Angiopoietin-1 requires IQ domain GTPase-activating protein 1 to activate Rac1 and promote endothelial barrier defense.
Angiopoietin-1 requires IQ domain GTPase-activating protein 1 to activate Rac1 and promote endothelial barrier defense.
复制标题
DOI：
10.1161/atvbaha.111.233189
复制
发表时间：
2011-11
期刊：
影响因子：
--
通讯作者：
Parikh SM
中科院分区：
文献类型：
--
作者：
David S;Ghosh CC;Mukherjee A;Parikh SM
IQ结构域GTP酶激活蛋白1（IQGAP 1）通过其支架特性和通过结合Rho GTP酶Rac 1以维持其活性而有助于上皮细胞中的细胞骨架网络调节。IQGAP 1在血管生成以外的内皮细胞中的功能仍不清楚。我们推测IQGAP 1参与了内皮屏障功能的调节。在人微血管内皮细胞（HMVEC）中沉默IQGAP 1导致粘附连接的破坏，内皮间间隙的形成和屏障功能的降低。此外，IQGAP 1的沉默消除了血管生成素-1（Angpt-1）的屏障增强作用，并消除了Angpt-1对凝血酶刺激细胞的屏障稳定作用。免疫共沉淀检测到内源性IQGAP 1与Rac 1在基线时的结合，Rac 1激活时结合较强，失活时结合较弱。GTP结合的Rac 1的测量显示，Angpt-1不仅不能激活Rac 1，如果IQGAP 1沉默，而且如果细胞转染Rac 1结合失能的突变体（T1050 AX 2）。此外，显性活性Rac 1足以完全逆转IQGAP 1减少诱导的形态和功能变化。这些实验是IQGAP 1在任何细胞类型中调节屏障功能的首次证明。此外，我们的数据显示Angpt-1需要IQGAP 1作为Rac 1的不可或缺的激活剂。
IQ domain GTPase-activating protein 1 (IQGAP1) contributes to cytoskeletal network regulation in epithelial cells by its scaffolding properties and by binding the Rho GTPase Rac1 to maintain its activity. The functions of IQGAP1 in endothelial cells beyond angiogenesis remain unclear. We hypothesized that IQGAP1 participates in the regulation of endothelial barrier function. Silencing IQGAP1 in human microvascular ECs (HMVECs) resulted in a disruption of adherens junctions, formation of inter-endothelial gaps, and a reduction in barrier function. Furthermore, silencing of IQGAP1 abrogated Angiopoietin-1's (Angpt-1) barrier enhancement effect and abolished the barrier-stabilizing effect of Angpt-1 on thrombin-stimulated cells. Co-immunoprecipitation detected binding of endogenous IQGAP1 with Rac1 at baseline that was stronger when Rac1 was activated and weaker when deactivated. Measurement of GTP-bound Rac1 revealed that Angpt-1 not only failed to activate Rac1 if IQGAP1 was silenced, but also if cells were transfected with a mutant disabled in Rac1 binding (T1050AX2). Furthermore, a dominant-active Rac1 was sufficient to completely reverse the morphological and functional changes induced by reduction in IQGAP1. These experiments are the first demonstration of IQGAP1 regulating barrier function in any cell type. Further, our data show that Angpt-1 requires IQGAP1 as an indispensable activator of Rac1.