Bidirectional regulation of monocyte chemoattractant protein-1 gene at distinct sites of its promoter by nitric oxide in vascular smooth muscle cells.

We have previously reported that chronic activation of phosphatidylinositol 3-kinase (PI3-kinase) by the overexpression of membrane-targeted p110CAAX induced proinflammatory gene expression in rat vascular smooth muscle cells (VSMCs) through the induction of CCAAT/enhancer binding protein-beta (C/EBP-beta) and C/EBP-delta. To examine the anti-inflammatory effect of nitric oxide (NO) on proinflammatory gene expression, we have investigated the effects of sodium nitroprusside (SNP) on the monocyte chemoattractant protein-1 (MCP-1) gene expression in VSMCs under chronic activation of PI3-kinase. At low concentrations (0.05 mM) of SNP, but not at high concentrations (0.5-1.0 mM), MCP-1 mRNA and protein expression as well as its transcriptional activity were significantly reduced. 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 PI3-kinase 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 PI3-kinase. However, the overexpression of CHOP alone upregulated MCP-1 promoter activity in a dose-dependent manner up to high concentrations. 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 using CHOP-RE as a decoy, we significantly suppressed the increase in promoter activity of MCP-1 induced by either CHOP or SNP. Thus CHOP induced by an NO donor has bidirectional effects on MCP-1 gene expression: it decreases gene expression by inhibition of C/EBPs, and it increases the gene expression through CHOP-RE.