLVVGLCTGQIK \nP21550 \nENOB \n1486 \n6.77 \n0.000 \n\nNYKPQEEYPDLSKHNNHMA \nP07310 \nKCRM \n2315 \n4.843 \n0.024 \n\nEEIEEDAGLGNGGLGRLAAC \nQ9WUB3 \nPYGM \n2030 \n4.411 \n0.004 \n\nGQCGDVLRALGQNPTQAEV \nP09542 \nMYL3 \n2013 \n4.077 \n0.033 \n\nAGPLCLQEVDEPPQHAL \nP70296 \nPEBP1 \n1873 \n4.038 \n0.004 \n\nEAFTVIDQNRDGIID \nP97457 \nMLRS \n1705 \n3.947 \n0.026 \n\nDLFDPIIQDRHGGY \nP07310 \nKCRM \n1645 \n3.668 \n0.022 \n\nKDLFDPIIQD \nP07310 \nKCRM \n1203 \n3.282 \n0.000 \n\nLDDVIQTGVDNPGHP \nP07310 \nKCRM \n1576 \n3.265 \n0.001 \n\nSEIIDVSSKAEEVK \nA2ASS6 \nTITIN \n1533 \n3.134 \n0.002 \n\nVIACIGEKLDE \nP17751 \nTPIS \n1246 \n2.976 \n0.003 \n\nGTNPTNAEVKKVLGNPSNEEM \nQ545G5 \nMYL1 \n2180 \n2.838 \n0.006 \n\nADRLGSSEVEQVQLV \nP07310 \nKCRM \n1629 \n2.804 \n0.001 \n\nIGQGTPIPDLPEVKRV \nA2AQA9 \nNEB \n1719 \n2.739 \n0.002 \n\nSTTAPPIQSPLPVIPHQK \nE9PYJ9 \nLDB3 \n1910 \n2.685 \n0.023 \n\nKADDGRPFPQVI \nP05064 \nALDOA \n1342 \n2.644 \n0.021 \n\nKSMTEQEQQQLIDD \nP07310 \nKCRM \n1692 \n2.442 \n0.001 \n\nGGGASLELLEGKVLPGVDA \nP09411 \nPGK1 \n1781 \n2.264 \n0.028 \n\nLEGTLLKPNMVTPGHA \nP05064 \nALDOA \n1677 \n2.208 \n0.010 \n\nVMVGMGQKDSYVGDEAQSK \nP68134 \nACTS \n2028 \n2.168 \n0.021 \n\nDLEEATLQHEATAAALR \nQ02566 \nMYH6 \n1838 \n2.07 \n0.038 \n\nAELQEVQITEEKPLLPG \nQ9QYG0 \nNDRG2 \n1935 \n2.043 \n0.048 \n\nSEGKCAELEEELKTVTNNL \nP58771 \nTPM1 \n2163 \n2.038 \n0.049 \n\nRSIKGYTLPPHC \nP07310 \nKCRM \n1428 \n1.967 \n0.001 \n\nNSNSHSSTFDAGAGIALNDNFVK \nP16858 \nG3P \n2365 \n1.935 \n0.038 \n\nTGKATSEASVSTPEETAPEPAKVPT \nP70402 \nMYBPH \n2484 \n1.919 \n0.003 \n\nDNEYGYSNRVVDL \nP16858 \nG3P \n1543 \n1.898 \n0.004 \n\nEELDAMMKEASGPINF \nP97457 \nMLRS \n1781 \n1.853 \n0.012 \n\nSNNKDQGGYEDFVEGLRV \nQ545G5 \nMYL1 \n2026 \n1.816 \n0.016 \n\nAEQIKHILANF \nP63028 \nTCTP \n1283 \n1.787 \n0.044 \n\nGADPEDVITGAFK \nP97457 \nMLRS \n1319 \n1.782 \n0.031 \n\nTSIEDAPITVQSKINQ \nA2AQA9 \nNEB \n1743 \n1.739 \n0.028 \n\nLKGGDDLDPNYVLS \nP07310 \nKCRM \n1505 \n1.732 \n0.040 \n\nIQTGVDNPGHPF \nQ6P8J7 \nKCRS \n1281 \n1.600 \n0.045 \n\nAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n1957 \n1.595 \n0.009 \n\nGQEQWEEGDLYDKEKQQKPF \nE9Q8K5 \nTITIN \n2481 \n1.586 \n0.036 \n\nAGTNGETTTQGLDGLSERCA \nP05064 \nALDOA \n2037 \n1.582 \n0.033 \n\nRVPTPNVSVVDLTCRLEKPAK \nP16858 \nG3P \n2378 \n1.531 \n0.022 \n\nKVPEKPEVVEKVEPAPLK \nF7CR78 \nF7CR78 \n2015 \n1.527 \n0.026 \n\nGETTTQGLDGLSERCAQ \nP05064 \nALDOA \n1822 \n1.526 \n0.042 \n\nDown-regulated peptides \n\nKIEFTPEQIEEF \nP09542 \nMYL3 \n1509 \n0.612 \n0.021 \n\nDLSAIKIEFSKEQQEDF \nP05977 \nMYL1 \n2026 \n0.600 \n0.019 \n\nHELYPIAKGDNQSPI \nP16015 \nCAH3 \n1681 \n0.559 \n0.038 \n\nNSLTGEFKGKY \nP07310 \nKCRM \n1243 \n0.545 \n0.027 \n\nDEPKPYPYPNLDDF \nQ3V1D3 \nAMPD1 \n1709 \n0.543 \n0.034 \n\nASHHPADFTPAVHA \nQ91VB8 \nHBA-A1 \n1457 \n0.537 \n0.016 \n\n(Continued) \n\nFrontiers in Endocrinology | www.frontiersin.org ",
"\nTABLE 1 |\n1ContinuedPeptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nSQVGDVLRALGTNPTNAE \nQ545G5 \nMYL1 \n1841 \n0.516 \n0.047 \n\nADEIAKAQVAQPGGDTI \nP70349 \nHINT1 \n1725 \n0.515 \n0.038 \n\nDKETPSGFTLDDV \nP07310 \nKCRM \n1423 \n0.478 \n0.037 \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n2654 \n0.467 \n0.038 \n\nPHPYPALTPEQKK \nP05064 \nALDOA \n1505 \n0.407 \n0.028 \n\nQLVVDGVKLM \nP07310 \nKCRM \n1101 \n0.400 \n0.001 \n\nKDLFDPIIQDRHG \nP07310 \nKCRM \n1553 \n0.395 \n0.022 \n\nKGVVPLAGTNGETTTQGLDGLSER \nP05064 \nALDOA \n2399 \n0.367 \n0.015 \n\nRVFDKEGNGTVMGAELR \nQ545G5 \nMYL1 \n1878 \n0.341 \n0.043 \n\nNADEVGGEALGRL \nA8DUK4 \nHBB-BS \n1300 \n0.339 \n0.037 \n\nNEHLGYVLTCPS \nP07310 \nKCRM \n1389 \n0.328 \n0.031 \n\nASHTEEEVSVSVPEVQKKTVTEEK \nE9Q8K5 \nTITIN \n2669 \n0.253 \n0.023 \n\nNEEDHLRV \nP07310 \nKCRM \n1010 \n0.212 \n0.027 \n\nVLTCPSNLGTGLRG \nP07310 \nKCRM \n1444 \n0.201 \n0.018 \n\nGGYKPTDKHKTDL \nP07310 \nKCRM \n1459 \n0.127 \n0.006 \n\nobservation was also demonstrated by other peptidomic studies. \nFrom Steven W. Taylor group' results, several identified peptides \nin the human islet cultures were derived from intracellular and \ncytoskeletal proteins such as microtubule-associated protein 4 \nand ubiquitin, which may result from a greater level of cellular \nstress ",
"\nTABLE 2 |\n2Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported asPeptide sequence \nProtein \nLocation \nDomain \nDescription \n\nUp-regualted peptides \n\nAVGAVFDISNADRLGSSEVEQ \nKCRM \n329-349 \n125-367 \nPhosphagen kinase C-terminal \n\nTNGAFTGEISPGMIKDLGATWV \nTPIS \n121-142 \n57-295 \nTIM \n\nIADLVVGLCTGQIK \nENOB \n381-394 \n142-432 \nEnolase C \n\nNYKPQEEYPDLSKHNNHMA \nKCRM \n13-31 \n11-98 \nPhosphagen kinase N-terminal \n\nEEIEEDAGLGNGGLGRLAAC \nPYGM \n124-143 \n113-828 \nPhosphorylase \n\nGQCGDVLRALGQNPTQAEV \nMYL3 \n83-101 \n58-95 \nEF-hand 1 \n\nEAFTVIDQNRDGIID \nMLRS \n32-46 \n25-60 \nEF-hand 1 \n\nDLFDPIIQDRHGGY \nKCRM \n87-100 \n11-98 \nPhosphagen kinase N-terminal \n\nKDLFDPIIQD \nKCRM \n86-95 \n11-98 \nPhosphagen kinase N-terminal \n\nLDDVIQTGVDNPGHP \nKCRM \n53-67 \n11-98 \nPhosphagen kinase N-terminal \n\nVIACIGEKLDE \nTPIS \n174-184 \n57-295 \nTIM \n\nGTNPTNAEVKKVLGNPSNEEM \nMYL1 \n39-59 \n6-41 \nEF-hand \n\nADRLGSSEVEQVQLV \nKCRM \n339-353 \n125-367 \nPhosphagen kinase C-terminal \n\nKADDGRPFPQVI \nALDOA \n87-98 \n15-364 \nGlycolytic \n\nKSMTEQEQQQLIDD \nKCRM \n177-190 \n125-367 \nPhosphagen kinase C-terminal \n\nGGGASLELLEGKVLPGVDA \nPGK1 \n395-413 \n9-406 \nPGK \n\nLEGTLLKPNMVTPGHA \nALDOA \n224-239 \n15-364 \nGlycolytic \n\nVMVGMGQKDSYVGDEAQSK \nACTS \n45-63 \n4-377 \nActin \n\nDLEEATLQHEATAAALR \nMYH6 \n1,179-1,195 \n845-1,926 \nMyosin_tail_1 \n\nSEGKCAELEEELKTVTNNL \nTPM1 \n186-204 \n1-284 \nCoiled coili \n\nRSIKGYTLPPHC \nKCRM \n135-146 \n125-367 \nPhosphagen kinase C-terminal \n\nSNNKDQGGYEDFVEGLRV \nMYL1 \n77-94 \n83-118 \nEF-hand \n\nAEQIKHILANF \nTCTP \n119-129 \n1-172 \nTCTP \n\nGADPEDVITGAFK \nMLRS \n93-105 \n95-130 \nEF-hand 2 \n\nLKGGDDLDPNYVLS \nKCRM \n115-128 \n125-367 \nPhosphagen kinase C-terminal \n\nGQEQWEEGDLYDKEKQQKPF \nTITIN \n1,691-1,710 \n1,709-1,799 \nIg-like \n\nAGTNGETTTQGLDGLSERCA \nALDOA \n117-136 \n15-364 \nGlycolytic \n\nRVPTPNVSVVDLTCRLEKPAK \nGAPDH \n232-252 \n243-248 \n[IL]-x-C-x-x-[DE] motif \n\nGETTTQGLDGLSERCAQ \nALDOA \n121-137 \n15-364 \nGlycolytic \n\nDown-regualted peptides \n\nKIEFTPEQIEEF \nMYL3 \n52-63 \n58-95 \nEF-hand 1 \n\nDLSAIKIEFSKEQQEDF \nMYL1 \n33-49 \n44-79 \nEF-hand 1 \n\nHELYPIAKGDNQSPI \nCAH3 \n17-31 \n3-259 \nAlpha-carbonic anhydrase \n\nNSLTGEFKGKY \nKCRM \n163-173 \n125-367 \nPhosphagen kinase C-terminal \n\nASHHPADFTPAVHA \nHBA-A1 \n111-124 \n3-142 \nGLOBIN \n\nDKETPSGFTLDDV \nKCRM \n44-56 \n11-98 \nPhosphagen kinase N-terminal \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nMYL1 \n76-100 \n83-118 \nEF-hand \n\nQLVVDGVKLM \nKCRM \n351-360 \n125-367 \nPhosphagen kinase C-terminal \n\nKDLFDPIIQDRHG \nKCRM \n86-98 \n11-98 \nPhosphagen kinase N-terminal \n\nKGVVPLAGTNGETTTQGLDGLSER \nALDOA \n111-134 \n15-364 \nGlycolytic \n\nRVFDKEGNGTVMGAELR \nMYL1 \n147-163 \n133-150 \nEF-hand \n\nNADEVGGEALGRL \nHBB-BS \n20-32 \n2-147 \nGLOBIN \n\nNEHLGYVLTCPS \nKCRM \n274-285 \n125-367 \nPhosphagen kinase C-terminal \n\nNEEDHLRV \nKCRM \n230-237 \n125-367 \nPhosphagen kinase C-terminal \n\nVLTCPSNLGTGLRG \nKCRM \n280-293 \n125-367 \nPhosphagen kinase C-terminal \n\n",
"\nTABLE 3 |\n3Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.Protein name \nDescription \nPeptide number \nAssociation score \nwith obesity # \n\nAssociation score with \ndiabetes mellitus # \n\nTPI1 \nTriosephosphate isomerase \n2 \n0.012 \n0.012 \n\nKCRM \nCreatine kinase M-type \n17 \n0.022 \n0.017 \n\nTTN \nTitin \n1 \n0.026 \n0.063 \n\nGAPDH \nGlyceraldehyde-3-phosphate dehydrogenase \n1 \n0.071 \n0.117 \n\nHBA-A1 \nAlpha globin 1 \n1 \n0.014 \n0.038 \n\nENOB \nBeta-enolase \n1 \n0.040 \n0.048 \n\nCAH3 \nCarbonic anhydrase 3 \n1 \n0.074 \n0.094 \n\nTCTP \nTranslationally-controlled tumor protein \n1 \n0.028 \n0.040 \n\nHBB-BS \nBeta-globin \n1 \n0.008 \n0.020 \n\nACTS \nActin, alpha skeletal muscle \n1 \n0.006 \n0.188 \n\n# ",
"\nTable S4 |\nS4The matching results of the precursor proteins from STRING database.",
"\nTable S5 |\nS5Precursor proteins-to-precursor proteins interaction from STRING database.",
"\nTable S6 |\nS6The information of candidate peptides for functional evaluation."
] | [
"At the indicated time, C2C12 myotubes were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Proteins were loaded and separated by 8%-10% (v/v) SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% milk. The membrane was incubated with the determined primary antibodies, respectively overnight at 4 • C as follows: Insulin receptor substrate 1 (Irs-1) (1:1000 in dilution, Cat No: 2382; Cell Signaling Technology, Danvers, MA, USA), Phospho-Irs-1(ser307) (1:1000 in dilution, Cat No: 2381; CST), RAC-alpha serine/threonine-protein kinase (Akt)(1:1000 in dilution, Cat No: 4685; CST), Phospho-Akt (Ser473)(1:1000 in dilution, Cat No: 4060; CST), Pgc1α(1:1000 in dilution, Cat No: ab54481; Abcam, Cambridge, UK), Tublin (1:1000 in dilution, Cat No: 10094-1-AP; Proteintech, Rosemont, USA). Then the membrane was incubated with goat anti-rabbit HRP secondary antibody (1:5000 in dilution, CAT: BL003A; Biosharp, Hefei, China). Proteins bands were visualized using a chemiluminescence kit and analyzed using Image J software.",
"Differentially secreted peptides from the cultured skeletal muscle tissues from db/db and Control mice. (A) Heatmaps highlight the peptides intensity patterns differentially secreted from the medium supernatants of cultured skeletal muscle tissues from control (con) vs. db/db (db) groups, n = 4 per group.(B) Volcano plot after two-sample t test for peptides detected in medium supernatants derived from the cultured skeletal muscle from control and db/db mice. Red color indicates peptides we defined as up-regulated peptides and blue indicates down-regulated peptides.",
"Charactrization of the basic feature of differentially secreted peptides. (A) Histogram displaying the Mr distribution of differentially secreted peptides from skeletal muscle peptidomics. (B) Histogram displaying the pI distribution of differentially secreted peptides from skeletal muscle peptidomics. (C) Scatter plot of Mr vs. pI.",
"Comprehensive location and functional assessment of precursor proteins of these differential peptides. (A) GO analysis of precursor proteins and TOP 20 GO terms in the biological process categories. (B) Canonical pathways analysis of precursor proteins and TOP 14 pathways terms in the biological process categories. (C) Distribution of intercellular location of precursor proteins. (D)",
"Analysis of cleavage pattern of differentially secreted peptides. (A) Distribution of the four cleavage sites in the identified differentially secreted peptides. (B) Peptides derived from the same precursor protein KCRM or ALDOA.",
"Effect of the candidate peptides on insulin and mitochondrial-related pathways. The differentiated C2C12 motes were treated with the candidate peptides (50 uM) named P1-P7 groups or solvent termed untreated groups. After incubation with or without 100 nM insulin for 30 min, cell lysates were analyzed by Western blot. (A,C) Total protein and phosphorylation of Irs1 at ser307 (p-Irs1) and Akt at ser473 (p-Akt). (B,D) Quantification of the protein levels of p-Irs1 (ser307) and p-Akt (ser473) relative to total protein, respectively. (E) Mitochondrial-related protein Pgc1α was detected and Tublin was used as internal control. (F) Quantification of the protein levels of Pgc1α relative to Tublin. Values are the means ± SD of three separate experiments. (G,H) Real-time quantitative PCR detection of the mRNA levels of Glut4 and Pgc1 relative to internal control PPIA, respectively. Values are the means ± SD of three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.",
"The up-regulated peptides were AVGAVFDISNADRLGSSEVEQ (KCRM/P07310), ADRLGSSEVEQVQLV (KCRM/P07310), GADPEDVITGAFK (MLRS/P97457), RVPTPNVSVVDLTCRLEKPAK (GAPDH/P16858), whereas the down-regulated peptides were DKETPSGFTLDDV (KCRM/P07310), NEHLGYVLTCPS (KCRM/P07310), VLTCPSNLGTGLRG (KCRM/P07310) as listed in",
"413), ALDOA-derived peptides (87-KADDGRPFPQVI-98, 224-LEGTLLKPNMVTPGHA-239, 117-AGTNGETTTQGLDGLSERCA-136, 121-GETTTQGLDGLSERCAQ-137),",
"This study was supported by National Natural Science Foundation of China (Grant No. 81600687, 81700738, 81770866, 81770837, 81870546), the Jiangsu Provincial Key Research and Development Program (BE2016619), Jiangsu Provincial Medical Innovation Team Program, 333 high level talents training project of Jiangsu Province, Jiangsu Provincial Medical Youth Talent, Jiangsu Province Natural Science",
"Functional assessment of precursor proteins deriving up-and down-regulated peptides, respectively. (A,B) GO analysis of precursor proteins deriving up-and down-regulated peptides, respectively. (C,D) Canonical pathways analysis of precursor proteins deriving up-and down-regulated peptides, respectively.",
"Differentially expressed peptides secreted from the medium supernatants of cultured skeletal muscle tissues from db/db and control mice.",
"Continued",
"Differentially secreted peptides located in functional domain or region based on Uniprot or Pfam database. peptides (n = 9) were located in the EF-hand domain from sarcomeric proteins (MYL1, MYL3 and MLRS). Generally, all EF-hand proteins display regulatory effect in two ways(85), calcium sensors for translating the signal to various responses and calcium buffers for controlling free Ca2 + ions level in the cytoplasm. On the other side, Ca2 + binding could induce a change of structural dynamics in the EF-hand motif, resulting in the activation or inactivation of target proteins(86,87). Importantly, Ca2 + signal participates a variety of physiological processes in skeletal muscle, especially acting as second messengers for GLUT4 translocation mediated by contraction(88) and insulin treatment(89). Additionally, GAPDH-derived peptide (231-RVPTPNVSVVDLTCRLEKPAK−252) contained [IL]-x-C-x-x-[DE] motif (243-248), which has been reported as",
"Protein precursors of peptides which are both associated with obesity and diabtetes (Score >0). The association score come from open targets platform database.",
"The matching results of the precursor proteins from STRING database.",
"Precursor proteins-to-precursor proteins interaction from STRING database.",
"The information of candidate peptides for functional evaluation."
] | [
"Figure S1",
"Figures 1A,B",
"(Figure 2A)",
"Figure 2B)",
"Figure 2C",
"Figure 3A)",
"Figure 3A",
"Figure 3B)",
"Figure 3B",
"Figure S2",
"Figure 3C",
"Figure 3D",
"Figure 4A)",
"(Figure 4B",
"(Figure 4B)",
"Figures 5A,B,",
"Figures 5E,F",
"Figures 5C,D.",
"Figures 5E,F",
"Figure 5G",
"Figure 5H",
"Figures 1A,B.",
"(Figures 2A,B)",
"(Figures 3A,B)",
"(Figures 3A,B)",
"Figures 5A,B",
"Figures 5C,D.",
"Figures 5E,F",
"Figures 5E,F,H",
"Figure S1"
] | [] | [
"Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis (1). Accordingly, the dysregulation of skeletal muscle metabolism also arise a number of metabolic disorders such as hyperinsulinaemia, excessive hepatic gluconeogenesis (2), abnormal lipid accumulation (3), impaired glucose uptake and metabolic inflexibility (4). Furthermore, disorders in skeletal muscle play a central role in the development of type 2 diabetic mellitus (T2DM), obesity and lead to other related complications (5). Thus, it is of great interest to deeply characterize the pathogenesis of dysregulation on skeletal muscle glucose/lipid homeostasis to whole-body endocrine and metabolic functions.",
"As an important secretory organ, skeletal muscle has drawn attention to be a potential target tissue for treating metabolic disorders (6). Thus, analysis of skeletal muscle secretome opens up a novel route for comprehending the communication of this tissue with other tissues such as adipose tissue (7), bone (8), liver (9) and pancreas (10,11). In light of recently reported experimental evidence, a variety of proteins generated by muscles fibers and released into the circulation are classified as myokines (12), most of which have autocrine, paracrine, and endocrine effects not only in muscle fiber growth (13) but also systemic metabolism (14). The first identified myokine IL-6 presented a vital locally muscular effects such as skeletal muscle growth and glucose/lipid metabolism (15, 16). Furthermore, IL-6 also could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues (17)(18)(19). Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM (20). These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. However, knowledge of the skeletal muscle secretome is scarcely reported under pathophysiology of metabolic diseases such as T2DM and obesity. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established.",
"Peptides in length of 3-50 amino acids residues, which are widely characterized in mouse and human, are termed as a sort of compounds produced or secreted by endocrine gland tissues as well as certain types of cells (21,22). And Abbreviations: T2DM, type 2 diabetic mellitus; GO, gene ontology; KCRM, creatine kinase m-type; Aldo A, fructose-bisphosphate aldolase A; Mr, relative molecular mass; pI, isoelectric point; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FA, formic acid; DDA, data dependent acquisition; FDR, false discovery rate; PSPEP, Proteomics System Performance Evaluation Pipeline; RIPA, Radio Immunoprecipitation Assay; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SD, standard deviation; TPIS, triosephosphate isomerase; ENOB, beta-enolase; PYGM, glycogen phosphorylase, muscle form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; MLRS, myosin regulatory light chain 2, skeletal muscle isoform; MYH6, myosin heavy chain 6; MYH7, myosin heavy chain 7; MYBPH, myosin-binding protein H; MYL3, myosin light chain 3; MYL1; LDH, myosin light chain 1; lactate dehydrogenase. these endogenous peptides have important physiological action, including neuroregulation (23), cell differentiation (24) and energy metabolism (25) and dysregulation of peptide hormone signaling have been implicated in a wide range of diseases (26,27). In view of that, further insight into identification of novel peptides is of major importance. Benefited from the progresses in peptide extraction method and application of modern analytical methods, (U)HPLC, nano-LC and CE, hyphenated with tandem mass spectrometry (MS/MS) technology (28,29), various techniques used for quantitative peptidomics have been applied to address the challenging question of identifying peptides with potential bioactive under the physiological or disease condition (30). More importantly, the peptidomics is widely used to identify biological markers (31), discover new drug (32) and therapeutic targets (33). Recently, quantative peptidomics has also been conducted in endocrine studies (22,34,35), however, the secreted peptidomics from skeletal muscle under the insulin-resistant condition was not fully characterized.",
"Herein, we performed liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to help characterize the secretome from cultured skeletal muscle tissues of db/db mice at peptides level and identify putative bioactive peptides. A global secreted peptides were established and bioinformatics analysis of precursor proteins provided a possible relationship of differential peptides with T2DM or insulin resistance. Additionally, the biological effects of these secreted peptides on C2C12 myotubes elucidated a possible regulatory role in insulin signaling-and mitochondrial-related genes expression. Taken together, these observations will encourage us to investigate function of these secreted peptides from cultured skeletal muscle tissues with other tissues under the diabetic state, thus representing a promising strategy for prevention and treatment of insulin resistance as well as the associated metabolic disorders.",
"All the studies involving mice acquired approval from the Ethical Committee of Nanjing Medical University. All procedure involving mice were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval Number: IACUC-1812053).",
"Twelve-week-old male C57BLKS/J db/db mice (n = 8, db/db group) and age-matched WT controls (n = 8, NC group) were purchased from the Model Animal Research Center of Nanjing Medical University. After adaptive raising for one week, mice were sacrificed by cervical dislocation and skeletal muscle tissues were isolated from the left hind leg (each mice of 100 ∼ 150 mg). Subsequent operations were carried out under a laminar airflow hood to decrease contamination. The visible blood vessels and connective tissue were removed from the tissue. After rinsed with PBS, the skeletal muscle tissues were cut into small pieces (3-4 mm 3 ) with scissors as described by an established protocol (36,37). Tissue cutting will lead to release of damaged cells slowly into the medium. Additionally, a small amount of serum proteins in the tissue pieces will diffuse out during culture period. Therefore, necessary washing procedures during culture were adopted to obtain medium samples (referred to as secretome) containing mainly skeletal muscle tissue-derived secreted components as previously reported (38,39). Tissue fragments were placed in a 10 cm plate (200∼300 mg from two mice as one sample) containing 10 mL serum/phenol red free DMEM/F12 medium (Gibco, Grand Island, CA, USA). After incubation in a humidified incubator at 37 • C under 95% O2 and 5% CO2 for 48 h, the medium was immediately supplemented with protease inhibitors and centrifuged (845 g, 10 min, 4 • C) to wipe off cell debris and dead cell. Supernatant samples from each group (NC or db/db group) were harvested from four individual tissue culture dishes independently (n = 4 per group). Lactate dehydrogenase (LDH) (36,40,41) and IL-6 (42) expression levels were detected to evaluate skeletal muscle vitality and capability during the in vitro culture. Then the supernatant samples obtained were stored at −80 • C until further processing.",
"First, both supernatant samples were concentrated to 1-2 mL by centrifuges for speed vacuum (LaboGene, Allerød, Denmark). Then equal volume of U2 solution containing 8 M urea and 100 mM tetraethyl-ammonium bromide in pH 8.0 was added to the concentrated supernatant for denaturation. Followed by centrifugation for 30 min (13,000 g, 30 • C), the medium supernatant was transferred to a new centrifuge tube. Subsequently, proteins were reduced by 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide successively. The protein concentrations of supernatant from cultured skeletal muscle samples were detected by Bradford method (43) and integrity of these samples were evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) combined with silver staining. Afterwards the peptides were separated from samples by treatment with Amicon R Ultra Centrifugal Filters in 10-kDa (Merck Millipore, Billerica, MA, USA) according to the manufacturer's instruction as previously described (37). The \"peptidome\" present in the filtrates was desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and the desalted peptide solution was vacuum-dried with centrifuges for speed vacuum (SCAN SPEED 40, LaboGene) as previously described (44) and immediately frozen at −80 • C until the following processing.",
"The LC-MS/MS analysis was conducted similarly to the previous protocols (45). The peptide samples were redissolved in 2% (v/v) acetonitrile/0.1% formic acid (FA) (v/v) and 5 µL solution was injected into an A Triple TOFTM 5600 mass spectrometer (AB Sciex, Redwood City, CA, USA) coupled to a ekspert TM nanoLC400 liquid chromatography (AB Sciex) via a nanosource electrospray interface equipped with distal coated SilicaTip emitters (New Objective, Woburn, MA, USA). First, load peptide onto a C18 trap column (5 µm, 100 µm × 20 mm, AB Sciex) and elute at 300 nL/min onto a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Shanghai, China) in the gradient as long as 120 min. These two mobile phases included buffer A containing 2% acetonitrile/ 0.1% FA/ 98% H2O (v/v) and buffer B containing 98% acetonitrile/0.1% FA/2% H2O (v/v). Then peptide mixture was eluted at a flow rate of 0.3 µL/min in a gradient generated with Solvent A containing 98% water and 2% acetonitrile containing 0.1% FA (v/v) and Solvent B containing 2% water and 98% acetonitrile containing 0.1% FA (v/v) according to the previously described reports (45,46). The mass spectrometer was operated in positive mode with a spray voltage of 2500 V, 206.84kPa for the curtain gas, 41.37kPa for the nebulizer gas and 150 • C as temperature. A data dependent acquisition (DDA) method was applied and a full scan MS spectrum (300-1500 m/z) with accumulation time of 0.25 s was adopted. Top 30 precursor ions for fragmentation based on the highest intensity were selected. The collections of MS1 spectra were in the range 350-1500 m/z, and MS2 spectra were in the range of 100-1500 m/z. A total of 47,709 MS/MS were collected from all LC-MS/MS analyses.",
"Protein Pilot Software (https://sciex.com.cn/products/software/ proteinpilot-software, version4.5,AB SCIEX) was adopted to analyze the original MS/MS file data (47). For peptides identification, the Paragon algorithm was employed against the Mus_musculus SwissProt sequence database (a total of 85210 items, updated in January 2019). The following parameters were installed: The parameters were set as follows: 1) cysteine modified with iodoacetamide; 2) biological modifications were selected as ID focus. The strategy of the automatic decoy database search was employed to estimate false discovery rate (FDR) calculation using the Proteomics System Performance Evaluation Pipeline (PSPEP) Software integrated in protein pilot-software. Only unique peptides (global FDR values < 1%) were considered for further analysis. Skyline v4.2 software was employed for MS1 filtering and ion chromatogram extractions for peptides label-free quantification (48,49). And the parameters setting for skyline MS1 filtering were according to previous studies (48). Using the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.",
"The relative molecular mass and isoelectric point of each peptide were calculated by the online tool (http://web.expasy. org/compute.pi/). All the precursors protein of differentially expressed peptides as one group were imported into for GO (http://www.geneontology.org/) (50) and pathway analysis (https://www.kegg.jp/) for predicting potential functions. The online tools UniProt Database (http://www.uniprot.org/) and Pfam (http://pfam.xfam.org/) were adopted to explore if the peptides' sequences were positioned in the conserved structural domains or regions of their precursors. The Open Targets Platform database (www.targetvalidation.org/) was adopted to investigate precursors associated with diabetes and obesity as previously reported (24). For visualization, clustergram and volcano plot graphs in this study were drawn with R language (http://www.r-project.org/). For determination of differentially expressed peptides, fold change was computed as the average values of biological duplication (n = 4).",
"All the peptides used in this study were custom-synthesized and HPLC-purified by Science Peptide Biological Technology Co., Ltd. (Shanghai, China) through the solid-phase method as described reported (51). The purity in 95% for each peptide was confirmed by HPLC-MS method. All the used peptides were stored in lyophilization at−20 • C until dissolved with sterile water immediately for treatment with cells in vitro.",
"C2C12 cells, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China) at 37 • C with 5% CO2. On the fourth day of cell differentiation, the C2C12 cells were pre-treated with synthesized peptides or solvent for 48 h at the same concentration of 50 µM, and then starved for 24 h with serumfree L-DMEM (Gibco). Subsequently, myotubes were incubated in L-DMEM in the presence or absence of 100 nM of insulin for 30 min. At the indicated time, the cells were collected for the following analysis. ",
"Total RNA was isolated using trizol reagent (Life Technologies, Carlsbad, CA, USA). And the cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was determined by real-time quantitative PCR (ABI ViiA7, Applied Biosystems, Foster City, California, USA) using the SYBR Green array. The relative gene expression was analyzed based on the 2 − CT method with normalization of the data to PPIA. The primers used for the real-time quantitative PCR were listed in Table S1.",
"Data were analyzed with GraphPad Prism 7 (San Diego, CA, USA), and the results were shown as the mean ± standard deviation (SD). Peptides with a fold change larger than 1.5 or <0.67 with a Student's t-test p-value <0.05 were selected as differently expressed peptides.",
"Considering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity. LDH and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period. LDH content of culture medium was assessed as an indicator of cell lysis at 0 h∼24 h, 24 h∼48 h and 48 h∼72 h; no significant increased in LDH occurred from 48 h to 72 h (not shown in the manuscript). Secretion of IL-6 remained stable from 24 h to 48 h in culture by ELISA (not shown in the manuscript). Therefore, we chose the 24 h∼48 h culture as an optimal time point. The protein composition and integrity of the secreted samples were evaluated by SDS-PAGE with silver staining in Figure S1. After validation of skeletal muscle culture system and samples assessment, peptides from four different culture dishes per group (control and db/db mice) were individually extracted from supernatants and analyzed via label-free mass spectrometry based quantification. To gain more insights into biological differences between control and db/db mice skeletal muscle, we employed LC-MS/MS technology and identified 3384 peptides, of which 2664 peptides showed valid quantitative values in the detection. A total of 63 peptides were identified differentially secreted, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 peptides were down-regulated (fold change < 0.67, P < 0.05) in medium supernatants from insulin resistant-skeletal muscle tissues. Visualization methods such as hierarchical clustering and volcano plot showed distinct peptides secretion profiles as shown in Figures 1A,B. The differentially secreted peptides were summarized in Table 1.",
"To characterize the general feature of differentially secreted peptides, we further analyzed the relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs. pI. The results indicated that the Mr varied from 1011.1 Da to 2670.9 Da with 70% between 1,300 and 2,100 Da (Figure 2A), and pI varied from 3.7 to 9.7, with 51% between 4 and 7 ( Figure 2B). Additionally, distribution of pI vs. Mr could divide these peptides into three groups around pI4, pI6 and pI10 ( Figure 2C).",
"To make comprehensive functional assessment of these differential peptides, we consider all the precursor proteins as one group and performed GO and Pathways analysis of them. First of all, the precursor proteins of these differential peptides identified from the cultured control and db/db skeletal muscle were classified using Gene Ontology categories, which revealed the majority of proteins were associated with striated muscle contraction and glucose metabolic process ( Figure 3A). The top 20 GO terms were listed in Figure 3A. The precursor proteins annotated involved in these GO terms were presented in Table S2. Subsequent pathway analysis in the KEGG database revealed a significant enrichment in metabolic pathway and glycolysis/gluconeogenesis process ( Figure 3B). The top 14 pathway terms were listed in Figure 3B. The precursor proteins annotated involved in these pathway terms were presented in Table S3. We further discriminated the up-and down-regulated peptides and performed GO and Pathways analysis of these two groups of precursor proteins. This analysis may bring overlapping terms such as phosphorylation, phosphagen metabolic process and phosphorus metabolic process in up-or down-regulated peptides, and a smaller number of Search tool for the retrieval of interacting genes/proteins (STRING) was used to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides. The size of node represents the number of interacting proteins and the color of node represents the bitscore of matching results from STRING database. The thicker the line (edge), the higher the reliability (evaluated by combined score >0.4).",
"terms in Pathway analysis (deriving up-regulated peptides). These analysis were presented in Figure S2. Additionally, the major intracellular locations of the precursor proteins were considered from literature sources, illustrating that 64% proteins were cytoplasmic and 15% proteins were cytoskeletal seen in Figure 3C. Based on above analysis, we found that a vast of precursor proteins were assigned to glucose metabolic process include ALDO A, triosephosphate isomerase (TPIS), beta-enolase (ENOB), glycogen phosphorylase, muscle form (PYGM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1(PGK1), specifically assigned to glycolysis/gluconeogenesis process. Another kind of proteins enriched in regulation of striated muscle contraction terms (GO) and regulation of actin cytoskeleton (Pathway) were myosin regulatory light chain 2, skeletal muscle isoform(MLRS), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin-binding protein H (MYBPH) and myosin light chain 3 (MYL3),both belonging to sarcomeric proteins. Subsequently, we used search tool for the retrieval of interacting genes/proteins (STRING) to construct a graphical network for describing the interaction between these precursor proteins corresponding to the identified peptides in Figure 3D. Based on protein-protein interaction and co-occurrence in KEGG pathways and literature mining, this network was constructed from 25 proteins that matched to the STRING database and the matching results were listed in Table S4. As shown from this network, 22 nodes representing precursor protein constituted the interaction diagram, which could form 93 reliable a-to-b interaction relationship by combined score (more than 0.4) as shown in Table S5. ",
"It is widely postulated that most peptides can be attributed mainly to the proteolytic enzymes as well as their type and level (cleavage specificity and activity), which can differ during disease (52)(53)(54). Briefly, the N-terminal pre-cleavage site, N terminus, C terminus, and C-terminal post-cleavage site of the identified peptides were commonly used to investigate the nature of proteolytic enzymes within serum (55) or tissues (56). Thus, we analyzed the distribution of peptide cleavage site and found that Lysine (K) was the most frequent cleavage site of N-terminal amino acid of identified peptides and Alanine (A) was the most frequent cleavage site of C-terminal amino acid of identified peptides. Lysine (K) was the most frequent cleavage site of Nterminal amino acid of preceding peptides, while Leucine (L) was the commonest C-terminal amino acid of preceding peptides ( Figure 4A). These data indicate that the pattern of cleavage sites may represent the specificity of cleavage and activity of proteolytic enzymes under the diabetic condition. Additionally, we also tried to align peptide sequences on the same precursor sequence to construct a \"peptide alignment map\". Searched from our samples, the largest number of identified peptides (n = 1 7) (Figure 4B) originated from KCRM and the second largest number of identified peptides (n = 6) came from ALDO A (Figure 4B), elucidating that these peptides were easily cleaved by certain kind of endogenous enzymes.",
"To check for specific domain structures or patterns in the identified peptides comparison with its precursor proteins, we retrieved domain information from the UniProt and Pfam database. In order to validate that if the peptides exerted important roles in the related metabolic diseases, we adopted the Open Targets Platform database to investigate protein precursors. In our searching results, most peptides (n = 44) from the identified pepetides (n = 63) were located in the functional domains ( Table 2). Additionally, 27 out of 44 peptides located in the functional domain were predicted closely related with both obesity and diabetes ( Table 3). These observations will encourage us to further investigate properties of the putative secreted peptides in skeletal muscle under the diabetic state.",
"To explicit the putative function of differentially secreted peptides, 7 differentially secreted peptides, which had already been annotated derived from authentic protein from Uniprot database, located in the functional motifs and showed relatively high abundance from MS detection, were chosen for further analysis. Table S6. Sequentially, we termed these candidated peptides P1-P7 in order. To use C2C12 myotubes as cell models in vitro, the results of changes in inuslin signaling revealed that up-regulated peptides P2-P4 addition could both significantly decrease the phosphorylation of Irs1 (Ser-307) and Akt (Ser-473) under insulin stimulation (100 nM, 30 min) as shown in Figures 5A,B, in spite of bringing a modestly up-regulation of p-Irs1 (Ser-307) at the basal status. Moreover, mitochondrial-related marker protein Pgc1α was significantly attenuated in the insulin-stimulated C2C12 cells by up-regulated peptides P1-P4 as shown in Figures 5E,F. Among these downregulated peptides, only peptide 7 presented a promotion on p-Akt (Ser-473) expression level both under the basal and insulin stimulation status as shown in Figures 5C,D. And these up-regulated peptide 1-4 could decrease Pgc1α protein level and down-regulated peptide 6 and 7 administration enhanced Pgc1α protein level by the stimulation of insulin presented in Figures 5E,F. We further evaluated Gut 4 (a key gene for glucose transport) and Pgc1α mRNA levels by peptides treatment upon insulin stimulation. The results also indicated that Peptide 3 and 4 modestly decreased Pgc1αexpression at mRNA levels seen in Figure 5G, while Peptide 6 and 7 significantly up-regulated both Glut4 and Pgc1α expression at mRNA levels seen in Figure 5H. These observations revealed that these candidated peptides (P1-P7) may affect the expressions of insulin signaling or mitochondrial-related genes in skeletal muscle cells.",
"Emerging as a widely known secrete organ, skeletal muscle tissues have been extensively discussed using a wide range of comparative and quantitative proteomic methods (57). Proteomic scannings of the medium supernatant from skeletal muscle cells have already afford approaches to identify a large number of secreted proteins (58)(59)(60)(61)(62)(63). Particularly, one such research reported the alteration of insulin effect on the secretome profile of skeletal muscle cells, revealing the changes of protein levels secreted from skeletal muscle during activation of insulin signaling pathway (58). A recent study also generated a comprehensive secretome analysis of skeletal muscle cells under palmitic acid-induced insulin resistance (61). In addition to these proteins, many other muscle-secreted compounds have been also come to light, including exosome (64,65), metabolites (66), and miRNAs (67). These studies have greatly expanded our knowledge of skeletal muscle secretome and might afford possibilities for exploring novel molecular targets in the maintenance of skeletal muscle physiology and even whole-body metabolism. However, to date few studies has focused on the peptides present in either skeletal muscle tissues or cells. Therefore, we attempted to comprehensively profile peptides that may play roles in regulating insulin sensitivity and offer enormous promise for exploring molecular mechanisms underlying insulin resistance.",
"In present study, a total of 63 peptides were differentially secreted in medium supernatants from cultured skeletal muscle tissues of db/db mice, of which 42 peptides were up-regulated (fold change > 1.5, P < 0.05) and 21 were down-regulated (fold change < 0.67, P < 0.05) shown in Figures 1A,B. The differences of dysregulation peptides may indeed reflect the changes between control and insulin-resistant mice in some extent. However, db/db mouse is a well-established leptin receptordeficient animal model (68,69). Despite no studies revealed the association between leptin receptor deficiency and peptide dysregulation/protein degradation so far, the possible effect by this mutation deserves to be taken into account and other nongenetic mice models could be adopted in the future studies. Based on our previous study (70), 10-kDa filters method was used to extract peptides in order to intercept proteins secreted by skeletal muscle to the conditional media. Characterization of the basic feature (Mr and pI) of these identified peptides (Figures 2A,B) only reflected the differences of distribution in the diabetic skeletal muscle tissues, but also proved the reliability of the used peptide extraction method.",
"Subsequently, GO and Pathway analysis revealed that the precursors protein of these peptides were mostly involved in muscle contraction and metabolism processes (Figures 3A,B). An interesting finding from our study was that most of the differentially identified peptides derived from cytosolic, cytoskeletal and mitochondrial proteins (Figures 3A,B), which differed from the secretory pathway peptides (71). This (34). Similarly, another peptidomic anaylsis of brain slices cultures and media also pointed that vast majority of secreted peptides arose from intracellular proteins (72). There does also exist some evidence that these identified N-or C-terminus protein yielding peptides, rather than internal fragments, raised the possibility that they are produced by selective processing rather than protein degradation (73). Taken together with previous researches, the current results show us meaningful hints that these intracellular peptides may be secreted via nonclassical mechanisms. Actually, another important origin of peptides is proteolytic degradation processes in body fluid under the physiological or pathologically processes. Most regulatory peptides were efficiently degraded by plasma enzymes once secreted into the bloodstream, which exerted anti-diabetic therapeutic function (74,75) or were identified as disease markers (76). Recently, Federico Aletti et al. (77) used protease activity detection and specific enzymes analysis to explain a large presence of circulating peptides under hemorrhagic shock, which gave us a possible way to evaluate peptides origin. Thus, a fundamental validation is whether the proteins-derived peptides are actually secreted from skeletal muscle cells per se or proteolytic degradation and are of biologically active. Among the differentially secreted peptides from cultured skeletal muscle tissues of db/db mice, we found that three peptides derived from GAPDH, four peptides derived from ALDO A and one peptides derived from PYGM were upregulated in the conditional medium from db/db groups as shown in Table 1. As described by Dustin S. Hittel et.al, a global proteomic survey of skeletal muscle revealed a statistically significant up-regulation in glycolytic enzymes GAPDH and ALDO A protein levels in obese/overweight patients (78). Another quantitative protein profile also identified a more abundant levels of GAPDH and PYGM in skeletal muscle from T2DM groups compared with the control groups (79). In addition to pointing the importance of the mitochondrial numbers and impairments under the insulin-resistant states (80)(81)(82), several studies noted that glycolytic capacity is higher in skeletal muscle of patients with T2DM or obesity (83). Notably, stronger changes of peptides derived from sarcomeric proteins such as myosin light chain 1 (MYL1) and MYL3 were also observed in the conditional medium from diabetic muscles as shown in Table 1. MYL1 and MYL3 are representive markers of fast-muscle and slow-muscle respectively, which were both regulated by insulin stimulation (79). And previous studies observed that property of T2DM individuals muscle was shifted to a fast-twitch glycolytic phenotype (84). In fact, deficiency in sarcomeric proteins in skeletal muscle also suggested their importance in skeletal muscle physiologic and pathological processes. On the whole, our results of increased glycolytic enzyme-or sarcomeric proteins-derived peptides secreted from insulin resistant skeletal muscle may support the hypothesis that altered glycolytic capacities or fiber types under the diabetic status contribute to this difference.",
"Till now, the putative function of these peptides are not clear. Therefore, we evaluated whether these peptides originated from functional domains of the corresponding precursor protein using the UniProt and Pfam database. In our searching results, most peptides (n = 44) from the identified peptides (n = 63) were located in the functional domains as listed in Table 2. Specifically, most peptides derived from functional enzymes were located in the enzymatic activity region, including TPI- TCTP-derived peptide (119-AEQIKHILANF-129) and CAH3-derived peptide . We also found another kind of S-Nitrosylation modifying sites for affecting GAPDH enzymatic activity (90). Therefore, future efforts need to be established for investigating the potential role of peptides on insulin sensitive cells in vitro or whole-body metabolism in vivo.",
"The above analyses provided a possibility to evaluate the biological effects of these differentially secreted peptides. As widely reported, insulin resistance in skeletal muscle is tightly connected with the deficit in insulin signaling (91). Consequently, the role of phosphorylation of Irs1 and Akt in signaling pathways is very crucial in anti-hyperglycemia and insulin sensitivity (92). In this result, we found these up-regulated peptides 1-4 both exerted a significantly attenuated insulin action in C2C12 cells, evaluated by a decreased level of p-Irs1 (Ser-307) and p-Akt (Ser-473) seen in Figures 5A,B, whereas only one down-regulated peptide P7 could remarkably promote insulin signaling only via Irs1 signaling pathway seen in Figures 5C,D. Peroxisome proliferator-activated receptor γ coactiva-tor-1(Pgc-1), which displays a dominant role through tight modulation of mitochondrial biogenesis and respiration, has also been demonstrated to participate in skeletal muscle insulin signaling and metabolic homeostasis (93). The up-regulated peptides 1-4 also brought out a decreased protein level of Pgc-1α in C2C12 cells as shown in Figures 5E,F, and down-regulated peptide 6 and 7 administration also gave rise to the increased Pgc1α mRNA and protein level in Figures 5E,F,H. Taken together, these selected peptides secreted from db/db mice skeletal muscle presented a promotive or inhibitory effect on insulin and mitochondrialrelated pathways in skeletal muscle cells by an autocrine manner. Notably, peptide 4 (231-RVPTPNVSVVDLTCRLEKPAK−252) derived from GAPDH displayed a most significant inhibitory effect toward these candidate peptides. However, the relationship between GAPDH-derived peptide and its precursor protein is to be determined. On the other hand, more methods need to be employed for the wider cell signaling screen and further research is required to explore the biologic function of skeletal muscle-secreted peptides on adipocytes or liver cells.",
"To our knowledge, no large-scale quantitative peptidomic analysis has been performed on skeletal muscle to elucidate secreted peptides profiles under the diabetic status. The present study identified and quantified changes with a label-free discovery using LC-MS/MS technology to construct a global secreted peptides picture. Further bioinformatics analysis of precursors comprehensively provided an atlas of peptides that may exist roles in regulating insulin sensitivity. This represented a new perspective toward exploring insulin resistance pathogenesis. Additionally, the detailed biological effects of these secreted peptides on skeletal muscle insulin resistance or cross-talk with other tissues remained to be elucidated in the future study.",
"The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.",
"The protocol has been approved by the Institutional Animal Care and Use Committee of Nanjing medical university (Approval Number: IACUC-1812053).",
"YWu and MH performed experiments and interpreted results of experiments. YWa and YG prepared the figures. XC and PX analyzed the data. CJ and TZ participated discussion. YZ helped write the manuscript with providing assistance. LY conceived and designed experiments, provided funding to regents, and approved final version of manuscript. All authors read and approved the final manuscript. ",
"The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2019.00741/full#supplementary-material Figure S1 | SDS-PAGE associated with silver staining of the medium supernatants from cultured skeletal muscle tissues (Con vs. db groups). Table S1 | Primers used in this study. Table S2 | GO terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. Table S3 | Pathway terms corresponded to the precursor proteins relative to these differentially secreted peptides in skeletal muscle from db/db mice. "
] | [] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Ethics Statement",
"Animal Experiments and Sample Preparation",
"Peptide Extraction and Desalting",
"Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)",
"Peptide Identification and Quantitative Analysis",
"Bioinformatics and Annotations",
"Synthetic Peptides",
"Cell Culture and Peptide Treatment",
"Western Blot Analysis and Antibodies",
"Reverse Transcription and Real-Time Quantitative PCR",
"Statistical Analysis",
"RESULTS",
"Identification of Secreted Peptides From Cultured db/db Skeletal Muscle Tissues",
"Charactrization of the Basic Feature of Differentially Secreted Peptides",
"Comprehensive Functional Assessment and Intercellular Location of Precursor Proteins of These Differential Peptides",
"Cleavage Pattern of Differentially Secreted Peptides",
"Putative Bioactive Peptides Associated With Diabetes and Obesity",
"The Effect of Candidated Peptides (P1-P7) on Insulin and Mitochondrial-Related Pathways in Skeletal Muscle Cells",
"DISCUSSION",
"DATA AVAILABILITY STATEMENT",
"ETHICS STATEMENT",
"AUTHOR CONTRIBUTIONS",
"SUPPLEMENTARY MATERIAL",
"FIGURE 1 |",
"FIGURE 2 |",
"FIGURE 3 |",
"FIGURE 4 |",
"FIGURE 5 |",
"FUNDING",
"Figure S2 |",
"TABLE 1 |",
"TABLE 1 |",
"TABLE 2 |",
"TABLE 3 |",
"Table S4 |",
"Table S5 |",
"Table S6 |"
] | [
"Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nUp-regulated peptides \n\nAVGAVFDISNADRLGSSEVEQ \nP07310 \nKCRM \n2163 \n9.728 \n0.005 \n\nTNGAFTGEISPGMIKDLGATWV \nP17751 \nTPIS \n2264 \n8.019 \n0.035 \n\nIADLVVGLCTGQIK \nP21550 \nENOB \n1486 \n6.77 \n0.000 \n\nNYKPQEEYPDLSKHNNHMA \nP07310 \nKCRM \n2315 \n4.843 \n0.024 \n\nEEIEEDAGLGNGGLGRLAAC \nQ9WUB3 \nPYGM \n2030 \n4.411 \n0.004 \n\nGQCGDVLRALGQNPTQAEV \nP09542 \nMYL3 \n2013 \n4.077 \n0.033 \n\nAGPLCLQEVDEPPQHAL \nP70296 \nPEBP1 \n1873 \n4.038 \n0.004 \n\nEAFTVIDQNRDGIID \nP97457 \nMLRS \n1705 \n3.947 \n0.026 \n\nDLFDPIIQDRHGGY \nP07310 \nKCRM \n1645 \n3.668 \n0.022 \n\nKDLFDPIIQD \nP07310 \nKCRM \n1203 \n3.282 \n0.000 \n\nLDDVIQTGVDNPGHP \nP07310 \nKCRM \n1576 \n3.265 \n0.001 \n\nSEIIDVSSKAEEVK \nA2ASS6 \nTITIN \n1533 \n3.134 \n0.002 \n\nVIACIGEKLDE \nP17751 \nTPIS \n1246 \n2.976 \n0.003 \n\nGTNPTNAEVKKVLGNPSNEEM \nQ545G5 \nMYL1 \n2180 \n2.838 \n0.006 \n\nADRLGSSEVEQVQLV \nP07310 \nKCRM \n1629 \n2.804 \n0.001 \n\nIGQGTPIPDLPEVKRV \nA2AQA9 \nNEB \n1719 \n2.739 \n0.002 \n\nSTTAPPIQSPLPVIPHQK \nE9PYJ9 \nLDB3 \n1910 \n2.685 \n0.023 \n\nKADDGRPFPQVI \nP05064 \nALDOA \n1342 \n2.644 \n0.021 \n\nKSMTEQEQQQLIDD \nP07310 \nKCRM \n1692 \n2.442 \n0.001 \n\nGGGASLELLEGKVLPGVDA \nP09411 \nPGK1 \n1781 \n2.264 \n0.028 \n\nLEGTLLKPNMVTPGHA \nP05064 \nALDOA \n1677 \n2.208 \n0.010 \n\nVMVGMGQKDSYVGDEAQSK \nP68134 \nACTS \n2028 \n2.168 \n0.021 \n\nDLEEATLQHEATAAALR \nQ02566 \nMYH6 \n1838 \n2.07 \n0.038 \n\nAELQEVQITEEKPLLPG \nQ9QYG0 \nNDRG2 \n1935 \n2.043 \n0.048 \n\nSEGKCAELEEELKTVTNNL \nP58771 \nTPM1 \n2163 \n2.038 \n0.049 \n\nRSIKGYTLPPHC \nP07310 \nKCRM \n1428 \n1.967 \n0.001 \n\nNSNSHSSTFDAGAGIALNDNFVK \nP16858 \nG3P \n2365 \n1.935 \n0.038 \n\nTGKATSEASVSTPEETAPEPAKVPT \nP70402 \nMYBPH \n2484 \n1.919 \n0.003 \n\nDNEYGYSNRVVDL \nP16858 \nG3P \n1543 \n1.898 \n0.004 \n\nEELDAMMKEASGPINF \nP97457 \nMLRS \n1781 \n1.853 \n0.012 \n\nSNNKDQGGYEDFVEGLRV \nQ545G5 \nMYL1 \n2026 \n1.816 \n0.016 \n\nAEQIKHILANF \nP63028 \nTCTP \n1283 \n1.787 \n0.044 \n\nGADPEDVITGAFK \nP97457 \nMLRS \n1319 \n1.782 \n0.031 \n\nTSIEDAPITVQSKINQ \nA2AQA9 \nNEB \n1743 \n1.739 \n0.028 \n\nLKGGDDLDPNYVLS \nP07310 \nKCRM \n1505 \n1.732 \n0.040 \n\nIQTGVDNPGHPF \nQ6P8J7 \nKCRS \n1281 \n1.600 \n0.045 \n\nAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n1957 \n1.595 \n0.009 \n\nGQEQWEEGDLYDKEKQQKPF \nE9Q8K5 \nTITIN \n2481 \n1.586 \n0.036 \n\nAGTNGETTTQGLDGLSERCA \nP05064 \nALDOA \n2037 \n1.582 \n0.033 \n\nRVPTPNVSVVDLTCRLEKPAK \nP16858 \nG3P \n2378 \n1.531 \n0.022 \n\nKVPEKPEVVEKVEPAPLK \nF7CR78 \nF7CR78 \n2015 \n1.527 \n0.026 \n\nGETTTQGLDGLSERCAQ \nP05064 \nALDOA \n1822 \n1.526 \n0.042 \n\nDown-regulated peptides \n\nKIEFTPEQIEEF \nP09542 \nMYL3 \n1509 \n0.612 \n0.021 \n\nDLSAIKIEFSKEQQEDF \nP05977 \nMYL1 \n2026 \n0.600 \n0.019 \n\nHELYPIAKGDNQSPI \nP16015 \nCAH3 \n1681 \n0.559 \n0.038 \n\nNSLTGEFKGKY \nP07310 \nKCRM \n1243 \n0.545 \n0.027 \n\nDEPKPYPYPNLDDF \nQ3V1D3 \nAMPD1 \n1709 \n0.543 \n0.034 \n\nASHHPADFTPAVHA \nQ91VB8 \nHBA-A1 \n1457 \n0.537 \n0.016 \n\n(Continued) \n\nFrontiers in Endocrinology | www.frontiersin.org ",
"Peptide sequence \nProtein ID \nProtein name \nMr(KDa) \nFold change \nP-value \n\nSQVGDVLRALGTNPTNAE \nQ545G5 \nMYL1 \n1841 \n0.516 \n0.047 \n\nADEIAKAQVAQPGGDTI \nP70349 \nHINT1 \n1725 \n0.515 \n0.038 \n\nDKETPSGFTLDDV \nP07310 \nKCRM \n1423 \n0.478 \n0.037 \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nQ545G5 \nMYL1 \n2654 \n0.467 \n0.038 \n\nPHPYPALTPEQKK \nP05064 \nALDOA \n1505 \n0.407 \n0.028 \n\nQLVVDGVKLM \nP07310 \nKCRM \n1101 \n0.400 \n0.001 \n\nKDLFDPIIQDRHG \nP07310 \nKCRM \n1553 \n0.395 \n0.022 \n\nKGVVPLAGTNGETTTQGLDGLSER \nP05064 \nALDOA \n2399 \n0.367 \n0.015 \n\nRVFDKEGNGTVMGAELR \nQ545G5 \nMYL1 \n1878 \n0.341 \n0.043 \n\nNADEVGGEALGRL \nA8DUK4 \nHBB-BS \n1300 \n0.339 \n0.037 \n\nNEHLGYVLTCPS \nP07310 \nKCRM \n1389 \n0.328 \n0.031 \n\nASHTEEEVSVSVPEVQKKTVTEEK \nE9Q8K5 \nTITIN \n2669 \n0.253 \n0.023 \n\nNEEDHLRV \nP07310 \nKCRM \n1010 \n0.212 \n0.027 \n\nVLTCPSNLGTGLRG \nP07310 \nKCRM \n1444 \n0.201 \n0.018 \n\nGGYKPTDKHKTDL \nP07310 \nKCRM \n1459 \n0.127 \n0.006 \n\nobservation was also demonstrated by other peptidomic studies. \nFrom Steven W. Taylor group' results, several identified peptides \nin the human islet cultures were derived from intracellular and \ncytoskeletal proteins such as microtubule-associated protein 4 \nand ubiquitin, which may result from a greater level of cellular \nstress ",
"Peptide sequence \nProtein \nLocation \nDomain \nDescription \n\nUp-regualted peptides \n\nAVGAVFDISNADRLGSSEVEQ \nKCRM \n329-349 \n125-367 \nPhosphagen kinase C-terminal \n\nTNGAFTGEISPGMIKDLGATWV \nTPIS \n121-142 \n57-295 \nTIM \n\nIADLVVGLCTGQIK \nENOB \n381-394 \n142-432 \nEnolase C \n\nNYKPQEEYPDLSKHNNHMA \nKCRM \n13-31 \n11-98 \nPhosphagen kinase N-terminal \n\nEEIEEDAGLGNGGLGRLAAC \nPYGM \n124-143 \n113-828 \nPhosphorylase \n\nGQCGDVLRALGQNPTQAEV \nMYL3 \n83-101 \n58-95 \nEF-hand 1 \n\nEAFTVIDQNRDGIID \nMLRS \n32-46 \n25-60 \nEF-hand 1 \n\nDLFDPIIQDRHGGY \nKCRM \n87-100 \n11-98 \nPhosphagen kinase N-terminal \n\nKDLFDPIIQD \nKCRM \n86-95 \n11-98 \nPhosphagen kinase N-terminal \n\nLDDVIQTGVDNPGHP \nKCRM \n53-67 \n11-98 \nPhosphagen kinase N-terminal \n\nVIACIGEKLDE \nTPIS \n174-184 \n57-295 \nTIM \n\nGTNPTNAEVKKVLGNPSNEEM \nMYL1 \n39-59 \n6-41 \nEF-hand \n\nADRLGSSEVEQVQLV \nKCRM \n339-353 \n125-367 \nPhosphagen kinase C-terminal \n\nKADDGRPFPQVI \nALDOA \n87-98 \n15-364 \nGlycolytic \n\nKSMTEQEQQQLIDD \nKCRM \n177-190 \n125-367 \nPhosphagen kinase C-terminal \n\nGGGASLELLEGKVLPGVDA \nPGK1 \n395-413 \n9-406 \nPGK \n\nLEGTLLKPNMVTPGHA \nALDOA \n224-239 \n15-364 \nGlycolytic \n\nVMVGMGQKDSYVGDEAQSK \nACTS \n45-63 \n4-377 \nActin \n\nDLEEATLQHEATAAALR \nMYH6 \n1,179-1,195 \n845-1,926 \nMyosin_tail_1 \n\nSEGKCAELEEELKTVTNNL \nTPM1 \n186-204 \n1-284 \nCoiled coili \n\nRSIKGYTLPPHC \nKCRM \n135-146 \n125-367 \nPhosphagen kinase C-terminal \n\nSNNKDQGGYEDFVEGLRV \nMYL1 \n77-94 \n83-118 \nEF-hand \n\nAEQIKHILANF \nTCTP \n119-129 \n1-172 \nTCTP \n\nGADPEDVITGAFK \nMLRS \n93-105 \n95-130 \nEF-hand 2 \n\nLKGGDDLDPNYVLS \nKCRM \n115-128 \n125-367 \nPhosphagen kinase C-terminal \n\nGQEQWEEGDLYDKEKQQKPF \nTITIN \n1,691-1,710 \n1,709-1,799 \nIg-like \n\nAGTNGETTTQGLDGLSERCA \nALDOA \n117-136 \n15-364 \nGlycolytic \n\nRVPTPNVSVVDLTCRLEKPAK \nGAPDH \n232-252 \n243-248 \n[IL]-x-C-x-x-[DE] motif \n\nGETTTQGLDGLSERCAQ \nALDOA \n121-137 \n15-364 \nGlycolytic \n\nDown-regualted peptides \n\nKIEFTPEQIEEF \nMYL3 \n52-63 \n58-95 \nEF-hand 1 \n\nDLSAIKIEFSKEQQEDF \nMYL1 \n33-49 \n44-79 \nEF-hand 1 \n\nHELYPIAKGDNQSPI \nCAH3 \n17-31 \n3-259 \nAlpha-carbonic anhydrase \n\nNSLTGEFKGKY \nKCRM \n163-173 \n125-367 \nPhosphagen kinase C-terminal \n\nASHHPADFTPAVHA \nHBA-A1 \n111-124 \n3-142 \nGLOBIN \n\nDKETPSGFTLDDV \nKCRM \n44-56 \n11-98 \nPhosphagen kinase N-terminal \n\nLGTNPTNAEVKKVLGNPSNEEMNAK \nMYL1 \n76-100 \n83-118 \nEF-hand \n\nQLVVDGVKLM \nKCRM \n351-360 \n125-367 \nPhosphagen kinase C-terminal \n\nKDLFDPIIQDRHG \nKCRM \n86-98 \n11-98 \nPhosphagen kinase N-terminal \n\nKGVVPLAGTNGETTTQGLDGLSER \nALDOA \n111-134 \n15-364 \nGlycolytic \n\nRVFDKEGNGTVMGAELR \nMYL1 \n147-163 \n133-150 \nEF-hand \n\nNADEVGGEALGRL \nHBB-BS \n20-32 \n2-147 \nGLOBIN \n\nNEHLGYVLTCPS \nKCRM \n274-285 \n125-367 \nPhosphagen kinase C-terminal \n\nNEEDHLRV \nKCRM \n230-237 \n125-367 \nPhosphagen kinase C-terminal \n\nVLTCPSNLGTGLRG \nKCRM \n280-293 \n125-367 \nPhosphagen kinase C-terminal \n\n",
"Protein name \nDescription \nPeptide number \nAssociation score \nwith obesity # \n\nAssociation score with \ndiabetes mellitus # \n\nTPI1 \nTriosephosphate isomerase \n2 \n0.012 \n0.012 \n\nKCRM \nCreatine kinase M-type \n17 \n0.022 \n0.017 \n\nTTN \nTitin \n1 \n0.026 \n0.063 \n\nGAPDH \nGlyceraldehyde-3-phosphate dehydrogenase \n1 \n0.071 \n0.117 \n\nHBA-A1 \nAlpha globin 1 \n1 \n0.014 \n0.038 \n\nENOB \nBeta-enolase \n1 \n0.040 \n0.048 \n\nCAH3 \nCarbonic anhydrase 3 \n1 \n0.074 \n0.094 \n\nTCTP \nTranslationally-controlled tumor protein \n1 \n0.028 \n0.040 \n\nHBB-BS \nBeta-globin \n1 \n0.008 \n0.020 \n\nACTS \nActin, alpha skeletal muscle \n1 \n0.006 \n0.188 \n\n# "
] | [
"Table S1",
"Table 1",
"Table S2",
"Table S3",
"Table S4",
"Table S5",
"Table 2)",
"Table 3)",
"Table S6",
"Table 1",
"Table 1",
"Table 2",
"Table S2",
"Table S3"
] | [
"A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice",
"A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice"
] | [
"Frontiers in Endocrinology | www.frontiersin.org"
] |
13,635,771 | 2022-03-19T02:41:50Z | CCBY | https://www.mdpi.com/1422-0067/13/7/7902/pdf | GOLD | 25cf22b87121018681c2d8cd096ca798727de852 | null | null | null | null | 10.3390/ijms13077902 | 2085991986 | 22942680 | 3430211 |
A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways
2012
Xu Hou
Department of Orthodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
Yuqin Shen
Department of Periodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
Chao Zhang
Department of Orthodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
Liru Zhang [email protected].
Yanyan Qin
Department of Orthodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
Department of Periodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
Yongli Yu [email protected]
Department of Immunology, Medical College of Norman Bethune
Jilin University
130021ChangchunChina
Liying Wang
Department of Molecular Biology, Medical College of Norman Bethune
Jilin University
130021ChangchunChina
Xinhua Sun
Department of Orthodontics
School of Stomatology
Jilin University
1500 Qinghua Road130021ChangchunChina
A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways
Int. J. Mol. Sci
13201210.3390/ijms13077902Received: 10 May 2012; in revised form: 11 May 2012 / Accepted: 15 June 2012 /International Journal of Molecular Sciences * Author to whom correspondence should be addressed; E-Mail: [email protected];oligodeoxynucleotideosteoblastdifferentiationERK1/2 MAPKp38 MAPK
A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.OPEN ACCESSInt. J. Mol. Sci. 2012, 13 7903
Introduction
Osteoblasts play an extremely important role in bone formation and remodeling processes. Osteoblasts are derived from mesenchymal stem cells, and can differentiate into mature and functional osteoblasts that produce extracellular matrix proteins and regulators of matrix mineralization [1]. Runx2 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Runx2 −/− mice show a complete lack of both intramembranous and endochondral ossification owing to maturation arrest of osteoblasts [2,3]. However, Runx2 can regulate the expression of osteoblast-specific genes including ALP, type I collagen and osteocalcin. To date, numerous reports have shown that signaling pathways and transcription factors could participate in osteoblastogenesis by regulating the production or activity of Runx2 [4]. Furthermore, Runx2 is necessary throughout life to promote the differentiation of new osteoblasts during bone remodeling [5].
Oligodeoxynucleotides (ODNs) containing unmethylated nucleotide motifs are immunostimulatory in vertebrates, and some ODNs containing CpG motifs are used for treating cancer, virus-associated diseases, and infections [6][7][8][9]. Recently, specific ODNs were found to have an effect on modulating osteoclast-and osteoblast-lineage cells [10]. In addition, CpG-ODN with a phosphorothioate backbone inhibits the BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines [11]. Chang et al. demonstrated that a novel CpG-ODN enhances the inhibitory effects on osteoclast differentiation by downregulation of TREM-2 [12]. In our previous studies, we have shown that ODN MT01, a synthetic 27-mer single stranded ODN with a design based on human mitochondrial DNA, promotes the proliferation and activation of osteoblasts [13], and stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts [14]. However, the detailed signaling pathway in which ODN MT01 modulates the differentiation of osteoblasts has not been fully elucidated.
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific protein kinases, including p38, JNK and ERK1/2 (p44/p42). These molecules play important roles in cell differentiation, proliferation and death [15], and are activated by phosphorylation of tyrosine/threonine residues in signal transduction cascades. Previous studies have shown that the p38 MAPK and ERK1/2 pathways are involved in the proliferation and differentiation of osteoblasts [16][17][18]. Therefore, we hypothesized that ODN MT01 might regulate the differentiation of osteoblastic cells via the MAPKs pathway.
This study aimed to investigate the regulation of MAPKs in response to ODN MT01, and to elucidate the involvement of the MAPK signaling pathway in regulation of Runx2, osteocalcin, type I collagen and ALP expression in ODN MT01-treated MG 63 cells.
Results and Discussion
Internalization Analysis of ODNs
The design of ODN MT01, a 27-mer ODN, was based on the motif sequence 5'-ACCCCCTCTACCCCCTCT-3' in human mitochondrial DNA. MT01 is a cytosine-rich ODN and contains five successive cytosines in each of its 9-mer motifs (5'-ACCCCCTCT-3') [19]. To investigate whether the effects of an ODN required internalization into cells, Cy5-labeled ODN MT01 was synthesized to trace the sites of ODN actions. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, and is temperature-and energy-dependent [20]. This result was consistent with our observations, as shown in Figure 1, ODN MT01 entered cells by endocytosis after incubation with MG 63 cells for 3 h. The fluorescence intensity was enhanced over time, and the intensity at 6 h was significantly higher than that at 3 h, and the intensity at 12 h was higher than that at 3 and 6 h. It was apparent that ODN MT01 stimulation of osteoblasts required internalization.
Effects of ODN MT01 on MAPK Signaling Proteins ERK1/2 and p38
To investigate the effect of ODN MT01 on MAPKs, both total and phosphorylated levels of MAPKs were investigated by Western blotting after 0, 30, 60 min, 24 h and 48 h of treatment with 1 µg/mL ODN MT01. To better demonstrate that the effects of MT01 in MG 63 cells were in response to the specific sequence of MT01 we tested another ODN sequence, ODN FC003, a 24-mer ODN(5'-TCTCTCTCTCTCTCTCTCTCTCTC-3'). The design of ODN FC003 was also based on human mitochondrial DNA, and contained successive thymidine-cytosine nucleotides. In our previous studies [13,14], we found that ODN FC003 is inactive in the regulation of osteoblasts and bone marrow mesenchymal stem cells. Therefore, we chose this sequence as a negative control. As shown in Figure 2A, phosphorylation of ERK1/2 was observed after 30 min of treatment with ODN MT01, which increased by 13.1-fold after 60 min compared with that at 0 min. Furthermore, the active effect lasted up to 48 h (8.9-fold increase), while the total level of ERK1/2 protein remained unchanged. For p38, there was no obvious change in the phosphorylation level before 30 min. However, p38 phosphorylation increased after 60 min of treatment with MT01 ( Figure 2B), and showed an increase of 4.7 fold at 24 h, which lasted up to 48 h. As shown in Figure 2 A and B, the FC003 sequence had no active effect on MG 63 cells via ERK1/2 or p38 MAPKs. Thus, these findings indicated that ODN MT01 induced the phosphorylation of ERK1/2 and p38 MAPK in MG 63 cells.
To investigate the mechanisms by which ODNs regulate the differentiation of osteoblasts, it is important to identify the functional role of signaling pathways activated by ODNs. At present, several signaling pathways have been shown to be involved in osteogenesis, such as MAPKs, BMP-2-Smad and NF-κB [21][22][23]. Among them, p38 and ERK1/2 MAPKs have been reported to be important for early osteoblast differentiation in various cell lines [24]. In this study, we found that MT01 was capable of activating ERK1/2 and p38 MAPK pathways indicating that MT01 induced the phosphorylation of MAPKs at the early stage of osteoblast differentiation. Previous reports have shown that ERK and p38 are phosphorylated in response to CpG-ODN, and p38 is phosphorylated to a lesser extent [25]. Thus, our findings were consistent with previous reports. ERK1/2 reacted more rapidly than p38 MAPK, and activation of both lasted up to 48 h after treatment with MT01. Once MAPK is activated by ODNs, transcription factors and other kinases may be phosphorylated to initiate events such as gene expression and post-translational protein modifications.
Effects of ERK and p38 Inhibitors on Up-Regulation of ALP Activity Induced by ODN MT01
To investigate the involvement of ERK and p38 MAPK in modulation of ALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38 (U0126 and SB203580, respectively) were used. Cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. As shown in Figure 2, compared with the control (phosphate-buffered saline (PBS) treatment), ALP activity was significantly up-regulated after ODN MT01 treatment at 48 and 72 h. Notably, U0126 and SB203580 significantly inhibited the up-regulation induced by ODN MT01.
The activity of osteogenic differentiation marker ALP is an important index to indicate osteogenesis at an early stage. As shown in Figure 3, after 24 h of treatment with ODN MT01, the activity of ALP was increased, compared with that in the control, but the difference was not significant. The density of cells increased after 48 and 72 h, and ODN MT01 exhibited an obvious promoting effect on osteogenic differentiation. In addition, inhibitors significantly downregulated ALP activity induced by ODN MT01 after 48 and 72 h. These results indicated that the ERK and p38 MAPK pathways were probably involved in ODN MT01-induced promotion of ALP activity.
Effect of ERK and p38 Inhibitors on Runx2 Expression in Response to ODN MT01
The above results confirmed that ODN MT01 induced the phosphorylation of ERK and p38 MAPK and further activated these MAPK signaling pathways. To investigate the role of Runx2, which is one of the most important factors for osteoblast differentiation, we used the ERK inhibitor U0126 and p38 inhibitor SB203580 to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 for 72 h. The total and phosphorylated protein levels of Runx2 were detected using Western blotting. As shown in Figure 4, there were obvious differences in the phosphorylation levels of ERK and p38 between the MT01-treatment and control groups; p-ERK and p-p38 were significantly inhibited in the presence of U0126 and SB203580, which were induced by MT01. For Runx2, there was no obvious change in protein levels in these groups, but MT01 induced notable phosphorylation of Runx2 (1.8-fold increase compared with the control). However, the up-regulated phosphorylation of Runx2 induced by MT01 was inhibited in the presence of U0126 and SB203580.
Runx2 is an essential transcription factor for osteoblast differentiation and bone formation, and inactivation of one Runx2 allele will cause cleidocranial dysplasia syndrome, a disease characterized by delayed osteoblast differentiation for bone formation through intramembranous ossification in mice and humans [3,26]. Previous reports have shown that extracellular matrix production can induce osteoblast differentiation by increasing Runx2 transcriptional activity, but not the mRNA or protein levels [27][28][29], which concurs with our results. Runx2 activity is controlled by various extracellular signaling pathways, and post-translational modifications (phosphorylation, acetylation and ubiquitination) can affect its stability and activity [30,31]. Among the post-translational modifications, the role of Runx2 phosphorylation has been well characterized. Ge et al. found that Runx2 is regulated by ERK1/2 and p38 MAPK-mediated phosphorylation [32], and ERK is more important for Runx2 phosphorylation than p38 [33]. Interestingly, in the present study, we found the same results in which inhibition of ERK showed a stronger effect on the expression of p-Runx2 (1-fold increase compared with the control) than that of p38 inhibition (1.2-fold increase compared with the control). These results suggested that Runx2 is phosphorylated on a complex set of sites that partially overlap between ERK and p38. Another study found that phosphorylation of p38 MAPK induces activation of Runx2 via TAK1 and MEK3 signaling pathways during early osteoblastic differentiation [21]. Here, we showed that MT01 greatly increased Runx2 phosphorylation, which was potently repressed by inhibitors of ERK and p38. Taken together, our results were consistent with other findings and demonstrated that MT01-induced phosphorylation of Runx2 was mediated by activation of both ERK1/2 and p38 MAPK signaling pathways, and ERK MAPK may be more important.
Effects of ERK and p38 Inhibitors on the Expression of Osteocalcin and Type I Collagen
Runx2 has been shown to regulate the expression of osteoblast-specific genes, such as ALP, osteocalcin and type I collagen [34]. To further confirm our results, we analyzed the expression of subsets of osteogenic marker genes including osteocalcin and type I collagen. We used ERK and p38 inhibitors to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 or ODN FC003 for 15 days. Then, the mRNA and protein expression of osteocalcin and type I collagen were analyzed by real-time PCR and Western blot, respectively. As shown in Figure 5A, compared with the control g, the protein expression of osteocalcin was increased by ODN MT01 treatment. Compared with ODN MT01 treatment, the protein level of osteocalcin was significantly decreased when cells were pretreated with the ERK and p38 inhibitors. The expression level of type I collagen exhibited a similar tendency with osteocalcin. As shown in Figure 5B,C, the mRNA levels were concurrent with the protein expression. Osteocalcin protein expression marks the late phase of osteogenic differentiation, and is only produced by mineralizing cells types. In addition, osteocalcin is considered as a specific marker of osteoblast differentiation and maturity. Type I collagen is the major product of osteoblasts, and accounts for 90% of the matrix protein content [35]. Type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life. Osteocalcin and type I collagen protein expression induced by ODN MT01 was increased at late stages of osteoblast differentiation, suggesting that ODN MT01 controll of the expression of osteocalcin and type I collagen in these cells could also control osteoblast differentiation and function. Furthermore, ERK and p38 MAPK inhibitors obviously decreased protein expression induced by MT01. Therefore, ODN MT01 induced the expression of osteocalcin and type I collagen via ERK and p38 MAPK signaling pathways.
Experimental Section
Materials
ODNs MT01 (5'-ACCCCCTCTACCCCCTCTACCCCCTCT-3') and FC003 (5'-TCTCTCTCTCTC TCTCTCTCTCTC-3') were synthesized by TaKaRa (Dalian, China), and were dissolved in axenic PBS. The human osteoblastic cell line MG 63 was obtained from the American Type Culture Collection (CRL-1427). An Alkaline phosphatase kit and micro-BCA assay kit were obtained from Jiancheng Biological Reagent Co. (Nanjing, China). A real-time PCR kit was purchased from TaKaRa (Tokyo, Japan). Monoclonal antibodies against p38, p-p38, p44/p42 and p-p44/42, were purchased from Santa Cruz Biotech. and those against Runx2, osteocalcin and type I collagen were purchased from Abcam (UK). The anti-phospho-Runx2 antibody was purchased from Abcam Co (UK). The phosphorylation site was Ser533.
Cell Culture
MG 63 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO 2 . Cells were seeded at an initial density of 2 × 10 5 cells/mL. To investigate the effect of ODN MT01 on MAPKs, cells were treated with 1 µg/mL ODN MT01 for 0, 30, 60 min, 24 and 48 h. To investigate activation of Runx2, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 72 h. To investigate the effects of ERK and p38 inhibitors on the mRNA and protein levels of osteocalcin and type I collagen, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 15 days. Medium was changed every 3 days, and ODNs and inhibitors were added at the same time.
Fluorescent Labeling of MT01
Cy5 was conjugated to the 5'-end of MT01, and then dissolved with PBS. Cells were cultured on 24 × 24 mm coverslips at a density of 1 × 10 4 cells/well overnight. Then, cells were treated with 1 µg/mL Cy5-labeled MT01 for 3, 6 and 12 h. Cells were washed three times with PBS according to the time points. While protected from light, Hoechst 33342 was added at 1 µg/mL for 5 min to stain nuclei. A laser scanning confocal microscope (Olympus IX81, Japan) was used to observe cell climb-chips. Stained cells were imaged with Olympus Fluoview FV1000 Viewer (Version1.7.c; Olympus Corporation: Tokyo, Japan, 2007).
Western Blot Analysis
Western blotting was used to evaluate total and phosphorylated protein levels of p38 and p44/42. MG 63 cells were cultured in 10-cm dishes and treated with 1 µg/mL ODN MT01 for the indicated times. To evaluate the protein levels of Runx2 and p-Runx2, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without ODN MT01 for 72 h. To investigate the levels of osteocalcin and type I collagen, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without ODN MT01 for 15 days. Then cells were collected, washed twice with cold Tris-buffered saline and resuspended in lysis buffer (50 mM Tris, pH 7.6, 0.01% EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/mL leupeptin). The protein concentration was measured using a BCA Protein Assay Reagent kit. Protein samples (40 μg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Then membranes were incubated with secondary antibodies at 20 °C for 2 h, and developed using an ECL chemiluminescent system. Loading differences were normalized using a GAPDH antibody.
ALP Activity Assay
Cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. Then, the cells were collected and lysed to measure ALP activity. The assay was conducted using an Alkaline Phosphatase Kit, according to the manufacturer's instructions. The protein concentration of cell lysates was measured using a micro-BCA assay kit, and ALP activity was normalized to the total protein concentration. Values were the averages of triplicate measurements.
Real-Time PCR
To quantify mRNA expression levels, real-time PCR was performed with cDNA samples. Primers were designed using qPrimerDepot(Nucleic Acids Res. 2007), a primer database for quantitative real-time PCR. Real-time PCR was performed using an ABI Steponeplus (ABI PRISM, Carlsbad, NM, USA), which allowed real-time monitoring of increases in PCR product concentrations after each cycle based on the fluorescence of the double-stranded DNA specific dye SYBR green. The number of cycles required to produce a detectable product above background was measured for each sample. These cycle numbers were then used to calculate fold differences in the initial mRNA level for each sample using the following method. First, the cycle number difference for GAPDH, a housekeeping gene, was determined in the control sample and appropriate ODN MT01-treated sample. Values were the averages of triplicate measurements. PCR primers used were as follows: GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'; Type I collagen forward, 5'-AGGGCCAAGACGAAGACATC-3' and reverse, 5'-AGATCACGTCATCGCACAACA-3'; OC forward, 5'-TGAGAGCCCTCACACTCCTC-3' and reverse, 5'-GCCGTAGAAGCGCCGATAGGC-3'.
Statistical Analysis
All experiments were performed with triplicate independent samples and repeated at least twice. Results were expressed as the mean ± SD. ANOVA and the Bonferroni post-hoc test were used to compare the differences between the ODN MT01-treatment group and other groups. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed with SAS software (version 8.0, SAS: Cary, NC, USA).
Conclusions
In this study, a specific ODN, ODN MT01, was endocytosed by osteoblasts and found to up-regulate the expression level of osteocalcin, type I collagen and ALP in MG 63 cells via the ERK1/2 and p38 MAPK pathways. This study provides further insight into the use of ODN MT01 for in vitro experimentation, and supports the potential use of ODN MT01 to regulate the rebuilding of bone.
Figure 1 .
1Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.
Figure 1 .
1Cont
Figure 2 .
2Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.
Figure 3 .
3Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72
Figure 4 .
4Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.
Figure 5 .
5MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD (
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgmentsThis research was supported by the Natural Science Funds of Jilin Province, China (No. 201015203). Quanshun Li is gratefully acknowledged for his correction and proofreading of the manuscript.
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| [
"A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.OPEN ACCESSInt. J. Mol. Sci. 2012, 13 7903"
] | [
"Xu Hou \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n",
"Yuqin Shen \nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n",
"Chao Zhang \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n",
"Liru Zhang [email protected]. ",
"Yanyan Qin \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n\nDepartment of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n",
"Yongli Yu [email protected] \nDepartment of Immunology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n",
"Liying Wang \nDepartment of Molecular Biology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina\n",
"Xinhua Sun \nDepartment of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina\n"
] | [
"Department of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina",
"Department of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina",
"Department of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina",
"Department of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina",
"Department of Periodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina",
"Department of Immunology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina",
"Department of Molecular Biology, Medical College of Norman Bethune\nJilin University\n130021ChangchunChina",
"Department of Orthodontics\nSchool of Stomatology\nJilin University\n1500 Qinghua Road130021ChangchunChina"
] | [
"Xu",
"Yuqin",
"Chao",
"Liru",
"Yanyan",
"Yongli",
"Liying",
"Xinhua"
] | [
"Hou",
"Shen",
"Zhang",
"Zhang",
"Qin",
"Yu",
"Wang",
"Sun"
] | [
"E D Jensen, ",
"R Gopalakrishnan, ",
"J J Westendorf, ",
"F Otto, ",
"A P Thornell, ",
"T Crompton, ",
"A Denzel, ",
"K C Gilmour, ",
"I R Rosewell, ",
"G W Stamp, ",
"R S Beddington, ",
"S Mundlos, ",
"B R Olsen, ",
"S Mundlos, ",
"F Otto, ",
"C Mundlos, ",
"J B Mulliken, ",
"A S Aylsworth, ",
"S Albright, ",
"D Lindhout, ",
"W G Cole, ",
"W Henn, ",
"J H Knoll, ",
"G S Stein, ",
"J B Lian, ",
"A J Van Wijnen, ",
"J L Stein, ",
"M Montecino, ",
"A Javed, ",
"S K Zaidi, ",
"D W Young, ",
"J Y Choi, ",
"S M Pockwinse, ",
"P Ducy, ",
"M Starbuck, ",
"M Priemel, ",
"J Shen, ",
"G Pinero, ",
"V Geoffroy, ",
"M Amling, ",
"G Karsenty, ",
"P Cerkovnik, ",
"B Jezersek Novakovic, ",
"V Stegel, ",
"S Novakovic, ",
"C S Liu, ",
"Y Sun, ",
"Y H Hu, ",
"L Sun, ",
"M Bao, ",
"Y Zhang, ",
"M Wan, ",
"L Dai, ",
"X Hu, ",
"X Wu, ",
"L Wang, ",
"P Deng, ",
"J Wang, ",
"J Chen, ",
"D Klinman, ",
"H Shirota, ",
"D Tross, ",
"T Sato, ",
"S Klaschik, ",
"A Amcheslavsky, ",
"H Hemmi, ",
"S Akira, ",
"N N Norgaard, ",
"T Holien, ",
"S Jonsson, ",
"H Hella, ",
"T Espevik, ",
"A Sundan, ",
"T Standal, ",
"J H Chang, ",
"E J Chang, ",
"H H Kim, ",
"S K Kim, ",
"Z Feng, ",
"Y Shen, ",
"L Wang, ",
"L Cheng, ",
"J Wang, ",
"Q Li, ",
"W Shi, ",
"X Sun, ",
"Y Shen, ",
"Z Feng, ",
"C Lin, ",
"X Hou, ",
"X Wang, ",
"J Wang, ",
"Y Yu, ",
"L Wang, ",
"X Sun, ",
"A M Bennett, ",
"N K Tonks, ",
"C Ge, ",
"Q Yang, ",
"G Zhao, ",
"H Yu, ",
"K L Kirkwood, ",
"R T Franceschi, ",
"H M Jeong, ",
"E H Han, ",
"Y H Jin, ",
"Y P Hwang, ",
"H G Kim, ",
"B H Park, ",
"J Y Kim, ",
"Y C Chung, ",
"K Y Lee, ",
"H G Jeong, ",
"D Reddi, ",
"S J Brown, ",
"G N Belibasakis, ",
"G Yang, ",
"M Wan, ",
"Y Zhang, ",
"L Sun, ",
"R Sun, ",
"D Hu, ",
"X Zhou, ",
"L Wang, ",
"X Wu, ",
"Y Yu, ",
"A M Krieg, ",
"M B Greenblatt, ",
"J H Shim, ",
"W Zou, ",
"D Sitara, ",
"M Schweitzer, ",
"D Hu, ",
"S Lotinun, ",
"Y Sano, ",
"R Baron, ",
"J M Park, ",
"M Kawano, ",
"W Ariyoshi, ",
"K Iwanaga, ",
"T Okinaga, ",
"M Habu, ",
"I Yoshioka, ",
"K Tominaga, ",
"T Nishihara, ",
"D Zhang, ",
"H Zheng, ",
"J Zhao, ",
"L Lin, ",
"C Li, ",
"J Liu, ",
"Y Pan, ",
"A Suzuki, ",
"J Guicheux, ",
"G Palmer, ",
"Y Miura, ",
"Y Oiso, ",
"J P Bonjour, ",
"J Caverzasio, ",
"W Zou, ",
"A Amcheslavsky, ",
"E F Wagner, ",
"G Karsenty, ",
"G Xiao, ",
"Y Cui, ",
"P Ducy, ",
"G Karsenty, ",
"R T Franceschi, ",
"G Xiao, ",
"D Jiang, ",
"P Thomas, ",
"M D Benson, ",
"K Guan, ",
"G Karsenty, ",
"R T Franceschi, ",
"R T Franceschi, ",
"C Ge, ",
"G Xiao, ",
"H Roca, ",
"D Jiang, ",
"S C Bae, ",
"Y H Lee, ",
"E J Jeon, ",
"K Y Lee, ",
"N S Choi, ",
"M H Lee, ",
"H N Kim, ",
"Y H Jin, ",
"H M Ryoo, ",
"J Y Choi, ",
"M Yoshida, ",
"N Nishino, ",
"C Ge, ",
"G Xiao, ",
"D Jiang, ",
"Q Yang, ",
"N E Hatch, ",
"H Roca, ",
"R T Franceschi, ",
"C Ge, ",
"Q Yang, ",
"G Zhao, ",
"H Yu, ",
"K L Kirkwood, ",
"R T Franceschi, ",
"A Yamaguchi, ",
"T Komori, ",
"T Suda, ",
"B Kern, ",
"J Shen, ",
"M Starbuck, ",
"G Karsenty, "
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] | [
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"Westendorf",
"Otto",
"Thornell",
"Crompton",
"Denzel",
"Gilmour",
"Rosewell",
"Stamp",
"Beddington",
"Mundlos",
"Olsen",
"Mundlos",
"Otto",
"Mundlos",
"Mulliken",
"Aylsworth",
"Albright",
"Lindhout",
"Cole",
"Henn",
"Knoll",
"Stein",
"Lian",
"Van Wijnen",
"Stein",
"Montecino",
"Javed",
"Zaidi",
"Young",
"Choi",
"Pockwinse",
"Ducy",
"Starbuck",
"Priemel",
"Shen",
"Pinero",
"Geoffroy",
"Amling",
"Karsenty",
"Cerkovnik",
"Jezersek Novakovic",
"Stegel",
"Novakovic",
"Liu",
"Sun",
"Hu",
"Sun",
"Bao",
"Zhang",
"Wan",
"Dai",
"Hu",
"Wu",
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"Deng",
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"Chen",
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"Zhao",
"Yu",
"Kirkwood",
"Franceschi",
"Jeong",
"Han",
"Jin",
"Hwang",
"Kim",
"Park",
"Kim",
"Chung",
"Lee",
"Jeong",
"Reddi",
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"Belibasakis",
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"Sun",
"Hu",
"Zhou",
"Wang",
"Wu",
"Yu",
"Krieg",
"Greenblatt",
"Shim",
"Zou",
"Sitara",
"Schweitzer",
"Hu",
"Lotinun",
"Sano",
"Baron",
"Park",
"Kawano",
"Ariyoshi",
"Iwanaga",
"Okinaga",
"Habu",
"Yoshioka",
"Tominaga",
"Nishihara",
"Zhang",
"Zheng",
"Zhao",
"Lin",
"Li",
"Liu",
"Pan",
"Suzuki",
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] | [
"Regulation of gene expression in osteoblasts. E D Jensen, R Gopalakrishnan, J J Westendorf, Biofactors. 36Jensen, E.D.; Gopalakrishnan, R.; Westendorf, J.J. Regulation of gene expression in osteoblasts. Biofactors 2010, 36, 25-32.",
"Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. F Otto, A P Thornell, T Crompton, A Denzel, K C Gilmour, I R Rosewell, G W Stamp, R S Beddington, S Mundlos, B R Olsen, Cell. 89Otto, F.; Thornell, A.P.; Crompton, T.; Denzel, A.; Gilmour, K.C.; Rosewell, I.R.; Stamp, G.W.; Beddington, R.S.; Mundlos, S.; Olsen, B.R.; et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997, 89, 765-771.",
"Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. S Mundlos, F Otto, C Mundlos, J B Mulliken, A S Aylsworth, S Albright, D Lindhout, W G Cole, W Henn, J H Knoll, Cell. 89Mundlos, S.; Otto, F.; Mundlos, C.; Mulliken, J.B.; Aylsworth, A.S.; Albright, S.; Lindhout, D.; Cole, W.G.; Henn, W.; Knoll, J.H.; et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997, 89, 773-779.",
"Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. G S Stein, J B Lian, A J Van Wijnen, J L Stein, M Montecino, A Javed, S K Zaidi, D W Young, J Y Choi, S M Pockwinse, Oncogene. 23Stein, G.S.; Lian, J.B.; van Wijnen, A.J.; Stein, J.L.; Montecino, M.; Javed, A.; Zaidi, S.K.; Young, D.W.; Choi, J.Y.; Pockwinse, S.M. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 2004, 23, 4315-4329.",
"A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. P Ducy, M Starbuck, M Priemel, J Shen, G Pinero, V Geoffroy, M Amling, G Karsenty, Ducy, P.; Starbuck, M.; Priemel, M.; Shen, J.; Pinero, G.; Geoffroy, V.; Amling, M.; Karsenty, G. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.",
". Genes Dev. 13Genes Dev. 1999, 13, 1025-1036.",
"Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. P Cerkovnik, B Jezersek Novakovic, V Stegel, S Novakovic, Int. J. Oncol. 38Cerkovnik, P.; Jezersek Novakovic, B.; Stegel, V.; Novakovic, S. Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells. Int. J. Oncol. 2011, 38, 1749-1758.",
"Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. C S Liu, Y Sun, Y H Hu, L Sun, Vaccine. 28Liu, C.S.; Sun, Y.; Hu, Y.H.; Sun, L. Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. Vaccine 2010, 28, 4153-4161.",
"Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. M Bao, Y Zhang, M Wan, L Dai, X Hu, X Wu, L Wang, P Deng, J Wang, J Chen, Clin. Immunol. 118Bao, M.; Zhang, Y.; Wan, M.; Dai, L.; Hu, X.; Wu, X.; Wang, L.; Deng, P.; Wang, J.; Chen, J.; et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. Clin. Immunol. 2006, 118, 180-187.",
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"Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. A Amcheslavsky, H Hemmi, S Akira, J. Bone Miner. Res. 20Amcheslavsky, A.; Hemmi, H.; Akira, S.; Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. J. Bone Miner. Res. 2005, 20, 1692-1699.",
"CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. N N Norgaard, T Holien, S Jonsson, H Hella, T Espevik, A Sundan, T Standal, J. Immunol. 185Norgaard, N.N.; Holien, T.; Jonsson, S.; Hella, H.; Espevik, T.; Sundan, A.; Standal, T. CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis. J. Immunol. 2010, 185, 3131-3139.",
"Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. J H Chang, E J Chang, H H Kim, S K Kim, Biochem. Biophys. Res. Commun. 389Chang, J.H.; Chang, E.J.; Kim, H.H.; Kim, S.K. Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. Biochem. Biophys. Res. Commun. 2009, 389, 28-33.",
"An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Z Feng, Y Shen, L Wang, L Cheng, J Wang, Q Li, W Shi, X Sun, Int. J. Mol. Sci. 12Feng, Z.; Shen, Y.; Wang, L.; Cheng, L.; Wang, J.; Li, Q.; Shi, W.; Sun, X. An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast. Int. J. Mol. Sci. 2011, 12, 2543-2555.",
"An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Y Shen, Z Feng, C Lin, X Hou, X Wang, J Wang, Y Yu, L Wang, X Sun, Int. J. Mol. Sci. 13Shen, Y.; Feng, Z.; Lin, C.; Hou, X.; Wang, X.; Wang, J.; Yu, Y.; Wang, L.; Sun, X. An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis. Int. J. Mol. Sci. 2012, 13, 2877-2892.",
"Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. A M Bennett, N K Tonks, Science. 278Bennett, A.M.; Tonks, N.K. Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science 1997, 278, 1288-1291.",
"Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, 10.1002/jbmr.561J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2011, 27, doi: 10.1002/jbmr.561.",
"Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. H M Jeong, E H Han, Y H Jin, Y P Hwang, H G Kim, B H Park, J Y Kim, Y C Chung, K Y Lee, H G Jeong, Food Chem. Toxicol. 48Jeong, H.M.; Han, E.H.; Jin, Y.H.; Hwang, Y.P.; Kim, H.G.; Park, B.H.; Kim, J.Y.; Chung, Y.C.; Lee, K.Y.; Jeong, H.G. Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation. Food Chem. Toxicol. 2010, 48, 3362-3368.",
"Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. D Reddi, S J Brown, G N Belibasakis, Microb. Pathog. 51Reddi, D.; Brown, S.J.; Belibasakis, G.N. Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK. Microb. Pathog. 2011, 51, 415-420.",
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"Therapeutic potential of Toll-like receptor 9 activation. A M Krieg, Nat. Rev. Drug. Discov. 5Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug. Discov. 2006, 5, 471-484.",
"The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. M B Greenblatt, J H Shim, W Zou, D Sitara, M Schweitzer, D Hu, S Lotinun, Y Sano, R Baron, J M Park, J. Clin. Invest. 120Greenblatt, M.B.; Shim, J.H.; Zou, W.; Sitara, D.; Schweitzer, M.; Hu, D.; Lotinun, S.; Sano, Y.; Baron, R.; Park, J.M.; et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J. Clin. Invest. 2010, 120, 2457-2473.",
"Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. M Kawano, W Ariyoshi, K Iwanaga, T Okinaga, M Habu, I Yoshioka, K Tominaga, T Nishihara, Biochem. Biophys. Res. Commun. 405Kawano, M.; Ariyoshi, W.; Iwanaga, K.; Okinaga, T.; Habu, M.; Yoshioka, I.; Tominaga, K.; Nishihara, T. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid. Biochem. Biophys. Res. Commun. 2011, 405, 575-580.",
"Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. D Zhang, H Zheng, J Zhao, L Lin, C Li, J Liu, Y Pan, J. Periodontal. Res. 46Zhang, D.; Zheng, H.; Zhao, J.; Lin, L.; Li, C.; Liu, J.; Pan, Y. Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway. J. Periodontal. Res. 2011, 46, 31-38.",
"Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. A Suzuki, J Guicheux, G Palmer, Y Miura, Y Oiso, J P Bonjour, J Caverzasio, 30BoneSuzuki, A.; Guicheux, J.; Palmer, G.; Miura, Y.; Oiso, Y.; Bonjour, J.P.; Caverzasio, J. Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. Bone 2002, 30, 91-98.",
"Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. W Zou, A Amcheslavsky, J. Biol. Chem. 278Zou, W.; Amcheslavsky, A.; Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. J. Biol. Chem. 2003, 278, 16732-16740.",
"Genetic control of skeletal development. E F Wagner, G Karsenty, Curr. Opin. Genet. Dev. 11Wagner, E.F.; Karsenty, G. Genetic control of skeletal development. Curr. Opin. Genet. Dev. 2001, 11, 527-532.",
"Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. G Xiao, Y Cui, P Ducy, G Karsenty, R T Franceschi, Mol. Endocrinol. 11Xiao, G.; Cui, Y.; Ducy, P.; Karsenty, G.; Franceschi, R.T. Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. Mol. Endocrinol. 1997, 11, 1103-1113.",
"MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. G Xiao, D Jiang, P Thomas, M D Benson, K Guan, G Karsenty, R T Franceschi, J. Biol. Chem. 275Xiao, G.; Jiang, D.; Thomas, P.; Benson, M.D.; Guan, K.; Karsenty, G.; Franceschi, R.T. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J. Biol. Chem. 2000, 275, 4453-4459.",
"Transcriptional regulation of osteoblasts. R T Franceschi, C Ge, G Xiao, H Roca, D Jiang, Cells Tissues Organs. 189Franceschi, R.T.; Ge, C.; Xiao, G.; Roca, H.; Jiang, D. Transcriptional regulation of osteoblasts. Cells Tissues Organs 2009, 189, 144-152.",
"Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. S C Bae, Y H Lee, Gene. 366Bae, S.C.; Lee, Y.H. Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation. Gene 2006, 366, 58-66.",
". E J Jeon, K Y Lee, N S Choi, M H Lee, H N Kim, Y H Jin, H M Ryoo, J Y Choi, M Yoshida, N Nishino, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylationJeon, E.J.; Lee, K.Y.; Choi, N.S.; Lee, M.H.; Kim, H.N.; Jin, Y.H.; Ryoo, H.M.; Choi, J.Y.; Yoshida, M.; Nishino, N.; et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation.",
". J. Biol. Chem. 281J. Biol. Chem. 2006, 281, 16502-16511.",
"Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor. C Ge, G Xiao, D Jiang, Q Yang, N E Hatch, H Roca, R T Franceschi, Ge, C.; Xiao, G.; Jiang, D.; Yang, Q.; Hatch, N.E.; Roca, H.; Franceschi, R.T. Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor.",
". J. Biol. Chem. 284J. Biol. Chem. 2009, 284, 32533-32543.",
"Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. C Ge, Q Yang, G Zhao, H Yu, K L Kirkwood, R T Franceschi, J. Bone Miner. Res. 27Ge, C.; Yang, Q.; Zhao, G.; Yu, H.; Kirkwood, K.L.; Franceschi, R.T. Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 2012, 27, 538-551.",
"Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. A Yamaguchi, T Komori, T Suda, Endocr. Rev. 21Yamaguchi, A.; Komori, T.; Suda, T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 2000, 21, 393-411.",
"Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. B Kern, J Shen, M Starbuck, G Karsenty, J. Biol. Chem. 276Kern, B.; Shen, J.; Starbuck, M.; Karsenty, G. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J. Biol. Chem. 2001, 276, 7101-7107."
] | [
"[1]",
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"[4]",
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"[7]",
"[8]",
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"[11]",
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"[35]"
] | [
"Regulation of gene expression in osteoblasts",
"Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development",
"Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia",
"Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression",
"Tumor vaccine composed of CpG ODN class C and irradiated tumor cells up-regulates the expression of genes characteristic of mature dendritic cells and of memory cells",
"Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity",
"Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide",
"Synthetic oligonucleotides as modulators of inflammation",
"Bar-Shavit, Z. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis",
"CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: Effects on myeloma cell apoptosis and in vitro osteoblastogenesis",
"Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation",
"An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast",
"An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis",
"Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases",
"Interactions between extracellular signal-regulated kinase 1/2 and P38 map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity",
"Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK-and ERK-dependent RUNX2 activation",
"Porphyromonas gingivalis induces RANKL in bone marrow stromal cells: Involvement of the p38 MAPK",
"Inhibition of a C-rich oligodeoxynucleotide on activation of immune cells in vitro and enhancement of antibody response in mice",
"Therapeutic potential of Toll-like receptor 9 activation",
"The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice",
"Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid",
"Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-κB pathway, but not through the p38 MAPK pathway",
"Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9",
"Genetic control of skeletal development",
"Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: Requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence",
"MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1",
"Transcriptional regulation of osteoblasts",
"Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation",
"Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity",
"Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1",
"Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes"
] | [
"Biofactors",
"Cell",
"Cell",
"Oncogene",
"A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development",
"Genes Dev",
"Int. J. Oncol",
"Vaccine",
"Clin. Immunol",
"J. Leukoc Biol",
"J. Bone Miner. Res",
"J. Immunol",
"Biochem. Biophys. Res. Commun",
"Int. J. Mol. Sci",
"Int. J. Mol. Sci",
"Science",
"J. Bone Miner. Res",
"Food Chem. Toxicol",
"Microb. Pathog",
"Immunology",
"Nat. Rev. Drug. Discov",
"J. Clin. Invest",
"Biochem. Biophys. Res. Commun",
"J. Periodontal. Res",
"Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation",
"J. Biol. Chem",
"Curr. Opin. Genet. Dev",
"Mol. Endocrinol",
"J. Biol. Chem",
"Cells Tissues Organs",
"Gene",
"J. Biol. Chem",
"Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor",
"J. Biol. Chem",
"J. Bone Miner. Res",
"Endocr. Rev",
"J. Biol. Chem"
] | [
"\nFigure 1 .\n1Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.",
"\nFigure 1 .\n1Cont",
"\nFigure 2 .\n2Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.",
"\nFigure 3 .\n3Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72",
"\nFigure 4 .\n4Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.",
"\nFigure 5 .\n5MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD ("
] | [
"Confocal fluorescence images of MG 63 cells treated with Cy5-labeled MT01. (A,C,E) Merged images of Cy5-labeled MT01 and cells with Hoechst 33342-labeled nuclei; (B,D,F) Cy5-labeled MT01; (A,B) Cells were treated with MT01-Cy5 for 3 h; (C,D) Cells were treated with MT01-Cy5 for 6 h; (E,F) Cells were treated with MT01-Cy5 for 12 h. Red: Cy5-labeled MT01, Blue: nuclei. The fluorescence intensity of Cy5 was analyzed (G), * p < 0.05, # p < 0.01.",
"Cont",
"Western blot analysis of phosphorylated and total ERK1/2 (A), and total p38 (B). MG 63 cells were cultured in the presence or absence of 1 µg/mL ODN MT01 or ODN FC003. Cell lysates were obtained at 0, 30, 60 min, 24 h and 48 h after ODN treatment.",
"Effect of the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) on ALP activity in MG 63 cells after ODN MT01 (1 µg/mL) treatment for 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (SD) (n = 6). # p < 0.05 vs. the corresponding value of ODN MT01 treatment at 48 h. * p < 0.05 vs. the corresponding value of ODN MT01 treatment at 72",
"Effect of ERK and p38 inhibitors on Runx2 expression in response to ODN MT01. MG 63 cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 72 h. The protein expression of Runx2, p-Runx2, p-ERK and p-p38 was analyzed by Western blotting.",
"MG 63 cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 15 d. Another group was treated with 1 µg/mL ODN FC003. Medium was changed every 3 days, and ODNs and inhibitors were added as the same time. (A) Western blot analysis of osteocalcin (OC) and type I collagen protein expression; (B) Real-time PCR analysis of type I collagen mRNA expression; (C) Real-time PCR analysis of OC mRNA expression. The gene expression of type I collagen and OC was measured by real-time PCR and results were normalized against the average housekeeping gene expression in each sample. One-way ANOVA and the Bonferroni post-hoc test were used to compare differences between the ODN MT01-treated group and other groups. * p < 0.05 vs. the corresponding value of ODN; ∆ p < 0.05 vs. the corresponding value of ODN+U0126; # p < 0.05 vs. the corresponding value of ODN+SB203580. Data are expressed as the mean ± SD ("
] | [
"Figure 1",
"Figure 2A",
"Figure 2B",
"Figure 2",
"Figure 2",
"Figure 3",
"Figure 4",
"Figure 5A",
"Figure 5B",
"(Nanjing, China)"
] | [] | [
"Osteoblasts play an extremely important role in bone formation and remodeling processes. Osteoblasts are derived from mesenchymal stem cells, and can differentiate into mature and functional osteoblasts that produce extracellular matrix proteins and regulators of matrix mineralization [1]. Runx2 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Runx2 −/− mice show a complete lack of both intramembranous and endochondral ossification owing to maturation arrest of osteoblasts [2,3]. However, Runx2 can regulate the expression of osteoblast-specific genes including ALP, type I collagen and osteocalcin. To date, numerous reports have shown that signaling pathways and transcription factors could participate in osteoblastogenesis by regulating the production or activity of Runx2 [4]. Furthermore, Runx2 is necessary throughout life to promote the differentiation of new osteoblasts during bone remodeling [5].",
"Oligodeoxynucleotides (ODNs) containing unmethylated nucleotide motifs are immunostimulatory in vertebrates, and some ODNs containing CpG motifs are used for treating cancer, virus-associated diseases, and infections [6][7][8][9]. Recently, specific ODNs were found to have an effect on modulating osteoclast-and osteoblast-lineage cells [10]. In addition, CpG-ODN with a phosphorothioate backbone inhibits the BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines [11]. Chang et al. demonstrated that a novel CpG-ODN enhances the inhibitory effects on osteoclast differentiation by downregulation of TREM-2 [12]. In our previous studies, we have shown that ODN MT01, a synthetic 27-mer single stranded ODN with a design based on human mitochondrial DNA, promotes the proliferation and activation of osteoblasts [13], and stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts [14]. However, the detailed signaling pathway in which ODN MT01 modulates the differentiation of osteoblasts has not been fully elucidated.",
"Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific protein kinases, including p38, JNK and ERK1/2 (p44/p42). These molecules play important roles in cell differentiation, proliferation and death [15], and are activated by phosphorylation of tyrosine/threonine residues in signal transduction cascades. Previous studies have shown that the p38 MAPK and ERK1/2 pathways are involved in the proliferation and differentiation of osteoblasts [16][17][18]. Therefore, we hypothesized that ODN MT01 might regulate the differentiation of osteoblastic cells via the MAPKs pathway.",
"This study aimed to investigate the regulation of MAPKs in response to ODN MT01, and to elucidate the involvement of the MAPK signaling pathway in regulation of Runx2, osteocalcin, type I collagen and ALP expression in ODN MT01-treated MG 63 cells.",
"The design of ODN MT01, a 27-mer ODN, was based on the motif sequence 5'-ACCCCCTCTACCCCCTCT-3' in human mitochondrial DNA. MT01 is a cytosine-rich ODN and contains five successive cytosines in each of its 9-mer motifs (5'-ACCCCCTCT-3') [19]. To investigate whether the effects of an ODN required internalization into cells, Cy5-labeled ODN MT01 was synthesized to trace the sites of ODN actions. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, and is temperature-and energy-dependent [20]. This result was consistent with our observations, as shown in Figure 1, ODN MT01 entered cells by endocytosis after incubation with MG 63 cells for 3 h. The fluorescence intensity was enhanced over time, and the intensity at 6 h was significantly higher than that at 3 h, and the intensity at 12 h was higher than that at 3 and 6 h. It was apparent that ODN MT01 stimulation of osteoblasts required internalization. ",
"To investigate the effect of ODN MT01 on MAPKs, both total and phosphorylated levels of MAPKs were investigated by Western blotting after 0, 30, 60 min, 24 h and 48 h of treatment with 1 µg/mL ODN MT01. To better demonstrate that the effects of MT01 in MG 63 cells were in response to the specific sequence of MT01 we tested another ODN sequence, ODN FC003, a 24-mer ODN(5'-TCTCTCTCTCTCTCTCTCTCTCTC-3'). The design of ODN FC003 was also based on human mitochondrial DNA, and contained successive thymidine-cytosine nucleotides. In our previous studies [13,14], we found that ODN FC003 is inactive in the regulation of osteoblasts and bone marrow mesenchymal stem cells. Therefore, we chose this sequence as a negative control. As shown in Figure 2A, phosphorylation of ERK1/2 was observed after 30 min of treatment with ODN MT01, which increased by 13.1-fold after 60 min compared with that at 0 min. Furthermore, the active effect lasted up to 48 h (8.9-fold increase), while the total level of ERK1/2 protein remained unchanged. For p38, there was no obvious change in the phosphorylation level before 30 min. However, p38 phosphorylation increased after 60 min of treatment with MT01 ( Figure 2B), and showed an increase of 4.7 fold at 24 h, which lasted up to 48 h. As shown in Figure 2 A and B, the FC003 sequence had no active effect on MG 63 cells via ERK1/2 or p38 MAPKs. Thus, these findings indicated that ODN MT01 induced the phosphorylation of ERK1/2 and p38 MAPK in MG 63 cells.",
"To investigate the mechanisms by which ODNs regulate the differentiation of osteoblasts, it is important to identify the functional role of signaling pathways activated by ODNs. At present, several signaling pathways have been shown to be involved in osteogenesis, such as MAPKs, BMP-2-Smad and NF-κB [21][22][23]. Among them, p38 and ERK1/2 MAPKs have been reported to be important for early osteoblast differentiation in various cell lines [24]. In this study, we found that MT01 was capable of activating ERK1/2 and p38 MAPK pathways indicating that MT01 induced the phosphorylation of MAPKs at the early stage of osteoblast differentiation. Previous reports have shown that ERK and p38 are phosphorylated in response to CpG-ODN, and p38 is phosphorylated to a lesser extent [25]. Thus, our findings were consistent with previous reports. ERK1/2 reacted more rapidly than p38 MAPK, and activation of both lasted up to 48 h after treatment with MT01. Once MAPK is activated by ODNs, transcription factors and other kinases may be phosphorylated to initiate events such as gene expression and post-translational protein modifications. ",
"To investigate the involvement of ERK and p38 MAPK in modulation of ALP activity induced by ODN MT01, specific chemical inhibitors of ERK and p38 (U0126 and SB203580, respectively) were used. Cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. As shown in Figure 2, compared with the control (phosphate-buffered saline (PBS) treatment), ALP activity was significantly up-regulated after ODN MT01 treatment at 48 and 72 h. Notably, U0126 and SB203580 significantly inhibited the up-regulation induced by ODN MT01.",
"The activity of osteogenic differentiation marker ALP is an important index to indicate osteogenesis at an early stage. As shown in Figure 3, after 24 h of treatment with ODN MT01, the activity of ALP was increased, compared with that in the control, but the difference was not significant. The density of cells increased after 48 and 72 h, and ODN MT01 exhibited an obvious promoting effect on osteogenic differentiation. In addition, inhibitors significantly downregulated ALP activity induced by ODN MT01 after 48 and 72 h. These results indicated that the ERK and p38 MAPK pathways were probably involved in ODN MT01-induced promotion of ALP activity. ",
"The above results confirmed that ODN MT01 induced the phosphorylation of ERK and p38 MAPK and further activated these MAPK signaling pathways. To investigate the role of Runx2, which is one of the most important factors for osteoblast differentiation, we used the ERK inhibitor U0126 and p38 inhibitor SB203580 to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 for 72 h. The total and phosphorylated protein levels of Runx2 were detected using Western blotting. As shown in Figure 4, there were obvious differences in the phosphorylation levels of ERK and p38 between the MT01-treatment and control groups; p-ERK and p-p38 were significantly inhibited in the presence of U0126 and SB203580, which were induced by MT01. For Runx2, there was no obvious change in protein levels in these groups, but MT01 induced notable phosphorylation of Runx2 (1.8-fold increase compared with the control). However, the up-regulated phosphorylation of Runx2 induced by MT01 was inhibited in the presence of U0126 and SB203580.",
"Runx2 is an essential transcription factor for osteoblast differentiation and bone formation, and inactivation of one Runx2 allele will cause cleidocranial dysplasia syndrome, a disease characterized by delayed osteoblast differentiation for bone formation through intramembranous ossification in mice and humans [3,26]. Previous reports have shown that extracellular matrix production can induce osteoblast differentiation by increasing Runx2 transcriptional activity, but not the mRNA or protein levels [27][28][29], which concurs with our results. Runx2 activity is controlled by various extracellular signaling pathways, and post-translational modifications (phosphorylation, acetylation and ubiquitination) can affect its stability and activity [30,31]. Among the post-translational modifications, the role of Runx2 phosphorylation has been well characterized. Ge et al. found that Runx2 is regulated by ERK1/2 and p38 MAPK-mediated phosphorylation [32], and ERK is more important for Runx2 phosphorylation than p38 [33]. Interestingly, in the present study, we found the same results in which inhibition of ERK showed a stronger effect on the expression of p-Runx2 (1-fold increase compared with the control) than that of p38 inhibition (1.2-fold increase compared with the control). These results suggested that Runx2 is phosphorylated on a complex set of sites that partially overlap between ERK and p38. Another study found that phosphorylation of p38 MAPK induces activation of Runx2 via TAK1 and MEK3 signaling pathways during early osteoblastic differentiation [21]. Here, we showed that MT01 greatly increased Runx2 phosphorylation, which was potently repressed by inhibitors of ERK and p38. Taken together, our results were consistent with other findings and demonstrated that MT01-induced phosphorylation of Runx2 was mediated by activation of both ERK1/2 and p38 MAPK signaling pathways, and ERK MAPK may be more important. ",
"Runx2 has been shown to regulate the expression of osteoblast-specific genes, such as ALP, osteocalcin and type I collagen [34]. To further confirm our results, we analyzed the expression of subsets of osteogenic marker genes including osteocalcin and type I collagen. We used ERK and p38 inhibitors to pre-treat cells for 1 h followed by treatment with 1 µg/mL ODN MT01 or ODN FC003 for 15 days. Then, the mRNA and protein expression of osteocalcin and type I collagen were analyzed by real-time PCR and Western blot, respectively. As shown in Figure 5A, compared with the control g, the protein expression of osteocalcin was increased by ODN MT01 treatment. Compared with ODN MT01 treatment, the protein level of osteocalcin was significantly decreased when cells were pretreated with the ERK and p38 inhibitors. The expression level of type I collagen exhibited a similar tendency with osteocalcin. As shown in Figure 5B,C, the mRNA levels were concurrent with the protein expression. Osteocalcin protein expression marks the late phase of osteogenic differentiation, and is only produced by mineralizing cells types. In addition, osteocalcin is considered as a specific marker of osteoblast differentiation and maturity. Type I collagen is the major product of osteoblasts, and accounts for 90% of the matrix protein content [35]. Type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life. Osteocalcin and type I collagen protein expression induced by ODN MT01 was increased at late stages of osteoblast differentiation, suggesting that ODN MT01 controll of the expression of osteocalcin and type I collagen in these cells could also control osteoblast differentiation and function. Furthermore, ERK and p38 MAPK inhibitors obviously decreased protein expression induced by MT01. Therefore, ODN MT01 induced the expression of osteocalcin and type I collagen via ERK and p38 MAPK signaling pathways.",
"ODNs MT01 (5'-ACCCCCTCTACCCCCTCTACCCCCTCT-3') and FC003 (5'-TCTCTCTCTCTC TCTCTCTCTCTC-3') were synthesized by TaKaRa (Dalian, China), and were dissolved in axenic PBS. The human osteoblastic cell line MG 63 was obtained from the American Type Culture Collection (CRL-1427). An Alkaline phosphatase kit and micro-BCA assay kit were obtained from Jiancheng Biological Reagent Co. (Nanjing, China). A real-time PCR kit was purchased from TaKaRa (Tokyo, Japan). Monoclonal antibodies against p38, p-p38, p44/p42 and p-p44/42, were purchased from Santa Cruz Biotech. and those against Runx2, osteocalcin and type I collagen were purchased from Abcam (UK). The anti-phospho-Runx2 antibody was purchased from Abcam Co (UK). The phosphorylation site was Ser533.",
"MG 63 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO 2 . Cells were seeded at an initial density of 2 × 10 5 cells/mL. To investigate the effect of ODN MT01 on MAPKs, cells were treated with 1 µg/mL ODN MT01 for 0, 30, 60 min, 24 and 48 h. To investigate activation of Runx2, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 72 h. To investigate the effects of ERK and p38 inhibitors on the mRNA and protein levels of osteocalcin and type I collagen, cells were pre-treated with or without the ERK inhibitor U0126 (10 μM) and p38 inhibitor SB203580 (10 μM) for 1 h, and then treated with or without ODN MT01 for 15 days. Medium was changed every 3 days, and ODNs and inhibitors were added at the same time.",
"Cy5 was conjugated to the 5'-end of MT01, and then dissolved with PBS. Cells were cultured on 24 × 24 mm coverslips at a density of 1 × 10 4 cells/well overnight. Then, cells were treated with 1 µg/mL Cy5-labeled MT01 for 3, 6 and 12 h. Cells were washed three times with PBS according to the time points. While protected from light, Hoechst 33342 was added at 1 µg/mL for 5 min to stain nuclei. A laser scanning confocal microscope (Olympus IX81, Japan) was used to observe cell climb-chips. Stained cells were imaged with Olympus Fluoview FV1000 Viewer (Version1.7.c; Olympus Corporation: Tokyo, Japan, 2007).",
"Western blotting was used to evaluate total and phosphorylated protein levels of p38 and p44/42. MG 63 cells were cultured in 10-cm dishes and treated with 1 µg/mL ODN MT01 for the indicated times. To evaluate the protein levels of Runx2 and p-Runx2, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h and then treated with or without ODN MT01 for 72 h. To investigate the levels of osteocalcin and type I collagen, cells were pre-treated with or without 10 μM U0126 or SB203580 for 1 h, and then treated with or without ODN MT01 for 15 days. Then cells were collected, washed twice with cold Tris-buffered saline and resuspended in lysis buffer (50 mM Tris, pH 7.6, 0.01% EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/mL leupeptin). The protein concentration was measured using a BCA Protein Assay Reagent kit. Protein samples (40 μg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Then membranes were incubated with secondary antibodies at 20 °C for 2 h, and developed using an ECL chemiluminescent system. Loading differences were normalized using a GAPDH antibody.",
"Cells were pre-treated with or without 10 μM U0126 and SB203580 for 1 h and then treated with or without 1 µg/mL ODN MT01 for 24, 48 and 72 h. Then, the cells were collected and lysed to measure ALP activity. The assay was conducted using an Alkaline Phosphatase Kit, according to the manufacturer's instructions. The protein concentration of cell lysates was measured using a micro-BCA assay kit, and ALP activity was normalized to the total protein concentration. Values were the averages of triplicate measurements.",
"To quantify mRNA expression levels, real-time PCR was performed with cDNA samples. Primers were designed using qPrimerDepot(Nucleic Acids Res. 2007), a primer database for quantitative real-time PCR. Real-time PCR was performed using an ABI Steponeplus (ABI PRISM, Carlsbad, NM, USA), which allowed real-time monitoring of increases in PCR product concentrations after each cycle based on the fluorescence of the double-stranded DNA specific dye SYBR green. The number of cycles required to produce a detectable product above background was measured for each sample. These cycle numbers were then used to calculate fold differences in the initial mRNA level for each sample using the following method. First, the cycle number difference for GAPDH, a housekeeping gene, was determined in the control sample and appropriate ODN MT01-treated sample. Values were the averages of triplicate measurements. PCR primers used were as follows: GAPDH forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3'; Type I collagen forward, 5'-AGGGCCAAGACGAAGACATC-3' and reverse, 5'-AGATCACGTCATCGCACAACA-3'; OC forward, 5'-TGAGAGCCCTCACACTCCTC-3' and reverse, 5'-GCCGTAGAAGCGCCGATAGGC-3'.",
"All experiments were performed with triplicate independent samples and repeated at least twice. Results were expressed as the mean ± SD. ANOVA and the Bonferroni post-hoc test were used to compare the differences between the ODN MT01-treatment group and other groups. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed with SAS software (version 8.0, SAS: Cary, NC, USA).",
"In this study, a specific ODN, ODN MT01, was endocytosed by osteoblasts and found to up-regulate the expression level of osteocalcin, type I collagen and ALP in MG 63 cells via the ERK1/2 and p38 MAPK pathways. This study provides further insight into the use of ODN MT01 for in vitro experimentation, and supports the potential use of ODN MT01 to regulate the rebuilding of bone."
] | [] | [
"Introduction",
"Results and Discussion",
"Internalization Analysis of ODNs",
"Effects of ODN MT01 on MAPK Signaling Proteins ERK1/2 and p38",
"Effects of ERK and p38 Inhibitors on Up-Regulation of ALP Activity Induced by ODN MT01",
"Effect of ERK and p38 Inhibitors on Runx2 Expression in Response to ODN MT01",
"Effects of ERK and p38 Inhibitors on the Expression of Osteocalcin and Type I Collagen",
"Experimental Section",
"Materials",
"Cell Culture",
"Fluorescent Labeling of MT01",
"Western Blot Analysis",
"ALP Activity Assay",
"Real-Time PCR",
"Statistical Analysis",
"Conclusions",
"Figure 1 .",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 ."
] | [] | [] | [
"A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways",
"A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways"
] | [
"Int. J. Mol. Sci"
] |
195,064,701 | 2022-02-06T16:46:01Z | null | https://doi.org/10.1172/jci.insight.127748 | GOLD | 7763ded8126fd66d292080ffad0b7de65f640443 | null | null | null | null | 10.1172/jci.insight.127748 | 2950028168 | 31211696 | null |
6-18-2019
School of Medicine
Washington University
Washington University School of Medicine Digital Commons@Becker Digital Commons@Becker
Department of Cell Biology and Physiology
Division of Nephrology
Department of Pediatrics
School of Medicine
Washington University
St. LouisMissouriUSA
Department of Molecular and Cellular Biology
Henry M. Goldman School of Dental Medicine
Boston University
BostonMassachusettsUSA
Cardiovascular Division
Department of Medicine
School of Medicine
Washington University
St. LouisMissouriUSA
School of Medicine
Department of Cell Biology and Physiology
Washington University
Campus, 660 South Euclid AvenueBox 822863110St. LouisMissouriUSA
6-18-201910.1172/jci.insight.127748
Intracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress
Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.
Introduction
Thoracic aortic aneurysm and dissection (TAAD) is a major cardiovascular health problem that arises from mutations that alter vascular extracellular matrix (ECM), smooth muscle cell function, or growth factor signaling pathways (1). In contrast to abdominal aortic aneurysms, TAAD has a strong genetic component, with pathogenic variants typically inherited in an autosomal dominant manner (2). A single highly penetrant, pathogenic mutation is responsible for disease in only one-fourth of individuals with TAAD, indicating that other undiscovered genetic factors influence disease onset and progression. In addition to the known pathogenic gene mutations, low-penetrant risk variants interact with other genetic or environmental factors to trigger disease or to increase the risk for developing disease but are not themselves pathogenic (2). Significant risk factors for TAAD, primarily when occurring in the background of low-penetrant disease-predisposing variants, are hypertension and bicuspid aortic valve (1).
A common histopathological feature of TAAD in humans is fragmented elastin in the aortic medial region (3). Elastin is the most abundant ECM protein in the aorta, where it plays a central role in the vessel's ability to expand and recoil in response to pulsatile blood flow. Smooth muscle cells (SMCs) synthesize and organize the protein into fenestrated sheets (lamellae) that separate smooth muscle cell layers. The secreted form of elastin (tropoelastin) undergoes extensive crosslinking outside the cell to form an elastic polymer, which is the functional form of the protein. The enzyme responsible for initiating crosslink formation is lysyl oxidase (LOX), a copper-binding amine oxidase that oxidizes lysine ε-amino groups in elastin and fibrillar collagens to aldehydes, which then spontaneously react to form covalent linkages between and within proteins (4,5). Approximately 90% of the lysine residues in elastin undergo crosslink formation, which makes it one of the most highly crosslinked and most stable proteins in the body (4). Inactivation of the Lox gene Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.
in mice results in early postnatal death due to ruptured aortic aneurysms, emphasizing the critical role of fully crosslinked, functional elastin in maintaining aorta integrity and function (6,7). The gene inactivation studies also show that other LOX isoforms cannot substitute for the loss of LOX in large, elastin-rich vessels.
LOX in vertebrates belongs to a family of 5 copper-dependent enzymes (LOX, LOXL1-4) that show a high degree of homology in their carboxy-terminal catalytic domain but differ in their amino-terminal sequences. LOXL2-4 form a subgroup that shares 4 amino-terminal scavenger receptor cysteine-rich domains that influence substrate binding but appear to have little role in regulating catalytic activity (8,9). LOX and LOXL1, in contrast, are synthesized without scavenger receptor sequences but have a propeptide domain that keeps the enzymes in a latent state until proteolytically processed outside the cell by BMP1/Tolloid-like metalloproteinases (10,11). Substrate specificity for each of the lysyl oxidases is still under investigation, but gene inactivation studies and phylogenetic analysis suggest that LOX and LOXL1 contribute to crosslinking of elastin and fibrillar collagens, whereas LOXL2-4 have different substrate specificities (12,13). All 5 LOX isoforms are synthesized in the ER and traffic to the Golgi, where the inactive apoenzyme binds copper and forms the cofactor lysyl tyrosine quinone (LTQ); both copper and LTQ are essential active site components required for catalytic activity (14).
Mutations within LOX in families with TAAD were recently identified by whole exome sequencing (3,15). Aortic disease in these families followed autosomal dominant inheritance, suggesting that 1 mutant LOX allele is sufficient to influence disease initiation or progression. Previously, we identified a LOX catalytic site mutation (p.Met298Arg) in a family with a history of TAAD (3). By introducing this human mutation into the homologous position in the mouse genome using CRISPR/Cas9 genome editing technology (c.857T>G encoding p.M292R; hereafter referred to as Mu), we confirmed that this amino acid change caused ruptured TAAD and perinatal death when both alleles were mutated (Lox Mu/Mu ) (3). In contrast to humans who developed TAAD when heterozygous for this LOX variant, aneurysms or other arterial disease were not observed in mice with a similar genotype (Lox +/Mu ) unless they were challenged with hypertension. In this report, we show that the nature of the functional loss associated with the methionine-to-arginine change in LOX results from a secretion defect, where the protein is retained in the ER and degraded through an autophagy/proteasome pathway. We also show that the vessel wall alterations associated with LOX functional haploinsufficiency increase the likelihood of developing vascular disease in association with environmental factors that increase wall stress.
Results
Lox +/Mu adult animals are predisposed to aortic dilation in response to hypertension. The generation and characterization of mice expressing the M292R mutation were described previously (3). Mice heterozygous for this Lox missense variant appear grossly normal, have normal blood pressure, and show no increased mortality through 2 years of age. Aortic diameter and circumferential wall stiffness at physiological blood pressure in Lox +/Mu animals are normal, but ascending aortic length measured from the aortic root to the brachiocephalic artery is 10% longer than in WT mice (3).
Hypertension is a known risk factor associated with aortic dilation (16). To test whether mice expressing mutant LOX are predisposed to aneurysm formation as a consequence of increased wall stress, Lox +/Mu mice were made hypertensive by subcutaneous delivery of angiotensin II (Ang II) via Alzet osmotic pumps. Pumps containing saline served as the control. Ang II infusion is a well-established model for inducing vessel wall changes in mice and other animals (17,18). Blood pressure measurements using radiotelemetry in awake, freely moving Lox +/+ and Lox +/Mu mice showed an increase of approximately 30 mmHg in systolic pressure and 25-30 mmHg in mean blood pressure in both genotypes 7-10 days after pump implantation (data not shown), consistent with what others have found using this model (19). After 4 weeks of treatment, all animals receiving Ang II had cardiac hypertrophy, indicating altered cardiac physiology consistent with Ang II-induced hypertension ( Figure 1A). However, only Lox +/Mu mice showed substantial dilation and elongation of the ascending aorta in response to Ang II; no change in aortic diameter was observed in Ang II-treated Lox +/+ mice or in Lox +/Mu mice infused with saline alone ( Figure 1B). Biaxial pressure-diameter testing showed the ascending aorta in Ang II-treated Lox +/Mu animals to be approximately 25% larger than the aorta from WT or saline-treated controls at mean physiological blood pressure ( Figure 1C). The shape of the pressure-diameter curves suggests that Ang II treatment did not affect aortic mechanical properties in either mutant or WT animals. Other than in the ascending aorta, no dilation or change in vessel mechanics occurred in the left common carotid artery for either Lox +/+ or Lox +/Mu animals with saline or Ang II treatment ( Figure 1D), nor were dilations or areas of stenosis found in the aortic arch, descending aorta, or abdominal aorta.
Ang II treatment leads to vessel wall thickening and changes in vascular cellularity. On a structural level, Ang II treatment resulted in ascending and descending aortic wall thickening in Lox +/Mu animals, but there were no changes in elastic lamellar number or lamellar organization, quantified by measurement of medial area ( Figure 2A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.127748DS1). Elastic lamellar fragmentation, already elevated in Lox +/Mu compared with Lox +/+ animals (3), did not increase in either genotype following Ang II treatment (data not shown). We analyzed elastin crosslinking using an ELISA assay for desmosine, a major crosslink in elastin. We found no difference in desmosine levels in aortic tissue from Lox +/+ and Lox +/Mu mice, confirming normal elastin levels and that elastin crosslinking is not affected in animals heterozygous for the Lox mutation (Supplemental Figure 1C). Hydroxyproline levels (as a measure of total collagen) trended higher in vessels from the mutant animals but were not significantly different from WT when normalized to the change in total protein (Supplemental Figure 1D). Higher protein quantities are consistent with the increase in wall thickness that occurred with Ang II treatment (Supplemental Figure 1B).
Changes in the number and types of cells were observed in the thickened aortic wall of the hypertensive animals. The most substantial change occurred in the adventitia of both the ascending and descending aorta, which underwent considerable expansion in the Lox +/+ and Lox +/Mu animals as a result of Ang II exposure ( Figure 2A). In the medial layer, Ang II infusion led to a decrease in the number of SMCs in Lox +/+ animals but an increase in SMCs in Lox +/Mu mice ( Figure 2B). There was also an increase in intralamellar area in both genotypes, although the change was most notable in Lox +/Mu mice. The nuclei of the intralamellar cells in the mutant media were rounder than cells in Lox +/+ vessels, suggesting a less polarized Lox +/+ and Lox +/Mu animals developed significant cardiac hypertrophy following 4 weeks of treatment with 2 μg/kg/ min Ang II compared with saline-treated controls. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. ****P < 0.0001. (B) The ascending aorta in Lox +/Mu animals is longer than in Lox +/+ animals and undergoes substantial dilation following Ang II treatment. (C) Pressure-diameter measurements at intraluminal pressures ranging from 0 to 175 mmHg. The ascending aorta from Ang II-treated Lox +/Mu animals has a larger diameter over the entire range of pressures compared with Lox +/+ animals treated with Ang II or with saline controls. (D) There was no difference in the diameter or mechanical properties of the left common carotid artery in either genotype with or without Ang II treatment. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each pressure level. Data are presented as mean ± SD. # P < 0.05, Lox +/+ with Ang II vs. Lox +/Mu with Ang II; *P < 0.05, **P < 0.01, Lox +/Mu with saline vs. Lox +/Mu with Ang II.
phenotype and noncircumferential orientation. Immunostaining for smooth muscle α-actin demonstrated uniform reactivity throughout the medial intralamellar spaces, whereas cell staining in the adventitia was heterogeneous (not shown).
Staining for the monocyte/macrophage marker CD68 in descending aorta showed an increased number of CD68 + cells in the media and adventitia of both Lox +/+ and Lox +/Mu descending aorta following Ang II treatment ( Figure 2C). These results are consistent with previous studies showing that infusion of Ang II promotes macrophage accumulation within the aortic wall (20). While the adventitia showed the highest level of immune cell infiltration, macrophage accretion in the medial layer was also observed, but the spatial localization of the CD68 + cells was different for the 2 genotypes. In Ang II-treated Lox +/+ vessels, medial CD68 + cells were associated with the first 2 elastic layers nearest to the intima. Medial CD68 + cells in the aorta of Lox +/Mu animals treated with Ang II, however, were in the outer layers of the media closest to the adventitia ( Figure 2C).
Evaluation of factors that adversely affect vessel wall integrity and SMC function. An explanation for why Lox +/Mu mice are more sensitive than WT animals to Ang II infusion was explored by first checking for possible differences in Ang II receptor expression using quantitative PCR (qPCR). In ascending aorta from Lox +/Mu mice, all 3 Ang II receptors (Agtr1a, Agtr1b, and Agtr2) were detected, and there was no difference in their expression levels . Macrophages (stained green, arrowheads) were detected with an antibody to CD68, and cell nuclei were visualized with DAPI. Red is elastin autofluorescence. The vessel wall was thicker in both Lox +/+ and Lox +/Mu animals following Ang II treatment, with more DAPI + and CD68 + cells in the adventitia of both genotypes. (B) In the medial layer, there were fewer SMCs in the aorta of WT animals treated with Ang II but more cells in Lox +/Mu aorta. Shown are the mean ± SD for cell number counts between the internal and external elastic lamina on 7 tissue sections from each group. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.01. (C) Medial CD68 + cells were mostly located on the luminal side of the media in the Lox +/+ aorta but nearer the adventitia in Lox +/Mu aorta after Ang II treatment.
compared with WT controls (Supplemental Figure 2A). Thus, the potential for Ang II receptor-mediated signaling is similar in both genotypes.
Many Ang II-induced responses on SMCs are attributable to stimulation of NADPH oxidase with the subsequent generation of ROS (21,22). We used dihydroethidium, a cell-permeable compound that interacts with superoxide, to assess oxidant levels in sections of WT and mutant ascending aorta and found no difference between the 2 genotypes (Supplemental Figure 2B). Thus, differences in levels of oxidative stress do not appear to be a factor in the Ang II susceptibility of Lox +/Mu mice.
In many models of aortic disease, the presence of proteases that degrade elastin can initiate and propagate disease by degrading the elastic fiber system in the vessel wall. We therefore investigated whether 3 elastolytic MMPs commonly linked to vascular remodeling -MMP2, MMP9, and MMP12 -were differentially expressed in mutant versus WT aorta. There were no differences in Mmp2 and Mmp9 mRNA expression levels, while expression of Mmp12 was diminished in the mutant aorta (Supplemental Figure 2C).
Active LOX is absent in Lox Mu/Mu mouse embryonic fibroblast conditioned medium. To elucidate how the M292R modification affects LOX function, we used mouse embryonic fibroblasts (MEFs) from WT, KO, and mutant animals to characterize LOX secretion and activity. Mutant cells were indistinguishable from WT cells in morphology and in their rate of proliferation (data not shown). LOX activity ( Figure 3A), assessed using an Amplex Red assay, was not detected in cell layer extracts of any MEF genotype (Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-), which was expected, since the full-length intracellular form of LOX (pro-LOX) is an inactive zymogen. LOX enzymatic activity was readily detected in the conditioned medium from Lox +/+ MEFs and showed linear enzyme kinetics over the period of the Amplex Red assay. Conditioned medium from Lox Mu/Mu MEFs, however, had no detectable activity, similar to conditioned medium from cells with inactivated Lox alleles (Lox -/-) ( Figure 3B). Activity was detected in conditioned medium from Lox +/Mu cells, where the linearity and rate of substrate oxidation were comparable to those seen in Lox +/+ cells, indicating that the mutant form of the protein does not adversely affect the enzymatic activity of WT LOX and hence does not function in a dominant negative fashion.
M292R LOX is poorly secreted and is retained in the ER. LOX is maintained as an inactive 50-kDa proenzyme through every stage of the synthetic and secretory pathway. Activation requires secretion into the extracellular space, where proteolytic removal of the N-terminal propeptide portion of the protein generates the 30-kDa active catalytic domain. To determine whether LOX activity is absent in M292R MEF conditioned medium because mutant LOX is not secreted, we performed Western blots using an anti-LOX antibody on Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF cell layers and conditioned medium. Immunoblotting showed increased levels of 50-kDa intracellular pro-LOX protein in cell extracts from Lox Mu/Mu MEFs compared with Lox +/+ cells ( Figure 4A). Conversely, there was abundant 30-kDa mature (processed) LOX in conditioned medium from WT cells but barely detectable levels in medium from Lox Mu/Mu MEFs ( Figure 4B). These findings indicate that the mutant protein is poorly secreted and is retained intracellularly as the proenzyme. Immunofluorescence imaging with an antibody to LOX showed minimal intracellular staining in Lox +/+ cells, confirming that WT LOX is secreted efficiently ( Figure 5A). Staining of Lox Mu/Mu cells, however, showed abundant intracellular staining, consistent with the immunoblot results described above. The staining pattern for intracellular LOX was typical of accumulation within the ER, which was confirmed by colocalization of LOX with calnexin, an ER-resident protein ( Figure 5, B and C).
A direct interaction between LOX and calnexin was shown using coimmunoprecipitation from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF lysates. Lox -/cells served as the negative control. Cell lysates were immunoprecipitated with an anti-calnexin antibody, followed by immunoblotting using an antibody to LOX. LOX protein was detected in calnexin immunoprecipitates from all genotypes except Lox -/-, confirming an interaction between the 2 proteins ( Figure 5D).
M292R LOX has an altered protein conformation. The high level of LOX detected in Lox Mu/Mu MEF calnexin immunoprecipitates suggests that M292R LOX is misfolded and trapped in the ER bound to calnexin. To determine whether M292R LOX is conformationally different from the WT enzyme, we explored possible structural changes by assessing differential sensitivity to proteolytic degradation. Changes in protein structure frequently expose or mask protease binding sites, leading to altered degradation products and a different degradation fingerprint. Immunoblotting of cell lysate from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs incubated at 37°C for 6 hours to exploit protein degradation by endogenous enzymes within the cell lysate showed time-dependent degradation of WT but not M292R LOX ( Figure 6). These results suggest that WT and M292R pro-LOX have different susceptibilities to degradation arising from cell-associated proteases. Differential degradation was also observed when cell lysates were incubated with pancreatic elastase. In digests of Lox +/+ cell lysate, there was a 25-kDa fragment evident with all doses of elastase that was absent in Lox Mu/Mu samples (Supplemental Figure 3, arrow). When treated with the highest dose of elastase, pro-LOX from Lox +/+ and Lox +/Mu samples degraded predominantly to a 30-kDa fragment. LOX from Lox Mu/Mu samples, in contrast, degraded further to mainly a 10-kDa fragment. It is important to note that pancreatic elastase does not cleave after methionine or arginine, so the Met-to-Arg mutation does not create or negate a protease cleavage site for this enzyme. Together, the conformational difference between the WT and mutant proteins suggests that M292R LOX is misfolded, which is consistent with its protracted interaction with calnexin in the ER.
M292R LOX does not elicit an ER stress response. Accumulation of misfolded proteins in the ER generally leads to ER stress, which subsequently activates the unfolded protein response (UPR) (23). UPR triggers 3 primary stress sensors, PERK, IRE1, and ATF6. Under resting conditions, BIP/Grp78 maintains these sensors in an inactive state. Upon ER stress, BIP binds to misfolded proteins, thereby allowing for sensor activation. To determine whether the continuous presence of misfolded mutant LOX induces ER stress, we assessed Bip transcript levels by qPCR. We also measured mRNA levels of 20% change was modest compared with the ~2-fold and ~4-fold increase in Atf4 and Chop, respectively, when ER stress was stimulated by treatment of MEFs with lopinavir/ritonavir, which served as a positive control (data not shown). Electron microscopic analysis of WT and mutant cells showed that ER volume was not markedly different in the 2 cell types (Supplemental Figure 4), suggesting that mutant protein does not accumulate to levels that would produce dilated saccules. Together, these results suggest that LOX protein containing the M292R mutation does not induce substantial ER stress.
Increased activation of autophagy in Lox Mu/Mu cells. The absence of ER stress in Lox Mu/Mu cells was surprising given that M292R LOX does not leave the ER. To determine whether mutant LOX is being removed from the ER for degradation through the lysosomal pathway, we costained cells for LOX and the lysosomal marker LAMP-2. No colocalization of the 2 proteins was observed, suggesting that the mutant protein was not trafficked through lysosomes (Supplemental Figure 5).
Autophagy is a general mechanism for clearing proteins accumulating in cells. Clearing of misfolded proteins from the ER, a process termed ER-phagy, plays a critical role in ER homeostasis (24). To determine whether mutant LOX directly activates autophagy-mediated protein clearance from the ER, we measured 2 autophagy activation markers, p62 and microtubule-associated protein 1A/1B, light chain 3 (LC3), in WT and Lox Mu/Mu MEFs. Following pretreatment with bafilomycin A1 (BafA1) to inhibit organelle acidification-induced degradation of p62 and LC3, only Lox Mu/Mu cells with and without BafA1 pretreatment had significantly increased p62 expression compared with Lox +/+ cells without BafA1 pretreatment ( Figure 8, A and B). The autophagy activation markers LC3-I and -II were also elevated in mutant compared with WT cells (Figure 8, A, C, and D), providing more evidence for increased autophagy.
We also used electron microscopy to screen for the presence of autophagosome-like vesicles in WT and mutant cells (Supplemental Figure 4). We observed that multi-and single-membrane vesicles typical of autophagosomes were particularly abundant in the perinuclear region but were also present throughout the cytoplasm of Lox Mu/Mu cells. Such structures were rare in WT cells. Together, the existence of autophagic vesicles along with increased expression of autophagy activation markers in mutant cells is consistent with the clearance of misfolded mutant LOX from the ER through an autophagy/proteasome pathway.
Mutant LOX does not influence the secretion of the WT protein.
To assess whether the secretion of WT LOX is affected by the presence of M292R mutant protein, we transiently expressed plasmids containing WT LOX, and M292R LOX fused to the fluorescent protein mApple, in Lox -/-MEFs. Successful transfection was confirmed by intracellular mApple expression using fluorescence imaging (data not shown). In the conditioned medium of Lox -/cells transfected with empty plasmid, no LOX was observed by Western blot analysis ( Figure 9A). In Lox -/-MEFs transfected with 2.5 μg WT Lox-mApple plasmid, an immunoreactive protein band at approximately 60 kDa was detected in the conditioned media, corresponding to the size of active LOX (30 kDa) fused to mApple (27 kDa). In Lox -/-MEFs transfected with 2.5 μg of M292R Lox-mApple plasmid, LOX protein was observed inside the cell by fluorescence microscopy, but no LOX protein was detected in conditioned medium by Western blotting, consistent with the inability of M292R LOX to be secreted. To determine whether mutant LOX alters the secretion of the WT protein, or if the WT protein acts as a chaperone to facilitate the secretion of the mutant protein, we cotransfected cells with 1.25 μg each of WT and mutant plasmid, which is half as much as was used for single plasmid transfection. If mutant LOX does not affect the secretion of the WT protein, then LOX should be detected in conditioned medium, but at half the level of the single plasmid transfection. If the mutant protein has an adverse effect on WT LOX secretion, then levels will be lower than 50%. If WT LOX acts as a chaperone to facilitate secretion of the mutant protein, then the combination of both secreted proteins would be higher than 50% and possibly equivalent to the WT transfection (1.25 + 1.25 = 2.5 μg). Figure 9 shows that LOX protein was detected in conditioned medium of doubly transfected cells at half the level of singly transfected cells, confirming that the mutant protein does not affect the secretion of WT LOX.
M292R propeptide processing. Our experimental results are consistent with the M292R mutation being a loss-of-function phenotype due to a secretion defect. The final activation step of the proenzyme occurs outside the cell through proteolytic separation of the propeptide and catalytic domains. To determine whether M292R pro-LOX could be processed to the 30-kDa mature form if it were secreted, we incubated cell lysates from WT and mutant MEFs with recombinant BMP1 (rBMP1), the enzyme responsible for propeptide cleavage. Western blot analysis showed that BMP1 appropriately processed both WT and M292R pro-LOX to the 30-kDa mature form (Supplemental Figure 6). Thus, the mutation did not inhibit propeptide processing. The processed mutant protein was not assayed for oxidase activity because intracellular LOX in M292R MEFs does not have bound copper and cannot be enzymatically active.
Discussion
LOX plays a critical role in vascular ECM maturation by catalyzing the crosslinking of elastin and fibrillar collagens, the 2 major ECM protein classes in the aortic wall. Phylogenic studies suggest that the current form of vertebrate LOX coevolved with elastin and helped facilitate the appearance of a closed circulatory system (12,25). Therefore, it is no surprise that mutations in LOX give rise to vascular insufficiency and disease. All LOX mutations associated with TAAD in humans show autosomal dominant inheritance, and most result in loss of function. We know from mouse studies that inactivation of both Lox alleles is lethal and that other members of the LOX family cannot assume LOX's critical role in vascular development and maintenance of the vessel wall. In this report, we utilize a mouse model of a well-characterized human mutation to explore how LOX haploinsufficiency leads to vascular dilation and aneurysm formation. Mice heterozygous for the Lox M292R variant (Lox +/Mu ) have increased elastic lamellar fragmentation in the wall of the ascending aorta (3), but do not develop aortic dilation under normal conditions. The absence of aneurysmal disease was surprising given that humans heterozygous for this same mutation are prone to aneurysm formation and vessel wall changes (3). Variability in the age of disease onset and disease severity in humans, however, suggested that a secondary challenge is a factor in disease initiation and progression. Here we show that hypertension induced by Ang II infusion resulted in vessel wall changes in both Lox +/+ and Lox +/Mu mice, but only Lox +/Mu animals showed aortic dilation localized to the ascending aorta. Several groups have reported that the effects of Ang II are most pronounced in the ascending aorta region in this model (26)(27)(28), but the underlying factors governing this vessel specificity are unknown. Differences in the embryonic origin of the cells that populate the aortic wall are one explanation (29,30), but the composition and maturation of the aortic ECM are also factors. In Lox +/Mu mice, changes in ECM crosslinking associated with LOX insufficiency could have more negative consequences for this vessel segment because of high collagen and elastin levels and the high mechanical stress experienced by the aortic wall during the cardiac cycle.
While several Ang II-induced vascular changes were expected (19,27) and common to WT and mutant animals, there were also significant differences. For instance, medial expansion was evident in both genotypes, but the effect was most notable in mutant mice, in which there was an increase in the number of smooth muscle cells between the elastic lamellae. This response is particularly unusual, since the intralamellar space is typically populated by a single smooth muscle cell layer, as can be seen in vessels from saline-treated Lox +/+ and Lox +/Mu animals ( Figure 2). The opposite change occurred in WT vessels, where there were fewer smooth muscle cells in response to Ang II, suggesting Ang II-related apoptosis or growth suppression (26), which we did not assess. mice. Oneway ANOVA with Tukey's multiple-comparisons test was used to assess differences, which were minimal between the 3 genotypes. Data are presented as mean ± SD. ***P < 0.001.
Another cellular change in the ascending and descending aorta following Ang II treatment was the accumulation of macrophages in the aortic wall of both WT and Lox +/Mu mice. CD68 + cells were found in the adventitia of both genotypes, with fewer cells in the medial layer. In Ang II-treated WT mice, CD68 + cells accumulated in the intima and inner elastic layers of the media and did not localize to breaks in elastic lamella, as has been reported in other models (18,20). Their location within the wall, however, shows that some cells were able to penetrate past the internal elastic lamina into the first or second layer of smooth muscle cells. Medial CD68 + cells in Lox +/Mu mice exposed to Ang II, in contrast, were localized to the outer aspect of the media and occurred at higher numbers than in WT vessels. These results suggest that the cellular origin of the CD68 + cells or the route of inflammatory cell infiltration is different in Lox +/+ and Lox +/Mu vessels as a response to wall stress.
The location of macrophages in the outer region of the wall provides a mechanistic explanation for why Lox +/Mu , but not WT, aorta dilates following Ang II treatment. ECM degradation is a primary factor leading to vessel wall dilation. Elastases and other matrix-degrading enzymes secreted by macrophages destabilize the vessel wall, particularly when matrix degradation occurs in or near the adventitia -the vessel compartment essential for maintaining the structural integrity of the artery (31). The medial cell hypertrophy and inflammatory cell accumulation in the Lox +/Mu aorta in response to hemodynamic stress are similar in several ways to vascular changes in mouse models of Marfan syndrome with mutations in fibrillin-1, a key elastic fiber protein. In Marfan mice, inflammatory cell accumulation at the medial-adventitial interface correlates with increased elastin degradation and a loss C and D) Also, LC3 was elevated in Lox Mu/Mu cells compared with Lox +/+ cells. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.001.
of structural integrity that leads to vessel wall dilation (32). As in the Ang II-treated Lox +/Mu animals, only the ascending aorta is affected in Marfan mice.
Our previous studies showed that mice with the Lox M292R mutation share numerous phenotypic traits with Lox +/animals (3), suggesting that M292R is a loss-of-function mutation in mice and humans. Even though the amino acid change is in the catalytic domain of LOX close to the copper binding site, it was not clear whether the mutation results in a catalytically dead enzyme or if some other aspect of LOX processing is negatively affected by the mutation. Our data show that the answer fits both possibilities: the mutant protein is retained in the ER and not secreted, and the enzyme is catalytically dead because of its inability to traffic to the Golgi, where it needs to bind copper and form the redox quinone cofactor LTQ required for catalytic activity (33). Even though we found that the propeptide of mutant pro-LOX could be cleaved by rBMP1 similarly to WT LOX, full-length LOX in the ER cannot be catalytically active even if propeptide processing does occur.
Why mutant LOX is retained in the ER is not clear, but our data point to protein misfolding as a probable cause. ER retention is characteristic of incompletely folded proteins. Folding intermediates of most secreted proteins interact with the calnexin/calreticulin protein complex in the ER to achieve the proper native conformation required for secretion (34). The calnexin/calreticulin protein complex recognizes N-linked glycan structures (LOX has 3 N-linked glycosylation sites in the propeptide region; ref. 11) that undergo cycles of sugar residue removal and addition. Once folded correctly, calnexin and calreticulin dissociate, and the folded proteins are transported from the ER to the Golgi apparatus. Mutant LOX colocalized intracellularly with calnexin and was isolated bound to calnexin in cellular extracts, suggesting that the protein remains misfolded despite repeated folding interactions with calnexin. Furthermore, the differential susceptibility to proteolytic degradation of mutant compared with WT LOX is consistent with structural differences between the 2 proteins.
It was interesting that mutant LOX accumulation in the ER did not induce an appreciable UPR. We saw no upregulation in mutant cells of the ER stress-related markers Bip (an ER luminal chaperone protein and critical ER stress sensor), Atf4 (a transcription factor downstream of PERK that plays a crucial role in the adaptation to ER stress), or Chop (a transcription factor regulated by ATF4 that drives proapoptotic gene transcription). Nor could we detect in mutant cells an increase in ER volume frequently associated with ER stress. One pathway whereby ER stress is mitigated is removal from the ER of mutant protein and degradation at the lysosome (ERAD-II) or proteasome (ERAD-I) (24). We were unable to localize LOX within lysosomes but did find increased expression of the autophagy markers p62 and LC3. Electron microscopy also identified an increased number of autophagic vesicles in Lox Mu/Mu cells. These results suggest that misfolded mutant LOX is being cleared from the ER through an autophagy/proteasome pathway and would explain why mutant protein levels never reached a critical threshold to sustain an ER stress response. Furthermore, there is mounting evidence that the autophagy and secretory pathways are coregulated and intimately linked (35), which may represent a mechanistic connection into the LOX trafficking defect that we identified. It should be noted that all the experiments probing the fate of the mutant protein were done in cultured cells. It is possible, but unlikely, that in vivo, in tissues, and at developmental stages relevant to the TAAD phenotype, LOX folding, secretion, ER retention, and stress occur differently than in MEFs.
Guo et al. (15) described 2 other LOX active site variants (S280R and S348R) that segregate with disease in families with TAAD. When expressed from transgenes in HeLa cells and examined in cell lysates, a portion of the pro-LOX protein was processed to its active form and was able to oxidize substrate at rates approximately equivalent to the WT enzyme (S348R) or with approximately 50% lower activity (S280R) (2,15). Enzymatic activity in conditioned medium from the transfected cells was not evaluated, so it is not known whether these LOX variants are efficiently secreted. However, the fact that the proteins are catalytically active implies trafficking through the Golgi and proteolytic removal of the propeptide domain. Thus, unlike the M292R mutation, which is retained in the ER, the S348R and S280R variants appear capable of traversing the secretory pathway. How these proteins, which have full (or somewhat reduced) catalytic activity, cause thoracic aortic disease is an interesting question. An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.
Methods
Antibodies. Antibodies used in this study are described in Supplemental Table 1.
Mouse models and cell isolation. A knockin mouse strain bearing the Lox M292R substitution was generated in the C57BL/6 background using CRISPR/Cas9 genome editing technology as previously described (3). Lox +/mice in the same background are described in ref. 6. Primary MEFs were isolated from the various mouse lines by digestion of E14.5 mouse embryos with trypsin/EDTA at 37°C for 30 minutes in DMEM (38). Cells were collected by centrifugation and grown in DMEM, 10% FBS, 1% penicillin/streptomycin, and 1 mM l-glutamine.
Ang II treatment and blood pressure measurements. Telemetry transmitters (Data Sciences International, model PA-C10) were implanted surgically in 3-to 4-month-old mice under isoflurane anesthesia. The catheter was inserted in the left common carotid artery and advanced to the aortic arch, and the transmitter was placed subcutaneously along the abdomen. Animals were allowed to recover for 7 days prior to activation of transmitters, and continuous recording of heart rate and arterial pressure was obtained, with sampling every 5 minutes for 15-second intervals for 3 days. Data were collected and stored using Dataquest ART (Data Sciences International).
After baseline blood pressure measurements were obtained, mice were implanted with osmotic pumps (Alzet model 1004, DURECT Corp.) that delivered Ang II (MilliporeSigma) subcutaneously at a dose of 2 μg/kg/min in the interscapular region, according to published procedures (39,40). Mice infused with saline were used as controls. After a week of Ang II treatment, arterial pressure and heart rate were again continuously recorded using the telemeters, with sampling every 5 minutes for 15-second intervals for 3 days. Animals were fed a standard laboratory chow diet. After 28 days, animals were sacrificed under isoflurane anesthesia, and vessels were collected for compliance studies and histological characterization.
Vessel compliance measurement. For vessel compliance measurements, the ascending aorta and left common carotid artery were excised and placed in a physiological saline solution. Vessels were then cleaned of surrounding fat and connective tissue and mounted onto a pressure arteriograph (Danish Myo Technology). Experiments were done at 37°C in an organ bath containing physiological saline. The mounted vessels were visualized using a computerized imaging system, allowing continuous recording of vessel diameters. Vessel outer diameters were recorded while intravascular pressure was increased from 0 to 175 mmHg by increments of 25 mmHg (12 seconds per step). The average of 3 outer diameter measurements was taken at each pressure (40,41).
Protein quantification, hydroxyproline, and desmosine assays. Each ascending aorta was hydrolyzed at 105°C overnight with 20 μL of 6 N hydrochloric acid (Thermo Fisher Scientific) and dried under vacuum in a SpeedVac. The dried pellet was dissolved in 400 μL water and filtered with a 0.45-μm filter. Total protein, hydroxyproline, and desmosine levels were determined as described previously (42).
Dihydroethidium staining for superoxide detection in aortic sections. Superoxide in aortic sections was detected using dihydroethidum (DHE) staining. In the presence of superoxide, DHE is oxidized to 2-hydroxyethidium, which is highly fluorescent and intercalates within the DNA, staining the nucleus a fluorescent red (43,44). After euthanasia and thoracotomy, the right atrium was clipped, and 5 mL cold (4°C) 1× PBS was flushed through the left ventricle to clear the blood. Ascending aorta was then dissected, placed in Tissue-Tek O.C.T. Compound (Sakura Finetek), and flash frozen in liquid nitrogen. Four-micrometer cryosections were obtained using a Leica CM1850 cryostat and placed on charged microscope slides (Thermo Fisher Scientific). A 10-mM stock solution of DHE (Life Technologies) in DMSO was diluted to 10 μM using 1× PBS. The 10-μM DHE solution was applied to unfixed aortic sections and incubated at 37°C for 30 minutes in the dark. After staining with DHE, sections were then rinsed with 1× PBS for 5 minutes at room temperature (RT) and mounted with a coverslip using ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were obtained on the Zeiss Axioscope fluorescence microscope with a QImaging MicroPublisher 3.3 RTV camera using QCapture software (QImaging). The entire procedure (from mouse euthanasia to image acquisition) occurred within 8 hours.
Immunofluorescence on aortic sections and area measurements. Ascending and descending aortas were dissected as above and placed in O.C.T. compound, and the blocks were frozen on dry ice. Four-micrometer-thick cryosections were placed on glass slides and stored at -80°C. The sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS at 4°C for 10 minutes, washed twice with 1× PBS (5 minutes each) at RT, and blocked with a solution containing 1% BSA (MilliporeSigma), 1% fish gelatin (MilliporeSigma), and 0.05% Triton-X (Acros Organics) in 1× PBS for 1 hour at RT. The aortic sections were then incubated with rat anti-mouse CD68 antibody (Bio-Rad) at a 1:1000 dilution in blocking solution at 4°C overnight. The next day, sections were washed 3 times with 1× PBS (5 minutes each) and incubated in goat anti-rat IgG Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) diluted 1:2000 and DAPI diluted 1:3000 in blocking solution at RT for 1 hour. The sections were washed twice with 1× PBS (5 minutes each) at RT, mounted with a coverslip using ProLong Diamond Antifade Mountant, and imaged using the Axioscope fluorescence microscope equipped with the QImaging MicroPublisher 3.3 RTV camera using QCapture software as above. For area measurements, frozen sections from the descending aorta from 3-month-old mice were prepared as described above. The area between the internal and external elastic laminae was traced in each ×40 image using ImageJ software (NIH) and measured in pixels 2 .
LOX activity assay. MEFs were grown in 10-cm dishes and maintained as above until fully confluent. Twenty-four hours before collection, culture medium was replaced with DMEM lacking phenol red and FBS, supplemented with 50 μg/mL ascorbic acid and 0.1% BSA. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. Cell lysates were collected by scraping in 1× PBS containing protease inhibitors. LOX activity in cell lysate and concentrated culture medium, incubated with and without the LOX inhibitor β-aminopropionitrile (BAPN), was quantified using the Amplex Red assay as previously described (3,45). Resorufin fluorescence, the product of Amplex Red oxidation, was measured at excitation and emission wavelengths (540 and 600 nm, respectively) every 30 minutes for 150 minutes using a BioTek H4 Hybrid Reader. LOX activity was calculated as the difference between total activity and activity in the presence of BAPN. Differences in relative fluorescent units were tested using 2-way ANOVA with Tukey's multiple-comparisons test.
Immunofluorescence imaging of primary cells. Primary MEFs were seeded in 4-chamber glass slides at 3 × 10 5 cells per/well. After 3 days in culture, the cells were fixed with cold methanol (2 washes, 5 seconds each) followed by 3 washes with blocking solution containing 1× PBS, 1% BSA, 1% fish gelatin (Milli-poreSigma), and 0.05% Triton-X100 at pH 7.4, then incubated in blocking solution for 30 minutes at RT. The cells were then incubated with a primary antibody to LOX at 1:1000 in blocking solution overnight at 4°C. After washing the cells with blocking solution to remove the primary antibody, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor 568 secondary antibody at 1:2000 (Thermo Fisher Scientific) and anti-calnexin primary antibody directly labeled with Alexa Fluor 488 at 1:4000 in blocking solution for 30 minutes at RT. The cells were washed with blocking solution 3 times, then mounted using ProLong Diamond Antifade Mountant (Invitrogen) and coverslipped. The samples were imaged using the Axioscope fluorescence microscope and QCapture Pro software.
Protein extraction from cell culture and aortic tissue. Cultured MEFs were switched to serum-free medium 24 hours before protein extraction. MEFs in 10-cm dishes were incubated in 1 mL lysis (RIPA) buffer (MilliporeSigma) containing protease inhibitors for 30 minutes at 4°C. The lysates were scraped from the dish into microfuge tubes and centrifuged at 17,500 g for 5 minutes, and the supernatant was collected. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. The protein concentration in cell lysates and concentrated media was measured using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Laemmli buffer with and without DTT was added to samples for SDS-PAGE and native gel electrophoresis, respectively. Only the samples with DTT were boiled at 100°C for 5 minutes.
RNA extraction and qPCR. RNA was extracted from confluent cell cultures using TRIzol (Thermo Fisher Scientific) following the manufacturer's protocol. For RNA isolation from aortic tissue, ascending aorta from adult Lox +/+ and Lox +/Mu mice were each homogenized in 500 μl TRIzol using QIAGEN TissueLyser. The manufacturer's protocol was followed for RNA isolation. At the time of RNA precipitation, 10 μg glycogen (Mil-liporeSigma) was added as a carrier to each sample. Each RNA pellet was dissolved in 10 μL molecular-grade water. The RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). One microgram of the isolated RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Reverse transcription was then carried out using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). One microliter of cDNA was used for qPCR using TaqMan Fast Universal PCR Master Mix and TaqMan assay primer/probes (Life Technologies) for Mmp12 (Mm00500554_m1), and Gapdh (Mm9999999915_ g1), which was used as an internal control. Ten-microliter reactions were performed in duplicate using a ViiA 7 Real-Time PCR System for Bip, Atf4, and Chop and QuantStudio 3 Real-Time PCR system (Applied Biosystems) for Agtr1a, Agtr1b, Agtr2, Mmp2, Mmp9, and Mmp12. All mRNA levels were normalized to Gapdh expression. rBMP1 treatment. Protein was extracted from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs as described above. Fifty microliters of cell lysates from cultured MEFs containing 50 μg total protein were incubated with 30, 60, and 90 ng rBMP1 (R&D Systems) for 45 minutes at 37°C in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5). The enzyme reaction was stopped by adding Laemmli buffer with DTT and incubated at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.
Bip (Mm00517691_m1), Atf4 (Mm00515325_g1), Chop (Mm01135937_g1), Agtr1a (Mm01957722_s1), Agtr1b (Mm02620758_s1), Agtr2 (Mm00431727_91), Mmp2 (Mm00439498_m1), Mmp9 (Mm00442991_m1),
Western blotting. Protein extracts from cells, concentrated condition medium, or tissues (generally 50 μg) were analyzed using 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) run at 100 mV for 1 hour. The protein was then transferred to ProBlott Membrane (Applied Biosystems). The blots were incubated in 5% nonfat dried milk in 0.1% 1× PBS-Tween for 1 hour at RT, then incubated with a primary antibody overnight at 4°C. The next day, the blots were washed with 0.1% 1× PBS-Tween and incubated in ECL anti-rabbit or anti-mouse IgG HRP-Linked Secondary Antibody (GE Healthcare) for 1 hour at RT. The blots were then washed and the immunoreactive bands detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The blots were stripped by incubating in 2% SDS, 75 mM Tris-HCl, and 100 mM β-mercaptoethanol for 1 hour at 65°C while shaking. The blot was then incubated in 0.1% 1× PBS-Tween wash buffer for 1 hour at RT before immunodetection of β-actin (MilliporeSigma) at 1:20,000 in wash buffer, which was used as a loading control. For protein samples from concentrated conditioned media, membranes were stained with Ponceau S solution (MilliporeSigma) before blocking and used as a loading control. Alternatively, stain-free images of Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) taken following the manufacturer's protocol served as a loading control.
Coimmunoprecipitation. Cell lysates were prepared from MEFs 5 days after confluency as described above. Immobilized protein A anti-rabbit IgG beads (50 μL), preequilibrated by washing with lysis buffer, were then added to 250 μL cell extract. After incubation on a rocking platform for 60 minutes at 4°C, the sample was centrifuged at 2500 g for 3 minutes, and the supernatant was incubated with 8 μg immunoprecipitation antibody for 1 hour at 4°C. Fifty microliters of precleared protein A beads was then added, and the samples incubated overnight at 4°C with rocking. Immune complexes bound to the protein A beads were collected by centrifugation and washed with lysis buffer prior to boiling at 100°C for 10 minutes with Laemmli buffer containing DTT. The supernatant was collected by centrifugation, and proteins were separated by SDS-PAGE and immunoblotted following the same protocol described above.
Protein degradation analysis. Confluent cultures of WT and mutant MEFs were treated with 5 μg/mL Brefeldin A (MilliporeSigma) to inhibit protein transport from ER to the Golgi apparatus. This ensured that WT LOX remained in the cells so that the starting amount of protein was the same across genotypes. After 4 hours, the cell lysate was extracted using lysis buffer placed at 37°C to initiate protein degradation. Every hour, 50 μL aliquot of cell lysate was collected, then immediately boiled with Laemmli sample buffer at 100°C for 5 minutes prior to SDS-PAGE analysis. For exogenous protease-mediated degradation, human pancreatic elastase in increasing concentrations (0.625, 1.25, 2.5, 5 ng/μL) was added to cell lysates containing 1 mg/ mL protein at 4°C for 30 minutes. After treatment, elastase activity was inhibited with protease inhibitor cocktail, and samples were boiled with Laemmli sample buffer at 100°C for 5 minutes for SDS-PAGE analysis.
Autophagy markers. Confluent MEFs in 6-well dishes were pretreated with 100 mM BafA1 (MilliporeSigma) for 3 hours before cell lysates were collected. The protein samples were separated on 4%-15% gradient gels for SDS-PAGE analysis. Immunoblotting was performed following the same procedure as above.
Lox-mApple plasmid cloning and expression. The mouse Lox cDNA in a mammalian expression vector driven by a pCMV promoter was purchased from GE Healthcare. The Lox open reading frame was PCR amplified using a 5′ primer bearing an XhoI restriction enzyme site (5′-CAGATCTCGAGAGCGTTTCGCCTGGGCTGTGC-3′) and a 3′ primer designed to remove the Lox stop codon bearing a BamHI restriction enzyme site (5′-GGATCCA-CATACGGTGAAATTGTGCAGCCTGAG-3′). The PCR product and the pGEMT shuttle vector (Promega) were digested with XhoI and BamHI, then ligated together using T4 ligase. The mutant Lox plasmid bearing the 893 T>G base pair change was generated using the Lox-pGEMT plasmid as the template, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific), following the manufacturer's protocol (5′-TTACCA-CAGCAGGGACGAATTCAGCCACTATG-3′ and 5′-TGTTGGTGACAGCTGTGCCACTCCC-3′). The WT and mutant Lox-pGEMT plasmid sequences were confirmed using Sanger sequencing performed at the Washington University Protein and Nucleic Acid Chemistry Laboratory. To generate the Lox-mApple plasmids, WT and mutant Lox pGEMT plasmids and the mApple plasmid (46), provided by David W. Piston (Washington University School of Medicine in St. Louis), were digested with XhoI and BamHI restriction enzymes. The WT and mutant mLox cDNA inserts were then ligated into mApple vectors to generate the mLox-mApple fusion plasmids and sequenced to confirm. To express the WT and mutant Lox-mApple plasmids in MEFs, Lipofectamine 3000 reagents (Thermo Fisher Scientific) were used following the manufacturer's protocol.
Transmission electron microscopy. MEFs from Lox +/+ , Lox +/Mu , and Lox Mu/Mu mice, cultured on glass coverslips, were washed in cacodylate buffer (0.15 M cacodylate in 2 mM CaCl 2 , pH 7.4) and fixed for 5 minutes with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer prewarmed to 37°C. The samples were then incubated at RT for an additional hour, washed with cacodylate buffer, and treated with 1% OsO 4 /1.5% potassium ferrocyanide for 1 hour in the dark. After washing with ultrapure water, en bloc staining was performed in 2% uranyl acetate for 1 hour in the dark. The samples were washed again in ultrapure water before dehydration in a graded acetone series. Finally, the samples were embedded in Epon resin and cured in a 60°C oven for 48 hours. The processed samples were imaged using a JEOL 1400 electron microscope.
Statistics. One-way ANOVA with Tukey's multiple-comparisons test was used to determine the significance, if any, between different groups, particularly genotypes. When 2 variables were present, e.g., genotype and treatment or genotype and gene, 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences. If the same sample was measured repeatedly (e.g., at different pressures in the pressure-diameter curves in Figure 1, C and D, or at different time points in Figure 3), then repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was performed. Prism 8 for Mac OS X (GraphPad Software) was used to run statistical analyses. In all numerical figures, data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical test used and significant differences are noted in each figure legend.
Study approval. All animal experiments were carried out following protocols approved by Washington University School of Medicine Institutional Animal Care and Use Committee.
Author contributions
VSL, CMH, TJB, and PCT participated in experimental design and acquired and analyzed primary data. NOS helped with data analysis, and RPM designed the research study and analyzed primary data. VSL wrote the initial draft of the manuscript, to which all authors contributed edits.
Vivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.
Figure 1 .
1The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both
Figure 2 .
2Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)
Figure 3 .
3LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .
Atf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the
Figure 4 .
4Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.
Figure 5 .
5Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.
Figure 6 .
6M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.
Figure 7 .
7M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu
Figure 8 .
8Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (
Figure 9 .
9Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A.
insight.jci.org https://doi.org/10.1172/jci.insight.127748
AcknowledgmentsThis work was supported by NIH grants R01HL53325, HL105314 (RPM), R21AR072748 (PCT), and R01HL131961 (NOS). Funds were also provided to RPM by the Ines Mandl Research Foundation. VSL was supported by training grant T32 HL125241 and a Predoctoral Individual National Research Service Award (F31HL136073). CMH was supported by NIH grant K08 HL135400. We thank Robyn Roth for assistance with electron microscopy and gratefully acknowledge assistance from James Fitzpatrick and staff at the Washington University Center for Cellular Imaging (WUCCI), which is supported by Washington University School of Medicine, the Children's Discovery Institute of Washington University and St. Louis Children's Hospital (CDI-CORE-2015-505), and the Foundation for Barnes-Jewish Hospital (grant no. 3770). Additionally, we thank Michael Wallendorf of the Washington University Division of Biostatistics for assistance with statistical data analysis.
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"Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. D M Milewicz, S K Prakash, F Ramirez, 10.1146/annurev-med-100415-022956Annu Rev Med. 68Milewicz DM, Prakash SK, Ramirez F. Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models. Annu Rev Med. 2017;68:51-67.",
"Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \"risk variants. C S Kwartler, 10.1016/j.ajhg.2018.05.012Am J Hum Genet. 1031Kwartler CS, et al. Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low pene- trant \"risk variants.\" Am J Hum Genet. 2018;103(1):138-143.",
"Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. V S Lee, 10.1073/pnas.1601442113Proc Natl Acad Sci U S A. 11331Lee VS, et al. Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans. Proc Natl Acad Sci U S A. 2016;113(31):8759-8764.",
"B A Kozel, R P Mecham, Rosenbloom J Elastin, Biology of Extracellular Matrix: An Overview. Berlin-Heidelberg. Mecham RPGermanySpringer-VerlagKozel BA, Mecham RP, Rosenbloom J. Elastin. In: Mecham RP, ed. Biology of Extracellular Matrix: An Overview. Berlin-Heidel- berg, Germany: Springer-Verlag; 2011:267-301.",
"Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. P C Trackman, 10.1016/j.matbio.2016.01.001Matrix Biol. Trackman PC. Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. Matrix Biol. 2016;52-54:7-18.",
"Lysyl oxidase is required for vascular and diaphragmatic development in mice. I K Hornstra, S Birge, B Starcher, A J Bailey, R P Mecham, S D Shapiro, 10.1074/jbc.M210144200J Biol Chem. 27816Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem. 2003;278(16):14387-14393.",
"Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. J M Mäki, 10.1161/01.CIR.0000038109.84500.1ECirculation. 10619Mäki JM, et al. Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation. 2002;106(19):2503-2509.",
"Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. A J López-Jiménez, T Basak, R M Vanacore, 10.1074/jbc.M117.798603J Biol Chem. 29241López-Jiménez AJ, Basak T, Vanacore RM. Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV. J Biol Chem. 2017;292(41):16970-16982.",
"Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. J M Mäki, H Tikkanen, K I Kivirikko, 10.1016/S0945-053X(01)00157-3Matrix Biol. 207Mäki JM, Tikkanen H, Kivirikko KI. Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. Matrix Biol. 2001;20(7):493-496.",
"Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. A Borel, 10.1074/jbc.M109499200J Biol Chem. 27652Borel A, et al. Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. J Biol Chem. 2001;276(52):48944-48949.",
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"Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. M Bignon, 10.1182/blood-2010-10-313296Blood. 11814Bignon M, et al. Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood. 2011;118(14):3979-3989.",
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"Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. K Saraff, F Babamusta, L A Cassis, A Daugherty, 10.1161/01.ATV.0000085631.76095.64Arterioscler Thromb Vasc Biol. 239Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23(9):1621-1626.",
"Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. A E Dikalova, M C Góngora, D G Harrison, J D Lambeth, S Dikalov, K K Griendling, 10.1152/ajpheart.00242.2010Am J Physiol Heart Circ Physiol. 2993Dikalova AE, Góngora MC, Harrison DG, Lambeth JD, Dikalov S, Griendling KK. Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. Am J Physiol Heart Circ Physiol. 2010;299(3):H673-H679.",
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"Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. A P Owens, 10.1161/CIRCRESAHA.109.212837Circ Res. 1063Owens AP, et al. Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. Circ Res. 2010;106(3):611-619.",
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"Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. R Pyo, 10.1172/JCI8931J Clin Invest. 10511Pyo R, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest. 2000;105(11):1641-1649.",
"Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. L Pereira, 10.1073/pnas.96.7.3819Proc Natl Acad Sci U S A. 967Pereira L, et al. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci U S A. 1999;96(7):3819-3823.",
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"Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. J X Zhang, I Braakman, K E Matlack, A Helenius, 10.1091/mbc.8.10.1943Mol Biol Cell. 810Zhang JX, Braakman I, Matlack KE, Helenius A. Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell. 1997;8(10):1943-1954.",
"The link between autophagy and secretion: a story of multitasking proteins. H Farhan, M Kundu, S Ferro-Novick, 10.1091/mbc.e16-11-0762Mol Biol Cell. 289Farhan H, Kundu M, Ferro-Novick S. The link between autophagy and secretion: a story of multitasking proteins. Mol Biol Cell. 2017;28(9):1161-1164.",
"Functional importance of lysyl oxidase family propeptide regions. P C Trackman, 10.1007/s12079-017-0424-4J Cell Commun Signal. 121Trackman PC. Functional importance of lysyl oxidase family propeptide regions. J Cell Commun Signal. 2018;12(1):45-53.",
"Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. S D Vallet, 10.1038/s41598-018-30190-6Sci Rep. 8111768Vallet SD, et al. Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners. Sci Rep. 2018;8(1):11768.",
"Isolation and handling of mouse embryonic fibroblasts. K Jain, P J Verma, J Liu, 10.1007/978-1-4939-1215-5_13Methods Mol Biol. 1194Jain K, Verma PJ, Liu J. Isolation and handling of mouse embryonic fibroblasts. Methods Mol Biol. 2014;1194:247-252.",
"Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. A Daugherty, M W Manning, L A Cassis, 10.1172/JCI7818J Clin Invest. 10511Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000;105(11):1605-1612.",
"Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. C M Halabi, T J Broekelmann, R H Knutsen, L Ye, R P Mecham, B A Kozel, Am J Physiol Heart Circ Physiol. 3095Halabi CM, Broekelmann TJ, Knutsen RH, Ye L, Mecham RP, Kozel BA. Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness. Am J Physiol Heart Circ Physiol. 2015;309(5):H1008-H1016.",
"Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. G Faury, 10.1172/JCI19028J Clin Invest. 1129Faury G, et al. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency. J Clin Invest. 2003;112(9):1419-1428.",
"Measurement of elastin, collagen, and total protein levels in tissues. I Stoilov, B C Starcher, R P Mecham, T J Broekelmann, 10.1016/bs.mcb.2017.08.008Methods Cell Biol. 143Stoilov I, Starcher BC, Mecham RP, Broekelmann TJ. Measurement of elastin, collagen, and total protein levels in tissues. Methods Cell Biol. 2018;143:133-146.",
"Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity. H Cai, S Dikalov, K K Griendling, D G Harrison, 10.1007/978-1-59745-571-8_20Methods Mol Med. 139Cai H, Dikalov S, Griendling KK, Harrison DG. Detection of reactive oxygen species and nitric oxide in vascular cells and tis- sues: comparison of sensitivity and specificity. Methods Mol Med. 2007;139:293-311.",
"Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. B Fink, K Laude, L Mccann, A Doughan, D G Harrison, S Dikalov, 10.1152/ajpcell.00028.2004Am J Physiol Cell Physiol. 2874Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S. Detection of intracellular superoxide formation in endothe- lial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol. 2004;287(4):C895-C902.",
"Measurement of lysyl oxidase activity from small tissue samples and cell cultures. P C Trackman, M V Bais, 10.1016/bs.mcb.2017.08.009Methods Cell Biol. 143Trackman PC, Bais MV. Measurement of lysyl oxidase activity from small tissue samples and cell cultures. Methods Cell Biol. 2018;143:147-156.",
"Photoconversion in orange and red fluorescent proteins. G J Kremers, K L Hazelwood, C S Murphy, M W Davidson, D W Piston, 10.1038/nmeth.1319Nat Methods. 65Kremers GJ, Hazelwood KL, Murphy CS, Davidson MW, Piston DW. Photoconversion in orange and red fluorescent proteins. Nat Methods. 2009;6(5):355-358."
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"Therapeutics targeting drivers of thoracic aortic aneurysms and acute aortic dissections: insights from predisposing genes and mouse models",
"Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant \"risk variants",
"Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans",
"Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone",
"Lysyl oxidase is required for vascular and diaphragmatic development in mice",
"Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice",
"Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV",
"Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains",
"Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1",
"Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor",
"Origin and evolution of lysyl oxidases",
"Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane",
"LOX mutations predispose to thoracic aortic aneurysms and dissections",
"Central artery stiffness and thoracic aortopathy",
"Aortic aneurysms",
"Angiotensin II-mediated development of vascular diseases",
"Origin of Matrix-producing cells that contribute to aortic fibrosis in hypertension",
"Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice",
"Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling",
"Redox stress in Marfan syndrome: dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm",
"The unfolded protein response and cell fate control",
"Vascular extracellular matrix and arterial mechanics",
"Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3",
"Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/-mice",
"Ascending aortic aneurysm in angiotensin II-infused mice: formation, progression, and the role of focal dissections",
"Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall",
"Developmental basis of vascular smooth muscle diversity",
"Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms",
"Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1",
"Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors",
"Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations",
"The link between autophagy and secretion: a story of multitasking proteins",
"Functional importance of lysyl oxidase family propeptide regions",
"Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners",
"Isolation and handling of mouse embryonic fibroblasts",
"Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice",
"Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness",
"Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency",
"Measurement of elastin, collagen, and total protein levels in tissues",
"Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity",
"Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay",
"Measurement of lysyl oxidase activity from small tissue samples and cell cultures",
"Photoconversion in orange and red fluorescent proteins"
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"Trends Cardiovasc Med",
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"\n\nVivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.",
"\nFigure 1 .\n1The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both",
"\nFigure 2 .\n2Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)",
"\nFigure 3 .\n3LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .",
"\n\nAtf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the",
"\nFigure 4 .\n4Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.",
"\nFigure 5 .\n5Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.",
"\nFigure 6 .\n6M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.",
"\nFigure 7 .\n7M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu",
"\nFigure 8 .\n8Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (",
"\nFigure 9 .\n9Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A."
] | [
"Vivian S. Lee, … , Nathan O. Stitziel, Robert P. Mecham JCI Insight. 2019;4(15):e127748. https://doi.org/10.1172/jci.insight.127748.",
"The ascending aorta in Lox +/Mu animals undergoes aortic dilation in response to hypertension. (A) Both",
"Ang II treatment leads to changes in vascular cellularity and infiltration of immune cells. (A) Frozen sections of descending aorta from Lox +/+ and Lox +/Mu animals following 28 days of treatment with Ang II or saline (asterisks indicate vessel lumen; scale bars: 5 μm)",
"LOX enzymatic activity is absent in Lox Mu/Mu cells. (A) LOX activity in cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/mouse embryonic fibroblasts measured using the Amplex Red assay over 150 minutes and expressed as relative fluorescence units (RFU). As expected, LOX activity was not detected in any of the cell extracts, as intracellular LOX is catalytically inactive until proteolytically processed outside of cells. (B) LOX activity in conditioned medium from the same cells as in A. LOX activity was detected in Lox +/+ and Lox +/Mu cells, but was absent in conditioned medium from Lox Mu/Mu MEFs. Cells from Lox -/animals served as a negative control. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each time point. Values are mean ± SD. *P < 0.05, ****P < 0.0001, Lox +/+ vs. Lox Mu/Mu .",
"Atf4 and Chop, downstream transcriptional activators of the PERK pathway. Atf4 and Chop were not elevated in Lox Mu/Mu MEFs compared with Lox +/+ cells (Figure 7, A and B). While there was a statistically significant increase in Bip mRNA in mutant cells(Figure 7C), the",
"Mutant LOX is retained inside the cell. (A) Immunoblotting of MEF lysate with an antibody to LOX shows increased amounts of pro-LOX (50 kDa) in Lox Mu/Mu cells compared with Lox +/+ MEFs. (B) Immunoblots of conditioned medium demonstrate less processed LOX (30 kDa) associated with Lox Mu/Mu cells, which is consistent with LOX retention within the cell. β-Actin-and Ponceau S-stained gel blots served as loading controls.",
"Intracellular mutant LOX interacts with ER-resident protein calnexin. (A) Immunofluorescence imaging of Lox +/+ , Lox +/Mu , and Lox Mu/Mu mouse embryonic fibroblasts (MEFs) using an anti-LOX antibody showed intracellular accumulation of mutant but not WT LOX protein. (B) Imaging using an antibody to calnexin showed a uniform distribution of intracellular calnexin staining in all cells. (C) Merged images of LOX and calnexin staining established colocalization of intracellular mutant LOX with calnexin. Scale bars: 1 μm (D) Cell lysates from Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-MEFs were immunoprecipitated with anti-calnexin antibody, separated by SDS-PAGE, then immunoblotted with an anti-LOX antibody. Both WT and mutant LOX were immunoprecipitated with calnexin, but the level of calnexin-bound mutant LOX was much higher than that of WT LOX. Cell lysate from Lox -/cells served as a negative control.",
"M292R Lox mutation leads to altered protein conformation. (A) Cell lysates from Lox +/+ and Lox Mu/Mu mouse embryonic fibroblasts were incubated at 37°C, and samples were collected every hour for 6 hours. Immunoblotting with an anti-LOX antibody showed more extensive degradation of WT LOX compared with the mutant protein. (B) Quantitation of band density from immunoblot in A.",
"M292R LOX does not elicit an ER stress response. qPCR showing transcript levels of the ER stress markers (A) Atf4, (B) Chop, and (C) Bip normalized to Gapdh expression in mouse embryonic fibroblasts from Lox +/+ , Lox +/Mu , and Lox Mu/Mu",
"Intracellular mutant LOX activates the autophagy markers p62 and LC3. (A) Mouse embryonic fibroblasts (MEFs) were pretreated with bafilomycin A1 (BafA1) to prevent organelle acidification-induced degradation of autophagy activation markers. Immunoblots were then used to detect P62 and LC3 isoforms in MEF lysates. (B) Quantification of the detected proteins showed a significant increase in p62 only in cell lysates of Lox Mu/Mu MEFs with and without BafA1 treatment compared with Lox +/+ MEFs without BafA1. (",
"Mutant LOX does not interfere with secretion of the WT protein. Lox -/mouse embryonic fibroblasts were transiently transfected with WT or mutant LOX fused to the fluorescent reporter mApple, and protein in conditioned medium from transfected cells was separated by SDS-PAGE. (A) Immunoblotting using an antibody to LOX showed that protein from the WT LOX construct is properly secreted, while mutant (Mu) LOX is not. When the WT and mutant LOX constructs were cotransfected into Lox -/cells at half the level of the single transfections, the amount of secreted protein that was detected was equivalent to that expected from the WT allele, suggesting that the presence of mutant LOX does not alter the secretion of the WT protein. The bottom panel is an image of the stained gel to document equal sample loading. (B) Quantification of the LOX bands in A."
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] | [
"Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.",
"Thoracic aortic aneurysm and dissection (TAAD) is a major cardiovascular health problem that arises from mutations that alter vascular extracellular matrix (ECM), smooth muscle cell function, or growth factor signaling pathways (1). In contrast to abdominal aortic aneurysms, TAAD has a strong genetic component, with pathogenic variants typically inherited in an autosomal dominant manner (2). A single highly penetrant, pathogenic mutation is responsible for disease in only one-fourth of individuals with TAAD, indicating that other undiscovered genetic factors influence disease onset and progression. In addition to the known pathogenic gene mutations, low-penetrant risk variants interact with other genetic or environmental factors to trigger disease or to increase the risk for developing disease but are not themselves pathogenic (2). Significant risk factors for TAAD, primarily when occurring in the background of low-penetrant disease-predisposing variants, are hypertension and bicuspid aortic valve (1).",
"A common histopathological feature of TAAD in humans is fragmented elastin in the aortic medial region (3). Elastin is the most abundant ECM protein in the aorta, where it plays a central role in the vessel's ability to expand and recoil in response to pulsatile blood flow. Smooth muscle cells (SMCs) synthesize and organize the protein into fenestrated sheets (lamellae) that separate smooth muscle cell layers. The secreted form of elastin (tropoelastin) undergoes extensive crosslinking outside the cell to form an elastic polymer, which is the functional form of the protein. The enzyme responsible for initiating crosslink formation is lysyl oxidase (LOX), a copper-binding amine oxidase that oxidizes lysine ε-amino groups in elastin and fibrillar collagens to aldehydes, which then spontaneously react to form covalent linkages between and within proteins (4,5). Approximately 90% of the lysine residues in elastin undergo crosslink formation, which makes it one of the most highly crosslinked and most stable proteins in the body (4). Inactivation of the Lox gene Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta, although in both ascending and descending aortae, changes in vessel wall structure, smooth muscle cell number, and inflammatory cell recruitment differed between WT and mutant animals. Studies with isolated cells revealed that M292R-mutant LOX is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi, where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss of function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.",
"in mice results in early postnatal death due to ruptured aortic aneurysms, emphasizing the critical role of fully crosslinked, functional elastin in maintaining aorta integrity and function (6,7). The gene inactivation studies also show that other LOX isoforms cannot substitute for the loss of LOX in large, elastin-rich vessels.",
"LOX in vertebrates belongs to a family of 5 copper-dependent enzymes (LOX, LOXL1-4) that show a high degree of homology in their carboxy-terminal catalytic domain but differ in their amino-terminal sequences. LOXL2-4 form a subgroup that shares 4 amino-terminal scavenger receptor cysteine-rich domains that influence substrate binding but appear to have little role in regulating catalytic activity (8,9). LOX and LOXL1, in contrast, are synthesized without scavenger receptor sequences but have a propeptide domain that keeps the enzymes in a latent state until proteolytically processed outside the cell by BMP1/Tolloid-like metalloproteinases (10,11). Substrate specificity for each of the lysyl oxidases is still under investigation, but gene inactivation studies and phylogenetic analysis suggest that LOX and LOXL1 contribute to crosslinking of elastin and fibrillar collagens, whereas LOXL2-4 have different substrate specificities (12,13). All 5 LOX isoforms are synthesized in the ER and traffic to the Golgi, where the inactive apoenzyme binds copper and forms the cofactor lysyl tyrosine quinone (LTQ); both copper and LTQ are essential active site components required for catalytic activity (14).",
"Mutations within LOX in families with TAAD were recently identified by whole exome sequencing (3,15). Aortic disease in these families followed autosomal dominant inheritance, suggesting that 1 mutant LOX allele is sufficient to influence disease initiation or progression. Previously, we identified a LOX catalytic site mutation (p.Met298Arg) in a family with a history of TAAD (3). By introducing this human mutation into the homologous position in the mouse genome using CRISPR/Cas9 genome editing technology (c.857T>G encoding p.M292R; hereafter referred to as Mu), we confirmed that this amino acid change caused ruptured TAAD and perinatal death when both alleles were mutated (Lox Mu/Mu ) (3). In contrast to humans who developed TAAD when heterozygous for this LOX variant, aneurysms or other arterial disease were not observed in mice with a similar genotype (Lox +/Mu ) unless they were challenged with hypertension. In this report, we show that the nature of the functional loss associated with the methionine-to-arginine change in LOX results from a secretion defect, where the protein is retained in the ER and degraded through an autophagy/proteasome pathway. We also show that the vessel wall alterations associated with LOX functional haploinsufficiency increase the likelihood of developing vascular disease in association with environmental factors that increase wall stress.",
"Lox +/Mu adult animals are predisposed to aortic dilation in response to hypertension. The generation and characterization of mice expressing the M292R mutation were described previously (3). Mice heterozygous for this Lox missense variant appear grossly normal, have normal blood pressure, and show no increased mortality through 2 years of age. Aortic diameter and circumferential wall stiffness at physiological blood pressure in Lox +/Mu animals are normal, but ascending aortic length measured from the aortic root to the brachiocephalic artery is 10% longer than in WT mice (3).",
"Hypertension is a known risk factor associated with aortic dilation (16). To test whether mice expressing mutant LOX are predisposed to aneurysm formation as a consequence of increased wall stress, Lox +/Mu mice were made hypertensive by subcutaneous delivery of angiotensin II (Ang II) via Alzet osmotic pumps. Pumps containing saline served as the control. Ang II infusion is a well-established model for inducing vessel wall changes in mice and other animals (17,18). Blood pressure measurements using radiotelemetry in awake, freely moving Lox +/+ and Lox +/Mu mice showed an increase of approximately 30 mmHg in systolic pressure and 25-30 mmHg in mean blood pressure in both genotypes 7-10 days after pump implantation (data not shown), consistent with what others have found using this model (19). After 4 weeks of treatment, all animals receiving Ang II had cardiac hypertrophy, indicating altered cardiac physiology consistent with Ang II-induced hypertension ( Figure 1A). However, only Lox +/Mu mice showed substantial dilation and elongation of the ascending aorta in response to Ang II; no change in aortic diameter was observed in Ang II-treated Lox +/+ mice or in Lox +/Mu mice infused with saline alone ( Figure 1B). Biaxial pressure-diameter testing showed the ascending aorta in Ang II-treated Lox +/Mu animals to be approximately 25% larger than the aorta from WT or saline-treated controls at mean physiological blood pressure ( Figure 1C). The shape of the pressure-diameter curves suggests that Ang II treatment did not affect aortic mechanical properties in either mutant or WT animals. Other than in the ascending aorta, no dilation or change in vessel mechanics occurred in the left common carotid artery for either Lox +/+ or Lox +/Mu animals with saline or Ang II treatment ( Figure 1D), nor were dilations or areas of stenosis found in the aortic arch, descending aorta, or abdominal aorta.",
"Ang II treatment leads to vessel wall thickening and changes in vascular cellularity. On a structural level, Ang II treatment resulted in ascending and descending aortic wall thickening in Lox +/Mu animals, but there were no changes in elastic lamellar number or lamellar organization, quantified by measurement of medial area ( Figure 2A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.127748DS1). Elastic lamellar fragmentation, already elevated in Lox +/Mu compared with Lox +/+ animals (3), did not increase in either genotype following Ang II treatment (data not shown). We analyzed elastin crosslinking using an ELISA assay for desmosine, a major crosslink in elastin. We found no difference in desmosine levels in aortic tissue from Lox +/+ and Lox +/Mu mice, confirming normal elastin levels and that elastin crosslinking is not affected in animals heterozygous for the Lox mutation (Supplemental Figure 1C). Hydroxyproline levels (as a measure of total collagen) trended higher in vessels from the mutant animals but were not significantly different from WT when normalized to the change in total protein (Supplemental Figure 1D). Higher protein quantities are consistent with the increase in wall thickness that occurred with Ang II treatment (Supplemental Figure 1B).",
"Changes in the number and types of cells were observed in the thickened aortic wall of the hypertensive animals. The most substantial change occurred in the adventitia of both the ascending and descending aorta, which underwent considerable expansion in the Lox +/+ and Lox +/Mu animals as a result of Ang II exposure ( Figure 2A). In the medial layer, Ang II infusion led to a decrease in the number of SMCs in Lox +/+ animals but an increase in SMCs in Lox +/Mu mice ( Figure 2B). There was also an increase in intralamellar area in both genotypes, although the change was most notable in Lox +/Mu mice. The nuclei of the intralamellar cells in the mutant media were rounder than cells in Lox +/+ vessels, suggesting a less polarized Lox +/+ and Lox +/Mu animals developed significant cardiac hypertrophy following 4 weeks of treatment with 2 μg/kg/ min Ang II compared with saline-treated controls. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. ****P < 0.0001. (B) The ascending aorta in Lox +/Mu animals is longer than in Lox +/+ animals and undergoes substantial dilation following Ang II treatment. (C) Pressure-diameter measurements at intraluminal pressures ranging from 0 to 175 mmHg. The ascending aorta from Ang II-treated Lox +/Mu animals has a larger diameter over the entire range of pressures compared with Lox +/+ animals treated with Ang II or with saline controls. (D) There was no difference in the diameter or mechanical properties of the left common carotid artery in either genotype with or without Ang II treatment. Repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences between groups at each pressure level. Data are presented as mean ± SD. # P < 0.05, Lox +/+ with Ang II vs. Lox +/Mu with Ang II; *P < 0.05, **P < 0.01, Lox +/Mu with saline vs. Lox +/Mu with Ang II.",
"phenotype and noncircumferential orientation. Immunostaining for smooth muscle α-actin demonstrated uniform reactivity throughout the medial intralamellar spaces, whereas cell staining in the adventitia was heterogeneous (not shown).",
"Staining for the monocyte/macrophage marker CD68 in descending aorta showed an increased number of CD68 + cells in the media and adventitia of both Lox +/+ and Lox +/Mu descending aorta following Ang II treatment ( Figure 2C). These results are consistent with previous studies showing that infusion of Ang II promotes macrophage accumulation within the aortic wall (20). While the adventitia showed the highest level of immune cell infiltration, macrophage accretion in the medial layer was also observed, but the spatial localization of the CD68 + cells was different for the 2 genotypes. In Ang II-treated Lox +/+ vessels, medial CD68 + cells were associated with the first 2 elastic layers nearest to the intima. Medial CD68 + cells in the aorta of Lox +/Mu animals treated with Ang II, however, were in the outer layers of the media closest to the adventitia ( Figure 2C).",
"Evaluation of factors that adversely affect vessel wall integrity and SMC function. An explanation for why Lox +/Mu mice are more sensitive than WT animals to Ang II infusion was explored by first checking for possible differences in Ang II receptor expression using quantitative PCR (qPCR). In ascending aorta from Lox +/Mu mice, all 3 Ang II receptors (Agtr1a, Agtr1b, and Agtr2) were detected, and there was no difference in their expression levels . Macrophages (stained green, arrowheads) were detected with an antibody to CD68, and cell nuclei were visualized with DAPI. Red is elastin autofluorescence. The vessel wall was thicker in both Lox +/+ and Lox +/Mu animals following Ang II treatment, with more DAPI + and CD68 + cells in the adventitia of both genotypes. (B) In the medial layer, there were fewer SMCs in the aorta of WT animals treated with Ang II but more cells in Lox +/Mu aorta. Shown are the mean ± SD for cell number counts between the internal and external elastic lamina on 7 tissue sections from each group. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.01. (C) Medial CD68 + cells were mostly located on the luminal side of the media in the Lox +/+ aorta but nearer the adventitia in Lox +/Mu aorta after Ang II treatment.",
"compared with WT controls (Supplemental Figure 2A). Thus, the potential for Ang II receptor-mediated signaling is similar in both genotypes.",
"Many Ang II-induced responses on SMCs are attributable to stimulation of NADPH oxidase with the subsequent generation of ROS (21,22). We used dihydroethidium, a cell-permeable compound that interacts with superoxide, to assess oxidant levels in sections of WT and mutant ascending aorta and found no difference between the 2 genotypes (Supplemental Figure 2B). Thus, differences in levels of oxidative stress do not appear to be a factor in the Ang II susceptibility of Lox +/Mu mice.",
"In many models of aortic disease, the presence of proteases that degrade elastin can initiate and propagate disease by degrading the elastic fiber system in the vessel wall. We therefore investigated whether 3 elastolytic MMPs commonly linked to vascular remodeling -MMP2, MMP9, and MMP12 -were differentially expressed in mutant versus WT aorta. There were no differences in Mmp2 and Mmp9 mRNA expression levels, while expression of Mmp12 was diminished in the mutant aorta (Supplemental Figure 2C).",
"Active LOX is absent in Lox Mu/Mu mouse embryonic fibroblast conditioned medium. To elucidate how the M292R modification affects LOX function, we used mouse embryonic fibroblasts (MEFs) from WT, KO, and mutant animals to characterize LOX secretion and activity. Mutant cells were indistinguishable from WT cells in morphology and in their rate of proliferation (data not shown). LOX activity ( Figure 3A), assessed using an Amplex Red assay, was not detected in cell layer extracts of any MEF genotype (Lox +/+ , Lox +/Mu , Lox Mu/Mu , and Lox -/-), which was expected, since the full-length intracellular form of LOX (pro-LOX) is an inactive zymogen. LOX enzymatic activity was readily detected in the conditioned medium from Lox +/+ MEFs and showed linear enzyme kinetics over the period of the Amplex Red assay. Conditioned medium from Lox Mu/Mu MEFs, however, had no detectable activity, similar to conditioned medium from cells with inactivated Lox alleles (Lox -/-) ( Figure 3B). Activity was detected in conditioned medium from Lox +/Mu cells, where the linearity and rate of substrate oxidation were comparable to those seen in Lox +/+ cells, indicating that the mutant form of the protein does not adversely affect the enzymatic activity of WT LOX and hence does not function in a dominant negative fashion.",
"M292R LOX is poorly secreted and is retained in the ER. LOX is maintained as an inactive 50-kDa proenzyme through every stage of the synthetic and secretory pathway. Activation requires secretion into the extracellular space, where proteolytic removal of the N-terminal propeptide portion of the protein generates the 30-kDa active catalytic domain. To determine whether LOX activity is absent in M292R MEF conditioned medium because mutant LOX is not secreted, we performed Western blots using an anti-LOX antibody on Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF cell layers and conditioned medium. Immunoblotting showed increased levels of 50-kDa intracellular pro-LOX protein in cell extracts from Lox Mu/Mu MEFs compared with Lox +/+ cells ( Figure 4A). Conversely, there was abundant 30-kDa mature (processed) LOX in conditioned medium from WT cells but barely detectable levels in medium from Lox Mu/Mu MEFs ( Figure 4B). These findings indicate that the mutant protein is poorly secreted and is retained intracellularly as the proenzyme. Immunofluorescence imaging with an antibody to LOX showed minimal intracellular staining in Lox +/+ cells, confirming that WT LOX is secreted efficiently ( Figure 5A). Staining of Lox Mu/Mu cells, however, showed abundant intracellular staining, consistent with the immunoblot results described above. The staining pattern for intracellular LOX was typical of accumulation within the ER, which was confirmed by colocalization of LOX with calnexin, an ER-resident protein ( Figure 5, B and C).",
"A direct interaction between LOX and calnexin was shown using coimmunoprecipitation from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEF lysates. Lox -/cells served as the negative control. Cell lysates were immunoprecipitated with an anti-calnexin antibody, followed by immunoblotting using an antibody to LOX. LOX protein was detected in calnexin immunoprecipitates from all genotypes except Lox -/-, confirming an interaction between the 2 proteins ( Figure 5D).",
"M292R LOX has an altered protein conformation. The high level of LOX detected in Lox Mu/Mu MEF calnexin immunoprecipitates suggests that M292R LOX is misfolded and trapped in the ER bound to calnexin. To determine whether M292R LOX is conformationally different from the WT enzyme, we explored possible structural changes by assessing differential sensitivity to proteolytic degradation. Changes in protein structure frequently expose or mask protease binding sites, leading to altered degradation products and a different degradation fingerprint. Immunoblotting of cell lysate from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs incubated at 37°C for 6 hours to exploit protein degradation by endogenous enzymes within the cell lysate showed time-dependent degradation of WT but not M292R LOX ( Figure 6). These results suggest that WT and M292R pro-LOX have different susceptibilities to degradation arising from cell-associated proteases. Differential degradation was also observed when cell lysates were incubated with pancreatic elastase. In digests of Lox +/+ cell lysate, there was a 25-kDa fragment evident with all doses of elastase that was absent in Lox Mu/Mu samples (Supplemental Figure 3, arrow). When treated with the highest dose of elastase, pro-LOX from Lox +/+ and Lox +/Mu samples degraded predominantly to a 30-kDa fragment. LOX from Lox Mu/Mu samples, in contrast, degraded further to mainly a 10-kDa fragment. It is important to note that pancreatic elastase does not cleave after methionine or arginine, so the Met-to-Arg mutation does not create or negate a protease cleavage site for this enzyme. Together, the conformational difference between the WT and mutant proteins suggests that M292R LOX is misfolded, which is consistent with its protracted interaction with calnexin in the ER.",
"M292R LOX does not elicit an ER stress response. Accumulation of misfolded proteins in the ER generally leads to ER stress, which subsequently activates the unfolded protein response (UPR) (23). UPR triggers 3 primary stress sensors, PERK, IRE1, and ATF6. Under resting conditions, BIP/Grp78 maintains these sensors in an inactive state. Upon ER stress, BIP binds to misfolded proteins, thereby allowing for sensor activation. To determine whether the continuous presence of misfolded mutant LOX induces ER stress, we assessed Bip transcript levels by qPCR. We also measured mRNA levels of 20% change was modest compared with the ~2-fold and ~4-fold increase in Atf4 and Chop, respectively, when ER stress was stimulated by treatment of MEFs with lopinavir/ritonavir, which served as a positive control (data not shown). Electron microscopic analysis of WT and mutant cells showed that ER volume was not markedly different in the 2 cell types (Supplemental Figure 4), suggesting that mutant protein does not accumulate to levels that would produce dilated saccules. Together, these results suggest that LOX protein containing the M292R mutation does not induce substantial ER stress.",
"Increased activation of autophagy in Lox Mu/Mu cells. The absence of ER stress in Lox Mu/Mu cells was surprising given that M292R LOX does not leave the ER. To determine whether mutant LOX is being removed from the ER for degradation through the lysosomal pathway, we costained cells for LOX and the lysosomal marker LAMP-2. No colocalization of the 2 proteins was observed, suggesting that the mutant protein was not trafficked through lysosomes (Supplemental Figure 5).",
"Autophagy is a general mechanism for clearing proteins accumulating in cells. Clearing of misfolded proteins from the ER, a process termed ER-phagy, plays a critical role in ER homeostasis (24). To determine whether mutant LOX directly activates autophagy-mediated protein clearance from the ER, we measured 2 autophagy activation markers, p62 and microtubule-associated protein 1A/1B, light chain 3 (LC3), in WT and Lox Mu/Mu MEFs. Following pretreatment with bafilomycin A1 (BafA1) to inhibit organelle acidification-induced degradation of p62 and LC3, only Lox Mu/Mu cells with and without BafA1 pretreatment had significantly increased p62 expression compared with Lox +/+ cells without BafA1 pretreatment ( Figure 8, A and B). The autophagy activation markers LC3-I and -II were also elevated in mutant compared with WT cells (Figure 8, A, C, and D), providing more evidence for increased autophagy.",
"We also used electron microscopy to screen for the presence of autophagosome-like vesicles in WT and mutant cells (Supplemental Figure 4). We observed that multi-and single-membrane vesicles typical of autophagosomes were particularly abundant in the perinuclear region but were also present throughout the cytoplasm of Lox Mu/Mu cells. Such structures were rare in WT cells. Together, the existence of autophagic vesicles along with increased expression of autophagy activation markers in mutant cells is consistent with the clearance of misfolded mutant LOX from the ER through an autophagy/proteasome pathway.",
"Mutant LOX does not influence the secretion of the WT protein.",
"To assess whether the secretion of WT LOX is affected by the presence of M292R mutant protein, we transiently expressed plasmids containing WT LOX, and M292R LOX fused to the fluorescent protein mApple, in Lox -/-MEFs. Successful transfection was confirmed by intracellular mApple expression using fluorescence imaging (data not shown). In the conditioned medium of Lox -/cells transfected with empty plasmid, no LOX was observed by Western blot analysis ( Figure 9A). In Lox -/-MEFs transfected with 2.5 μg WT Lox-mApple plasmid, an immunoreactive protein band at approximately 60 kDa was detected in the conditioned media, corresponding to the size of active LOX (30 kDa) fused to mApple (27 kDa). In Lox -/-MEFs transfected with 2.5 μg of M292R Lox-mApple plasmid, LOX protein was observed inside the cell by fluorescence microscopy, but no LOX protein was detected in conditioned medium by Western blotting, consistent with the inability of M292R LOX to be secreted. To determine whether mutant LOX alters the secretion of the WT protein, or if the WT protein acts as a chaperone to facilitate the secretion of the mutant protein, we cotransfected cells with 1.25 μg each of WT and mutant plasmid, which is half as much as was used for single plasmid transfection. If mutant LOX does not affect the secretion of the WT protein, then LOX should be detected in conditioned medium, but at half the level of the single plasmid transfection. If the mutant protein has an adverse effect on WT LOX secretion, then levels will be lower than 50%. If WT LOX acts as a chaperone to facilitate secretion of the mutant protein, then the combination of both secreted proteins would be higher than 50% and possibly equivalent to the WT transfection (1.25 + 1.25 = 2.5 μg). Figure 9 shows that LOX protein was detected in conditioned medium of doubly transfected cells at half the level of singly transfected cells, confirming that the mutant protein does not affect the secretion of WT LOX.",
"M292R propeptide processing. Our experimental results are consistent with the M292R mutation being a loss-of-function phenotype due to a secretion defect. The final activation step of the proenzyme occurs outside the cell through proteolytic separation of the propeptide and catalytic domains. To determine whether M292R pro-LOX could be processed to the 30-kDa mature form if it were secreted, we incubated cell lysates from WT and mutant MEFs with recombinant BMP1 (rBMP1), the enzyme responsible for propeptide cleavage. Western blot analysis showed that BMP1 appropriately processed both WT and M292R pro-LOX to the 30-kDa mature form (Supplemental Figure 6). Thus, the mutation did not inhibit propeptide processing. The processed mutant protein was not assayed for oxidase activity because intracellular LOX in M292R MEFs does not have bound copper and cannot be enzymatically active.",
"LOX plays a critical role in vascular ECM maturation by catalyzing the crosslinking of elastin and fibrillar collagens, the 2 major ECM protein classes in the aortic wall. Phylogenic studies suggest that the current form of vertebrate LOX coevolved with elastin and helped facilitate the appearance of a closed circulatory system (12,25). Therefore, it is no surprise that mutations in LOX give rise to vascular insufficiency and disease. All LOX mutations associated with TAAD in humans show autosomal dominant inheritance, and most result in loss of function. We know from mouse studies that inactivation of both Lox alleles is lethal and that other members of the LOX family cannot assume LOX's critical role in vascular development and maintenance of the vessel wall. In this report, we utilize a mouse model of a well-characterized human mutation to explore how LOX haploinsufficiency leads to vascular dilation and aneurysm formation. Mice heterozygous for the Lox M292R variant (Lox +/Mu ) have increased elastic lamellar fragmentation in the wall of the ascending aorta (3), but do not develop aortic dilation under normal conditions. The absence of aneurysmal disease was surprising given that humans heterozygous for this same mutation are prone to aneurysm formation and vessel wall changes (3). Variability in the age of disease onset and disease severity in humans, however, suggested that a secondary challenge is a factor in disease initiation and progression. Here we show that hypertension induced by Ang II infusion resulted in vessel wall changes in both Lox +/+ and Lox +/Mu mice, but only Lox +/Mu animals showed aortic dilation localized to the ascending aorta. Several groups have reported that the effects of Ang II are most pronounced in the ascending aorta region in this model (26)(27)(28), but the underlying factors governing this vessel specificity are unknown. Differences in the embryonic origin of the cells that populate the aortic wall are one explanation (29,30), but the composition and maturation of the aortic ECM are also factors. In Lox +/Mu mice, changes in ECM crosslinking associated with LOX insufficiency could have more negative consequences for this vessel segment because of high collagen and elastin levels and the high mechanical stress experienced by the aortic wall during the cardiac cycle.",
"While several Ang II-induced vascular changes were expected (19,27) and common to WT and mutant animals, there were also significant differences. For instance, medial expansion was evident in both genotypes, but the effect was most notable in mutant mice, in which there was an increase in the number of smooth muscle cells between the elastic lamellae. This response is particularly unusual, since the intralamellar space is typically populated by a single smooth muscle cell layer, as can be seen in vessels from saline-treated Lox +/+ and Lox +/Mu animals ( Figure 2). The opposite change occurred in WT vessels, where there were fewer smooth muscle cells in response to Ang II, suggesting Ang II-related apoptosis or growth suppression (26), which we did not assess. mice. Oneway ANOVA with Tukey's multiple-comparisons test was used to assess differences, which were minimal between the 3 genotypes. Data are presented as mean ± SD. ***P < 0.001.",
"Another cellular change in the ascending and descending aorta following Ang II treatment was the accumulation of macrophages in the aortic wall of both WT and Lox +/Mu mice. CD68 + cells were found in the adventitia of both genotypes, with fewer cells in the medial layer. In Ang II-treated WT mice, CD68 + cells accumulated in the intima and inner elastic layers of the media and did not localize to breaks in elastic lamella, as has been reported in other models (18,20). Their location within the wall, however, shows that some cells were able to penetrate past the internal elastic lamina into the first or second layer of smooth muscle cells. Medial CD68 + cells in Lox +/Mu mice exposed to Ang II, in contrast, were localized to the outer aspect of the media and occurred at higher numbers than in WT vessels. These results suggest that the cellular origin of the CD68 + cells or the route of inflammatory cell infiltration is different in Lox +/+ and Lox +/Mu vessels as a response to wall stress.",
"The location of macrophages in the outer region of the wall provides a mechanistic explanation for why Lox +/Mu , but not WT, aorta dilates following Ang II treatment. ECM degradation is a primary factor leading to vessel wall dilation. Elastases and other matrix-degrading enzymes secreted by macrophages destabilize the vessel wall, particularly when matrix degradation occurs in or near the adventitia -the vessel compartment essential for maintaining the structural integrity of the artery (31). The medial cell hypertrophy and inflammatory cell accumulation in the Lox +/Mu aorta in response to hemodynamic stress are similar in several ways to vascular changes in mouse models of Marfan syndrome with mutations in fibrillin-1, a key elastic fiber protein. In Marfan mice, inflammatory cell accumulation at the medial-adventitial interface correlates with increased elastin degradation and a loss C and D) Also, LC3 was elevated in Lox Mu/Mu cells compared with Lox +/+ cells. Two-way ANOVA with Tukey's multiple-comparisons test, with the 2 variables being genotype and treatment, was used to assess differences. Data are presented as mean ± SD. *P < 0.05, **P < 0.001.",
"of structural integrity that leads to vessel wall dilation (32). As in the Ang II-treated Lox +/Mu animals, only the ascending aorta is affected in Marfan mice.",
"Our previous studies showed that mice with the Lox M292R mutation share numerous phenotypic traits with Lox +/animals (3), suggesting that M292R is a loss-of-function mutation in mice and humans. Even though the amino acid change is in the catalytic domain of LOX close to the copper binding site, it was not clear whether the mutation results in a catalytically dead enzyme or if some other aspect of LOX processing is negatively affected by the mutation. Our data show that the answer fits both possibilities: the mutant protein is retained in the ER and not secreted, and the enzyme is catalytically dead because of its inability to traffic to the Golgi, where it needs to bind copper and form the redox quinone cofactor LTQ required for catalytic activity (33). Even though we found that the propeptide of mutant pro-LOX could be cleaved by rBMP1 similarly to WT LOX, full-length LOX in the ER cannot be catalytically active even if propeptide processing does occur.",
"Why mutant LOX is retained in the ER is not clear, but our data point to protein misfolding as a probable cause. ER retention is characteristic of incompletely folded proteins. Folding intermediates of most secreted proteins interact with the calnexin/calreticulin protein complex in the ER to achieve the proper native conformation required for secretion (34). The calnexin/calreticulin protein complex recognizes N-linked glycan structures (LOX has 3 N-linked glycosylation sites in the propeptide region; ref. 11) that undergo cycles of sugar residue removal and addition. Once folded correctly, calnexin and calreticulin dissociate, and the folded proteins are transported from the ER to the Golgi apparatus. Mutant LOX colocalized intracellularly with calnexin and was isolated bound to calnexin in cellular extracts, suggesting that the protein remains misfolded despite repeated folding interactions with calnexin. Furthermore, the differential susceptibility to proteolytic degradation of mutant compared with WT LOX is consistent with structural differences between the 2 proteins.",
"It was interesting that mutant LOX accumulation in the ER did not induce an appreciable UPR. We saw no upregulation in mutant cells of the ER stress-related markers Bip (an ER luminal chaperone protein and critical ER stress sensor), Atf4 (a transcription factor downstream of PERK that plays a crucial role in the adaptation to ER stress), or Chop (a transcription factor regulated by ATF4 that drives proapoptotic gene transcription). Nor could we detect in mutant cells an increase in ER volume frequently associated with ER stress. One pathway whereby ER stress is mitigated is removal from the ER of mutant protein and degradation at the lysosome (ERAD-II) or proteasome (ERAD-I) (24). We were unable to localize LOX within lysosomes but did find increased expression of the autophagy markers p62 and LC3. Electron microscopy also identified an increased number of autophagic vesicles in Lox Mu/Mu cells. These results suggest that misfolded mutant LOX is being cleared from the ER through an autophagy/proteasome pathway and would explain why mutant protein levels never reached a critical threshold to sustain an ER stress response. Furthermore, there is mounting evidence that the autophagy and secretory pathways are coregulated and intimately linked (35), which may represent a mechanistic connection into the LOX trafficking defect that we identified. It should be noted that all the experiments probing the fate of the mutant protein were done in cultured cells. It is possible, but unlikely, that in vivo, in tissues, and at developmental stages relevant to the TAAD phenotype, LOX folding, secretion, ER retention, and stress occur differently than in MEFs.",
"Guo et al. (15) described 2 other LOX active site variants (S280R and S348R) that segregate with disease in families with TAAD. When expressed from transgenes in HeLa cells and examined in cell lysates, a portion of the pro-LOX protein was processed to its active form and was able to oxidize substrate at rates approximately equivalent to the WT enzyme (S348R) or with approximately 50% lower activity (S280R) (2,15). Enzymatic activity in conditioned medium from the transfected cells was not evaluated, so it is not known whether these LOX variants are efficiently secreted. However, the fact that the proteins are catalytically active implies trafficking through the Golgi and proteolytic removal of the propeptide domain. Thus, unlike the M292R mutation, which is retained in the ER, the S348R and S280R variants appear capable of traversing the secretory pathway. How these proteins, which have full (or somewhat reduced) catalytic activity, cause thoracic aortic disease is an interesting question. An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.",
"Antibodies. Antibodies used in this study are described in Supplemental Table 1.",
"Mouse models and cell isolation. A knockin mouse strain bearing the Lox M292R substitution was generated in the C57BL/6 background using CRISPR/Cas9 genome editing technology as previously described (3). Lox +/mice in the same background are described in ref. 6. Primary MEFs were isolated from the various mouse lines by digestion of E14.5 mouse embryos with trypsin/EDTA at 37°C for 30 minutes in DMEM (38). Cells were collected by centrifugation and grown in DMEM, 10% FBS, 1% penicillin/streptomycin, and 1 mM l-glutamine.",
"Ang II treatment and blood pressure measurements. Telemetry transmitters (Data Sciences International, model PA-C10) were implanted surgically in 3-to 4-month-old mice under isoflurane anesthesia. The catheter was inserted in the left common carotid artery and advanced to the aortic arch, and the transmitter was placed subcutaneously along the abdomen. Animals were allowed to recover for 7 days prior to activation of transmitters, and continuous recording of heart rate and arterial pressure was obtained, with sampling every 5 minutes for 15-second intervals for 3 days. Data were collected and stored using Dataquest ART (Data Sciences International).",
"After baseline blood pressure measurements were obtained, mice were implanted with osmotic pumps (Alzet model 1004, DURECT Corp.) that delivered Ang II (MilliporeSigma) subcutaneously at a dose of 2 μg/kg/min in the interscapular region, according to published procedures (39,40). Mice infused with saline were used as controls. After a week of Ang II treatment, arterial pressure and heart rate were again continuously recorded using the telemeters, with sampling every 5 minutes for 15-second intervals for 3 days. Animals were fed a standard laboratory chow diet. After 28 days, animals were sacrificed under isoflurane anesthesia, and vessels were collected for compliance studies and histological characterization.",
"Vessel compliance measurement. For vessel compliance measurements, the ascending aorta and left common carotid artery were excised and placed in a physiological saline solution. Vessels were then cleaned of surrounding fat and connective tissue and mounted onto a pressure arteriograph (Danish Myo Technology). Experiments were done at 37°C in an organ bath containing physiological saline. The mounted vessels were visualized using a computerized imaging system, allowing continuous recording of vessel diameters. Vessel outer diameters were recorded while intravascular pressure was increased from 0 to 175 mmHg by increments of 25 mmHg (12 seconds per step). The average of 3 outer diameter measurements was taken at each pressure (40,41).",
"Protein quantification, hydroxyproline, and desmosine assays. Each ascending aorta was hydrolyzed at 105°C overnight with 20 μL of 6 N hydrochloric acid (Thermo Fisher Scientific) and dried under vacuum in a SpeedVac. The dried pellet was dissolved in 400 μL water and filtered with a 0.45-μm filter. Total protein, hydroxyproline, and desmosine levels were determined as described previously (42).",
"Dihydroethidium staining for superoxide detection in aortic sections. Superoxide in aortic sections was detected using dihydroethidum (DHE) staining. In the presence of superoxide, DHE is oxidized to 2-hydroxyethidium, which is highly fluorescent and intercalates within the DNA, staining the nucleus a fluorescent red (43,44). After euthanasia and thoracotomy, the right atrium was clipped, and 5 mL cold (4°C) 1× PBS was flushed through the left ventricle to clear the blood. Ascending aorta was then dissected, placed in Tissue-Tek O.C.T. Compound (Sakura Finetek), and flash frozen in liquid nitrogen. Four-micrometer cryosections were obtained using a Leica CM1850 cryostat and placed on charged microscope slides (Thermo Fisher Scientific). A 10-mM stock solution of DHE (Life Technologies) in DMSO was diluted to 10 μM using 1× PBS. The 10-μM DHE solution was applied to unfixed aortic sections and incubated at 37°C for 30 minutes in the dark. After staining with DHE, sections were then rinsed with 1× PBS for 5 minutes at room temperature (RT) and mounted with a coverslip using ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were obtained on the Zeiss Axioscope fluorescence microscope with a QImaging MicroPublisher 3.3 RTV camera using QCapture software (QImaging). The entire procedure (from mouse euthanasia to image acquisition) occurred within 8 hours.",
"Immunofluorescence on aortic sections and area measurements. Ascending and descending aortas were dissected as above and placed in O.C.T. compound, and the blocks were frozen on dry ice. Four-micrometer-thick cryosections were placed on glass slides and stored at -80°C. The sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS at 4°C for 10 minutes, washed twice with 1× PBS (5 minutes each) at RT, and blocked with a solution containing 1% BSA (MilliporeSigma), 1% fish gelatin (MilliporeSigma), and 0.05% Triton-X (Acros Organics) in 1× PBS for 1 hour at RT. The aortic sections were then incubated with rat anti-mouse CD68 antibody (Bio-Rad) at a 1:1000 dilution in blocking solution at 4°C overnight. The next day, sections were washed 3 times with 1× PBS (5 minutes each) and incubated in goat anti-rat IgG Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) diluted 1:2000 and DAPI diluted 1:3000 in blocking solution at RT for 1 hour. The sections were washed twice with 1× PBS (5 minutes each) at RT, mounted with a coverslip using ProLong Diamond Antifade Mountant, and imaged using the Axioscope fluorescence microscope equipped with the QImaging MicroPublisher 3.3 RTV camera using QCapture software as above. For area measurements, frozen sections from the descending aorta from 3-month-old mice were prepared as described above. The area between the internal and external elastic laminae was traced in each ×40 image using ImageJ software (NIH) and measured in pixels 2 .",
"LOX activity assay. MEFs were grown in 10-cm dishes and maintained as above until fully confluent. Twenty-four hours before collection, culture medium was replaced with DMEM lacking phenol red and FBS, supplemented with 50 μg/mL ascorbic acid and 0.1% BSA. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. Cell lysates were collected by scraping in 1× PBS containing protease inhibitors. LOX activity in cell lysate and concentrated culture medium, incubated with and without the LOX inhibitor β-aminopropionitrile (BAPN), was quantified using the Amplex Red assay as previously described (3,45). Resorufin fluorescence, the product of Amplex Red oxidation, was measured at excitation and emission wavelengths (540 and 600 nm, respectively) every 30 minutes for 150 minutes using a BioTek H4 Hybrid Reader. LOX activity was calculated as the difference between total activity and activity in the presence of BAPN. Differences in relative fluorescent units were tested using 2-way ANOVA with Tukey's multiple-comparisons test.",
"Immunofluorescence imaging of primary cells. Primary MEFs were seeded in 4-chamber glass slides at 3 × 10 5 cells per/well. After 3 days in culture, the cells were fixed with cold methanol (2 washes, 5 seconds each) followed by 3 washes with blocking solution containing 1× PBS, 1% BSA, 1% fish gelatin (Milli-poreSigma), and 0.05% Triton-X100 at pH 7.4, then incubated in blocking solution for 30 minutes at RT. The cells were then incubated with a primary antibody to LOX at 1:1000 in blocking solution overnight at 4°C. After washing the cells with blocking solution to remove the primary antibody, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor 568 secondary antibody at 1:2000 (Thermo Fisher Scientific) and anti-calnexin primary antibody directly labeled with Alexa Fluor 488 at 1:4000 in blocking solution for 30 minutes at RT. The cells were washed with blocking solution 3 times, then mounted using ProLong Diamond Antifade Mountant (Invitrogen) and coverslipped. The samples were imaged using the Axioscope fluorescence microscope and QCapture Pro software.",
"Protein extraction from cell culture and aortic tissue. Cultured MEFs were switched to serum-free medium 24 hours before protein extraction. MEFs in 10-cm dishes were incubated in 1 mL lysis (RIPA) buffer (MilliporeSigma) containing protease inhibitors for 30 minutes at 4°C. The lysates were scraped from the dish into microfuge tubes and centrifuged at 17,500 g for 5 minutes, and the supernatant was collected. Conditioned medium from each dish was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) to a final volume of 1 mL. The protein concentration in cell lysates and concentrated media was measured using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Laemmli buffer with and without DTT was added to samples for SDS-PAGE and native gel electrophoresis, respectively. Only the samples with DTT were boiled at 100°C for 5 minutes.",
"RNA extraction and qPCR. RNA was extracted from confluent cell cultures using TRIzol (Thermo Fisher Scientific) following the manufacturer's protocol. For RNA isolation from aortic tissue, ascending aorta from adult Lox +/+ and Lox +/Mu mice were each homogenized in 500 μl TRIzol using QIAGEN TissueLyser. The manufacturer's protocol was followed for RNA isolation. At the time of RNA precipitation, 10 μg glycogen (Mil-liporeSigma) was added as a carrier to each sample. Each RNA pellet was dissolved in 10 μL molecular-grade water. The RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). One microgram of the isolated RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Reverse transcription was then carried out using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). One microliter of cDNA was used for qPCR using TaqMan Fast Universal PCR Master Mix and TaqMan assay primer/probes (Life Technologies) for Mmp12 (Mm00500554_m1), and Gapdh (Mm9999999915_ g1), which was used as an internal control. Ten-microliter reactions were performed in duplicate using a ViiA 7 Real-Time PCR System for Bip, Atf4, and Chop and QuantStudio 3 Real-Time PCR system (Applied Biosystems) for Agtr1a, Agtr1b, Agtr2, Mmp2, Mmp9, and Mmp12. All mRNA levels were normalized to Gapdh expression. rBMP1 treatment. Protein was extracted from Lox +/+ , Lox +/Mu , and Lox Mu/Mu MEFs as described above. Fifty microliters of cell lysates from cultured MEFs containing 50 μg total protein were incubated with 30, 60, and 90 ng rBMP1 (R&D Systems) for 45 minutes at 37°C in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5). The enzyme reaction was stopped by adding Laemmli buffer with DTT and incubated at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.",
"Western blotting. Protein extracts from cells, concentrated condition medium, or tissues (generally 50 μg) were analyzed using 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) run at 100 mV for 1 hour. The protein was then transferred to ProBlott Membrane (Applied Biosystems). The blots were incubated in 5% nonfat dried milk in 0.1% 1× PBS-Tween for 1 hour at RT, then incubated with a primary antibody overnight at 4°C. The next day, the blots were washed with 0.1% 1× PBS-Tween and incubated in ECL anti-rabbit or anti-mouse IgG HRP-Linked Secondary Antibody (GE Healthcare) for 1 hour at RT. The blots were then washed and the immunoreactive bands detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The blots were stripped by incubating in 2% SDS, 75 mM Tris-HCl, and 100 mM β-mercaptoethanol for 1 hour at 65°C while shaking. The blot was then incubated in 0.1% 1× PBS-Tween wash buffer for 1 hour at RT before immunodetection of β-actin (MilliporeSigma) at 1:20,000 in wash buffer, which was used as a loading control. For protein samples from concentrated conditioned media, membranes were stained with Ponceau S solution (MilliporeSigma) before blocking and used as a loading control. Alternatively, stain-free images of Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) taken following the manufacturer's protocol served as a loading control.",
"Coimmunoprecipitation. Cell lysates were prepared from MEFs 5 days after confluency as described above. Immobilized protein A anti-rabbit IgG beads (50 μL), preequilibrated by washing with lysis buffer, were then added to 250 μL cell extract. After incubation on a rocking platform for 60 minutes at 4°C, the sample was centrifuged at 2500 g for 3 minutes, and the supernatant was incubated with 8 μg immunoprecipitation antibody for 1 hour at 4°C. Fifty microliters of precleared protein A beads was then added, and the samples incubated overnight at 4°C with rocking. Immune complexes bound to the protein A beads were collected by centrifugation and washed with lysis buffer prior to boiling at 100°C for 10 minutes with Laemmli buffer containing DTT. The supernatant was collected by centrifugation, and proteins were separated by SDS-PAGE and immunoblotted following the same protocol described above.",
"Protein degradation analysis. Confluent cultures of WT and mutant MEFs were treated with 5 μg/mL Brefeldin A (MilliporeSigma) to inhibit protein transport from ER to the Golgi apparatus. This ensured that WT LOX remained in the cells so that the starting amount of protein was the same across genotypes. After 4 hours, the cell lysate was extracted using lysis buffer placed at 37°C to initiate protein degradation. Every hour, 50 μL aliquot of cell lysate was collected, then immediately boiled with Laemmli sample buffer at 100°C for 5 minutes prior to SDS-PAGE analysis. For exogenous protease-mediated degradation, human pancreatic elastase in increasing concentrations (0.625, 1.25, 2.5, 5 ng/μL) was added to cell lysates containing 1 mg/ mL protein at 4°C for 30 minutes. After treatment, elastase activity was inhibited with protease inhibitor cocktail, and samples were boiled with Laemmli sample buffer at 100°C for 5 minutes for SDS-PAGE analysis.",
"Autophagy markers. Confluent MEFs in 6-well dishes were pretreated with 100 mM BafA1 (MilliporeSigma) for 3 hours before cell lysates were collected. The protein samples were separated on 4%-15% gradient gels for SDS-PAGE analysis. Immunoblotting was performed following the same procedure as above.",
"Lox-mApple plasmid cloning and expression. The mouse Lox cDNA in a mammalian expression vector driven by a pCMV promoter was purchased from GE Healthcare. The Lox open reading frame was PCR amplified using a 5′ primer bearing an XhoI restriction enzyme site (5′-CAGATCTCGAGAGCGTTTCGCCTGGGCTGTGC-3′) and a 3′ primer designed to remove the Lox stop codon bearing a BamHI restriction enzyme site (5′-GGATCCA-CATACGGTGAAATTGTGCAGCCTGAG-3′). The PCR product and the pGEMT shuttle vector (Promega) were digested with XhoI and BamHI, then ligated together using T4 ligase. The mutant Lox plasmid bearing the 893 T>G base pair change was generated using the Lox-pGEMT plasmid as the template, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific), following the manufacturer's protocol (5′-TTACCA-CAGCAGGGACGAATTCAGCCACTATG-3′ and 5′-TGTTGGTGACAGCTGTGCCACTCCC-3′). The WT and mutant Lox-pGEMT plasmid sequences were confirmed using Sanger sequencing performed at the Washington University Protein and Nucleic Acid Chemistry Laboratory. To generate the Lox-mApple plasmids, WT and mutant Lox pGEMT plasmids and the mApple plasmid (46), provided by David W. Piston (Washington University School of Medicine in St. Louis), were digested with XhoI and BamHI restriction enzymes. The WT and mutant mLox cDNA inserts were then ligated into mApple vectors to generate the mLox-mApple fusion plasmids and sequenced to confirm. To express the WT and mutant Lox-mApple plasmids in MEFs, Lipofectamine 3000 reagents (Thermo Fisher Scientific) were used following the manufacturer's protocol.",
"Transmission electron microscopy. MEFs from Lox +/+ , Lox +/Mu , and Lox Mu/Mu mice, cultured on glass coverslips, were washed in cacodylate buffer (0.15 M cacodylate in 2 mM CaCl 2 , pH 7.4) and fixed for 5 minutes with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer prewarmed to 37°C. The samples were then incubated at RT for an additional hour, washed with cacodylate buffer, and treated with 1% OsO 4 /1.5% potassium ferrocyanide for 1 hour in the dark. After washing with ultrapure water, en bloc staining was performed in 2% uranyl acetate for 1 hour in the dark. The samples were washed again in ultrapure water before dehydration in a graded acetone series. Finally, the samples were embedded in Epon resin and cured in a 60°C oven for 48 hours. The processed samples were imaged using a JEOL 1400 electron microscope.",
"Statistics. One-way ANOVA with Tukey's multiple-comparisons test was used to determine the significance, if any, between different groups, particularly genotypes. When 2 variables were present, e.g., genotype and treatment or genotype and gene, 2-way ANOVA with Tukey's multiple-comparisons test was used to assess differences. If the same sample was measured repeatedly (e.g., at different pressures in the pressure-diameter curves in Figure 1, C and D, or at different time points in Figure 3), then repeated measures 2-way ANOVA with Tukey's multiple-comparisons test was performed. Prism 8 for Mac OS X (GraphPad Software) was used to run statistical analyses. In all numerical figures, data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical test used and significant differences are noted in each figure legend.",
"Study approval. All animal experiments were carried out following protocols approved by Washington University School of Medicine Institutional Animal Care and Use Committee.",
"VSL, CMH, TJB, and PCT participated in experimental design and acquired and analyzed primary data. NOS helped with data analysis, and RPM designed the research study and analyzed primary data. VSL wrote the initial draft of the manuscript, to which all authors contributed edits."
] | [] | [
"Intracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress",
"Introduction",
"Results",
"Discussion",
"Methods",
"Author contributions",
"Figure 1 .",
"Figure 2 .",
"Figure 3 .",
"Figure 4 .",
"Figure 5 .",
"Figure 6 .",
"Figure 7 .",
"Figure 8 .",
"Figure 9 ."
] | [] | [
"Table 1"
] | [] | [] |
201,836,719 | 2022-02-04T21:02:50Z | CCBY | http://www.jlr.org/content/60/11/1807.full.pdf | HYBRID | ad5e3dbde1c06e288b88d8a128bb0cb660702320 | null | null | null | null | 10.1194/jlr.m092379 | 2971567338 | 31484694 | null |
Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling
2019
Soke Chee Kwong
Faculty of Medicine
School of Medicine
Faculty of Health and Medical Sciences
Pharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §
University of Malaya
50603Kuala LumpurMalaysia;
§ Taylor's University
Subang Jaya47500Lakeside Campus, SelangorMalaysia
Amira Hajirah
Abd Jamil
Anthony Rhodes
Nur Aishah Taib
§
Ivy Chung [email protected]
Faculty of Medicine
School of Medicine
Faculty of Health and Medical Sciences
Pharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §
University of Malaya
50603Kuala LumpurMalaysia;
§ Taylor's University
Subang Jaya47500Lakeside Campus, SelangorMalaysia
Faculty of Medicine
Departments of Pharmacology
the Translational Core Laboratory
University of Malaya
Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling
Journal of Lipid Research
601807201910.1194/jlr.M092379Manuscript received 7 January 2019 and in revised form 6 August 2019.This article is available online at This work was supported by University of Malaya High Impact Research Grant UM.C/HIR/MOHE/06; University of Malaya Research Grant RP019-b; and The authors declare no conflicts of interest. Published, JLR Papers in Press, September 4, 2019 DOI https://doi.org/10.1194/jlr.M092379 1 To whom correspondence should be addressed.Abbreviations: CPT1A, carnitine palmitoyltransferase 1AER, estro- gen receptorFABP, fatty acid binding proteinFAO, FA oxidationGEO, Gene Expression OmnibusGLS1, glutaminase 1GLUT1, glu- cose transporter 1HER2, human epidermal growth factor receptor 2MCAD, medium-chain acyl-CoA dehydrogenaseMTT, methyl thiazolyl tetrazoliumOCR, oxygen consumption rateOD, optical densityPR, progesterone receptorTNBC, triple-negative breast cancer
ERs) and progesterone receptors (PRs), as well as human epidermal growth factor receptor 2 (HER2). It is the most aggressive subtype of breast cancer, affecting about 15-20% of total breast cancer cases. Compared with other subtypes, TNBC tumors are associated with higher histologic grade and larger tumor size (1, 2). The current standard regimen for TNBC patients is a combination of surgery and chemotherapy (3), which often fails to effectively slow down the tumor progression. Hence, TNBC patients have lower overall survival (81% vs. 91%) and disease-free survival (72% vs. 86%) compared with non-TNBC patients (4). Given the lack of ER and HER2 in this subtype, there is no molecular characterization that allows this subtype to be targeted.Uncontrolled proliferation of cancer cells is metabolically demanding. The metabolic pathways utilized by cancer cells to sustain high-energy demands and biosynthesis differ from those employed by healthy cells (5). Challenged by hostile environments such as hypoxia and acute interruptions in nutrient availability, cancer cells typically develop metabolic plasticity, which enables the utilization of available nutrients as bioenergetic substrates. This metabolic flexibility allows maintained ATP production under varying physiological and pathological conditions and is primarily regulated by substrate concentration, hormone levels, blood flow, oxygen supply, and workload(6).Cancer-associated changes in cellular metabolism may also be a direct consequence of oncogenic signal transduction.Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, partly due to the lack of targeted therapy available. Cancer cells heavily reprogram their metabolism and acquire metabolic plasticity to satisfy the high-energy demand due to uncontrolled proliferation. Accumulating evidence shows that deregulated lipid metabolism affects cancer cell survival, and therefore we sought to understand the function of fatty acid binding protein 7 (FABP7), which is expressed predominantly in TNBC tissues. As FABP7 was not detected in the TNBC cell lines tested, Hs578T and MDA-MB-231 cells were transduced with lentiviral particles containing either FABP7 open reading frame or red fluorescent protein. During serum starvation, when lipids were significantly reduced, FABP7 decreased the viability of Hs578T, but not of MDA-MB-231, cells. FABP7overexpressing Hs578T (Hs-FABP7) cells failed to efficiently utilize other available bioenergetic substrates such as glucose to sustain ATP production, which led to S/G2 phase arrest and cell death. We further showed that this metabolic phenotype was mediated by PPAR- signaling, despite the lack of fatty acids in culture media, as Hs-FABP7 cells attempted to survive. This study provides imperative evidence of metabolic vulnerabilities driven by FABP7 via PPAR- signaling.-Kwong, S. C., A. H. A. Jamil, A. Rhodes, N. A. Taib, and I. Chung. Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling. J. Lipid Res. 2019. 60: 1807-1817.Supplementary key words fatty acid binding protein • peroxisome proliferator-activated receptor alpha • cancer • nutrient deprivation • fatty acid metabolism • metabolic adaptation Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptors
AKT activation augments the Warburg effect and renders the cancer cells addicted to glucose (7). Myc protein promotes mitochondrial glutaminolysis and glutamine addiction by shunting glucose away from mitochondrial metabolism (8). Cancer cells that fail to demonstrate metabolic flexibility during nutrient deprivation could become vulnerable in their survival (9). Hence, metabolic vulnerabilities exhibited by cancer cells can be targeted for cancer management (6,10).
Alterations in lipid-and cholesterol-associated pathways encountered in tumors are now increasingly recognized and more frequently described (11,12), but not completely understood. Many cancer types show a strong lipid avidity, in which increasing uptake of exogenous lipid sources (13) or overactivation of endogenous lipid synthesis (14) is observed. Fatty acid binding protein 7 (FABP7), a member of the FABP intracellular lipid chaperone family, has been shown to be upregulated in TNBC compared with other breast cancer subtypes (15,16). FABPs regulates lipid metabolism by increasing fatty acid uptake (17), fatty acid oxidation (FAO) (18), and lipolysis (19). Yet, in TNBC, correlation of FABP7 expression and disease prognosis is debatable. While one study showed that FABP7 expression correlates with lower overall and recurrence-free survival (20), several have shown that FABP7-positive basal tumors (synonymous with TNBC) are associated with better prognosis (21,22). It is unclear, at this point, how FABP7-governed pathways impact the survival of TNBC.
In this study, we explored the roles of FABP7 in adaptation to nutrient depletion in TNBC cell lines. We showed that overexpression of FABP7 decreased the viability of Hs578T cells during serum starvation. This led to cell-cycle arrest and a significant increase in cell death. We further showed that this phenotype was mediated by PPAR-regulated genes.
MATERIALS AND METHODS
Cell culture
All cell lines used in this study were purchased from American Type Culture Collection (Gaithersburg, MD). They were cultured in medium according to the manufacturer's protocol and supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), unless specified otherwise. Hs578T, MCF7, MDA-MB-231, and MDA-MB-435S were maintained in DMEM high glucose while BT474 was cultured in MEM. The cell lines were incubated at 37°C and 5% CO 2 atmosphere.
For nutrient-deprivation experiments, the cells were first seeded in complete medium (10% FBS). The next day (hereafter referred to as 0 h), the cells were washed once with PBS and replaced with nutrient-deprived medium. For the glucose-and glutamine-starvation experiments, 10% FBS were added into the basal medium. Basal medium used for glucose starvation was glucose-free DMEM (Life Technologies, catalog no. 11966-025) that contained 4 mM l-glutamine. For the glutamine-starvation experiment, glutamine-free DMEM (Life Technologies, catalog no. 11960-044) containing 25 mM glucose was used. The serumstarvation experiment mimicked a culture condition with deprived lipids. The cells were challenged with serum-free DMEM (Life Technologies, catalog no. 11965-092) that contains high glucose (25 mM) with l-glutamine (4 mM), but without sodium pyruvate, HEPES, lipids, and growth factors.
The PPAR- antagonist (GW6471) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a nonlethal concentration (2 M).
Data mining at GEO database
A microarray profile of selected TNBC was obtained from the publicly available Gene Expression Omnibus (GEO) database. The search keyword used was "triple negative breast cancer," and the results were filtered with "Homo sapiens." The search was conducted from September 1 to 30, 2018.
Generation of FABP7-transduced TNBC cell lines
TNBC cell lines Hs578T and MDA-MB-231 were transduced with FABP7 gene using Thermo Scientific Precision LentiORF constructs at MOI 10 (Clone ID: PLOHS_100004067). For their control counterparts, the cells were transduced with Precision LentiORF RFP (catalog no. OHS5833). The transduced cells were selected with blasticidin for 30 days to generate stable cell line.
MTT assay
Cells were seeded in 96-well plates and subjected to different growth conditions. At time of termination, 10 l of methyl thiazolyl tetrazolium (MTT) solution (5 mg/ml) (Sigma, St. Louis, MO) was added into each well for 4 h, before the addition of 100 l of 10% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals overnight. Absorbance was measured at 575 nm with reference to 650 nm. All experiments were performed in triplicate and repeated three times.
RNA extraction and qRT-PCR
Total RNA extraction was carried out using Trizol (Invitrogen, Carlsbad, CA) following the recommended protocol. About 500 ng of RNA was converted into cDNA using DyNAmo cDNA synthesis kit (Finnzymes, Vantaa, Finland). For quantitative RT-PCR (qRT-PCR), 5 ng/l cDNA template was added into pool solution containing 5× HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Tartu, Estonia), 10 pmol/l forward and reverse primers, and UltraPure distilled water. ABI StepOne Plus (Applied Biosystem, Foster City, CA) was used, and 40 cycles of amplification were performed. The expression of metabolic genes was normalized to housekeeping genes 18S rRNA and ribosomal protein L13a. Sequence of the primers used is described in supplemental Table S1.
Protein extraction and Western blotting
Protein lysate was harvested by scraping the cells in cold PBS. After centrifugation, PBS was discarded, and the cells were incubated in lysis buffer (0.1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1× phosphatase, and 1× protease inhibitors) for 30 min on ice. The mixture was centrifuged for 20 min, and supernatant was collected as protein lysate. Protein concentration was quantified using Bradford assay (Bio-Rad, Hercules, CA). A total of 30 g of protein was resolved on 15% SDS-PAGE prior to transferring on PVDF membrane. Target proteins were detected using primary Abs FABP7 (Cell Signaling Technology) and ECL prime Western blotting detection reagent (Amersham, GE Healthcare Lifesciences, Sweden) before being visualized with gel-documentation system (Biospectrum 410, UVP).
BrdU cell-proliferation assay
The BrdU cell-proliferation assay kit (Cell Signaling Technology, Beverly, MA) was used to measure the cell-proliferation rate at 24, 48, and 72 h after serum starvation. The data were normalized to their counterparts cultured in complete medium. The cells were incubated with 10X BrdU solution for three h, prior to fixation for 30 min and staining with 1× BrdU Detection Antibody for 1 h. The stained cells were washed and incubated with 1× HRPconjugated secondary antibody solution for 30 min, before TMB solution was added. STOP solution was added after 15 min, and absorbance was read at 450 nm.
Cell-cycle analysis with propidium iodide
Cell-cycle analysis was conducted on the cells at 0, 24, 48, and 72 h after serum starvation. The cells were fixed with 70% ethanol at 4°C for 30 min, before two washes with PBS. Enzymatic removal of RNA was achieved by incubating the cells with 100 g/ml RNase for 15 min before DNA staining with 50 g/ml propidium iodide. The cells were analyzed using a BD FACSCanto II flow cytometer, and the cell-cycle distribution of the cell was analyzed with Modfit LT software. The samples were run in triplicates, and the mean value was calculated.
Annexin V apoptosis assay
Cells were harvested for apoptosis assay at 0, 24, 48, and 72 h after serum starvation. Floating cells in the culture medium and the trypsinized cells were collected and washed once with ice-cold PBS, before they were resuspended in 100 l of assay buffer (1 part buffer: 9 parts distilled water) with 5 l of annexin V-PE and 5 l of 7AAD (BD Bioscience, Franklin Lakes, NJ). The cells were vortexed gently and kept in the dark for 15 min, before adding 400 l of buffer. The staining was analyzed using a BD FACSCanto II flow cytometer and viewed using FACS DiVa Software (BD Bioscience, San Jose, CA). All samples were run in triplicates, and mean value was calculated.
Metabolic activity assays
GAPDH, glutaminase (GSL), and FAO enzyme activities were measured using calorimetric assay kits from Biomedical Research Service Center, State University of New York (Buffalo, NY). All samples were harvested using 1× Cell Lysis Solution. Protein concentration of the samples was assessed with a Bradford assay and normalized to 1 mg/ml. GAPDH activity was measured using GAPDH enzyme assay kit (catalog no. E-101). Briefly, 10 l of sample or water (as blank) was incubated in 50 l of GAPDH assay solution. After gentle agitation, the plate was kept in a non-CO 2 incubator at 37°C for 60 min. For GSL assay (catalog no. E-133), 10 l of sample was incubated in either 40 l of glutamine solution or water. Following a 2 h incubation period in a non-CO 2 incubator at 37°C, 50 l of TA assay solution was added, and the plate was further incubated for another 1 h. For FAO enzyme assay (catalog no. E-141), 50 l of FAO Assay Solution or control solution was added to 10 l of the protein sample. The plate was kept in a non-CO 2 incubator at 37°C for 60 min. All experiments were terminated by adding 50 l of 3% acetic acid, and the plate was read at optical density of 492 nm (OD 492) with a spectrophotometer. Blank reading was subtracted from the sample reading. GAPDH activity in IU/l unit was determined by multiplying OD by 16.98. Glutaminase (GLS) activity in IU/l unit = mol/ (l·min) = (OD × 1,000 × 150 l) ÷ (120 min × 0.6 cm × 18 × 10 l) = OD × 11.58. For FAO assay, the subtracted OD represents the FAO activity of the sample.
Measurement of OCR
Basal oxygen consumption rate (OCR) was carried out with a Mito Fuel Flex Test Kit on XFe96 Bioanalyzer (Agilent). Briefly, 4,000 cells were seeded on a Seahorse XF96-well assay plate in complete medium. The cells grown in complete medium were terminated at 24 h after seeding. For the serum-starvation experiment, the cells were left incubated overnight before they were washed with PBS and incubated in serum-free medium for 24 h. Upon termination, all wells were replaced with assay medium, and baseline OCR was immediately measured for 18 min. The basal OCR was normalized to protein concentration of the cells.
Immunofluorescence staining
The cells were seeded on a coverslip in complete medium. After overnight incubation, the cells were either harvested or challenged with serum-free medium for 24 h. The cells were fixed with 4% formaldehyde for 30 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 1 h. The cells were incubated with FABP7 primary antibody (1:2,000; Cell Signaling catalog no. 13347S) and PPAR- primary antibody (1:400; Santa Cruz Biotechnology, catalog no. sc-398394), followed by Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific). The incubation time was 90 min for primary antibody and 60 min for secondary antibody. The cells were washed three times with PBS after staining with each antibody. All the procedures were carried out on ice. The coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories Cat#H-1200) and analyzed with a Leica TCS SP5 II laser microscope (Leica, Heidelberg, Germany) using 60× objective.
Statistical analysis
Statistical analysis assessing the difference between the means of two groups was performed using Student's t-test with Graph-Pad-Prism (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.
RESULTS
FABP7 overexpression decreased viability of Hs578T, but not MDA-MB-231, cells in serum starvation
The expression of FABP7 was determined in human breast cancer tissues and TNBC cell lines. Analysis from the GEO database (dataset GSE65194) revealed that FABP7 expression was significantly higher in TNBC (n = 55) compared with non-TNBC (n = 98) subtypes (Fig. 1A). When examined in a panel of breast cancer cell lines, FABP7 showed low expression in all cells including MCF7 (luminal A), BT474 (luminal B), MDA-MB-231, and Hs578T (TNBC) cells, when compared with MDA-MB-435S, a melanoma cell line reported to express high FABP7 (Fig. 1B). Correspondingly, FABP7 protein was not detected in these breast cancer cell lines (Fig. 1C).
To understand the functional role of FABP7 in TNBC cells, we established FABP7-overexpressing Hs578T and MDA-MB-231 TNBC cells by transduction using lentiviral particles containing FABP7 open reading frame (Hs-FABP7 and 231-FABP7, respectively) and their control counterparts using lentiviral particles containing red fluorescent protein (Hs-RFP and 231-RFP, respectively) (Fig. 1D). When the cells were cultured in complete medium for 72 h, there was no difference in cell growth between cells expressing FABP7 and their respective RFP controls (Fig. 2). However, when cultured in glucose-or glutamine-deprived condition, both FABP7-overexpressing cells and RFP controls demonstrated substantial reduction in cell viability with greater effect observed in glucose-deprived than glutamine-deprived medium ( Fig. 2A,B).
Interestingly, when lipids were significantly reduced during serum starvation, overexpression of FABP7 resulted in decreased viability of Hs578T cells (Fig. 2C). At 24 h after serum starvation, the cell viability of Hs-FABP7 cells decreased by 10%, which progressively decreased to approximately 70% at 72 h after starvation (P < 0.0001 vs. Hs-RFP cells). Cell viability of Hs-RFP cells was not affected by serum starvation, indicating that the decreased viability in Hs-FABP7 cells may be related to FABP7 expression. This phenotype appeared to be specific to Hs578T cells, as FABP7 expression did not affect the viability of MDA-MB-231 cells in serum-free medium (Fig. 2C).
Decreased viability of Hs-FABP7 cells in serum starvation was due to reduced proliferation, cell-cycle arrest, and cell death
The reduction in cell viability of Hs-FABP7 cells during serum starvation resulted from a decrease in cell proliferation. Hs-FABP7 cells demonstrated a 70% reduction in BrdU uptake as early as 24 h after serum starvation, whereas the control Hs-RFP cells only showed a decrease by 20% (Fig. 3A). The nonresponsive MDA-MB-231 cells maintained their proliferation rate in the range of 85-89% during serum starvation, regardless of FABP7 expression.
Flow-cytometry analysis showed that FABP7 induced cellcycle arrest in Hs578T cells during serum starvation ( Fig. 3B and supplemental Fig. S1). Hs-RFP cells showed a higher distribution of cells (80%) in G1 phase throughout serum starvation, indicating that the cells were able to complete the cell cycle and proliferate, parallel to increased cell viability over time. In contrast, Hs-FABP7 cells demonstrated a consistently higher percentage of cells in S and G2 phase upon serum starvation. For instance, at 48 h after serum starvation, 24% of Hs-FABP7 cells were accumulated in S phase and 8.23% in G2 phase, whereas only 5.15% and 1.99% of Hs-RFP cells were detected at S and G2 phase (P = 0.0052 for S phase and P = 0.0241 for G2 phase). The percentage of cells in S/G2 phase increased to 40.15% in Hs-FABP7 cells compared with 14.29% in Hs-RFP cells at 72 h after serum starvation. Cell arrest in S/G2 phase in Hs-FABP7 cells may partly contribute to the decreased cell viability during serum starvation. This effect was not observed in MDA-MB-231 cells (Fig. 3B).
The decrease in Hs-FABP7 cell viability during serum starvation was potentially attributed to apoptosis and necrosis (Fig. 3C). More necrotic cell death and late apoptosis was observed in Hs-FABP7 cells (34.26% and 21.3%) than in Hs-RFP cells (18.83% and 4.63%) at 72 h after serum starvation (P < 0.0001). Upon serum starvation, increasing cell death was observed in MDA-MB-231 cells, but with no significant difference observed between 231-RFP and 231-FABP7 cells. Taken together, the decreased cell viability in Hs-FABP7 cells during serum starvation was due to reduced proliferation rate, cell cycle arrest at S and G2 phase, and cell death.
Perturbations in the metabolism and mitochondrial oxygen consumption in Hs-FABP7 cells during serum starvation
Given the role of FABP7 in fatty acid metabolism, we investigated the mechanism of FABP7-induced cell death by exploring a panel of genes and enzyme activities involved in the metabolism of glucose, glutamine, and fatty acid ( Fig. 4 and supplemental Figs. S2, S3). In complete medium, despite the increase in glucose transporter 1 (GLUT1) mRNA expression, glycolysis-related genes phosphofructokinase 1 (PFK1) and GAPDH remained unchanged in Hs-FABP7 cells, whereas the genes regulating FAO, carnitine palmitoyltransferase 1A (CPT1A), and medium-chain acyl-CoA dehydrogenase (MCAD) were markedly upregulated (Fig. 4A). In Hs-FABP7 cells, the expression of GLS1, encoding the rate-limiting enzyme for glutaminolysis, was not significantly different than in Hs-RFP cells. When cultured in complete medium, Hs-FABP7 cells did not show any differences in GAPDH, GLS, and FAO activities when compared with Hs-RFP cells 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001. ( Fig. 4B-D). Subsequently, mitochondrial OCR was similar in Hs-FABP7 cells and in the control Hs-RFP cells (Fig. 4E).
However, when challenged with serum starvation, a widespread downregulation of glucose and glutamine metabolism-related genes were observed in Hs-FABP7 cells when compared with Hs-RFP cells. Interestingly, despite the absence of serum (lipids) during serum starvation, the gene expression of CPT1A and MCAD was about 2-fold higher in Hs-FABP7 cells than Hs-RFP cells (Fig. 4A). Serum starvation generally decreased glycolytic enzyme activity in Hs578T cells, but FABP7-overexpressing cells had a further 30% reduction (P = 0.0121) in GAPDH activity compared with Hs-RFP cells (Fig. 4B). Although GLS activity remained not significantly different in both cells during serum starvation, GLS activity was significantly lower (P = 0.0296) in Hs-FABP7 cells cultured in serum starvation than in complete medium (Fig. 4C). Interestingly, FAO activity in Hs-FABP7 remained similar with the control Hs-RFP cells (Fig. 4D), despite upregulation of FAO-related genes. This may be explained by the lack of fatty acid in the culture medium to activate the metabolic pathway. Consequently, mitochondrial oxidative capacity of both Hs-RFP and Hs-FABP7 cells was generally lower during serum starvation (P = 0.0001 for both cells); however, Hs-FABP7 cells had much lower OCR (16.69 pmol/min/µg) compared with Hs-RFP cells (21.7 pmol/min/µg) (P < 0.05) (Fig. 4E). These data indicate a lower mitochondrial oxidative capacity particularly in Hs-FABP7 cells, probably due to limited bioenergetic substrates during serum starvation.
It is worthwhile to note that, unlike Hs578T, MDA-MB-231 cells showed a rather different metabolic profile, as indicated by the expression of metabolic genes ( Fig. 4A and supplemental Fig. S2B). When cultured in complete medium, although FABP7 was not affecting the cell viability, glycolysis was upregulated in 231-FABP7 cells, with about a 1.7-fold increase in GAPDH expression. Meanwhile, a 4-fold decrease in GLS1 expression was observed, indicative of a downregulation of the glutaminolysis pathway. Upon serum starvation, 231-FABP7 cells demonstrated a marked increase in glucose and glutamine metabolismrelated genes, with more than 4-fold increase in GLUT1 and GLS1 expression.
Taken together, our data show that Hs-FABP7 cells, when challenged with a serum-starved condition, were not able to metabolically adapt and effectively utilize other available energetic substrates, especially glucose, and, as a consequence, could not survive.
PPAR- signaling mediated FABP7-induced cell death in Hs578T cells under serum starvation
The upregulation of CPT1A and MCAD mRNA, accompanied with a downregulation of Glut1 mRNA in Hs-FABP7 cells, indicated a possible role of PPAR- signaling, as these genes are direct targets of PPAR- transcription factor. Immunofluorescence staining showed that the expression of PPAR- protein was increased in the nucleus during serum starvation ( Fig. 5A and supplemental Fig. S4A). Similarly, FABP7 protein was observed to translocate into the nucleus (Fig. 5A and supplemental Fig. S4B) in Hs-FABP7 cells upon serum starvation, potentially acting as fatty acid chaperones to deliver ligands to PPAR- protein (23). This suggests that nuclear localization of FABP7 enhanced the action of PPAR- transcriptional activity during serum starvation in Hs-FABP7 cells.
To investigate whether PPAR- activation was responsible for FABP7-mediated inhibition of cell viability in Hs-FABP7 cells, we treated the cells with a PPAR--specific inhibitor (GW6471). In both Hs578T and MDA-MB-231 cells, treatment of GW6471 at a concentration of 2 M in complete medium had no significant effect on cell viability (Fig. 5B). Yet, the same concentration of GW6471 reversed the cell-growth inhibition observed in Hs-FABP7 cells when exposed to serum-starved conditions (Fig. 5B and supplemental Fig. S5). Subsequent gene-expression analysis revealed that PPAR- target genes were downregulated (CPT1A: 2.43-fold; MCAD: 1.54-fold), when compared with vehicle-treated cells (Fig. 5C). A significant 2-fold increase of GLUT1, PFK1, glucose-6-phosphate dehydrogenase (G6PD), and isocitrate dehydrogenase 1 (IDH1) mRNA expression was observed in GW6471-treated Hs-FABP7 cells (Fig. 5C), indicating that the rescue of cell viability may be due to the switch from lipid to glucose as bioenergetic substrates in these cells. Taken together, FABP7-induced cell death in Hs578T cells during serum starvation is due to activated PPAR- signaling.
DISCUSSION
In this study, we showed that overexpression of FABP7 disabled TNBC cell line Hs578T to metabolically adapt during serum starvation. FABP7-overexpressing cells failed to efficiently utilize glucose as bioenergetics substrates, when challenged with serum starvation. This was accompanied by increased expression of genes controlling FAO as it attempts to utilize free fatty acids liberated by lipolysis from the lipid storage that resulted from preexposure to complete medium (24). However, as free fatty acid storage was depleted, serum-starved FABP7-overexpressing cells were not able to further metabolically adapt and sustain ATP production. The failure of the cells to efficiently utilize glucose for ATP generation leads to cell death. We further showed that the metabolic impact of FABP7 in Hs578T cells during serum starvation is mediated by PPAR- signaling.
FABP7 is predominantly expressed in the nervous system and mammary gland. In the mammary gland, FABP7 inhibits proliferation and promotes differentiation of mammary cells (25). Similarly, in the nervous system, FABP7 is involved in the differentiation of neurons and development of the cortex (26,27). The expression of FABP7 at the edge of astrocytes also suggested that FABP7 may regulate the motility of astrocytes by increasing the uptake of polyunsaturated fatty acids (28). Yet, the evidence of FABP7 expression in TNBC cell lines was rather limited (20,29). A previous study demonstrating the function of endogenous FABP7 in TNBC was reported using MDA-MB-435S, a melanoma cell line formerly misidentified as a TNBC cell line (30). Our study, however, provides evidence on the role of FABP7 as a mediator for tumor cell death during nutrient-deprived conditions. This finding indicates an anti-tumorigenic role of FABP7 in TNBC, which is mediated by specific nutrient availability.
The metabolic vulnerabilities of FABP7-overexpressing Hs578T cells resulted in S/G2-phase arrest, which subsequently led to cell death. Serum starvation typically induces cell-cycle arrest at G1 phase (31,32), which is observed in Hs-RFP and MDA-MB-231 cells. These cells were able to proliferate, despite a lower proliferation rate, indicating that the lower mitochondrial activity in these cells during serum starvation was sufficient to overcome the metabolic checkpoint and complete the cell cycle (33). Hs-FABP7 cells, however, arrested at the S/G2 phase. G2 arrest has been described as another growth-restricting mechanism in mitogen-starved conditions, likely due to elevated E2F activity (34). Cell arrest could potentially lead to cell death, in which both apoptosis and necrosis were observed in Hs-FABP7 cells upon serum starvation. Apoptosis is a cellular suicide program triggered by stress signals such as nutrient deprivation, which results in the execution of caspasemediated cell death (35). Serum starvation has been shown to induce apoptosis in hepatocellular carcinoma cells (36) and MCF7 breast cancer cells (37), in which the latter was associated with an altered ratio of apoptosis regulating proteins Bax and Bcl-2. Necrosis, on the other hand, may occur when the minimal bioenergetic demands are not met (38), a condition similar to our observations where Hs-FABP7 cells may have limited bioenergetic substrates during serum starvation. In fact, there are common mediators of apoptosis and necrosis, which could potentially contribute to the two modes of cell death observed in our model (39). It is worth noting that FABP7 did not induce cell death in MDA-MB-231 cells during serum starvation, indicating that this cell line is probably more resilient to nutrient deprivation than Hs578T cells. The disparity in the response of Hs578T and MDA-MB-231 cells to serum starvation may also be attributed to differences in their metabolic profiles (40). Hence, it would be worthwhile to examine other TNBC cells with differing metabolic profiles, in delineating the molecular heterogeneity involved in cell death induced by metabolic vulnerabilities.
During serum starvation, cancer cells sense and adapt to nutrient deprivation by upregulating glucose metabolism Fig. 5. FABP7-induced cell death in Hs578T cells during serum starvation was mediated by the action of PPAR- signaling. A: Confocal microscopy of immunofluorescence staining of FABP7 (green) and PPAR- (red) proteins in Hs578T and MDA-MB-231 cells after culture in either complete medium or serum-free medium for 24 h. Blue, DAPI. Scale bar, 50 m. Data shown are representative of two independent experiments. B: Cells were treated with PPAR- inhibitor GW6471 (2 M) in complete or serum-starved medium for 72 h, before analyzing for cell viability using an MTT assay. Data shown are mean ± SEM of triplicates; this is a representative of three independent experiments. *** P < 0.0001. C: mRNA expression of key metabolic genes after treatment with 2 M GW6471 in Hs-FABP7 cells for 24 h in serum starvation when compared with vehicle-treated cells. The genes were normalized to housekeeping gene 18S rRNA. CM, complete medium; SFM, serum-free medium. (41,42), besides releasing fatty acids from the lipid droplet through lipolysis for oxidation (24). Central to such adaption, PPAR- is known to be a lipid sensor that maintains cellular energy homeostasis. It is a ligand-activated class of nuclear hormone receptor that binds to peroxisome proliferator response elements (PPREs) to induce transcription of genes such as lipid processing. Its activation aims at increasing lipid combustion in order to yield more energy production, especially in a challenged environment (43). It was shown in rat hepatocytes that PPAR- expression is increased by fasting-and starvation-induced glucocorticoids (44), suggesting the role of PPAR- to maintain cell survival.
In our model, increased PPAR- expression was observed when the cells were serum-starved, possibly as an adaptation strategy of the cells. The activation of PPAR- was observed in Hs-FABP7 cells during serum starvation, as reflected in the increase of PPAR- target genes, CPT1A and MCAD, which are involved in fatty acid mitochondrial uptake and oxidation (45). Our observation may indicate the usage of fatty acids liberated from lipid droplets as the substrates for FAO. However, the attempt of the cells to survive serum starvation failed as the lipid storage was limited and fatty acids were absent in the serum-free media. In addition, PPAR- activation also potentially inhibited glucose metabolism by targeting the consensus PPRE motif in the promoter region of Glut1, hence repressing its transcription (46). As a result, Hs-FABP7 cells could not efficiently utilize glucose as bioenergetic substrates when serumstarved, which eventually led to cell death.
Our data suggest that PPAR--induced changes are FABP7-dependent. Literature evidence shows that in Cos-7 cells where FABPs and PPAR- are not detected, transfection of the proteins showed that PPAR- activity is amplified by FABP1 and FABP2 (47). In another study, PPAR- transactivation is proportional to levels of L-FABP in human hepatoma HepG2 cells line (48). In our model, upon serum starvation, increased nuclear FABP7 expression was observed, suggesting the role of FABP7 as fatty acids chaperones to transport PPAR- ligands into the nucleus for PPAR- activation. In murine L cells, transduction with FABP1 had dramatically enhanced the uptake of fatty acid into the nucleus (49). Incubation of rat liver nuclei with fluorescence-tagged FABP1 together with fatty acids demonstrated a strong fluorescence response in the nuclei, which otherwise did not happen when fatty acids were absent (50). There has been evidence indicating physical interaction between FABP1 and PPAR- in primary hepatocytes (51). Ablation of L-FABP gene expression significantly impaired fatty acid distribution in the nucleus and affected PPAR- activation in these cells (52). Hence, whether FABP7 and PPAR- require direct contact to induce cell death in TNBC needs to be further investigated. In short, our data suggest that FABP7 facilitates PPAR- activation in serum-starved condition by transporting its ligand (fatty acids) into the nucleus, before enhancing its transcriptional activity.
Our study provides an understanding on the role of FABP7 in regulating the metabolic adaptation of TNBC cell lines. Transcriptional activation of PPAR- in FABP7overexpressing cell can limit their metabolic plasticity when challenged with serum starvation and resulted in reduced survival of these cells. Hence, manipulating metabolic stress in cells expressing FABP7 protein may potentially be an effective targeted therapeutic strategy in FABP7-positive TNBC patients.
Fig. 1 .
1Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.
Fig. 2 .
2The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.
Fig.
Fig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.
Fig. 4 .
4FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.
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Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes. A L Mcintosh, B P Atshaves, H A Hostetler, H Huang, J Davis, O I Lyuksyutova, D Landrock, A B Kier, F Schroeder, Arch. Biochem. Biophys. 485McIntosh, A. L., B. P. Atshaves, H. A. Hostetler, H. Huang, J. Davis, O. I. Lyuksyutova, D. Landrock, A. B. Kier, and F. Schroeder. 2009. Liver type fatty acid binding protein (L-FABP) gene ablation re- duces nuclear ligand distribution and peroxisome proliferator- activated receptor- activity in cultured primary hepatocytes. Arch. Biochem. Biophys. 485: 160-173.
| [
"ERs) and progesterone receptors (PRs), as well as human epidermal growth factor receptor 2 (HER2). It is the most aggressive subtype of breast cancer, affecting about 15-20% of total breast cancer cases. Compared with other subtypes, TNBC tumors are associated with higher histologic grade and larger tumor size (1, 2). The current standard regimen for TNBC patients is a combination of surgery and chemotherapy (3), which often fails to effectively slow down the tumor progression. Hence, TNBC patients have lower overall survival (81% vs. 91%) and disease-free survival (72% vs. 86%) compared with non-TNBC patients (4). Given the lack of ER and HER2 in this subtype, there is no molecular characterization that allows this subtype to be targeted.Uncontrolled proliferation of cancer cells is metabolically demanding. The metabolic pathways utilized by cancer cells to sustain high-energy demands and biosynthesis differ from those employed by healthy cells (5). Challenged by hostile environments such as hypoxia and acute interruptions in nutrient availability, cancer cells typically develop metabolic plasticity, which enables the utilization of available nutrients as bioenergetic substrates. This metabolic flexibility allows maintained ATP production under varying physiological and pathological conditions and is primarily regulated by substrate concentration, hormone levels, blood flow, oxygen supply, and workload(6).Cancer-associated changes in cellular metabolism may also be a direct consequence of oncogenic signal transduction.Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, partly due to the lack of targeted therapy available. Cancer cells heavily reprogram their metabolism and acquire metabolic plasticity to satisfy the high-energy demand due to uncontrolled proliferation. Accumulating evidence shows that deregulated lipid metabolism affects cancer cell survival, and therefore we sought to understand the function of fatty acid binding protein 7 (FABP7), which is expressed predominantly in TNBC tissues. As FABP7 was not detected in the TNBC cell lines tested, Hs578T and MDA-MB-231 cells were transduced with lentiviral particles containing either FABP7 open reading frame or red fluorescent protein. During serum starvation, when lipids were significantly reduced, FABP7 decreased the viability of Hs578T, but not of MDA-MB-231, cells. FABP7overexpressing Hs578T (Hs-FABP7) cells failed to efficiently utilize other available bioenergetic substrates such as glucose to sustain ATP production, which led to S/G2 phase arrest and cell death. We further showed that this metabolic phenotype was mediated by PPAR- signaling, despite the lack of fatty acids in culture media, as Hs-FABP7 cells attempted to survive. This study provides imperative evidence of metabolic vulnerabilities driven by FABP7 via PPAR- signaling.-Kwong, S. C., A. H. A. Jamil, A. Rhodes, N. A. Taib, and I. Chung. Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling. J. Lipid Res. 2019. 60: 1807-1817.Supplementary key words fatty acid binding protein • peroxisome proliferator-activated receptor alpha • cancer • nutrient deprivation • fatty acid metabolism • metabolic adaptation Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptors"
] | [
"Soke Chee Kwong \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n",
"Amira Hajirah ",
"Abd Jamil ",
"Anthony Rhodes ",
"Nur Aishah Taib ",
"§ ",
"Ivy Chung [email protected] \nFaculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;\n\n§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia\n",
"\nFaculty of Medicine\nDepartments of Pharmacology\nthe Translational Core Laboratory\nUniversity of Malaya\n\n"
] | [
"Faculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;",
"§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia",
"Faculty of Medicine\nSchool of Medicine\nFaculty of Health and Medical Sciences\nPharmacy, † Pathology,** and Surgery † † and University of Malaya Cancer Research Institute, § §\nUniversity of Malaya\n50603Kuala LumpurMalaysia;",
"§ Taylor's University\nSubang Jaya47500Lakeside Campus, SelangorMalaysia",
"Faculty of Medicine\nDepartments of Pharmacology\nthe Translational Core Laboratory\nUniversity of Malaya\n"
] | [
"Soke",
"Amira",
"Abd",
"Anthony",
"Nur",
"Aishah",
"§",
"Ivy"
] | [
"Chee Kwong",
"Hajirah",
"Jamil",
"Rhodes",
"Taib",
"Chung"
] | [
"S K Kanapathy Pillai, ",
"A Tay, ",
"S Nair, ",
"C-O Leong, ",
"R Dent, ",
"M Trudeau, ",
"K I Pritchard, ",
"W M Hanna, ",
"H K Kahn, ",
"C A Sawka, ",
"L A Lickley, ",
"E Rawlinson, ",
"P Sun, ",
"S A Narod, ",
"E Rodler, ",
"L Korde, ",
"J Gralow, ",
"H G Kaplan, ",
"J A Malmgren, ",
"Y Zhang, ",
"J-M Yang, ",
"E Obre, ",
"R Rossignol, ",
"R L Elstrom, ",
"D E Bauer, ",
"M Buzzai, ",
"R Karnauskas, ",
"M H Harris, ",
"D R Plas, ",
"H Zhuang, ",
"R M Cinalli, ",
"A Alavi, ",
"C M Rudin, ",
"D R Wise, ",
"R J Deberardinis, ",
"A Mancuso, ",
"N Sayed, ",
"X-Y. Zhang, ",
"H K Pfeiffer, ",
"I Nissim, ",
"E Daikhin, ",
"M Yudkoff, ",
"S B Mcmahon, ",
"M Buzzai, ",
"D E Bauer, ",
"R G Jones, ",
"R J Deberardinis, ",
"G Hatzivassiliou, ",
"R L Elstrom, ",
"C B Thompson, ",
"K A Olson, ",
"J C Schell, ",
"J Rutter, ",
"S Beloribi-Djefaflia, ",
"S Vasseur, ",
"F Guillaumond, ",
"F Baenke, ",
"B Peck, ",
"H Miess, ",
"A Schulze, ",
"J Zhao, ",
"Z Zhi, ",
"C Wang, ",
"H Xing, ",
"G Song, ",
"X Yu, ",
"Y Zhu, ",
"X Wang, ",
"X Zhang, ",
"Y Di, ",
"T Mashima, ",
"H Seimiya, ",
"T Tsuruo, ",
"X Y Tang, ",
"S Umemura, ",
"H Tsukamoto, ",
"N Kumaki, ",
"Y Tokuda, ",
"R Y Osamura, ",
"Y E Shi, ",
"J Ni, ",
"G Xiao, ",
"Y E Liu, ",
"A Fuchs, ",
"G Yu, ",
"J Su, ",
"J M Cosgrove, ",
"L Xing, ",
"M Zhang, ",
"V L Spitsberg, ",
"E Matitashvili, ",
"R C Gorewit, ",
"R E Burrier, ",
"C R Manson, ",
"P Brecher, ",
"N R Coe, ",
"M A Simpson, ",
"D A Bernlohr, ",
"R Z Liu, ",
"K Graham, ",
"D D Glubrecht, ",
"R Lai, ",
"J R Mackey, ",
"R Godbout, ",
"A T Alshareeda, ",
"E A Rakha, ",
"C C Nolan, ",
"I O Ellis, ",
"A R Green, ",
"H Zhang, ",
"E A Rakha, ",
"G Ball, ",
"I Spiteri, ",
"M Aleskandarany, ",
"E Paish, ",
"D G Powe, ",
"R D Macmillan, ",
"C Caldas, ",
"I O Ellis, ",
"R Mita, ",
"M J Beaulieu, ",
"C Field, ",
"R Godbout, ",
"A S Rambold, ",
"S Cohen, ",
"J Lippincott-Schwartz, ",
"Y Yang, ",
"E Spitzer, ",
"N Kenney, ",
"W Zschiesche, ",
"M Li, ",
"A Kromminga, ",
"T Müller, ",
"F Spener, ",
"A Lezius, ",
"J H Veerkamp, ",
"S G Kuhar, ",
"L Feng, ",
"S Vidan, ",
"M E Ross, ",
"M E Hatten, ",
"N Heintz, ",
"Y Arai, ",
"N Funatsu, ",
"K Numayama-Tsuruta, ",
"T Nomura, ",
"S Nakamura, ",
"N Osumi, ",
"M Kipp, ",
"T Clarner, ",
"S Gingele, ",
"F Pott, ",
"S Amor, ",
"P Van Der, ",
"C Valk, ",
"Beyer, ",
"K Bensaad, ",
"E Favaro, ",
"C A Lewis, ",
"B Peck, ",
"S Lord, ",
"J M Collins, ",
"K E Pinnick, ",
"S Wigfield, ",
"F M Buffa, ",
"J L Li, ",
"M Lacroix, ",
"J-S Shin, ",
"S-W Hong, ",
"S-L O Lee, ",
"T-H Kim, ",
"I-C Park, ",
"S -K. An, ",
"W K Lee, ",
"J S Lim, ",
"K I Kim, ",
"Y Yang, ",
"Y Huang, ",
"Z Fu, ",
"W Dong, ",
"Z Zhang, ",
"J Mu, ",
"J Zhang, ",
"S M Schieke, ",
"J P Mccoy, ",
"Jr , ",
"T Finkel, ",
"F Foijer, ",
"H Te Riele, ",
"K Matsuura, ",
"K Canfield, ",
"W Feng, ",
"M Kurokawa, ",
"X Kou, ",
"Y Jing, ",
"W Deng, ",
"K Sun, ",
"Z Han, ",
"F Ye, ",
"G Yu, ",
"Q Fan, ",
"L Gao, ",
"Q Zhao, ",
"Tovar Sepulveda, ",
"V A , ",
"X Shen, ",
"M Falzon, ",
"S Jin, ",
"R S Dipaola, ",
"R Mathew, ",
"E White, ",
"V Nikoletopoulou, ",
"M Markaki, ",
"K Palikaras, ",
"N Tavernarakis, ",
"N J Lanning, ",
"J P Castle, ",
"S J Singh, ",
"A N Leon, ",
"E A Tovar, ",
"A Sanghera, ",
"J P Mackeigan, ",
"F V Filipp, ",
"C R Graveel, ",
"N Zheng, ",
"K Wang, ",
"J He, ",
"Y Qiu, ",
"G Xie, ",
"M Su, ",
"W Jia, ",
"H Li, ",
"S Dai, ",
"A Gocher, ",
"L Euscher, ",
"A Edelman, ",
"S R Pyper, ",
"N Viswakarma, ",
"S Yu, ",
"J K Reddy, ",
"M Viana Abranches, ",
"F C Esteves De Oliveira, ",
"J Bressan, ",
"S Mandard, ",
"M Müller, ",
"S Kersten, ",
"M You, ",
"J Jin, ",
"Q Liu, ",
"Q Xu, ",
"J Shi, ",
"Y Hou, ",
"M L Hughes, ",
"B Liu, ",
"M L Halls, ",
"K M Wagstaff, ",
"R Patil, ",
"T Velkov, ",
"D A Jans, ",
"N W Bunnett, ",
"M J Scanlon, ",
"C J Porter, ",
"C Wolfrum, ",
"C M Borrmann, ",
"T Börchers, ",
"F Spener, ",
"H Huang, ",
"O Starodub, ",
"A Mcintosh, ",
"A B Kier, ",
"F Schroeder, ",
"J W Lawrence, ",
"D J Kroll, ",
"P I Eacho, ",
"H A Hostetler, ",
"A L Mcintosh, ",
"B P Atshaves, ",
"S M Storey, ",
"H R Payne, ",
"A B Kier, ",
"F Schroeder, ",
"A L Mcintosh, ",
"B P Atshaves, ",
"H A Hostetler, ",
"H Huang, ",
"J Davis, ",
"O I Lyuksyutova, ",
"D Landrock, ",
"A B Kier, ",
"F Schroeder, "
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] | [
"Kanapathy Pillai",
"Tay",
"Nair",
"Leong",
"Dent",
"Trudeau",
"Pritchard",
"Hanna",
"Kahn",
"Sawka",
"Lickley",
"Rawlinson",
"Sun",
"Narod",
"Rodler",
"Korde",
"Gralow",
"Kaplan",
"Malmgren",
"Zhang",
"Yang",
"Obre",
"Rossignol",
"Elstrom",
"Bauer",
"Buzzai",
"Karnauskas",
"Harris",
"Plas",
"Zhuang",
"Cinalli",
"Alavi",
"Rudin",
"Wise",
"Deberardinis",
"Mancuso",
"Sayed",
"Zhang",
"Pfeiffer",
"Nissim",
"Daikhin",
"Yudkoff",
"Mcmahon",
"Buzzai",
"Bauer",
"Jones",
"Deberardinis",
"Hatzivassiliou",
"Elstrom",
"Thompson",
"Olson",
"Schell",
"Rutter",
"Beloribi-Djefaflia",
"Vasseur",
"Guillaumond",
"Baenke",
"Peck",
"Miess",
"Schulze",
"Zhao",
"Zhi",
"Wang",
"Xing",
"Song",
"Yu",
"Zhu",
"Wang",
"Zhang",
"Di",
"Mashima",
"Seimiya",
"Tsuruo",
"Tang",
"Umemura",
"Tsukamoto",
"Kumaki",
"Tokuda",
"Osamura",
"Shi",
"Ni",
"Xiao",
"Liu",
"Fuchs",
"Yu",
"Su",
"Cosgrove",
"Xing",
"Zhang",
"Spitsberg",
"Matitashvili",
"Gorewit",
"Burrier",
"Manson",
"Brecher",
"Coe",
"Simpson",
"Bernlohr",
"Liu",
"Graham",
"Glubrecht",
"Lai",
"Mackey",
"Godbout",
"Alshareeda",
"Rakha",
"Nolan",
"Ellis",
"Green",
"Zhang",
"Rakha",
"Ball",
"Spiteri",
"Aleskandarany",
"Paish",
"Powe",
"Macmillan",
"Caldas",
"Ellis",
"Mita",
"Beaulieu",
"Field",
"Godbout",
"Rambold",
"Cohen",
"Lippincott-Schwartz",
"Yang",
"Spitzer",
"Kenney",
"Zschiesche",
"Li",
"Kromminga",
"Müller",
"Spener",
"Lezius",
"Veerkamp",
"Kuhar",
"Feng",
"Vidan",
"Ross",
"Hatten",
"Heintz",
"Arai",
"Funatsu",
"Numayama-Tsuruta",
"Nomura",
"Nakamura",
"Osumi",
"Kipp",
"Clarner",
"Gingele",
"Pott",
"Amor",
"Van Der",
"Valk",
"Beyer",
"Bensaad",
"Favaro",
"Lewis",
"Peck",
"Lord",
"Collins",
"Pinnick",
"Wigfield",
"Buffa",
"Li",
"Lacroix",
"Shin",
"Hong",
"Lee",
"Kim",
"Park",
"-K. An",
"Lee",
"Lim",
"Kim",
"Yang",
"Huang",
"Fu",
"Dong",
"Zhang",
"Mu",
"Zhang",
"Schieke",
"Mccoy",
"Finkel",
"Foijer",
"Te Riele",
"Matsuura",
"Canfield",
"Feng",
"Kurokawa",
"Kou",
"Jing",
"Deng",
"Sun",
"Han",
"Ye",
"Yu",
"Fan",
"Gao",
"Zhao",
"Sepulveda",
"Shen",
"Falzon",
"Jin",
"Dipaola",
"Mathew",
"White",
"Nikoletopoulou",
"Markaki",
"Palikaras",
"Tavernarakis",
"Lanning",
"Castle",
"Singh",
"Leon",
"Tovar",
"Sanghera",
"Mackeigan",
"Filipp",
"Graveel",
"Zheng",
"Wang",
"He",
"Qiu",
"Xie",
"Su",
"Jia",
"Li",
"Dai",
"Gocher",
"Euscher",
"Edelman",
"Pyper",
"Viswakarma",
"Yu",
"Reddy",
"Viana Abranches",
"Esteves De Oliveira",
"Bressan",
"Mandard",
"Müller",
"Kersten",
"You",
"Jin",
"Liu",
"Xu",
"Shi",
"Hou",
"Hughes",
"Liu",
"Halls",
"Wagstaff",
"Patil",
"Velkov",
"Jans",
"Bunnett",
"Scanlon",
"Porter",
"Wolfrum",
"Borrmann",
"Börchers",
"Spener",
"Huang",
"Starodub",
"Mcintosh",
"Kier",
"Schroeder",
"Lawrence",
"Kroll",
"Eacho",
"Hostetler",
"Mcintosh",
"Atshaves",
"Storey",
"Payne",
"Kier",
"Schroeder",
"Mcintosh",
"Atshaves",
"Hostetler",
"Huang",
"Davis",
"Lyuksyutova",
"Landrock",
"Kier",
"Schroeder"
] | [
"Triplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. S K Kanapathy Pillai, A Tay, S Nair, C-O Leong, BMC Clin. Pathol. 1218Kanapathy Pillai, S. K., A. Tay, S. Nair, and C-O. Leong. 2012. Triple- negative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women. BMC Clin. Pathol. 12: 18.",
"Triple-negative breast cancer: clinical features and patterns of recurrence. R Dent, M Trudeau, K I Pritchard, W M Hanna, H K Kahn, C A Sawka, L A Lickley, E Rawlinson, P Sun, S A Narod, Clin. Cancer Res. 13Dent, R., M. Trudeau, K. I. Pritchard, W. M. Hanna, H. K. Kahn, C. A. Sawka, L. A. Lickley, E. Rawlinson, P. Sun, and S. A. Narod. 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res. 13: 4429-4434.",
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] | [
"Triplenegative breast cancer is associated with EGFR, CK5/6 and c-KIT expression in Malaysian women",
"Triple-negative breast cancer: clinical features and patterns of recurrence",
"Current treatment options in triple negative breast cancer",
"Impact of triple negative phenotype on breast cancer prognosis",
"Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention",
"Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy",
"Akt stimulates aerobic glycolysis in cancer cells",
"Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction",
"The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid -oxidation",
"Pyruvate and metabolic flexibility: illuminating a path toward selective cancer therapies",
"Lipid metabolic reprogramming in cancer cells",
"Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development",
"Exogenous lipids promote the growth of breast cancer cells via CD36",
"De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy",
"Overexpression of fatty acid binding protein-7 correlates with basal-like subtype of breast cancer",
"Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene",
"Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland",
"Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis",
"Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels",
"A fatty acid-binding protein 7/RXR pathway enhances survival and proliferation in triple-negative breast cancer",
"Fatty acid binding protein 7 expression and its subcellular localization in breast cancer",
"The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer",
"Brain Fatty Acid-binding Protein and -3/-6 fatty acids: mechanistic insight into malignant glioma cell migration",
"Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics",
"Members of the fatty acid binding protein family are differentiation factors for the mammary gland",
"Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation",
"Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex",
"Brain lipid binding protein (FABP7) as modulator of astrocyte function",
"Fatty acid uptake and lipid storage induced by HIF-1 contribute to cell growth and survival after hypoxia-reoxygenation",
"MDA-MB-435 cells are from melanoma, not from breast cancer",
"Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells",
"Serum starvation-induces down-regulation of Bcl-2/Bax confers apoptosis in tongue coating-related cells in vitro",
"Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression",
"Restriction beyond the restriction point: mitogen requirement for G2 passage",
"Metabolic regulation of apoptosis in cancer",
"Tumor necrosis factor- attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells",
"Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells",
"Metabolic catastrophe as a means to cancer cell death",
"Crosstalk between apoptosis, necrosis and autophagy",
"Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities",
"Effects of ADMA on gene expression and metabolism in serum-starved LoVo cells",
"Serum starvation induces a rapid increase of Akt phosphorylation in ovarian cancer cells",
"PPAR: energy combustion, hypolipidemia, inflammation and cancer",
"Peroxisome proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus",
"Peroxisome proliferator-activated receptor target genes",
"PPAR promotes cancer cell Glut1 transcription repression",
"Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor in a ligand-selective manner",
"Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors -and -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus",
"Liver fatty acid-binding protein targets fatty acids to the nucleus. Real time confocal and multiphoton fluorescence imaging in living cells",
"Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus",
"L-FABP directly interacts with PPAR in cultured primary hepatocytes",
"Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferatoractivated receptor- activity in cultured primary hepatocytes"
] | [
"BMC Clin. Pathol",
"Clin. Cancer Res",
"Breast Dis",
"Breast J",
"Cancer Biol. Ther",
"Int. J. Biochem. Cell Biol",
"Cancer Res",
"Proc. Natl. Acad. Sci. USA",
"Oncogene",
"Trends Biochem. Sci",
"Oncogenesis",
"Dis. Model. Mech",
"Oncol. Rep",
"Br. J. Cancer",
"Pathol. Res. Pract",
"MRG. Cancer Res",
"Eur. J. Biochem",
"Biochim. Biophys. Acta",
"J. Lipid Res",
"J. Pathol",
"Breast Cancer Res. Treat",
"Breast Cancer Res. Treat",
"J. Biol. Chem",
"Dev. Cell",
"J. Cell Biol",
"Development",
"J. Neurosci",
"Physiol. Res",
"Cell Reports",
"Cancer Chemother. Pharmacol",
"Int. J. Oncol",
"Mol. Med. Rep",
"Cell Cycle",
"Cell Div",
"Int. Rev. Cell. Mol. Biol",
"BMC Cancer",
"Endocrinology",
"J. Cell Sci",
"Biochim. Biophys. Acta",
"Cancer Metab",
"Sci. Rep",
"FASEB J",
"Nucl. Recept. Signal",
"Nutr. Hosp",
"Cell. Mol. Life Sci",
"J. Cell. Biochem",
"J. Biol. Chem",
"Proc. Natl. Acad. Sci. USA",
"J. Biol. Chem",
"J. Lipid Res",
"J. Lipid Res",
"Arch. Biochem. Biophys"
] | [
"\nFig. 1 .\n1Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.",
"\nFig. 2 .\n2The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.",
"\nFig.\nFig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.",
"\nFig. 4 .\n4FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium."
] | [
"Generation of FABP7-overexpressing TNBC cells. A: The result shows data mining on FABP7 expression in non-TNBC versus TNBC tumors from online database Geodataset. *** P < 0.0001. FABP7 mRNA (B) and protein expressions (C) of breast cancer cell lines were analyzed using qRT-PCR and Western blotting, respectively. D: Western blot was performed for FABP7 protein detection to confirm FABP7 expression posttransduction in the selected TNBC cell lines. Data represent the mean ± SEM of duplicates and are representative of two independent experiments.",
"The effect of FABP7 on TNBC cell viability in various nutrient-deprived conditions. MTT assay was used to investigate the viability of Hs578T and MDA-MB-231 cells in glucose-starved (A), glutamine-starved (B), and serum-starved (C) conditions. The cells were cultured in either complete medium or nutrient-deprived medium. Data represent the mean ± SEM of triplicates and are representative of three independent experiments. ** P < 0.001; *** P < 0.0001. CM, complete medium; SFM, serum-free medium.",
"Fig. 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001.",
"FABP7 altered the metabolism in Hs578T cells during serum starvation. A: Diagram showing genes involved in glycolysis, glutaminolysis, and FA metabolism, as highlighted in the box. The heatmap compares the expression of metabolic genes in FABP7-overexpressing cells to control cells cultured in complete medium and serum-free medium. The genes were normalized to housekeeping gene 18S rRNA. Data represent the mean ± SEM of duplicates and are representative of two independent experiments. Enzymatic assays evaluating GAPDH (B), GLS (C), and FAO (D) activities were conducted. Data shown are mean ± SEM of duplicates. E: Basal OCR of the cells (n = 18 for each group) was measured with a Seahorse Mito Fuel Flex Test Kit. Data shown are mean ± SEM of replicates, a representative of two experiments. * P < 0.05; ** P < 0.01; *** P < 0.0001. CM, complete medium; SFM, serum-free medium."
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"Fig. 3B and supplemental Fig. S1",
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"Fig. 4 and supplemental Figs. S2, S3)",
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"Fig. 4A and supplemental Fig. S2B",
"Fig. 5A and supplemental Fig. S4A)",
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"AKT activation augments the Warburg effect and renders the cancer cells addicted to glucose (7). Myc protein promotes mitochondrial glutaminolysis and glutamine addiction by shunting glucose away from mitochondrial metabolism (8). Cancer cells that fail to demonstrate metabolic flexibility during nutrient deprivation could become vulnerable in their survival (9). Hence, metabolic vulnerabilities exhibited by cancer cells can be targeted for cancer management (6,10).",
"Alterations in lipid-and cholesterol-associated pathways encountered in tumors are now increasingly recognized and more frequently described (11,12), but not completely understood. Many cancer types show a strong lipid avidity, in which increasing uptake of exogenous lipid sources (13) or overactivation of endogenous lipid synthesis (14) is observed. Fatty acid binding protein 7 (FABP7), a member of the FABP intracellular lipid chaperone family, has been shown to be upregulated in TNBC compared with other breast cancer subtypes (15,16). FABPs regulates lipid metabolism by increasing fatty acid uptake (17), fatty acid oxidation (FAO) (18), and lipolysis (19). Yet, in TNBC, correlation of FABP7 expression and disease prognosis is debatable. While one study showed that FABP7 expression correlates with lower overall and recurrence-free survival (20), several have shown that FABP7-positive basal tumors (synonymous with TNBC) are associated with better prognosis (21,22). It is unclear, at this point, how FABP7-governed pathways impact the survival of TNBC.",
"In this study, we explored the roles of FABP7 in adaptation to nutrient depletion in TNBC cell lines. We showed that overexpression of FABP7 decreased the viability of Hs578T cells during serum starvation. This led to cell-cycle arrest and a significant increase in cell death. We further showed that this phenotype was mediated by PPAR-regulated genes.",
"All cell lines used in this study were purchased from American Type Culture Collection (Gaithersburg, MD). They were cultured in medium according to the manufacturer's protocol and supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), unless specified otherwise. Hs578T, MCF7, MDA-MB-231, and MDA-MB-435S were maintained in DMEM high glucose while BT474 was cultured in MEM. The cell lines were incubated at 37°C and 5% CO 2 atmosphere.",
"For nutrient-deprivation experiments, the cells were first seeded in complete medium (10% FBS). The next day (hereafter referred to as 0 h), the cells were washed once with PBS and replaced with nutrient-deprived medium. For the glucose-and glutamine-starvation experiments, 10% FBS were added into the basal medium. Basal medium used for glucose starvation was glucose-free DMEM (Life Technologies, catalog no. 11966-025) that contained 4 mM l-glutamine. For the glutamine-starvation experiment, glutamine-free DMEM (Life Technologies, catalog no. 11960-044) containing 25 mM glucose was used. The serumstarvation experiment mimicked a culture condition with deprived lipids. The cells were challenged with serum-free DMEM (Life Technologies, catalog no. 11965-092) that contains high glucose (25 mM) with l-glutamine (4 mM), but without sodium pyruvate, HEPES, lipids, and growth factors.",
"The PPAR- antagonist (GW6471) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a nonlethal concentration (2 M).",
"A microarray profile of selected TNBC was obtained from the publicly available Gene Expression Omnibus (GEO) database. The search keyword used was \"triple negative breast cancer,\" and the results were filtered with \"Homo sapiens.\" The search was conducted from September 1 to 30, 2018.",
"TNBC cell lines Hs578T and MDA-MB-231 were transduced with FABP7 gene using Thermo Scientific Precision LentiORF constructs at MOI 10 (Clone ID: PLOHS_100004067). For their control counterparts, the cells were transduced with Precision LentiORF RFP (catalog no. OHS5833). The transduced cells were selected with blasticidin for 30 days to generate stable cell line.",
"Cells were seeded in 96-well plates and subjected to different growth conditions. At time of termination, 10 l of methyl thiazolyl tetrazolium (MTT) solution (5 mg/ml) (Sigma, St. Louis, MO) was added into each well for 4 h, before the addition of 100 l of 10% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals overnight. Absorbance was measured at 575 nm with reference to 650 nm. All experiments were performed in triplicate and repeated three times.",
"Total RNA extraction was carried out using Trizol (Invitrogen, Carlsbad, CA) following the recommended protocol. About 500 ng of RNA was converted into cDNA using DyNAmo cDNA synthesis kit (Finnzymes, Vantaa, Finland). For quantitative RT-PCR (qRT-PCR), 5 ng/l cDNA template was added into pool solution containing 5× HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Tartu, Estonia), 10 pmol/l forward and reverse primers, and UltraPure distilled water. ABI StepOne Plus (Applied Biosystem, Foster City, CA) was used, and 40 cycles of amplification were performed. The expression of metabolic genes was normalized to housekeeping genes 18S rRNA and ribosomal protein L13a. Sequence of the primers used is described in supplemental Table S1.",
"Protein lysate was harvested by scraping the cells in cold PBS. After centrifugation, PBS was discarded, and the cells were incubated in lysis buffer (0.1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1× phosphatase, and 1× protease inhibitors) for 30 min on ice. The mixture was centrifuged for 20 min, and supernatant was collected as protein lysate. Protein concentration was quantified using Bradford assay (Bio-Rad, Hercules, CA). A total of 30 g of protein was resolved on 15% SDS-PAGE prior to transferring on PVDF membrane. Target proteins were detected using primary Abs FABP7 (Cell Signaling Technology) and ECL prime Western blotting detection reagent (Amersham, GE Healthcare Lifesciences, Sweden) before being visualized with gel-documentation system (Biospectrum 410, UVP).",
"The BrdU cell-proliferation assay kit (Cell Signaling Technology, Beverly, MA) was used to measure the cell-proliferation rate at 24, 48, and 72 h after serum starvation. The data were normalized to their counterparts cultured in complete medium. The cells were incubated with 10X BrdU solution for three h, prior to fixation for 30 min and staining with 1× BrdU Detection Antibody for 1 h. The stained cells were washed and incubated with 1× HRPconjugated secondary antibody solution for 30 min, before TMB solution was added. STOP solution was added after 15 min, and absorbance was read at 450 nm.",
"Cell-cycle analysis was conducted on the cells at 0, 24, 48, and 72 h after serum starvation. The cells were fixed with 70% ethanol at 4°C for 30 min, before two washes with PBS. Enzymatic removal of RNA was achieved by incubating the cells with 100 g/ml RNase for 15 min before DNA staining with 50 g/ml propidium iodide. The cells were analyzed using a BD FACSCanto II flow cytometer, and the cell-cycle distribution of the cell was analyzed with Modfit LT software. The samples were run in triplicates, and the mean value was calculated.",
"Cells were harvested for apoptosis assay at 0, 24, 48, and 72 h after serum starvation. Floating cells in the culture medium and the trypsinized cells were collected and washed once with ice-cold PBS, before they were resuspended in 100 l of assay buffer (1 part buffer: 9 parts distilled water) with 5 l of annexin V-PE and 5 l of 7AAD (BD Bioscience, Franklin Lakes, NJ). The cells were vortexed gently and kept in the dark for 15 min, before adding 400 l of buffer. The staining was analyzed using a BD FACSCanto II flow cytometer and viewed using FACS DiVa Software (BD Bioscience, San Jose, CA). All samples were run in triplicates, and mean value was calculated.",
"GAPDH, glutaminase (GSL), and FAO enzyme activities were measured using calorimetric assay kits from Biomedical Research Service Center, State University of New York (Buffalo, NY). All samples were harvested using 1× Cell Lysis Solution. Protein concentration of the samples was assessed with a Bradford assay and normalized to 1 mg/ml. GAPDH activity was measured using GAPDH enzyme assay kit (catalog no. E-101). Briefly, 10 l of sample or water (as blank) was incubated in 50 l of GAPDH assay solution. After gentle agitation, the plate was kept in a non-CO 2 incubator at 37°C for 60 min. For GSL assay (catalog no. E-133), 10 l of sample was incubated in either 40 l of glutamine solution or water. Following a 2 h incubation period in a non-CO 2 incubator at 37°C, 50 l of TA assay solution was added, and the plate was further incubated for another 1 h. For FAO enzyme assay (catalog no. E-141), 50 l of FAO Assay Solution or control solution was added to 10 l of the protein sample. The plate was kept in a non-CO 2 incubator at 37°C for 60 min. All experiments were terminated by adding 50 l of 3% acetic acid, and the plate was read at optical density of 492 nm (OD 492) with a spectrophotometer. Blank reading was subtracted from the sample reading. GAPDH activity in IU/l unit was determined by multiplying OD by 16.98. Glutaminase (GLS) activity in IU/l unit = mol/ (l·min) = (OD × 1,000 × 150 l) ÷ (120 min × 0.6 cm × 18 × 10 l) = OD × 11.58. For FAO assay, the subtracted OD represents the FAO activity of the sample.",
"Basal oxygen consumption rate (OCR) was carried out with a Mito Fuel Flex Test Kit on XFe96 Bioanalyzer (Agilent). Briefly, 4,000 cells were seeded on a Seahorse XF96-well assay plate in complete medium. The cells grown in complete medium were terminated at 24 h after seeding. For the serum-starvation experiment, the cells were left incubated overnight before they were washed with PBS and incubated in serum-free medium for 24 h. Upon termination, all wells were replaced with assay medium, and baseline OCR was immediately measured for 18 min. The basal OCR was normalized to protein concentration of the cells.",
"The cells were seeded on a coverslip in complete medium. After overnight incubation, the cells were either harvested or challenged with serum-free medium for 24 h. The cells were fixed with 4% formaldehyde for 30 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 1 h. The cells were incubated with FABP7 primary antibody (1:2,000; Cell Signaling catalog no. 13347S) and PPAR- primary antibody (1:400; Santa Cruz Biotechnology, catalog no. sc-398394), followed by Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific). The incubation time was 90 min for primary antibody and 60 min for secondary antibody. The cells were washed three times with PBS after staining with each antibody. All the procedures were carried out on ice. The coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories Cat#H-1200) and analyzed with a Leica TCS SP5 II laser microscope (Leica, Heidelberg, Germany) using 60× objective.",
"Statistical analysis assessing the difference between the means of two groups was performed using Student's t-test with Graph-Pad-Prism (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.",
"The expression of FABP7 was determined in human breast cancer tissues and TNBC cell lines. Analysis from the GEO database (dataset GSE65194) revealed that FABP7 expression was significantly higher in TNBC (n = 55) compared with non-TNBC (n = 98) subtypes (Fig. 1A). When examined in a panel of breast cancer cell lines, FABP7 showed low expression in all cells including MCF7 (luminal A), BT474 (luminal B), MDA-MB-231, and Hs578T (TNBC) cells, when compared with MDA-MB-435S, a melanoma cell line reported to express high FABP7 (Fig. 1B). Correspondingly, FABP7 protein was not detected in these breast cancer cell lines (Fig. 1C).",
"To understand the functional role of FABP7 in TNBC cells, we established FABP7-overexpressing Hs578T and MDA-MB-231 TNBC cells by transduction using lentiviral particles containing FABP7 open reading frame (Hs-FABP7 and 231-FABP7, respectively) and their control counterparts using lentiviral particles containing red fluorescent protein (Hs-RFP and 231-RFP, respectively) (Fig. 1D). When the cells were cultured in complete medium for 72 h, there was no difference in cell growth between cells expressing FABP7 and their respective RFP controls (Fig. 2). However, when cultured in glucose-or glutamine-deprived condition, both FABP7-overexpressing cells and RFP controls demonstrated substantial reduction in cell viability with greater effect observed in glucose-deprived than glutamine-deprived medium ( Fig. 2A,B).",
"Interestingly, when lipids were significantly reduced during serum starvation, overexpression of FABP7 resulted in decreased viability of Hs578T cells (Fig. 2C). At 24 h after serum starvation, the cell viability of Hs-FABP7 cells decreased by 10%, which progressively decreased to approximately 70% at 72 h after starvation (P < 0.0001 vs. Hs-RFP cells). Cell viability of Hs-RFP cells was not affected by serum starvation, indicating that the decreased viability in Hs-FABP7 cells may be related to FABP7 expression. This phenotype appeared to be specific to Hs578T cells, as FABP7 expression did not affect the viability of MDA-MB-231 cells in serum-free medium (Fig. 2C).",
"The reduction in cell viability of Hs-FABP7 cells during serum starvation resulted from a decrease in cell proliferation. Hs-FABP7 cells demonstrated a 70% reduction in BrdU uptake as early as 24 h after serum starvation, whereas the control Hs-RFP cells only showed a decrease by 20% (Fig. 3A). The nonresponsive MDA-MB-231 cells maintained their proliferation rate in the range of 85-89% during serum starvation, regardless of FABP7 expression.",
"Flow-cytometry analysis showed that FABP7 induced cellcycle arrest in Hs578T cells during serum starvation ( Fig. 3B and supplemental Fig. S1). Hs-RFP cells showed a higher distribution of cells (80%) in G1 phase throughout serum starvation, indicating that the cells were able to complete the cell cycle and proliferate, parallel to increased cell viability over time. In contrast, Hs-FABP7 cells demonstrated a consistently higher percentage of cells in S and G2 phase upon serum starvation. For instance, at 48 h after serum starvation, 24% of Hs-FABP7 cells were accumulated in S phase and 8.23% in G2 phase, whereas only 5.15% and 1.99% of Hs-RFP cells were detected at S and G2 phase (P = 0.0052 for S phase and P = 0.0241 for G2 phase). The percentage of cells in S/G2 phase increased to 40.15% in Hs-FABP7 cells compared with 14.29% in Hs-RFP cells at 72 h after serum starvation. Cell arrest in S/G2 phase in Hs-FABP7 cells may partly contribute to the decreased cell viability during serum starvation. This effect was not observed in MDA-MB-231 cells (Fig. 3B).",
"The decrease in Hs-FABP7 cell viability during serum starvation was potentially attributed to apoptosis and necrosis (Fig. 3C). More necrotic cell death and late apoptosis was observed in Hs-FABP7 cells (34.26% and 21.3%) than in Hs-RFP cells (18.83% and 4.63%) at 72 h after serum starvation (P < 0.0001). Upon serum starvation, increasing cell death was observed in MDA-MB-231 cells, but with no significant difference observed between 231-RFP and 231-FABP7 cells. Taken together, the decreased cell viability in Hs-FABP7 cells during serum starvation was due to reduced proliferation rate, cell cycle arrest at S and G2 phase, and cell death.",
"Given the role of FABP7 in fatty acid metabolism, we investigated the mechanism of FABP7-induced cell death by exploring a panel of genes and enzyme activities involved in the metabolism of glucose, glutamine, and fatty acid ( Fig. 4 and supplemental Figs. S2, S3). In complete medium, despite the increase in glucose transporter 1 (GLUT1) mRNA expression, glycolysis-related genes phosphofructokinase 1 (PFK1) and GAPDH remained unchanged in Hs-FABP7 cells, whereas the genes regulating FAO, carnitine palmitoyltransferase 1A (CPT1A), and medium-chain acyl-CoA dehydrogenase (MCAD) were markedly upregulated (Fig. 4A). In Hs-FABP7 cells, the expression of GLS1, encoding the rate-limiting enzyme for glutaminolysis, was not significantly different than in Hs-RFP cells. When cultured in complete medium, Hs-FABP7 cells did not show any differences in GAPDH, GLS, and FAO activities when compared with Hs-RFP cells 3. Decreased viability of Hs-FABP7 cells in serum starvation was due to decreased proliferation, cellcycle arrest, and cell death. A: Proliferation of Hs578T and MDA-MB-231 cells cultured in serum-starved conditions and in complete medium for 24, 48, and 72 h was measured using BrdU assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. B: Effects of FABP7 on cell cycle in serum starvation were detected by flow cytometry using propidium iodide staining. The analysis on cell-cycle distribution were performed using Modfit LT software. Data shown are the mean ± SEM of triplicates and are representative of three independent experiments; coefficient of variation (CV) values shown were also the average of the triplicates. C: Percentage of necrotic and apoptotic cells was measured using an annexin V/7-AAD apoptosis assay. Data shown are the mean ± SEM of triplicates and are representative of two independent experiments. The P value represents the statistical significance when the Hs-FABP7 group was compared with the control group. * P < 0.05; ** P < 0.001; *** P < 0.0001. ( Fig. 4B-D). Subsequently, mitochondrial OCR was similar in Hs-FABP7 cells and in the control Hs-RFP cells (Fig. 4E).",
"However, when challenged with serum starvation, a widespread downregulation of glucose and glutamine metabolism-related genes were observed in Hs-FABP7 cells when compared with Hs-RFP cells. Interestingly, despite the absence of serum (lipids) during serum starvation, the gene expression of CPT1A and MCAD was about 2-fold higher in Hs-FABP7 cells than Hs-RFP cells (Fig. 4A). Serum starvation generally decreased glycolytic enzyme activity in Hs578T cells, but FABP7-overexpressing cells had a further 30% reduction (P = 0.0121) in GAPDH activity compared with Hs-RFP cells (Fig. 4B). Although GLS activity remained not significantly different in both cells during serum starvation, GLS activity was significantly lower (P = 0.0296) in Hs-FABP7 cells cultured in serum starvation than in complete medium (Fig. 4C). Interestingly, FAO activity in Hs-FABP7 remained similar with the control Hs-RFP cells (Fig. 4D), despite upregulation of FAO-related genes. This may be explained by the lack of fatty acid in the culture medium to activate the metabolic pathway. Consequently, mitochondrial oxidative capacity of both Hs-RFP and Hs-FABP7 cells was generally lower during serum starvation (P = 0.0001 for both cells); however, Hs-FABP7 cells had much lower OCR (16.69 pmol/min/µg) compared with Hs-RFP cells (21.7 pmol/min/µg) (P < 0.05) (Fig. 4E). These data indicate a lower mitochondrial oxidative capacity particularly in Hs-FABP7 cells, probably due to limited bioenergetic substrates during serum starvation.",
"It is worthwhile to note that, unlike Hs578T, MDA-MB-231 cells showed a rather different metabolic profile, as indicated by the expression of metabolic genes ( Fig. 4A and supplemental Fig. S2B). When cultured in complete medium, although FABP7 was not affecting the cell viability, glycolysis was upregulated in 231-FABP7 cells, with about a 1.7-fold increase in GAPDH expression. Meanwhile, a 4-fold decrease in GLS1 expression was observed, indicative of a downregulation of the glutaminolysis pathway. Upon serum starvation, 231-FABP7 cells demonstrated a marked increase in glucose and glutamine metabolismrelated genes, with more than 4-fold increase in GLUT1 and GLS1 expression.",
"Taken together, our data show that Hs-FABP7 cells, when challenged with a serum-starved condition, were not able to metabolically adapt and effectively utilize other available energetic substrates, especially glucose, and, as a consequence, could not survive.",
"The upregulation of CPT1A and MCAD mRNA, accompanied with a downregulation of Glut1 mRNA in Hs-FABP7 cells, indicated a possible role of PPAR- signaling, as these genes are direct targets of PPAR- transcription factor. Immunofluorescence staining showed that the expression of PPAR- protein was increased in the nucleus during serum starvation ( Fig. 5A and supplemental Fig. S4A). Similarly, FABP7 protein was observed to translocate into the nucleus (Fig. 5A and supplemental Fig. S4B) in Hs-FABP7 cells upon serum starvation, potentially acting as fatty acid chaperones to deliver ligands to PPAR- protein (23). This suggests that nuclear localization of FABP7 enhanced the action of PPAR- transcriptional activity during serum starvation in Hs-FABP7 cells.",
"To investigate whether PPAR- activation was responsible for FABP7-mediated inhibition of cell viability in Hs-FABP7 cells, we treated the cells with a PPAR--specific inhibitor (GW6471). In both Hs578T and MDA-MB-231 cells, treatment of GW6471 at a concentration of 2 M in complete medium had no significant effect on cell viability (Fig. 5B). Yet, the same concentration of GW6471 reversed the cell-growth inhibition observed in Hs-FABP7 cells when exposed to serum-starved conditions (Fig. 5B and supplemental Fig. S5). Subsequent gene-expression analysis revealed that PPAR- target genes were downregulated (CPT1A: 2.43-fold; MCAD: 1.54-fold), when compared with vehicle-treated cells (Fig. 5C). A significant 2-fold increase of GLUT1, PFK1, glucose-6-phosphate dehydrogenase (G6PD), and isocitrate dehydrogenase 1 (IDH1) mRNA expression was observed in GW6471-treated Hs-FABP7 cells (Fig. 5C), indicating that the rescue of cell viability may be due to the switch from lipid to glucose as bioenergetic substrates in these cells. Taken together, FABP7-induced cell death in Hs578T cells during serum starvation is due to activated PPAR- signaling.",
"In this study, we showed that overexpression of FABP7 disabled TNBC cell line Hs578T to metabolically adapt during serum starvation. FABP7-overexpressing cells failed to efficiently utilize glucose as bioenergetics substrates, when challenged with serum starvation. This was accompanied by increased expression of genes controlling FAO as it attempts to utilize free fatty acids liberated by lipolysis from the lipid storage that resulted from preexposure to complete medium (24). However, as free fatty acid storage was depleted, serum-starved FABP7-overexpressing cells were not able to further metabolically adapt and sustain ATP production. The failure of the cells to efficiently utilize glucose for ATP generation leads to cell death. We further showed that the metabolic impact of FABP7 in Hs578T cells during serum starvation is mediated by PPAR- signaling.",
"FABP7 is predominantly expressed in the nervous system and mammary gland. In the mammary gland, FABP7 inhibits proliferation and promotes differentiation of mammary cells (25). Similarly, in the nervous system, FABP7 is involved in the differentiation of neurons and development of the cortex (26,27). The expression of FABP7 at the edge of astrocytes also suggested that FABP7 may regulate the motility of astrocytes by increasing the uptake of polyunsaturated fatty acids (28). Yet, the evidence of FABP7 expression in TNBC cell lines was rather limited (20,29). A previous study demonstrating the function of endogenous FABP7 in TNBC was reported using MDA-MB-435S, a melanoma cell line formerly misidentified as a TNBC cell line (30). Our study, however, provides evidence on the role of FABP7 as a mediator for tumor cell death during nutrient-deprived conditions. This finding indicates an anti-tumorigenic role of FABP7 in TNBC, which is mediated by specific nutrient availability.",
"The metabolic vulnerabilities of FABP7-overexpressing Hs578T cells resulted in S/G2-phase arrest, which subsequently led to cell death. Serum starvation typically induces cell-cycle arrest at G1 phase (31,32), which is observed in Hs-RFP and MDA-MB-231 cells. These cells were able to proliferate, despite a lower proliferation rate, indicating that the lower mitochondrial activity in these cells during serum starvation was sufficient to overcome the metabolic checkpoint and complete the cell cycle (33). Hs-FABP7 cells, however, arrested at the S/G2 phase. G2 arrest has been described as another growth-restricting mechanism in mitogen-starved conditions, likely due to elevated E2F activity (34). Cell arrest could potentially lead to cell death, in which both apoptosis and necrosis were observed in Hs-FABP7 cells upon serum starvation. Apoptosis is a cellular suicide program triggered by stress signals such as nutrient deprivation, which results in the execution of caspasemediated cell death (35). Serum starvation has been shown to induce apoptosis in hepatocellular carcinoma cells (36) and MCF7 breast cancer cells (37), in which the latter was associated with an altered ratio of apoptosis regulating proteins Bax and Bcl-2. Necrosis, on the other hand, may occur when the minimal bioenergetic demands are not met (38), a condition similar to our observations where Hs-FABP7 cells may have limited bioenergetic substrates during serum starvation. In fact, there are common mediators of apoptosis and necrosis, which could potentially contribute to the two modes of cell death observed in our model (39). It is worth noting that FABP7 did not induce cell death in MDA-MB-231 cells during serum starvation, indicating that this cell line is probably more resilient to nutrient deprivation than Hs578T cells. The disparity in the response of Hs578T and MDA-MB-231 cells to serum starvation may also be attributed to differences in their metabolic profiles (40). Hence, it would be worthwhile to examine other TNBC cells with differing metabolic profiles, in delineating the molecular heterogeneity involved in cell death induced by metabolic vulnerabilities.",
"During serum starvation, cancer cells sense and adapt to nutrient deprivation by upregulating glucose metabolism Fig. 5. FABP7-induced cell death in Hs578T cells during serum starvation was mediated by the action of PPAR- signaling. A: Confocal microscopy of immunofluorescence staining of FABP7 (green) and PPAR- (red) proteins in Hs578T and MDA-MB-231 cells after culture in either complete medium or serum-free medium for 24 h. Blue, DAPI. Scale bar, 50 m. Data shown are representative of two independent experiments. B: Cells were treated with PPAR- inhibitor GW6471 (2 M) in complete or serum-starved medium for 72 h, before analyzing for cell viability using an MTT assay. Data shown are mean ± SEM of triplicates; this is a representative of three independent experiments. *** P < 0.0001. C: mRNA expression of key metabolic genes after treatment with 2 M GW6471 in Hs-FABP7 cells for 24 h in serum starvation when compared with vehicle-treated cells. The genes were normalized to housekeeping gene 18S rRNA. CM, complete medium; SFM, serum-free medium. (41,42), besides releasing fatty acids from the lipid droplet through lipolysis for oxidation (24). Central to such adaption, PPAR- is known to be a lipid sensor that maintains cellular energy homeostasis. It is a ligand-activated class of nuclear hormone receptor that binds to peroxisome proliferator response elements (PPREs) to induce transcription of genes such as lipid processing. Its activation aims at increasing lipid combustion in order to yield more energy production, especially in a challenged environment (43). It was shown in rat hepatocytes that PPAR- expression is increased by fasting-and starvation-induced glucocorticoids (44), suggesting the role of PPAR- to maintain cell survival.",
"In our model, increased PPAR- expression was observed when the cells were serum-starved, possibly as an adaptation strategy of the cells. The activation of PPAR- was observed in Hs-FABP7 cells during serum starvation, as reflected in the increase of PPAR- target genes, CPT1A and MCAD, which are involved in fatty acid mitochondrial uptake and oxidation (45). Our observation may indicate the usage of fatty acids liberated from lipid droplets as the substrates for FAO. However, the attempt of the cells to survive serum starvation failed as the lipid storage was limited and fatty acids were absent in the serum-free media. In addition, PPAR- activation also potentially inhibited glucose metabolism by targeting the consensus PPRE motif in the promoter region of Glut1, hence repressing its transcription (46). As a result, Hs-FABP7 cells could not efficiently utilize glucose as bioenergetic substrates when serumstarved, which eventually led to cell death.",
"Our data suggest that PPAR--induced changes are FABP7-dependent. Literature evidence shows that in Cos-7 cells where FABPs and PPAR- are not detected, transfection of the proteins showed that PPAR- activity is amplified by FABP1 and FABP2 (47). In another study, PPAR- transactivation is proportional to levels of L-FABP in human hepatoma HepG2 cells line (48). In our model, upon serum starvation, increased nuclear FABP7 expression was observed, suggesting the role of FABP7 as fatty acids chaperones to transport PPAR- ligands into the nucleus for PPAR- activation. In murine L cells, transduction with FABP1 had dramatically enhanced the uptake of fatty acid into the nucleus (49). Incubation of rat liver nuclei with fluorescence-tagged FABP1 together with fatty acids demonstrated a strong fluorescence response in the nuclei, which otherwise did not happen when fatty acids were absent (50). There has been evidence indicating physical interaction between FABP1 and PPAR- in primary hepatocytes (51). Ablation of L-FABP gene expression significantly impaired fatty acid distribution in the nucleus and affected PPAR- activation in these cells (52). Hence, whether FABP7 and PPAR- require direct contact to induce cell death in TNBC needs to be further investigated. In short, our data suggest that FABP7 facilitates PPAR- activation in serum-starved condition by transporting its ligand (fatty acids) into the nucleus, before enhancing its transcriptional activity.",
"Our study provides an understanding on the role of FABP7 in regulating the metabolic adaptation of TNBC cell lines. Transcriptional activation of PPAR- in FABP7overexpressing cell can limit their metabolic plasticity when challenged with serum starvation and resulted in reduced survival of these cells. Hence, manipulating metabolic stress in cells expressing FABP7 protein may potentially be an effective targeted therapeutic strategy in FABP7-positive TNBC patients."
] | [] | [
"MATERIALS AND METHODS",
"Cell culture",
"Data mining at GEO database",
"Generation of FABP7-transduced TNBC cell lines",
"MTT assay",
"RNA extraction and qRT-PCR",
"Protein extraction and Western blotting",
"BrdU cell-proliferation assay",
"Cell-cycle analysis with propidium iodide",
"Annexin V apoptosis assay",
"Metabolic activity assays",
"Measurement of OCR",
"Immunofluorescence staining",
"Statistical analysis",
"RESULTS",
"FABP7 overexpression decreased viability of Hs578T, but not MDA-MB-231, cells in serum starvation",
"Decreased viability of Hs-FABP7 cells in serum starvation was due to reduced proliferation, cell-cycle arrest, and cell death",
"Perturbations in the metabolism and mitochondrial oxygen consumption in Hs-FABP7 cells during serum starvation",
"PPAR- signaling mediated FABP7-induced cell death in Hs578T cells under serum starvation",
"DISCUSSION",
"Fig. 1 .",
"Fig. 2 .",
"Fig.",
"Fig. 4 ."
] | [] | [
"Table S1"
] | [
"Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling",
"Metabolic role of fatty acid binding protein 7 in mediating triple-negative breast cancer cell death via PPAR- signaling"
] | [
"Journal of Lipid Research"
] |
236,209,571 | 2022-01-11T02:58:22Z | CCBY | https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/jcmm.16803 | GOLD | ba785ecf67234bc1c401929c79e39a7028e5082e | null | null | null | null | 10.1111/jcmm.16803 | null | 34302427 | 8419198 |
O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis
2021
Shanshan Dong
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Qi Tian
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Tengfei Zhu
Department of Critical Care Medicine
Shenzhen Third People's Hospital
ShenzhenGuangdongChina
| Kangli Wang
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Ganting Lei
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Yanling Liu
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Haofeng Xiong
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
| Lu Shen
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Meng Wang
Rongjuan Zhao
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Huidan Wu
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
| Bin Li
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
National Clinical Research Center for Geriatric Disorders
Department of Geriatrics
Xiangya Hospital
Central South University
ChangshaHunanChina
Qiumeng Zhang
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Yujun Yao
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
| Hui Guo
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
| Kun Xia
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Hunan Key Laboratory of Molecular Precisional Medicine
Central South University
ChangshaHunanChina
| Lu Xia
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
| Zhengmao Hu
Center for Medical Genetics
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
ChangshaHunanChina
Center for Medical Genetics
Hunan Key Laboratory of Animal Models for Human Diseases
Central South University
Correspondence Zhengmao Hu and Lu XiaChangshaHunanChina
School of Life Sciences
Hunan Key Laboratory of Medical Genetics
Central South University
410078ChangshaHunanChina
O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis
J Cell Mol Med
202110.1111/jcmm.16803Received: 10 February 2021 | Revised: 13 May 2021 | Accepted: 6 July 20218432 |
High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGFβ pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGFβ signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGFβ signalling and ECM synthesis in the KO cells. The fact that TGFβ signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGFβ signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGFβ signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGFβ signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.K E Y W O R D SECM, high myopia, SLC39A5, Smad, TGFβ signalling, zinc
| INTRODUC TI ON
Myopia is a condition of refractive error causing light to focus in front of the retina, with axial elongation as its most common pathology. High myopia, clinically defined as a refractive error of at least −6.00 diopters (D) or an axial length >26 mm, 1 is one of the leading causes of visual impairment worldwide 2,3 with a significantly increased prevalence. 3,4 Thus, the prevention of high myopia has become a global public health problem. Pedigree analyses and twin studies identified high myopia highly heritable. 5,6 Even though numerous loci (1q41, 4q25, 8p23, etc.) and causative genes (ZNF644, CCDC111, SCO2, etc.) have been identified, 7 the pathogenic mechanism of high myopia remains unclear.
Recently, scleral extracellular matrix (ECM) synthesis or remodelling has been proposed as an underlying cause of high myopia. [8][9][10] According to this hypothesis, scleral collagen synthesis is regulated during myopia progression, 9 while scleral ECM is remodelled in myopic animal. 10 ECM synthesis and remodelling are targets of the TGFβ signalling pathway, one of the most reproducibly dysregulated pathways in myopia development. [10][11][12][13] Total or isoform-specific scleral TGFβ is frequently altered during myopia progression. 9,10,14 In the previous study, we first identified SLC39A5 as a high myopia-associated gene, and mutations of SLC39A5 dysregulated the BMP/TGFβ pathway. 15 Further research confirmed SLC39A5 as one of the top three genes contributing to nonsyndromic high myopia. 16 This study aimed to investigate the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia. We found that SLC39A5 played a key role in maintaining intracellular zinc homeostasis. SLC39A5 depletion-induced zinc deficiency directly destabilized Smad proteins, and Smads instability further impaired TGFβ signalling-mediated ECM synthesis, thus contributing to the pathogenesis of high myopia.
| MATERIAL S AND ME THODS
| Cell culture
| SLC39A5 knockout cell line construction
According to the protocols from Zhang laboratory (http://www. genom e-engin eering.org/gecko/), target-specific sgRNA was cloned into the lentiCRISPRv2 vector (sgRNAs are listed in Table S1). Then, lentiviruses were packaged in HEK293T cells by co-transfection of CRISPR-sgRNA/pVSVG/pspAX2. The supernatants containing viral particles were collected 48 h after transfection and filtered (0.22 μm pore size). Then, HEK293 cells were transduced with viral supernatant supplemented and selected with 1 μg/ml puromycin.
After transfection, cells were detached and seeded in 10 ml Petri dishes at a density of 200 cells per plate. Puromycin (2 µg/ml) was added to allow the selection of positive clones during the following two weeks. Positive clones were isolated and transferred to sixwell plates. The SLC39A5 knockout clones were validated by direct Sanger sequencing (primers are listed in Table S2). The negative control cell line was generated from HEK293 transformed with the identical lentiviral vector lacking specific sgRNAs.
| Zinc quantification
Zinc quantification assay (Abcam Cat# ab102507) was performed for accurate measurement of zinc levels according to the manufacturer's instructions. In brief, cells were harvested with NP-40 lysis buffer (EDTA-free), and proteins were deproteinized by an equal volume of 7% TCA Solution Buffer. The supernatants were transferred to new clean tubes after centrifugation. 200 μl Zinc Reaction Buffer was applied to 50 μl tested samples in a 96-well plate. After a 10-min incubation at room temperature, the OD 560 was measured in a microplate reader. The zinc standard curve was plotted after subtracting the background value, and the tested sample concentration was calculated from the standard curve.
| RNA-seq and analysis
Total RNA for RNA-seq library was extracted using TRIzol (Invitrogen Cat# AM9738), validated on a NanoDrop 1000 Spectrophotometer (Thermo) and quantitated using Qubit (Thermo). RNA-seq libraries were ing data were filtered with SOAPnuke (v1.5.2), then mapped to the reference genome using HISAT2 (v2.0.4) and aligned to the reference coding gene set using Bowtie2 (v2.2.5). Differential gene expression analysis between the target and reference sets of treatments was determined using DESeq2 (https://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq.html). Enrichment analysis of annotated different expressed genes was performed with the Phyper (https://en.wikip edia.org/wiki/ Hyper geome tric_distr ibution) based on the hypergeometric test. Table S3). All samples were run in triplicate. qRT-PCR data were analysed by LightCycler 96 software. Relative candidate gene mRNA levels were normalized to those of β-actin or GAPDH. A value of p < 0.05 was considered to be statistically significant.
| qRT-PCR validation
| Plasmid construction and transient transfection
The Smad1 zinc-binding site substitutions were generated to assess the correlation between zinc-binding capacity and protein stability. Smad1 WT plasmid was brought from OriGene (OriGene Cat# RC200299), and the four zinc-binding site substitutions (C64A, C109A, C121A and H126A) were separately generated using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in Table S4). Table S5).
Finally, the rescue sequence was cloned into the pLVX-IRES-puro vector to construct the KO rescue plasmid.
SLC30A1 shRNA interference plasmids were also constructed to rescue the poor intracellular zinc level of SLC39A5 KO cells by suppressing the zinc efflux baseline. The three targeting sequences were obtained from the siRNA sequence database (Thermo) and then generated into the pFUGW-lentiviral vector between the XbaI and BamHI restriction sites, respectively (primers are listed in Table S6).
| Immunoblotting
| Immunofluorescence
Cells were seeded on specific coverslips in 12-well plates.
The cell slides were fixed in 4% paraformaldehyde for 10 min.
Permeabilization was performed with 0.1% PBST (phosphatebuffered saline and 0.1% Triton) for 15 min. After blocking with 5% BSA for 1 h, the cell slides were incubated with primary antibody at recommended dilutions overnight in 4°C and stained with Alex Fluor 488-conjugated second antibody (Jackson Cat# 111-545-144) for 1 h protected from light. DAPI was applied for 1 min as a nuclear marker. Images were acquired with an Eclipse TCS-SP5 inverted confocal microscope (Leica).
| Wound healing capacity test
HEK293 cells were seeded into the 2-well culture insert (Ibidi Cat# 81176) pre-coated by Matrigel. After cell attachment for approximately 24 h to form an optically confluent monolayer, the culture insert was removed to create the wound gap and the cells were then cultured with serum-free medium. The wound gap closure was monitored by taking pictures with an Eclipse inverted microscope (Leica) at different time points.
| Statistical analysis
GraphPad Prism was used to calculate and plot the mean ± SEM of measured quantities. Significances were assessed by two-way analysis of variance (ANOVA)/Mann-Whitney U test (nonparametric)/Student's t test (parametric), p-value < 0.05 was considered to indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
| RE SULTS
| SLC39A5 depletion impairs ECM synthesis
Two SLC39A5 knockout (KO) cell lines were developed using the CRISPR-Cas9 system in the human embryonic kidney cell line (HEK293) ( Figure 1A). Cell morphology anomalies were markedly observed in the SLC39A5-depleted cells. After regular growth for 2 days, the KO cells revealed a tendency to form small cell aggregates instead of a typical optically confluent monolayer ( Figure 1B).
This phenotype suggested a potential impairment or abnormality of ECM generation. Considering the influence of ECM dynamics on cell migration, invasion and morphogenesis, we immediately investigated the cellular motility and migratory capacity of the SLC39A5depleted HEK293 cells. Scratch assay was performed in both groups.
A 2-well culture insert generated a uniform gap in the confluent monolayer, and wound healing was imaged at different time points.
The results showed that the gap in the control group was completely healed after 24 h, while the wound gap in the KO group was not able to close ( Figure S1; Figure 1C).
To investigate the overall alteration caused by SLC39A5 depletion, RNA-sequencing (RNA-seq) was performed using a Immunofluorescence also verified the decrease in these ECM proteins ( Figures S3 and S4).
| SLC39A5 depletion suppresses TGFβ signalling
The transcription of collagens, FN and LN is closely related to TGFβ signalling, and we have already determined that mutations of SLC39A5 dysregulated TGFβ signalling previously. 15 Considering the transcriptional downregulation of collagens, FN and LN in SLC39A5 KO cells, we hypothesized that reduced SLC39A5 expression would inhibit the TGFβ signalling pathway. Hence, we examined the expression of Smad proteins, which lie on the centre of the TGFβ signalling pathway. The results showed an evident upregulation of total Receptor-Smads (R-Smads) (Smad1, Smad2/3) expression along with a significant decrease in phosphorylated Smad expression (pSmad1/5/9, pSmad2/3) ( Figure S5). Additionally, Co-Smad (Smad4) expression was also increased, while inhibitor-Smad (Smad7) was not affected (Figure S5), suggesting a suppressed activation of the TGFβ signalling, which was confirmed through immunofluorescence ( Figures S6 and S7). The qRT-PCR performed to assess the transcriptional levels of Smad1, Smad2 and Smad4 in KO cells showed increased mRNA expression of the tested proteins ( Figure S8). To validate the phenotype of inhibited TGFβ signalling activation and insufficient ECM synthesis resulting from SLC39A5 depletion, rescue experiments were carefully carried out. As shown in Figure 3, re-expression of SLC39A5 restored intracellular zinc level ( Figure 3A). TGFβ signalling activation and ECM synthesis were also rescued ( Figure
| SLC39A5 depletion induces zinc deficiency and destabilizes Smad protein
The TGFβ pathway is a zinc-regulated pathway ( Figure S9), while SLC39A5 is a zinc transporter. 17,18 These facts prompted us to confirm the involvement of zinc homeostasis in TGFβ signalling alteration in SLC39A5 KO cells. Thus, zinc quantification was performed to detect intracellular zinc levels. As shown in Thus, we selected the most widely expressed zinc efflux protein SLC30A1 (20), as a target. Surprisingly, silencing SLC30A1 counterbalanced the influence of SLC39A5 depletion, which significantly reverted intracellular zinc level close to normal ( Figure S10) and normalized the TGFβ signalling pathway and ECM synthesis ( Figure S11).
| DISCUSS ION
SLC39A5 was first identified as a high myopia pathogenic gene in our previous study 15 and was later confirmed as one of the top three genes contributing to nonsyndromic high myopia. 16,[20][21][22][23] However, the mechanisms by which SLC39A5 contributes to high myopia are not well understood. Here, we demonstrated that SLC39A5 may play a role in the pathogenesis of high myopia by modulating TGFβ signalling-mediated ECM synthesis through the regulation of intracellular zinc homeostasis. SLC39A5 depletion significantly lowered the intracellular zinc level, which destabilized Smad proteins, and further inhibited TGFβ signalling-mediated ECM synthesis, which ultimately contributed to the pathogenesis of high myopia.
Zinc homeostasis is crucial for all organisms as it serves as a catalytic or structural cofactor for multiple types of proteins. Zinc homeostasis depends on the transport of zinc in both directions across the plasma membrane and both in and out of various vesicular compartments. 24 The SLC39 family facilitates zinc transport into the cytoplasm in the opposite direction to SLC30 family members. 25 Within this network, SLC39A5 is a specific zinc transporter. 17,18,26 Our work confirmed the zinc uptake characteristic of SLC39A5, in which SLC39A5 depletion caused an intracellular decline in zinc levels in HEK293 or lymphocyte cells. Once zinc homeostasis was disrupted, the catalytic or structural stability of proteins was impaired, suggesting that SLC39A5 depletion, indirectly, caused this impairment.
Zinc has a profound effect on stabilization of zinc-binding proteins, 27-31 since the proteins responsible for cellular zinc buffering will bind zinc transiently; therefore, their function will strongly depend on the cellular zinc status. 32 It has been proposed that zinc chelation affects protein stability through various mechanisms. The human SOD1 protein requires zinc binding to facilitate the restructuring of apoproteins, 33 while the putative protease of Bacteroides thetaiotaomicron (ppBat) requires zinc chelation to link the β-strands to α-helices in protein structures. 34 In our study, the R-Smads and Co-Smad, which are pivotal to the BMP/TGFβ signalling pathway, 35 F I G U R E 2 SLC39A5 depletion impairs extracellular matrix (ECM) synthesis. (A) shows the pathway enrichment analysis for the differentially expressed genes between the control and KO subpopulations of HEK293 cells. Pathway analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics Resources, with an enrichment p-value cut-off of 0.01. (B) shows the qRT-PCR validation of the differentially expressed genes of the ECM members indicated by RNA-seq. (C-E) shows the expression of several ECM components (COL1, COL2, COL4, FN and LN) in the supernatant, whole-cell lysate or intracellular lysate (trypsin digested) of wild-type (WT) and SLC39A5 knockout (KO) HEK293 cells. All tested ECM components were decreased in both the whole lysate and intracellular lysate. (B) and (C) show statistical analyses of the ECM components in the whole lysate and intracellular lysate, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are zinc-binding proteins. We found that Smad proteins were rapidly degraded during SLC39A5 deletion-induced zinc deficiency.
Further results suggested that loose zinc coupling, facilitated by a mutant ion-binding site, directly destabilized the exogenous Smad1 proteins, which clearly confirms the necessity of zinc chelation in maintaining the protein stability of Smads, while disturbances in zinc homeostasis induce protein instability.
Zinc binding has also been implicated in protein phosphorylation and subcellular translocation. 36 Non-zinc-bound protein kinase C delta (PKCδ) showed a significantly different structure near its phosphorylation site, compared with when it was zinc-bound. Subcellular translocation of PKCδ was also influenced by intracellular zinc. These findings expand our understanding of the phosphorylation decrease in R-Smads during SLC39A5-depleted conditions.
Since R-Smads are directly phosphorylated by upstream receptor kinases, which then form heteromeric complexes with Smad4 to translocate into the nucleus, 37 the zinc deficiency induced by SLC39A5 depletion may barely maintain the Smad protein in a favourable structure for phosphorylation, and may lead to nucleus translocation retention, further impairing the TGFβ signalling.
It is well recognized that TGFβ signalling directly affects ECM dynamics or triggers it when mediating other biological processes. [38][39][40][41] Herein, suppressed TGFβ signalling in the zinc-deficient conditions restrained ECM synthesis in HEK293. Recently, ECM synthesis and remodelling have attracted significant attention in high myopia aetiology studies. [8][9][10]42 We found that the transcription of all ECM proteins tested (COL1, COL2, COL4, FN and LN) was decreased under zinc-deficient conditions. ECM-induced cell morphogenesis According to this hypothesis, the SLC39A5-induced zinc deficiency suppresses TGFβ signalling, leading to insufficient ECM synthesis, which may ultimately contribute to high myopia development. We further validated this hypothesis by performing rescue experiments.
As expected, of SLC39A5 re-expression, which recovers zinc influx, and SLC30A1 silencing, which blocks zinc efflux, could identically rescue the alteration of R-Smad phosphorylation and ECM synthesis in SLC39A5-depleted HEK293 cells, by re-establishing zinc homeostasis.
Upon reviewing the relationship between zinc and high myopia, we found plenty of indications of zinc involvement in high myopia development. 43 First, zinc is present abundantly in the ocular system, including the retina and choroid; thus, zinc deficiency produces a variety of ocular manifestations. Furthermore, a number of high myopia gene products are zinc-related proteins.
Among them, ZNF644, ZFHX1B, ZC3H11B and SCO2 are zinc finger proteins, and HGF and IGF1 are positively regulated by intracellular zinc concentration. Additionally, certain pathogenic genes (LAMA1 and TGFB1) may even be regulated by intracellular zinc, according to the zinc-TGFβ signalling-ECM synthesis flow.
Finally, multiple small molecules that serve as chaperones for disease-related genes or pathways are zinc-related proteins. Smad proteins, together with their anchors (SARA and HGS), are either zinc-binding or zinc finger proteins. These findings strongly highlight the correlation between zinc and high myopia.
In summary, our data suggest that depletion of SLC39A5 induces zinc deficiency, which restrains TGFβ signalling-mediated ECM synthesis, thus possibly contributing to high myopia pathogenesis. Zinc homeostasis appears to be a dominant aetiological factor of high myopia development.
ACK N OWLED G EM ENTS
CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.
AUTH O R CO NTR I B UTI O N
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
O RCI D
Shanshan Dong
https://orcid.org/0000-0002-1605-7066
Zhengmao Hu https://orcid.org/0000-0002-3921-8205
R E FE R E N C E S
HEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.
prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-
Total RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in
SLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in
Cells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.
SLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.
3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).
Figure 4 ,
4SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.
in the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.
This work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].
Shanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead).
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Additional supporting information may be found online in the Supporting Information section. How to cite this article. S U Pp O Rti N G I N Fo R M Ati O N ; Dong, S Tian, Q Zhu, T , S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Dong S, Tian Q, Zhu T, et al.
SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. 10.1111/jcmm.16803J Cell Mol Med. 25SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. J Cell Mol Med. 2021;25:8432- 8441. https://doi.org/10.1111/jcmm.16803
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"High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGFβ pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGFβ signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGFβ signalling and ECM synthesis in the KO cells. The fact that TGFβ signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGFβ signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGFβ signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGFβ signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.K E Y W O R D SECM, high myopia, SLC39A5, Smad, TGFβ signalling, zinc"
] | [
"Shanshan Dong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Qi Tian \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Tengfei Zhu \nDepartment of Critical Care Medicine\nShenzhen Third People's Hospital\nShenzhenGuangdongChina\n",
"| Kangli Wang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Ganting Lei \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Yanling Liu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Haofeng Xiong \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"| Lu Shen \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Meng Wang ",
"Rongjuan Zhao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Huidan Wu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"| Bin Li \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nNational Clinical Research Center for Geriatric Disorders\nDepartment of Geriatrics\nXiangya Hospital\nCentral South University\nChangshaHunanChina\n",
"Qiumeng Zhang \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"Yujun Yao \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"| Hui Guo \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"| Kun Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nHunan Key Laboratory of Molecular Precisional Medicine\nCentral South University\nChangshaHunanChina\n",
"| Lu Xia \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n",
"| Zhengmao Hu \nCenter for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina\n\nCenter for Medical Genetics\nHunan Key Laboratory of Animal Models for Human Diseases\nCentral South University\nCorrespondence Zhengmao Hu and Lu XiaChangshaHunanChina\n\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\n410078ChangshaHunanChina\n"
] | [
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Department of Critical Care Medicine\nShenzhen Third People's Hospital\nShenzhenGuangdongChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"National Clinical Research Center for Geriatric Disorders\nDepartment of Geriatrics\nXiangya Hospital\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Hunan Key Laboratory of Molecular Precisional Medicine\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nSchool of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\nChangshaHunanChina",
"Center for Medical Genetics\nHunan Key Laboratory of Animal Models for Human Diseases\nCentral South University\nCorrespondence Zhengmao Hu and Lu XiaChangshaHunanChina",
"School of Life Sciences\nHunan Key Laboratory of Medical Genetics\nCentral South University\n410078ChangshaHunanChina"
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"T L Young, ",
"S M Ronan, ",
"L A Drahozal, ",
"I G Morgan, ",
"K Ohno-Matsui, ",
"Saw Sm Myopia, ",
"B A Holden, ",
"T R Fricke, ",
"D A Wilson, ",
"E Dolgin, ",
"C J Hammond, ",
"H Snieder, ",
"C E Gilbert, ",
"T D Spector, ",
"A P Klein, ",
"B Suktitipat, ",
"P Duggal, ",
"X B Cai, ",
"S R Shen, ",
"D F Chen, ",
"Q Zhang, ",
"Z B Jin, ",
"J A Rada, ",
"S Shelton, ",
"T T Norton, ",
"A I Jobling, ",
"M Nguyen, ",
"A Gentle, ",
"N A Mcbrien, ",
"A I Jobling, ",
"R Wan, ",
"A Gentle, ",
"B V Bui, ",
"N A Mcbrien, ",
"B Y Chen, ",
"C Y Wang, ",
"W Y Chen, ",
"J X Ma, ",
"D S Lam, ",
"W S Lee, ",
"Y F Leung, ",
"Y Seko, ",
"H Shimokawa, ",
"T Tokoro, ",
"P C Wu, ",
"C L Tsai, ",
"G M Gordon, ",
"H Guo, ",
"Jin X Zhu, ",
"T , ",
"X B Cai, ",
"Y H Zheng, ",
"D F Chen, ",
"J Dufner-Beattie, ",
"Y M Kuo, ",
"J Gitschier, ",
"G K Andrews, ",
"F Wang, ",
"B E Kim, ",
"M J Petris, ",
"D J Eide, ",
"J Chai, ",
"J W Wu, ",
"N Yan, ",
"J Massague, ",
"N P Pavletich, ",
"Y Shi, ",
"C Y Feng, ",
"X Q Huang, ",
"X W Cheng, ",
"R H Wu, ",
"F Lu, ",
"Z B Jin, ",
"Z B Jin, ",
"J Wu, ",
"X F Huang, ",
"L Wan, ",
"B Deng, ",
"Z Wu, ",
"X Chen, ",
"D Jiang, ",
"J Li, ",
"X Xiao, ",
"R D Palmiter, ",
"L Huang, ",
"D J Eide, ",
"J Jeong, ",
"D J Eide, ",
"W Kou, ",
"H S Kolla, ",
"A Ortiz-Acevedo, ",
"D C Haines, ",
"M Junker, ",
"G R Dieckmann, ",
"L H Nguyen, ",
"T T Tran, ",
"Ltn Truong, ",
"H H Mai, ",
"T T Nguyen, ",
"D Pretzer, ",
"B Schulteis, ",
"Vander Velde, ",
"D G Smith, ",
"C D Mitchell, ",
"J W Manning, ",
"M C , ",
"T Mabrouk, ",
"G Lemay, ",
"C Iannuzzi, ",
"M Adrover, ",
"R Puglisi, ",
"R Yan, ",
"P A Temussi, ",
"A Pastore, ",
"K Kluska, ",
"J Adamczyk, ",
"A Krężel, ",
"S Z Potter, ",
"H Zhu, ",
"B F Shaw, ",
"T Knaus, ",
"M K Uhl, ",
"S Monschein, ",
"S Moratti, ",
"K Gruber, ",
"P Macheroux, ",
"J Massagué, ",
"J Seoane, ",
"D Wotton, ",
"K G Slepchenko, ",
"J M Holub, ",
"Y V Li, ",
"Y Shi, ",
"J Massagué, ",
"J Tan, ",
"Z H Deng, ",
"S Z Liu, ",
"J T Wang, ",
"C Huang, ",
"N Malachkova, ",
"D Yatsenko, ",
"G Ljudkevich, ",
"V Shkarupa, ",
"Y Mo, ",
"Y Wang, ",
"B Cao, ",
"J Zhang, ",
"G Ren, ",
"T Yang, ",
"H J Lin, ",
"C C Wei, ",
"C Y Chang, ",
"T Fukada, ",
"N Civic, ",
"T Furuichi, ",
"M Fedor, ",
"B Urban, ",
"K Socha, ",
"S U Pp O Rti N G I N Fo R M Ati O N ; Dong, ",
"S Tian, ",
"Q Zhu, ",
"T , "
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] | [
"Evidence that a locus for familial high myopia maps to chromosome 18p. T L Young, S M Ronan, L A Drahozal, Am J Hum Genet. 63Young TL, Ronan SM, Drahozal LA, et al. Evidence that a locus for familial high myopia maps to chromosome 18p. Am J Hum Genet. 1998;63:109-119.",
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"Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. T Knaus, M K Uhl, S Monschein, S Moratti, K Gruber, P Macheroux, Biochem Biophys Acta. 1844Knaus T, Uhl MK, Monschein S, Moratti S, Gruber K, Macheroux P. Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron. Biochem Biophys Acta. 2014;1844:2298-2305.",
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"Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). K G Slepchenko, J M Holub, Y V Li, Cell Signal. 44Slepchenko KG, Holub JM, Li YV. Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ). Cell Signal. 2018;44:148-157.",
"Mechanisms of TGF-β signaling from cell membrane to the nucleus. Y Shi, J Massagué, Cell. 113Shi Y, Massagué J. Mechanisms of TGF-β signaling from cell mem- brane to the nucleus. Cell. 2003;113:685-700.",
"TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. J Tan, Z H Deng, S Z Liu, J T Wang, C Huang, Curr Eye Res. 35Tan J, Deng ZH, Liu SZ, Wang JT, Huang C. TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro. Curr Eye Res. 2010;35:37-44.",
"Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia. N Malachkova, D Yatsenko, G Ljudkevich, V Shkarupa, Oftalmologicheskii Zhurnal. 76Malachkova N, Yatsenko D, Ljudkevich G, Shkarupa V. Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myo- pia. Oftalmologicheskii Zhurnal. 2018;76:45-48.",
"Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. Y Mo, Y Wang, B Cao, J Zhang, G Ren, T Yang, J Tradit Chin Med Sci. 3Mo Y, Wang Y, Cao B, Zhang J, Ren G, Yang T. Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia. J Tradit Chin Med Sci. 2016;3:124-132.",
"Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. H J Lin, C C Wei, C Y Chang, EBioMedicine. 10Lin HJ, Wei CC, Chang CY, et al. Role of chronic inflammation in myopia progression: clinical evidence and experimental validation. EBioMedicine. 2016;10:269-281.",
"The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. T Fukada, N Civic, T Furuichi, PLoS One. 33642Fukada T, Civic N, Furuichi T, et al. The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways. PLoS One. 2008;3:e3642.",
"Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia. M Fedor, B Urban, K Socha, J Ophthalmol. 2019Fedor M, Urban B, Socha K, et al. Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and ado- lescents with myopia. J Ophthalmol. 2019;2019:1-7.",
"Additional supporting information may be found online in the Supporting Information section. How to cite this article. S U Pp O Rti N G I N Fo R M Ati O N ; Dong, S Tian, Q Zhu, T , S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Dong S, Tian Q, Zhu T, et al.",
"SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. 10.1111/jcmm.16803J Cell Mol Med. 25SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis. J Cell Mol Med. 2021;25:8432- 8441. https://doi.org/10.1111/jcmm.16803"
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] | [
"Evidence that a locus for familial high myopia maps to chromosome 18p",
"Global prevalence of myopia and high myopia and temporal trends from",
"The myopia boom",
"Genes and environment in refractive error: the twin eye study",
"Heritability analysis of spherical equivalent, axial length, corneal curvature, and anterior chamber depth in the Beaver Dam Eye Study",
"An overview of myopia genetics",
"The sclera and myopia",
"Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression",
"Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function",
"Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model",
"TGFbeta-induced factor: a candidate gene for high myopia",
"Expression of bFGF and TGFbeta 2 in experimental myopia in chicks",
"Chondrogenesis in scleral stem/ progenitor cells and its association with form-deprived myopia in mice",
"SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia",
"Expanding the phenotypic and genotypic landscape of nonsyndromic high myopia: a crosssectional study in 731 Chinese patients",
"The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5",
"The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells",
"Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding",
"Mutational screening of SLC39A5, LEPREL1 and LRPAP1 in a cohort of 187 high myopia patients",
"Trio-based exome sequencing arrests de novo mutations in early-onset high myopia",
"Exome sequencing study of 20 patients with high myopia",
"Detection of mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 families with earlyonset high myopia by exome sequencing",
"Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers",
"Zinc transporters and the cellular trafficking of zinc",
"The SLC39 family of zinc transporters",
"Modulation of zinc-and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36",
"Overcharging of the zinc ion in the structure of the zinc-finger protein is needed for DNA binding stability",
"Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom",
"Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability",
"The role of zinc in the stability of the marginally stable IscU scaffold protein",
"Metal binding properties, stability and reactivity of zinc fingers",
"Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein",
"Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron",
"Smad transcription factors",
"Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ)",
"Mechanisms of TGF-β signaling from cell membrane to the nucleus",
"TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro",
"Polymorphism of TGF-β1 (rs1800469) in children with different degrees of myopia",
"Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi tablets in guinea pigs with form-deprivation myopia",
"Role of chronic inflammation in myopia progression: clinical evidence and experimental validation",
"The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways",
"Concentration of zinc, copper, selenium, manganese, and Cu/Zn ratio in hair of children and adolescents with myopia",
"SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis"
] | [
"Am J Hum Genet",
"Lancet",
"Ophthalmology",
"Nature",
"Invest Ophthalmol Vis Sci",
"Arch Ophthalmol",
"Exp Eye Res",
"Exp Eye Res",
"J Biol Chem",
"Exp Eye Res",
"Graefes Arch Clin Exp Ophthalmol",
"Invest Ophthalmol Vis Sci",
"Invest Ophthalmol Vis Sci",
"Mol Vis",
"J Med Genet",
"Invest Ophthalmol Vis Sci",
"J Biol Chem",
"J Biol Chem",
"J Biol Chem",
"Sci Rep",
"Proc Natl Acad Sci",
"PeerJ",
"Invest Ophthalmol Vis Sci",
"Pflugers Arch",
"Biochem Biophys Acta",
"Mol Aspects Med",
"J Biol Inorg Chem",
"Biochemistry",
"Pharm Res",
"J Virol",
"Protein Sci",
"Coord Chem Rev",
"J Am Chem Soc",
"Biochem Biophys Acta",
"Genes Dev",
"Cell Signal",
"Cell",
"Curr Eye Res",
"Oftalmologicheskii Zhurnal",
"J Tradit Chin Med Sci",
"EBioMedicine",
"PLoS One",
"J Ophthalmol",
"Additional supporting information may be found online in the Supporting Information section. How to cite this article",
"J Cell Mol Med"
] | [
"\n\nHEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.",
"\n\nprepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-",
"\n\nTotal RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in",
"\n\nSLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in",
"\n\nCells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.",
"\n\nSLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.",
"\n\n3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).",
"\nFigure 4 ,\n4SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.",
"\n\nin the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.",
"\n\nThis work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].",
"\n\nShanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead)."
] | [
"HEK293 cells and HEK293T cells were cultured inDulbecco's modified Eagle's medium (DMEM) (Gibco Cat# C11995500BT) containing 10% foetal bovine serum (FBS) (Gibco Cat# 10099-141). Human lymphocyte cells (M16345, M16346, M16349, M16344, M19118 and M21932) from a high myopia pedigree carrying SLC39A5 mutations reported previously 15 were cultured in Roswell Park Memorial Institute (RPMI) (Gibco Cat# C11875500BT) containing 20% FBS. All cells were maintained at 37°C with 5% CO 2 in a humidified incubator and passaged every 2-3 days. All members were recruited for blood collection after providing informed consent. The study was approved by the Institutional Review Board of the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics and adhered to the tenets of the Declaration of Helsinki.",
"prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat# RS-122-2001) according to the standard Illumina library preparation procedure. In brief, purified RNA was poly-A-selected and fragmented, followed by first and second strand cDNA synthesis. Double-stranded cDNA was processed from end repair to PCR amplification according to the library construction steps. Libraries were purified using AMPure XP beads (Beckman Coulter Cat# A63882), then validated for the appropriate size on an Agilent 2100 Bioanalyzer (Agilent) and quantitated using Quantitative Real-Time PCR (qRT-PCR) (TaqMan Probe). Library pools were clustered and run on an Illumina HiSeq 4000 platform (Illumina), according to the manufacturer's recommended protocol. The sequenc-",
"Total RNA was extracted by TRIzol (Invitrogen Cat# 15596018) according to the manufacturer's instructions. The cDNA was obtained by reverse transcription-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Cat# K1622). qRT-PCR was conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Cat# K0251) (primers are listed in",
"SLC39A5 rescue plasmids were projected to restore the SLC39A5 expression of the KO cells. The pcDNA3.1-SLC39A5-Flag plasmid used previously 15 was introduced with an NGG synonymous mutation in the PAM sequence using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in",
"Cells were lysed by Nonidet P-40 (NP-40) buffer (Beyotime Biotechnology Cat# P0013F) for collagen immunoblot or by SDS lysis buffer for other immunoblots. polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk (5% BSA for phospho-antibody) in 1% PBST (phosphatebuffered saline and 1% Triton). After incubated with primary antibodies (Collagen Ⅰ: Abcam Cat# ab34710; Collagen Ⅱ: Abcam Cat# ab34712; Collagen Ⅳ: Abcam Cat# ab6586; fibronectinTechnology Cat# 8828S) at recommended dilutions overnight in 4°C, the membranes were washed and incubated with the secondary antibody at 1:10,000 dilution for 1 h. Finally, the blots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Cat# P36599) detection system.",
"SLC39A5 knockout cell line (KO-1). In total, 810 upregulated and 1207 downregulated transcripts were predicted. All differentially expressed genes were subjected to Gene Ontology (GO) functional annotation analysis using the Database for AnnotationVisualization and Integrated Discovery (DAVID) (https://david. ncifc rf.gov/). As shown in Figure 2, the significantly enriched genes and pathways were mainly associated with ECM organization, cell adhesion, wound healing and collagen fibril organization (Figure 2A). The qRT-PCR results also validated the RNA-seq predictions (Figure 2B), identifying transcriptional downregulation of certain ECM members under SLC39A5 depletion conditions. Later, immunoblot assay was used to assess the expression of Collagen I (COL1), Collagen Ⅱ (COL2), Collagen Ⅳ (COL4), fibronectin (FN) and laminin (LN) based on their robust expression in the ocular system. The results showed that contents of the candidate proteins in both whole-cell lysate and intracellular lysate (trypsin digested) were significantly decreased (Figure 2C-E) (the bands of COL4/FN in the supernatant were found to be false positives (Figure S2)), suggesting that SLC39A5 depletion dysregulated the ECM constituents.",
"3B,C), wherein the total contents of R-Smads and Smad4, the phosphorylation state of R-Smads, and the expression of COL1, COL2, COL4, FN and LN were back to normal as expected. The case of total increase and phosphorylation decrease in the content of Smad proteins prompted an examination of the stability. We used cycloheximide (CHX) to evaluate the half-life of candidate Smads. The results showed that all Smad proteins tested revealed an accelerated degradation rate even with higher primary expression, suggesting a shorter half-life of Smad proteins under SLC39A5depleted conditions (Figure 3D-G).",
"SLC39A5 depletion significantly decreased the average intracellular zinc levels in the KO group (Figure 4A). We further evaluated the zinc level in lymphocyte cells generated from a high myopia pedigree, previously reported to harbour a SLC39A5 mutation 15 and also observed lower average intracellular zinc levels in the patient cohort, similar to that observed in the KO cells (Figure 4B). DNA-binding capacity of R-Smad requires zinc chelation, 19 and the shorter half-life of Smad proteins caused by SLC39A5 depletion indicated that zinc may also affect the Smad protein stability. To evaluate protein stability, four recombinant Smad1 plasmids, each carrying a single binding site mutant (C64A, C109A, C121A and H126A), were constructed and transferred to HEK293 cells, along with a wild-type (WT) plasmid. As expected, the CHX assay showed a similar shorter half-life of the mutant Smad1 proteins and F I G U R E 1 SLC39A5 depletion induces abnormal cell morphogenesis and migration. (A) shows the validation of the homozygous knockout (KO) HEK293 cell lines. Direct Sanger sequencing indicated the KO-1 protein was early terminated when an A was inserted at position 141 (c.141insA, p.Y47*) or a 9-bp insertion after a 7-bp deletion from position 141 to 147 (c.141_147delinsATGCCAAAC, p.Y47*). KO-2 harboured a 16-bp deletion from position 439 to 454 (c.439_454del16, p.D147fs3*), inducing early termination after two missense threonine amino acids. (B) shows the typical morphology of the three SLC39A5-related cell lines under 5×, 10× or 40× magnification, respectively. Two KO cell lines with substantial differences in the optically confluent monolayers were observed. (C) shows the relative scratch open area of the wound healing process of the three SLC39A5-related cell lines. Fitting curves were obtained based on the scratch area images at different time points within 48 h. Compared with the control group, KO cells exhibited an evidently impaired wound healing ability a faster degradation rate (Figure 4C,D), confirming the hypothesis that zinc chelation is required for Smad protein stability. According to this hypothesis, the reestablishment of zinc homeostasis would be a novel strategy to rescue the phenotypes in SLC39A5 KO cells.",
"in the knockout (KO) cells after SLC39A5 re-expression via lentivirus infection. (D-G) show the degradation of cycloheximide (CHX)-treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E-G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001 and migration were also affected. Overall, these results indicate the presence of a zinc-TGFβ signalling-ECM synthesis regulation flow.",
"This work was supported by the National Natural Science Foundation of China [81671122 to Zhengmao Hu, 81730036 to Kun Xia and 81871079 to Hui Guo]; and the Hunan Provincial Grants [2019SK2051, 2019JJ70002 and 2018DK2016 to Zhengmao Hu, and 2018SK1030 to Kun Xia].",
"Shanshan Dong: Conceptualization (equal); Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing-original draft (lead). Qi Tian: Data curation (supporting); Resources (supporting). Tengfei Zhu: Data curation F I G U R E 4 SLC39A5 depletion induces zinc deficiency and destabilizes Smad proteins. (A) shows zinc quantification of the SLC39A5related HEK293 cell lines. The average intracellular zinc level was lower in knockout (KO) cells than that in the control group. (B) shows zinc quantification of the lymphocyte cells from a high myopia pedigree carrying SLC39A5 mutations reported previously. 15 The average intracellular zinc levels of the lymphocytes of patients (M16345, M16346 and M16349) were markedly lower than those of the normal controls (M16344, M19118 and M21932). (C,D) show the degradation of the cycloheximide (CHX)-treated wild-type or zinc-binding site mutant Smad1 protein at different time points within 8 h of CHX treatment. Degradation curves (D) were created based on statistical analyses of the Western blotting results (C). **p < 0.01 (supporting); Resources (supporting). Kangli Wang: Formal analysis (supporting). Ganting Lei: Formal analysis (supporting). Yanling Liu: Formal analysis (supporting). Haofeng Xiong: Methodology (supporting). Lu Shen: Methodology (supporting). Meng Wang: Methodology (supporting). Rongjuan Zhao: Methodology (supporting). Huidan Wu: Methodology (supporting). Bin Li: Writing-review & editing (supporting). Qiumeng Zhang: Methodology (supporting). Yujun Yao: Writing-review & editing (supporting). Hui Guo: Funding acquisition (supporting); Writing-review & editing (supporting). Kun Xia: Conceptualization (supporting); Funding acquisition (supporting); Writing-review & editing (supporting). Lu Xia: Conceptualization (lead); Writing-review & editing (lead). Zhengmao Hu: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing-review & editing (lead)."
] | [
"Figure 1A",
"Figure 1B)",
"Figure S1",
"Figure 1C",
"Figures S3 and S4",
"Figure S5",
"(Figure S5)",
"Figures S6 and S7)",
"Figure S8",
"Figure 3",
"Figure 3A",
"Figure",
"Figure S9)",
"Figure S10)",
"Figure S11"
] | [] | [
"Myopia is a condition of refractive error causing light to focus in front of the retina, with axial elongation as its most common pathology. High myopia, clinically defined as a refractive error of at least −6.00 diopters (D) or an axial length >26 mm, 1 is one of the leading causes of visual impairment worldwide 2,3 with a significantly increased prevalence. 3,4 Thus, the prevention of high myopia has become a global public health problem. Pedigree analyses and twin studies identified high myopia highly heritable. 5,6 Even though numerous loci (1q41, 4q25, 8p23, etc.) and causative genes (ZNF644, CCDC111, SCO2, etc.) have been identified, 7 the pathogenic mechanism of high myopia remains unclear.",
"Recently, scleral extracellular matrix (ECM) synthesis or remodelling has been proposed as an underlying cause of high myopia. [8][9][10] According to this hypothesis, scleral collagen synthesis is regulated during myopia progression, 9 while scleral ECM is remodelled in myopic animal. 10 ECM synthesis and remodelling are targets of the TGFβ signalling pathway, one of the most reproducibly dysregulated pathways in myopia development. [10][11][12][13] Total or isoform-specific scleral TGFβ is frequently altered during myopia progression. 9,10,14 In the previous study, we first identified SLC39A5 as a high myopia-associated gene, and mutations of SLC39A5 dysregulated the BMP/TGFβ pathway. 15 Further research confirmed SLC39A5 as one of the top three genes contributing to nonsyndromic high myopia. 16 This study aimed to investigate the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia. We found that SLC39A5 played a key role in maintaining intracellular zinc homeostasis. SLC39A5 depletion-induced zinc deficiency directly destabilized Smad proteins, and Smads instability further impaired TGFβ signalling-mediated ECM synthesis, thus contributing to the pathogenesis of high myopia. ",
"According to the protocols from Zhang laboratory (http://www. genom e-engin eering.org/gecko/), target-specific sgRNA was cloned into the lentiCRISPRv2 vector (sgRNAs are listed in Table S1). Then, lentiviruses were packaged in HEK293T cells by co-transfection of CRISPR-sgRNA/pVSVG/pspAX2. The supernatants containing viral particles were collected 48 h after transfection and filtered (0.22 μm pore size). Then, HEK293 cells were transduced with viral supernatant supplemented and selected with 1 μg/ml puromycin.",
"After transfection, cells were detached and seeded in 10 ml Petri dishes at a density of 200 cells per plate. Puromycin (2 µg/ml) was added to allow the selection of positive clones during the following two weeks. Positive clones were isolated and transferred to sixwell plates. The SLC39A5 knockout clones were validated by direct Sanger sequencing (primers are listed in Table S2). The negative control cell line was generated from HEK293 transformed with the identical lentiviral vector lacking specific sgRNAs.",
"Zinc quantification assay (Abcam Cat# ab102507) was performed for accurate measurement of zinc levels according to the manufacturer's instructions. In brief, cells were harvested with NP-40 lysis buffer (EDTA-free), and proteins were deproteinized by an equal volume of 7% TCA Solution Buffer. The supernatants were transferred to new clean tubes after centrifugation. 200 μl Zinc Reaction Buffer was applied to 50 μl tested samples in a 96-well plate. After a 10-min incubation at room temperature, the OD 560 was measured in a microplate reader. The zinc standard curve was plotted after subtracting the background value, and the tested sample concentration was calculated from the standard curve.",
"Total RNA for RNA-seq library was extracted using TRIzol (Invitrogen Cat# AM9738), validated on a NanoDrop 1000 Spectrophotometer (Thermo) and quantitated using Qubit (Thermo). RNA-seq libraries were ing data were filtered with SOAPnuke (v1.5.2), then mapped to the reference genome using HISAT2 (v2.0.4) and aligned to the reference coding gene set using Bowtie2 (v2.2.5). Differential gene expression analysis between the target and reference sets of treatments was determined using DESeq2 (https://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq.html). Enrichment analysis of annotated different expressed genes was performed with the Phyper (https://en.wikip edia.org/wiki/ Hyper geome tric_distr ibution) based on the hypergeometric test. Table S3). All samples were run in triplicate. qRT-PCR data were analysed by LightCycler 96 software. Relative candidate gene mRNA levels were normalized to those of β-actin or GAPDH. A value of p < 0.05 was considered to be statistically significant.",
"The Smad1 zinc-binding site substitutions were generated to assess the correlation between zinc-binding capacity and protein stability. Smad1 WT plasmid was brought from OriGene (OriGene Cat# RC200299), and the four zinc-binding site substitutions (C64A, C109A, C121A and H126A) were separately generated using Fast multiSite Mutagenesis System (Transgen Biotech Cat# FM201-01) (primers are listed in Table S4). Table S5).",
"Finally, the rescue sequence was cloned into the pLVX-IRES-puro vector to construct the KO rescue plasmid.",
"SLC30A1 shRNA interference plasmids were also constructed to rescue the poor intracellular zinc level of SLC39A5 KO cells by suppressing the zinc efflux baseline. The three targeting sequences were obtained from the siRNA sequence database (Thermo) and then generated into the pFUGW-lentiviral vector between the XbaI and BamHI restriction sites, respectively (primers are listed in Table S6). ",
"Cells were seeded on specific coverslips in 12-well plates.",
"The cell slides were fixed in 4% paraformaldehyde for 10 min.",
"Permeabilization was performed with 0.1% PBST (phosphatebuffered saline and 0.1% Triton) for 15 min. After blocking with 5% BSA for 1 h, the cell slides were incubated with primary antibody at recommended dilutions overnight in 4°C and stained with Alex Fluor 488-conjugated second antibody (Jackson Cat# 111-545-144) for 1 h protected from light. DAPI was applied for 1 min as a nuclear marker. Images were acquired with an Eclipse TCS-SP5 inverted confocal microscope (Leica).",
"HEK293 cells were seeded into the 2-well culture insert (Ibidi Cat# 81176) pre-coated by Matrigel. After cell attachment for approximately 24 h to form an optically confluent monolayer, the culture insert was removed to create the wound gap and the cells were then cultured with serum-free medium. The wound gap closure was monitored by taking pictures with an Eclipse inverted microscope (Leica) at different time points.",
"GraphPad Prism was used to calculate and plot the mean ± SEM of measured quantities. Significances were assessed by two-way analysis of variance (ANOVA)/Mann-Whitney U test (nonparametric)/Student's t test (parametric), p-value < 0.05 was considered to indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.",
"Two SLC39A5 knockout (KO) cell lines were developed using the CRISPR-Cas9 system in the human embryonic kidney cell line (HEK293) ( Figure 1A). Cell morphology anomalies were markedly observed in the SLC39A5-depleted cells. After regular growth for 2 days, the KO cells revealed a tendency to form small cell aggregates instead of a typical optically confluent monolayer ( Figure 1B).",
"This phenotype suggested a potential impairment or abnormality of ECM generation. Considering the influence of ECM dynamics on cell migration, invasion and morphogenesis, we immediately investigated the cellular motility and migratory capacity of the SLC39A5depleted HEK293 cells. Scratch assay was performed in both groups.",
"A 2-well culture insert generated a uniform gap in the confluent monolayer, and wound healing was imaged at different time points.",
"The results showed that the gap in the control group was completely healed after 24 h, while the wound gap in the KO group was not able to close ( Figure S1; Figure 1C).",
"To investigate the overall alteration caused by SLC39A5 depletion, RNA-sequencing (RNA-seq) was performed using a Immunofluorescence also verified the decrease in these ECM proteins ( Figures S3 and S4).",
"The transcription of collagens, FN and LN is closely related to TGFβ signalling, and we have already determined that mutations of SLC39A5 dysregulated TGFβ signalling previously. 15 Considering the transcriptional downregulation of collagens, FN and LN in SLC39A5 KO cells, we hypothesized that reduced SLC39A5 expression would inhibit the TGFβ signalling pathway. Hence, we examined the expression of Smad proteins, which lie on the centre of the TGFβ signalling pathway. The results showed an evident upregulation of total Receptor-Smads (R-Smads) (Smad1, Smad2/3) expression along with a significant decrease in phosphorylated Smad expression (pSmad1/5/9, pSmad2/3) ( Figure S5). Additionally, Co-Smad (Smad4) expression was also increased, while inhibitor-Smad (Smad7) was not affected (Figure S5), suggesting a suppressed activation of the TGFβ signalling, which was confirmed through immunofluorescence ( Figures S6 and S7). The qRT-PCR performed to assess the transcriptional levels of Smad1, Smad2 and Smad4 in KO cells showed increased mRNA expression of the tested proteins ( Figure S8). To validate the phenotype of inhibited TGFβ signalling activation and insufficient ECM synthesis resulting from SLC39A5 depletion, rescue experiments were carefully carried out. As shown in Figure 3, re-expression of SLC39A5 restored intracellular zinc level ( Figure 3A). TGFβ signalling activation and ECM synthesis were also rescued ( Figure ",
"The TGFβ pathway is a zinc-regulated pathway ( Figure S9), while SLC39A5 is a zinc transporter. 17,18 These facts prompted us to confirm the involvement of zinc homeostasis in TGFβ signalling alteration in SLC39A5 KO cells. Thus, zinc quantification was performed to detect intracellular zinc levels. As shown in Thus, we selected the most widely expressed zinc efflux protein SLC30A1 (20), as a target. Surprisingly, silencing SLC30A1 counterbalanced the influence of SLC39A5 depletion, which significantly reverted intracellular zinc level close to normal ( Figure S10) and normalized the TGFβ signalling pathway and ECM synthesis ( Figure S11).",
"SLC39A5 was first identified as a high myopia pathogenic gene in our previous study 15 and was later confirmed as one of the top three genes contributing to nonsyndromic high myopia. 16,[20][21][22][23] However, the mechanisms by which SLC39A5 contributes to high myopia are not well understood. Here, we demonstrated that SLC39A5 may play a role in the pathogenesis of high myopia by modulating TGFβ signalling-mediated ECM synthesis through the regulation of intracellular zinc homeostasis. SLC39A5 depletion significantly lowered the intracellular zinc level, which destabilized Smad proteins, and further inhibited TGFβ signalling-mediated ECM synthesis, which ultimately contributed to the pathogenesis of high myopia.",
"Zinc homeostasis is crucial for all organisms as it serves as a catalytic or structural cofactor for multiple types of proteins. Zinc homeostasis depends on the transport of zinc in both directions across the plasma membrane and both in and out of various vesicular compartments. 24 The SLC39 family facilitates zinc transport into the cytoplasm in the opposite direction to SLC30 family members. 25 Within this network, SLC39A5 is a specific zinc transporter. 17,18,26 Our work confirmed the zinc uptake characteristic of SLC39A5, in which SLC39A5 depletion caused an intracellular decline in zinc levels in HEK293 or lymphocyte cells. Once zinc homeostasis was disrupted, the catalytic or structural stability of proteins was impaired, suggesting that SLC39A5 depletion, indirectly, caused this impairment.",
"Zinc has a profound effect on stabilization of zinc-binding proteins, 27-31 since the proteins responsible for cellular zinc buffering will bind zinc transiently; therefore, their function will strongly depend on the cellular zinc status. 32 It has been proposed that zinc chelation affects protein stability through various mechanisms. The human SOD1 protein requires zinc binding to facilitate the restructuring of apoproteins, 33 while the putative protease of Bacteroides thetaiotaomicron (ppBat) requires zinc chelation to link the β-strands to α-helices in protein structures. 34 In our study, the R-Smads and Co-Smad, which are pivotal to the BMP/TGFβ signalling pathway, 35 F I G U R E 2 SLC39A5 depletion impairs extracellular matrix (ECM) synthesis. (A) shows the pathway enrichment analysis for the differentially expressed genes between the control and KO subpopulations of HEK293 cells. Pathway analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics Resources, with an enrichment p-value cut-off of 0.01. (B) shows the qRT-PCR validation of the differentially expressed genes of the ECM members indicated by RNA-seq. (C-E) shows the expression of several ECM components (COL1, COL2, COL4, FN and LN) in the supernatant, whole-cell lysate or intracellular lysate (trypsin digested) of wild-type (WT) and SLC39A5 knockout (KO) HEK293 cells. All tested ECM components were decreased in both the whole lysate and intracellular lysate. (B) and (C) show statistical analyses of the ECM components in the whole lysate and intracellular lysate, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are zinc-binding proteins. We found that Smad proteins were rapidly degraded during SLC39A5 deletion-induced zinc deficiency.",
"Further results suggested that loose zinc coupling, facilitated by a mutant ion-binding site, directly destabilized the exogenous Smad1 proteins, which clearly confirms the necessity of zinc chelation in maintaining the protein stability of Smads, while disturbances in zinc homeostasis induce protein instability.",
"Zinc binding has also been implicated in protein phosphorylation and subcellular translocation. 36 Non-zinc-bound protein kinase C delta (PKCδ) showed a significantly different structure near its phosphorylation site, compared with when it was zinc-bound. Subcellular translocation of PKCδ was also influenced by intracellular zinc. These findings expand our understanding of the phosphorylation decrease in R-Smads during SLC39A5-depleted conditions.",
"Since R-Smads are directly phosphorylated by upstream receptor kinases, which then form heteromeric complexes with Smad4 to translocate into the nucleus, 37 the zinc deficiency induced by SLC39A5 depletion may barely maintain the Smad protein in a favourable structure for phosphorylation, and may lead to nucleus translocation retention, further impairing the TGFβ signalling.",
"It is well recognized that TGFβ signalling directly affects ECM dynamics or triggers it when mediating other biological processes. [38][39][40][41] Herein, suppressed TGFβ signalling in the zinc-deficient conditions restrained ECM synthesis in HEK293. Recently, ECM synthesis and remodelling have attracted significant attention in high myopia aetiology studies. [8][9][10]42 We found that the transcription of all ECM proteins tested (COL1, COL2, COL4, FN and LN) was decreased under zinc-deficient conditions. ECM-induced cell morphogenesis According to this hypothesis, the SLC39A5-induced zinc deficiency suppresses TGFβ signalling, leading to insufficient ECM synthesis, which may ultimately contribute to high myopia development. We further validated this hypothesis by performing rescue experiments.",
"As expected, of SLC39A5 re-expression, which recovers zinc influx, and SLC30A1 silencing, which blocks zinc efflux, could identically rescue the alteration of R-Smad phosphorylation and ECM synthesis in SLC39A5-depleted HEK293 cells, by re-establishing zinc homeostasis.",
"Upon reviewing the relationship between zinc and high myopia, we found plenty of indications of zinc involvement in high myopia development. 43 First, zinc is present abundantly in the ocular system, including the retina and choroid; thus, zinc deficiency produces a variety of ocular manifestations. Furthermore, a number of high myopia gene products are zinc-related proteins.",
"Among them, ZNF644, ZFHX1B, ZC3H11B and SCO2 are zinc finger proteins, and HGF and IGF1 are positively regulated by intracellular zinc concentration. Additionally, certain pathogenic genes (LAMA1 and TGFB1) may even be regulated by intracellular zinc, according to the zinc-TGFβ signalling-ECM synthesis flow.",
"Finally, multiple small molecules that serve as chaperones for disease-related genes or pathways are zinc-related proteins. Smad proteins, together with their anchors (SARA and HGS), are either zinc-binding or zinc finger proteins. These findings strongly highlight the correlation between zinc and high myopia.",
"In summary, our data suggest that depletion of SLC39A5 induces zinc deficiency, which restrains TGFβ signalling-mediated ECM synthesis, thus possibly contributing to high myopia pathogenesis. Zinc homeostasis appears to be a dominant aetiological factor of high myopia development. ",
"The authors confirm that there are no conflicts of interest. ",
"The data that support the findings of this study are available from the corresponding author upon reasonable request.",
"https://orcid.org/0000-0002-1605-7066",
"Zhengmao Hu https://orcid.org/0000-0002-3921-8205"
] | [] | [
"| INTRODUC TI ON",
"| MATERIAL S AND ME THODS",
"| Cell culture",
"| SLC39A5 knockout cell line construction",
"| Zinc quantification",
"| RNA-seq and analysis",
"| qRT-PCR validation",
"| Plasmid construction and transient transfection",
"| Immunoblotting",
"| Immunofluorescence",
"| Wound healing capacity test",
"| Statistical analysis",
"| RE SULTS",
"| SLC39A5 depletion impairs ECM synthesis",
"| SLC39A5 depletion suppresses TGFβ signalling",
"| SLC39A5 depletion induces zinc deficiency and destabilizes Smad protein",
"| DISCUSS ION",
"ACK N OWLED G EM ENTS",
"CO N FLI C T O F I NTE R E S T",
"AUTH O R CO NTR I B UTI O N",
"DATA AVA I L A B I L I T Y S TAT E M E N T",
"O RCI D",
"Shanshan Dong",
"R E FE R E N C E S",
"Figure 4 ,"
] | [] | [
"Table S1",
"Table S2",
"Table S3",
"Table S4",
"Table S5",
"Table S6"
] | [
"O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis",
"O R I G I N A L A R T I C L E SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis"
] | [
"J Cell Mol Med"
] |
220,888,721 | 2022-01-23T02:19:35Z | CCBY | https://nutritionandmetabolism.biomedcentral.com/track/pdf/10.1186/s12986-020-00480-w | GOLD | 01ce14faf78d6b8948a0ecf411868189efe5ecaf | null | null | null | null | 10.1186/s12986-020-00480-w | 3046886996 | 32774437 | 7395973 |
Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial
Xiaomin Sun
Xiao-Kai Ma
Lin Zhang
Zhen-Bo Cao
Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial
10.1186/s12986-020-00480-wR E S E A R C H Open Access
Background: Previous studies indicated that serum 25-hydroxyvitamin D [25(OH)D] concentrations are positively associated with physical activity levels independent of sun exposure. However, the effect of resistance training on serum 25(OH) D concentrations remains unclear. Thus, this study aimed to examine the effect of chronic resistance training on serum 25(OH) D concentrations and determine whether 25(OH) D concentration variations are influenced by body composition changes. Methods: Eighteen young men aged 19-39 years were randomly divided into a 12-week resistance training group (RT, n = 9) and non-exercise control group (CON, n = 9). The trial was undertaken in Shanghai University of Sport in Shanghai, China. Randomization and allocation to trial group were carried out by a central computer system. Serum 25(OH) D and intact parathyroid hormone concentrations were measured using commercially available enzymelinked immunosorbent assay kits. Body composition was measured by dual-energy X-ray absorptiometry.Results: The average serum 25(OH) D concentrations were 26.6 nmol/L at baseline. After the 12-week intervention program, serum 25(OH) D concentrations significantly increased in both groups. Serum 25(OH) D concentrations at midpoint (6-week) increased significantly only in the CON group (P < 0.01). From training midpoint to endpoint, a significantly greater increase in serum 25(OH) D concentrations was noted in the RT group (P-interaction = 0.043); 25(OH) D concentration changes (end-pre) were negatively related to fat-free mass (mid-pre) (r = − 0.565, P = 0.015) and muscle mass (mid-pre) (r = − 0.554, P = 0.017).Conclusions: There were no beneficial effects of the 12-week resistance training on serum 25(OH) D concentration in vitamin D deficient young men, and an indication that seasonal increase in serum 25(OH) D concentrations during the early phase of resistance training was transiently inhibited, which may partly be attributed to resistance training-induced muscle mass gain.
Introduction
Circulating 25-hydroxyvitamin D [25(OH)D] is the major form of vitamin D, which is used in vitamin D status assessment. Accumulated studies indicated that vitamin D not only regulates calcium and phosphorus metabolism but also plays an important role in modifying cardiovascular diseases risk, and improving immune function and physical fitness [1][2][3][4].
Previous studies indicated that higher levels of physical activity or cardiorespiratory fitness were related to higher circulating 25(OH) D concentrations [5][6][7]. Although individuals who were physically active outdoors are more likely to have higher circulating 25(OH) D due to increased sun exposure [8,9], this relationship was still observed after adjustment for sunlight exposure [6,10]. Scragg et al. [5] found that higher levels of physical activity were associated with higher serum 25(OH) D concentrations not only during summer but also during winter, when the vitamin D synthesis from sun exposure is extremely limited [11]. Consistently, several intervention studies suggested that endurance exercise could increase circulating 25(OH) D or prevent its seasonal reduction [12][13][14]. These findings indicated that physical activity could directly increase 25(OH) D concentrations.
Resistance training (RT) promotes health benefits through increased skeletal muscle mass and qualitative adaptations [15]. In addition to adiposity tissue, muscle tissue was suggested to serve as an another important site to store 25(OH) D, and then it returns to the circulating when needed [16,17]. Nevertheless, Makanae and colleagues reported that intramuscular expression of CYP27B1, which catalyses the hydroxylation and activation of 25(OH) D, was significantly increased at 1 h and 3 h after resistance exercise, which may enhance local vitamin D metabolism in skeletal muscle [18]. It is well known that compared with endurance exercise, RT could not only reduce fat mass, but also it is more effective in increasing muscle mass and strength [19,20], which may largely affect vitamin D metabolism. However, whether serum 25(OH) D concentrations are affected by RT remains unclear.
This study aimed to evaluate the effect of chronic resistance training on serum 25(OH) D concentrations and to determine whether the effects are attributable to changes in body composition in healthy young men.
Methods
Participants
Eighteen healthy men with no chronic diseases (aged 19 to 39 years) were included in this study, which was conducted from March to July 2016. Participants using vitamin D supplements or sunscreen regularly were excluded. Assessments were performed at 0 week (pre), 6 weeks (midpoint), and 12 weeks (endpoint). All subjects provided informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). The trial has been retrospectively registered at Chinese Clinical Trial Registry website (ChiCTR2000030876).
Experimental protocol
Eighteen participants were randomly allocated to the following two groups by using a computer-generated random number sequence: resistance training group (RT) and no-exercise control group (CON). Participants in the RT group were instructed to exercise 2-3 times a week in a gymnasium. A supervised training session was performed in the afternoon between 1630 h and 2000 h during the 12 weeks of progressive resistance training. In the first 2 weeks, the participants were taught how to properly perform each movement. The resistance workload gradually changed from light to heavy, and the participants were instructed to complete 10 repetitions for each set of the following exercises: leg press, preacher curl, chest press, leg curl, and iso row, with 60-120-s rest period between two sets; additionally, they also performed three sets of 15-25 repetitions of abdominal crunch and back extension with their own body weight, with a 60-120-s rest period between two sets. At the first training session of the third week, the participants were instructed to perform strength testing, and 12 repetitions maximum (12RM) for each exercise were performed, except back extension and abdominal crunch. The workload for the next resistance training was calculated from the participants' previously determined 12RM. The participants were encouraged to perform back extension and abdominal crunch exercises 30 times from the first training session of the third week. When the strength training equipment at our gymnasium had been upgraded at the eighth week of training, the resistance training program was revised to include the following: leg extension, standing one-arm cable curl, standing cable chest press, leverage shoulder press, leverage high row, back extension, and abdominal crunch. These new components of the resistance training program were selected based on the principle of using similar muscle groups. The order of resistance exercise was randomized; however, training of the same muscle groups continuously was avoided. The participants were instructed not to undertake any formal exercise or change their levels of general physical activity and dietary habits.
Anthropometric measurements
Height was measured with the participants wearing light clothing and being barefoot (HK6000-ST, HENGKANG JIAYE Technology Co., Ltd. CHN). Dual-energy X-ray absorptiometry (DXA Prodigy, GE Lunar Corp., Madison, WI, USA) was used for the measurement of whole and regional body composition, including total body mass, muscle mass, fat mass, fat percentage, and bone mineral density. Body mass index was calculated as body mass (kg) divided by height (m 2 ). Fat-free mass (FFM) was calculated as body mass minus fat mass.
Physical activity and sun exposure
Physical activity was measured using Actigraph GT3X+ accelerometers (ActiGraph, LLC, Pensacola, FL, USA). Participants were instructed to wear the accelerometers on their right hip at all times, except during water activities (swimming, showering) or sleeping, for 7 consecutive days at baseline. A wear time of ≥480 min/day was used as the criterion for a valid day, and ≥ 2 weekdays and 1 weekend day was used as the criteria for a valid 7-day period of accumulated data. Non-wear time was defined as consecutive periods of ≥60 min of zero accelerometer counts and excluded from the analyses. Data were recorded in 60-s epochs. A pragmatic cutoff of ≥2690 cpm was used to categorize moderatevigorous physical activity time [21,22]. The 7-day data collection for all subjects was fixed within 2 weeks. Outdoor time from 0900 h to 1600 h for 7 consecutive days was recorded and used as an alternative for sun exposure in the analysis.
Blood analysis
Blood samples were obtained after at least an 8-h overnight fasting period and subsequently centrifuged at 3000 rpm at 4°C for 10 min. Serum was collected and stored at − 80°C until analysis. Serum 25(OH) D and intact parathyroid hormone (iPTH) concentrations were measured in duplicate using commercially available enzyme-linked immunosorbent assay kits (25(OH) D, IDS, Bolton, UK; iPTH, DRG, Marburg, Germany) according to the manufacturer's instructions. The intraassay and inter-assay coefficients of variation were 9.1 and 7.7%, respectively, for serum 25(OH) D and 9.6 and 3.6%, respectively, for iPTH. Vitamin D deficiency is defined as 25(OH) D concentration < 50 nmol/L, and vitamin D insufficiency as 25(OH) D concentration < 75 nmol/L [23].
Statistical analysis
All statistical analyses were performed using IBM SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were assessed for normality using a Kolmogorov-Smirnov test prior to all statistical analyses. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables were presented as median (interquartile range), unless otherwise indicated. The Student t test (for normally distributed variables) or the Mann-Whitney U test (for non-normally distributed variables) was used to evaluate the differences between groups. Two-factor repeat-measured analysis of variance (ANOVA) (group × time) was used to determine the effect of exercise on 25(OH) D, iPTH, and body composition indicators. A post hoc test with Bonferroni correction was performed to identify statistically significant differences among the mean values when a considerable interaction was identified. Pearson correlation and partial correlation coefficients were computed between 25(OH) D concentration changes and body composition indicators. The level of statistical significance was set at P < 0.05.
Results
The participants' characteristics and blood parameters at baseline are presented in Table 1. The average serum 25(OH) D concentrations were 26.6 nmol/L, and all participants showed vitamin D deficiency. No significant difference in either body composition indicators or blood parameters was found between the groups (P > 0.05).
As shown in Table 2, significant interactions of group × time were found for FFM (P = 0.004) and muscle mass (P = 0.003). After resistance training, the FFM values (mid-pre, 2.7%, P = 0.003; end-pre, 3.0%, P = 0.004) and muscle mass (mid-pre, 3.0%, P = 0.002; end-pre, 3.4%, P = 0.004) significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in the CON group. In addition, we observed borderline significant interactions in body fat percentage, arm fat percentage, leg fat percentage, and trunk fat percentage ( Table 2).
As shown in Table 2 and Fig. 1, a significant group × time interaction was observed for serum 25(OH) D concentrations (P = 0.025). After the 12-week intervention, the mean serum 25(OH) D concentrations significantly increased to 36.7 ± 2.7 nmol/L in the RT group and 40.7 ± 2.7 nmol/L in the CON group. Moreover, in the CON group, higher serum 25(OH) D concentrations were observed at midpoint compared with the values at baseline (P < 0.01), whereas no notable differences were found in the RT group. From midpoint to endpoint, although serum 25(OH) D concentrations significantly increased in both groups, the increase was significantly greater in the RT group (25.8%) than in the CON group (9.4%) (P-interaction =0.043).
Correlation analyses were performed to identify the factors associated with the changes in 25(OH) D concentrations (Table 3). 25(OH) D concentration changes (end-pre) were negatively related to FFM (mid-pre) (P = 0.015) and muscle mass (mid-pre) (P = 0.017) changes (Fig. 2); a borderline association was observed between 25(OH) D concentration changes (mid-pre) and FFM (mid-pre) (P = 0.058) and muscle mass (mid-pre) (P = 0.057) changes. In addition, significant correlations of 25(OH) D concentration changes (end-pre) with FFM (mid-pre) (P = 0.039) and muscle mass (mid-pre) (P = 0.043) changes were observed after adjustment for group, age, and changes in sun exposure (end-pre), body weight (end-pre), and body fat percentage (end-pre).
Discussion
In this study, we found that the seasonal increase in serum 25(OH) D concentrations in young men was transiently inhibited during the early phase of RT; subsequently, serum 25(OH) D concentrations increased in both RT and CON groups, and the increase was observed to be greater in RT group than in CON group. The transient inhibition could be partly attributed to muscle mass and fat free mass gain induced by RT in the early phase.
Previous studies have reported significant positive associations of serum 25(OH) D concentrations with physical activity levels independent of sun exposure [6,10]. Bell et al. [24] found that 25(OH) D concentrations were higher in participants who had engaged in regular muscle-building exercise for at least 1 year compared to those in the control group. Consistently, our recent studies also observed that exercise training could increase serum 25(OH) D concentrations or prevent its seasonal reduction in young and old men [13,14]. However, the effect of RT on serum 25(OH) D concentrations remains unclear. Compared with endurance training, RT is more effective in increasing muscle mass and strength [24], which was also supported by our results. In the present study, we observed that after resistance training, body mass was reduced, while FFM and muscle mass were significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in CON group.
In addition to adiposity tissues, muscle tissues are also served as another important storage for 25(OH) D, which could enable optimal vitamin D status to maintain its physiological function during winter months [17]. Makanae and colleagues observed that intramuscular CYP27B1 was significantly increased after resistance exercise in skeletal muscle, which may enhance local vitamin D metabolism [18]. Srikuea et al. [25] also found that the CYP27B1 expression in the muscle increased significantly during skeletal muscle regeneration from injury. Thus, we speculated that the transient inhibition in serum 25(OH) D levels in RT group during the early phase of resistance training could be partly due to absorption of serum 25(OH) D in the increasing muscle mass and its use in muscle activity. 25(OH) D consumption in muscle may induce a compensatory increase in serum 25(OH) D concentrations; however, the concentrations were still lower than that in control group, which may due to the lack of vitamin D storage and short-term intervention period. Previous studies report that serum 25(OH) D concentrations gradually increase from around March to August [26]. This could partly explain the reason why serum 25(OH) D concentration was transiently inhibited in early Spring and followed by a marked increase in early Summer in our study. Future studies that aim at exploring the mechanism underlying the effect of resistance training on serum 25(OH) D concentrations are warranted. This study has several limitations. First, our study included only men with relatively lower serum 25(OH) D concentrations. Therefore, it remains to be established whether these findings are applicable to other subjects with higher 25(OH) D concentrations. Second, considering the seasonal variation of serum 25(OH) D concentration, the findings in our study have seasonal limitations. Third, at baseline resistance training group had relatively lower values of muscle mass and body fat percentage, although they are not statistically significant, which should be interpreted with caution. Fourth, we couldn't observe any relationship between muscle mass and 25(OH) D at three points, which may due to the small sample size. However, the muscle mass changes were significantly related with 25(OH) D concentration changes. Finally, although our sample size was relatively small, a post hoc power calculation would still give us a 90% chance, with an effect size of 0.58, to demonstrate the interaction effect of serum 25(OH) D concentrations, which was considered statistically significant at P < 0.05 at the endpoints.
Conclusions
In summary, serum 25(OH) D concentrations in vitamin D deficient young men was observed to be transiently inhibited during the early phase of resistance training and subsequently increased in the later phase. This finding could be partly attributed to the muscle mass and fat free mass gain induced by resistance training during the early phase. Our findings indicated that adequate administration of oral supplements or increased daily dietary vitamin D intake are encouraged to increase serum 25(OH) D concentrations, especially for those who exercise regularly.
Fig. 1
1Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05
25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum
Table 1
1Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activityVariable
Resistance training group (RT) (n = 9)
Non-exercise control group (CON) (n = 9)
Age (y)
24.2 ± 3.1
26.7 ± 6.2
Height (cm)
1.74 ± 0.03
1.75 ± 0.06
Weight (kg)
68.5 ± 9.7
68.7 ± 7.6
BMI (kg/m 2 )
22.5 ± 2.9
22.3 ± 1.9
FFM (kg)
54.2 (49.2 -56.4 )
57.5 (51.1 -58.8 )
Muscle mass (kg)
51.4 (46.6 -53.3 )
54.9 (48.4 -55.8 )
Body fat percentage (%)
21.9 ± 6.9
18.8 ± 6.0
Arms fat percentage (%)
18.7 ± 7.1
14.4 ± 5.1
Legs fat percentage (%)
20.5 ± 5.6
18.1 ± 5.2
Trunk fat percentage (%)
25.1 ± 8.3
21.6 ± 7.3
BMD (g/cm 2 )
1.16 ± 0.07
1.18 ± 0.07
25(OH) D (nmol/L)
26.9 ± 4.7
26.2 ± 4.1
iPTH (pg/mL)
54.9 ± 19.8
44.6 ± 23.8
MVPA (min/day)
69.8 ± 32.2
65.8 ± 16.3
Sun exposure (min/day)
40.0 ± 27.9
50.0 ± 30.7
Table 2
2Characteristics of the participants at baseline, midpoint and endpointVariable
Resistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P
Baseline
Midpoint
Endpoint
Baseline
Midpoint
Endpoint
Time Group Time × Group interaction
Weight (kg) a
68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0
68.8 ± 3.1
68.7 ± 3.1
68.6 ± 3.0
0.156 0.958 0.762
BMI (kg/m 2 ) a
22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8
22.3 ± 0.9
22.2 ± 0.9
22.2 ± 0.8
0.273 0.768 0.455
FFM (kg) a
52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7
56.1 ± 1.8
55.8 ± 1.8
0.347 0.341 0.004
Muscle mass (kg) a
49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6
53.2 ± 1.7
53.0 ± 1.7
0.399 0.348 0.003
Body fat percentage a
22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0
18.3 ± 2.0
18.0 ± 1.9
18.3 ± 2.0
0.643 0.327 0.058
Arms fat percentage a
19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7
13.7 ± 1.8
13.4 ± 1.8
13.6 ± 1.7
0.687 0.091 0.077
Legs fat percentage a
20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7
17.7 ± 1.7
17.2 ± 1.6
17.6 ± 1.7
0.846 0.378 0.064
Trunk fat percentage a
25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4
21.1 ± 2.5
20.8 ± 2.4
21.1 ± 2.4
0.484 0.397 0.075
BMD (g/cm 2 ) a
1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03
0.016 0.538 0.235
25(OH) D (nmol/L) b
26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8
37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025
iPTH (pg/mL) b
61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6
37.6 ± 8.3
48.3 ± 6.1
51.5 ± 5.6
0.110 0.131 0.242
Sun exposure (min/day) 40.0± 9.8
76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8
73.5 ± 13.5 50.5 ± 15.2
0.015 0.889 0.493
Data are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact
parathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end.
c
Change is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup.
Boldface indicates significance (P < 0.05)
Table 3
3Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free massVariables
25(OH)D mid-pre
25(OH)D end-pre
r
P
r
P
Body fat percentage
mid-pre
0.014
0.956
0.101
0.690
end-mid
−0.217
0.387
−0.004
0.986
end-pre
−0.090
0.722
0.064
0.800
FFM
mid-pre
−0.455
0.058
−0.565
0.015
end-mid
0.121
0.632
0.037
0.883
end-pre
−0.289
0.244
−0.424
0.080
Muscle mass
mid-pre
−0.456
0.057
−0.554
0.017
end-mid
0.142
0.575
0.037
0.884
end-pre
−0.280
0.261
−0.415
0.087
AcknowledgementsThe authors would like to thank all the participants who took part in the study.Authors' contributionsThe study was designed by ZBC and XKM; data were collected and analyzed by XKM and XS; data interpretation and manuscript preparation were undertaken by ZBC, XKM, XS, and LZ. All authors read and approved the final manuscript.FundingThis study was supported in part by a grant from the National Natural Science Foundation of China (Nos. 31571226 and 81703220).Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Ethics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). All subjects provided informed consent for inclusion before they participated in the study.Consent for publicationNot applicable.
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| [
"Background: Previous studies indicated that serum 25-hydroxyvitamin D [25(OH)D] concentrations are positively associated with physical activity levels independent of sun exposure. However, the effect of resistance training on serum 25(OH) D concentrations remains unclear. Thus, this study aimed to examine the effect of chronic resistance training on serum 25(OH) D concentrations and determine whether 25(OH) D concentration variations are influenced by body composition changes. Methods: Eighteen young men aged 19-39 years were randomly divided into a 12-week resistance training group (RT, n = 9) and non-exercise control group (CON, n = 9). The trial was undertaken in Shanghai University of Sport in Shanghai, China. Randomization and allocation to trial group were carried out by a central computer system. Serum 25(OH) D and intact parathyroid hormone concentrations were measured using commercially available enzymelinked immunosorbent assay kits. Body composition was measured by dual-energy X-ray absorptiometry.Results: The average serum 25(OH) D concentrations were 26.6 nmol/L at baseline. After the 12-week intervention program, serum 25(OH) D concentrations significantly increased in both groups. Serum 25(OH) D concentrations at midpoint (6-week) increased significantly only in the CON group (P < 0.01). From training midpoint to endpoint, a significantly greater increase in serum 25(OH) D concentrations was noted in the RT group (P-interaction = 0.043); 25(OH) D concentration changes (end-pre) were negatively related to fat-free mass (mid-pre) (r = − 0.565, P = 0.015) and muscle mass (mid-pre) (r = − 0.554, P = 0.017).Conclusions: There were no beneficial effects of the 12-week resistance training on serum 25(OH) D concentration in vitamin D deficient young men, and an indication that seasonal increase in serum 25(OH) D concentrations during the early phase of resistance training was transiently inhibited, which may partly be attributed to resistance training-induced muscle mass gain."
] | [
"Xiaomin Sun ",
"Xiao-Kai Ma ",
"Lin Zhang ",
"Zhen-Bo Cao "
] | [] | [
"Xiaomin",
"Xiao-Kai",
"Lin",
"Zhen-Bo"
] | [
"Sun",
"Ma",
"Zhang",
"Cao"
] | [
"M Wacker, ",
"M F Holick, ",
"S Christakos, ",
"M Hewison, ",
"D G Gardner, ",
"C L Wagner, ",
"I N Sergeev, ",
"E Rutten, ",
"X Sun, ",
"Z B Cao, ",
"Y Zhang, ",
"Y Ishimi, ",
"I Tabata, ",
"M Higuchi, ",
"X Sun, ",
"Z B Cao, ",
"K Tanisawa, ",
"T Ito, ",
"S Oshima, ",
"M Higuchi, ",
"R Scragg, ",
"I Holdaway, ",
"R Jackson, ",
"T Lim, ",
"D Scott, ",
"L Blizzard, ",
"J Fell, ",
"C Ding, ",
"T Winzenberg, ",
"G Jones, ",
"A Ardestani, ",
"B Parker, ",
"S Mathur, ",
"P Clarkson, ",
"L S Pescatello, ",
"H J Hoffman, ",
"A Burgaz, ",
"A Akesson, ",
"A Oster, ",
"K Michaelsson, ",
"A Wolk, ",
"M G Kimlin, ",
"R M Lucas, ",
"S L Harrison, ",
"I Van Der Mei, ",
"B K Armstrong, ",
"D C Whiteman, ",
"M A Kluczynski, ",
"M J Lamonte, ",
"J A Mares, ",
"J Wactawski-Wende, ",
"A W Smith, ",
"C D Engelman, ",
"M F Holick, ",
"L Maïmoun, ",
"D Simar, ",
"C Caillaud, ",
"E Peruchon, ",
"C Sultan, ",
"M Rossi, ",
"X Sun, ",
"Z B Cao, ",
"H Taniguchi, ",
"K Tanisawa, ",
"M Higuchi, ",
"X Sun, ",
"Z B Cao, ",
"K Tanisawa, ",
"H Taniguchi, ",
"T Kubo, ",
"M Higuchi, ",
"W L Westcott, ",
"M Abboud, ",
"D A Puglisi, ",
"B N Davies, ",
"M Rybchyn, ",
"N P Whitehead, ",
"K E Brock, ",
"R S Mason, ",
"M S Rybchyn, ",
"M Abboud, ",
"T C Brennan-Speranza, ",
"D R Fraser, ",
"Y Makanae, ",
"R Ogasawara, ",
"K Sato, ",
"Y Takamura, ",
"K Matsutani, ",
"K Kido, ",
"L H Willis, ",
"C A Slentz, ",
"L A Bateman, ",
"A T Shields, ",
"L W Piner, ",
"C W Bales, ",
"B Strasser, ",
"U Siebert, ",
"W Schobersberger, ",
"H Jarrett, ",
"L Fitzgerald, ",
"A C Routen, ",
"J E Sasaki, ",
"D John, ",
"P S Freedson, ",
"M F Holick, ",
"N C Binkley, ",
"H A Bischoff-Ferrari, ",
"C M Gordon, ",
"D A Hanley, ",
"R P Heaney, ",
"N H Bell, ",
"R N Godsen, ",
"D P Henry, ",
"J Shary, ",
"S Epstein, ",
"R Srikuea, ",
"X Zhang, ",
"O K Park-Sarge, ",
"K A Esser, ",
"T Kobayashi, ",
"T Okano, ",
"S Shida, ",
"K Okada, ",
"T Suginohara, ",
"H Nakao, "
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"Matsutani",
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"Gordon",
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"Bell",
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"Epstein",
"Srikuea",
"Zhang",
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"Esser",
"Kobayashi",
"Okano",
"Shida",
"Okada",
"Suginohara",
"Nakao"
] | [
"Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. M Wacker, M F Holick, Nutrients. 51Wacker M, Holick MF. Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation. Nutrients. 2013;5(1):111-48.",
"Vitamin D: beyond bone. S Christakos, M Hewison, D G Gardner, C L Wagner, I N Sergeev, E Rutten, Ann N Y Acad Sci. 1287Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al. Vitamin D: beyond bone. Ann N Y Acad Sci. 2013;1287:45-58.",
"Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. X Sun, Z B Cao, Y Zhang, Y Ishimi, I Tabata, M Higuchi, Nutrients. 61Sun X, Cao ZB, Zhang Y, Ishimi Y, Tabata I, Higuchi M. Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults. Nutrients. 2014;6(1):221-30.",
"The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. X Sun, Z B Cao, K Tanisawa, T Ito, S Oshima, M Higuchi, Nutrients. 71Sun X, Cao ZB, Tanisawa K, Ito T, Oshima S, Higuchi M. The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men. Nutrients. 2014;7(1):91-102.",
"Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. R Scragg, I Holdaway, R Jackson, T Lim, Ann Epidemiol. 25Scragg R, Holdaway I, Jackson R, Lim T. Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population. Ann Epidemiol. 1992;2(5):697-703.",
"A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. D Scott, L Blizzard, J Fell, C Ding, T Winzenberg, G Jones, Clin Endocrinol. 735Scott D, Blizzard L, Fell J, Ding C, Winzenberg T, Jones G. A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults. Clin Endocrinol. 2010;73(5): 581-7.",
"Relation of vitamin D level to maximal oxygen uptake in adults. A Ardestani, B Parker, S Mathur, P Clarkson, L S Pescatello, H J Hoffman, Am J Cardiol. 1078Ardestani A, Parker B, Mathur S, Clarkson P, Pescatello LS, Hoffman HJ, et al. Relation of vitamin D level to maximal oxygen uptake in adults. Am J Cardiol. 2011;107(8):1246-9.",
"Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. A Burgaz, A Akesson, A Oster, K Michaelsson, A Wolk, Am J Clin Nutr. 865Burgaz A, Akesson A, Oster A, Michaelsson K, Wolk A. Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. Am J Clin Nutr. 2007;86(5):1399-404.",
"The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. M G Kimlin, R M Lucas, S L Harrison, I Van Der Mei, B K Armstrong, D C Whiteman, Am J Epidemiol. 1797Kimlin MG, Lucas RM, Harrison SL, van der Mei I, Armstrong BK, Whiteman DC, et al. The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study. Am J Epidemiol. 2014;179(7):864-74.",
"Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women. M A Kluczynski, M J Lamonte, J A Mares, J Wactawski-Wende, A W Smith, C D Engelman, Ann Epidemiol. 216Kluczynski MA, Lamonte MJ, Mares JA, Wactawski-Wende J, Smith AW, Engelman CD, et al. Duration of physical activity and serum 25- hydroxyvitamin D status of postmenopausal women. Ann Epidemiol. 2011; 21(6):440-9.",
"Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. M F Holick, Clin Rev Bone Miner Metabol. 71Holick MF. Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D. Clin Rev Bone Miner Metabol. 2009;7(1):2-19.",
"Effect of antioxidants and exercise on bone metabolism. L Maïmoun, D Simar, C Caillaud, E Peruchon, C Sultan, M Rossi, J Sports Sci. 263Maïmoun L, Simar D, Caillaud C, Peruchon E, Sultan C, Rossi M, et al. Effect of antioxidants and exercise on bone metabolism. J Sports Sci. 2008;26(3): 251-8.",
"Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. X Sun, Z B Cao, H Taniguchi, K Tanisawa, M Higuchi, J Clin Endocrinol Metab. 10211Sun X, Cao ZB, Taniguchi H, Tanisawa K, Higuchi M. Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults. J Clin Endocrinol Metab. 2017;102(11):3937-44.",
"Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. X Sun, Z B Cao, K Tanisawa, H Taniguchi, T Kubo, M Higuchi, Endocrine. 592Sun X, Cao ZB, Tanisawa K, Taniguchi H, Kubo T, Higuchi M. Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men. Endocrine. 2018;59(2):330-7.",
"Resistance training is medicine: effects of strength training on health. W L Westcott, Curr Sports Med Rep. 114Westcott WL. Resistance training is medicine: effects of strength training on health. Curr Sports Med Rep. 2012;11(4):209-16.",
"Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells. M Abboud, D A Puglisi, B N Davies, M Rybchyn, N P Whitehead, K E Brock, Endocrinology. 1549Abboud M, Puglisi DA, Davies BN, Rybchyn M, Whitehead NP, Brock KE, et al. Evidence for a specific uptake and retention mechanism for 25- hydroxyvitamin D (25OHD) in skeletal muscle cells. Endocrinology. 2013; 154(9):3022-30.",
"The role of skeletal muscle in maintaining vitamin D status in Winter. R S Mason, M S Rybchyn, M Abboud, T C Brennan-Speranza, D R Fraser, Curr Dev Nutr. 31087Mason RS, Rybchyn MS, Abboud M, Brennan-Speranza TC, Fraser DR. The role of skeletal muscle in maintaining vitamin D status in Winter. Curr Dev Nutr. 2019;3(10):nzz087.",
"Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Y Makanae, R Ogasawara, K Sato, Y Takamura, K Matsutani, K Kido, Exp Physiol. 10010Makanae Y, Ogasawara R, Sato K, Takamura Y, Matsutani K, Kido K, et al. Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle. Exp Physiol. 2015;100(10):1168-76.",
"Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. L H Willis, C A Slentz, L A Bateman, A T Shields, L W Piner, C W Bales, J Appl Physiol. 113121831Willis LH, Slentz CA, Bateman LA, Shields AT, Piner LW, Bales CW, et al. Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults. J Appl Physiol. 2012;113(12):1831.",
"Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. B Strasser, U Siebert, W Schobersberger, Sports Med. 405Strasser B, Siebert U, Schobersberger W. Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism. Sports Med. 2010;40(5):397-415.",
"Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. H Jarrett, L Fitzgerald, A C Routen, J Phys Act Health. 123Jarrett H, Fitzgerald L, Routen AC. Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults. J Phys Act Health. 2014;12(3):382-7.",
"Validation and comparison of ActiGraph activity monitors. J E Sasaki, D John, P S Freedson, J Sci Med Sport. 145Sasaki JE, John D, Freedson PS. Validation and comparison of ActiGraph activity monitors. J Sci Med Sport. 2011;14(5):411-6.",
"Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. M F Holick, N C Binkley, H A Bischoff-Ferrari, C M Gordon, D A Hanley, R P Heaney, J Clin Endocrinol Metab. 967Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96(7):1911-30.",
"The effects of musclebuilding exercise on vitamin D and mineral metabolism. N H Bell, R N Godsen, D P Henry, J Shary, S Epstein, J Bone Miner Res. 34Bell NH, Godsen RN, Henry DP, Shary J, Epstein S. The effects of muscle- building exercise on vitamin D and mineral metabolism. J Bone Miner Res. 1988;3(4):369-73.",
"VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. R Srikuea, X Zhang, O K Park-Sarge, K A Esser, Am J Physiol Cell Physiol. 3034Srikuea R, Zhang X, Park-Sarge OK, Esser KA. VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. Am J Physiol Cell Physiol. 2012;303(4): C396-405.",
"Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. T Kobayashi, T Okano, S Shida, K Okada, T Suginohara, H Nakao, J Nutr Sci Vitaminol (Tokyo). 293Kobayashi T, Okano T, Shida S, Okada K, Suginohara T, Nakao H, et al. Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects. J Nutr Sci Vitaminol (Tokyo). 1983;29(3):271-81.",
"Publisher's Note. Publisher's Note",
"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."
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"Vitamin D -effects on skeletal and extraskeletal health and the need for supplementation",
"Vitamin D: beyond bone",
"Association between serum 25-hydroxyvitamin D and inflammatory cytokines in healthy adults",
"The relationship between serum 25-hydroxyvitamin D concentration, cardiorespiratory fitness, and insulin resistance in Japanese men",
"Plasma 25-hydroxyvitamin D3 and its relation to physical activity and other heart disease risk factors in the general population",
"A prospective study of the associations between 25-hydroxy-vitamin D, sarcopenia progression and physical activity in older adults",
"Relation of vitamin D level to maximal oxygen uptake in adults",
"Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter",
"The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD study",
"Duration of physical activity and serum 25-hydroxyvitamin D status of postmenopausal women",
"Vitamin D and Health: Evolution, Biologic Functions, and Recommended Dietary Intakes for Vitamin D",
"Effect of antioxidants and exercise on bone metabolism",
"Effect of an acute bout of endurance exercise on serum 25(OH) D concentrations in young adults",
"Effects of chronic endurance exercise training on serum 25(OH) D concentrations in elderly Japanese men",
"Resistance training is medicine: effects of strength training on health",
"Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells",
"The role of skeletal muscle in maintaining vitamin D status in Winter",
"Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle",
"Effects of aerobic and/or resistance training on body mass and fat mass in overweight or obese adults",
"Resistance training in the treatment of the metabolic syndrome: a systematic review and meta-analysis of the effect of resistance training on metabolic clustering in patients with abnormal glucose metabolism",
"Inter-instrument reliability of the Actigraph GT3X+ ambulatory activity monitor during free-living conditions in adults",
"Validation and comparison of ActiGraph activity monitors",
"Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline",
"The effects of musclebuilding exercise on vitamin D and mineral metabolism",
"VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation",
"Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects"
] | [
"Nutrients",
"Ann N Y Acad Sci",
"Nutrients",
"Nutrients",
"Ann Epidemiol",
"Clin Endocrinol",
"Am J Cardiol",
"Am J Clin Nutr",
"Am J Epidemiol",
"Ann Epidemiol",
"Clin Rev Bone Miner Metabol",
"J Sports Sci",
"J Clin Endocrinol Metab",
"Endocrine",
"Curr Sports Med Rep",
"Endocrinology",
"Curr Dev Nutr",
"Exp Physiol",
"J Appl Physiol",
"Sports Med",
"J Phys Act Health",
"J Sci Med Sport",
"J Clin Endocrinol Metab",
"J Bone Miner Res",
"Am J Physiol Cell Physiol",
"J Nutr Sci Vitaminol (Tokyo)",
"Publisher's Note",
"Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations"
] | [
"\nFig. 1\n1Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05",
"\n\n25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum",
"\nTable 1\n1Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activityVariable \nResistance training group (RT) (n = 9) \nNon-exercise control group (CON) (n = 9) \n\nAge (y) \n24.2 ± 3.1 \n26.7 ± 6.2 \n\nHeight (cm) \n1.74 ± 0.03 \n1.75 ± 0.06 \n\nWeight (kg) \n68.5 ± 9.7 \n68.7 ± 7.6 \n\nBMI (kg/m 2 ) \n22.5 ± 2.9 \n22.3 ± 1.9 \n\nFFM (kg) \n54.2 (49.2 -56.4 ) \n57.5 (51.1 -58.8 ) \n\nMuscle mass (kg) \n51.4 (46.6 -53.3 ) \n54.9 (48.4 -55.8 ) \n\nBody fat percentage (%) \n21.9 ± 6.9 \n18.8 ± 6.0 \n\nArms fat percentage (%) \n18.7 ± 7.1 \n14.4 ± 5.1 \n\nLegs fat percentage (%) \n20.5 ± 5.6 \n18.1 ± 5.2 \n\nTrunk fat percentage (%) \n25.1 ± 8.3 \n21.6 ± 7.3 \n\nBMD (g/cm 2 ) \n1.16 ± 0.07 \n1.18 ± 0.07 \n\n25(OH) D (nmol/L) \n26.9 ± 4.7 \n26.2 ± 4.1 \n\niPTH (pg/mL) \n54.9 ± 19.8 \n44.6 ± 23.8 \n\nMVPA (min/day) \n69.8 ± 32.2 \n65.8 ± 16.3 \n\nSun exposure (min/day) \n40.0 ± 27.9 \n50.0 ± 30.7 \n\n",
"\nTable 2\n2Characteristics of the participants at baseline, midpoint and endpointVariable \nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \n\nBaseline \nMidpoint \nEndpoint \nBaseline \nMidpoint \nEndpoint \nTime Group Time × Group interaction \n\nWeight (kg) a \n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \n68.8 ± 3.1 \n68.7 ± 3.1 \n68.6 ± 3.0 \n0.156 0.958 0.762 \n\nBMI (kg/m 2 ) a \n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \n22.3 ± 0.9 \n22.2 ± 0.9 \n22.2 ± 0.8 \n0.273 0.768 0.455 \n\nFFM (kg) a \n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \n56.1 ± 1.8 \n55.8 ± 1.8 \n0.347 0.341 0.004 \n\nMuscle mass (kg) a \n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \n53.2 ± 1.7 \n53.0 ± 1.7 \n0.399 0.348 0.003 \n\nBody fat percentage a \n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \n18.3 ± 2.0 \n18.0 ± 1.9 \n18.3 ± 2.0 \n0.643 0.327 0.058 \n\nArms fat percentage a \n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \n13.7 ± 1.8 \n13.4 ± 1.8 \n13.6 ± 1.7 \n0.687 0.091 0.077 \n\nLegs fat percentage a \n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \n17.7 ± 1.7 \n17.2 ± 1.6 \n17.6 ± 1.7 \n0.846 0.378 0.064 \n\nTrunk fat percentage a \n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \n21.1 ± 2.5 \n20.8 ± 2.4 \n21.1 ± 2.4 \n0.484 0.397 0.075 \n\nBMD (g/cm 2 ) a \n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \n0.016 0.538 0.235 \n\n25(OH) D (nmol/L) b \n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \n\niPTH (pg/mL) b \n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \n37.6 ± 8.3 \n48.3 ± 6.1 \n51.5 ± 5.6 \n0.110 0.131 0.242 \n\nSun exposure (min/day) 40.0± 9.8 \n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \n73.5 ± 13.5 50.5 ± 15.2 \n0.015 0.889 0.493 \n\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \n\nc \n\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \nBoldface indicates significance (P < 0.05) \n",
"\nTable 3\n3Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free massVariables \n25(OH)D mid-pre \n25(OH)D end-pre \n\nr \nP \nr \nP \n\nBody fat percentage \n\nmid-pre \n0.014 \n0.956 \n0.101 \n0.690 \n\nend-mid \n−0.217 \n0.387 \n−0.004 \n0.986 \n\nend-pre \n−0.090 \n0.722 \n0.064 \n0.800 \n\nFFM \n\nmid-pre \n−0.455 \n0.058 \n−0.565 \n0.015 \n\nend-mid \n0.121 \n0.632 \n0.037 \n0.883 \n\nend-pre \n−0.289 \n0.244 \n−0.424 \n0.080 \n\nMuscle mass \n\nmid-pre \n−0.456 \n0.057 \n−0.554 \n0.017 \n\nend-mid \n0.142 \n0.575 \n0.037 \n0.884 \n\nend-pre \n−0.280 \n0.261 \n−0.415 \n0.087 \n\n"
] | [
"Effects of resistance training on serum 25(OH) D concentrations. The analysis was adjusted according to age, MVPA, and changes in sun exposure and body fat percentage (End-Pre). 25(OH) D, 25-hydroxyvitamin D; RT, resistance training; CON, non-exercise control; MVPA, moderate to vigorous physical activity. *, compared with pre, P < 0.05; †, compared with mid, P < 0.05",
"25(OH)D: 25-hydroxyvitamin D; BMD: Bone mineral density; BMI: Body mass index; FFM: Fat-free mass; iPTH: parathyroid hormone; MVPA: Moderate and vigorous physical activity; RM: Repetition maximum",
"Participant characteristics according to groups at baselineValues are presented as mean ± standard deviation, median (interquartile range), or percentage Student t test (for normally distributed variables) or the Mann-Witney U test (for nonnormally distributed variables) was used to evaluate the differences. Significant difference, P < 0.05 BMI body mass index, FFM fat free mass, 25(OH) D 25-hydroxyvitamin D, iPTH intact parathyroid hormone, MVPA moderate-vigorous physical activity",
"Characteristics of the participants at baseline, midpoint and endpoint",
"Correlations between changes in indicators of body composition and changes in serum 25(OH) D concentrations 25(OH) D 25-hydroxyvitamin D, FFM fat-free mass"
] | [
"Fig. 1",
"(Fig. 2)"
] | [] | [
"Circulating 25-hydroxyvitamin D [25(OH)D] is the major form of vitamin D, which is used in vitamin D status assessment. Accumulated studies indicated that vitamin D not only regulates calcium and phosphorus metabolism but also plays an important role in modifying cardiovascular diseases risk, and improving immune function and physical fitness [1][2][3][4].",
"Previous studies indicated that higher levels of physical activity or cardiorespiratory fitness were related to higher circulating 25(OH) D concentrations [5][6][7]. Although individuals who were physically active outdoors are more likely to have higher circulating 25(OH) D due to increased sun exposure [8,9], this relationship was still observed after adjustment for sunlight exposure [6,10]. Scragg et al. [5] found that higher levels of physical activity were associated with higher serum 25(OH) D concentrations not only during summer but also during winter, when the vitamin D synthesis from sun exposure is extremely limited [11]. Consistently, several intervention studies suggested that endurance exercise could increase circulating 25(OH) D or prevent its seasonal reduction [12][13][14]. These findings indicated that physical activity could directly increase 25(OH) D concentrations.",
"Resistance training (RT) promotes health benefits through increased skeletal muscle mass and qualitative adaptations [15]. In addition to adiposity tissue, muscle tissue was suggested to serve as an another important site to store 25(OH) D, and then it returns to the circulating when needed [16,17]. Nevertheless, Makanae and colleagues reported that intramuscular expression of CYP27B1, which catalyses the hydroxylation and activation of 25(OH) D, was significantly increased at 1 h and 3 h after resistance exercise, which may enhance local vitamin D metabolism in skeletal muscle [18]. It is well known that compared with endurance exercise, RT could not only reduce fat mass, but also it is more effective in increasing muscle mass and strength [19,20], which may largely affect vitamin D metabolism. However, whether serum 25(OH) D concentrations are affected by RT remains unclear.",
"This study aimed to evaluate the effect of chronic resistance training on serum 25(OH) D concentrations and to determine whether the effects are attributable to changes in body composition in healthy young men.",
"Eighteen healthy men with no chronic diseases (aged 19 to 39 years) were included in this study, which was conducted from March to July 2016. Participants using vitamin D supplements or sunscreen regularly were excluded. Assessments were performed at 0 week (pre), 6 weeks (midpoint), and 12 weeks (endpoint). All subjects provided informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shanghai University of Sport (2016001). The trial has been retrospectively registered at Chinese Clinical Trial Registry website (ChiCTR2000030876).",
"Eighteen participants were randomly allocated to the following two groups by using a computer-generated random number sequence: resistance training group (RT) and no-exercise control group (CON). Participants in the RT group were instructed to exercise 2-3 times a week in a gymnasium. A supervised training session was performed in the afternoon between 1630 h and 2000 h during the 12 weeks of progressive resistance training. In the first 2 weeks, the participants were taught how to properly perform each movement. The resistance workload gradually changed from light to heavy, and the participants were instructed to complete 10 repetitions for each set of the following exercises: leg press, preacher curl, chest press, leg curl, and iso row, with 60-120-s rest period between two sets; additionally, they also performed three sets of 15-25 repetitions of abdominal crunch and back extension with their own body weight, with a 60-120-s rest period between two sets. At the first training session of the third week, the participants were instructed to perform strength testing, and 12 repetitions maximum (12RM) for each exercise were performed, except back extension and abdominal crunch. The workload for the next resistance training was calculated from the participants' previously determined 12RM. The participants were encouraged to perform back extension and abdominal crunch exercises 30 times from the first training session of the third week. When the strength training equipment at our gymnasium had been upgraded at the eighth week of training, the resistance training program was revised to include the following: leg extension, standing one-arm cable curl, standing cable chest press, leverage shoulder press, leverage high row, back extension, and abdominal crunch. These new components of the resistance training program were selected based on the principle of using similar muscle groups. The order of resistance exercise was randomized; however, training of the same muscle groups continuously was avoided. The participants were instructed not to undertake any formal exercise or change their levels of general physical activity and dietary habits.",
"Height was measured with the participants wearing light clothing and being barefoot (HK6000-ST, HENGKANG JIAYE Technology Co., Ltd. CHN). Dual-energy X-ray absorptiometry (DXA Prodigy, GE Lunar Corp., Madison, WI, USA) was used for the measurement of whole and regional body composition, including total body mass, muscle mass, fat mass, fat percentage, and bone mineral density. Body mass index was calculated as body mass (kg) divided by height (m 2 ). Fat-free mass (FFM) was calculated as body mass minus fat mass.",
"Physical activity was measured using Actigraph GT3X+ accelerometers (ActiGraph, LLC, Pensacola, FL, USA). Participants were instructed to wear the accelerometers on their right hip at all times, except during water activities (swimming, showering) or sleeping, for 7 consecutive days at baseline. A wear time of ≥480 min/day was used as the criterion for a valid day, and ≥ 2 weekdays and 1 weekend day was used as the criteria for a valid 7-day period of accumulated data. Non-wear time was defined as consecutive periods of ≥60 min of zero accelerometer counts and excluded from the analyses. Data were recorded in 60-s epochs. A pragmatic cutoff of ≥2690 cpm was used to categorize moderatevigorous physical activity time [21,22]. The 7-day data collection for all subjects was fixed within 2 weeks. Outdoor time from 0900 h to 1600 h for 7 consecutive days was recorded and used as an alternative for sun exposure in the analysis.",
"Blood samples were obtained after at least an 8-h overnight fasting period and subsequently centrifuged at 3000 rpm at 4°C for 10 min. Serum was collected and stored at − 80°C until analysis. Serum 25(OH) D and intact parathyroid hormone (iPTH) concentrations were measured in duplicate using commercially available enzyme-linked immunosorbent assay kits (25(OH) D, IDS, Bolton, UK; iPTH, DRG, Marburg, Germany) according to the manufacturer's instructions. The intraassay and inter-assay coefficients of variation were 9.1 and 7.7%, respectively, for serum 25(OH) D and 9.6 and 3.6%, respectively, for iPTH. Vitamin D deficiency is defined as 25(OH) D concentration < 50 nmol/L, and vitamin D insufficiency as 25(OH) D concentration < 75 nmol/L [23].",
"All statistical analyses were performed using IBM SPSS 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were assessed for normality using a Kolmogorov-Smirnov test prior to all statistical analyses. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables were presented as median (interquartile range), unless otherwise indicated. The Student t test (for normally distributed variables) or the Mann-Whitney U test (for non-normally distributed variables) was used to evaluate the differences between groups. Two-factor repeat-measured analysis of variance (ANOVA) (group × time) was used to determine the effect of exercise on 25(OH) D, iPTH, and body composition indicators. A post hoc test with Bonferroni correction was performed to identify statistically significant differences among the mean values when a considerable interaction was identified. Pearson correlation and partial correlation coefficients were computed between 25(OH) D concentration changes and body composition indicators. The level of statistical significance was set at P < 0.05.",
"The participants' characteristics and blood parameters at baseline are presented in Table 1. The average serum 25(OH) D concentrations were 26.6 nmol/L, and all participants showed vitamin D deficiency. No significant difference in either body composition indicators or blood parameters was found between the groups (P > 0.05).",
"As shown in Table 2, significant interactions of group × time were found for FFM (P = 0.004) and muscle mass (P = 0.003). After resistance training, the FFM values (mid-pre, 2.7%, P = 0.003; end-pre, 3.0%, P = 0.004) and muscle mass (mid-pre, 3.0%, P = 0.002; end-pre, 3.4%, P = 0.004) significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in the CON group. In addition, we observed borderline significant interactions in body fat percentage, arm fat percentage, leg fat percentage, and trunk fat percentage ( Table 2).",
"As shown in Table 2 and Fig. 1, a significant group × time interaction was observed for serum 25(OH) D concentrations (P = 0.025). After the 12-week intervention, the mean serum 25(OH) D concentrations significantly increased to 36.7 ± 2.7 nmol/L in the RT group and 40.7 ± 2.7 nmol/L in the CON group. Moreover, in the CON group, higher serum 25(OH) D concentrations were observed at midpoint compared with the values at baseline (P < 0.01), whereas no notable differences were found in the RT group. From midpoint to endpoint, although serum 25(OH) D concentrations significantly increased in both groups, the increase was significantly greater in the RT group (25.8%) than in the CON group (9.4%) (P-interaction =0.043).",
"Correlation analyses were performed to identify the factors associated with the changes in 25(OH) D concentrations (Table 3). 25(OH) D concentration changes (end-pre) were negatively related to FFM (mid-pre) (P = 0.015) and muscle mass (mid-pre) (P = 0.017) changes (Fig. 2); a borderline association was observed between 25(OH) D concentration changes (mid-pre) and FFM (mid-pre) (P = 0.058) and muscle mass (mid-pre) (P = 0.057) changes. In addition, significant correlations of 25(OH) D concentration changes (end-pre) with FFM (mid-pre) (P = 0.039) and muscle mass (mid-pre) (P = 0.043) changes were observed after adjustment for group, age, and changes in sun exposure (end-pre), body weight (end-pre), and body fat percentage (end-pre).",
"In this study, we found that the seasonal increase in serum 25(OH) D concentrations in young men was transiently inhibited during the early phase of RT; subsequently, serum 25(OH) D concentrations increased in both RT and CON groups, and the increase was observed to be greater in RT group than in CON group. The transient inhibition could be partly attributed to muscle mass and fat free mass gain induced by RT in the early phase.",
"Previous studies have reported significant positive associations of serum 25(OH) D concentrations with physical activity levels independent of sun exposure [6,10]. Bell et al. [24] found that 25(OH) D concentrations were higher in participants who had engaged in regular muscle-building exercise for at least 1 year compared to those in the control group. Consistently, our recent studies also observed that exercise training could increase serum 25(OH) D concentrations or prevent its seasonal reduction in young and old men [13,14]. However, the effect of RT on serum 25(OH) D concentrations remains unclear. Compared with endurance training, RT is more effective in increasing muscle mass and strength [24], which was also supported by our results. In the present study, we observed that after resistance training, body mass was reduced, while FFM and muscle mass were significantly increased at the midpoint and endpoint compared to the baseline values, whereas no significant changes were observed in CON group.",
"In addition to adiposity tissues, muscle tissues are also served as another important storage for 25(OH) D, which could enable optimal vitamin D status to maintain its physiological function during winter months [17]. Makanae and colleagues observed that intramuscular CYP27B1 was significantly increased after resistance exercise in skeletal muscle, which may enhance local vitamin D metabolism [18]. Srikuea et al. [25] also found that the CYP27B1 expression in the muscle increased significantly during skeletal muscle regeneration from injury. Thus, we speculated that the transient inhibition in serum 25(OH) D levels in RT group during the early phase of resistance training could be partly due to absorption of serum 25(OH) D in the increasing muscle mass and its use in muscle activity. 25(OH) D consumption in muscle may induce a compensatory increase in serum 25(OH) D concentrations; however, the concentrations were still lower than that in control group, which may due to the lack of vitamin D storage and short-term intervention period. Previous studies report that serum 25(OH) D concentrations gradually increase from around March to August [26]. This could partly explain the reason why serum 25(OH) D concentration was transiently inhibited in early Spring and followed by a marked increase in early Summer in our study. Future studies that aim at exploring the mechanism underlying the effect of resistance training on serum 25(OH) D concentrations are warranted. This study has several limitations. First, our study included only men with relatively lower serum 25(OH) D concentrations. Therefore, it remains to be established whether these findings are applicable to other subjects with higher 25(OH) D concentrations. Second, considering the seasonal variation of serum 25(OH) D concentration, the findings in our study have seasonal limitations. Third, at baseline resistance training group had relatively lower values of muscle mass and body fat percentage, although they are not statistically significant, which should be interpreted with caution. Fourth, we couldn't observe any relationship between muscle mass and 25(OH) D at three points, which may due to the small sample size. However, the muscle mass changes were significantly related with 25(OH) D concentration changes. Finally, although our sample size was relatively small, a post hoc power calculation would still give us a 90% chance, with an effect size of 0.58, to demonstrate the interaction effect of serum 25(OH) D concentrations, which was considered statistically significant at P < 0.05 at the endpoints.",
"In summary, serum 25(OH) D concentrations in vitamin D deficient young men was observed to be transiently inhibited during the early phase of resistance training and subsequently increased in the later phase. This finding could be partly attributed to the muscle mass and fat free mass gain induced by resistance training during the early phase. Our findings indicated that adequate administration of oral supplements or increased daily dietary vitamin D intake are encouraged to increase serum 25(OH) D concentrations, especially for those who exercise regularly. "
] | [] | [
"Introduction",
"Methods",
"Participants",
"Experimental protocol",
"Anthropometric measurements",
"Physical activity and sun exposure",
"Blood analysis",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Fig. 1",
"Table 1",
"Table 2",
"Table 3"
] | [
"Variable \nResistance training group (RT) (n = 9) \nNon-exercise control group (CON) (n = 9) \n\nAge (y) \n24.2 ± 3.1 \n26.7 ± 6.2 \n\nHeight (cm) \n1.74 ± 0.03 \n1.75 ± 0.06 \n\nWeight (kg) \n68.5 ± 9.7 \n68.7 ± 7.6 \n\nBMI (kg/m 2 ) \n22.5 ± 2.9 \n22.3 ± 1.9 \n\nFFM (kg) \n54.2 (49.2 -56.4 ) \n57.5 (51.1 -58.8 ) \n\nMuscle mass (kg) \n51.4 (46.6 -53.3 ) \n54.9 (48.4 -55.8 ) \n\nBody fat percentage (%) \n21.9 ± 6.9 \n18.8 ± 6.0 \n\nArms fat percentage (%) \n18.7 ± 7.1 \n14.4 ± 5.1 \n\nLegs fat percentage (%) \n20.5 ± 5.6 \n18.1 ± 5.2 \n\nTrunk fat percentage (%) \n25.1 ± 8.3 \n21.6 ± 7.3 \n\nBMD (g/cm 2 ) \n1.16 ± 0.07 \n1.18 ± 0.07 \n\n25(OH) D (nmol/L) \n26.9 ± 4.7 \n26.2 ± 4.1 \n\niPTH (pg/mL) \n54.9 ± 19.8 \n44.6 ± 23.8 \n\nMVPA (min/day) \n69.8 ± 32.2 \n65.8 ± 16.3 \n\nSun exposure (min/day) \n40.0 ± 27.9 \n50.0 ± 30.7 \n\n",
"Variable \nResistance training (RT) group (n = 9) Non-exercise control (CON) group (n = 9) P \n\nBaseline \nMidpoint \nEndpoint \nBaseline \nMidpoint \nEndpoint \nTime Group Time × Group interaction \n\nWeight (kg) a \n68.4 ± 3.1 68.6 ± 3.1 68.5 ± 3.0 \n68.8 ± 3.1 \n68.7 ± 3.1 \n68.6 ± 3.0 \n0.156 0.958 0.762 \n\nBMI (kg/m 2 ) a \n22.6 ± 0.9 22.6 ± 0.9 22.6 ± 0.8 \n22.3 ± 0.9 \n22.2 ± 0.9 \n22.2 ± 0.8 \n0.273 0.768 0.455 \n\nFFM (kg) a \n52.5 ± 1.7 53.9 ± 1.8 c 54.1 ± 1.8 c 55.9 ± 1.7 \n56.1 ± 1.8 \n55.8 ± 1.8 \n0.347 0.341 0.004 \n\nMuscle mass (kg) a \n49.7 ± 1.6 51.2 ± 1.7 c 51.4 ± 1.7 c 53.1 ± 1.6 \n53.2 ± 1.7 \n53.0 ± 1.7 \n0.399 0.348 0.003 \n\nBody fat percentage a \n22.4 ± 2.0 20.6 ± 1.9 20.1 ± 2.0 \n18.3 ± 2.0 \n18.0 ± 1.9 \n18.3 ± 2.0 \n0.643 0.327 0.058 \n\nArms fat percentage a \n19.3 ± 1.8 17.9 ± 1.8 17.2 ± 1.7 \n13.7 ± 1.8 \n13.4 ± 1.8 \n13.6 ± 1.7 \n0.687 0.091 0.077 \n\nLegs fat percentage a \n20.8 ± 1.7 19.3 ± 1.6 19.2 ± 1.7 \n17.7 ± 1.7 \n17.2 ± 1.6 \n17.6 ± 1.7 \n0.846 0.378 0.064 \n\nTrunk fat percentage a \n25.7 ± 2.5 23.6 ± 2.4 22.8 ± 2.4 \n21.1 ± 2.5 \n20.8 ± 2.4 \n21.1 ± 2.4 \n0.484 0.397 0.075 \n\nBMD (g/cm 2 ) a \n1.16 ± 0.02 1.16 ± 0.02 1.17 ± 0.03 1.18 ± 0.02 1.18 ± 0.02 1.20 ± 0.03 \n0.016 0.538 0.235 \n\n25(OH) D (nmol/L) b \n26.0 ± 1.8 29.2 ± 2.7 36.7 ± 2.7 cd 27.2 ± 1.8 \n37.2 ± 2.7 c 40.7 ± 2.7 cd 0.807 0.261 0.025 \n\niPTH (pg/mL) b \n61.9 ± 8.3 58.7 ± 6.1 63.4 ± 5.6 \n37.6 ± 8.3 \n48.3 ± 6.1 \n51.5 ± 5.6 \n0.110 0.131 0.242 \n\nSun exposure (min/day) 40.0± 9.8 \n76.5 ± 13.5 63.8 ± 15.2 50.0 ± 9.8 \n73.5 ± 13.5 50.5 ± 15.2 \n0.015 0.889 0.493 \n\nData are presented as the adjusted mean ± SE. BMI body mass index, FFM fat free mass, BMD bone mineral density, 25(OH) D 25-hydroxyvitamin D, iPTH intact \nparathyroid hormone. a Adjusted by age, MVPA. b Adjusted by age, MVPA, baseline test time, changes of sun exposure and body fat percent from pre to end. \n\nc \n\nChange is significantly different from the baseline value within a subgroup. d Change is significantly different from the endpoint to midpoint within a subgroup. \nBoldface indicates significance (P < 0.05) \n",
"Variables \n25(OH)D mid-pre \n25(OH)D end-pre \n\nr \nP \nr \nP \n\nBody fat percentage \n\nmid-pre \n0.014 \n0.956 \n0.101 \n0.690 \n\nend-mid \n−0.217 \n0.387 \n−0.004 \n0.986 \n\nend-pre \n−0.090 \n0.722 \n0.064 \n0.800 \n\nFFM \n\nmid-pre \n−0.455 \n0.058 \n−0.565 \n0.015 \n\nend-mid \n0.121 \n0.632 \n0.037 \n0.883 \n\nend-pre \n−0.289 \n0.244 \n−0.424 \n0.080 \n\nMuscle mass \n\nmid-pre \n−0.456 \n0.057 \n−0.554 \n0.017 \n\nend-mid \n0.142 \n0.575 \n0.037 \n0.884 \n\nend-pre \n−0.280 \n0.261 \n−0.415 \n0.087 \n\n"
] | [
"Table 1",
"Table 2",
"Table 2)",
"Table 2",
"(Table 3"
] | [
"Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial",
"Effects of resistance training on serum 25(OH) D concentrations in young men: a randomized controlled trial"
] | [] |
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