Active Ras-induced effects on skeletal myoblast differentiation and apoptosis are independent of constitutive PI3-kinase activity

23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts. Surprisingly, while expression of either G12V:H-Ras or F156L:H-Ras results in constitutive signaling through PI3-kinase, only cells expressing G12V:H-Ras additionally possess constitutive signaling through MAPK, and NFkappaB. Pharmacological abrogation of the Ras-induced constitutive PI3-kinase signal, however, is not responsible for the impaired differentiation or apoptosis in either A2:G12V:H-Ras myoblasts or A2:F156L:H-Ras myoblasts. Thus, our data suggest that a pathway distinct from those that signals through MAPK, NFkappaB or PI3-kinase is responsible for the impaired differentiation and apoptosis in 23A2 skeletal myoblasts expressing constitutively active Ras.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

Serum withdrawal-induced accumulation of phosphoinositide 3-kinase lipids in differentiating 3T3-L6 myoblasts: distinct roles for Ship2 and PTEN

Phosphoinositide 3-kinase (PI3K) activation and synthesis of phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃) lipids mediate growth factor signaling that leads to cell proliferation, migration, and survival. PI3K-dependent activation of Akt is critical for myoblast differentiation induced by serum withdrawal, suggesting that in these cells PI3K signaling is activated in an unconventional manner. Here we investigate the mechanisms by which PI3K signaling and Akt are regulated during myogenesis. We report that PI-3,4-P₂ and PI-3,4,5-P₃ accumulated in the plasma membranes of serum-starved 3T3-L6 myoblasts due to de novo synthesis and increased lipid stability. Surprisingly, only newly synthesized lipids were capable of activating Akt. Knockdown of the lipid phosphatase PTEN moderately increased PI3K lipids but significantly increased Akt phosphorylation and promoted myoblast differentiation. Knockdown of the lipid phosphatase Ship2, on the other hand, dramatically increased the steady-state levels of PI-3,4,5-P₃ but did not affect Akt phosphorylation and increased apoptotic cell death. Together, these results reveal the existence of two distinct pools of PI3K lipids in differentiating 3T3-L6 myoblasts: a pool of nascent lipids that is mainly dephosphorylated by PTEN and is capable of activating Akt and promoting myoblast differentiation and a stable pool that is dephosphorylated by Ship2 and is unable to activate Akt.     

Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways

Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. However, its role in their differentiation remains ambiguous. Here, we provide evidence that Shh promotes the proliferation and differentiation of primary cultures of chicken adult myoblasts (also termed satellite cells) and mouse myogenic C2 cells. These effects are reversed by cyclopamine, a specific chemical inhibitor of the Shh pathway. In addition, we show that Shh and its downstream molecules are expressed in adult myoblast cultures and localize adjacent to Pax7 in muscle sections. These gene expressions are regulated during postnatal muscle growth in chicks. Most importantly, we report that Shh induces MAPK/ERK and phosphoinositide 3-kinase (PI3K)-dependent Akt phosphorylation and that activation of both signaling pathways is essential for Shh's signaling in muscle cells. However, the effect of Shh on Akt phosphorylation is more robust than that on MAPK/ERK, and data suggest that Shh influences these pathways in a manner similar to IGF-I. By exploiting specific chemical inhibitors of the MAPK/ERK and PI3K/Akt signaling pathways, UO126 and Ly294002, respectively, we demonstrate that Shh-induced Akt phosphorylation, but not that of MAPK/ERK, is required for its promotive effects on muscle cell proliferation and differentiation. Taken together, we suggest that Shh acts in an autocrinic manner in adult myoblasts, and provide first evidence of a role for PI3K/Akt in Shh signaling during myoblast differentiation.     

Involvement of type I protein kinase A in the differentiation of L6 myoblast in conjunction with phosphatidylinositol 3-kinase

To investigate the role of protein kinase A (PKA) (EC 2.7.1.37) in myogenesis, PKA activity was closely monitored during the differentiation of L6 rat skeletal myoblasts. As the differentiation proceeded, total PKA activity increased about 2-3 fold, and the protein levels of PKA RIalpha and Calpha subunits increased about 3-4 fold. We then looked at the effect of the specific inhibitor for PKA, N-[2-(p-bromocinnamy-lamino)-ethyl]-5-isoquinoline-sulfonamide (H89), on the differentiation of L6 myoblasts. H89 completely blocked the myotube formation and abolished the up-regulation of RIalpha and Ca. This inhibitory effect of H89 was dose-dependent and could be reversed upon removal of H89 from the culture medium. Furthermore, we demonstrated that specific inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin, and LY294002 blocked the myotube formation and abolished the increase of PKA activity, which normally accompanied the differentiation of myoblasts. These results suggest that type I PKA may play a functional role(s) in the differentiation of myoblast as a putative downstream effector of the PI3K signaling pathway.     

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.     

La PI3 kinase,une activité critique pour la différenciation des cellules musculaires IGFs dépendante

Evidence has accumulated that suggests that insulin-like growth factors (IGFs) exert a positive influence on myoblast differentiation. We have undertaken to study the signalling events required for differentiation resulting from type-1 IGF receptor stimulation in C2 myoblasts, where autocrine production of IGF-II was abolished by means of antisense RNA. Exposure of the cells to IGFs leads to a rapid and sustained activation of phosphatidyl-inositol 3-kinase followed by the expression of Myod, myogenin and differentiation. The fungal metabolite, wortmannin, inhibits both PI 3-kinase and muscle differentiation with an IC 50 in the nanomolar range. IGFs are also known to cause a rapid activation of MAP kinase. However, the synthetic inhibitor of MEK, PD098059, which prevents MAP kinase activation, does not affect myoblast differentiation. These results provide evidence that PI3-kinase, but not MAP kinase, is required for insulin-like growth factor receptor-dependent differentiation of muscle cells.     

Direct interaction between the cytoplasmic tail of ADAM 12 and the Src homology 3 domain of p85alpha activates phosphatidylinositol 3-kinase in C2C12 cells

ADAM 12, a member of the ADAM family of transmembrane metalloprotease-disintegrins, has been implicated previously in the differentiation of skeletal myoblasts. In the present study, we show that the cytoplasmic tail of mouse ADAM 12 interacts in vitro and in vivo with the Src homology 3 domain of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. By site-directed mutagenesis, we have identified three p85alpha-binding sites in ADAM 12 involving PXXP motifs located at amino acids 825-828, 833-836, and 884-887. Using green fluorescent protein (GFP)-pleckstrin homology (PH) domain fusion protein as a probe for PI 3-kinase lipid products, we have further demonstrated that expression of ADAM 12 in C2C12 cells resulted in translocation of GFP-PH to the plasma membrane. This suggests that transmembrane ADAM 12, by providing docking sites for the Src homology 3 domain of p85alpha, activates PI 3-kinase by mediating its recruitment to the membrane. Because PI 3-kinase is critical for terminal differentiation of myoblasts, and because expression of ADAM 12 is up-regulated at the onset of the differentiation process, ADAM 12-mediated activation may constitute one of the regulatory mechanisms for PI 3-kinase during myoblast differentiation.     

Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging

Mice lacking superoxide dismutase-2 (SOD2 or MnSOD) die during embryonic or early neonatal development, with diffuse superoxide-induced mitochondrial damage. Although stem and progenitor cells are exquisitely sensitive to oxidant stress, they have not been well studied in MnSOD2-manipulated mouse models. Patterns of proliferation and differentiation of cultured myoblasts (muscle progenitor cells), PI3-Akt signaling during differentiation, and the maintenance of mitochondrial mass with aging using myoblasts from young (3–4 week old) and aged (27–29 months old) MnSOD2-overexpressing ( Sod2- Tg) and heterozygote ( Sod2 ^{+/ −}) mice were characterized by us. Overexpression of MnSOD2 in myoblasts had a protective effect on mitochondrial DNA abundance and some aspects of mitochondrial function with aging, and preservation of differentiation potential. Sod2 deficiency resulted in defective signaling in the PI3-Akt pathway, specifically impaired phosphorylation of Akt at Ser473 and Thr308 in young myoblasts, and decreased differentiation potential. Compared with young myoblasts, aged myoblast Akt was constitutively phosphorylated, unresponsive to mitogen signaling, and indifferent to MnSOD2 levels. These data suggest that specific sites in the PI3K-Akt pathway are more sensitive to increased superoxide levels than to the increased hydrogen peroxide levels generated in Sod2 -transgenic myoblasts. In wild-type myoblasts, aging was associated with significant loss of mitochondrial DNA relative to chromosomal DNA, but MnSOD2 overexpression was associated with maintained myoblast mitochondrial DNA with aging.     

Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture.

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.     

Muscle-specific overexpression of the type 1 IGF receptor results in myoblast-independent muscle hypertrophy via PI3K, and not calcineurin, signaling

The insulin-like growth factors (IGF-I and IGF-II), working through the type 1 IGF receptor (IGF-1R), are key mediators of skeletal muscle fiber growth and hypertrophy. These processes are largely dependent on stimulation of proliferation and differentiation of muscle precursor cells, termed myoblasts. It has not been rigorously determined whether the IGFs can also mediate skeletal muscle hypertrophy in a myoblast-independent fashion. Similarly, although the phosphatidylinositol 3-kinase (PI3K) and calcineurin signaling pathways have been implicated in skeletal muscle hypertrophy, these pathways are also involved in skeletal myoblast differentiation. To determine whether the IGFs can stimulate skeletal muscle hypertrophy in a myoblast-independent fashion, we developed and validated a retroviral expression vector that mediated overexpression of the human IGF-1R in rat L6 skeletal myotubes (immature muscle fibers), but not in myoblasts. L6 myotubes transduced with this vector accumulated significantly higher amounts of myofibrillar proteins, in a ligand- and receptor-dependent manner, than controls and demonstrated significantly increased rates of protein synthesis. Stimulation of myotube hypertrophy was independent of myoblast contributions, inasmuch as these cultures did not exhibit increased levels of myoblast proliferation or differentiation. Experiments with PI3K and calcineurin inhibitors indicated that myoblast-independent myotube hypertrophy was mediated by PI3K, but not calcineurin, signaling. This study demonstrates that IGF can mediate skeletal muscle hypertrophy in a myoblast-independent fashion and suggests that muscle-specific overexpression of the IGF-1R or stimulation of its signaling pathways could be used to develop strategies to ameliorate muscle wasting without stimulating proliferative pathways leading to carcinogenesis or other pathological sequelae.     

Ca/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca²⁺/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

Induction of cyclins E and A in response to mitogen removal: a basic alteration associated with the arrest of differentiation of C2 myoblasts transformed by simian virus 40 large T antigen.

We previously showed that C2 myoblasts transformed by simian virus 40 large T antigen (SVLT) stop the myogenic process after the induction of myogenin and of high Rb levels; the induced Rb, however, becomes notably phosphorylated. We have analyzed the protein levels and activities of cyclin-dependent kinases (cdks) in untransformed C2 cells and in transformants of either SVLT or the cytoplasmic mutant NKT1 (which permits differentiation) upon a shift from growth medium (GM) to mitogen-poor differentiation medium (DM). After the shift, cdk4 levels remained constant and cdk6 levels decreased in all cell types; cdk2 minimally increased only in SVLT cells. Cyclin D1 was downregulated in DM in all cell types, and cyclin D3 was upregulated (albeit less strongly in SVLT cells than in the others). In contrast, a dramatic difference between SVLT cells and the other cells was observed for cyclins E and A, which essentially disappeared (as protein and RNA) in normal C2 and NKT1 cells upon the shift from GM to DM, whereas they increased in SVLT cells. Concurrently, cdk2 activity ceased in C2 and NKT1 cells in DM, whereas it persisted at 20% of the GM level in SVLT cells. cdk4 activity was detectable in all cells only in GM. Cyclin E and A induction thus appeared to sustain enough Rb phosphorylation to interfere with tissue-specific expression, with cdk activity not high enough to activate cyclin self-regulation. In DM, cdk2 complexed to D3 was underphosphorylated in all cells, and SVLT allowed strong inductions of p21 and p27 without affecting their complexes with cdks.     

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.     

Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway.

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.     

A cell surface phosphoprotein of 48 kDa specific for myoblast fusion

The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.