ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.     

PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

Transforming Growth Factor-��-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-β-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.     

Stem cell antigen-1 localizes to lipid microdomains and associates with insulin degrading enzyme in skeletal myoblasts

Stem cell antigen-1 (Sca-1, Ly6A/E) is a glycosylphosphotidylinositol-anchored protein that identifies many tissue progenitor cells. We originally identified Sca-1 as a marker of myogenic precursor cells and subsequently demonstrated that Sca-1 regulates proliferation of activated myoblasts, suggesting an important role for Sca-1 in skeletal muscle homeostasis. Beyond its functional role in regulating proliferation, however, little is known about the mechanism(s) that drive Sca-1-mediated events. We now report that lipid microdomain organization is essential for normal myogenic differentiation, and that Sca-1 constitutively localizes to these domains during myoblast proliferation and differentiation. We also demonstrate that Sca-1 associates with insulin degrading enzyme (IDE), a catalytic protein responsible for the cleavage of mitogenic peptides, in differentiating myoblasts. We show that chemical inhibition of IDE as well as RNAi knockdown of IDE mRNA recapitulates the phenotype of Sca-1 interference, that is, sustained myoblast proliferation and delayed myogenic differentiation. These findings identify the first signaling protein that physically and functionally associates with Sca-1 in myogenic precursor cells, and suggest a potential pathway for Sca-1-mediated signaling. Future efforts to manipulate this pathway may lead to new strategies for augmenting the myogenic proliferative response, and ultimately muscle repair.     

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: when primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo+ cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo + myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo- cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

Involvement of p38 MAPK-mediated signaling in the calpeptin-mediated suppression of myogenic differentiation and fusion in C2C12 cells

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of μ-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways

p38 mitogen-activated protein kinase (MAPK) is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.     

Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding

The receptor for advanced glycation end products (RAGE), a multiligand receptor of the immunoglobulin superfamily, has been implicated in the inflammatory response, diabetic angiopathy and neuropathy, neurodegeneration, cell migration, tumor growth, neuroprotection, and neuronal differentiation. We show here that (i) RAGE is expressed in skeletal muscle tissue and its expression is developmentally regulated and (ii) RAGE engagement by amphoterin (HMGB1), a RAGE ligand, in rat L6 myoblasts results in stimulation of myogenic differentiation via activation of p38 mitogen-activated protein kinase (MAPK), up-regulation of myogenin and myosin heavy chain expression, and induction of muscle creatine kinase. No such effects were detected in myoblasts transfected with a RAGE mutant lacking the transducing domain or myoblasts transfected with a constitutively inactive form of the p38 MAPK upstream kinase, MAPK kinase 6, Cdc42, or Rac-1. Moreover, amphoterin counteracted the antimyogenic activity of the Ca(2+)-modulated protein S100B, which was reported to inhibit myogenic differentiation via inactivation of p38 MAPK, and basic fibroblast growth factor (bFGF), a known inhibitor of myogenic differentiation, in a manner that was inversely related to the S100B or bFGF concentration and directly related to the extent of RAGE expression. These data suggest that RAGE and amphoterin might play an important role in myogenesis, accelerating myogenic differentiation via Cdc42-Rac-1-MAPK kinase 6-p38 MAPK.     

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.     

干扰Sema7A抑制C2C12成肌细胞的增殖和分化

为研究脑信号蛋白家族（Semaphorins）成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA（small interfering RNA,siRNA）,用此siRNA转染C2C12成肌细胞．通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,real-time qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高．免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低．Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降．进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调．以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.     

Androgen receptor enhances myogenin expression and accelerates differentiation

Animal and clinical studies indicated that the androgen-AR signaling pathway is required for appropriate development of sexually dimorphic skeletal muscles and increases lean muscle mass, muscle strength, and muscle protein synthesis. However, the detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development need further study at the molecular level. C2C12 myoblast cells stably transfected with the Flag-tagged AR were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. The results indicate that the androgen-AR signaling pathway may suppress skeletal myoblast cell growth and accelerate myoblast cell differentiation via enhanced myogenin expression. This is a first report showing the role of androgen-AR signaling pathway in regulation of myoblast cell growth and myogenic regulatory factors.     

Rac1 inhibits myogenic differentiation by preventing the complete withdrawal of myoblasts from the cell cycle

The small GTPase protein Rac1 is involved in a wide range of biological processes, yet its role in cell differentiation is mostly unknown. Here we show that Rac1 activity is high in proliferating myoblasts and decreases during the differentiation process. To analyze the involvement of Rac1 in muscle differentiation, different forms of the protein were expressed in muscle cells. A constitutively activated form of Rac1 (Rac1Q61L) inhibited the activity of MyoD in promoting muscle differentiation, whereas a dominant negative form of Rac1 (Rac1T17N) induced the activity of MyoD in promoting muscle differentiation. Expression of Rac1T17N imposed myogenic differentiation on myoblasts growing under mitogenic conditions. In inquiring whether Rac1 affected the withdrawal of myoblasts from the cell cycle, we analyzed the expression of cyclin D1 and p21(WAF1) and the phosphorylation state of the retinoblastoma protein. According to these markers and bromodeoxyuridine incorporation, C2 myoblasts expressing Rac1T17N exited the cell cycle earlier than control C2 cells. Myoblasts expressing Rac1Q61L did not permanently withdraw from the cell cycle. An indication of the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in Rac1-mediated myoblast proliferation was obtained by the use of MAPK kinase inhibitors U0126 and PD098059. These inhibitors arrested C2-Rac1Q61L cell cycling. Taken together, our results show that Rac1 activation interferes with myoblast exit from the cell cycle via or in concert with the MAPK pathway.     

Cooperation between Shh and IGF-I in promoting myogenic proliferation and differentiation via the MAPK/ERK and PI3K/Akt pathways requires Smo activity

Sonic Hedgehog (Shh) has been shown to promote adult myoblast proliferation and differentiation and affect Akt phosphorylation via its effector Smoothened (Smo). Here, the relationship between Shh and insulin-like growth factor I (IGF-I) was examined with regard to myogenic differentiation via signaling pathways which regulate this process. Each factor enhanced Akt and MAPK/ERK (p42/44) phosphorylation and myogenic factor expression levels in a dose-responsive manner, while combinations of Shh and IGF-I showed additive effects. Blockage of the IGF-I effects by neutralizing antibody partially reduced Shh's effects on signaling pathways, suggesting that IGF-I enhances, but is not essential for Shh effects. Addition of cyclopamine, a Smo inhibitor, reduced Shh- and IGF-I-induced Akt phosphorylation in a similar manner, implying that Shh affects gain of the IGF-I signaling pathway. This implication was also examined via a genetic approach. In cultures derived from Smo(mut) (MCre;Smo(flox/flox)) mice lacking Smo expression specifically in hindlimb muscles, IGF-I-induced Akt and p42/44 phosphorylation was significantly reduced compared to IGF-I's effect on Smo(cont) cells. Moreover, remarkable inhibition of the stimulatory effect of IGF-I on myogenic differentiation was observed in Smo(mut) cultures, implying that intact Smo is required for IGF-I effects in myoblasts. Immunoprecipitation assays revealed that tyrosine-phosphorylated proteins, including the regulatory unit of PI3K (p85), are recruited to Smo in response to Shh. Moreover, IGF-IR was found to associate with Smo in response to Shh and to IGF-I, suggesting that Shh and IGF-I are already integrated at the receptor level, a mechanism by which their signaling pathways interact in augmenting their effects on adult myoblasts.     

Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage

The proliferation, differentiation, and fusion of a small number of myogenic precursor cells must be precisely regulated during development to ensure the proper size, organization, and function of the limb musculature. We have examined the role of Sonic hedgehog (Shh) in these processes by both augmentation and inhibition of the Shh-mediated signaling pathway. Our data show that Shh regulates muscle development by repressing the terminal differentiation of early myogenic precursor cells and does not function as a myoblast mitogen. Shh function in hypaxial muscle appears to be spatially restricted to the early myoblast population within the ventral muscles of the posterior region of the limb. Furthermore, Shh appears to act as a permissive, rather than an inductive, signal for slow MyHC expression in myoblasts. Our data thus provide the foundation for a new hypothesis for Shh function in hypaxial skeletal muscle development.