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. 2010 Jul 7;24(8):1637–1649. doi: 10.1210/me.2010-0043

Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17β-Estradiol

Neetu Sud 1, Dean A Wiseman 1, Stephen M Black 1
PMCID: PMC2940462  PMID: 20610538

Abstract

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERα) within the plasma membrane of endothelial cells (EC). ERα is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERα to the plasma membrane. However, the role played by caveolin 1 in regulating 17β-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17β-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERα localization to the plasma membrane in EC. Further, before estrogen stimulation ERα associates with caveolin 1, whereas stimulation promotes a pp60Src-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERα-PI3 kinase interactions and disrupting caveolin 1-ERα interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERα/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62–73 of caveolin 1 as the ERα-binding site. Delivery of a synthetic peptide based on this sequence decreased ERα plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17β-estradiol-induced NO production by promoting ERα to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.

We show that caveolin 1 regulates ERα plasma membrane trafficking in endothelial cells and identify amino acids 62–73 of caveolin 1 as the ERα binding site.

Estrogen induces both rapid and delayed effects on the cardiovascular system. Delayed effects (e.g. remodeling or lipid alterations) typically require hours to days to occur and require transcriptional effects with subsequent modulation of protein expression (1,2,3,4). Conversely, early effects of estrogen stimulation are so rapid that they do not involve activation of RNA and protein synthesis. These rapid actions are known as nongenomic actions and have been linked to membrane-associated estrogen receptor (ER), which make up approximately 3% of the total ER pool (4). The ability of 17β-estradiol to induce immediate vasodilatory effects that are both endothelial and NO dependent has been demonstrated in several systems. It has been shown that 17β-estradiol can induce rapid NO release by activating the PI3 kinase/Akt pathway in human endothelial cells (EC) (5,6,7). Additionally, acute activation of endothelial nitric oxide synthase (eNOS) by 17β-estradiol to produce NO in uterine aortic EC acts at least partially through an ERK pathway, possibly via ER localized on the plasma membrane (8). However, the molecular mechanisms of this process are still incompletely understood.

Caveolae are plasma membrane-attached vesicular organelles present in most cell types, but are particularly abundant in adipocytes, EC, fibroblasts, and smooth muscle cells (9). Caveolin 1, a 21- to 24-kDa protein, is a principal integral membrane component of caveolae (10,11). Caveolin 1 appears to organize the association of signaling molecules within caveolae (12), and caveolin 1 expression has been shown to promote ERα translocation to the plasma membrane in breast cancer cell line, MCF7 (13). Within rat lung EC, the colocalization of ERα with caveolin 1 has also been detected by confocal immunofluorescence microscopy (13). In vascular smooth muscle cells, recent reports suggest there is relatively little association of ERα and caveolin 1 in unstimulated cells, but, in response to estrogen, association increases (13). In contrast, in MCF7 cells there appears to be a relatively strong association of these two proteins within the plasma membrane under basal conditions whereas estrogen reduces the level of interaction (13). These previous studies led us to analyze the role of caveolin 1 in 17β-estradiol-mediated NO signaling in EC. In this current study, we examine the significance of caveolin 1 in 17β-estradiol-mediated eNOS activation in EC. We also determined the effect of modulating caveolin 1 protein levels and ERα-caveolin 1 protein interactions on 17β-estradiol signaling leading to eNOS activation and identified the region within caveolin 1 that is required to traffic ERα to the plasma membrane.

Results

To decrease caveolin 1 protein levels, pulmonary artery endothelial cells (PAEC) were transfected with a caveolin 1-specific small interfering RNA (siRNA) or a scrambled siRNA (as a control). Forty-eight hours after transfection, caveolin 1 expression was significantly reduced (−70%; Fig. 1, A and B). Further, although total ERα expression levels were unchanged in the caveolin 1 siRNA-transfected cells, the amount of ERα localized to the plasma membrane was significantly reduced (Fig. 1, C and D). Reprobing for 5′-nucleotidase confirmed equal loading of the plasma membrane fractions (Fig. 1C). We also observed a significant reduction in 17β-estradiol-mediated phosphorylation of eNOS at serine1177 in the caveolin 1 siRNA-transfected cells (Fig. 1, E and F), as well as an attenuation in NO generation (Fig. 1I). However, caveolin 1 depletion did not alter plasma membrane-localized eNOS (Fig. 1, G and H) or the generation of NO to shear stress (Fig. 1J) or vascular endothelial growth factor (VEGF) (Fig. 1K), suggesting that effect of caveolin 1 on NO signaling is specific to 17β-estradiol.

Figure 1.

Figure 1

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Decreasing caveolin 1 expression attenuates 17β-estradiol-mediated activation of eNOS. PAEC were transfected with either a caveolin 1 siRNA or a scrambled siRNA (as a control). Immunoblot analysis demonstrates a significant decrease in caveolin 1 protein levels in caveolin 1 siRNA-transfected cells (A and B; *, P < 0.05 vs. scrambled siRNA). Plasma membrane fractions were isolated and subjected to immunoblotting with either an antibody against ERα or eNOS. Decreasing caveolin 1 expression results in a significant reduction in ERα localization to the plasma membrane (C and D; *, P < 0.05 vs. scrambled siRNA) although total ERα levels are unchanged (C). The siRNA-mediated knockdown of caveolin 1 does not alter eNOS localized to the plasma membrane (E and F), Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). The siRNA-mediated knockdown of caveolin 1 significantly inhibits 17β-estradiol-stimulated (100 nm, 30 min) eNOS phosphorylation at serine 1177 (G and H) and NO generation (I). The siRNA-mediated knockdown of caveolin 1 does not inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel J) or VEGF (100 ng/ml, 15min; panel K). Data are mean ± sem, n = 3–6. *, P < 0.05 vs. no 17β-estradiol; †, P < 0.05 vs. 17β-estradiol-treated scrambled siRNA.

Previous studies suggest that estrogen-mediated stimulation of eNOS requires pp60Src signaling (5) and that pp60Src is capable of phosphorylating caveolin 1 (14). Thus, we determined whether pp60Src-mediated phosphorylation of caveolin 1 is involved in the estrogen-mediated activation of eNOS. We found that acute 17β-estradiol stimulation induced significant caveolin 1 phosphorylation at tyrosine-14 (Fig. 2, A and B), and this was blocked in cells pretreated with the specific pp60Src inhibitor, PP2 (10 μm, Fig. 2, A and B) with no change in total caveolin 1 protein levels (Fig. 2, A and B). The inhibition of pp60Src also caused a decrease in eNOS phosphorylation at serine 1177 (Fig. 2, C and D) and attenuated 17β-estradiol-mediated NO production (Fig. 2E). These data indicate that caveolin 1 is phosphorylated by pp60Src during 17β-estradiol stimulation and that the increases in NO signaling are pp60Src dependent. To confirm that the effect on NO signaling was ER dependent, we also used the ER antagonist ICI 182,780 and found that both 17β-estradiol-mediated increases in eNOS ser1117 phosphorylation (Fig. 2, C and D) and NO generation (Fig. 2E) were attenuated. To determine whether caveolin 1 phosphorylation is involved in regulating ERα localization to the plasma membrane, adenoviral expression constructs of wild-type caveolin 1, and a caveolin 1 mutant deficient in phosphorylation at tyrosine 14 were transduced into PAEC (Fig. 3, A and B). Our data indicate that the fraction of ERα localized to the plasma membrane increased in cells expressing either wild-type caveolin 1 or mutant Y14F caveolin 1 (Fig. 3, C and D). The overexpression of the Y14F mutant of caveolin 1 did not alter the levels of plasma membrane-localized eNOS (Fig. 3, E and F). Reprobing for 5′-nucleotidase confirmed equal loading of the plasma membrane fractions (Fig. 3, C and E). For further confirmation, PAEC were cotransfected with green fluorescent protein (GFP)-ERα, stained with a plasma membrane-specific marker, Alexa Fluor 594 wheat germ agglutinin (WGA), and colocalization analyzed using high-resolution fluorescence microscopy. The intensity of yellow fluorescence (colocalized red and green channel fluorescence) was significantly higher in cells transduced with either wild-type or Y14F mutant caveolin 1 (Fig. 3, G and H). These data suggest that caveolin 1 promotes the localization of ERα to the plasma membrane in a phosphorylation-independent fashion.

Figure 2.

Figure 2

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17β-Estradiol stimulates caveolin 1 phosphorylation through the activation of pp60Src. PAEC were exposed to pp60Src inhibitor, PP2 (10 μm, 30 min), before 17β-estradiol (100 nm, 30 min). 17β-Estradiol significantly increases phospho-Tyr14 caveolin 1, and PP2 inhibits this phosphorylation although total caveolin 1 levels are unchanged (A and B). 17β-Estradiol also produces significantly higher eNOS phosphorylation at serine 1177 compared with untreated cells, and pretreatment with the ER antagonist, ICI 182 780 (10 μm, 30 min) or PP2 significantly attenuates eNOS phosphorylation at serine 1177 (C and D). 17β-Estradiol treatment also induces a significant increase in NO generation, and pretreatment with ICI 182,780 or PP2 results in significant reduction in NO production (E). Data are mean ± sem n = 3–4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. 17β-estradiol treated.

Figure 3.

Figure 3

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Caveolin 1 phosphorylation is not required for the localization of ERα to the plasma membrane. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav) or a tyrosine 14-deficient mutant (AdY14FCav) indicates significant overexpression of AdWTCav and AdY14Cav (A and B). There is significantly higher ERα in the plasma membrane in both AdWTCav- and AdY14Cav-transduced cells (C and D). AdY14Cav overexpression did not alter eNOS localization to the plasma membrane (E and F). Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). Immunofluorescence analysis of cells transduced with AdGFP, AdWTCav-1, or AdY14FCav-1 were then transfected with an ERα-GFP construct. The plasma membrane of these cells was labeled with Alexa Fluor 594 WGA (red). The extent of membrane localization of ERα was then determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA and green fluorescence of ERα-GFP (G); the white bar in each individual panel represents 30 μm). The intensity of yellow fluorescence is significantly higher in cells transduced with either AdWTCav or AdY14Cav (H). Data are mean ± sem n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

Estrogen can induce rapid NO release by activating the PI3 kinase/Akt pathway (5,6,7). Thus, we next determined whether the phosphorylation of caveolin 1 modulates ERα/PI3 kinase interactions. ERα was immunoprecipitated from PAEC overexpressing wild-type or Y14F-mutant caveolin 1 and the interactions of caveolin 1 and PI3 kinase with ERα analyzed in the presence and absence of 17β-estradiol. Under basal conditions, caveolin 1 is associated with ERα, and acute exposure to 17β-estradiol disrupts this interaction (Fig. 4, A and C). Moreover, the ERα/caveolin 1 association is significantly higher in wild-type caveolin 1 overexpressing cells under basal conditions, and the disruption of this complex by 17β-estradiol is enhanced (Fig. 4, A and C). However, 17β-estradiol fails to disrupt the ERα-caveolin 1 complex in cells overexpressing the Y14F caveolin 1 mutant protein (Fig. 4, A and C). We next investigated the interaction of ERα with PI3 kinase after 17β-estradiol stimulation. 17β-Estradiol stimulated the interaction of ERα with PI3 kinase (Fig. 4, A and B), and this interaction was significantly enhanced in wild-type caveolin 1 overexpressing cells (Fig. 4, A and B). Conversely, the 17β-estradiol-mediated increase in ERα-PI3 kinase complex formation was significantly attenuated in cells overexpressing Y14F caveolin 1 (Fig. 4, A and B). Because Akt is known to be the downstream effector of PI3 kinase leading to eNOS activation (15,16), we next analyzed Akt activity. We found that 17β-estradiol increased Akt activity (Fig. 5, A and B) and this was significantly enhanced in cells overexpressing wild-type caveolin 1 (Fig. 5, A and B). However, 17β-estradiol failed to induce Akt activity in cells overexpressing Y14F caveolin 1 (Fig. 5, A and B). Further, in response to 17β-estradiol stimulation, wild-type caveolin 1 overexpression resulted in higher eNOS ser1177 phosphorylation and NO generation (Fig. 5E), whereas Y14F-mutant caveolin 1 overexpression attenuated 17β-estradiol-induced eNOS serine 1177 phosphorylation (Fig. 5, C and D) and NO generation (Fig. 5E). The overexpression of either wild-type caveolin 1 (Fig. 5, F and G) or the Y14F mutant of caveolin 1 (Fig. 5, H and I) did not inhibit NO generation in response to shear stress or VEGF. Together these data suggest that caveolin Y14 phosphorylation is important for 17β-estradiol induced Akt activation.

Figure 4.

Figure 4

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Caveolin 1 phosphorylation enhances the interaction of ERα with PI3 kinase. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the interaction of ERα with caveolin 1 (A and C) or PI3 kinase (A and B) was determined. 17β-Estradiol exposure causes a dissociation of the ERα-caveolin 1 complex (A and C). This dissociation was enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1 (A and C). 17β-Estradiol also significantly increases the interaction of ERα with PI3 kinase (A and B). Transduction with AdWTCav potentiates the 17β-estradiol-mediated interaction (A and B) whereas AdY14FCav attenuates this effect (A and B). Data are mean ± sem; n = 3. *, P < 0.05 vs. untreated cells; †, P < 0.05 vs. AdGFP with 17β-estradiol. IB, Immunoblotting; IP, immunoprecipitation.

Figure 5.

Figure 5

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Caveolin 1 phosphorylation enhances Akt activation and NO signaling in response to 17β-estradiol. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the effect on Akt activity (A and B), eNOS phosphorylation at serine1177 (C and D), and NO generation (E) was determined. The 17β-estradiol-mediated increase in Akt activity (A and B), eNOS phosphorylation at serine 1177 (C and D), and NO generation (E) is enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1. The overexpression of wild-type caveolin 1 (F and G) or AdY14F (H and I) did not significantly attenuate the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min) or VEGF (100 ng/ml, 15min). Data are mean ± sem; n =3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. AdGFP with 17β-estradiol.

Previous studies have suggested that the scaffold domain of caveolin 1 is important for the trafficking of ER to the plasma membrane (17). Thus, we next determined whether the overexpression of a caveolin 1 mutant in which the scaffold domain was removed could effect ERα trafficking to the plasma membrane. Our data indicate that the overexpression of the caveolin 1 scaffold domain mutant (CavΔ; Fig. 6, A and B) reduced both ERα (Fig. 6, C and D) and caveolin 1 (Fig. 6, C and E) localization to the plasma membrane. However, when total cell extracts were subjected to immunoprecipitation, we found that the amount of caveolin 1 bound to the caveolin 1 scaffold domain mutant was increased to the same extent as wild-type caveolin 1 overexpression (Fig. 6, F and G), suggesting that there is another site on caveolin 1 that is responsible for ERα binding.

Figure 6.

Figure 6

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The scaffold domain of caveolin 1 is required for ERα trafficking to the plasma membrane but not for protein-protein interaction. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav), or a caveolin 1 mutant lacking the scaffold domain (AdCavΔ) indicates significant overexpression of AdWTCav and AdCavΔ (A and B). There is significantly higher ERα (C and D) and caveolin 1 (C and E) in the plasma membrane in the AdWTCav-transduced cells. However, both ERα (C and D) and caveolin 1 (C and E) are significantly decreased in the AdCavΔ-transduced cells (C and E). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C). Immunoprecipitation (IP) of caveolin 1 from whole-cell extracts followed by IB with ERα identified a significant increase in ERα bound to caveolin 1 in both AdWTCav- and AdCavΔ-transduced cells (F and G). Data are mean ± sem; n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

To elucidate the amino acid sequence in caveolin 1 crucial for its interaction with ERα, SPOT analysis was used to screen a library of overlapping peptides (12 amino acids), each peptide shifted by five amino acids across the entire sequence of caveolin 1. Of 26 peptides in the array, only one peptide reacted with recombinant ERα (Fig. 7A). The amino acid sequence of this peptide corresponded to residues 62–73 of caveolin 1 (Fig. 7B). Using this amino acid sequence, we created a synthetic peptide fused at the N terminus to the protein transduction domain of the HIV (TAT, R-K-K-R-R-Q-R-R-R, TAT-Cav62–73; Fig. 7B). When this fusion peptide was delivered to cells, we observed a significant reduction in ERα-caveolin 1 association compared with cells exposed to a scrambled version of the peptide, TAT-Cav62–73Scr (Fig. 7, C and D). Similarly, Western blot analysis demonstrated that TAT-Cav62–73 significantly reduced ERα localization to the plasma membrane fraction (Fig. 7, E and F). Reprobing for 5′-nucleotidase confirmed equal loading of the plasma membrane fractions (Fig. 7E). Fluorescent microscopy demonstrated that TAT-Cav62–73 significantly reduced the colocalization of ERα-GFP with the plasma membrane-specific marker, Alexa 594 WGA (Fig. 7, G and H). To determine whether the inhibition of the ERα-caveolin 1 interaction altered ERα-PI3 kinase interactions, ERα was immunoprecipitated from 17β-estradiol-stimulated cells pretreated with TAT-Cav62–73 or TAT-Cav62–73scr. The relative level of PI3 kinase coimmunoprecipitated with ERα was significantly reduced in cells exposed to TAT-Cav62–73 (Fig. 8, A and B), indicating reduced interaction. Cells pretreated with TAT-Cav62–73 also showed significant reductions in both eNOS phosphorylation at serine 1177 (Fig. 8, C and D) and NO generation (Fig. 8E). However, the TAT-Cav62–73 peptide did not alter NO generation in response to shear stress (Fig. 8F) or VEGF (Fig. 8G).

Figure 7.

Figure 7

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Amino acids 62–73 of caveolin 1 span the ERα interaction region. Probing a peptide array representing the entire sequence of human caveolin 1 with recombinant human ERα (100 ng/ml) identified a single highly reactive peptide spanning amino acids 62–73 (shown by arrow in panel A). TAT-Cav62–73, along with a scrambled version containing a similar amino acid composition (TAT-Cav62–73scr), was synthesized as a fusion with the N-terminal TAT protein transduction domain from the HIV (B). PAEC were exposed to each peptide (100 ng/ml). Immunopreciptation (IP) analyses demonstrate that TAT-Cav62–73 significantly attenuates the association of ERα with caveolin 1 (C and D). Western blot analysis also demonstrates that TAT-Cav62–73 significantly attenuates ERα levels in the plasma membrane without altering total ERα levels (E and F; *, P < 0.05 vs. TAT-Cav62–73scr). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (E). TAT-Cav62–73 and TAT-Cav62–73scr peptide-treated cells were transfected with an ERα-GFP construct and the plasma membrane labeled with Alexa Fluor 594 WGA (red). The extent of plasma membrane localization of ERα was determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA, and green fluorescence of ERα-GFP). TAT-Cav62–73 significantly attenuates the yellow fluorescence (G and H, the white bar in each panel represents 30 μm). TAT-Cav62–73 did not alter eNOS localization to the plasma membrane (I and J). n = 3; *, P < 0.05 vs. TAT-Cav62–73scr.

Figure 8.

Figure 8

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Disrupting ERα-caveolin 1 interactions attenuates the interaction of ERα with PI3 kinase and inhibits downstream NO signaling. TAT-Cav62–73 significantly attenuates the 17β-estradiol-mediated association of ERα with PI3 kinase (A and B), the increase in eNOS phosphorylation at serine 1177 (C and D), and the increase in NO generation (E). TAT-Cav62–73 did inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel F) or VEGF (100 ng/ml, 15min; panel G). Data are mean ± sem; n = 3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. TAT-Cav62–73scr with 17β-estradiol.

Discussion

Previous studies examining the role of caveolin 1 in the regulation of eNOS activity have centered on caveolin 1 interacting with eNOS and inhibiting calcium-calmodulin binding (18). However, our data demonstrate, for the first time, a positive role for caveolin 1 in 17β-estradiol-induced NO signaling in EC, and suggest that the role of caveolin 1 in NO signaling is more complex than previously appreciated. Down-regulating caveolin 1 protein levels in PAEC using siRNA reduced the ability of 17β-estradiol to induce eNOS activation whereas 17β-estradiol stimulated the phosphorylation of caveolin 1 at tyrosine 14 in a pp60Src -dependent manner, consistent with previous studies (19). Furthermore, we found that the overexpression of wild-type caveolin 1 resulted in higher eNOS phosphorylation at serine1177 and NO production after 17β-estradiol stimulation. Conversely, overexpression of a phosphorylation-deficient mutant of caveolin 1, in which tyrosine 14 was replaced with phenylalanine, blocked the estrogen-induced increase in eNOS serine1177 phosphorylation as well as NO production. These results emphasize the key role played by caveolin 1 in promoting eNOS activation in response to 17β-estradiol and identify caveolin 1 phosphorylation as a critical step in this process.

This study also reveals, for the first time, that caveolin 1 potentiates ERα localization to the plasma membrane in EC, as illustrated by our data showing that modulating caveolin 1 protein levels results in altered levels of plasma membrane-localized ERα. Further our data confirm previous studies in MCF7 cells that caveolin 1 regulates ER plasma membrane localization (20). Interestingly, overexpression of the Y14F mutant of caveolin 1 also augmented ERα localization to the plasma membrane, suggesting that caveolin 1 Y14 phosphorylation is not required for ERα plasma membrane localization. In addition, we observed that basally, caveolin 1 complexes with ERα on the plasma membrane, and 17β-estradiol stimulation disrupts this interaction. Although it has been previously demonstrated that caveolin 1 expression facilitates ERα translocation to the plasma membrane in MCF7 breast cancer cells (13), this is the first study demonstrating that a similar event occurs in EC. Our data are also consistent with the MCF7 study in the observation that there is a strong association of ERα with caveolin 1, and 17β-estradiol signaling reduces this interaction (13). However, in vascular smooth muscle cells, it has been shown that there is little association of ER and caveolin 1 in the basal state but in response to 17β-estradiol, the association increased (13). This suggests that the ER-caveolin 1 interaction may be regulated in a cell type-specific manner. Another important finding of our study is that although caveolin 1 phosphorylation is not involved in targeting ERα to the plasma membrane, it is required to induce the dissociation of caveolin 1 from ERα in response to 17β-estradiol. This is supported by our data showing that in cells overexpressing the Y14F caveolin 1 mutant, 17β-estradiol fails to promote dissociation of ERα from caveolin 1, and this is accompanied by attenuated Akt activation, reduced serine 1177 eNOS phosphorylation, and reduced NO generation. It is also worth pointing out that our studies on caveolin 1 phosphorylation have focused on its effect on its interaction with ERα. However, because caveolin 1 can also inhibit eNOS activity, it is possible that, in addition to the effects we have observed of phosphorylation on ERα-caveolin 1 interactions, that phosphorylation of caveolin 1 could result in its release from eNOS, which could also lead to increased NO signaling. However, further studies will be required to test this hypothesis.

It should be noted that our study has focused on the ability of caveolin 1 to modulate ERα translocation and signaling. However, a second type of ER, ERβ, is also expressed in EC. Like ERα, ERβ can be involved in mediating 17β-estradiol signaling in EC (6), and our recent data indicate that 17β-estradiol stimulates NO signaling equally through ERα and ERβ in these PAEC (21). Further studies will be required to determine whether caveolin 1 can also promote trafficking of ERβ to the plasma membrane and whether modulation ERβ translocation to the plasma membrane affects its ability to activate NO signaling. Another interesting aspect of this study is the finding that estrogen-mediated disruption of the ERα-caveolin 1 protein-protein interaction is required for ERα to interact with PI3 kinase and that pp60Src-mediated phosphorylation of caveolin 1 is crucial in promoting the dissociation of ERα and caveolin 1 to allow ERα to bind to PI3 kinase. This is shown in cells overexpressing the Y14F mutant of caveolin 1, in which ERα-caveolin 1 complexes persist despite stimulation with 17β-estradiol, and ERα interactions with PI3 kinase fail to occur. Conversely, overexpression of wild-type caveolin 1 augmented ERα-PI3 kinase interactions in response to 17β-estradiol. This enhanced ERα interaction with PI3 kinase resulted in increased NO generation as a consequence of enhanced Akt activation and phosphorylation of eNOS at serine 1177. Our data indicate that there were no changes in NO generation caveolin 1 knockdown or overexpression of wild-type- or Y14D mutant-caveolin 1 in response to either shear stress or VEGF. However, the NO released in response to 17β-estradiol was significantly enhanced by the overexpression of caveolin 1 and attenuated by caveolin1 knockdown or Y14F mutant overexpression. However, Lisanti and co-workers (22) have shown that eNOS caveolin-deficient mice have an increased response to acetylcholine indicative of enhanced NO generation. These apparent discrepant data are likely due to the experimental protocol used. In the caveolin 1 knockout mouse study, the data were obtained in a situation in which there was a chronic loss of caveolin 1 signaling. However, in our studies we examined acute effects that are occurring in the 15- to 30-min range. Thus, our studies do not allow us to determine what would be the long-term effect on either eNOS localization or NO signaling if the modulation of caveolin 1 signaling were maintained. It is also important to note that existing literature indicates that caveolin 1 can exert two independent modes of posttranslational regulation on eNOS activity (23,24,25). Caveolin 1 enables the enrichment of eNOS in caveolae and compartmentalizes the enzyme for optimal activation (compartmentation effect) (26). However, the direct interaction between caveolin 1 and eNOS holds the enzyme in an inactivated state (clamp effect) (27,28,29). Thus, it is possible to affect one pathway without altering the other. This may also explain why in previous studies the overexpression of caveolin 1, although increasing ER plasma membrane localization, inhibited 17β-estradiol-mediated activation of ERK (13), because the clamp effect (ER-caveolin 1 interaction) may overcome the activation effect induced by 17β-estradiol. In addition, it is also possible that there are cell-specific effects of caveolin 1 on ER and/or eNOS plasma membrane localization. However, future studies will be required to test this possibility. In addition, there are other caveolar localized proteins, such as SR-BI, that also regulate eNOS (30,31) and may involve estrogen. These studies have shown that the caveolar location of SR-BI is important for its activation of eNOS. Thus, caveolin 1 may also indirectly alter eNOS activation via estrogen through its ability to localize SR-BI to the caveolus.

Our observations that caveolin 1 is required to traffic ERα to the plasma membrane prompted us to investigate this interaction using peptide array analysis (32,33,34,35). Using a library of 12 mer overlapping peptides, each shifted by five amino acids across the entire sequence of caveolin 1 SPOT synthesized on cellulose membrane, we successfully identified residues 62–73 in caveolin 1 as the ERα-binding region. To analyze the significance of this interaction, we designed a synthetic peptide (TAT-Cav62–73) encompassing this sequence and fused to TAT sequence and determined its effects on estrogen-induced NO signaling. Treatment of cells with TAT-Cav62–73 significantly decreased ERα-caveolin 1 interaction and reduced ERα localization to the plasma membrane. This led us to investigate whether the TAT-Cav62–73 could also affect 17β-estradiol-mediated NO production. We found that this peptide decreased in ERα-caveolin 1 interactions, which in turn significantly reduced ERα-PI3 kinase interactions, serine 1177 phosphorylation of eNOS, and NO production in response to 17β-estradiol. Previous studies have shown that caveolin 1 has multiple domains (36). The region of caveolin 1 (amino acids 62–73) that we identified is different from the region corresponding to amino acids 82–101, which has been termed the “scaffolding domain” shown to interact with a broad range of signal transduction factors, including tyrosine kinase receptors, eNOS, and heterotrimeric G proteins, and is implicated in mediating the membrane attachment of caveolin 1 (12). Caveolin 1 lacking this scaffolding domain is incapable of reaching the cell surface (36). The scaffolding domain of caveolin 1 has previously been demonstrated to promote membrane localization of ERα (13) and to be responsible for the physical interaction of ERα with caveolin 1 (17). However, this study was carried out in vitro, and our data indicate that, at last in PAEC, the scaffold domain is required to traffic ERα to the plasma membrane but that even when the scaffold domain is deleted, ERα can still interact with caveolin 1. Further, we show, for the first time, that amino acids 62–73 of caveolin 1 are also important both for interaction between ERα and caveolin 1 and in facilitating ERα localization to the plasma membrane. Interestingly, this region of caveolin 1 encompasses the region between amino acids 66 and 70, which is in the most conserved region of the molecule and that has been suggested to be necessary for the exit of caveolin 1 from the endoplasmic reticulum (36). It has been shown that the substitution of amino acids 66–70 with alanine resulted in caveolin 1 accumulating in the endoplasmic reticulum (36). Thus, the interaction of caveolin 1 with ERα with a region of caveolin 1 that is important for the exit of caveolin 1 from endoplasmic reticulum suggests that ERα and caveolin 1 may traffic together through the endoplasmic reticulum to reach the plasma membrane. However, further studies will be required to test this hypothesis.

We conclude that caveolin 1 increases ERα plasma membrane localization in PAEC, and this localization appears to be dependent on amino acid sequence 62–73 of caveolin 1; this is separate from the scaffolding domain of caveolin 1 (amino acids 83–101), which appears to be necessary for the ER-caveolin 1 complex to reach the plasma membrane but not involved in the interaction of caveolin 1 with ERα. We further conclude that ERα translocation to the plasma membrane is a prerequisite for 17β-estradiol-induced NO signaling. This signal transduction pathway is manifested by an increase in pp60Src phosphorylation-mediated dissociation of caveolin 1 from ERα, permitting the ERα-PI3 kinase complex to form. This, in turn, leads to Akt activation, eNOS activation at serine 1177, and increased NO production. We believe pp60Src-mediated caveolin 1 phosphorylation is a critical event in this signaling cascade, as evidenced by the fact that overexpression of the caveolin 1 Y14F mutant abrogates ERα-caveolin 1 dissociation, preventing formation of ER-PI3 kinase complex, reducing Akt activation, and ultimately blunting endothelial cell NO production in response to 17β-estradiol.

Materials and Methods

Cell culture

Primary cultures of ovine PAEC were isolated as described elsewhere (37). Cells were maintained in DMEM containing phenol red supplemented with 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT), antibiotics, and antimycotics (MediaTech, Inc., Manassas, VA) at 37 C in a humidified atmosphere with 5% CO2-95% air. Cells were used between passages 3 and 10, seeded at approximately 50% confluence, and used when fully confluent. In all experiments where cells were exposed to 17β-estradiol, cells were seeded onto glass coverslips and incubated overnight in phenol red-free charcoal-stripped serum before exposure.

Detection of NOx

NO generated by PAEC in response to 17β-estradiol was measured using an NO-sensitive electrode with a 2-mm diameter tip connected to an Apollo 4000 Free Radical Analyzer (ISO-NOP; World Precision Instruments, Inc., Sarasota, FL) as described elsewhere (38).

Alternative methods of eNOS stimulation

PAEC were exposed to either VEGF or laminar shear stress to activate eNOS. Cells were exposed to 50 ng/ml of VEGF for 5 min. Laminar shear stress was applied using a cone-plate viscometer that accepts six-well tissue culture plates, as described previously (37,38). PAEC were exposed to 15 dyn/cm2 for 15 min. This represents a physiological level of laminar shear stress in the major human arteries, which is in the range of 5–20 dyn/cm2 (39) with localized increases to 30–100 dyn/cm2.

siRNA-mediated caveolin 1 knockdown

Cells were transfected with either an siRNA against caveolin 1 (QIAGEN, Chatsworth, CA; catalog no. SI00299642) or negative control siRNA, having no homology with ovine database (target sequence, 5′-AATTCTCCGAACGTGTCACGT-3′; catalog no. 1022076) using Hiperfect transfection reagent (QIAGEN) according to manufacturer’s instructions. Cells were harvested 48 h later, and the level of knockdown was evaluated by Western blotting using anticaveolin 1 antibody.

Overexpression of wild type and mutants of caveolin 1

Adenoviral constructs for wild-type (AdWTCav), a scaffold domain mutant of caveolin 1 (containing a deletion of amino acids 82–101, AdCavΔ), or caveolin 1 with the tyrosine at position 14 mutated to phenylalanine (AdCavY14F) were prepared using full-length human cDNA inserts obtained from the American Type Culture Collection (Manassas, VA). The scaffold domain deletion and the Y14F point mutation were generated commercially (Retrogen, Inc., San Diego, CA). The viruses were amplified in human embryonic kidney-293 cells and titered. EC at approximately 90% confluence were transduced using an approximate multiplicity of infection of 200:1. As a control, an adenovirus containing GFP was used to control for cellular effects due to adenoviral gene transduction. Immunoprecipitation and Western blotting were carried out as described elsewhere (38).

Localization of ERα to the plasma membrane

PAEC were transduced with AdGFP, AdWTCav, or AdY14FCav and transfected with ER-GFP, grown on coverslips, and then fixed with methanol. After three washes with PBS, cells were labeled with Alexa Fluor 594 (WGA, 5 μg/ml) for 10 min. Cells were washed with PBS and mounted with ProLong antifade reagent and subjected to fluorescent microscopy using a DeltaVision Personal DV fluorescent microscope (Applied Precision, Inc., Issaquah, WA). Membrane localization of ERα was determined by calculating the Pearson coefficient (40,41) between images (green for ERα-GFP and red for Alexa Fluor 594 WGA). ERα-GFP was a generous gift from Dr. Stavros Manolagas, University of Arkansas for Medical Sciences.

Plasma membrane isolation

Plasma membrane was isolated from cells using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce Chemical Co., Rockford, IL) according to manufacturer’s protocol and as we described previously (42).

Akt kinase assay

Akt activation was determined using a commercial kit (Cell Signaling Technology, Danvers, MA) according to manufacturer’s protocol. Briefly, Akt was immunoprecipitated from cell extracts using an Akt antibody cross-linked to agarose beads. An in vitro kinase assay was performed using a glycogen synthase kinase (GSK)-3 fusion protein as a substrate. Phosphorylation of GSK-3 was measured by Western blotting using a Phospho GSK-3 (ser 21/9) antibody.

Generation and analysis of a caveolin 1 peptide array

A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα. Bound ERα protein was identified using an anti-ERα antibody followed by detection with secondary antirabbit horseradish peroxidase-coupled antibody and visualized using SuperSignal West Femto Maximum Sensitivity Substrate Kit (Pierce) and a Kodak 440CF image station (Eastman Kodak, Rochester, NY).

Generation of a peptide to inhibit the ERα-caveolin 1 interaction

Scanning the peptide array of caveolin 1 with ERα identified a 12-amino acid sequence that strongly bound to recombinant ERα. A peptide corresponding to amino acids 62–73 of caveolin 1 (TAT-Cav62–73) or scrambled sequence (TAT-Cav62–73Scr) of this region was fused with the N-terminal protein transduction domain from the HIV (R-K-K-R-R-Q-R-R-R). To determine its effects on 17β-estradiol-induced NO production, cells were treated with 100 ng/ml of each peptide before stimulation.

Statistical analysis

Statistical calculations were performed using the GraphPad Prism version 4.01 software. (GraphPad Software, Inc., San Diego, CA) The mean ± sem was calculated for all samples, and significance was determined by either the unpaired t test or ANOVA. For the ANOVA analyses, Newman-Kuels post hoc testing was also performed. A value of P < 0.05 was considered significant.

Supplementary Material

[RETRACTION]
me.2010-0043_index.html (705B, html)

Footnotes

This work was supported by National Institutes of Health (NIH) Grants HL60190, HL67841, HL084739, and HD057406 (all to S.M.B.); Grant 0550133Z from the American Heart Association, Pacific Mountain Affiliates (to S.M.B.); and by a grant from the Fondation LeDucq (to S.M.B.). D.W. was supported in part by NIH Grant F32HL090198. N.S. was supported in part by an American Heart Association postdoctoral fellowship from the American Heart Association Southeast affiliates and by NIH Grant HL097153.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 7, 2010

Abbreviations: EC, Endothelial cells; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; GFP, green fluorescent protein; GSK, glycogen synthase kinase; PAEC, pulmonary artery endothelial cells; PI3 kinase, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; WGA, wheat germ agglutinin.

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