Cell Cycle Withdrawal Promotes Myogenic Induction of Akt, a Positive Modulator of Myocyte Survival

ABSTRACT During myogenesis, proliferating myoblasts withdraw from the cell cycle, acquire an apoptosis-resistant phenotype, and differentiate into myotubes. Previous studies indicate that myogenic induction of the cyclin-dependent kinase inhibitor p21 results in an inhibition of apoptotic cell death in addition to its role as a negative cell cycle regulator. Here we demonstrate that the protein encoded by theAkt proto-oncogene is induced in C2C12 cells during myogenic differentiation with a corresponding increase in kinase activity. In differentiating cultures, expression of dominant-negative forms of Akt increase the frequency of cell death whereas expression of wild-type Akt protects against death, indicating that Akt is a positive modulator of myocyte survival. Antisense oligonucleotides against p21 block cell cycle withdrawal, inhibit Akt induction, and enhance cell death in differentiating myocyte cultures. Adenovirus-mediated transfer of wild-type or constitutively active Akt constructs confer partial resistance to cell death under conditions where cell cycle exit is blocked by the antisense oligonucleotides. Collectively, these data indicate that cell cycle withdrawal facilitates the induction of Akt during myogenesis, promoting myocyte survival.

[1]  K. Walsh,et al.  Inhibition of Myogenesis by Multiple Cyclin-Cdk Complexes , 1997, The Journal of Biological Chemistry.

[2]  Andrius Kazlauskas,et al.  The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase , 1995, Cell.

[3]  J. Brugge,et al.  Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation , 1997, Molecular and cellular biology.

[4]  G. Hannon,et al.  Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD , 1995, Science.

[5]  C. Stewart,et al.  Insulin-like Growth Factor-II Is an Autocrine Survival Factor for Differentiating Myoblasts (*) , 1996, The Journal of Biological Chemistry.

[6]  Xin Lu,et al.  RB regulates the stability and the apoptotic function of p53 via MDM2. , 1999, Molecular cell.

[7]  P. Vogt,et al.  Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Elledge,et al.  p21 and p57 control muscle differentiation at the myogenin step , 1999 .

[9]  M. Goebl,et al.  Phosphorylation of Nuclear MyoD Is Required for Its Rapid Degradation , 1998, Molecular and Cellular Biology.

[10]  G. Kulik,et al.  Akt-Dependent and -Independent Survival Signaling Pathways Utilized by Insulin-Like Growth Factor I , 1998, Molecular and Cellular Biology.

[11]  Y. Nabeshima,et al.  Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts , 1996, The Journal of cell biology.

[12]  J. Testa,et al.  Structure, expression and chromosomal mapping of c-akt: relationship to v-akt and its implications. , 1993, Oncogene.

[13]  J. Florini,et al.  Hormones, growth factors, and myogenic differentiation. , 1991, Annual review of physiology.

[14]  P. Tsichlis,et al.  Akt Is a Direct Target of the Phosphatidylinositol 3-Kinase , 1996, The Journal of Biological Chemistry.

[15]  L. Tsai,et al.  Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[16]  David R. Kaplan,et al.  Regulation of Neuronal Survival by the Serine-Threonine Protein Kinase Akt , 1997, Science.

[17]  J. Woodgett,et al.  Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. , 1998, The Biochemical journal.

[18]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[19]  B. Gallie,et al.  pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis. , 1996, Genes & development.

[20]  J. Cheng,et al.  Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene. , 1995, Oncogene.

[21]  F. Graham,et al.  An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Wills,et al.  Rb functions to inhibit apoptosis during myocyte differentiation. , 1997, Cancer research.

[23]  N. Rosenthal,et al.  Specific, temporally regulated expression of the insulin-like growth factor II gene during muscle cell differentiation. , 1993, Endocrinology.

[24]  S. Elledge,et al.  p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells , 1995, Science.

[25]  P. Rotwein,et al.  Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation. , 1993, Molecular endocrinology.

[26]  S. Elledge,et al.  p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step. , 1999, Genes & development.

[27]  M. Abdellatif,et al.  Human E2F-1 reactivates cell cycle progression in ventricular myocytes and represses cardiac gene transcription. , 1996, Developmental biology.

[28]  Lewis C Cantley,et al.  PI3K: Downstream AKTion Blocks Apoptosis , 1997, Cell.

[29]  P. Nisen,et al.  Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery. , 1997, The Journal of clinical investigation.

[30]  F. McCormick,et al.  Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. , 1997, Science.

[31]  C. Pinset,et al.  Cell Cycle–regulated Expression of the Muscle Determination Factor Myf5 in Proliferating Myoblasts , 1998, The Journal of cell biology.

[32]  B. Hemmings.  Akt Signaling--Linking Membrane Events to Life and Death Decisions , 1997, Science.

[33]  H. Perlman,et al.  Cell cycle exit upon myogenic differentiation. , 1997, Current opinion in genetics & development.

[34]  John Calvin Reed,et al.  Regulation of cell death protease caspase-9 by phosphorylation. , 1998, Science.

[35]  W. Gu,et al.  Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells. , 1994, Science.

[36]  L. Peso,et al.  Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. , 1997, Science.

[37]  N. Tonks,et al.  Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. , 1997, Science.

[38]  J. Dominov,et al.  Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells , 1998, The Journal of cell biology.

[39]  A. Zantema,et al.  Cyclin D1 is an essential mediator of apoptotic neuronal cell death. , 1996, The EMBO journal.

[40]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[41]  J R Florini,et al.  "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. , 1991, The Journal of biological chemistry.

[42]  A GLUCKSMANN,et al.  Cell deaths in normal vertebrate ontogeny. , 1951, Biological reviews of the Cambridge Philosophical Society.

[43]  A. Ross,et al.  The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells , 1996, Molecular and cellular biology.

[44]  P. Rotwein,et al.  Coordinate expression of insulin-like growth factor II and its receptor during muscle differentiation. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[45]  G. Lyons,et al.  Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin , 1998, Oncogene.

[46]  R. Roth,et al.  Akt, a Pleckstrin Homology Domain Containing Kinase, Is Activated Primarily by Phosphorylation* , 1996, The Journal of Biological Chemistry.

[47]  K. Walsh,et al.  MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation , 1995, Molecular and cellular biology.

[48]  J. Labbé,et al.  cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity , 1999, Molecular and Cellular Biology.

[49]  D. Pot,et al.  A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain , 1997, Molecular and cellular biology.

[50]  J. Testa,et al.  Membrane translocation and activation of the Akt kinase in growth factor-stimulated hematopoietic cells. , 1997, Leukemia research.

[51]  W Baumeister,et al.  Proteasome from Thermoplasma acidophilum: a threonine protease. , 1995, Science.

[52]  A. Mahfoudi,et al.  p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene. , 1997, Genes & development.

[53]  S. R. Datta,et al.  Akt Phosphorylation of BAD Couples Survival Signals to the Cell-Intrinsic Death Machinery , 1997, Cell.

[54]  J. Cidlowski,et al.  Cell cycle regulation and apoptosis. , 1998, Annual review of physiology.

[55]  David R. Kaplan,et al.  Direct Regulation of the Akt Proto-Oncogene Product by Phosphatidylinositol-3,4-bisphosphate , 1997, Science.

[56]  R. Kitsis,et al.  Induction of DNA synthesis and apoptosis in cardiac myocytes by E1A oncoprotein , 1996, The Journal of cell biology.

[57]  T. Jacks,et al.  Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle , 1996, The Journal of cell biology.

[58]  K. Walsh,et al.  Resistance to Apoptosis Conferred by Cdk Inhibitors During Myocyte Differentiation , 1996, Science.

[59]  K. Walsh,et al.  Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis , 1996, The Journal of cell biology.

[60]  S. Shankland,et al.  Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27(Kip1). , 1999, The Journal of clinical investigation.

[61]  W. Sellers,et al.  Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. , 1998, Genes & development.