Li Jianyu, Zhao Xiangmin, Li Xinmei, Lerea Kenneth M, Olson Susan C
Dept. of Biochemistry, New York Medical College, Valhalla, NY 10595, USA.
Am J Physiol Cell Physiol. 2007 Jun;292(6):C2185-96. doi: 10.1152/ajpcell.00204.2006. Epub 2007 Feb 28.
We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G alpha(i3) dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT(2) antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G alpha(i3), Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT(2)-dependent increase in eNOS expression in PAECs.
我们之前曾报道，血管紧张素II（ANG II）通过一种对百日咳毒素敏感的2型受体刺激Src酪氨酸激酶，该受体进而激活丝裂原活化蛋白激酶（MAPK），导致肺动脉内皮细胞（PAECs）中一氧化氮合酶（NOS）表达增加。本研究旨在探究ANG II激活Src从而导致PAECs中ERK1/ERK2磷酸化增加以及NOS蛋白增加的信号通路。用Gα(i3)显性负性（DN）cDNA转染PAECs可阻断ANG II依赖的Src激活、ERK1/ERK2磷酸化以及NOS表达增加。ANG II刺激胶原蛋白序列同源性（Shc）的酪氨酸磷酸化增加（15分钟），当PAECs用Src抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并-[3,4-d]嘧啶（PP2）预处理时，这种增加被阻止。ANG II诱导Shc与生长因子受体结合蛋白2（Grb2）以及Grb2与七号染色体失活蛋白（Sos）之间的Src依赖性结合，两者在15分钟时均达到最大值。当PAECs用AT(2)拮抗剂PD-123319或PP2预处理，或用Src DN cDNA转染时，ANG II依赖的Ras GTP结合增加被阻止。当PAECs用Ras DN cDNA转染或用法尼基转移酶抑制剂FTI-277处理时，ANG II依赖的MAPK激活以及内皮型NOS（eNOS）增加被阻止。当PAECs用PD-123319和PP2预处理时，ANG II诱导的Raf-1磷酸化被阻止。Raf激酶抑制剂1可阻止ANG II依赖的eNOS表达增加。总体而言，这些数据表明Gα(i3)、Shc、Grb2、Ras和Raf-1将Src与PAECs中MAPK的激活以及AT(2)依赖的eNOS表达增加联系起来。
