Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells

Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+)), a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+) in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+)-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt) in maintaining de novo lipogenesis in prostate cancer (PCa) cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC) lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK) and phosphorylation of acetyl-CoA carboxylase (ACC). In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.     

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.     

Extension of human cell lifespan by nicotinamide phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.     

Inhibition of AMP-activated protein kinase α (AMPKα) by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts and cardiomyocytes: role of p53 and SIRT1

Doxorubicin, an anthracycline antibiotic, is widely used in cancer treatment. Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Although its side effects in non-carcinoma cells, especially in heart tissue, are well known, the molecular targets of doxorubicin are poorly characterized. Here, we report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) or H9C2 cardiomyocyte were exposed to doxorubicin at different doses and durations. Cell death and p53, SIRT1, and AMPK levels were examined by Western blot. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Genetic deletion of AMPKα1 reduced NAD(+) levels and SIRT1 activity and significantly increased the levels of p53 and cell death. Pre-activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside or transfection with an adenovirus encoding a constitutively active AMPK (AMPK-CA) markedly reduced the effects of doxorubicin in MEFs from Ampkα1 knock-out mice. Conversely, pre-inhibition of Ampk further sensitized MEFs to doxorubicin-induced cell death. Genetic knockdown of p53 protected both wild-type and Ampkα1(-/-) MEFs from doxorubicin-induced cell death. p53 accumulation in Ampkα1(-/-) MEFs was reversed by SIRT1 activation by resveratrol. Taken together, these data suggest that AMPK inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction in MEFs and further suggest that pharmacological activation of AMPK might alleviate the side effects of doxorubicin.     

A novel inverse relationship between metformin-triggered AMPK-SIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells

AMP-activated protein kinase (AMPK) and the NAD(+)-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.     

Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase

The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.     

AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

The link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic α subunits, α1 and α2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the α1 but not the α2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPKα1 and AMPKα2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPKα1-specific effect on myogenesis was likely due to the dominant expression of α1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPKα1 subunit for myogenesis in vivo, we prepared both DsRed AMPKα1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wild-type myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPKα1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPKα1.     

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.     

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.     

Insulin-like growth factors,hepatocyte growth factor and transforming growth factor-alpha in mouse tongue myogenesis

Many reports have shown that tongue striated muscles have several unique characteristics not found in other skeletal muscles such as limb and trunk. Several peptide growth factors are reported to play important roles in skeletal myogenesis. In this article, the roles of insulin-like growth factors (IGF), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha in mouse tongue myogenesis were studied using an organ culture system of the mandible or tongue obtained from mouse embryos. It was found that IGF-I promotes the differentiation of tongue myoblasts. HGF plays an essential role in the migration and proliferation of tongue myogenic cells, and inhibits the differentiation of tongue myoblasts. TGF-alpha does not play an essential role in the proliferation of tongue myogenic cells, but does promote the early differentiation of tongue myoblasts. The role of IGF-I in the differentiation of tongue myoblasts, and that of HGF in the migration, proliferation and differentiation of tongue myogenic cells appear to be almost identical to their roles in the myogenesis of limb and cultured myogenic cell lines. However, the role of TGF-alpha in the proliferation and differentiation of tongue myogenic cells appears to be different from its role in the myogenesis of limb and cultured myogenic cell lines such as C2 and L6.     

Differentiation-dependent regulation of skeletal myogenesis by neuregulin-1

Neuregulins comprise a group of growth factor proteins that regulate the differentiation of skeletal muscle. Here, we report that neuregulins are regulators of myogenic differentiation and stimulate mitogenesis in L6 skeletal myoblasts. The mitogenic response to neuregulin-1 was differentiation-dependent and observed only in aligned, differentiating cells. Treatment of these cells with neuregulin-1 increased [3H]thymidine incorporation and cell proliferation by 2- to 5-fold, while a minimal increase was seen in proliferating myoblasts. Neuregulin-1 did not induce DNA synthesis in fused, multinucleated myotubes. The increased DNA synthesis correlated with downregulation of myogenin and inhibition of myoblast fusion and myotube formation. These data suggest that neuregulins may regulate skeletal myogenesis in vivo and that this regulation is dependent on the state of differentiation of the myocytes.     

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV‐1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide⁺ (NAD⁺)‐dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the conversion of nicotinamide into NAD⁺, which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat‐regulated SIRT activity was studied in Hela‐CD4‐β‐gal (MAGI) cells. We demonstrated that Tat caused NAD⁺ depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat‐induced NAD⁺ depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat‐induced inhibition of SIRT1 activity was potentiated in Nampt‐knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat‐induced HIV‐1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat‐induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV‐1 infection. J. Cell. Biochem. 110: 1464–1470, 2010. © 2010 Wiley‐Liss, Inc.     

The microRNA-127-3p directly targeting Vamp2 in C2C12 myoblasts

MicroRNAs (miRNAs) have been reported that can regulate skeletal muscle growth and development. Previously, we demonstrated that miR-127-3p were differently expressed in skeletal muscle and muscle cells. However, the molecular mechanism of miR-127-3p regulation of skeletal myogenesis are not well elucidated. In this study, we transfected miR-127-3p into C2C12 cells, and found miR-127-3p induces myogenesis by targeting Vamp2. Moreover, the regulatory mechanism of Vamp2 in myoblasts proliferation and differentiation was further confirmed. In conclusion, our data providedevidences that miR-127-3p reciprocally regulated myoblasts proliferation and differentiation through directly targeting Vamp2.     

NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.

[Methods]

Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor’s changes were examined in normal or NAD metabolism related inhibitor treated cells.

[Results]

Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation.

[Conclusion]

In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5- bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.     

Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes

Nutrition availability is one of the major environmental signals influencing cell fate, such as proliferation, differentiation, and apoptosis, often functioning in concert with other humoral factors, including insulin. Herein, we show that low-serum-induced differentiation of C(2)C(12) myocytes is significantly hampered under low glucose (LG; 5 mM) compared with high glucose (HG; 22.5 mM) conditions, concurrently with nuclear accumulation of SIRT1, an NAD(+)-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending on glucose availability, with regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient glucose, whereas insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive upregulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the hampered differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinol-dependent restoration, indicating that insulin can function as a negative as well as a positive myogenic factor depending on glucose availability. Taken together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved, at least in part, through regulation of the cellular contents and localization of SIRT1 and FoxO3a in differentiating C(2)C(12) myocytes.     

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.