ERK Pathway Mediates the Activation of Cdk2 in IGF‐1–Induced Proliferation of Human Osteosarcoma MG‐63 Cells

Insulin‐like growth factor‐1 (IGF‐1) is a potent mitogen for osteoblasts. The primary signaling mechanism involved in mediating this proliferative effect of IGF‐1 is not well defined. The roles of extracellular signal‐regulated kinase 1 (ERK1) and cyclin‐dependent kinase 2 (Cdk2) kinases in the IGF‐1–induced proliferative signaling pathway of human osteosarcoma MG63 cells were investigated using a selective inhibitor of MEK, PD98059, and a Cdk inhibitor, olomoucine. Treatment of MG63 cells with PD98059 and olomoucine inhibited IGF‐1–stimulated proliferation of these cells and induced cell cycle arrest at G0/G1. PD98059 significantly abolished IGF‐1–stimulated kinase activity of ERK1 in a dose‐dependent manner. PD98059 also inhibited the kinase activity of Cdk2 in IGF‐1–stimulated cells, although the inhibition by olomoucine was much greater. The extent of inhibition of Cdk2 activity by PD98059 and olomoucine was consistent with their effects on cell proliferation and cell cycle. Cyclin A was complexed with Cdk2 in unstimulated MG63 cells, but Cdk2 kinase activity in the complex was up‐regulated only in IGF‐1–treated cells. This was consistent with an observed IGF‐1–stimulated hyperphosphorylation of retinoblastoma protein (pRb) with the possibility that the activated Cdk2 kinase is involved in phosphorylation of pRb in IGF‐1–induced cell proliferation. Taken together, these results suggest that the MEK/ERK pathway act in a positive regulatory fashion to activate Cdk2 in IGF‐1–induced mitogenesis in osteoblasts.

[1]  L. J. Korn,et al.  Ras p21 as a potential mediator of insulin action in Xenopus oocytes. , 1987, Science.

[2]  R. Davis,et al.  The mitogen-activated protein kinase signal transduction pathway. , 1993, The Journal of biological chemistry.

[3]  A. Wells,et al.  Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism. , 1996, Molecular biology of the cell.

[4]  Anne Fernandez,et al.  Cyclin a is required for the onset of DNA replication in mammalian fibroblasts , 1991, Cell.

[5]  E. Canalis,et al.  Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. , 1988, Endocrinology.

[6]  K. Pumiglia,et al.  Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Crews,et al.  The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. , 1992, Science.

[8]  J. Nevins,et al.  E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. , 1992, Science.

[9]  C. Sherr.  G1 phase progression: Cycling on cue , 1994, Cell.

[10]  D O Morgan,et al.  Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. , 1992, The EMBO journal.

[11]  A. L. Cahill,et al.  Nicotinic Agonists, Phorbol Esters, and Growth Factors Activate Two Extracellular Signal‐Regulated Kinases, ERK1 and ERK2, in Bovine Chromaffin Cells , 1992, Journal of neurochemistry.

[12]  S. Elledge,et al.  CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Maller,et al.  Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II , 1988, Nature.

[14]  K. M. Mulder,et al.  Transforming Growth Factor β Activation of p44mapk in Proliferating Cultures of Epithelial Cells (*) , 1995, The Journal of Biological Chemistry.

[15]  E. Canalis,et al.  Effect of insulinlike growth factor I on DNA and protein synthesis in cultured rat calvaria. , 1980, The Journal of clinical investigation.

[16]  R. Furlanetto,et al.  Insulin-like growth factor-I induces cyclin-D1 expression in MG63 human osteosarcoma cells in vitro. , 1994, Molecular endocrinology.

[17]  James M. Roberts,et al.  Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle , 1992 .

[18]  G. Johnson,et al.  Ras-dependent growth factor regulation of MEK kinase in PC12 cells. , 1994, Science.

[19]  S. Duursma,et al.  Effects of recombinant human insulin-like growth factor I and II (IGF-I/-II) and growth hormone (GH) on the growth of normal adult human osteoblast-like cells and human osteogenic sarcoma cells. , 1991, Growth regulation.

[20]  R. Weinberg,et al.  Regulation of retinoblastoma protein functions by ectopic expression of human cyclins , 1992, Cell.

[21]  R. Pepperkok,et al.  Cyclin A is required at two points in the human cell cycle. , 1992, The EMBO journal.

[22]  L. Chaudhary,et al.  Activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) by FGF-2 and PDGF-BB in normal human osteoblastic and bone marrow stromal cells: differences in mobility and in-gel renaturation of ERK1 in human, rat, and mouse osteoblastic cells. , 1997, Biochemical and biophysical research communications.

[23]  M. Benito,et al.  Involvement of Raf-1 kinase and protein kinase C zeta in insulin-like growth factor I-induced brown adipocyte mitogenic signaling cascades: inhibition by cyclic adenosine 3',5'-monophosphate. , 1996, Endocrinology.

[24]  C. Kahn,et al.  Insulin receptor substrate 1 mediates insulin and insulin-like growth factor I-stimulated maturation of Xenopus oocytes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[25]  U. Rapp,et al.  Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. , 1992, Genes & development.

[26]  G. Draetta.  Mammalian G1 cyclins. , 1994, Current opinion in cell biology.

[27]  E. Olson,et al.  Interplay between proliferation and differentiation within the myogenic lineage. , 1992, Developmental biology.

[28]  G L Johnson,et al.  Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. , 1994, Science.

[29]  D. Morgan,et al.  Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2 , 1995, Molecular and cellular biology.

[30]  S. Elledge,et al.  Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1 , 1993, Nature.

[31]  E. Canalis,et al.  Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures. , 1989, The Journal of clinical investigation.

[32]  J. Harper,et al.  Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro. , 1993, Molecular biology of the cell.

[33]  C. Rani,et al.  Cell Cycle Arrest Induced by an Inhibitor of Glucosylceramide Synthase , 1995, The Journal of Biological Chemistry.

[34]  R. Kerr.  German super-deep hole hits bottom. , 1994, Science.

[35]  S. Melmed,et al.  Insulin-like growth factor-1 activation of extracellular signal-related kinase-1 and -2 in growth hormone-secreting cells. , 1994, Molecular endocrinology.

[36]  M. Meyerson,et al.  Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase , 1991, Nature.

[37]  James M. Roberts,et al.  Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta. , 1993, Science.

[38]  H. Leonhardt,et al.  Reversal of terminal differentiation and control of DNA replication: Cyclin A and cdk2 specifically localize at subnuclear sites of DNA replication , 1993, Cell.

[39]  M. Kitagawa,et al.  cdc2-like kinase is associated with the retinoblastoma protein. , 1992, Oncogene.

[40]  E. Harlow,et al.  The retinoblastoma protein is phosphorylated during specific phases of the cell cycle , 1989, Cell.

[41]  Jonathan A. Cooper,et al.  Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. , 1995, Current opinion in cell biology.

[42]  D. Morgan,et al.  Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[43]  C. Marshall,et al.  Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation , 1995, Cell.