Redox regulation of human protease-activated receptor-2 by activated factor X

Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMCs) through the protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR-1 and PAR-2 in human vascular SMCs. FXa increased PAR-2 mRNA, protein, and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. PAR-2 regulation was not influenced by the thrombin inhibitor argatroban or PAR-1 siRNA. FXa increased dichlorofluorescein diacetate fluorescence and 8-isoprostane formation and induced expression of the NADPH oxidase subunit NOX-1. NOX-1 siRNA prevented FXa-stimulated PAR-2 regulation, as did ebselen and cell-permeative and impermeative forms of catalase. Exogenous H(2)O(2) increased PAR-2 expression and mitogenic activity. FXa promoted nuclear translocation and PAR-2/DNA binding of nuclear factor κB (NF-κB); NF-κB inhibition prevented PAR-2 regulation by FXa. FXa also promoted PAR-2 mRNA stabilization through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR siRNA abolished FXa-stimulated PAR-2 expression. Thus FXa induces functional expression of PAR-2 but not of PAR-1 in human SMCs, independent of thrombin formation, via a mechanism involving NOX-1-containing NADPH oxidase, H(2)O(2), NF-κB, and HuR.