Regulation of Human Vascular Protease-Activated Receptor-3 through mRNA Stabilization and the Transcription Factor Nuclear Factor of Activated T Cells (NFAT) Anke C. Rosenkranz, Bernhard H. Rauch, 1 Anke Doller, Wolfgang Eberhardt, Andreas Bo ¨ hm, Ellen Bretschneider, and Karsten Schro ¨r Institut fu ¨ r Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universita ¨ t Du ¨ sseldorf, Germany (A,C.R., B.H.R., A.B., K.S.); Pharmazentrum Frankfurt, Klinikum der J.W. Goethe Universita ¨ t, Institut fu ¨ r Allgemeine Pharmakologie und Toxikologie, Frankfurt/Main, Germany (A.D., W.E.); and Institut fu ¨ r Vaskula ¨ re Medizin, Medizinische Fakulta ¨ t, Friedrich-Schiller-Universita ¨ t, Jena, Germany (E.B.) Received April 8, 2011; accepted May 18, 2011 ABSTRACT Thrombin promotes vascular smooth muscle cell (SMC) prolif- eration and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynami- cally regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 ex- pression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the pres- ence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shut- tling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regu- lated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and pre- vented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury. Introduction The clinical success of venous bypass grafting is often limited by constrictive vascular remodelling. This involves vascular smooth muscle cell (SMC) proliferation, migration, and inflammation (Forrester et al., 1991), in which the clot- ting factor thrombin plays a central role (Schro ¨r et al., 2010). Thrombin acts via protease-activated receptors (PARs), which are proteolytically cleaved to create a new NH 2 -termi- nal domain that autoactivates the receptor (Coughlin, 2000). Of four known PARs, PAR-1, PAR-3, and PAR-4 are activated by thrombin, responding with EC 50 values of 50 pM, 0.2 nM, and 5 nM, respectively (Steinberg, 2005; Schro ¨r et al., 2010). PAR-1 is the prototypical receptor mediating most thrombin actions (Wilcox et al., 1994; Coughlin, 2000), whereas PAR-4 mediates mouse and human platelet activation (Coughlin, This study was supported in part by the Deutsche Forschungsgemeinschaft [SCH 194/11-10; SFB 612 Project B11], the Forschungskommission der Hei- nrich-Heine-Universita ¨t Du ¨ sseldorf [Grant 9772399]; the Ernst und Berta Grimmke-Stiftung [Grant 3/09]; and the Gesellschaft fu ¨ r Thrombose- und Ha ¨ mostase- Forschung [Rudolf-Marx Stipend]. 1 Current affiliation: Institut fu ¨ r Pharmakologie, Ernst-Moritz-Arndt-Uni- versita ¨ t Greifswald, Germany. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.111.072850. ABBREVIATIONS: SMC, smooth muscle cell; CsA, cyclosporin A; EPAC, exchange protein directly activated by cAMP; ERK1/2, extracellular- regulated kinase 1/2; HuR, human antigen R; IBMX, isobutyl-1-methylxanthine; IL-6, interleukin-6; NFAT, nuclear factor of activated T cells; PAR, protease-activated receptor; PGI 2 , prostacyclin; PKA, protein kinase A; PKI, myristoylated protein kinase A inhibitor; PMA, phorbol 12-myristate 13-acetate; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; UTR, untranslated region; IP, immunoprecipitation; siRNA, short interfering RNA; db-cAMP, dibutyryl cAMP; 8CPT- 2Me-cAMP, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, sodium salt; COX-2, cyclooxygenase-2. 0026-895X/11/8002-337–344$25.00 MOLECULAR PHARMACOLOGY Vol. 80, No. 2 Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics 72850/3705140 Mol Pharmacol 80:337–344, 2011 Printed in U.S.A. 337 at ASPET Journals on December 29, 2017 molpharm.aspetjournals.org Downloaded from