Abstract 1170: SHP2-dependent Dephosphorylation of p190A Rho GAP Induces RhoA Activation by Angiotensin II in Vascular Smooth Muscle Cells

Abstract

Angiotensin II (Ang II) is a major regulator of blood pressure, that essentially acts through activation of Ang II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R receptor activates numerous intracellular signaling pathways, including the small G protein RhoA known to control VSMC proliferation, migration, differentiation and contraction. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor, that replaces bound GDP by GTP and/or inhibition of Rho GTPase-activating-protein (GAP) that hydrolyzes GTP to GDP. Here we assess the involvement of the p190A Rho GAP in RhoA activation induced by Ang II. The introduction of small interfering RNA (siRNA) targeting p190A in VSMC from rat aorta increased basal RhoA-Rho kinase pathway activity (790 ± 11% of control, n = 3). Moreover p190A-siRNA abolished the early activation of RhoA-Rho kinase pathway induced after 5 min of AngII (0.1 μM) stimulation but not the delayed RhoA activation induced after 60 min. We then measured p190A tyrosine phosphorylation known to reflect its activity. In resting VSMC, p190A was basally phosphorylated. In the presence of the AT2R inhibitor PD123319 (1 μM), activation of AT1R induced p190A dephosphorylation that was maximal at 5 min of Ang II stimulation (26 ± 5% of control, n = 4). The activation of AT2R by Ang II in the presence of losartan (1 μM) had no effect, neither on RhoA activation nor on p190A phosphorylation. Expression of a p190A phosphomimetic mutant decreased the basal activity of RhoA-Rho kinase pathway. In contrast expression of catalytically inactive or phosphoresistant p190A mutants increased the basal activity of RhoA-Rho kinase pathway and inhibited RhoA activation by Ang II. Moreover, using siRNA, we show that the tyrosine phosphatase SHP2, known to be activated by AT1R stimulation, was necessary for Ang II-mediated p190A dephosphorylation and RhoA activation. Our work demonstrates that RhoA/Rho kinase activity is controlled by the p190A Rho GAP in VSMC. Activity of p190A is basally high and maintains a low level of RhoA-Rho kinase activity in resting VSMC. Dephosphorylation of p190A through SHP2-dependent process is required for AT1R-induced RhoA activation.