Role of receptor and non-receptor protein tyrosine kinases in vasoactive peptide-induced signaling

Endothelin-1 (ET-1) and angiotensin II (Ang II) play important roles in maintaining blood pressure and vascular homeostasis, and a heightened activity of these vasoactive peptides is thought to contribute to the development of vascular pathologies, such as hypertension, atherosclerosis, hypertrophy and restenosis. This is caused by an excessive activation of several growth and proliferative signaling pathways, which include members of the mitogen-activated protein kinase (MAPK) family, as well as the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) pathway. While the activation of these signaling pathways is well elucidated, the upstream elements responsible for ET-1 and Ang II-induced MAPK and PI3-K/PKB activation remain poorly understood. During the last several years, the concept of transactivation of receptor and/or non-receptor protein tyrosine kinases (PTK) in triggering vasoactive peptide-induced signaling events has gained much recognition. We have recently demonstrated that insulin-like growth factor-1 receptor (IGF-1R) plays a role in tranducing the effect of H2O2, leading to PKB phosphorylation. Since vasoactive peptides elicit their responses through generation of reactive oxygen species, including H2O2, we investigated whether IGF-1R transactivation plays a similar role in ET-1 and Ang II-induced PKB phosphorylation and hypertrophic responses in VSMC. AG-1024, a specific inhibitor of IGF-1R, attenuated both ET-1 and Ang II-induced PKB phosphorylation in a dose-dependent manner. ET-1 and Ang II treatment also induced the phosphorylation of tyrosine residues in the autophosphorylation sites of IGF-1R, which was blocked by AG-1024. In addition, both ET-1 and Ang II evoked tyrosine phosphorylation of c-Src, a non-receptor PTK, and pharmacological inhibition of c-Src PTK activity by PP-2, a specific inhibitor of Src-family tyrosine kinase, significantly reduced PKB phosphorylation as well as tyrosine phosphorylation of IGF-1R induced by the two vasoactive peptides. Furthermore, protein and DNA synthesis, markers of cell growth and proliferation, enhanced by ET-1 and Ang II were also attenuated by AG-1024 and PP-2.  While this work demonstrates the role of c-Src in ET-1 and Ang II-induced PKB phosphorylation, its role in ET-1-induced MAPK signaling and regulation of transcription factors, such as early growth response factor-1 (Egr-1), which was recently shown to be expressed in atherosclerotic plaque, remains controversial in VSMC. Therefore, we have also investigated the involvement of c-Src in ET-1 and Ang II-induced ERK 1/2, JNK and p38mapk activation, as well as Egr-1 regulation. ET-1 and Ang II-induced the phosphorylation of ERK 1/2, JNK and p38mapk, and enhanced the expression of Egr-1 in aortic VSMC. This increased phosphorylation was decreased by PP-2. Further proof for the role of c-Src in this process was obtained by using mouse embryonic fibroblasts (MEF) deficient in c-Src (Src -/- MEF). ET-1-induced Egr-1 expression, as well as MAPK activation, were found to be downregulated in Src -/- MEF, as compared to MEF expressing normal Src levels. In summary, these data demonstrate that IGF-1R and c-Src PTK play a critical role in mediating both PKB and MAPK phosphorylation and Egr-1 expression, as well as hypertrophic and proliferative responses induced by ET-1 and Ang II in VSMC.

Keyphrases: Endotheline-1, Angiotensin II, PKB, MAPK, IGF-1R, c-Src, VSMC, Egr-1, Proliferation, Hypertrophy, Endothelin-1