Urotensin-II Induces Vascular Smooth Muscle Cell Proliferation and Creb Phosporylation Through Store Operated Calcium Entry and EGFR Transactivation

Urotensin-II (U-II) is a vasoactive peptide with many effects in the cardiovascular system. It has been described to induce vascular smooth muscle cell (SMC) proliferation, and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling; however, its signalling pathway still remains unclear. The aim of this study was to elucidate the role of calcium (Ca2+)-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation, focussing our attention on store-operated Ca2+ entry (SOCE) pathway and epithelium growth factor receptor (EGFR) transactivation.We used 5-bromo-2-deoxyuridine (BrdU) labeling, molecular knockdown with small interfering RNA (siRNA) and immunofluorescence to study UII-promoted aortic SMC proliferation. Ca2+ mobilization assays were performed to study Ca2+ entry in cultured SMC.We found that UII enhanced intracellular Ca2+ concentration ([Ca2+]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as STIM1, Orai1, or TRPC1. Moreover, UII stimulated SMC proliferation and Ca2+/cAMP response element-binding protein (CREB) activation through SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Additionally, we determined that epithelium growth factor receptor (EGFR) transactivation, extracellular signal-regulated kinase (ERK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways were involved in UII-mediated Ca2+ influx, CREB activation and VSMCs proliferation.Our data show for the first time that UII-induced SMC proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1 and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.Acknowledgements: This study was supported by Spanish Ministry of Science and Innovation [BFU-2010-21043-C02-01; BFU-2010-21043-C02-02]; Instituto Carlos III [RD12/0042/0041, PI12/00941]; and The Andalusian Government [P10-CVI-6095; PI-0108-2012].