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The Insulin-like Growth Factor-Phosphatidylinositol 3-Kinase-Akt Signaling Pathway Regulates Myogenin Expression in Normal Myogenic Cells but Not in Rhabdomyosarcoma-derived RD Cells*

  • Qing Xu
    Affiliations
    Department of Biochemistry, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, Peoples Republic of China
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  • Zhenguo Wu
    Correspondence
    To whom correspondence should be addressed. Tel: 852-2358-8704; Fax: 852-2388-1552
    Affiliations
    Department of Biochemistry, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, Peoples Republic of China
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  • Author Footnotes
    * The work was supported by a start-up fund from Hong Kong University of Science & Technology and a grant from the Research Grant Council of Hong Kong Special Administrative Region (HKUST6205/00M).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 24, 2000DOI:https://doi.org/10.1074/jbc.M005030200
      Insulin-like growth factors (IGFs) can stimulate skeletal muscle differentiation. One of the molecular mechanisms underlying IGF-stimulated myogenesis is transcriptional induction of myogenin. The current work is aimed to elucidate the signaling pathways mediating the IGF effect on myogenin promoter in mouse C2C12 myogenic cells. We show that phosphatidylinositol 3-kinase (PI3K)/Akt and p70 S6K are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. We have identified three cis-elements, namely the E box, MEF2, and MEF3 sites, within the 133-base pair mouse proximal myogenin promoter that are under the control of the IGF/PI3K/Akt pathway. Simultaneous mutation of all three elements completely abolishes activation of the myogenin promoter by PI3K/Akt. We demonstrate that PI3K/Akt can increase both the MyoD and the MEF2-dependent reporter activity by enhancing the transcriptional activity of MyoD and MEF2. Interestingly, IGF1 does not enhance myogenin expression in Rhabdomyosarcoma-derived RD cells. Consistently, the constitutively active PI3K/Akt fail to activate the myogenic reporters, suggesting the IGF/PI3K/Akt pathway is defective in RD cells and the defect(s) is downstream to PI3K/Akt. This is the first time that a defect in the IGF/PI3K/Akt pathway has been revealed in RD cells which provides another clue to future therapeutic treatment of Rhabdomyosarcoma.
      MRF
      myogenic regulatory factor(s)
      bp
      base pair(s)
      IGF
      insulin-like growth factor
      PI3K
      phosphatidylinositol 3-kinase
      RMS
      Rhabdomyosarcoma
      DM
      differentiation medium
      GM
      growth medium
      Mammalian skeletal muscle differentiation has been a model system in the past decade for studying the molecular mechanisms that switch the cellular program from proliferation to differentiation. Intensive studies have led to the discovery and characterization of two families of transcription factors that play pivotal roles during differentiation (
      • Lassar A.B.
      • Skapek S.X.
      • Novitch B.
      ,
      • Molkentin J.D.
      • Olson E.N.
      ,
      • Yun K.
      • Wold B.
      ,
      • Arnold H.H.
      • Winter B.
      ). One of them consists of MyoD family proteins (also called myogenic regulatory factors or MRFs)1 which include four members: Myf5, MyoD, myogenin, and MRF4, all members of the basic helix-loop-helix superfamily and exclusively expressed in skeletal muscles. Knock-out and knock-in data reveal the existence of a genetic hierarchy in which MyoD and Myf5 act upstream to determine the myogenic fate of muscle precursor cells, while myogenin and MRF4 act downstream of Myf5 and MyoD to control the differentiation process (
      • Yun K.
      • Wold B.
      ,
      • Arnold H.H.
      • Winter B.
      ). The other group of transcription factors important for muscle differentiation consists of MEF2 proteins which also include four members: MEF2A, -2B, -2C, and -2D (
      • Black B.L.
      • Olson E.N.
      ). MRF and MEF2 members can physically interact with each other to synergistically activate many muscle-specific genes (
      • Molkentin J.D.
      • Olson E.N.
      ,
      • Molkentin J.D.
      • Black B.L.
      • Martin J.F.
      • Olson E.N.
      ).
      Among four MRFs, myogenin is critically involved in executing the differentiation program. Although myoblasts from myogenin null mice are present, they do not differentiate in vivo leading to severe muscle deficiency and perinatal death of the homozygous mice (
      • Hasty P.
      • Bradley A.
      • Morris J.H.
      • Edmondson D.G.
      • Venuti J.M.
      • Olson E.N.
      • Klein W.H.
      ,
      • Nabeshima Y.
      • Hanaoka K.
      • Hayasaka M.
      • Esumi E.
      • Li S.
      • Nonaka I.
      ). Expression of myogenin is considered one of the earliest molecular markers for cells committed to differentiation in vitro. The up-regulation of myogenin, in concomitant with induction of the cell cycle inhibitor p21 cip1 , indicates that cells have irreversibly withdrawn from the cell cycle and entered the differentiation program (
      • Lassar A.B.
      • Skapek S.X.
      • Novitch B.
      ). Regulation of myogenin has been extensively studied by both transfection analyses in cell culture systems and transgenic studies (
      • Cheng T.C.
      • Hanley T.A.
      • Mudd J.
      • Merlie J.P.
      • Olson E.N.
      ,
      • Yee S.P.
      • Rigby P.W.
      ,
      • Edmondson D.G.
      • Cheng T.C.
      • Cserjesi P.
      • Chakraborty T.
      • Olson E.N.
      ,
      • Buchberger A.
      • Ragge K.
      • Arnold H.H.
      ,
      • Malik S.
      • Huang C.F.
      • Schmidt J.
      ,
      • Johanson M.
      • Meents H.
      • Ragge K.
      • Buchberger A.
      • Arnold H.H.
      • Sandmoller A.
      ). A 133-bp myogenin proximal promoter has been found to contain sufficient cis-elements to correctly target a lacZ transgene to specific muscle-forming regions (
      • Yee S.P.
      • Rigby P.W.
      ). Furthermore, an E box, a MEF2 site, and a MEF3 site within this 133-bp proximal myogenin promoter have been identified as critical cis-elements regulating myogenin expression (
      • Cheng T.C.
      • Hanley T.A.
      • Mudd J.
      • Merlie J.P.
      • Olson E.N.
      ,
      • Yee S.P.
      • Rigby P.W.
      ,
      • Edmondson D.G.
      • Cheng T.C.
      • Cserjesi P.
      • Chakraborty T.
      • Olson E.N.
      ,
      • Buchberger A.
      • Ragge K.
      • Arnold H.H.
      ,
      • Malik S.
      • Huang C.F.
      • Schmidt J.
      ,
      • Johanson M.
      • Meents H.
      • Ragge K.
      • Buchberger A.
      • Arnold H.H.
      • Sandmoller A.
      ,
      • Spitz F.
      • Demignon J.
      • Porteu A.
      • Kahn A.
      • Concordet J.P.
      • Daegelen D.
      • Maire P.
      ). MyoD, MEF2, and homeodomain-containing Six (see “Discussion”) proteins have been implicated as the nuclear factors binding to these cis-elements. Despite the above progress, how exactly these nuclear factors are activated by various intracellular signaling pathways is less well understood at present. Nor is known about the relationships between these cis-elements/transacting factors and the IGF signaling pathway.
      Insulin-like growth factors (IGFs) have been shown to potently stimulate myogenesis in cultured myogenic cells and are required for normal skeletal muscle development during mouse embryogenesis (
      • Powell-Braxton L.
      • Hollingshead P.
      • Warburton C.
      • Dowd M.
      • Pitts-Meek S.
      • Dalton D.
      • Gillett N.
      • Stewart T.A.
      ,
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ). In rat L6 myogenic cells, IGFs display biphasic action profiles: initially they stimulate proliferation, then function as strong inducers of differentiation (
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ,
      • Rosenthal S.M.
      • Cheng Z.Q.
      ,
      • Engert J.C.
      • Berglund E.B.
      • Rosenthal N.
      ). One of the molecular mechanisms underlying the stimulatory myogenic effect of IGFs lies in their abilities to transcriptionally induce myogenin mRNA (
      • Florini J.R.
      • Ewton D.Z.
      • Roof S.L.
      ). It remains obscure how exactly this is achieved.
      Several intracellular signaling pathways have been identified that are activated in response to IGF stimulation. One of them is mitogen-activated protein kinase (MAPK)-mediated signaling pathway. It has been shown that the ERK subgroup of MAPKs can be activated by IGF treatment via the classical receptor tyrosine kinase/Grb2-Sos/Ras/Raf mediated pathway (
      • Petley T.
      • Graff K.
      • Jiang W.
      • Yang H.
      • Florini J.
      ). Activation of ERK may be partially responsible for the initial mitogenic effect of IGF (
      • Rosenthal S.M.
      • Cheng Z.Q.
      ,
      • Coolican S.A.
      • Samuel D.S.
      • Ewton D.Z.
      • McWade F.J.
      • Florini J.R.
      ). Another IGF-activated intracellular signaling pathway that has attracted much attention is mediated by phosphatidylinositol 3-kinase (PI3K). In response to IGF stimulation, activated PI3K converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate which results in subsequent activation of the pleckstrin homology domain-containing serine/threonine kinases PDK1 and Akt/PKB (
      • Marte B.M.
      • Downward J.
      ,
      • Alessi D.R.
      • Cohen P.
      ,
      • Leevers S.J.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Activated PI3K also leads to activation of p70 S6K , which is mediated mainly by mTOR, PDK1, and atypical PKCs (
      • Dufner A.
      • Thomas G.
      ).
      It has been demonstrated that PI3K mediates the stimulatory effect of IGFs on muscle differentiation (
      • Coolican S.A.
      • Samuel D.S.
      • Ewton D.Z.
      • McWade F.J.
      • Florini J.R.
      ,
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ). Specific interference of endogenous PI3K activity abolishes myogenic differentiation. Akt is shown to mediate the PI3K effect during muscle differentiation (
      • Jiang B.H.
      • Aoki M.
      • Zheng J.Z.
      • Li J.
      • Vogt P.K.
      ). Deliberate activation of either PI3K or Akt greatly enhances muscle differentiation (
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Jiang B.H.
      • Aoki M.
      • Zheng J.Z.
      • Li J.
      • Vogt P.K.
      ). However, the downstream targets that couple PI3K/Akt stimulatory signals to myogenic signaling pathways remain to be identified.
      The present study sought to identify the signaling pathways mediating the effect of IGF on myogenin expression in C2C12 cells, and to define the IGF-responsive cis-elements on myogenin promoter and the nuclear signal receivers/transcription factors that bind these cis-elements. We showed PI3K, Akt, and p70 S6K are critical signaling molecules mediating the IGF effect. Using IGF-mediated myogenin induction as a model, we demonstrated that the stimulatory effect of PI3K/Akt on myogenin promoter activation was mediated by the unique E box, MEF2, and MEF3 sites in the 133-bp proximal myogenin promoter. MyoD and MEF2 family proteins are implicated as downstream targets of PI3K/Akt. p70 S6K was also shown to mediate part of the PI3K signal along with Akt. We also carried out similar experiments in RD cells, a cell line derived from Rhabdomyosarcoma (RMS), a childhood malignant tumor expressing myogenic regulatory factors yet failing to undergo myogenic differentiation (
      • Anderson J.
      • Gordon A.
      • Pritchard-Jones K.
      • Shipley J.
      ,
      • Merlino G.
      • Helman L.J.
      ). We found that IGF1 failed to induce myogenin expression in RD cells. While IGF1 could activate PI3K leading to Akt phosphorylation and activation in RD, the constitutively active PI3K/Akt failed to activate the myogenic reporter genes suggesting the defect in the IGF/PI3K/Akt pathway in RD cells lies downstream to PI3K/Akt.

      Acknowledgement

      We thank Drs. S. P. Yee, P. L. Puri, J. Han, C. Makris, G. Natoli, and M. Nemer for providing various reagents. We also thank Carol Wong for technical assistance.

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