cAbl Tyrosine Kinase Mediates Reactive Oxygen Species– and Caveolin-Dependent AT ₁ Receptor Signaling in Vascular Smooth Muscle

Abstract

Abstract-Important output signals of the angiotensin subtype 1 receptor (AT 1 R) in vascular smooth muscle cells (VSMCs) are mediated by angiotensin II (Ang II)-stimulated transactivation of the epidermal growth factor receptor (EGF-R), which is critical for vascular hypertrophy. Ang II-induced EGF-R transactivation is mediated through cSrc, a proximal target of reactive oxygen species (ROS) derived from NAD(P)H oxidase (NOX) and is dependent on AT 1 R trafficking through caveolin1 (Cav1)-enriched lipid rafts. Underlying molecular mechanisms are incompletely understood. The nonreceptor tyrosine kinase, proto-oncogene cAbl is a substrate of Src and is a major mediator for ROS-dependent tyrosine phosphorylation of Cav1. We thus hypothesized that cAbl is important for ROS-, cSrc-, and Cav1-dependent growth-related AT 1 R signal transduction. Here we show that Ang II induces tyrosine phosphorylation of cAbl in rat VSMCs and mouse aorta, and that Ang II promotes association of cAbl with AT 1 R, both of which are Src-dependent. Pretreatment of rat VSMCs with the NOX inhibitor diphenylene iodonium or the antioxidants N-acetylcysteine or ebselen significantly inhibited Ang II-induced cAbl phosphorylation. Cell fractionation shows that both EGF-Rs and cAbl are found basally in Cav1-enriched membrane fractions. Knockdown of cAbl protein using small interference RNA inhibits Ang II-stimulated: (1) trafficking of AT 1 R into, and EGF-R out of, Cav1-enriched lipid rafts; (2) EGF-R transactivation; (3) appearance of the transactivated EGF-R and phospho-Cav1 at focal adhesions; and (4) vascular hypertrophy. These studies provide a novel role of cAbl in the spatial and temporal organization of growth-related AT 1 R signaling in VSMCs and suggest that cAbl may be generally important in signaling of G-protein coupled receptors. Key Words: cAbl Ⅲ caveolin-1 Ⅲ NAD(P)H oxidase Ⅲ angiotensin II Ⅲ vascular smooth muscle A ngiotensin II (Ang II) is important in mediating vascular remodeling and hypertrophy in hypertension. These effects are mediated, in large part, through the G-protein coupled angiotensin subtype 1 receptors (AT 1 Rs). The growth-related outputs of the AT 1 R are dependent on the transactivation (tyrosine phosphorylation) of the epidermal growth factor receptor (EGF-R) and its activation of mitogenactivated protein kinases and Akt, which are involved in vascular smooth muscle cell (VSMC) hypertrophy. 1,2 Many effects of Ang II are dependent on the AT 1 R stimulation of reactive oxygen species (ROS) production by NAD(P)H oxidase. 2-5 EGF-R transactivation in VSMCs is mediated through the activation of cSrc, a proximal ROS-dependent event. 5 Caveolae/lipid rafts are specialized membrane microdomains where multimolecular signaling molecule complexes are compartmentalized, in part, via interacting with caveolin1 (Cav1). We showed that in VSMCs, Ang II stimulation promotes AT 1 R association with Cav1 and its trafficking into Cav1-enriched lipid rafts, events which in turn are associated contemporaneously with egress o...

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

“…In VSM, Cav-1 plays a role in the coupling of ␣-adrenergic, ANG II, and endothelin receptors to VSM contraction and growth (34,38,39,43). In the endothelium, Cav-1 anchors eNOS to caveolae, thus preventing its activation and reducing NO production and vascular relaxation (3,7,33).…”

ADAM17 Mediates Epidermal Growth Factor Receptor Transactivation and Vascular Smooth Muscle Cell Hypertrophy Induced by Angiotensin II

“…15 In addition to these second messengers and ADAM17, multiple upstream components have been reported to participate in EGFR transactivation by Ang II. 2,6,7,26 However, the relationships between these distinct signal transduction components still remain unclear. Because ADAM phosphorylation 27,28 seems to be required for the activation of certain ADAM, it is possible that the upstream components may converge on an ADAM kinase that regulates ADAM17 activity.…”

“…2,4 Intracellular Ca 2ϩ elevation, protein kinase C activation or reactive oxygen species (ROS) production seems to be required for the upstream of EGFR transactivation induced by Ang II through the AT 1 receptor. [5][6][7] Recently, the mechanism of EGFR transactivation via several GPCRs has been shown to involve a proteolytic cleavage ("ecto-domain shedding") of a membrane-anchored EGF ligand precursor to release a biologically active growth factor mediated through ADAM (a disintegrin and metalloprotease) family metalloproteases. 8,9 Previously, we and others have shown that heparin-binding EGF (HB-EGF) shedding is essential for EGFR transactivation via the AT 1 receptor.…”

Caveolin-Dependent Angiotensin II Type 1 Receptor Signaling in Vascular Smooth Muscle

“…8,9 These results are consistent with a model that requires temporal dispersion and organization of the AT 1 R signaling repertoire in VSMCs. 7,10 Accumulating evidence suggests that receptors and the signaling molecules with which they associate are not randomly distributed in the cell membrane but are localized in specialized signaling domains. Functionally distinct microdomains, formed by the lateral packing of glycosphingolipids and cholesterol within the membrane bilayer, have been identified in plasma membranes.…”

“…7,10,12,13 The full expression of the AT 1 R signaling repertoire depends on caveolae/lipid rafts and reactive oxygen species (ROS) production by reduced nicotinamideadenine dinucleotide phosphate (NADPH) oxidase. 7,10,13,14 Several recent reviews have described the physiological and pathophysiological roles of caveolae and caveolins in the cardiovascular system generally. [15][16][17][18] The main focus of this review is to summarize the recent progress on the emerging understanding of the role of Cav1 as a central organizer for the spatially and temporally regulated, ROS-dependent, growth-related AT 1 R signaling in VSMCs.…”