Regulation of protease-activated receptor-1 by vasodilatory prostaglandins via NFAT

We recently reported that prostacyclin suppresses protease-activated receptor-1 (PAR-1) in human vascular smooth muscle cells (VSMC) via cyclic AMP and protein kinase A. This study examines the downstream mechanisms, particularly the role of nuclear factor of activated T-cells (NFAT).

This study provides the first evidence that NFAT2 contributes to the transcriptional control of PAR-1 in human VSMC and that PKA-dependent NFAT2 inhibition represents a mechanism by which vasodilatory prostaglandins regulate the vascular actions of thrombin.

Human saphenous vein VSMC were exposed to phorbol 12-myristate 13-acetate (PMA) to induce endogenous cyclooxygenase-2-dependent prostaglandin generation. This was found to attenuate PAR-1 expression; similar suppression was seen with the EP2-prostaglandin receptor agonist butaprost. Stimulation of the 'exchange protein directly activated by cyclic AMP' (EPAC) was without effect. The NFAT inhibitor cyclosporin A (CsA) or NFAT2 siRNA both reduced PAR-1 mRNA and protein expression and prevented the stimulatory effects of thrombin or PAR-1 activating peptide (TFLLRN) on ERK1/2 phosphorylation and interleukin-6 expression. CsA or mutation of the NFAT binding motif in the PAR-1 promoter also blunted PAR-1 promoter activity (luciferase reporter assay). These inhibitory actions of CsA were comparable to those of the prostacyclin-mimetic iloprost, and both CsA and iloprost similarly attenuated nuclear NFAT2 localization and binding to the PAR-1 promoter (chromatin immunoprecipitation assay). Conclusions: This study provides the first evidence that NFAT2 contributes to the transcriptional control of PAR-1 in human VSMC and that PKA-dependent NFAT2 inhibition represents a mechanism by which vasodilatory prostaglandins regulate the vascular actions of thrombin.