Redox regulation of human protease-activated receptor-2 by activated factor X |
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Authors: | Jobi Klaus Rauch Bernhard H Dangwal Seema Freidel Kerstin Doller Anke Eberhardt Wolfgang Fischer Jens W Schrör Karsten Rosenkranz Anke C |
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Institution: | a Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germanyb Institut für Pharmakologie, Ernst-Moritz-Arndt-Universität, Greifswald, Germanyc Institute for Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, Germanyd Pharmazentrum Frankfurt, Klinikum der J.W. Goethe Universität, Institut für Allgemeine Pharmakologie und Toxikologie, Frankfurt/Main, Germany |
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Abstract: | 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 H2O2 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, H2O2, NF-κB, and HuR. |
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Keywords: | ChIP chromatin immunoprecipitation DCF-DA 2&prime 7&prime -dichlorofluorescein diacetate DETC diethyldithiocarbamate FXa active factor X HuR human antigen R NF-κB nuclear factor κB PAR protease-activated receptor ROS reactive oxygen species SMC smooth muscle cell |
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