Nitric Oxide Inhibits Matrix Metalloproteinase-2 Expression Via the Induction of Activating Transcription Factor 3 in Endothelial Cells

Nitric oxide (NO) has been shown to inhibit migration of cells in which various matrix metalloproteinases (MMPs) are involved. The underlying molecular mechanisms of this inhibition remain elusive. Endothelial cells (ECs) constitutively produce MMP-2. The effect of NO on MMP-2 expression was examined. A dose-dependent inhibition of MMP-2 mRNA level was demonstrated in ECs treated with NO. ECs infected with adenovirus carrying endothelial NO synthase (Ade-NOS) reduced MMP-2 expression. The inhibitory effect of NO on MMP-2 expression was a transcriptional event because NO reduced MMP-2 promoter activity. NO treatment of ECs consequently suppressed MMP-2 secretion revealed by zymographic assay. Functional analysis of MMP-2 promoter (1716 base pairs) indicated that the p53-binding site (-1659 to -1629) was crucial for MMP-2 promoter activity. Activating transcription factor 3 (ATF3) has been reported to act as a transcriptional repressor for p53. ECs treated with NO induced ATF3 expression. Consistently, Ade-NOS-infected ECs showed an increase of ATF3 level. Moreover, ECs either over-expressed ATF3 or, when treated with an ATF3 activator (MG-132; carbobenzoxy-l-leucyl-l-leucyl-l-leucinal), resulted in a repression of MMP-2 promoter activity. Because of MMP-2 suppression by NO, ECs treated with NO inhibited endothelial migration, a phenomenon similar to that of ECs treated with MMP-2 antibody or MG-132. These results indicate that NO-attenuating endothelial migration is mediated at least in part by its reduction of MMP-2 expression via the up-regulation of ATF3. This study provides a molecular basis that supports the notion that NO acts as a negative regulator in endothelial migration.