Identification of the inhibitory factor for the transcription of inflammatory genes in vascular smooth muscle cells

我们已报道，通过膜靶向p110CAAX的过度表达慢性激活磷脂酰肌醇3-激酶(PI3K)，通过诱导CCAAT/增强子结合蛋白-β(C/eBP-β)和C/eBP-δ诱导大鼠血管平滑肌细胞(VSMC)的促炎基因表达。为探讨一氧化氮(NO)对促炎症基因表达的影响，我们观察了硝普钠(SNP)对PI3K慢性激活的VSMC单核细胞趋化蛋白-1(MCP-1)基因表达的影响。低浓度(0.05 mM)的SNP可显著降低MCP-1mRNA和蛋白的表达及其转录活性，而高浓度(0.5-1.0 mM)的SNP对MCP-1的转录活性无影响。我们发现，SNP诱导C/EBP同源蛋白(CHOP)表达，抑制C/EBP结合活性，并以剂量依赖的方式降低PI3K慢性激活所诱导的C/EBP活性，最大剂量可达1.0 mM。一致地，CHOP表达的增加显著降低了PI3K诱导的MCP-1启动子活性。然而，CHOP单独过表达可剂量依赖性地上调MCP-1启动子的活性，直至高浓度。MCP-1启动子缺失分析和凝胶迁移率改变分析表明，CHOP-RE位于MCP-1启动子-190~-179碱基之间。以CHOP-RE为诱饵，可显著抑制CHOP或SNP诱导的MCP-1启动子活性的升高。因此，NO供体诱导的CHOP对MCP-1基因表达具有双向作用：通过抑制C/EBPs降低基因表达，通过CHOP-RE促进基因表达。

We have reported that chronic activation of phosphatidylinositol 3-kinase (PI3K) by the overexpression of membrane-targeted p110CAAX induces proinflammatory gene expression in rat vascular smooth muscle cells (VSMCs) through the induction of CCAAT/enhancer binding protein-β (C/EBP-β) and C/EBP-δ. To examine the antiinflammatory effect of nitric oxide (NO) on the proinflammatory gene expression, we investigated the effects of sodium nitroprusside (SNP) on the monocyte chemoattractant protein-1 (MCP-1) gene expression in VSMCs under chronic activation of PI3K. At low concentration (0.05 mM) of SNP, but not at high concentration (0.5-1.0 mM), it significantly reduced MCP-1 mRNA and protein expression as well as its transcriptional activity. We found that SNP induced C/EBP homologous protein (CHOP) expression, which inhibited C/EBP binding activity and reduced the C/EBP activity induced by chronic activation of PI3K in a dose-dependent manner up to 1.0 mM. Consistently, the increase in CHOP expression significantly reduced the MCP-1 promoter activity induced by PI3K. However, the overexpression of CHOP alone up-regulated MCP-1 promoter activity in a dose-dependent manner up to high concentration. Deletion analysis of MCP-1 promoter and electrophoretic mobility shift assay identified the CHOP-response element (CHOP-RE) at the region between -190 and -179 bp of MCP-1 promoter. By CHOP-RE used as a decoy, the increase in promoter activity of MCP-1 induced by either CHOP or SNP was suppressed significantly. Thus, CHOP induced by an NO-donor has bidirectional effects on MCP-1 gene expression : it decreases the gene expression by inhibition of C/EBPs, and it increases the gene expression through the CHOP-RE.