Wang Dong, Paria Biman C, Zhang Qiuhua, Karpurapu Manjula, Li Quanyi, Gerthoffer William T, Nakaoka Yoshikazu, Rao Gadiparthi N
Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA.
Circ Res. 2009 May 8;104(9):1066-75. doi: 10.1161/CIRCRESAHA.109.196691. Epub 2009 Apr 9.
To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.
为了解表皮生长因子受体（EGFR）转活化在G蛋白偶联受体（GPCR）激动剂诱导的信号事件中的作用，我们研究了凝血酶激活血管平滑肌细胞（VSMC）中Gab1-SHP2的能力。凝血酶以EGFR依赖性方式激活Gab1和SHP2。同样，凝血酶诱导Rac1和Cdc42激活，当Gab1或SHP2刺激被阻断时，这些反应受到抑制。凝血酶还以时间和EGFR-Gab1-SHP2-Rac1/Cdc42依赖性方式诱导PAK1激活。通过药理学或遗传学方法抑制EGFR、Gab1、SHP2、Rac1、Cdc42或PAK1的激活可减弱凝血酶诱导的VSMC应力纤维形成和运动性。凝血酶在VSMC中以时间依赖性方式激活RhoA。发现LARG，一种RhoA特异性鸟嘌呤核苷酸交换因子（GEF），与Gab1相关，其水平的siRNA介导的消耗抑制了RhoA、Rac1和PAK1的激活。显性负突变体介导的RhoA激活干扰抑制了凝血酶诱导的VSMC中Rac1和PAK1刺激及其应力纤维形成和迁移。球囊损伤诱导PAK1活性，干扰其激活导致平滑肌细胞从介质向内膜迁移减弱，导致新生内膜形成减少和管腔大小增加。重组水蛭素对凝血酶信号的抑制也阻断了球囊损伤诱导的EGFR酪氨酸磷酸化和PAK1活性。这些结果表明，凝血酶介导的PAK1激活在血管壁重塑中起关键作用，它可能是针对这些血管病变的药物开发的潜在靶点。
