A novel effect of insulin on vascular smooth muscle cells through the transcriptional regulation ?the meaning of the activation of C/EBP

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Phosphatidylinositol 3-kinase (PI 3-kinase is a key molecule mediating signals of insulin in vascular smooth muscle cells (VSMCs). To examine the-effect of chronic activation of PI 3-kinase on the gene expression of VSMCs, membrane-targeted p110CAAX, a catalytic subunit of PI 3-kinase, was o overexpressed m rat SMCs by adenovirus-mediated gene transfer. Similar to insulin' s effects, cells overexpressing p110CAAX exhibited 10-to 15-fold increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression as compared with the control cells. Electrophoretic mobility shift assay showed that the overexpression of p110CAAX activated neither the NF-κB binding nor the activator protein (AP-1) binding activities. To investigate the mechanisms for the induction of MCP-1 gene expression by p110CAAX, 3.6 kb of the upstream region of MCP-1 gene was cloned and the promoter activity was analyzed by luciferase reporter assay. We found that two CCAAT/enhancer binding protein (C/EBP) binding sites located between 2.6 and 3.6 kb upstream of the MCP-1 gene were responsible for the induction by p110CAAX. The overexpression of C/EBP-β and C/EBP-δ but not C/EBP-α caused 6-to 8-fold induction of MCP-1 promoter activity. Consistently, the overexpression of p110CAAX induced mRNA expression and nuclear expression of CIEBP-β and C/EBP-δ in VSMCs. Insulin at 1 to 10 nM also increased nuclear expression of C/EBP-βand C/EBP-δ in VSMCs. These results clearly indicate that the activation of PI 3-kinase induced proinflammatory gene expression through activating C/EBP-β and CIEBP-δ but not NF-κB, which may explain the proinflammatory effect of insulin in the insulin-resistant state.