p63RhoGEF—a key mediator of angiotensin II-dependent signaling and processes in vascular smooth muscle cells

Abstract

The purpose of our study was to investigate the role of endogenous p63RhoGEF in G(q/11)-dependent RhoA activation and signaling in rat aortic smooth muscle cells (RASMCs). Therefore, we studied the expression and subcellular localization in freshly isolated RASMCs and performed loss of function experiments to analyze its contribution to RhoGTPase activation and functional responses such as proliferation and contraction. By this, we could show that p63RhoGEF is endogenously expressed in RASMCs and acts there as the dominant mediator of the fast angiotensin II (ANG II)-dependent but not of the sphingosine-1-phosphate (S(1)P)-dependent RhoA activation. p63RhoGEF is not an activator of the concomitant Rac1 activation and functions independently of caveolae. The knockdown of endogenous p63RhoGEF significantly reduced the mitogenic response of ANG II, abolished ANG II-induced stress fiber formation and cell elongation in 2-D culture, and impaired the ANG II-driven contraction in a collagen-based 3-D model. In conclusion, our data provide for the first time evidence that p63RhoGEF is an important mediator of ANG II-dependent RhoA activation in RASMCs and therewith a leading actor in the subsequently triggered cellular processes, such as proliferation and contraction.

The blocking of angiotensin II type 1 receptor and RhoA/Rho kinase activity in hypertensive patients: Effect of olmesartan medoxomil and implication with cardiovascular-renal remodeling

“…Given these effects of olmesartan on oxidative stress and on the improvement of endothelial dysfunction including inflammatory processes mediated by Ang II and given that RhoA/Rho kinase activation, as mentioned above, is mainly involved in the Ang II signaling following Ang II AT1R activation including oxidative stress, [1][2][3]9 it comes as no surprise that olmesartan may be able to downregulate the RhoA/Rho kinase pathway via AT1R blockade and reduction of oxidative stress effects. This is exactly what is reported by the results of the present study, which show in hypertensive patients on olmesartan treatment a reduced protein level of p63RhoGEF, the transducer of Ang II message to directly activate RhoA/Rho kinase pathway 5 and a reduced phosphorylation state of MYPT-1, the regulatory subunit of MLC phosphatase (MLCP), the target of Rho kinase-mediated inhibitory phosphorylation of MLCP, this latter resulting in the inhibition of the Rho kinase-induced increased activity of MLC kinase leading to reduced vascular smooth muscle contraction and proliferation. 2 Although the extrapolation of these data obtained in mononuclear cells to mechanisms that usually take place in endothelial and smooth muscle cells might be considered a limitation, it should be noted that circulating blood cells are widely used in vascular biology to study ex-vivo pathophysiological mechanisms of hypertension and remodeling.…”

“…1,9 A critical role for the transduction of Ang II signaling to directly activate the RhoA/ Rho kinase pathway has been provided for p63RhoGEF as demonstrated in vitro by studies of loss of function and functional responses 5,7 and supported by reports of its increased level in animal models of hypertension 20 and in hypertensive patients. 8,21 One of the processes triggered by RhoA/Rho kinase activation is the Ang II-mediated increase of oxidative stress via increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 10 with subsequent endothelial dysfunction, production of proinflammatory cytokines and growth factors ultimately leading to cardiovascular and renal remodeling, target organ damages of hypertension.…”

“…p63RhoGEF has been reported as a specific mediator transducing the Ang II message from activated AT1R via Gq protein to RhoA/ Rho kinase activation 5 via binding of the α subunit of Gq protein. 6,7 This leads to effects of Ang II-mediated activation of RhoA/Rho kinase on the cardiovascular system, such as vascular contraction, proliferation and cardiovascular remodeling.…”

“…19 This study aims to evaluate in mononuclear cells from essential hypertensive patients the effect of a relatively short-term treatment with olmesartan on the RhoA/Rho kinase pathway. To this end we have evaluated the protein expression of p63RhoGEF, a guanine nucleotide exchange factor, key mediator of the RhoA/Rho kinase activation by Ang II, [5][6][7] and the phosphorylation state of MYPT-1, a marker of Rho kinase. 1,2 …”

Does p63RhoGEF, a new key mediator of angiotensin II signalling, play a role in blood pressure regulation and cardiovascular remodelling in humans?

“…However, while the participation of p63RhoGEF in Ang II-induced RhoA activation in humans could be hypothesised based on the results obtained in vitro, 10 no direct supporting evidence is currently available in humans. Such a demonstration should be given, and the human model of Bartter's/Gitelman's (BS/GS) syndromes offers a good opportunity for such an investigation.…”