Insulin-like growth factor I-mediated activation of the transcription factor cAMP response element-binding protein in PC12 cells. Involvement of p38 mitogen-activated protein kinase-mediated pathway.

IGF-I is known to support growth and to prevent apoptosis in neuronal cells. Activation of the nuclear transcription factor cAMP response element-binding protein (CREB) has emerged as a central determinant in neuronal functions. In the present investigation, we examined the IGF-I-mediated phosphorylation and transcriptional activation of CREB in rat pheochromocytoma (PC12) cells, a cellular model for neuronal differentiation, and defined three distinct postreceptor signaling pathways important for this effect including the p38 mitogen-activated protein kinase (MAPK) pathway. CREB phosphorylation at serine 133 and its transcriptional activation as measured by a CREB-specific Gal4-CREB reporter and the neuroendocrine-specific gene chromogranin A was induced 2-3.3-fold by insulin-like growth factor (IGF)-I. This activation was significantly blocked (p < 0.001) by the dominant negative K-CREB or by mutation of the CRE site. IGF-I stimulated chromogranin A gene expression by Northern blot analysis 3.7-fold. Inhibition of MAPK kinase with PD98059, PI 3-kinase with wortmannin, and p38 MAPK with SB203580 blocked IGF-I-mediated phosphorylation and transcriptional activation of CREB by 30-50% (p < 0.001). Constitutively active and dominant negative regulators of the Ras and PI 3-kinase pathways confirmed the contribution of these pathways for CREB regulation by IGF-I. Cotransfection of PC12 cells with p38beta and constitutively active MAPK kinase 6 resulted in enhanced basal as well as IGF-I-stimulated chromogranin A promoter. IGF-I activated p38 MAPK, which was blocked by the inhibitor SB203580. This is the first description of a p38 MAPK-mediated nuclear signaling pathway for IGF-I leading to CREB-dependent neuronal specific gene expression.

Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3-L1 cell lines.

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf --> MEK pathway and has an impact on physiologically relevant genes in these cells.