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.     

Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.     

p130Cas-dependent actin remodelling regulates myogenic differentiation

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.     

A cyclic AMP-dependent pathway regulates the expression of acetylcholinesterase during myogenic differentiation of C2C12 cells

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.     

Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.     

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.     

敲减DTX4表达抑制C2C12细胞成肌分化

DTX4（Deltex 4 homolog）蛋白属于Deltex家族成员|Deltex家族是Notch信号通路的调节因子. 已知Notch信号通路在成肌分化中发挥重要作用. 然而,DTX4是否参与调控肌肉发育尚未有报道. 本研究探索DTX4对成肌分化的影响及作用机制. 实时定量PCR和蛋白质印迹分析揭示,伴随小鼠C2C12成肌细胞（myoblast）分化为肌管（myotube）过程,成肌分化标志蛋白肌球蛋白重链（myosin heavy-chain,MyHC）、肌细胞生成素(myogenin)表达逐渐升高,DTX4 mRNA及蛋白质表达水平也逐渐升高. 通过顺序专一的siRNA敲减DTX4表达后,C2C12成肌细胞肌管面积和肌管融合指数明显减少|MyHC、肌细胞生成素蛋白表达水平明显降低|但ERK信号通路未见明显变化.上述结果表明,敲减DTX4表达抑制C2C12细胞成肌分化.我们的结果提示,DTX4可能参与C2C12细胞成肌分化.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.     

Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes. Evidence for TWEAK-independent functions of Fn14 during myogenesis

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal alpha-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.     

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.     

Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation.

Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

MyoD binds to Mos and inhibits the Mos/MAP kinase pathway

When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.