Stress-activated Protein Kinase-2/p38 and a Rapamycin-sensitive Pathway Are Required for C2C12 Myogenesis

Abstract

The differentiation of C2C12 myoblasts to myotubes was found to be accompanied by a strong activation of p70 S6 kinase and the mitogen-activated protein kinase (MAPK) family member SAPK2/p38, without significant activation of p42 MAPK and only slight activation of SAPK1/JNK and protein kinase Bα. Consistent with these findings, SB 203580 (a specific inhibitor of SAPK2/p38) or rapamycin (which blocks the activation of p70 S6 kinase) prevented the formation of multinucleated myotubes, as well as the expression of muscle-specific proteins that included SAPK3 (another MAPK family member). PD 098059 (which prevents the activation of p42 MAPK) had no effect on myotube formation. Surprisingly, the slow activation of p70 S6 kinase during differentiation was not only prevented by rapamycin but also by SB 203580, and the activation of MAPKAP kinase-2 (anin vivo substrate of SAPK2/p38) was not only prevented by SB 203580 but also by rapamycin. In contrast, the acute activation of p70 S6 kinase in C2C12 myoblasts induced by phorbol esters was unaffected by SB 203580 and the acute activation of MAPKAP kinase-2 induced by anisomycin was unaffected by rapamycin. These results show for the first time that SAPK2/p38 plays an essential role in C2C12 cell differentiation. The differentiation of C2C12 myoblasts to myotubes was found to be accompanied by a strong activation of p70 S6 kinase and the mitogen-activated protein kinase (MAPK) family member SAPK2/p38, without significant activation of p42 MAPK and only slight activation of SAPK1/JNK and protein kinase Bα. Consistent with these findings, SB 203580 (a specific inhibitor of SAPK2/p38) or rapamycin (which blocks the activation of p70 S6 kinase) prevented the formation of multinucleated myotubes, as well as the expression of muscle-specific proteins that included SAPK3 (another MAPK family member). PD 098059 (which prevents the activation of p42 MAPK) had no effect on myotube formation. Surprisingly, the slow activation of p70 S6 kinase during differentiation was not only prevented by rapamycin but also by SB 203580, and the activation of MAPKAP kinase-2 (anin vivo substrate of SAPK2/p38) was not only prevented by SB 203580 but also by rapamycin. In contrast, the acute activation of p70 S6 kinase in C2C12 myoblasts induced by phorbol esters was unaffected by SB 203580 and the acute activation of MAPKAP kinase-2 induced by anisomycin was unaffected by rapamycin. These results show for the first time that SAPK2/p38 plays an essential role in C2C12 cell differentiation. The differentiation of skeletal muscle starts when myoblasts withdraw from the cell division cycle and is characterized morphologically by the alignment, elongation, and fusion of mononucleated myoblasts into multinucleated myotubes. Myogenesis is largely controlled by the myogenic basic helix-loop-helix family of transcription factors (MyoD, myogenin, myf5, and MRF4), and the myocyte enhancer factor-2 (1Lassar A.B. Skapek S.X. Novitch B. Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (312) Google Scholar, 2Olson E.N. Perry M. Schulz R.A. Dev. Biol. 1995; 172: 2-14Crossref PubMed Scopus (315) Google Scholar), which control the expression of many muscle-specific genes, such as the myosin heavy chain and creatine kinase. Cell division is prevented during muscle differentiation by the induction of cyclin-dependent protein kinase inhibitors (3Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar,4Franklin D.S. Xiong Y. Mol. Biol. Cell. 1996; 7: 1587-1599Crossref PubMed Scopus (105) Google Scholar). Skeletal muscle differentiation is negatively influenced either by treating myoblasts with serum or growth factors (e.g. basic fibroblast growth factor-2, transforming growth factor β1) or by oncogenes, such as c-myc, c-jun, c-fos, Ha-ras, and E1a (1Lassar A.B. Skapek S.X. Novitch B. Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (312) Google Scholar, 5Alema S. Tato F. Semin. Cancer Biol. 1994; 5: 147-156PubMed Google Scholar, 6Ludolph D.C. Konieczny S.F. FASEB J. 1995; 9: 1595-1604Crossref PubMed Scopus (218) Google Scholar), and differentiation can be induced by switching the cells to medium containing low concentrations of serum. Some differentiation inhibitors activate the classical mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MKP1, MAPK phosphatase-1; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein, IGF-1, insulin-like growth factor-1; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; MAPKAP-K2, MAPK activated protein kinase-2; FCS, fetal calf serum; PKB, protein kinase B; PI 3-kinase, phosphatidylinositide 3-kinase. pathway, which has been implicated in regulating proliferation and differentiation in a variety of cells (7Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar, 8Gotoh Y. Masuyama N. Suzuku A. Ueno N. Nishida E. EMBO J. 1995; 14: 2491-2498Crossref PubMed Scopus (134) Google Scholar). For this reason, the role of the MAPK pathway in regulating skeletal muscle differentiation has been studied extensively, but the results have been controversial. For example, the compound PD 098059 (which prevents the activation of MAPK by inhibiting the activation of MAPK kinase-1 (9Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar)) was reported to increase the rate of myotube formation in the presence of insulin-like growth factor-1 (IGF-I) in L6 (10Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar) and 23A2 cells (11Weyman C.M. Wolfman A. Endocrinology. 1998; 139: 1794-1800Crossref PubMed Scopus (55) Google Scholar), suggesting a negative role for MAPK in this process. In contrast, another laboratory reported that MAPK plays a positive role in myogenesis (12Gredinger E. Gerber A.N. Tamir Y. Tapscott S.J. Bengal E. J. Biol. Chem. 1998; 273: 10436-10444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). They reported that MAPK activity is induced concomitantly with muscle differentiation in either C2 myoblasts or muscle cells derived from MyoD-expressing 10T1/2 fibroblasts. They also reported that PD 098059 partially inhibited the fusion of myoblasts to multinucleated myotubes and the induction of MyoD expression that normally takes place during terminal differentiation, without affecting the expression of certain muscle-specific proteins. Another group reported that MAPK has a negative role at the early stages of myogenesis because myoblasts overexpressing MAPK phosphatase-1 (MKP1) showed enhanced production of muscle-specific genes. However, overexpression of MKP1 inhibited myotube formation at later times, and it was suggested that the MAPK pathway plays positive, as well as negative roles in muscle differentiation (13Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar). Other investigators reported that PD 098059 did not affect differentiation of C2 myoblasts, implying that the MAPK pathway is not required for this process (14Pinset C. Garcia A. Rousse S. Dubois C. Montarras D. C. R. Acad. Sci. (Paris). 1997; 320: 367-374Crossref PubMed Scopus (30) Google Scholar). They also reported that wortmannin, an inhibitor of phosphatidylinositide 3-kinase, inhibits the IGF-I-dependent differentiation of C2 myoblasts. MKP1 is not specific for MAPK but can also inactivate other MAPK family members in vitro, such as SAPK2a/p38α (15Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar) and SAPK1/JNK (16Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (373) Google Scholar). SAPK2a/p38α and SAPK1/JNK lie in distinct signal transduction pathways and, in many cells, become activated in response to cellular stresses (such as the protein synthesis inhibitor anisomycin), lipopolysaccharide, or proinflammatory cytokines (reviewed in Ref. 17Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). The effects of MKP1 on muscle differentiation (13Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar) could therefore be explained by the inhibition of other MAPK cascades. For this reason, we have investigated the potential role of SAPK2/p38 in skeletal muscle differentiation by studying the effect of SB 203580, a specific inhibitor of both SAPK2a/p38α and its close relative SAPK2b/p38β (collectively referred to as SAPK2/p38) (18Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys R.J. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 19Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar, 20Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar, 21Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar). We demonstrate that the SAPK2/p38 pathway is activated during C2C12 cell differentiation and that SB 203580 (but not PD 098059) blocks myotube formation and the expression of muscle-specific genes. Our results demonstrate for the first time that SAPK2/p38 is required for C2C12 myogenesis triggered by low concentrations of serum. Tissue culture reagents, myelin basic protein (MBP) and human IGF-1 were purchased from Life Technologies, Inc. (Paisley, UK). Protein G-Sepharose and GSH-agarose were from Amersham Pharmacia Biotech (Milton Keynes, UK). The N-terminal 194 residues of c-Jun (cJun-(1–194)), provided by Dr. R. Treisman (Imperial Cancer Research Fund, London), was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein and purified on GSH-agarose. Rapamycin, LY 294002, and SB 203580 were purchased from Calbiochem. PD 098059 was a gift from Dr. Alan Saltiel (Parke-Davis Pharmaceuticals, Ann Arbor, MI). The anti-myosin heavy chain antibody, the creatine kinase assay kit, anisomycin, and phorbol 12-myristate 13-acetate (PMA) were from Sigma (Poole, UK). Anti-p21 and anti-myogenin antibodies were from Santa Cruz Biotechnology. Anti-p42 MAPK, anti-MAPK-activated protein kinase-2 (MAPKAP-K2), anti-p70 S6 kinase, and anti-SAPK3 antibodies were raised and purified as described elsewhere (9Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar, 20Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar, 22Gould G.W. Cuenda A. Thomson F.J. Cohen P. Biochem. J. 1995; 311: 735-738Crossref PubMed Scopus (91) Google Scholar, 23Cuenda A. Alonso G. Morrice N. Jones M. Meier R. Cohen P. Nebreda A.R. EMBO J. 1996; 15: 4156-4164Crossref PubMed Scopus (115) Google Scholar). Other reagents were of analytical grade or better and purchased from BDH Chemicals or Sigma. The mouse C2C12 myoblasts (American Type Culture Collection) were cultured at 37 °C (in an atmosphere of 5% CO2) in Dulbecco's modified Eagle's medium supplemented with 20% (v/v) fetal calf serum (FCS), 0.5% (v/v) chick embryo extract, and antibiotics. To analyze the effect of different inhibitors of signal transduction pathways on C2C12 differentiation, confluent cells were washed with Dulbecco's modified Eagle's medium and differentiation medium was added without or with 10 μm SB 203580 (19Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar) or 100 nm rapamycin (24Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar), 10 μm LY 294002 (10Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar), or 50 μm PD 098059 (9Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar). All the inhibitors were dissolved in dimethyl sulfoxide (Me2SO) to give concentrations of 1–50 mm, and 1 μl was added per 1 ml of culture medium. The equivalent volume of Me2SO was added to control cultures in every experiment. The medium was replaced with fresh medium (with or without inhibitors) every 24 h. At various times, C2C12 cells were lysed in 50 mm Tris acetate, pH 7.5, 0.27 m sucrose, 1 mm EDTA, 1 mm EGTA, 1 mm orthovanadate, 10 mmsodium β-glycerophosphate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 1% (w/v) Triton X-100, 0.01% (w/v) Brij-35, 0.1% (v/v) 2-mercaptoethanol, 1 mmbenzamidine, 0.2 mm phenylmethylsulfonyl fluoride, and leupeptin (5 μg/ml). The extracts were centrifuged for 5 min at 13,000 × g, and the supernatant (termed lysate) was removed and either used immediately or quick frozen in liquid nitrogen and stored at −80 °C. MAPKAP-K2 (22Gould G.W. Cuenda A. Thomson F.J. Cohen P. Biochem. J. 1995; 311: 735-738Crossref PubMed Scopus (91) Google Scholar), p70 S6 kinase (25Baxter R.M. Cohen P. Obermeier A. Ullrich A. Downes C.P. Doza Y.N. Eur. J. Biochem. 1995; 234: 84-91Crossref PubMed Scopus (52) Google Scholar), protein kinase Bα (PKBα) (26Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich B.A. Hemmings Nature. 1995; 378: 785-789Crossref PubMed Scopus (4376) Google Scholar), and p42 MAPK (27Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar) were immunoprecipitated from 0.1 mg of C2C12 lysates as described previously for other cell lines. After immunoprecipitation, MAPKAP-K2 was assayed using the peptide KKLNRTLSVA (28Stokoe D. 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Biochem. 1996; PubMed Scopus (112) Google Scholar). of protein kinase is that which the of 1 of peptide or protein substrate in 1 C2C12 myoblasts were to and induced to by the medium from 20% to 5% serum and for to this the were of protein that lie in distinct signal transduction MAPKAP-K2, p70 S6 kinase, p42 MAPK, and We found that MAPKAP-K2, an in vivo substrate of SAPK2/p38 (15Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar, 19Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar), was activated during differentiation and activated as by the that no activation with anisomycin 1 p70 S6 kinase, activation is prevented by was activated during differentiation 1 activated 3 as by the of to increase activity 1 These also that the expression of MAPKAP-K2 and p70 S6 kinase not during differentiation. In to MAPKAP-K2 and p70 S6 kinase, p42 MAPK of the classical MAPK was not activated at during differentiation but was activated in response to 1 SAPK1/JNK was not activated to the first 3 of differentiation, at which multinucleated myotubes but was activated at when the myotubes were 1 The of myoblasts or myotubes with anisomycin a activation of SAPK1/JNK 1 which is activated in vivo a phosphatidylinositide pathway, was activated to but activation was only of that which for a with IGF-1 To the in differentiation, we studied the effects of inhibitors of signal transduction pathways on C2C12 cell during differentiation. the first 24 h of differentiation, the myoblasts and with other in the multinucleated myotubes were 10 μm SB 203580 was added to the differentiation cells and a in the and of myoblasts was 3 in differentiation medium not SB 203580 prevented the fusion of C2C12 myoblasts into multinucleated myotubes. formation was also largely (but not when 100 nm rapamycin or 10 μm LY 294002 inhibitor of PI was in the differentiation medium and Consistent with the MAPK pathway not activated during differentiation, 50 μm PD 098059 did not affect the rate of myotube formation the activation of MAPK was by PD 098059 We studied the effects of inhibitors on the expression or activity of proteins during muscle differentiation SB 203580, and LY 294002 inhibited the induction of creatine kinase, myogenin, and myosin heavy chain 3 In contrast, the induction of was only partially by SB 203580 or rapamycin but was prevented by LY PD 098059 did not the induction of of the differentiation studied but to increase creatine kinase The of p42 MAPK, which is not a muscle-specific protein, did not during C2C12 differentiation and was not by of the inhibitors The of myotube formation in the presence of 10 μm SB 203580 or 100 nm rapamycin and the strong activation of MAPKAP-K2 and p70 S6 kinase during the of C2C12 myoblasts to myotubes that SAPK2/p38 and a pathway required for differentiation. from in other cell SB 203580 inhibited the activation of MAPKAP-K2, and rapamycin inhibited the activation of p70 S6 kinase rapamycin also prevented the activation of MAPKAP-K2, SB 203580 prevented the activation of p70 S6 kinase during the differentiation process In contrast, rapamycin had no effect on the acute activation of MAPKAP-K2 induced by anisomycin and SB 203580 had no effect on the acute activation of p70 S6 kinase by PMA, in myotubes 5 Consistent with the effects of SB 203580 and rapamycin on the activation of MAPKAP-K2 and p70 S6 kinase, these inhibitors also prevented the induction of creatine kinase activity 4 The activation of MAPKAP-K2 and p70 S6 kinase, and the induction of creatine kinase during C2C12 differentiation, were also by LY 294002 acute activation of MAPKAP-K2 by anisomycin is not by and the activation of p70 S6 kinase is not by SB C2C12 myoblasts were incubated for min at 37 °C in the or presence of 10 μm or 100 nm rapamycin and for min with 10 anisomycin or for min with in the presence or of these The cells were and MAPKAP-K2 or p70 S6 kinase were immunoprecipitated from the lysates and assayed as described The results as for The in creatine kinase activity 4 during differentiation the in the activation of MAPKAP-K2 4 and p70 S6 kinase 4 The activation of MAPKAP-K2 and the induction of creatine kinase were both by SB 203580 with of μm The of C2C12 myoblasts to myotubes was by SB 203580 at a SAPK3 C. Ullrich A. Acad. Sci. S. A. 1996; PubMed Scopus Google Scholar) and Y. Biochem. 1996; PubMed Scopus Google Scholar)) is a MAPK family member when in cell can be activated by the cellular stresses and proinflammatory cytokines as SAPK2/p38 A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar). SAPK3 expression is induced during the differentiation of C2C12 cells, and overexpression of SAPK3 in C2C12 cells the fusion rate of the myoblasts C. Ullrich A. Acad. Sci. S. A. 1996; PubMed Scopus Google Scholar). In the we that the of SAPK3 during C2C12 myotube formation and also showed that induction is prevented by either SB 203580 or rapamycin but not by PD 098059 not suggested both positive and negative roles for the classical MAPK in the differentiation of cell to multinucleated myotubes the but in this we to for a role for p42 MAPK in C2C12 The differentiation of C2C12 cells was not accompanied by activation or inhibition of p42 MAPK 1 PD which prevents activation of the MAPK pathway, had no effect on myotube formation or on the expression of differentiation such as myogenin, or myosin heavy chain SB 203580, a specific inhibitor of SAPK2/p38, which is a MAPK family member that in a distinct protein kinase myotube formation and the expression of muscle-specific proteins and at concentrations to that SAPK2/p38 activity in 4 and SB 203580 also prevented the expression of a MAPK family member that is expressed in skeletal is that the of myotube formation that when the MAPK MKP1 is in C2C12 cells (13Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar) results from the of SAPK2/p38 of MAPK the formation and muscle-specific expression is also by rapamycin in C2C12 and or myoblasts (10Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). with protein and the inhibits the protein kinase of rapamycin Nature. 1994; PubMed Scopus Google Scholar). which protein synthesis by its from G.J. A. J.C. Science. 1997; PubMed Scopus Google Scholar). is also required for the activation of p70 S6 kinase V. C.M. Acad. Sci. S. A. PubMed Scopus Google Scholar), but it p70 S6 kinase or the activity of a p70 S6 kinase is LY 294002 is as an inhibitor of PI (24Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar), but it also inhibits other members of the PI which is inhibited by LY 294002 with an 5 μm G.J. C. G. J.C. EMBO J. 1996; 15: PubMed Scopus Google Scholar). The of C2C12 myogenesis by 10 μm LY 294002 could therefore be explained by the inhibition of the inhibition of PI 3-kinase. be with the slight activation of PI during differentiation, which is by the activation of 1 However, LY 294002 prevents the induction of rapamycin only has a effect LY 294002 therefore the induction of at in by a SAPK2/p38 is not inhibited by rapamycin not and rapamycin not the acute activation of SAPK2/p38 by anisomycin was therefore that the slow (but activation of SAPK2/p38 during C2C12 differentiation is by rapamycin that a pathway the expression of a factor that the activation of Another was that the slow (but activation of p70 S6 kinase during differentiation is not only by rapamycin but also by SB SB 203580 not because phorbol activation of p70 S6 kinase (which is by is unaffected by SB that the activation of SAPK2/p38 the expression of a factor that the activation of and p70 S6 kinase. is that SAPK2/p38 and both required to the which both pathways a positive that differentiation to the results that SAPK2/p38 and both essential roles in the differentiation of C2C12 myoblasts to myotubes. We Dr. G. and Dr. A. for with the and of C2C12