Caveolin-1 Is Essential for Activation of Rac1 and NAD(P)H Oxidase After Angiotensin II Type 1 Receptor Stimulation in Vascular Smooth Muscl
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动脉硬化血栓血管生物学 2005年第9期
From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.
Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Room 319, Atlanta, GA 30322. E-mail mfukai@emory.edu
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 (AT1R) 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 AT1R 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, H2O2 production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signal-regulated 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 AT1R 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.
Conclusions— Cav1 plays an essential role in AT1R targeting into Cav1-enriched lipid rafts and Rac1 activation, which are required for proper organization of ROS-dependent Ang II signaling linked to VSMC hypertrophy.
Angiotensin II (Ang II)–induced vascular hypertrophy is dependent on caveolae/lipid rafts and reactive oxygen species (ROS) derived from NAD(P)H oxidase. Using caveolin-1 siRNA, we demonstrate that caveolin-1 plays an essential role in AT1 receptor targeting into caveolae/lipid rafts and Rac1 activation, which are required for ROS-dependent, growth-related Ang II signaling.
Key Words: angiotensin II ? reactive oxygen species ? caveolin ? caveolae ? vascular hypertrophy ? vascular smooth muscle
Introduction
Angiotensin 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-protein–coupled Ang II type1 receptor (AT1R). Major outputs of the AT1R 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.3–6 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–/– mice.8,9 We showed that Ang II promotes association of AT1R with Cav1 and AT1R 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-rich microdomains such as caveolae/lipid rafts are essential for transactivation of EGF-R.12 Furthermore, Nox1, a homolog of the phagocytic NAD(P)H oxidase subunit gp91phox, has been shown to be found in caveolin-enriched fractions in VSMCs.13 However, a specific role of Cav1 in ROS-dependent, growth-related AT1R signaling in VSMCs is incompletely understood.
The small G-protein Rac1 is a critical component of NAD(P)H oxidase activated by Ang II,14 and its translocation to the plasma membrane is an essential step for activation of oxidase.15 We demonstrated previously that Ang II stimulation promotes Rac1 association with Cav1 and its translocation into caveolae/lipid rafts through microtubule-dependent manner.11 Activation of Rac is achieved through the exchange of bound GDP for GTP by a family of guanine nucleotide exchange factors (GEFs).16 GEFs that target Rac include Son of Sevenless (Sos-1), Vav1, –2, and –3; Tiam1, and ?Pix; and P-Rex1.17 All of these GEF proteins contain a pleckstrin homology domain, but Sos-1, Vav, and P-Rex1 also have caveolin-binding consensus sequences. Sos-1 has been identified in Cav1-enriched fractions in nonvascular systems.18 Little is known about GEFs activated by Ang II and its relationships with Cav1.
Here we demonstrate that knockdown Cav1 expression using a small interfering RNA (siRNA) results in inhibition of Ang II–stimulated AT1R targeting into caveolae/lipid rafts, GTP loading of Rac1, and its translocation to the plasma membrane. As its consequence, Cav1 siRNA inhibits Ang II–induced H2O2 production, its downstream EGF-R transactivation, and Akt phosphorylation, as well as vascular hypertrophy without affecting ROS-independent pathways. Moreover, Ang II stimulation induces tyrosine phosphorylation of Sos-1, which is inhibited by Cav1 siRNA, demonstrating a molecular mechanism by which Cav1 regulates activation of Rac1. These findings suggest an essential role of Cav1 in AT1 trafficking into Cav1-enriched lipid rafts and Rac1 activation, which is required for activation of NAD(P)H oxidase and redox-signaling linking to vascular hypertrophy.
Materials and Methods
Materials
Anti-EGFR, AT1 antibody, and Sos-1 antibodies were from Santa Cruz. Antibodies to Cav1, Cav3, and phosphotyrosine (PY20) were from BD Biosciences. Antibodies to phospho–Akt/protein kinase B (PKB) (pS473) and phospho–extracellular signal-regulated kinase 1/2 (ERK1/2) were from Cell Signaling. Other materials were purchased from Sigma.
Cell culture, synthetic siRNA and its transfection, intracellular H2O2 measurement, immunoprecipitation and immunoblotting, Rac activity assay, isolation of membrane and cytosolic fractions, purification of caveolae fractions, confocal immunofluorescence microscopy, [3H] leucine incorporation assay, and statistical analyses are described in the Materials and Methods section in the online data supplement available at http://atvb.ahajournals.org.
Results
Knockdown of Cav1 Protein by siRNA Inhibits EGF-R Transactivation by Ang II
EGF-R transactivation plays major roles in growth-related Ang II signaling in VSMCs.2,19 We reported previously that cholesterol-enriched microdomains such as caveolae/lipid rafts are involved in Ang II–induced EGF-R transactivation.12 We therefore determined specifically the role of Cav1 in this response by transfecting Cav1 siRNA into VSMCs. As shown in Figure 1A, Cav1 siRNA, but not scrambled siRNA, completely knocked down Cav1 protein in VSMCs without affecting Cav3, -tubulin, and AT1R protein expression, confirming the specificity of Cav1 siRNA. As shown in Figure 1B, Cav1 siRNA significantly inhibited Ang II–induced transactivation of EGF-R without affecting EGF-induced EGF-R autophosphorylation (Figure 1C), suggesting that Cav1 is involved in the pathways linking AT1R to the EGF-R activation.
Figure 1. Effects of knockdown of Cav1 protein by siRNA on EGF-R phosphorylation by Ang II and EGF. A, VSMCs were transfected with Cav1 or scrambled siRNA (50 nmol/L) or transfection reagent alone (control). At 48 hours after transfection, cells were stimulated with Ang II (100 nmol/L) for 2 minutes, and lysates were immunoblotted (IB) with anti-Cav1, Cav3, -tubulin, or AT1R antibody. The results are representative of 3 separate experiments. B and C, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L for 2 minutes; B) or EGF (100 ng/mL for 30 s; C). Lysates were immunoprecipitated (IP) with anti–EGF-R antibody and immunoblotted with anti-phosphotyrosine (pTyr) or EGF-R antibody. The graphs represent averaged data, corrected for total EGF-R loading, expressed as fold change over basal. *P<0.05 for increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Knockdown of Cav1 Protein by siRNA Inhibits Ang II–Stimulated AT1R Trafficking Into Cav1-Enriched Membrane Fractions
Because we have shown previously that Ang II stimulation promotes AT1R trafficking into a Cav1-enriched membrane fraction,10,11 which is required for EGF-R transactivation by Ang II,11,12 we examined whether Cav1 is involved in this response. Detergent-free OptiPrep gradient cell fractionation to isolate Cav1-enriched/lipid raft membranes20 showed that in unstimulated VSMCs, AT1R was not found in buoyant, lower-density fractions containing Cav1 and EGF-R (Figure 2, fractions 2 through 4). Ang II stimulation (5 minutes) promoted AT1R movement into Cav1-enriched fractions cotemporaneously with the egress of EGF-R from these fractions. This bidirectional receptor trafficking was almost completely inhibited by Cav1 siRNA (Figure 2A and 2B). These data suggest that Cav1-mediated EGF-R transactivation by Ang II is associated with the AT1R targeting into as well as the egress of EGF-R from caveolae/lipid rafts.
Figure 2. Effects of Cav1 siRNA on localization of AT1R and EGF-R in caveolin-enriched membrane fractions. VSMCs transfected with scrambled or Cav1 siRNA as described above were stimulated with Ang II (100 nmol/L) for 5 minutes. Caveolae fractions (1 through 8) were immunoblotted (IB) for Cav1, AT1R, or EGF-R (A). PC indicates positive control (VSMC lysates). Equal amounts of Cav1-enriched fraction 3 were immunoblotted with anti-AT1R, EGF-R, Cav1, or Cav3 antibody (B). The blots are representative of 3 separate experiments.
Cav1 Is Involved in Ang II–Induced ROS Production
Because EGF-R transactivation by Ang II is dependent on ROS derived from NAD(P)H oxidase,5 we examined whether Cav1 is involved in Ang II–stimulated ROS production. As shown in Figure 3A, Cav1 siRNA significantly inhibited Ang II–induced increase in H2O2 production without affecting basal levels, as measured by 2'7'-dicholorofluorescin diacetate (DCF-DA) fluorescence. In contrast, H2O2 production induced by phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C [PKC]) is not inhibited by Cav1 siRNA, indicating the specific involvement of Cav1 in the pathways transmitting the AT1R signal to the NAD(P)H oxidase but not in the PKC–NAD(P)H oxidase pathways.
Figure 3. Effects of Cav1 siRNA on Ang II–induced H2O2 production, Rac1 activation, and membrane translocation. A, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) or PMA (10 μmol/L) for 30 minutes and used for DCF-DA fluorescence measurement. Graphs represent averaged data expressed as fold increase in DCF fluorescence intensity over basal. *P<0.05 for the difference of fluorescence intensity. B, VSMCs were stimulated with Ang II (100 nmol/L), and lysates were immunoprecipitated (IP) with anti-Cav1 antibody and immunoblotted (IB) with anti-Rac1 or Cav1 antibody. The bottom graphs represent averaged data, expressed as a fold increase in the association over basal. *P<0.05 for changes in Rac1-caveolin complex induced by Ang II vs vehicle alone. C and D, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 5 minutes (C) or 0 to 15 minutes (D). C, Lysates bound to PAK-1 agarose were immunoblotted with anti-Rac antibody. Values are the means±SE for 3 independent experiments. D, Lysates were separated as cytoplasm and membrane fractions as described in Methods and then immunoblotted with anti-Rac1 antibody. Graphs represent averaged data expressed as fold increase in the expression of Rac1 over basal. *P<0.05 for difference of Rac1 expression induced by Ang II vs vehicle alone.
Cav1 Is Involved in Ang II–Induced Rac1 Activation and Membrane Translocation
Because Rac1 is involved in Ang II–stimulated NAD(P)H oxidase activation in VSMCs,14 we examined whether Cav1 is involved in Ang II–induced Rac1 activation and membrane translocation. Coimmunoprecipitation assays show that Ang II stimulation promoted Rac1 association with Cav1 within 5 minutes (Figure 3B). This interaction was accompanied by activation of Rac1 as measured by a pull-down assay that quantifies the GTP–Rac binding to its effector p21-activated kinase-1 (PAK-1) (Figure 3C). Cav1 siRNA significantly inhibited the Ang II–stimulated increase in the amount of GTP-bound Rac1 (Figure 3C). Because Rac GTP loading and translocation from the cytosol to plasma membranes are critical steps for activation of NAD(P)H oxidase, we examined whether Cav1 is involved in Rac1 translocation. As shown in Figure 3D, Ang II caused a rapid and significant increase in the amount of Rac1 in the membrane fraction that peaked at 2 minutes, which was significantly inhibited by Cav1 siRNA. Of note, knockdown of Cav1 itself increased association of Rac1 with membrane in the basal state. These results suggest that Cav1 is necessary for GTP loading of Rac1 and for proper localization and targeting of Rac1 to the membrane fractions, presumably through the Cav1–Rac1 interaction (Figure 3B). These events may contribute to the activation of NAD(P)H oxidase by Ang II in VSMCs.
Ang II Stimulates the Tyrosine Phosphorylation of Sos-1, Which Is Cav1 Dependent
Rac-GEF plays an important role in activation of Rac.16 Sos-1, a Rac-GEF, has a Cav1-binding consensus sequence and has been shown to be found in Cav1-enriched fractions in nonvascular systems.18 To gain insight into the molecular mechanisms by which Cav1 is involved in Rac1 activation, we tested the role of Cav1 in activation of Sos-1 by Ang II. As shown in Figure 4A, Ang II stimulation significantly increased tyrosine phosphorylation of Sos-1 within 2 minutes, with peak at 5 minutes. PMA had no effect on this response (data not shown). Moreover, Ang II stimulation promoted association of Cav1 with Sos-1 (Figure 4B) cotemporaneously with Sos-1 phosphorylation (Figure 5C). Cav1 siRNA significantly inhibited these responses (Figure 4C).
Figure 4. Effects of Cav1 siRNA on Ang II–induced tyrosine phosphorylation of Sos-1. A and B, VSMCs were stimulated with Ang II (100 nmol/L), and lysates were immunoprecipitated (IP) with anti–Sos-1 (A) or Cav1 (B) antibody and immunoblotted (IB) with anti-phosphotyrosine (pTyr) or Sos-1 antibody (A) or with anti–Sos-1 or Cav1 antibody (B). The bottom graphs represent averaged data expressed as a fold increase over basal. *P<0.05 for increase by Ang II vs vehicle alone. C, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 5 minutes. Lysates were immunoprecipitated with anti–Sos-1 antibody and immunoblotted with pTyr or Sos-1 antibody. The graphs represent averaged data, corrected for total Sos-1 loading, expressed as fold change over basal. *P<0.05 for increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Figure 5. Effects of Cav1 siRNA on Ang II–stimulated actin cytoskeletal reorganization. VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 2 minutes. A, Cells were fixed, permeabilized, and stained with Rhodamine Red-X–conjugated phalloidin (left) or with monoclonal mouse anti–-tubulin antibody, followed by Rhodamine Red-X–conjugated goat anti-mouse IgG (right).
Cav1 Is Involved in Ang II–Induced Actin Cytoskeletal Reorganization, ROS-Dependent Akt Phosphorylation, and VSMC Hypertrophy
Recent evidence suggests that an intact actin cytoskeleton is important in Ang II–stimulated NAD(P)H oxidase activation in VSMCs.21 Cav1 also interacts with actin cytoskeleton binding proteins,22,23 and Ang II induces actin stress fiber formation.24 We hypothesized that the roles of Cav1 in Ang II–induced ROS production and in actin cytoskeleton interactions might be interrelated. Thus, we examined the effect of Cav1 siRNA on Ang II–induced actin stress fiber formation using confocal microscopy. We found that it significantly reduced the phalloidin staining without affecting -tubulin staining (Figure 5). These results suggest that Cav1 is involved in Ang II–stimulated actin cytoskeletal reorganization in VSMCs, which may contribute to proper activation of NAD(P)H oxidase.
To assess further the functional role of Cav1 in ROS-dependent Ang II signaling linked to vascular hypertrophy, we examined the effects of Cav1 siRNA on phosphorylation of the ROS-dependent kinase Akt and the ROS-independent kinase ERK1/2. Akt and ERK1/2 are involved in Ang II–induced VSMC hypertrophy.3,4 As shown in Figure 6A and 6B, Cav1 siRNA significantly inhibited Ang II–induced Akt phosphorylation, whereas ERK1/2 phosphorylation was not affected. To determine the functional role of Cav1 in hypertrophy, we examined the effect of Cav1 siRNA on Ang II–stimulated [3H]leucine incorporation. As shown in Figure 6C, Cav1 siRNA significantly inhibited the basal and Ang II–induced responses. The inhibitory effects of Cav1 siRNA are not caused by toxic effects because the trypan blue exclusion test for cell viability indicated that cells transfected with siRNA were >98% viable. Thus, Cav1 plays an important role for Ang II–induced hypertrophy, at least in part through regulating ROS-dependent AT1R signaling pathways in VSMCs.
Figure 6. Effects of Cav1 siRNA on Akt and ERK1/2 phosphorylation as well as hypertrophy. A and B, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 2 minutes. Western analysis for phospho-Akt and total Akt (A) or phospho-ERK1/2 and total ERK1/2 (B) were shown. C, VSMCs transfected with scrambled or Cav1 siRNA as described were used for measurement of [3H]leucine incorporation induced by Ang II (100 nmol/L). The graphs represent averaged data expressed as fold change over basal. *P<0.05 for basal and increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Discussion
The present study using siRNA-mediated knockdown of Cav1 protein demonstrates that Cav1 is required for AT1R targeting into Cav-enriched/lipid rafts, Rac1 activation, ROS generation, EGF-R transactivation, Akt phosphorylation, as well as hypertrophy in VSMCs. In contrast, EGF-induced EGF-R autophosphorylation, PMA-induced ROS production, as well as Ang II–induced ROS-independent ERK1/2 phosphorylations are not affected by Cav1 siRNA. We also found that Cav1 is involved in Ang II–stimulated tyrosine phosphorylation of Sos-1 and actin cytoskeletal reorganization, which may contribute to the Rac1-dependent NAD(P)H oxidase activation in VSMCs.
Disruption of caveolae/lipid rafts with the cholesterol-depleting agent methyl-?-cyclodextrin, inhibits Ang II–induced EGF-R transactivation,12 a major output of growth-related AT1R signaling in VSMCs.19,25 However, a specific role of Cav1 in AT1R signaling has not been defined. Here, we show that Cav1 siRNA completely knocked down endogenous Cav1 protein without affecting Cav3, -tubulin, and AT1R protein expression, confirming the specificity of this gene-silencing approach. We found that Cav1 siRNA significantly inhibited Ang II–induced transactivation of EGF-R without affecting EGF-induced EGF-R autophosphorylation (Figure 1), which is consistent with our previous results obtained with cholesterol depletion.12 These results suggest that Cav1 is specifically involved in the pathways linking AT1R to the EGF-R activation, and that the EGF-R kinase itself is not a target of Cav1. However, use of siRNA can always be associated with off-target effects.26 Thus, it is important to examine whether re-expression of siRNA-resistant mutant Cav1 rescues the impairment of Ang II–induced EGF-R transactivation in future study.
We demonstrated previously that Ang II stimulation promotes AT1R association with Cav1 and its trafficking into the caveolae/lipid rafts in VSMCs.10,11 EGF-Rs are found basally in caveolae/lipid rafts and associate with Cav1.11,12 Ang II stimulates AT1R movement into these microdomains cotemporaneously with the release of EGF-R from the caveolae/lipid rafts as well as from Cav1. These events appear to be required for Ang II transactivation of EGF-R and downstream signaling events.11,12 The present study shows that Cav1 siRNA blocks these events. It was reported previously that the Cav1 consensus binding sequence within the AT1R is required for the receptor movement from the endoplasmic reticulum to the plasma membrane in COS-7 cells transfected with epitope-tagged AT1R.27 To our knowledge, the present study is the first demonstration that Cav1 may function as a scaffold protein or a chaperone for endogenous AT1R targeting into Cav1-enriched microdomains in VSMCs.
Major elements of the AT1R-mediated signaling repertoire in VSMCs are ROS dependent,1 and Rac1 is a critical component for Ang II–induced NAD(P)H oxidase activation.28 We reported that in VSMCs, Ang II promotes association of Rac1 with Cav1 and Rac1 translocation to the Cav1-enriched membrane fractions,11 where the Nox1 NAD(P)H oxidase is found.13 Consistent with our data, Rac1 has been shown to associate dynamically with caveolae/lipid rafts after agonist stimulation in other systems.29,30 Here, we show that Cav1 siRNA inhibits Ang II– but not PMA-stimulated increase in H2O2 production, indicating the specific involvement of Cav1 in the pathways linking AT1R signal to the NAD(P)H oxidase but not in the PKC-stimulated NAD(P)H oxidase pathways. Furthermore, Ang II–induced Rac1 activation and its translocation to the membrane are inhibited by Cav1 siRNA. Of note, knockdown of Cav1 caused mislocalization of Rac1 to the membrane fraction in the basal state. Given that Cav1 binds Rac1 slightly in basal state (Figure 3B) and that some Cav1 is also found in the cytosol,31 it is possible that Cav1 is involved in keeping Rac1 in active form in the cytosol before stimulation. These results suggest that Cav1 regulates not only GTP loading of Rac1 but also proper localization of Rac1 and its membrane translocation, presumably through its binding to Rac1. These events may contribute to the activation of NAD(P)H oxidase in VSMCs. Gonzalez et al32 reported that Cav1 siRNA enhances basal and sphingosine 1–phosphate-induced Rac1 activity in bovine aortic endothelial cells. This discordance may be attributable to differences of cell types and in their portfolios of expressed proteins or of coupling differences in the agonists being studied.32 Our results are consistent with the notion that Cav1 may play an important role in Rac1-related signal generation, which is required for triggering and sustaining activation of NAD(P)H oxidase in VSMCs.
Activation of Rac is regulated by Rac-GEFs,16 including Sos, Vav, Tiam1, Pix and ?Pix, and P-Rex1.17 Sos-1 in particular has been identified in caveolin-enriched fractions in other systems18 and exhibits the Cav1 consensus binding sequences. In the present study, we demonstrated that Ang II stimulates tyrosine phosphorylation of Sos-1 and association of Sos-1 with Cav1 (Figure 4) cotemporaneously with Rac1 activation by Ang II, whereas PMA has no effects on these responses. We found that Cav1 siRNA inhibits Ang II–induced tyrosine phosphorylation of Sos-1 (Figure 4C). Given that PMA-induced ROS production is not inhibited by Cav1 siRNA (Figure 3A), these results suggest that one of the molecular targets of Cav1 is Sos-1, thereby regulating Rac1 activity. To conclude that Rac1 and Sos-1 are key mediators of Ang II/Cav1 signaling, it is essential to examine the effects of knockdown of these proteins by specific siRNAs. This point requires further investigation. Moreover, Rac1 is a regulator of actin cytoskeleton,16 and Cav1 interacts with a variety of actin-binding cytoskeletal proteins.22,23 Actin cytoskeleton plays an important role in Ang II–induced NAD(P)H oxidase activation in VSMCs.21 We showed that Cav1 siRNA inhibits Ang II–stimulated formation of actin stress fibers without affecting microtubule structure. A role of Cav1 in regulating cytoskeletal structure has been demonstrated in other systems.32,33 Together, these results suggest that Cav1 is involved in Rac1 activation and actin cytoskeletal reorganization, which may contribute to efficient temporally and spatially specific NAD(P)H oxidase activation in VSMCs.
Our previous work demonstrates that Ang II activates the redox-sensitive kinase Akt and redox-insensitive kinase ERK1/2, which contribute to Ang II–induced hypertrophy.3,4 We show that Cav1 siRNA inhibits Ang II–stimulated ROS-dependent Akt phosphorylation and [3H]leucine incorporation without affecting ROS-independent ERK1/2 phosphorylation, suggesting that Cav1 is specifically involved in the ROS-dependent AT1R signaling events regulating VSMC hypertrophy. These results are consistent with those obtained by disruption of caveoale/lipid rafts with cholesterol binding reagents.11 Studies in other systems show an inhibitory or a stimulatory role of Cav1 in activation of ERK1/2 and Akt pathways.9,32 Thus, the characteristics of Cav1-mediated responses appear to be highly dependent on the molecular context and cell type, reflecting varying patterns of expression of Cav1 as well as of the multiple proteins with which it can interact. Some studies using Cav1–/– mice show that Cav1 functions as a negative regulator for cell growth.9 In contrast, Cav1–/–, apolipoprotein E–/– (apoE–/–) double-knockout mice are relatively protected from the development of atherosclerosis in the aorta compared with the apoE–/– genotype.34 Ang II is proatherogenic, as reflected by the exacerbation of the process by Ang II infusion in apoE–/– mice.35 Given that Cav1 functions as a signaling scaffold, it is reasonable that removal of compartmentalization of ROS-dependent signaling components by knockdown of Cav1 may inhibit proatherogenic stimuli that normally act through caveolae/lipid rafts.
In summary, we provide evidence that Cav1 plays an important role in AT1R trafficking into the Cav1-enriched lipid rafts, which is required for Rac1 activation, ROS production, ROS-dependent EGF-R transactivation, and Akt phosphorylation, as well as VSMC hypertrophy. We also found that Cav1 is involved in Ang II–stimulated tyrosine phosphorylation of Sos-1 and actin cytoskeletal reorganization, which may contribute to the efficient Rac1-dependent NAD(P)H oxidase activation in VSMCs. These findings suggest an essential role of Cav1 for the spatial–temporal organization of ROS-dependent AT1R signaling in VSMCs and provide additional support for the importance of Cav1 in cardiovascular regulation.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grant HL60728 (R.W.A., M.U.F.) and an American Heart Association national scientist development grant (M.U.F.). Immunofluorescence analysis was performed in the internal medicine imaging core supported by NIH grants HL058000 and HL075209.
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Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Room 319, Atlanta, GA 30322. E-mail mfukai@emory.edu
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 (AT1R) 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 AT1R 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, H2O2 production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signal-regulated 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 AT1R 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.
Conclusions— Cav1 plays an essential role in AT1R targeting into Cav1-enriched lipid rafts and Rac1 activation, which are required for proper organization of ROS-dependent Ang II signaling linked to VSMC hypertrophy.
Angiotensin II (Ang II)–induced vascular hypertrophy is dependent on caveolae/lipid rafts and reactive oxygen species (ROS) derived from NAD(P)H oxidase. Using caveolin-1 siRNA, we demonstrate that caveolin-1 plays an essential role in AT1 receptor targeting into caveolae/lipid rafts and Rac1 activation, which are required for ROS-dependent, growth-related Ang II signaling.
Key Words: angiotensin II ? reactive oxygen species ? caveolin ? caveolae ? vascular hypertrophy ? vascular smooth muscle
Introduction
Angiotensin 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-protein–coupled Ang II type1 receptor (AT1R). Major outputs of the AT1R 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.3–6 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–/– mice.8,9 We showed that Ang II promotes association of AT1R with Cav1 and AT1R 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-rich microdomains such as caveolae/lipid rafts are essential for transactivation of EGF-R.12 Furthermore, Nox1, a homolog of the phagocytic NAD(P)H oxidase subunit gp91phox, has been shown to be found in caveolin-enriched fractions in VSMCs.13 However, a specific role of Cav1 in ROS-dependent, growth-related AT1R signaling in VSMCs is incompletely understood.
The small G-protein Rac1 is a critical component of NAD(P)H oxidase activated by Ang II,14 and its translocation to the plasma membrane is an essential step for activation of oxidase.15 We demonstrated previously that Ang II stimulation promotes Rac1 association with Cav1 and its translocation into caveolae/lipid rafts through microtubule-dependent manner.11 Activation of Rac is achieved through the exchange of bound GDP for GTP by a family of guanine nucleotide exchange factors (GEFs).16 GEFs that target Rac include Son of Sevenless (Sos-1), Vav1, –2, and –3; Tiam1, and ?Pix; and P-Rex1.17 All of these GEF proteins contain a pleckstrin homology domain, but Sos-1, Vav, and P-Rex1 also have caveolin-binding consensus sequences. Sos-1 has been identified in Cav1-enriched fractions in nonvascular systems.18 Little is known about GEFs activated by Ang II and its relationships with Cav1.
Here we demonstrate that knockdown Cav1 expression using a small interfering RNA (siRNA) results in inhibition of Ang II–stimulated AT1R targeting into caveolae/lipid rafts, GTP loading of Rac1, and its translocation to the plasma membrane. As its consequence, Cav1 siRNA inhibits Ang II–induced H2O2 production, its downstream EGF-R transactivation, and Akt phosphorylation, as well as vascular hypertrophy without affecting ROS-independent pathways. Moreover, Ang II stimulation induces tyrosine phosphorylation of Sos-1, which is inhibited by Cav1 siRNA, demonstrating a molecular mechanism by which Cav1 regulates activation of Rac1. These findings suggest an essential role of Cav1 in AT1 trafficking into Cav1-enriched lipid rafts and Rac1 activation, which is required for activation of NAD(P)H oxidase and redox-signaling linking to vascular hypertrophy.
Materials and Methods
Materials
Anti-EGFR, AT1 antibody, and Sos-1 antibodies were from Santa Cruz. Antibodies to Cav1, Cav3, and phosphotyrosine (PY20) were from BD Biosciences. Antibodies to phospho–Akt/protein kinase B (PKB) (pS473) and phospho–extracellular signal-regulated kinase 1/2 (ERK1/2) were from Cell Signaling. Other materials were purchased from Sigma.
Cell culture, synthetic siRNA and its transfection, intracellular H2O2 measurement, immunoprecipitation and immunoblotting, Rac activity assay, isolation of membrane and cytosolic fractions, purification of caveolae fractions, confocal immunofluorescence microscopy, [3H] leucine incorporation assay, and statistical analyses are described in the Materials and Methods section in the online data supplement available at http://atvb.ahajournals.org.
Results
Knockdown of Cav1 Protein by siRNA Inhibits EGF-R Transactivation by Ang II
EGF-R transactivation plays major roles in growth-related Ang II signaling in VSMCs.2,19 We reported previously that cholesterol-enriched microdomains such as caveolae/lipid rafts are involved in Ang II–induced EGF-R transactivation.12 We therefore determined specifically the role of Cav1 in this response by transfecting Cav1 siRNA into VSMCs. As shown in Figure 1A, Cav1 siRNA, but not scrambled siRNA, completely knocked down Cav1 protein in VSMCs without affecting Cav3, -tubulin, and AT1R protein expression, confirming the specificity of Cav1 siRNA. As shown in Figure 1B, Cav1 siRNA significantly inhibited Ang II–induced transactivation of EGF-R without affecting EGF-induced EGF-R autophosphorylation (Figure 1C), suggesting that Cav1 is involved in the pathways linking AT1R to the EGF-R activation.
Figure 1. Effects of knockdown of Cav1 protein by siRNA on EGF-R phosphorylation by Ang II and EGF. A, VSMCs were transfected with Cav1 or scrambled siRNA (50 nmol/L) or transfection reagent alone (control). At 48 hours after transfection, cells were stimulated with Ang II (100 nmol/L) for 2 minutes, and lysates were immunoblotted (IB) with anti-Cav1, Cav3, -tubulin, or AT1R antibody. The results are representative of 3 separate experiments. B and C, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L for 2 minutes; B) or EGF (100 ng/mL for 30 s; C). Lysates were immunoprecipitated (IP) with anti–EGF-R antibody and immunoblotted with anti-phosphotyrosine (pTyr) or EGF-R antibody. The graphs represent averaged data, corrected for total EGF-R loading, expressed as fold change over basal. *P<0.05 for increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Knockdown of Cav1 Protein by siRNA Inhibits Ang II–Stimulated AT1R Trafficking Into Cav1-Enriched Membrane Fractions
Because we have shown previously that Ang II stimulation promotes AT1R trafficking into a Cav1-enriched membrane fraction,10,11 which is required for EGF-R transactivation by Ang II,11,12 we examined whether Cav1 is involved in this response. Detergent-free OptiPrep gradient cell fractionation to isolate Cav1-enriched/lipid raft membranes20 showed that in unstimulated VSMCs, AT1R was not found in buoyant, lower-density fractions containing Cav1 and EGF-R (Figure 2, fractions 2 through 4). Ang II stimulation (5 minutes) promoted AT1R movement into Cav1-enriched fractions cotemporaneously with the egress of EGF-R from these fractions. This bidirectional receptor trafficking was almost completely inhibited by Cav1 siRNA (Figure 2A and 2B). These data suggest that Cav1-mediated EGF-R transactivation by Ang II is associated with the AT1R targeting into as well as the egress of EGF-R from caveolae/lipid rafts.
Figure 2. Effects of Cav1 siRNA on localization of AT1R and EGF-R in caveolin-enriched membrane fractions. VSMCs transfected with scrambled or Cav1 siRNA as described above were stimulated with Ang II (100 nmol/L) for 5 minutes. Caveolae fractions (1 through 8) were immunoblotted (IB) for Cav1, AT1R, or EGF-R (A). PC indicates positive control (VSMC lysates). Equal amounts of Cav1-enriched fraction 3 were immunoblotted with anti-AT1R, EGF-R, Cav1, or Cav3 antibody (B). The blots are representative of 3 separate experiments.
Cav1 Is Involved in Ang II–Induced ROS Production
Because EGF-R transactivation by Ang II is dependent on ROS derived from NAD(P)H oxidase,5 we examined whether Cav1 is involved in Ang II–stimulated ROS production. As shown in Figure 3A, Cav1 siRNA significantly inhibited Ang II–induced increase in H2O2 production without affecting basal levels, as measured by 2'7'-dicholorofluorescin diacetate (DCF-DA) fluorescence. In contrast, H2O2 production induced by phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C [PKC]) is not inhibited by Cav1 siRNA, indicating the specific involvement of Cav1 in the pathways transmitting the AT1R signal to the NAD(P)H oxidase but not in the PKC–NAD(P)H oxidase pathways.
Figure 3. Effects of Cav1 siRNA on Ang II–induced H2O2 production, Rac1 activation, and membrane translocation. A, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) or PMA (10 μmol/L) for 30 minutes and used for DCF-DA fluorescence measurement. Graphs represent averaged data expressed as fold increase in DCF fluorescence intensity over basal. *P<0.05 for the difference of fluorescence intensity. B, VSMCs were stimulated with Ang II (100 nmol/L), and lysates were immunoprecipitated (IP) with anti-Cav1 antibody and immunoblotted (IB) with anti-Rac1 or Cav1 antibody. The bottom graphs represent averaged data, expressed as a fold increase in the association over basal. *P<0.05 for changes in Rac1-caveolin complex induced by Ang II vs vehicle alone. C and D, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 5 minutes (C) or 0 to 15 minutes (D). C, Lysates bound to PAK-1 agarose were immunoblotted with anti-Rac antibody. Values are the means±SE for 3 independent experiments. D, Lysates were separated as cytoplasm and membrane fractions as described in Methods and then immunoblotted with anti-Rac1 antibody. Graphs represent averaged data expressed as fold increase in the expression of Rac1 over basal. *P<0.05 for difference of Rac1 expression induced by Ang II vs vehicle alone.
Cav1 Is Involved in Ang II–Induced Rac1 Activation and Membrane Translocation
Because Rac1 is involved in Ang II–stimulated NAD(P)H oxidase activation in VSMCs,14 we examined whether Cav1 is involved in Ang II–induced Rac1 activation and membrane translocation. Coimmunoprecipitation assays show that Ang II stimulation promoted Rac1 association with Cav1 within 5 minutes (Figure 3B). This interaction was accompanied by activation of Rac1 as measured by a pull-down assay that quantifies the GTP–Rac binding to its effector p21-activated kinase-1 (PAK-1) (Figure 3C). Cav1 siRNA significantly inhibited the Ang II–stimulated increase in the amount of GTP-bound Rac1 (Figure 3C). Because Rac GTP loading and translocation from the cytosol to plasma membranes are critical steps for activation of NAD(P)H oxidase, we examined whether Cav1 is involved in Rac1 translocation. As shown in Figure 3D, Ang II caused a rapid and significant increase in the amount of Rac1 in the membrane fraction that peaked at 2 minutes, which was significantly inhibited by Cav1 siRNA. Of note, knockdown of Cav1 itself increased association of Rac1 with membrane in the basal state. These results suggest that Cav1 is necessary for GTP loading of Rac1 and for proper localization and targeting of Rac1 to the membrane fractions, presumably through the Cav1–Rac1 interaction (Figure 3B). These events may contribute to the activation of NAD(P)H oxidase by Ang II in VSMCs.
Ang II Stimulates the Tyrosine Phosphorylation of Sos-1, Which Is Cav1 Dependent
Rac-GEF plays an important role in activation of Rac.16 Sos-1, a Rac-GEF, has a Cav1-binding consensus sequence and has been shown to be found in Cav1-enriched fractions in nonvascular systems.18 To gain insight into the molecular mechanisms by which Cav1 is involved in Rac1 activation, we tested the role of Cav1 in activation of Sos-1 by Ang II. As shown in Figure 4A, Ang II stimulation significantly increased tyrosine phosphorylation of Sos-1 within 2 minutes, with peak at 5 minutes. PMA had no effect on this response (data not shown). Moreover, Ang II stimulation promoted association of Cav1 with Sos-1 (Figure 4B) cotemporaneously with Sos-1 phosphorylation (Figure 5C). Cav1 siRNA significantly inhibited these responses (Figure 4C).
Figure 4. Effects of Cav1 siRNA on Ang II–induced tyrosine phosphorylation of Sos-1. A and B, VSMCs were stimulated with Ang II (100 nmol/L), and lysates were immunoprecipitated (IP) with anti–Sos-1 (A) or Cav1 (B) antibody and immunoblotted (IB) with anti-phosphotyrosine (pTyr) or Sos-1 antibody (A) or with anti–Sos-1 or Cav1 antibody (B). The bottom graphs represent averaged data expressed as a fold increase over basal. *P<0.05 for increase by Ang II vs vehicle alone. C, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 5 minutes. Lysates were immunoprecipitated with anti–Sos-1 antibody and immunoblotted with pTyr or Sos-1 antibody. The graphs represent averaged data, corrected for total Sos-1 loading, expressed as fold change over basal. *P<0.05 for increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Figure 5. Effects of Cav1 siRNA on Ang II–stimulated actin cytoskeletal reorganization. VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 2 minutes. A, Cells were fixed, permeabilized, and stained with Rhodamine Red-X–conjugated phalloidin (left) or with monoclonal mouse anti–-tubulin antibody, followed by Rhodamine Red-X–conjugated goat anti-mouse IgG (right).
Cav1 Is Involved in Ang II–Induced Actin Cytoskeletal Reorganization, ROS-Dependent Akt Phosphorylation, and VSMC Hypertrophy
Recent evidence suggests that an intact actin cytoskeleton is important in Ang II–stimulated NAD(P)H oxidase activation in VSMCs.21 Cav1 also interacts with actin cytoskeleton binding proteins,22,23 and Ang II induces actin stress fiber formation.24 We hypothesized that the roles of Cav1 in Ang II–induced ROS production and in actin cytoskeleton interactions might be interrelated. Thus, we examined the effect of Cav1 siRNA on Ang II–induced actin stress fiber formation using confocal microscopy. We found that it significantly reduced the phalloidin staining without affecting -tubulin staining (Figure 5). These results suggest that Cav1 is involved in Ang II–stimulated actin cytoskeletal reorganization in VSMCs, which may contribute to proper activation of NAD(P)H oxidase.
To assess further the functional role of Cav1 in ROS-dependent Ang II signaling linked to vascular hypertrophy, we examined the effects of Cav1 siRNA on phosphorylation of the ROS-dependent kinase Akt and the ROS-independent kinase ERK1/2. Akt and ERK1/2 are involved in Ang II–induced VSMC hypertrophy.3,4 As shown in Figure 6A and 6B, Cav1 siRNA significantly inhibited Ang II–induced Akt phosphorylation, whereas ERK1/2 phosphorylation was not affected. To determine the functional role of Cav1 in hypertrophy, we examined the effect of Cav1 siRNA on Ang II–stimulated [3H]leucine incorporation. As shown in Figure 6C, Cav1 siRNA significantly inhibited the basal and Ang II–induced responses. The inhibitory effects of Cav1 siRNA are not caused by toxic effects because the trypan blue exclusion test for cell viability indicated that cells transfected with siRNA were >98% viable. Thus, Cav1 plays an important role for Ang II–induced hypertrophy, at least in part through regulating ROS-dependent AT1R signaling pathways in VSMCs.
Figure 6. Effects of Cav1 siRNA on Akt and ERK1/2 phosphorylation as well as hypertrophy. A and B, VSMCs transfected with scrambled or Cav1 siRNA as described were stimulated with Ang II (100 nmol/L) for 2 minutes. Western analysis for phospho-Akt and total Akt (A) or phospho-ERK1/2 and total ERK1/2 (B) were shown. C, VSMCs transfected with scrambled or Cav1 siRNA as described were used for measurement of [3H]leucine incorporation induced by Ang II (100 nmol/L). The graphs represent averaged data expressed as fold change over basal. *P<0.05 for basal and increase by Ang II in cells transfected with scrambled vs Cav1 siRNA.
Discussion
The present study using siRNA-mediated knockdown of Cav1 protein demonstrates that Cav1 is required for AT1R targeting into Cav-enriched/lipid rafts, Rac1 activation, ROS generation, EGF-R transactivation, Akt phosphorylation, as well as hypertrophy in VSMCs. In contrast, EGF-induced EGF-R autophosphorylation, PMA-induced ROS production, as well as Ang II–induced ROS-independent ERK1/2 phosphorylations are not affected by Cav1 siRNA. We also found that Cav1 is involved in Ang II–stimulated tyrosine phosphorylation of Sos-1 and actin cytoskeletal reorganization, which may contribute to the Rac1-dependent NAD(P)H oxidase activation in VSMCs.
Disruption of caveolae/lipid rafts with the cholesterol-depleting agent methyl-?-cyclodextrin, inhibits Ang II–induced EGF-R transactivation,12 a major output of growth-related AT1R signaling in VSMCs.19,25 However, a specific role of Cav1 in AT1R signaling has not been defined. Here, we show that Cav1 siRNA completely knocked down endogenous Cav1 protein without affecting Cav3, -tubulin, and AT1R protein expression, confirming the specificity of this gene-silencing approach. We found that Cav1 siRNA significantly inhibited Ang II–induced transactivation of EGF-R without affecting EGF-induced EGF-R autophosphorylation (Figure 1), which is consistent with our previous results obtained with cholesterol depletion.12 These results suggest that Cav1 is specifically involved in the pathways linking AT1R to the EGF-R activation, and that the EGF-R kinase itself is not a target of Cav1. However, use of siRNA can always be associated with off-target effects.26 Thus, it is important to examine whether re-expression of siRNA-resistant mutant Cav1 rescues the impairment of Ang II–induced EGF-R transactivation in future study.
We demonstrated previously that Ang II stimulation promotes AT1R association with Cav1 and its trafficking into the caveolae/lipid rafts in VSMCs.10,11 EGF-Rs are found basally in caveolae/lipid rafts and associate with Cav1.11,12 Ang II stimulates AT1R movement into these microdomains cotemporaneously with the release of EGF-R from the caveolae/lipid rafts as well as from Cav1. These events appear to be required for Ang II transactivation of EGF-R and downstream signaling events.11,12 The present study shows that Cav1 siRNA blocks these events. It was reported previously that the Cav1 consensus binding sequence within the AT1R is required for the receptor movement from the endoplasmic reticulum to the plasma membrane in COS-7 cells transfected with epitope-tagged AT1R.27 To our knowledge, the present study is the first demonstration that Cav1 may function as a scaffold protein or a chaperone for endogenous AT1R targeting into Cav1-enriched microdomains in VSMCs.
Major elements of the AT1R-mediated signaling repertoire in VSMCs are ROS dependent,1 and Rac1 is a critical component for Ang II–induced NAD(P)H oxidase activation.28 We reported that in VSMCs, Ang II promotes association of Rac1 with Cav1 and Rac1 translocation to the Cav1-enriched membrane fractions,11 where the Nox1 NAD(P)H oxidase is found.13 Consistent with our data, Rac1 has been shown to associate dynamically with caveolae/lipid rafts after agonist stimulation in other systems.29,30 Here, we show that Cav1 siRNA inhibits Ang II– but not PMA-stimulated increase in H2O2 production, indicating the specific involvement of Cav1 in the pathways linking AT1R signal to the NAD(P)H oxidase but not in the PKC-stimulated NAD(P)H oxidase pathways. Furthermore, Ang II–induced Rac1 activation and its translocation to the membrane are inhibited by Cav1 siRNA. Of note, knockdown of Cav1 caused mislocalization of Rac1 to the membrane fraction in the basal state. Given that Cav1 binds Rac1 slightly in basal state (Figure 3B) and that some Cav1 is also found in the cytosol,31 it is possible that Cav1 is involved in keeping Rac1 in active form in the cytosol before stimulation. These results suggest that Cav1 regulates not only GTP loading of Rac1 but also proper localization of Rac1 and its membrane translocation, presumably through its binding to Rac1. These events may contribute to the activation of NAD(P)H oxidase in VSMCs. Gonzalez et al32 reported that Cav1 siRNA enhances basal and sphingosine 1–phosphate-induced Rac1 activity in bovine aortic endothelial cells. This discordance may be attributable to differences of cell types and in their portfolios of expressed proteins or of coupling differences in the agonists being studied.32 Our results are consistent with the notion that Cav1 may play an important role in Rac1-related signal generation, which is required for triggering and sustaining activation of NAD(P)H oxidase in VSMCs.
Activation of Rac is regulated by Rac-GEFs,16 including Sos, Vav, Tiam1, Pix and ?Pix, and P-Rex1.17 Sos-1 in particular has been identified in caveolin-enriched fractions in other systems18 and exhibits the Cav1 consensus binding sequences. In the present study, we demonstrated that Ang II stimulates tyrosine phosphorylation of Sos-1 and association of Sos-1 with Cav1 (Figure 4) cotemporaneously with Rac1 activation by Ang II, whereas PMA has no effects on these responses. We found that Cav1 siRNA inhibits Ang II–induced tyrosine phosphorylation of Sos-1 (Figure 4C). Given that PMA-induced ROS production is not inhibited by Cav1 siRNA (Figure 3A), these results suggest that one of the molecular targets of Cav1 is Sos-1, thereby regulating Rac1 activity. To conclude that Rac1 and Sos-1 are key mediators of Ang II/Cav1 signaling, it is essential to examine the effects of knockdown of these proteins by specific siRNAs. This point requires further investigation. Moreover, Rac1 is a regulator of actin cytoskeleton,16 and Cav1 interacts with a variety of actin-binding cytoskeletal proteins.22,23 Actin cytoskeleton plays an important role in Ang II–induced NAD(P)H oxidase activation in VSMCs.21 We showed that Cav1 siRNA inhibits Ang II–stimulated formation of actin stress fibers without affecting microtubule structure. A role of Cav1 in regulating cytoskeletal structure has been demonstrated in other systems.32,33 Together, these results suggest that Cav1 is involved in Rac1 activation and actin cytoskeletal reorganization, which may contribute to efficient temporally and spatially specific NAD(P)H oxidase activation in VSMCs.
Our previous work demonstrates that Ang II activates the redox-sensitive kinase Akt and redox-insensitive kinase ERK1/2, which contribute to Ang II–induced hypertrophy.3,4 We show that Cav1 siRNA inhibits Ang II–stimulated ROS-dependent Akt phosphorylation and [3H]leucine incorporation without affecting ROS-independent ERK1/2 phosphorylation, suggesting that Cav1 is specifically involved in the ROS-dependent AT1R signaling events regulating VSMC hypertrophy. These results are consistent with those obtained by disruption of caveoale/lipid rafts with cholesterol binding reagents.11 Studies in other systems show an inhibitory or a stimulatory role of Cav1 in activation of ERK1/2 and Akt pathways.9,32 Thus, the characteristics of Cav1-mediated responses appear to be highly dependent on the molecular context and cell type, reflecting varying patterns of expression of Cav1 as well as of the multiple proteins with which it can interact. Some studies using Cav1–/– mice show that Cav1 functions as a negative regulator for cell growth.9 In contrast, Cav1–/–, apolipoprotein E–/– (apoE–/–) double-knockout mice are relatively protected from the development of atherosclerosis in the aorta compared with the apoE–/– genotype.34 Ang II is proatherogenic, as reflected by the exacerbation of the process by Ang II infusion in apoE–/– mice.35 Given that Cav1 functions as a signaling scaffold, it is reasonable that removal of compartmentalization of ROS-dependent signaling components by knockdown of Cav1 may inhibit proatherogenic stimuli that normally act through caveolae/lipid rafts.
In summary, we provide evidence that Cav1 plays an important role in AT1R trafficking into the Cav1-enriched lipid rafts, which is required for Rac1 activation, ROS production, ROS-dependent EGF-R transactivation, and Akt phosphorylation, as well as VSMC hypertrophy. We also found that Cav1 is involved in Ang II–stimulated tyrosine phosphorylation of Sos-1 and actin cytoskeletal reorganization, which may contribute to the efficient Rac1-dependent NAD(P)H oxidase activation in VSMCs. These findings suggest an essential role of Cav1 for the spatial–temporal organization of ROS-dependent AT1R signaling in VSMCs and provide additional support for the importance of Cav1 in cardiovascular regulation.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grant HL60728 (R.W.A., M.U.F.) and an American Heart Association national scientist development grant (M.U.F.). Immunofluorescence analysis was performed in the internal medicine imaging core supported by NIH grants HL058000 and HL075209.
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