Abstract 5496: Vasodilatory Prostaglandins Transcriptionally Regulate Protease-Activated Receptor-1 in Human Vascular Smooth Muscle Cells via Inhibition of Nuclear Factor of Activated T-Cells
Protease-activated receptor PAR-1 mediates the mitogenic effects of thrombin in vascular smooth muscle cells (VSMC) and contributes to neointima formation in vivo. We recently reported that prostacyclin downregulates PAR-1 in human VSMC via activation of cyclic AMP-dependent protein kinase A (PKA), and now investigated NFAT (nuclear factor of activated T-cells) as a potential regulatory target downstream of Gs-coupled prostaglandin receptors and PKA. In human saphenous vein VSMC, the calcineurin/NFAT inhibitor cyclosporin A (CsA) increased cytosolic vs. nuclear accumulation of NFAT1c, a measure of NFAT inactivation, and downregulated PAR-1 mRNA and protein in a time -and concentration-dependent manner (significantly at 3–10 μmol/L, 16–24h). CsA also reduced PAR-1 promoter activity in a luciferase reporter assay to a comparable extent as site-directed mutagenesis of an NFAT/AP1 concensus sequence in the promoter. Moreover, PAR-1 mRNA expression was significantly attenuated by siRNA against NFAT1c (10 nmol/L). The prostacyclin analog iloprost (10 nmol/L, 3h) significantly blunted PAR-1 promoter activity and enhanced cytosolic NFAT accumulation via PKA-dependent mechanisms, and reduced NFAT binding to the human PAR-1 promoter in an electrophoretic mobility shift assay. Induction of interleukin-6 (IL-6) mRNA in response to thrombin (3 IU/L, 30 min) or a PAR-1 activating peptide (TFLLRN, 200 μmol/L, 3h) was impaired by pretreatment (24h) of VSMC with either iloprost (10 nmol/L) or CsA (10 μmol/L) or by siRNA against NFAT1c (48h). The regulatory effects of iloprost on PAR-1 were mimicked by selective activators of Gs-coupled EP2 or EP4 prostaglandin receptors as well as by induction of cyclooxygenase-2 with phorbol 12-myristate 13-acetate (100 nmol/L) to promote endogenous prostaglandin generation. This study provides the first evidence that NFAT contributes to the constitutive regulation of PAR-1, and that endogenous prostaglandins acting via Gs-coupled prostanoid receptors control PAR-1 expression and function by suppressing the transcriptional activity of NFAT in human VSMC.