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

磷脂酰肌醇3-激酶(Pi3-Kinase，PI-3-Kinase)是血管平滑肌细胞(VSMCs)胰岛素信号传导的关键分子。为探讨PI3K的慢性激活对VSMC基因表达的影响，通过腺病毒介导的基因转移方法，在大鼠SMC中高表达PI3K催化亚基p110CAAX。与胰岛素的S效应相似，高表达p110CAAX的细胞中单核细胞趋化蛋白-1(MCP-1)的表达比对照细胞高10~15倍。凝胶迁移率改变分析表明，p110CAAX的过表达既不能激活NF-κB结合活性，也不能激活激活蛋白(AP-1)结合活性。为探讨p110CAAX诱导单核细胞趋化蛋白-1基因表达的机制，克隆了3.6kb的单核细胞趋化蛋白-1基因上游区域，并用荧光素酶报告基因分析其启动子活性。我们发现，位于MCP-1基因上游2.6-3.6kb之间的两个CCAAT/增强子结合蛋白(C/EBP)结合位点负责p110CAAX的诱导。过表达C/eBP-β和C/eBP-δ而不是C/eBP-α可诱导6-8倍的单核细胞趋化蛋白-1启动子活性。P110CAAX过表达可诱导VSMC中CIEBP-β和C/EBP-δ的基因表达和核表达。胰岛素浓度为1~10 nM时，VSMC中C/eBP-β和C/eBP-δ的核表达增加。这些结果清楚地表明，PI3K的激活通过激活C/eBP-β和CIEBP-δ而不是通过激活NF-κB来诱导促炎基因的表达，这可能解释了胰岛素在胰岛素抵抗状态下的促炎作用。

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.