Epidermal Growth Factor Receptor Transactivation by Angiotensin II Requires Reactive Oxygen Species in Vascular Smooth Muscle Cells

Abstract

Abstract-Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited (Ϸ80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca 2ϩ attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca 2ϩ mobilization or phosphorylation of Ca 2ϩ -dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II-induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II-stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease. A ngiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle growth, a cardinal feature of hypertension and atherosclerosis. Ang II exerts its effects via activation of a complex set of enzymatic cascades exhibiting considerable cross talk. Recently, attention has focused on the role of reactive oxygen species (ROS) in transducing growthrelated molecular signals and cardiovascular disease. Ang II rapidly increases superoxide and hydrogen peroxide (H 2 O 2 ) production in vascular smooth muscle cells (VSMCs) via activation of a p22phox-based NAD(P)H oxidase. 1-4 These ROS are required for activation of a specific set of downstream kinases, including p38 mitogen-activated protein kinase (p38 MAPK) and Akt. 4,5 Importantly, both of these pathways are required for the hypertrophic response by Ang II. 4,5 The most proximal mechanisms coupling these responses to the Ang II type 1 (AT 1 ) receptor remain unclear.

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

“…Indeed, disruption of the actin cytoskeleton and its associated focal adhesions with cytochalasin D completely inhibits Ang II-, but not EGF-, induced EGF-R phosphorylation. 2 This result supports the concept that an intact cytoskeleton and/or focal adhesions are required for Ang II-induced transactivation of the EGF-R, in VSMCs. The colocalization of activated EGF-Rs, paxillin and tyrosine-phosphorylated caveolin suggests that in focal adhesions, phosphorylated caveolin may function as a positive regulator for transactivation and may be relevant to the generation of active, spatially organized signaling molecules.…”

“…Several signaling mechanisms have been implicated in Ang II-induced EGF-R transactivation, including calcium and c-Src in VSMCs (2,19). Interestingly, cholesterol depletion had no effect on either of these early signaling events, suggesting that the intact cholesterol-enriched microdomain is not required for the initial activation of calcium and c-Src.…”

Nox4 NAD(P)H Oxidase Mediates Src-dependent Tyrosine Phosphorylation of PDK-1 in Response to Angiotensin II

“…We show that submillimolar concentrations of hydrogen peroxide lead to tyrosine phosphorylation and activation of Src or PDK-1, suggesting that these kinases are modulated by ROS. This is in agreement with previous published studies reporting the redox sensitivity of Src and PDK-1 in other cell types (30,43,45,48,53). These observations prompted us to test the hypothesis that endogenous ROS act as upstream intermediates in Ang II-mediated and Src-dependent activation of PDK-1.…”

Molecular Mechanism of the Inhibitory Effect of Aldosterone on Endothelial NO Synthase Activity

“…14 -16 Previous publications have suggested that ROS produced by NADPH oxidase mediate many angiotensin II effects in the cardiovascular system. 17,18 It was reported recently that Aldo also increases NADPH oxidase activity in the vasculature. 19,20 In the NADPH oxidase system, cytosolic component p47 phox was shown to have a pivotal role in the regulation of enzymatic activity.…”