Sp1 Transcription Factor as a Molecular Target for Nitric Oxide– and Cyclic Nucleotide–Mediated Suppression of cGMP-Dependent Protein Kinase-I&agr; Expression in Vascular Smooth Muscle Cells

cGMP-dependent protein kinase (PKG) expression is highly variable and decreases in cultured vascular smooth muscle cells (VSMCs), exposure of cells to nitric oxide (NO), or in response to balloon catheter injury in vivo. In this study, the mechanisms of human type I PKG-&agr; (PKG-I&agr;) gene expression were examined. Three structurally unrelated NO donors decreased PKG-I&agr; promoter activity after transfection of a promoter/luciferase construct in VSMCs. Promoter deletion analysis demonstrated that (1) a 120-bp promoter containing tandem Sp1 sites was sufficient to drive basal PKG-I&agr; promoter activity, and (2) NO was inhibitory at this site. Cyclic nucleotide analogues also suppressed PKG-I&agr; promoter activity with cAMP being more potent than cGMP. The effects of cyclic nucleotides to suppress PKG-I&agr; promoter activity were attenuated by a specific cAMP-dependent protein kinase (PKA) inhibitor. Single or double mutation of Sp1 binding sites abolished PKG-I&agr; expression. Moreover, Sp1 binding activity on the PKG-I&agr; promoter was detected in A7r5 cells, and this binding was inhibited by NO and cyclic nucleotides. These results indicate that PKG-I&agr; gene expression is driven by an Sp1 transcription mechanism, and that NO and cAMP inhibit Sp1-mediated PKG-I&agr; gene expression through separate mechanisms.

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