Caveolin-1 Is Essential for Activation of Rac1 and NAD(P)H Oxidase After Angiotensin II Type 1 Receptor Stimulation in Vascular Smooth Muscle Cells

Abstract

Objective-Angiotensin II (Ang II) is a potent mediator of vascular hypertrophy in vascular smooth muscle cells (VSMCs).These effects are mediated through the Ang II type 1 receptor (AT 1 R) and require its trafficking through caveolin-1 (Cav1)-enriched lipid rafts and reactive oxygen species (ROS) derived from Rac1-dependent NAD(P)H oxidase. The specific role(s) of Cav1 in AT 1 R signaling is incompletely understood. Methods and Results-Knockdown of Cav1 protein by small interfering RNA (siRNA) inhibits Ang II-stimulated Rac1 activation and membrane translocation, H 2 O 2 production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signalregulated kinase 1/2 phosphorylation. Ang II stimulates tyrosine phosphorylation of Sos-1, a Rac-guanine nucleotide exchange factor, which is inhibited by Cav1 siRNA, demonstrating involvement of Cav1 in Rac1 activation. Detergent-free fractionation showed that EGF-Rs are found basally in Cav1-enriched lipid raft membranes and associate with Cav1. Ang II stimulates AT 1 R movement into these microdomains contemporaneously with the egress of EGF-R. Both aspects of this bidirectional receptor trafficking are inhibited by Cav1 siRNA. Moreover, Cav1 siRNA inhibits Ang II-induced vascular hypertrophy. Key Words: angiotensin II Ⅲ reactive oxygen species Ⅲ caveolin Ⅲ caveolae Ⅲ vascular hypertrophy Ⅲ vascular smooth muscle A ngiotensin II (Ang II), the major effector peptide of the renin-angiotensin system, is a pleuripotent hormone in vascular smooth muscle cells (VSMCs) and stimulates arterial hypertrophy, a hallmark of remodeling in hypertension. These effects are mediated primarily through the G-proteincoupled Ang II type1 receptor (AT 1 R). Major outputs of the AT 1 R are dependent on the transactivation (tyrosine phosphorylation) of the epidermal growth factor receptor (EGF-R), leading to activation of downstream targets mitogen-activated protein kinases and Akt, which are important for the expression of the full hypertrophy-related Ang II signaling repertoire in VSMCs. 1,2 Many of these processes are mediated via generation of reactive oxygen species (ROS), which act as signaling molecules. [3][4][5][6] Emerging evidence indicates that NAD(P)H oxidase is a major source of vascular ROS in VSMCs. 1 Caveolae/lipid rafts are cholesterol-enriched, specialized membrane microdomains in which multimolecular complexes of signaling molecules such as EGF-R and Src are compartmentalized via interacting with caveolin-1 (Cav1). 7,8 The pathophysiological importance of Cav1 is reflected in the cardiovascular phenotype of Cav1 Ϫ/Ϫ mice. 8,9 We showed that Ang II promotes association of AT 1 R with Cav1 and AT 1 R trafficking into caveolin-enriched/lipid rafts in VSMCs. 10,11 This event is associated with egress of EGF-R from these specialized microdomains, where they are basally located. 11 Using cholesterol-binding agents such as ␤-cyclodextrin, we reported previously that cholesterol...

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).…”

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

“…34,35 In VSMCs, Ang II induces rapid (Ͻ2 minutes) translocation of a portion of the AT 1 R from heavy membrane fractions to Cav1-enriched lipid rafts, and AT 1 R and Cav1 become associated. 7,10,12 We explored the mechanisms involved in AT 1 R trafficking to the Cav1-enriched/lipid raft signaling locus and found that it depends on Cav1. 10 Moreover, Cav3 has been shown to act as a chaperone for the AT 1 R, allowing the receptor to traffic through the exocytic pathway and to localize at the cell membrane in transfection systems.…”

“…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.…”

“…In VSMCs, Ang II stimulation promotes AT 1 R association with Cav1 and trafficking of the receptor from heavy density membrane fractions into relatively buoyant Cav1-enriched lipid rafts, which is required for transactivation of EGF-R at focal adhesions, another membrane signaling domains associated with growth factor and integrin signaling. 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.…”

“…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.…”