From the Division of Cardiovascular Medicine, Department of Internal Medicine (T.T., T.F., K.-i.H.).
Division of Signal Transduction, Department of Biochemistry and Molecular Biology (M.M., K.N., Y.R.).
Arterioscler Thromb Vasc Biol. 2018 May;38(5):1159-1169. doi: 10.1161/ATVBAHA.118.310991. Epub 2018 Mar 29.
We previously reported that afadin, an actin filament-binding protein, regulated vascular endothelial growth factor-induced angiogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the mechanisms of how Rho-associated kinase is activated in afadin-knockdown human umbilical vein endothelial cells (HUVECs) and how its activation is involved in defects of vascular endothelial growth factor-induced network formation and migration of the cells.
Knockdown of afadin or ArhGAP29, a GTPase-activating protein for RhoA, increased Rho-associated kinase activity and reduced the vascular endothelial growth factor-induced network formation and migration of cultured HUVECs, accompanied by the defective formation of membrane protrusions, such as lamellipodia and peripheral ruffles. Treatment of the afadin- or ArhGAP29-knockdown HUVECs with Rho-associated kinase inhibitors, Y-27632 or fasudil, partially restored the reduced network formation and migration as well as the defective formation of membrane protrusions. ArhGAP29 bound to afadin and was colocalized with afadin at the leading edge of migrating HUVECs. The defective formation of membrane protrusions in ArhGAP29-knockdown HUVECs was restored by expression of mutant ArhGAP29 that bound to afadin and contained a RhoGAP domain but not mutant ArhGAP29 that could bind to afadin and lacked the RhoGAP domain or mutant ArhGAP29 that could not bind to afadin and contained the RhoGAP domain. This suggested the requirement of both the interaction of afadin with ArhGAP29 and RhoGAP activity of ArhGAP29 for migration of HUVECs.
Our results highlight a critical role of the afadin-ArhGAP29 axis for the regulation of Rho-associated kinase activity during vascular endothelial growth factor-induced network formation and migration of HUVECs.
我们之前报道过，连接肌动蛋白丝的蛋白 afadin 可调节血管内皮生长因子诱导的血管生成。然而，其潜在的分子机制尚不清楚。在此，我们研究了 afadin 敲低的人脐静脉内皮细胞（HUVEC）中 Rho 相关激酶激活的机制，以及其激活如何参与血管内皮生长因子诱导的网络形成和细胞迁移缺陷。
afadin 或 RhoA 的 GTP 酶激活蛋白 ArhGAP29 的敲低增加了 Rho 相关激酶的活性，并减少了培养的 HUVEC 中血管内皮生长因子诱导的网络形成和迁移，同时伴有膜突如片状伪足和周边皱襞的形成缺陷。用 Rho 相关激酶抑制剂 Y-27632 或法舒地尔处理 afadin 或 ArhGAP29 敲低的 HUVEC，部分恢复了网络形成和迁移减少以及膜突形成缺陷。ArhGAP29 与 afadin 结合，并与 afadin 在迁移的 HUVEC 的前缘共定位。在 ArhGAP29 敲低的 HUVEC 中，膜突形成缺陷通过表达与 afadin 结合并含有 RhoGAP 结构域的突变型 ArhGAP29 而得到恢复，但不通过表达可以与 afadin 结合但缺乏 RhoGAP 结构域的突变型 ArhGAP29 或不能与 afadin 结合且含有 RhoGAP 结构域的突变型 ArhGAP29 而得到恢复。这表明 afadin 与 ArhGAP29 的相互作用以及 ArhGAP29 的 RhoGAP 活性对于 HUVEC 的迁移都是必需的。
我们的结果强调了 afadin-ArhGAP29 轴在血管内皮生长因子诱导的 HUVEC 网络形成和迁移过程中对 Rho 相关激酶活性的调节的关键作用。
