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Insulin-induced egr-1 and c-fos Expression in 32D Cells Requires Insulin Receptor, Shc, and Mitogen-activated Protein Kinase, but Not Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Activation*

Open AccessPublished:November 22, 1996DOI:https://doi.org/10.1074/jbc.271.47.30222
      Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulin's effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N., Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37°C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 μM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.

      INTRODUCTION

      Insulin's effects are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1),
      The abbreviations used are: IRS-1
      insulin receptor substrate-1
      PI
      phosphatidylinositol
      MAP
      mitogen-activated protein
      CHO
      Chinese hamster ovary
      MEK
      mitogen-activated protein kinase kinase.
      IRS-2, or Shc, mainly through phosphotyrosine binding domains (reviewed in Ref.
      • Cheatham B.
      • Kahn C.R.
      ). These substrates bind to SH2 domains of several cytoplasmic signal proteins through their tyrosine phosphorylation sites. These proteins include the 85-kDa subunit of phosphatidylinositol (PI) 3′-kinase, GRB-2, or Syp (tyrosine phosphatase) (
      • Cheatham B.
      • Kahn C.R.
      ). Activation of these molecules and the following activation of other intracellular molecules, such as p21ras, Raf-1, mitogen-activated protein (MAP) kinase, or S6 kinase, are believed to be responsible for many of insulin's biological responses. However, many studies suggest that insulin utilizes multiple signal transduction pathways. The insulin signaling network is more complex than was thought a decade ago.
      Insulin has mitogenic effects as well as metabolic effects and affects nuclear events such as gene expression or cell growth (reviewed in Ref.
      • O'Brien R.M.
      • Granner D.K.
      ). One of insulin's effects on nuclear events is the stimulation or inhibition of a number of immediate-early genes (
      • Taub R.
      • Roy A.
      • Dieter R.
      • Koontz J.
      ,
      • Mohn K.L.
      • Laz T.M.
      • Hsu J.-C.
      • Melby A.E.
      • Bravo R.
      • Taub R.
      ). The immediate-early genes are a large and diverse group, and the mechanisms involved in their regulation are complex. The induction of c-fos transcription, one of the well characterized immediate-early genes, by insulin or other growth factors is believed to require receptor phosphorylation and p21ras activation (
      • Stumpo D.J.
      • Stewart T.N.
      • Gilman M.Z.
      • Blackshear P.J.
      ,
      • Medema R.H.
      • Wubbolts R.
      • Bos J.L.
      ). However, recent reports suggested that induction of expression of some immediate-early genes was independent of growth factor receptor autophosphorylation. For instance, Eldredge et al. (
      • Eldredge E.R.
      • Korf G.M.
      • Christensen T.A.
      • Connolly D.C.
      • Getz M.J.
      • Maihle N.J.
      ) reported that epidermal growth factor induced c-fos expression in cells expressing kinase-deficient epidermal growth factor receptors. Mundschau et al. (
      • Mundschau L.J.
      • Forman L.W.
      • Weng H.
      • Faller D.V.
      ) showed that induction of egr-1, but not c-fos, c-myc, and JE, was independent of platelet-derived growth factor receptor autophosphorylation using three different conditions in which platelet-derived growth factor receptor autophosphorylation was blocked. The early growth response gene egr-1, also known as NGF1A, Krox-24, zif 268, and T1S-8, encodes a protein with three zinc finger motifs, structures that are present in many DNA-binding transcription factors (
      • Huang R.-P.
      • Ngo L.
      • Okamura D.
      • Tucker M.
      • Adamson E.D.
      ). We recently demonstrated that insulin induced egr-1 mRNA transcription to a similar level as the maximum stimulation by serum in CHO cells expressing low numbers of insulin receptors (CHONEO cells) or tyrosine kinase-defective insulin receptors (CHOA1018K cells) as well as in cells expressing wild-type insulin receptors (CHOHIRc cells) (
      • Harada S.
      • Smith R.M.
      • Smith J.A.
      • Shah N.
      • Hu D.-Q.
      • Jarett L.
      ). These results indicate the existence of multiple signaling mechanisms, which may operate independently of insulin receptor and IRS-1 phosphorylation and affect some, but not all, nuclear responses to growth factor stimulation.
      A potential problem in interpreting the results with CHO cells is their low levels of endogenous insulin receptor or IRS-1. The argument could be made that undetectable levels of insulin receptor or IRS-1 phosphorylation could account for insulin's effects in the CHONEO and CHOA1018K cells, despite data to the contrary. 32D cells are mouse myeloid progenitor cells and are insensitive to insulin because they have very low levels of insulin receptors and insulin-like growth factor-1 receptors and no detectable IRS-1 or related molecules, e.g. IRS-2/4PS (
      • Wang L.-M.
      • Myers Jr., M.G.
      • Sun X.-J.
      • Aaronson S.A.
      • White M.F.
      • Pierce J.H.
      ). 32D cells overexpressing insulin receptors, IRS-1, or both have been used to investigate the requirement of these molecules in insulin signaling mechanisms. Previous studies using 32D cells demonstrated that insulin receptors or IRS-1 alone is not sufficient for insulin-stimulated mitogenesis (
      • Wang L.-M.
      • Myers Jr., M.G.
      • Sun X.-J.
      • Aaronson S.A.
      • White M.F.
      • Pierce J.H.
      ). IRS-1 is essential for insulin stimulation of PI 3-kinase and p70S6K (
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.-M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      ), whereas insulin receptors alone are sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and MAP kinase (
      • Myers Jr., M.G.
      • Wang L.-M.
      • Sun X.-J.
      • Zhang Y.
      • Yenush L.
      • Schlessinger J.
      • Pierce J.H.
      • White M.F.
      ). In this study, we assessed the effects of insulin on immediate-early gene expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Our data demonstrate that insulin-induced egr-1 and c-fos mRNA expression in 32D cell clones requires the insulin receptor and its phosphorylation, but not IRS-1 phosphorylation. Shc phosphorylation, Shc-GRB-2 association, and MAP kinase activation seem to be a pathway responsible for insulin-induced egr-1 and c-fos expression.

      DISCUSSION

      The insulin signaling network is complex and involves molecules that regulate each other. Adding to the complexity are the observations that different cell types may have different and cell-specific concentrations of these signaling molecules. Recently, several studies have shown that IRS-1 phosphorylation and PI 3-kinase activation, but not p21ras or MAP kinase activation, are necessary for insulin's metabolic effects, such as glucose transporter GLUT4 translocation in 3T3-L1 adipocytes (
      • Gould G.W.
      • Merrall N.W.
      • Martin S.
      • Jess T.J.
      • Campbell I.W.
      • Calderhead D.M.
      • Gibbs E.M.
      • Holman G.D.
      • Plevin R.J.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Haruta T.
      • Morris A.J.
      • Rose D.W.
      • Nelson J.G.
      • Mueckler M.
      • Olefsky J.M.
      ). Regulation of glycogen synthase activity by insulin involves a MAP kinase-independent and rapamycin-sensitive pathway (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cautrecasas P.
      • Saltiel A.R.
      ,
      • Azpiazu I.
      • Saltiel A.R.
      • Depaoli-Roach A.A.
      • Lawrence Jr., J.C.
      ). In contrast, p21ras or MAP kinase activation seems to be related to insulin's mitogenic effects (
      • Sale E.M.
      • Atkinson P.G.P.
      • Sale G.J.
      ,
      • Jhun B.H.
      • Meinkoth J.L.
      • Leitner J.W.
      • Draznin B.
      • Olefsky J.M.
      ). In this study, we have demonstrated that insulin-induced immediate-early gene egr-1 and c-fos mRNA expression requires insulin receptor phosphorylation, Shc phosphorylation, Shc-GRB-2 association, and MAP kinase activation. These effects appear to be independent of IRS-1 phosphorylation or PI 3-kinase activation. Previous studies with 32D cells showed that insulin receptors alone are sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and MAP kinase (
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.-M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      ). Taken together, these data suggest that the insulin receptor, Shc-GRB-2-SOS, p21ras, MEK, and MAP kinase constitute the pathway that leads to insulin-induced immediate-early gene expression.
      Others have shown that insulin-induced mitogenesis, measured by thymidine incorporation, requires IRS-1 phosphorylation (
      • Wang L.-M.
      • Myers Jr., M.G.
      • Sun X.-J.
      • Aaronson S.A.
      • White M.F.
      • Pierce J.H.
      ,
      • Yenush L.
      • Fernandez R.
      • Myers Jr., M.G.
      • Grammer T.C.
      • Sun X.J.
      • Blenis J.
      • Pierce J.H.
      • Schlessinger J.
      • White M.F.
      ), suggesting that an IRS-1 pathway is necessary for complete mitogenesis, which requires many other events, such as translation and activation of transcription factors. The mechanisms involved in translation are not completely understood. In one of the best characterized systems, phosphorylation of eukaryotic translation initiation factor 4E and its binding protein, PHAS-I (phosphorylated heat- and acid-stable protein), correlates with an increase in the rate of protein synthesis under a variety of in vivo conditions including stimulation by insulin. Recently, it has been shown that phosphorylation of eukaryotic translation initiation factor 4E and PHAS-I requires IRS-1-mediated stimulation of PI 3-kinase and p70S6K (
      • Azpiazu I.
      • Saltiel A.R.
      • Depaoli-Roach A.A.
      • Lawrence Jr., J.C.
      ,
      • Mendes P.
      • Myers Jr., M.G.
      • White M.F.
      • Rhoads R.T.
      ). Regulation of PHAS-I seems to be independent of MAP kinase activation or SH2 domain-containing protein-tyrosine phosphatase (
      • Azpiazu I.
      • Saltiel A.R.
      • Depaoli-Roach A.A.
      • Lawrence Jr., J.C.
      ,
      • Mendes P.
      • Myers Jr., M.G.
      • White M.F.
      • Rhoads R.T.
      ). These results suggest that insulin utilizes different pathways depending on the different actions of insulin. Induction of immediate-early genes, one of the earliest steps for mitogenesis, requires Shc and MAP kinase activation, whereas PI 3-kinase and p70S6K are necessary in protein synthesis.
      Interestingly, we saw more Shc phosphorylation in 32D/IR cells than in 32D/IR+IRS cells, although phosphorylation of the insulin receptor β-subunit was less in the former. These results suggest that Shc and IRS-1 may compete for the binding site on the insulin receptor. This speculation is supported by the data obtained with a yeast two-hybrid system showing that both IRS-1 and Shc bind to the same region of the insulin receptor β-subunit (
      • He W.
      • O'Neill T.J.
      • Gustafson T.A.
      ). Therefore, less insulin receptor is available for Shc to bind to in the cells overexpressing IRS-1. Another possibility is that an IRS-1 pathway and a Shc pathway may negatively regulate each other. A recent study demonstrated that constitutively active MEK (MAP kinase kinase) inhibited GLUT4 translocation by negative regulation of PI 3-kinase,
      E. Van Obberghen, unpublished data.
      suggesting that a MAP kinase pathway and a PI 3-kinase pathway negatively regulate each other. These interactions appear to be important for regulation of cell-specific insulin action. Our data showing that inhibition of PI 3-kinase in 32D/IR+IRS cells actually increased egr-1 expression are consistent with these findings.
      Finally, the results with 32D cells demonstrated in this study are different from our previous report with CHO cells, which demonstrated that insulin-induced egr-1, but not c-fos, mRNA expression was independent of phosphorylation of the insulin receptor or IRS-1 (
      • Harada S.
      • Smith R.M.
      • Smith J.A.
      • Shah N.
      • Hu D.-Q.
      • Jarett L.
      ). The reason may be because each cell type utilizes different and cell-specific signaling mechanisms. We saw an increase in tyrosine phosphorylation of 120-kDa proteins in CHO cells, whereas phosphorylation of these 120-kDa proteins was not regulated by insulin in 32D cells. Alternatively, CHO cells, even CHONEO cells, have considerable amounts of insulin receptor or insulin-like growth factor-1 receptor compared with 32D cells. So even though we could not detect receptor or IRS-1 phosphorylation, we cannot rule out the possibility that the downstream substrate may be more sensitive, and minute phosphorylation of the receptor may be enough to conduct insulin signaling.
      In summary, we demonstrated that insulin induced egr-1 and c-fos mRNA expression in a similar manner in 32D/IR cells and 32D/IR+IRS cells, but not in 32D cells or 32D/IRS cells. The signaling mechanisms involved seem to be insulin receptor phosphorylation, Shc phosphorylation, Shc-GRB-2 association, and MAP kinase activation. IRS-1 phosphorylation and PI 3-kinase activation were not involved in the pathway. These results clearly separate and identify the signaling mechanism for one of the many actions of insulin. Whether or not each cell type has different and cell-specific pathways and how to regulate this complicated insulin network will require further study.

      Acknowledgments

      We thank Dr. Alan R. Saltiel for providing PD 98059.

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