Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

Abstract

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

“…There are a variety of nonenzymatic and enzymatic sources for ROS production reported in vivo (43). Thus, there is accumulating evidence that the membrane NADPH oxidase is the predominant source of ROS production in vascular tissue, including SMCs (27,28,44). Lipoxygenases, such as 5-lipoxygenase, which is a key enzyme in leukotriene production, and 12/15-lipoxygenase, which is expressed in smooth muscle (45,46), are a family of lipidperoxidizing enzymes that are another source of ROS (31,32).…”

“…These include NADPH oxidase, a major source of ROS in response to vasoactive agonists such as angiotensin II, tumor necrosis factor-␣ , and thrombin (27,28), and lipoxygenases, such as 5-lipoxygenase, which is a key enzyme in leukotriene production (29,30), and 12/15-lipoxygenase, whose biologic functions are unknown (31,32). We found that NDGA and AA-861, both of which are 5-lipoxygenase inhibitors, markedly reduced OxLDL-induced ROS production.…”

From bedside to bench to bedside: role of renin-angiotensin-aldosterone system in remodeling of resistance arteries in hypertension

“…This process of transactivation has been demonstrated for EGF receptor, PDGF receptor, subtype ␤, and IGF-1 receptor (131,174). Processes underlying ANG II-induced receptor tyrosine kinase transactivation include activation of tyrosine kinases (Pyk2 and Src), redox-sensitive processes and possibly mulation of MMPs that release heparin-binding EGF (131,174,187).…”

Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease

“…There are relatively few reports of endogenous ROS regulating the MAP kinase cascade. In VSMCs, intracellular ROS are critical for Ang II-induced activation of p38MAPK, JNK and ERK5, whereas phosphorylation of ERK1/2 appears to be redox-insensitive (54). However, serotonin-mediated ERK1/2 activation in smooth muscle cells is redox-sensitive, but in fibroblasts, it is not (40).…”