Submitted on April 25, 2006
Accepted on February 16, 2007
Angiotensin II Type 2 Receptor-Dependent Increase in Nitric Oxide Synthase Expression in Pulmonary Endothelium is Mediated via a G
i3/Ras/Raf/MAPK Pathway
Jianyu Li1, Xiangmin Zhao1, Xinmei Li2, Kenneth M Lerea3, and Susan C Olson1*
1 Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, United States
2 Pediatrics, New York Medical College, Valhalla, New York, United States
3 Cell Biology and Anatomy, New York Medical College, Valhalla, New York, United States
* To whom correspondence should be addressed. E-mail: susan_olson{at}nymc.edu.
We have reported that angiotensin II (Ang II) stimulated Src tyrosine kinase via 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). This 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 Gαi3 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 Shc (15 min) that was prevented when PAECs were pretreated with PP2, a Src inhibitor. Ang II induced a Src-dependent association between Shc and Grb2 and between Grb2 and Sos, both of which were maximal at 15 min. Ang II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT2 antagonist, PD123319, with PP2, or were transfected with the Src DN cDNA. Ang II-dependent activation of MAPK and increase in eNOS was prevented when PAECs were transfected with a 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 PD123319 and PP2. The Raf kinase inhibitor 1 prevented the Ang II-dependent increase in eNOS expression. Collectively, these data suggest that Gαi3, Shc, Grb2, Ras and Raf-1 link Src to activation of MAPK and to the AT2-dependent increase in eNOS expression in PAECs.
