Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.
FASEB J. 2010 Dec;24(12):4865-76. doi: 10.1096/fj.10-155499. Epub 2010 Aug 25.
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.
我们的研究目的是探讨内源性 p63RhoGEF 在 G(q/11) 依赖性 RhoA 激活和信号转导中的作用在大鼠主动脉平滑肌细胞（RASMCs）中。因此，我们研究了在新鲜分离的 RASMCs 中的表达和亚细胞定位，并进行了功能丧失实验，以分析其对 RhoGTPase 激活和功能反应（如增殖和收缩）的贡献。通过这种方法，我们可以证明 p63RhoGEF 在内皮细胞中表达，并作为快速血管紧张素 II（ANG II）依赖性但不是鞘氨醇 1-磷酸（S(1)P）依赖性 RhoA 激活的主要介质发挥作用。p63RhoGEF 不是伴随的 Rac1 激活的激活剂，并且独立于 caveolae 发挥作用。内源性 p63RhoGEF 的敲低显著降低了 ANG II 的促有丝分裂反应，消除了 ANG II 在 2-D 培养中诱导的应力纤维形成和细胞伸长，并损害了基于胶原的 3-D 模型中 ANG II 驱动的收缩。总之，我们的数据首次提供证据表明，p63RhoGEF 是 RASMCs 中 ANG II 依赖性 RhoA 激活的重要介质，并且是随后触发的细胞过程（如增殖和收缩）中的主要参与者。
