Abstract 5278: Essential Role of the Rho Exchange Factor Arhgef1 for Angiotensin II-induced RhoA/Rho Kinase Activation and Arterial Tone Regulation

Abstract

Rho exchange factors (Rho-GEFs) are signaling molecules responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although over-activation of the small G protein RhoA and its effector Rho kinase is recognized as a critical component of the pathogenesis of hypertension in experimental models and in human, the molecular mechanisms and the RhoA-GEF(s) involved in this activation of RhoA signaling in vascular smooth muscle cells (VSMCs) are still unknown. Nevertheless, angiotensin II (Ang II) has been shown to play a key role in the over-activation of RhoA in hypertension. Here we identify the RhoA-GEF and the molecular mechanism responsible for the activation of RhoA and Rho kinase in response to Ang II type 1 receptor activation (AT1R) in rat aortic VSMCs. Systematic functional analysis by siRNA-mediated silencing of the 28 RhoA GEFs indicated that only Arhgef1 (p115, lsc) is involved in Ang II (0.1 μ M)-mediated RhoA/Rho kinase activation. Knock-down of Arhgef1 had no significant effect on the activation of RhoA/Rho kinase induced by the thromboxan analog U46619, endothelin-1 and noradrenaline. The potency and efficacy of noradrenaline, phenyl-ephrine, U46619 and endothelin-1 to elicit contraction of rat aortic rings were not modified by Arhgef1 silencing whereas the contractile effect of Ang II was abolished. Ang II-induced Arhgef1 activation was further confirmed by in vitro guanine nucleotide exchange assay on RhoA, and we demonstrated by the transfection of Arhgef1 mutants that Ang II-induced Arhgef1 activation was due to its phosphorylation on Tyr738 by the tyrosine kinase Jak2. To confirm the functional role of Arhgef1 in Ang II-mediated regulation of vascular tone and hypertension, we then determined the activity of Arhgef1 in arteries from Ang II treated rats by pull-down assay. Chronic infusion of Ang II (250 ng/kg/min, 14 days) through an osmotic minipump induced an increase in arterial pressure associated with enhanced RhoA/Rho kinase activity and activation of Arhgef1 in arteries. Our results thus show that Arhgef1 activation and its downstream RhoA signaling are essential for Ang II-induced contraction and identify Arhgef1 as a potential specific target for the treatment of Ang II-dependent hypertension.