M PP2 for 1 h, 100 nM ICI 182,780 for 30 min, 100 ng/ml. PTX for 17 h, or 5 mM ...... binding by the âpureâ antiestrogen ICI 164,384 appears to be mediated by ...
The FASEB Journal • Research Communication
Resveratrol stimulates nitric oxide production by increasing estrogen receptor ␣-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells Carolyn M. Klinge,1 Nalinie S. Wickramasinghe, Margarita M. Ivanova, and Susan M. Dougherty Department of Biochemistry and Molecular Biology and the Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky, USA Epidemiological studies correlate moderate red wine consumption to reduced incidence of cardiovascular disease. Resveratrol is a polyphenolic compound in red wine that has cardioprotective effects in rodents. Although endothelial cell (EC) studies indicate that micromolar resveratrol has diverse biological activities, these concentrations are not physiologically relevant because human oral ingestion provides only brief exposure to nanomolar plasma levels. Previously, we reported that nanomolar resveratrol activated ERK1/2 signaling in bovine aortic ECs (BAECs). The goal of this study was to determine the mechanisms by which nanomolar resveratrol rapidly activates endothelial nitric oxide synthase (eNOS) in human umbilical vein ECs (HUVECs). We report for the first time that resveratrol increased interaction between estrogen receptor ␣ (ER␣), caveolin-1 (Cav-1) and c-Src, and increased phosphorylation of Cav-1, c-Src, and eNOS. Pretreatment with the lipid raft disruptor beta-methyl cyclodextrin or G␣ inhibitor pertussis toxin blocked resveratrol- and E2-induced eNOS activation and NO production. Depletion of endogenous ER␣, not ER, by siRNA attenuated resveratrol- and E2-induced ERK1/2, Src, and eNOS phosphorylation. Our data demonstrate that nanomolar resveratrol induces ER␣Cav-1-c-SRC interaction, resulting in NO production through a G␣-protein-coupled mechanism. This study provides important new insights into mechanisms for the beneficial effects of resveratrol in ECs.—Klinge, C. M., Wickramasinghe, N. S., Ivanova, M. M., Dougherty, S. M. Resveratrol stimulates nitric oxide production by increasing estrogen receptor ␣-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells. FASEB J. 22, 2185–2197 (2008) ABSTRACT
Key Words: nongenomic estrogen action 䡠 membrane-associated estrogen receptor 䡠 red wine polyphenols Epidemiological studies have demonstrated that the consumption of polyphenolic-rich foods, i.e., fruits and vegetables, and drinking purple grape juice or moder0892-6638/08/0022-2185 © FASEB
ate amounts of red wine reduces the incidence of mortality and morbidity from coronary heart disease and improves endothelial function (1). Regular, moderate intake of wine (0.7–1.1 oz/day) or alcohol (1.1– 1.8 oz/day) has been reported to decrease coronary artery disease (2). A recent crossover study of 35 healthy premenopausal women found that two 100-ml glasses of red wine per day (20 g ethanol/day) for 4 wk resulted in a greater reduction in inflammatory biomarkers, cellular adhesion molecules (i.e., VCAM-1 and E-selectin), and monocyte adhesion to endothelial cells (ECs) compared to white wine (3). A key polyphenol implicated in the cardioprotective effects of red wine is trans-resveratrol, i.e., trans-3,5,4⬘-trihydroxystilbene (4). A study in healthy men of the oral absorption of 25 mg trans-resveratrol/70 kg administered in white wine, grape juice, and vegetable juice showed peak serum resveratrol concentrations of 40 nM, ⬃30 min after consumption (5). Notably, these serum levels are inconsistent with most reported cell-based studies in which the biological activities of resveratrol were detected between 5 and 100 M (5). However, circulating levels are consistent with our report that nanomolar resveratrol concentrations rapidly activated ERK1/2 (MAPK) in bovine aortic endothelial cells (BAECs) (6). Similar to estradiol (E2) (7), MAPK activation by resveratrol was dependent on the activities of estrogen receptor (ER), MEK-1, c-Src, matrix metalloproteinases (MMPs), and epidermal growth factor-receptor (EGFR), and, in turn, activated endothelial nitric oxide synthase (eNOS) (6). The resveratrol concentration that activated nongenomic/membrane-initiated ER signaling was an order of magnitude less than that required for ER genomic activity as we (8, 9) and others (10, 11) previously reported. These data coincide with the recent report that low doses (0.2 mg/kg/day) of a 1 Correspondence: Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292, USA. E-mail: carolyn.klinge@ louisville.edu doi: 10.1096/fj.07-103366
2185
red wine polyphenolic extract (RWPC) were proangiogenic in postischemic rats, whereas high doses (20 mg/kg/day) were antiangiogenic (12). E2 has proven cardioprotective activity in animal models (13). Estrogens have both genomic, i.e., transcriptional regulation, and “nongenomic,” i.e., membrane-initiated intracellular signaling, activities mediated by direct binding to ER␣ and ER (14). Although nongenomic E2 activity is mediated in part by GPR30 (15, 16), E2 does not activate MAPK in ECs from ER␣/ER double-knockout (DERKO) mice (17). Studies using carotid and femoral arteries isolated from ER␣-knockout (␣ERKO) and ER-knockout (ERKO) mice indicate that both ER␣ and ER are responsible for E2-induced vasodilation (18). Cumulative studies show that a subpopulation of intact ER␣ is associated with the endothelial plasma membrane and with caveolae (13). Recent electron microscopy studies revealed nuclear, cytoplasmic, and plasma membrane localization of both ER␣ and ER in human umbilical vein ECs (HUVECs) (19). Interestingly, there was no overlap of ER␣ and ER immunofluorescent signals, indicating that ER␣ and ER occupy discrete loci in HUVECs (19). Thus, we propose that ER␣ and ER may have different interaction partners and physiological effects in the vasculature, and the studies reported here address this hypothesis. Caveolin-1 (Cav-1) serves as a structural core for interaction of plasma-membrane-associated proteins, including the ␣-subunit of G-proteins, Ha-Ras, Srckinases, eNOS, EGF receptors, some protein kinase-C isoenzymes (20), and ER␣ (20, 21). Binding of E2 to ER␣ in caveolae leads to G␣i activation, MAPK and Akt signaling, and perturbation of the local Ca2⫹ environment, leading to eNOS phosphorylation and NO production (13). Endothelial plasma membrane-associated ER␣ is coupled via a G␣i to MAPK and eNOS (22). Striatin couples ER␣ to G␣i in EAhy926 immortalized ECs (23). The specific MEK inhibitor PD98059 blocked eNOS stimulation by E2 in HUVECs (24). Although no one has investigated endogenous ER-Cav-1 interaction in ECs, a recent study showed endogenous ER bound to overexpressed Cav-1 in human embryonic kidney293 cells, and this interaction resulted in E2-induced activation of MAPK, AP-1, and vitamin D receptor expression (25). Lung tissue membranes isolated from ERKO mice showed reduced levels of Cav-1, indicating a role for ER in regulating Cav-1 expression (26). However, whether ER␣ and ER differentially regulate the rapid, nongenomic effects of estrogens, including phytoestrogens that bind ER with higher affinity than ER␣ (27), in ECs remains to be addressed. The goal of the experiments reported in this paper was to elucidate the components of the signaling pathways involved in the ability of nanomolar concentrations of resveratrol to increase eNOS activity through a MAPK- and ER-dependent (6) mechanism in ECs. Specifically, we addressed the roles of ER␣- and ERcaveolar interactions in the resveratrol-activated MAPKeNOS pathway in ECs. In this study, we tested the 2186
Vol. 22
July 2008
hypotheses that 1) resveratrol increases ER␣- and/or ER-Src-Cav-1 interaction, 2) resveratrol induces Cav-1 and eNOS phosphorylation, and 3) caveolae are required for rapid resveratrol-induced MAPK and eNOS activation. As described below, our results confirm the role of ER␣ in these interactions and reveal that ER does not appear to play a role in the rapid signaling and interactions stimulated by either resveratrol or E2 in HUVECs.
MATERIALS AND METHODS Chemicals E2, pertussis toxin (PTX), and methyl--cyclodextrin (betaCD) were from Sigma (St. Louis, MO, USA). ICI 182,780 and PP2 were purchased from Tocris (Ellisville, MO, USA). Transresveratrol was generously provided by Royalmount Pharma (Montreal, QC, Canada). E2 and ICI 182,780 were dissolved in 100% ethanol (EtOH) and diluted in culture medium to a final 1:10,000 dilution (0.001% EtOH final). Antibodies Antibodies to Cav-1 and eNOS were obtained from BD Biosciences (San Jose, CA, USA); antibodies to Src, P-Tyr416Src, P-Ser-118-ER␣, P-Tyr14-Cav-1, MAPK (ERK1/2), phospho-p44/42 MAPK(P-ERK1/2), P-Ser-1177-eNOS, P-Thr495eNOS, AKT, and P-Thr308-AKT were obtained from Cell Signaling Technology (Beverly, MA, USA). ER␣ monoclonal antibody AER320 was obtained from Neomarkers/Lab Vision (Fremont, CA, USA). ER polyclonal antibody H150 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ER polyclonal PA1–311 and monoclonal MA1–32317 antibodies were purchased from Affinity BioReagents (Golden, CO, USA). ER monoclonal antibody 6A12 (MSERB11-PX2) was from GeneTex (San Antonio, TX, USA). Cell treatments HUVECs were purchased from Cambrex BioScience (Walkersville, MD, USA), used between P3– 8, and were maintained in EGM-2 (Cambrex). Cells were serum-starved for 24 h before each experiment and treated with vehicle (EtOH), 10 nM E2, 50 nM resveratrol, and other treatments, in EBM-2 medium without serum. Where indicated, cells were pretreated with 10 M PP2 for 1 h, 100 nM ICI 182,780 for 30 min, 100 ng/ml PTX for 17 h, or 5 mM beta-CD for 30 min before the addition of EtOH, 50 nM resveratrol, and 10 nM E2. The concentrations of the chemical inhibitors were selected to minimize off-target effects, based on published reports: PP2, the selective Src family kinase activity inhibitor (28); PTX, which inhibits G␣s and G␣i, but not G␣q (19); and methylbeta-CD, which disrupts caveolae by binding and sequestering cholesterol (29, 30). HUVECs were treated with each inhibitor, e.g., PP2, ICI 182,780, beta-CD, and PTX, alone as well as in combination with EtOH, E2, or resveratrol, and cells were monitored by Brightfield microscopy. At the times and concentrations of each inhibitor used, there were no obvious changes in the morphology of the cells (Fig. 8). siRNA knockdown HUVECs were transfected with siRNA duplexes obtained from New England Biolabs (Ipswich, MA, USA; ER␣ ShortCut
The FASEB Journal
KLINGE ET AL.
siRNA Mix) and Invitrogen (Carlsbad, CA, USA; ESR2 Stealth Select 3 RNAi). Control/nonspecific siRNA from each respective company was used in parallel with the ER subtype-specific siRNAs. The cells were transfected according to the manufacturer’s protocols. Forty-eight hours post-transfection, the cells were treated with 10 nM E2, 50 nM resveratrol, or EtOH for 10 min, and whole cell extracts (WCEs) were harvested for Western blot analysis.
EtOH, which was set to 1, to obviate any effect of the vehicle in which E2 and resveratrol were dissolved. Statistical analysis Statistical analyses were performed using Student’s 2-tailed t test or 1-way ANOVA followed by Dunnett’s multiple comparison with GraphPad Prism (GraphPad, San Diego, CA, USA). A value of P ⬍ 0.05 was considered statistically significant.
Measurement of NO NO production was assessed using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA; Sigma) (31). In brief, HUVECs were incubated in serum-free EBM-2 medium with 100 M l-arginine (Sigma) for 24 h prior to loading with 3 M DAF-2-DA for 30 min at 37°C. Cells were rinsed 3⫻ with HEPES buffered physiological salt solution (PSS). Cells were then treated with EtOH, 10 nM E2 or 50 nM resveratrol for 10 and 20 min. For the inhibitor studies, HUVECs were pretreated for 30 min with 100 M nitro-larginine methyl ester (L-NAME, Sigma), a NOS inhibitor; 5 mM beta-CD for 30 min, or 100 ng/ml PTX for 17 h before the cells were loaded with DAF-2 DA. After stimulation, the cells were fixed in 2% paraformaldehyde for 5 min at 4°C. The fixed cells were examined using a Zeiss Axiovert 200 inverted microscope (Carl Zeiss, Thornwood, NY, USA) with an ⫻10 objective lens, using an FL bulb and fluorescein isothiocyanate (FITC) filters with a peak excitation wavelength of 494 nm and a peak emission wavelength of 517 nm for DAF-2DA fluorescence. For Brightfield images, the light source was a halogen bulb and Brightfield filter in the same microscope. Images were captured using Axio Camera HRm and AxioVision Release 4.3 software (Carl Zeiss). All images were captured using identical microscope imaging settings with an exposure time of 236 s. Preparation of WCEs, coimmunoprecipitation, and Western blot analysis HUVECs were lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 0.5 mM EGTA; 1 mM EDTA; 1% Triton X-100; 1% Na cholate; 0.5 mM PMSF) supplemented with protease and phosphatase inhibitors (Sigma). Insoluble material was removed by centrifugation, and 150 g WCE was incubated with 1.5 g of caveolin-1 (Cav-1) antibody (BD Biosciences), ER␣, or ER antibodies overnight (17 h) with rotation at 4°C. Protein A-Sepharose 4B (Zymed, San Francisco, CA, USA) was added for 2 h with rotation at 4°C. The beads were sedimented at 10,000 g and washed with lysis buffer 4⫻, resuspended in 75 l of Laemmli buffer, and boiled; 30 l was separated on duplicate 10% SDS-PAGE gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, Pensacola, FL, USA) as described (6). Membranes were probed first for phosphoproteins (P-ER␣, P-Src, P-Cav-1, P-eNOS) and then stripped and reprobed for ER␣, ER, Src, Cav-1, and eNOS. Mouse IgG TrueBlot (eBioscience, San Diego, CA, USA) was used as secondary antibody for some experiments. Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) was used to detect protein bands. Immunoblots were scanned into Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA) using a Microtek ScanMaker III scanner (Microtek, Carson, CA, USA) and Un-Scan-It 5.1 (Silk Scientific, Orem, UT, USA) was used to digitize the data (6). The treatmentspecific P-protein pixels were divided by concordant nonphosphoprotein pixels and by Cav-1 in the same blot. In all experiments, ratios of P-protein/total protein were normalized to RESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
RESULTS Resveratrol and E2 induce ER␣-Cav-1 interaction and phosphorylation Although resveratrol is a low-affinity agonist for genomic ER␣ and ER activity (8 –11, 32–34), no one has examined whether resveratrol affects ER␣-interaction with Cav-1 in ECs. Serum-starved HUVECs were treated with 50 nM resveratrol or 10 nM E2 (positive control) for 2–30 min. Cellular proteins were immunoprecipitated with an antibody to Cav-1 and analyzed by immunoblotting with ER␣ and P-Ser-118-ER␣. Resveratrol increased ER␣-Cav-1 interaction with a peak at 5–20 min (Fig. 1A, B). Similar results were observed for E2 at the early time points, with a decrease in ER␣-Cav1 interaction at 30 min followed by a return to basal ER␣-Cav1 interaction by 45 min. Notably, the kinetics of direct ER␣-Cav-1 interaction in HUVECs was different in cells treated with E2 or resveratrol at the 30- and 45-min time points. The E2 data are in agreement with an E2-induced increase in ER␣-Cav-1 interaction in vascular smooth muscle cells (21). We examined whether the increase in ER␣-Cav-1 interaction triggered by resveratrol was associated with increased ER␣ phosphorylation on Ser-118, a target of MAPK (35). Figure 1C–E shows that resveratrol and E2 rapidly increased the amount of P-ER␣ associated with Cav-1 and increased the P-ER␣/ER␣ ratio. The amount of P-ER␣ associated with Cav-1 in resveratrol-treated HUVECs was increased between 5 and 30 min and then declined, with a second increase detected at 120 min. These data correlate with the ER␣/Cav-1 interaction data shown in Fig. 1A, B. Similar results were detected with E2. The ratio of P-ER␣/ER␣ after Cav-1 immunoprecipitation (IP) showed a different pattern with a peak at 5 min., followed by a decrease, and then resveratrol treatment resulted in a second peak at 20 min. (Fig. 1E). Resveratrol and E2 induce Src-Cav-1 interaction and phosphorylation Pretreatment of HUVECs for 6 h with 1 or 2.5 M resveratrol blocked subsequent VEGF-induced Src activation, Src-Cav-1 interaction, and Cav-1 phosphorylation (36), but no one has examined the rapid effects of nanomolar concentrations of resveratrol on Src-Cav-1 interaction. Figure 2 shows that resveratrol and E2 increased the amount of Src coimmunoprecipitated 2187
Figure 1. Resveratrol and E2 increase ER␣-Cav-1 and P- ER␣-Cav-1 interaction in HUVECs. Cells were treated with EtOH (control), E2 (10 nM), or resveratrol (50 nM) for the indicated times. A) ER␣-Cav-1 interaction was examined by immunoprecipitation (IP) with an antibody to Cav-1 followed by immunoblotting with ER␣ antibody. The membrane was stripped and reprobed for Cav-1. Representative Western blots are shown. B) Quantitation of the relative ER␣/Cav-1 ratio with the t0 ratio set to 1. C) Interactions of P-ER␣-Cav-1 and P-ER␣-ER␣ were examined by IP with an antibody to Cav-1 after 5 min of E2 or resveratrol. D, E) Quantitation of the relative P-ER␣/Cav-1 (D) and P-ER␣/ ER␣ ratios (E) with the t0 ratio set to 1. Values in B, D, and E are means ⫾ se from 3 separate experiments, except 60 min is from a single IP; *P ⬍ 0.05 vs. t0.
with Cav-1 by ⬃50% at the 5-min time point and that increased Src association with Cav-1 was sustained in resveratrol-treated HUVECs until after 20 min, whereas there was a decrease in Src-Cav-1 interaction at 10 min in E2-treated cells. Similarly, P-Tyr416-Src (active form of Src)-Cav-1 interaction peaked at 5 min in cells treated with either resveratrol or E2 and then decreased. Phosphorylation of c-Src kinase on Tyr416 results in disruption of the inhibitory/inactive conformation due to intramolecular and intermolecular interactions between the SH2 domain and P-Y527 and between its SH3 domain and the SH2-kinase linker domains and allows molecular recognition of c-Src substrates and binding partners (37). However, P-Src showed a second peak of interaction with Cav-1 at 20 min in resveratrol-treated cells. The ratio of P-Src/Src was increased after 2 min with either resveratrol or E2. The increase at the 20 min time point was not statistically significant. Pretreatment with the selective Src-kinase inhibitor PP2 blocked the increase in P-Src (Fig. 2D). 2188
Vol. 22
July 2008
Both resveratrol and E2 increased the P-Cav-1/Cav-1 ratio at 10 and 120 min, indicating a biphasic effect on Cav-1 phosphorylation (Fig. 3A). The data in Fig. 3B, C compare the time course of phosphoprotein-Cav-1 interaction in HUVECs after resveratrol or E2 treatment. These data indicate different time kinetics of P-ER␣Cav-1 interaction with resveratrol showing an extended P-ER␣-Cav-1 interaction compared to E2, which shows a peak at 5 min. Although the initial P-Src-Cav-1 peak at 5 min is similar for resveratrol and E2, resveratroltreated HUVECs show a second P-Src-Cav-1 peak at 20 min not seen in E2-treated cells. Figure 3B indicates that ER␣ in the ER␣-Cav-1 complex in resveratroltreated cells is maintained in the phosphorylated active form for an extended period of time, which may indicate that resveratrol has a sustained effect on e-NOS phosphorylation and NO production. Resveratrol increases eNOS Ser-1177 phosphorylation Phosphorylation of eNOS at Ser-1177 indicates activation, whereas P-thr495 indicates inactive eNOS
The FASEB Journal
KLINGE ET AL.
Figure 2. Resveratrol and E2 induce Src-Cav-1 interaction and phosphorylation. A) HUVECs were treated with EtOH (control), E2 (10 nM), or resveratrol (50 nM) for the indicated times. Where indicated, cells were pretreated with the Src kinase inhibitor PP2 (10 M) for 1 h. Src-Cav-1, P-Src-Cav-1, and P-Src-Src interactions were examined by IP as in Fig. 1. Representative Western blots at 5-min time point are shown. B–D) Quantitation of the Src/Cav-1 (B), P-Src/Cav-1 (C), and P-Src/Src ratios (D). Values are means ⫾ se from 3 separate experiments, except 60 min is from a single IP; *P ⬍ 0.05 vs. t0. †P ⬍ 0.05 vs. same treatment without PP2 pretreatment.
(38). The effect of resveratrol on eNOS phosphorylation is unknown. Here, we observed that resveratrol increased P-Ser-1177-eNOS in 5 min of treatment with a peak at 10 min for both resveratrol and E2 (Fig. 4A, B). All data were normalized for the vehicle EtOH, which did not increase P-Ser-1177-eNOS (Fig. 4A, and data not shown for other time points up to 45 min). Wortmannin and PD98059 inhibited resveratrol- and E2-induced increase in P-Ser-1177-
eNOS, indicating a role for both PI3K and MAPK pathways in eNOS activation. There was a concomitant decrease in P-thr495-eNOS after 10 min with resveratrol or cotreatment with E2 or resveratrol and wortmannin. In contrast, cotreatment with PD958059 increased P-thr495-eNOS (Fig. 4C). The increased P-Ser-1177-eNOS/eNOS at 5 and 10 min correspond to the time course of ER␣-Cav-1 interaction and Cav-1 phosphorylation.
Figure 3. Resveratrol and E2 increase phosphorylation of Cav-1, Src, and ER␣. HUVECs were treated with EtOH (control), E2 (10 nM), or resveratrol (50 nM) for the indicated times. P-Cav-1-Cav-1, P-ER␣-Cav-1, P-Src-Cav-1 interactions were examined by IP with an antibody to Cav-1 as in Fig. 1. A) A representative Western blot at the 10-min time point for P-Cav-1/Cav-1. B, C) Quantitation of the P-ER␣/Cav-1, P-Src/Cav-1, and PCav1/Cav-1 ratios in resveratrol-treated (B) and E2-treated cells (C). Values are means ⫾ se from 3 separate experiments, except 60 min is from a single IP; *P ⬍ 0.05 vs. t0. RESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
2189
Figure 4. Resveratrol and E2 activate eNOS in a wortmannin-, PD98059-, and PTX- sensitive manner. A–C) HUVECs were treated with EtOH, E2 (10 nM), or resveratrol (50 nM) for the indicated times. Where indicated, HUVECs were pretreated with 100 nM wortmannin (Wort) or 50 M PD98059 for 1 h prior to 10 min treatment with EtOH, resveratrol, or E2. WCEs were analyzed with anti-Ser-1177-eNOS (active eNOS; A, B), or anti-P-thr495-eNOS antibody (inactive eNOS; C). D, E) HUVECs were pretreated with 100 ng/ml PTX for 18 h prior to a 10 min treatment with EtOH, 10 nM E2, or 50 nM resveratrol. WCEs were analyzed using Western blot analysis with P-MAPK, P-Src, P-Ser-1177-eNOS antibodies, and reprobing the same blots for MAPK, Src, or e-NOS. Three representative Western blots are shown. Graphs show quantitation of the P-Ser-1177-eNOS/e-NOS (B), P-thr-495-eNOS/eNOS (C), and P-protein/protein ratios (E). Values are means ⫾ se from 3 separate experiments; *P ⬍ 0.05 vs. EtOH; †P ⬍ 0.05 vs. same treatment without Wort or PD98059 (B) or PTX pretreatment (E).
PTX blocks resveratrol-induced MAPK, Src, and eNOS phosphorylation E2 stimulated the direct interaction of ER␣ with G␣i (22) via ER␣-striatin interaction (23). To test the hypothesis that resveratrol’s activation of Src, MAPK, and eNOS is mediated by G␣i, HUVECs were pretreated with PTX (Fig. 4D, E). Pretreatment with PTX completely blocked resveratrol- and E2-stimulated Src, MAPK and eNOS activation, indicating that signaling involves PTX-sensitive G␣i or G␣s, but not G␣q (39) in HUVECs. Data are normalized to EtOH. Resveratrol did not act additively with E2 in activating Src, MAPK, or eNOS, as measured by the phosphoprotein/total protein ratio (Fig. 4E). Because we used individual concentrations of E2 and resveratrol at which MAPK activation is maximum (6), we suggest that the lack of additivity indicates that the response is mediated by a common pathway which is saturated. Pretreatment with ICI 182,780 inhibits recruitment of ER␣ to Cav-1 and Cav-1 phosphorylation To test the role of direct resveratrol-ER␣ binding in the rapid changes observed in Figs. 1–3, HUVECs were pretreated with 100 nM ICI 182,780 for 30 min before the addition of either 50 nM resveratrol or 10 nM E2. ICI 182,780 is a well-established antagonist of genomic ER that prevents coactivator recruitment (40), inhibits ER␣ dimerization (41), and enhances ER␣ proteasomal degradation after 6 h (40). In addition, ICI 182,780 inhibits the rapid E2 activation of membrane-associated ER␣ re2190
Vol. 22
July 2008
sponses (13, 42) but not GPR30-mediated activity (16). Pretreatment of HUVECs with ICI 182,780 inhibited the resveratrol- and E2-induced increases in the P-Src/Src, P-Cav-1/Cav-1, P-ER␣/Cav-1 and ER␣/Cav-1 ratios (Fig. 5A, B). These data indicate that the increased phosphoprotein/protein ratios and interactions induced by both resveratrol and E2 are at least in part ERmediated. Caveolae are required for resveratrol-induced ER␣-Cav1 interaction and eNOS activation To determine whether caveolae are required for resveratrol-induced ER␣-Cav-1 interaction and phosphorylation of Src, Cav-1, and ER␣, serum-starved HUVECs were pretreated with EtOH (vehicle control) or 5 mM beta-CD for 30 min to disrupt caveolae by binding and sequestering cholesterol (29). Figure 5C, D shows that beta-CD inhibited resveratrol- and E2-induced ER␣-Cav-1 interaction, and ER␣, Src, and eNOS-Ser-1177 phosphorylation. Together, the results demonstrate that caveolae are required for Cav-1-ER␣-Src interaction and resveratrol signaling, and disruption of caveolae with beta-CD inhibits ER␣-Src-Cav-1 interaction and resveratrol- and E2-induced ER␣ phosphorylation and eNOS activation. Resveratrol and E2 do not stimulate ER-Cav-1 interaction No one has examined the effect of E2 or resveratrol on endogenous ER-Cav-1 interaction and downstream sig-
The FASEB Journal
KLINGE ET AL.
Figure 5. Pretreatment of HUVECs with ICI 182,780 or methyl--cyclodextrin (CD) inhibits resveratrol- and E2-induced protein-protein interactions and phosphorylation. HUVECs were pretreated with 100 nM ICI 182,780 (A, B), or 5 mM CD (C, D) for 30 min, followed by either a 5 min (for the P-Src/Src data, because that is the time of maximal P-Src/Src) or 10 min treatment with EtOH (control), E2 (10 nM), or resveratrol (50 nM) (for all other analyses indicated). The indicated interactions were examined by IP with an antibody to Cav-1 followed by immunoblotting with P-Src, P-Cav-1, or P-ER␣ antibodies and reprobing the same blots for Src, Cav-1, or ER␣ as described in Fig. 1. The data for HUVECs not preincubated with the inhibitors are from Figs. 1–3 and are included for ease of visual comparison. Three representative Western blots are shown. Graphs show quantitation of the P-Src/Src, P-Src/Cav1, P-Cav1/Cav1, P-ER␣/ ER␣, P- ER␣/Cav1, and ER␣/Cav1 ratios (B, D). Values are means ⫾ se from 3 separate experiments; *P ⬍ 0.05 vs. EtOH; †P ⬍ 0.05 vs. same treatment without ICI, PTX, or CD pretreatment.
naling pathways in ECs. Quantitative Western blot analysis using recombinant human (rh) ER␣ and ER as standards revealed that HUVECs express 0.9 and 0.2 fmol/g WCE protein of ER␣ and ER, respectively (data not shown). Thus, HUVECs have ⬃4.5 times more ER␣ than ER. ER was shown to immunoprecipitate with Cav-1 (Fig. 6A). Because ER and some of its splice variants migrate ⬃53–59 kDa, Mouse IgG TrueBlot, which has low reactivity with mouse IgG heavy and light chains (55 and 23 kDa), was used to demonstrate the specificity of ER signal detection. In contrast to ER␣, neither resveratrol or E2 increased ER-Cav-1 interaction (Fig. 6A). Similar results were obtained using ER polyclonal antibodies H150 and PA1–311 (data not shown). In reverse IP experiments in which HUVEC WCEs were immunoprecipitated with ER and probed for Cav-1, the Cav-1/ER ratio decreased with a 10-min treatment of either resveratrol or E2 (Fig. 6B). Although ER␣ and ER form homodimers and heterodimers in isolated plasma membrane preparations from HUVECs (17), ER␣ did not coimmunoprecipitate with ER (Fig. 6B). The band detected at ⬃54 kDa is IgG heavy chain (Fig. 6B). Thus, E2 or resveratrol decreased ER-Cav-1 interaction (Fig. 6C), in contrast to the increased ER␣/Cav-1 ratio with E2 and resveratrol (Fig. 1B). In conclusion, although both ER␣ and ER interact with Cav-1 in HUVECs, resveratrol and E2 appear to have opposing effects on ER␣- and ERCav-1 interaction. Knockdown of ER␣ and not ER inhibits rapid activation of MAPK, Src, and eNOS by E2 and resveratrol To further examine the ER subtype specificity for the observed rapid stimulation of MAPK, Src, and eNOS RESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
phosphorylation in response to resveratrol and E2 in HUVECs, cells were transfected with control/nonspecific siRNA or siRNA targeting ER␣ or ER. Transfection of HUVECs with siRNA for ER␣ reduced ER␣ protein by 80% but had no significant effect on ER expression (Fig. 7A). Transfection of HUVECs with siRNA for ER reduced ER protein by 75%, but had no significant effect on ER␣ expression (Fig. 7B). Knockdown of ER␣ reduced the E2- or resveratrolinduced activation of MAPK, Src, and eNOS, as well as basal eNOS phosphorylation (Fig. 7C). In contrast, knockdown of ER did not significantly inhibit the E2or resveratrol-induced activation of MAPK, Src, or eNOS. Together, these data indicate that ER␣, not ER, plays the major role in the E2- and resveratrolinduced activation of MAPK, Src, and eNOS in HUVECs. Resveratrol stimulates NO production We examined the acute effects of resveratrol and E2 on NO production using DAF-2 fluorescent dye. Resveratrol- and E2-induced DAF-2 fluorescence, indicative of NO production, was maximal after 10 min and was sustained after 20 min (Fig. 8). To verify that the increase in green fluorescence reflected NO production, HUVECs were pretreated with L-NAME, a NOS inhibitor. As expected, L-NAME blocked the increase in green fluorescence. Pretreatment of HUVECs with beta-CD and PTX also completely blocked resveratrolor E2-induced NO production (Fig. 8). These data agree with resveratrol-induced Ser-1177 eNOS phosphorylation (Fig. 4A). 2191
Figure 6. Resveratrol and E2 decrease ER interaction with Cav-1 in HUVECs. A) HUVECs were treated for 10 min with EtOH, 10 nM E2, or 50 nM resveratrol (lanes 1–3), and WCEs (150 g protein) were immunoprecipitated with Cav-1 followed by immunoblotting with ER monoclonal antibody 6A12 and Cav-1. These membranes used mouse IgG TrueBlot HRP-conjugated secondary antibody to minimize interference by the heavy and light chains of the immunoprecipitating antibody, as described in Materials and Methods. For lane 4, Cav-1 antibody was incubated with protein A-sepharose 4B overnight, rinsed with lysis buffer, and boiled in Laemmli buffer, as described in Materials and Methods. Laemmli buffer (25 l) was loaded in lane 4; Cav-1 antibody (0.5 g) was diluted in Laemmli buffer, boiled, and loaded into lane 5. Lanes 6 and 7 include 15 g WCEs from EtOH- or E2-treated HUVECs, respectively. Baculovirus-expressed rhER (14 fmol) was included as a control (lane 8). B) Reverse IP: ER-Cav-1 interaction was examined by IP (as in A) with an antibody to ER antibody (MA-23217, lanes 1–3) and Western blot analysis for Cav-1. Baculovirus-expressed rhER␣ (558 and 1395 fmol, lanes 4 and 5) or rhER (355 and 710 fmol, lanes 6 and 7) were separated in parallel. Molecular weight (MW) marker size is indicated in kilodaltons. Antibody specificity is demonstrated: ER antibody H150 did not recognize ER␣ and ER␣ antibody AER320 likewise did not bind ER. C) Quantitation of the ER/Cav1 and Cav-1/ER ratios from 3 separate experiments for each IP (IP-Cav-1, probe ER; IP-ER, probe Cav-1). Values are means ⫾ se[scap]; *P ⬍ 0.05 vs. EtOH (Student’s t test, 2-tailed). D) Western blot for ER␣ in WCEs of HUVECs: 40 g WCE from MCF-7 (untreated) or HUVECs, untreated, or treated for 10 min with EtOH, 10 nM E2 or 50 nM resveratrol, was separated on a 10% SDS-PAGE gel. The indicated amount (fmol) of rhER␣ was added as a loading control. MW marker size is indicated in kilodaltons. The membrane was first probed with AER320 ER␣ antibody and then stripped and reprobed for ␣-tubulin. There is no evidence of the ⬃54 kDa band detected by AER320 in the IP for ER in B. We suggest that this is mouse IgG.
DISCUSSION Red wine and its key constituent resveratrol have a variety of activities associated with cardioprotective effects (2). Nonetheless, the exact mechanisms for red wine’s ability to prevent coronary artery disease and stroke is not fully elucidated. One major area of concern in studying resveratrol’s effects is the discrepancy between the high concentrations apparently needed to achieve biological activities in vitro or in animal models with the epidemiological effects seen at moderate red wine consumption that would achieve only nanomolar or lower concentrations of bioactive polyphenolics in vivo for a short time (5). The primary goal of this study was to determine the molecular mechanisms by which nanomolar concentrations of resveratrol, concentrations compatible with oral consumption (5), rapidly activate NO production in ECs by examining endogenous rather than overexpressed proteins. Here, we demonstrated that nanomolar concentrations of resveratrol, like E2 (43), increase ER␣-Cav-1-Src interaction in lipid rafts/caveolae in HUVECs in a timedependent manner leading to Cav-1, Src, ERK1/2, and eNOS phosphorylation, and NO production. Furthermore, we show that resveratrol’s activation of eNOS appears to be mediated by activation of G␣i and/or G␣s proteins and that ER does not appear to be critical for 2192
Vol. 22
July 2008
these signaling events, since siRNA knockdown of ER did not prevent resveratrol or E2 activation of Src, MAPK, or eNOS in HUVECs. Previously, we reported that nanomolar concentrations of resveratrol activated NO production in BAECs in an ER-, Src-, MEK1/ERK1/2-, MMP-, and EGF-Rdependent manner (6). However, the exact mechanisms by which resveratrol-ER interaction activated eNOS remained undefined. ER␣ localizes in caveolae of ECs (13), and the signal transduction mechanisms by which E2 rapidly (i.e., nongenomically) activates eNOS have been explored (reviewed in ref. 44). ER␣ interacts directly with c-Src, Cav-1, hsp90, and other proteins in caveolae of ECs and breast cancer cells (13, 20, 45– 47). In response to E2, Src is rapidly activated, inducing formation of a complex that contains ER␣, c-Src, and the p85 subunit of PI3K. These interactions are required for activation of eNOS (13, 42, 43). Our data are consistent with these reports, as we demonstrated that resveratrol, like E2, rapidly induces ER␣-Cav-1 association, whereas neither resveratrol nor E2 stimulates ER interaction with Cav-1 interaction. Resveratrol, like E2 (43, 48), increased ER␣ and ER interaction with c-Src. Further, our data showing that ER␣ did not coimmunoprecipitate with ER, and vice versa, is also consistent with recent electron microscopy studies indicating that
The FASEB Journal
KLINGE ET AL.
Figure 7. Knockdown of ER␣ attenuates resveratrol- and E2-induced activation of Src, MAPK, and eNOS. HUVECs were transfected with control siRNA (siControl), siER␣ (A) or siER (B) for 48 h. ER␣ and ER expression was examined in 40 g WCE. The membrane was stripped and reprobed for -actin for normalization. Values below the blots are percentages of ER␣/-actin or ER/-actin in control cells. HUVECs transfected (48 h) with siControl, siER␣ (A), or siER (B) were treated with EtOH, 10 nM E2, or 50 nM resveratrol for 10 min. WCEs (40 g) were Western blotted with the indicated phosphoantibodies, then stripped and reprobed for total eNOS, ERK1/2 (MAPK), and Src. (C) Quantitation of the P-MAPK/MAPK, P-Src/Src, P-Ser-1177-eNOS/e-NOS ratios. Values were normalized for EtOH with siControl and are means ⫾ se from 3 separate experiments; *P ⬍ 0.05 vs. same treatment in HUVECs transfected with siControl.
ER␣ and ER occupy discrete loci in HUVECs (19). Thus, we conclude that ER␣ and ER have different interaction partners in HUVECs and speculate that this may apply to physiological interactions in the vasculature as well. Support for this idea at the genomic level is borne out by recent microarray profiling of genes regulated by E2 in aortas from ovariectomized wild-type vs. ER␣ or ER knockout mice (49). ER␣ was responsible for the up-regulation of most of the identified E2 target genes in the aorta. In contrast, E2-ER actively repressed the transcription of nuclear-encoded mitochondrial respiratory chain genes. Because the animals were treated for 1 wk with E2, these data reflect not only primary target genes, but secondary and tertiary genes as well as integration of nongenomic and genomic actions of E2 in the aorta. That ER␣ and ER have selective actions in a cell-type-dependent manner is likewise indicated by the report that E2 and genistein, a phytoestrogen that is a selective ER agonist, but not propyl-pyrazole-triol, a selective ER␣ agonist, increased eNOS and decreased nNOS at the mRNA and protein levels in the rat hypothalamic paraventricular nucleus (50). Moreover, the ER-selective agonist diarylpropionitrile rapidly increased NO production, and E2stimulated NO was inhibited by the selective ER antagonist R,R-tetrahydrochrysene (51). In addition to E2 and synthetic ER ligands, a variety of phytochemicals and endocrine disruptors initiate second messengertriggered signal cascades emanating from the plasma membrane that are mediated by ER␣, ER, and/or GPR30 (52–57). One novel finding in the present study is that nanomolar levels of resveratrol, like E2, caused a rapid association of Cav-1 with Src (Fig. 2B). However, resRESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
veratrol elicited a more sustained interaction (5–20 min), whereas E2 caused only transient interactions peaking at 5 and 20 min. Indeed, the chronological order of protein-protein interactions and phosphorylation stimulated by resveratrol and E2, while largely overlapping, is somewhat different (Fig. 3B, C). For example, E2 more rapidly increased ER␣-Cav-1 interaction compared to resveratrol (Fig. 1B). Further, the E2-induced ER␣-Cav-1 interaction was reduced below basal at 30 min, whereas the resveratrol-activated interaction was reduced at 45 min. The mechanism for the differences in chronology and the more sustained Cav-1-Src triggered by resveratrol interaction is unknown but may involve differences in the identity or kinetics of proteins in addition to those tested here and that are involved in the signalosome complex, such as MNAR/PELP and the p85 regulatory subunit of PI3K (58). Further studies are required to identify the proteins involved in differences between resveratrol and E2. Importantly, Src kinase activity appears to be involved in resveratrol- and E2-induced eNOS activation, since pretreatment with the selective Src kinase inhibitor PP2 completely blocked resveratrol- or E2-induced association of Src/Cav-1 (Fig. 2D). These data are consistent with previous reports on E2 activation of the Src-Cav-1-eNOS signaling pathway in rat lung ECs and MCF-7 human breast cancer cells (21, 59). Another novel observation is that the rapid effects of resveratrol are mediated by Cav-1-associated full-length ER␣ (66 kDa). Although the 46-kDa isoform of ER␣ lacking the N terminus (A and B domains) was reported to be preferentially associated with the plasma membrane in EA.hy926 cells (immortalized human ECs) (37, 60), our data show that full-length ER␣ and 2193
Figure 8. Resveratrol and E2 increase NO production in HUVECs. HUVECs were serum-starved and loaded with DAF-2 DA before treatment with EtOH, 10 nM E2, or 50 nM resveratrol for 10 or 20 min, as indicated. Where indicated, cells were pretreated for 30 min with 100 M l-NAME or 5 mM -CD, or for 17 h with 100 ng/ml PTX. Cells were fixed and viewed as described in Materials and Methods. Emission of green light (510 nm) from cells excited at 480 nm is indicative of NO production. Scale bars ⫽ 100 m.
ER1 (61) were immunoprecipitated with Cav-1 in HUVECs. It is possible that the ER␣ isoforms present in the plasma membrane differ in different ECs. Notably, pretreatment with ICI 182,780 before the addition of resveratrol or E2 and siRNA against ER␣, not ER, blocked the P-Src/Src, P-Src/Cav-1, P-Cav-1/Cav-1, PER␣/ER␣, P-ER␣/Cav-1, and ER␣/Cav-1 interactions (Fig. 5B) and phosphorylation of Src, Cav-1, eNOS, and ER␣ (Fig. 7). Cumulatively, our data suggest that resveratrol and E2 activate a similar nongenomic ER pathway (Fig. 9) that accounts for rapid E2 activation of eNOS in ECs (22, 43, 45, 46). In contrast to our previous study in BAECs in which subtype-specific ligands indicated roles for both ER␣ and ER in eNOS activity (6), siRNA knockdown studies in HUVECs indicate that ER␣ plays a predominant role in the resveratrol and E2-activation of MAPK, Src, and eNOS. Coincidentally, while resvera2194
Vol. 22
July 2008
trol and E2 increased ER␣-Cav-1 interaction within 5 min, ER-Cav-1 interaction was decreased by ligand treatment (Fig. 6). Thus, it is tempting to speculate, on the basis of numerous studies in a variety of tissues/cell types establishing that ER is a down-regulator of responses up-regulated by ER␣ (62–71), that the decrease in ER-Cav-1 interaction may be part of the stimulatory mechanism. Cav-1 is a substrate for Src kinase that phosphorylates Cav-1 on Tyr-14 (72). Increased P-Cav-1 has been suggested as an indicator of a reduced interaction between Cav-1 and other signaling proteins (20). Our data agree with these findings, as indicated by the inverse association of Cav-1 phosphorylation, i.e., at 10 min, with reduced P-Src-Cav-1 and P-ER␣-Cav-1 association. However, when P-Cav-1/Cav-1 peaked again at 60 min and remained high up to 120 min in resveratroltreated cells, P-ER␣/Cav-1 was also high (Fig. 3B). The distinct difference between E2 and resveratrol induced effects in HUVECs is the extended P-ER␣-Cav-1 association in resveratrol-, but not E2-treated cells (Fig. 3C). Notably, beta-CD, which disrupts caveolae-like structures by binding to and sequestering cholesterol from the plasma membrane of intact cells (73), abolished resveratrol- and E2-induced protein-Cav-1 associations, eNOS phosphorylation, and NO production. These observations indicate that intact caveolar structure is required for resveratrol’s rapid effects on ER␣-Cav-1 interaction, eNOS activation, and NO production in HUVECs, similar to our observations reported here using E2 as a positive control established in ECs by other investigators (13, 20). Because direct interactions between plasma membrane-associated ER␣ and G␣i have been implicated in
Figure 9. Signaling pathways by which resveratrol rapidly activates eNOS and increased NO production in HUVECs. Resveratrol, like E2, activates ER␣ that is localized in a “signalsome complex” within caveolae that includes Cav-1, which binds Src, Grb7, MEK, G proteins, and eNOS (47, 82). This model is based primarily on studies in breast cancer cells and BAECs from E. Levin’s group (7, 82– 85) and our present and previously reported research (6). Data reported here indicate that ER-Cav-1 interaction is reduced by E2 and resveratrol, suggesting the possibility that ER leaving the plasma membrane may be part of the stimulatory mechanism leading to downstream phosphorylation events and NO production. Chemicals that inhibit the activity of steps in the pathway used in experiments reported here are indicated.
The FASEB Journal
KLINGE ET AL.
eNOS activation and NO production in COS-7 cells transfected with ER␣ and specific G␣i proteins (22), we investigated whether G␣i is involved in resveratrolinduced e-NOS activation in HUVECs. Our data demonstrate that pretreatment with PTX, a G␣ inhibitor, blocked completely resveratrol’s activation of Src, MAPK, and eNOS, indicating that G␣ plays an important role in rapid resveratrol signaling in HUVECs. These findings are similar to E2 as shown here and reported by others (13, 20, 44, 74). Previous studies showed that high concentrations (micromoles or even millimoles) of resveratrol have a variety of beneficial activities in cardiovascular tissues (75). For example, 10 M resveratrol produced a powerful relaxation response in endothelium-containing rings of rat aorta (76). Likewise, 10 M resveratrol caused rapid coronary vasodilation and increased coronary flow in Langendorff-perfused rat hearts (77). Recently, a 5-min treatment with 30 or 100 M resveratrol suppressed ischemia-reperfusion-induced ventricular arrhythmias in perfused rat hearts (78). These studies are limited by nonphysiologically relevant resveratrol concentrations used (5). In addition to differences in resveratrol effects due to high concentrations used experimentally, we hypothesize that because of its rapid metabolism (79 – 81), resveratrol’s acute effects in the vasculature may contribute to its vascular/cardioprotective effects through different mechanisms than chronic/high dose effects. The resveratrol concentration used here is an order of magnitude less than that required for ER genomic activity as reported by us (8, 9) and others (10, 11). In conclusion, the data presented here demonstrate that nutritionally relevant (nanomolar) concentrations of resveratrol rapidly activate plasma membrane-associated ER␣ in caveolae of HUVECs, leading to eNOS activation and NO production via activation of G␣i, Cav-1, Src, and MAPK in a manner similar to that elicited by E2. These studies imply that dietary intake of resveratrol may offer possible vascular protective effects in vivo.
4. 5. 6.
7. 8. 9. 10.
11.
12.
13. 14.
15.
16.
17.
We thank Royalmount Pharma (Montreal, QC, Canada) for providing trans-resveratrol for our study. We thank Dr. Wasana Sumanasekera for her assistance in the initial IP experiments. This research was supported by American Heart Association grant 0555270B to C.M.K. We thank Dr. Barbara J. Clark for her insightful suggestions on this manuscript.
18.
19.
REFERENCES 1.
Stoclet, J. C., Chataigneau, T., Ndiaye, M., Oak, M. H., El Bedoui, J., Chataigneau, M., and Schini-Kerth, V. B. (2004) Vascular protection by dietary polyphenols. Eur. J. Pharmacol. 500, 299 –313 2. Cordova, A. C., Jackson, L. S. M., Berke-Schlessel, D. W., and Sumpio, B. E. (2005) The cardiovascular protective effect of red wine. J. Am. Coll. Surg. 200, 428 – 439 3. Sacanella, E., Vazquez-Agell, M., Mena, M. P., Antunez, E., Fernandez-Sola, J., Nicolas, J. M., Lamuela-Raventos, R. M., Ros, E., and Estruch, R. (2007) Down-regulation of adhesion molecules and other inflammatory biomarkers after moderate wine
RESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
20.
21.
22.
consumption in healthy women: a randomized trial. Am. J. Clin. Nutr. 86, 1463–1469 Sato, M., Maulik, N., and Das, D. K. (2002) Cardioprotection with alcohol: role of both alcohol and polyphenolic antioxidants. Ann. N. Y. Acad. Sci. 957, 122–135 Goldberg, D. M., Yan, J., and Soleas, G. J. (2003) Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clin. Biochem. 36, 79 – 87 Klinge, C. M., Blankenship, K. A., Risinger, K. E., Bhatnagar, S., Noisin, E. L., Sumanasekera, W. K., Brey, D. M., and Keynton, R. S. (2005) Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells. J. Biol. Chem. 280, 7460 –7468 Razandi, M., Pedram, A., Park, S. T., and Levin, E. R. (2003) Proximal events in signaling by plasma membrane estrogen receptors. J. Biol. Chem. 278, 2701–2712 Bowers, J. L., Tyulmenkov, V. V., Jernigan, S. C., and Klinge, C. M. (2000) Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 141, 3657–3667 Klinge, C. M., Risinger, K. E., Watts, M. B., Beck, V., Eder, R., and Jungbauer, A. (2003) Estrogenic activity in white and red wine extracts. J. Agric. Food Chem. 51, 1850 –1857 Gehm, B. D., McAndrews, J. M., Chien, P. Y., and Jameson, J. L. (1997) Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc. Natl. Acad. Sci. U. S. A. 94, 14138 –14143 Levenson, A. S., Svoboda, K. M., Pease, K. M., Kaiser, S. A., Chen, B., Simons, L. A., Jovanovic, B. D., Dyck, P. A., and Jordan, V. C. (2002) Gene expression profiles with activation of the estrogen receptor alpha-selective estrogen receptor modulator complex in breast cancer cells expressing wild-type estrogen receptor. Cancer Res. 62, 4419 – 4426 Baron-Menguy, C., Bocquet, A., Guihot, A. L., Chappard, D., Amiot, M.-J., Andriantsitohaina, R., Loufrani, L., and Henrion, D. (2007) Effects of red wine polyphenols on postischemic neovascularization model in rats: low doses are proangiogenic, high doses anti-angiogenic. FASEB J. 21, 3511–3521 Chambliss, K. L., and Shaul, P. W. (2002) Estrogen modulation of endothelial nitric oxide synthase. Endocr. Rev. 23, 665– 686 Ihionkhan, C. E., Chambliss, K. L., Gibson, L. L., Hahner, L. D., Mendelsohn, M. E., and Shaul, P. W. (2002) Estrogen causes dynamic alterations in endothelial estrogen receptor expression. Circ. Res. 91, 814 – 820 Filardo, E., Quinn, J., Pang, Y., Graeber, C., Shaw, S., Dong, J., and Thomas, P. (2007) Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane. Endocrinology 148, 3236 –3245 Filardo, E. J., Quinn, J. A., Frackelton, A. R., Jr., and Bland, K. I. (2002) Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol. Endocrinol. 16, 70 – 84 Razandi, M., Pedram, A., Merchenthaler, I., Greene, G. L., and Levin, E. R. (2004) Plasma membrane estrogen receptors exist and functions as dimers. Mol. Endocrinol. 18, 2854 –2865 Guo, X., Razandi, M., Pedram, A., Kassab, G., and Levin, E. R. (2005) Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta. J. Biol. Chem. 280, 19704 –19710 Joy, S., Siow, R. C. M., Rowlands, D. J., Becker, M., Wyatt, A. W., Aaronson, P. I., Coen, C. W., Kallo, I., Jacob, R., and Mann, G. E. (2006) The isoflavone equol mediates rapid vascular relaxation: Ca2⫹-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells. J. Biol. Chem. 281, 27335–27345 Kiss, A. L., Turi, A., Mullner, N., Kovacs, E., Botos, E., and Greger, A. (2005) Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localisation: An in vivo study. Mol. Cell. Endocrinol. 245, 128 –137 Razandi, M., Oh, P., Pedram, A., Schnitzer, J., and Levin, E. R. (2002) ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol. Endocrinol. 16, 100 –115 Wyckoff, M. H., Chambliss, K. L., Mineo, C., Yuhanna, I. S., Mendelsohn, M. E., Mumby, S. M., and Shaul, P. W. (2001) Plasma membrane estrogen receptors are coupled to endothe-
2195
23.
24.
25.
26.
27.
28. 29. 30. 31.
32.
33.
34. 35. 36.
37.
38.
39. 40.
2196
lial nitric-oxide synthase through Galpha(i). J. Biol. Chem. 276, 27071–27076 Lu, Q., Pallas, D. C., Surks, H. K., Baur, W. E., Mendelsohn, M. E., and Karas, R. H. (2004) Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor ␣. Proc. Natl. Acad. Sci. U. S. A. 101, 17126 –17131 Hisamoto, K., Ohmichi, M., Kanda, Y., Adachi, K., Nishio, Y., Hayakawa, J., Mabuchi, S., Takahashi, K., Tasaka, K., Miyamoto, Y., Taniguchi, N., and Murata, Y. (2001) Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells. J. Biol. Chem. 276, 47642– 47649 Gilad, L. A., and Schwartz, B. (2007) Association of estrogen receptor  with plasma-membrane caveola components: implication in control of vitamin D receptor. J. Mol. Endocrinol. 38, 603– 618 Morani, A., Barros, R. P. A., Imamov, O., Hultenby, K., Arner, A., Warner, M., and Gustafsson, J. A. (2006) Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ER-/-) mice. Proc. Natl. Acad. Sci. U. S. A. 103, 7165–7169 Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J., Safe, S. H., van der Saag, P., van der Burg, B., and Gustafsson, J. A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252– 4263 Greger, J. G., Guo, Y., Henderson, R., Ross, J. F., and Cheskis, B. J. (2006) Characterization of MNAR expression. Steroids 71, 317–322 Van Deurs, B., Roepstorff, K., Hommelgaard, A. M., and Sandvig, K. (2003) Caveolae: anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol. 13, 92–100 Zhuang, L., Lin, J., Lu, M. L., Solomon, K. R., and Freeman, M. R. (2002) Cholesterol-rich lipid rafts mediate Akt-regulated survival in prostate cancer cells. Cancer Res. 62, 2227–2231 Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y., and Nagano, T. (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem. 70, 2446 –2453 Ashby, J., Tinwell, H., Pennie, W., Brooks, A. N., Lefevre, P. A., Beresford, N., and Sumpter, J. P. (1999) Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects. J. Appl. Toxicol. 19, 39 – 45 Klinge, C. M., Jernigan, S. C., Risinger, K. E., Lee, J. E., Tyulmenkov, V. V., Falkner, K. C., and Prough, R. A. (2001) Short heterodimer partner (SHP) orphan nuclear receptor inhibits the transcriptional activity of aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT). Arch. Biochem. Biophys. 390, 64 –70 Signorelli, P., and Ghidoni, R. (2005) Resveratrol as an anticancer nutrient: molecular basis, open questions and promises. J. Nutr. Biochem. 16, 449 – 466 Lannigan, D. A. (2003) Estrogen receptor phosphorylation. Steroids 68, 1–9 Lin, M. T., Yen, M. L., Lin, C. Y., and Kuo, M. L. (2003) Inhibition of vascular endothelial growth factor-induced angiogenesis by resveratrol through interruption of Src-dependent vascular endothelial cadherin tyrosine phosphorylation. Mol. Pharmacol. 64, 1029 –1036 Li, L., Hisamoto, K., Kim, K. H., Haynes, M. P., Bauer, P. M., Sanjay, A., Collinge, M., Baron, R., Sessa, W. C., and Bender, J. R. (2007) Variant estrogen receptor c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation. Proc. Natl. Acad. Sci. 104, 16468 –16473 Chen, Z.-P., Mitchelhill, K. I., Michell, B. J., Stapleton, D., Rodriguez-Crespo, I., Witters, L. A., Power, D. A., Ortiz de Montellano, P. R., and Kemp, B. E. (1999) AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett. 443, 285–289 Fields, T. A., and Casey, P. J. (1997) Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. Biochem. J. 321, 561–571 Wijayaratne, A. L., and McDonnell, D. P. (2001) The human estrogen receptor-␣ is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J. Biol. Chem. 276, 35684 –35692
Vol. 22
July 2008
41.
42. 43. 44. 45.
46.
47. 48.
49.
50.
51.
52. 53. 54.
55.
56.
57.
58.
59.
60.
Fawell, S. E., White, R., Hoare, S., Sydenham, M., Page, M., and Parker, M. G. (1990) Inhibition of estrogen receptor-DNA binding by the “pure” antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization. Proc. Natl. Acad. Sci. U. S. A. 87, 6883– 6887 Levin, E. R. (2002) Cellular functions of plasma membrane estrogen receptors. Steroids 67, 471– 475 Kim, K. H., and Bender, J. R. (2005) Rapid, estrogen receptormediated signaling: why is the endothelium so special? [online] Sci. STKE 2005, pe28 Mineo, C., and Shaul, P. W. (2006) Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae. Cardiovasc. Res. 70, 31– 41 Chambliss, K. L., Simon, L., Yuhanna, I. S., Mineo, C., and Shaul, P. W. (2005) Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ER␣ domains with known nuclear functions. Mol. Endocrinol. 19, 277–289 Chambliss, K. L., and Shaul, P. W. (2002) Rapid activation of endothelial NO synthase by estrogen: evidence for a steroid receptor fast-action complex (SRFC) in caveolae. Steroids 67, 413– 419 Moriarty, K., Kim, K. H., and Bender, J. R. (2006) Estrogen receptor-mediated rapid signaling. Endocrinology 147, 5557–5563 Reineri, S., Bertoni, A., Sanna, E., Baldassarri, S., Sarasso, C., Zanfa, M., Canobbio, I., Torti, M., and Sinigaglia, F. (2007) Membrane lipid rafts coordinate estrogen-dependent signaling in human platelets. Biochim. Biophys. Acta 1773, 273–278 O’Lone, R., Knorr, K., Jaffe, I. Z., Schaffer, M. E., Martini, P. G. V., Karas, R. H., Bienkowska, J., Mendelsohn, M. E., and Hansen, U. (2007) Estrogen receptors ␣ and  mediate distinct pathways of vascular gene expression, including genes involved in mitochondrial electron transport and generation of reactive oxygen species. Mol. Endocrinol. 21, 1281–1296 Gingerich, S., and Krukoff, T. L. (2005) Estrogen modulates endothelial and neuronal nitric oxide synthase expression via an estrogen receptor -dependent mechanism in hypothalamic slice cultures. Endocrinology 146, 2933–2941 Gingerich, S., and Krukoff, T. L. (2006) Estrogen in the paraventricular nucleus attenuates L-glutamate-induced increases in mean arterial pressure through estrogen receptor  and NO. Hypertension 48, 1130 –1136 Watson, C. S., Alyea, R. A., Jeng, Y. J., and Kochukov, M. Y. (2007) Nongenomic actions of low concentration estrogens and xenoestrogens on multiple tissues. Mol. Cell. Endocrinol. 274, 1–7 Watson, C. S., and Lange, C. A. (2005) Steadying the boat: integrating mechanisms of membrane and nuclear-steroid-receptor signalling. EMBO Rep. 6, 116 –119 Watson, C. S., Bulayeva, N. N., Wozniak, A. L., and Finnerty, C. C. (2005) Signaling from the membrane via membrane estrogen receptor-␣: Estrogens, xenoestrogens, and phytoestrogens. Steroids 70, 364 –371 Bulayeva, N. N., Wozniak, A., Lash, L. L., and Watson, C. S. (2005) Mechanisms of membrane estrogen receptor-␣-mediated rapid stimulation of Ca2⫹ levels and prolactin release in a pituitary cell line. Am. J. Physiol. Endocrinol. Metab. 288, E388 –E397 Bulayeva, N. N., and Watson, C. S. (2004) Xenoestrogeninduced ERK-1 and ERK-2 activation via multiple membraneinitiated signaling pathways. Environ. Health Perspect. 112, 1481– 1487 Bulayeva, N. N., Gametchu, B., and Watson, C. S. (2004) Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways. Steroids 69, 181–192 Greger, J. G., Fursov, N., Cooch, N., McLarney, S., Freedman, L. P., Edwards, D. P., and Cheskis, B. J. (2007) Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase. Mol. Cell. Biol. 27, 1904 –1913 Razandi, M., Alton, G., Pedram, A., Ghonshani, S., Webb, P., and Levin, E. R. (2003) Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane. Mol. Cell. Biol. 23, 1633–1646 Li, L., Haynes, M. P., and Bender, J. R. (2003) Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 100, 4807– 4812
The FASEB Journal
KLINGE ET AL.
61.
62. 63.
64.
65.
66.
67.
68. 69.
70.
71.
72. 73.
Scobie, G. A., Macpherson, S., Millar, M. R., Groome, N. P., Romana, P. G., and Saunders, P. T. (2002) Human oestrogen receptors: differential expression of ER␣ and  and the identification of ER variants. Steroids 67, 985–992 Foley, E. F., Jazaeri, A. A., Shupnik, M. A., Jazaeri, O., and Rice, L. W. (2000) Selective loss of estrogen receptor beta in malignant human colon. Cancer Res. 60, 245–248 Fournier, B., Gutzwiller, S., Dittmar, T., Matthias, G., Steenbergh, P., and Matthias, P. (2001) Estrogen rceptor (ER)-␣, but not ER-, mediates regulation of the insulin-like growth factor I gene by antiestrogens. J. Biol. Chem. 276, 35444 –35449 Frasor, J., Barnett, D. H., Danes, J. M., Hess, R., Parlow, A. F., and Katzenellenbogen, B. S. (2003) Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ER in the uterus. Endocrinology 144, 3159 –3166 Hartman, J., Lindberg, K., Morani, A., Inzunza, J., Strom, A., and Gustafsson, J.-A. (2006) Estrogen receptor  inhibits angiogenesis and growth of T47D breast cancer xenografts. Cancer Res. 66, 11207–11213 Matthews, J., Wihlen, B., Tujague, M., Wan, J., Strom, A., and Gustafsson, J.-A. (2006) Estrogen receptor (ER)  modulates ER␣-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol. Endocrinol. 20, 534 –543 Paruthiyil, S., Parmar, H., Kerekatte, V., Cunha, G. R., Firestone, G. L., and Leitman, D. C. (2004) Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G(2) cell cycle arrest. Cancer Res. 64, 423– 428 Weihua, Z., Andersson, S., Cheng, G., Simpson, E. R., Warner, M., and Gustafsson, J. A. (2003) Update on estrogen signaling. FEBS Lett. 546, 17–24 Weihua, Z., Makela, S., Andersson, L. C., Salmi, S., Saji, S., Webster, J. I., Jensen, E. V., Nilsson, S., Warner, M., and Gustafsson, J. A. (2001) A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc. Natl. Acad. Sci. U. S. A. 98, 6330 – 6335 Weihua, Z., Saji, S., Makinen, S., Cheng, G., Jensen, E. V., Warner, M., and Gustafsson, J. A. (2000) Estrogen receptor (ER) beta, a modulator of ER␣ in the uterus. Proc. Natl. Acad. Sci. U. S. A. 97, 5936 –5941 Sladek, C. D., and Somponpun, S. J. (2008) Estrogen receptors: Their roles in regulation of vasopressin release for maintenance of fluid and electrolyte homeostasis. Front. Neuroendocrinol. 29, 114 –127. Aoki, T., Nomura, R., and Fujimoto, T. (1999) Tyrosine phosphorylation of caveolin-1 in the endothelium. Exp. Cell. Res. 253, 629 – 636 Podar, K., Shringarpure, R., Tai, Y. T., Simoncini, M., Sattler, M., Ishitsuka, K., Richardson, P. G., Hideshima, T., Chauhan,
RESVERATROL ACTIVATES ER␣-CAV-1 SIGNALING
74. 75. 76.
77.
78. 79.
80.
81.
82. 83.
84. 85.
D., and Anderson, K. C. (2004) Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib. Cancer Res. 64, 7500 –7506 Revankar, C. M., Cimino, D. F., Sklar, L. A., Arterburn, J. B., and Prossnitz, E. R. (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307, 1625–1630 Perviaz, S. (2003) Resveratrol: from grapevines to mammalian biology. FASEB J. 17, 1975–1985 Orallo, F., Alvarez, E., Camina, M., Leiro, J. M., Gomez, E., and Fernandez, P. (2002) The possible implication of trans-resveratrol in the cardioprotective effects of long-term moderate wine consumption. Mol. Pharmacol. 61, 294 –302 Bradamante, S., Barenghi, L., Piccinini, F., Bertelli, A. A., De Jonge, R., Beemster, P., and De Jong, J. W. (2003) Resveratrol provides late-phase cardioprotection by means of a nitric oxideand adenosine-mediated mechanism. Eur. J. Pharmacol 465, 115–123 Chen, W.-P., Su, M.-J., and Hung, L.-M. (2007) In vitro electrophysiological mechanisms for antiarrhythmic efficacy of resveratrol, a red wine antioxidant. Eur. J. Pharmacol. 554, 196 –204 Niles, R. M., Cook, C. P., Meadows, G. G., Fu, Y.-M., McLaughlin, J. L., and Rankin, G. O. (2006) Resveratrol is rapidly metabolized in athymic (Nu/Nu) mice and does not inhibit human melanoma xenograft tumor growth. J. Nutr. 136, 2542–2546 Yu, C., Shin, Y. G., Chow, A., Li, Y., Kosmeder, J. W., Lee, Y. S., Hirschelman, W. H., Pezzuto, J. M., Mehta, R. G., and van Breemen, R. B. (2002) Human, rat, and mouse metabolism of resveratrol. Pharm. Res. 19, 1907–1914 Marier, J. F., Vachon, P., Gritsas, A., Zhang, J., Moreau, J. P., and Ducharme, M. P. (2002) Metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. J. Pharmacol. Exp. Ther. 302, 369 –373 Levin, E. R. (2005) Integration of the extranuclear and nuclear actions of estrogen. Mol. Endocrinol. 19, 1951–1959 Pedram, A., Razandi, M., Sainson, R. C. A., Kim, J. K., Hughes, C. C., and Levin, E. R. (2007) A conserved mechanism for steroid receptor translocation to the plasma membrane. J. Biol. Chem. 282, 22278 –22288 Pedram, A., Razandi, M., and Levin, E. R. (2006) Nature of functional estrogen receptors at the plasma membrane. Mol. Endocrinol. 20, 1996 –2009 Kim, J. K., and Levin, E. R. (2006) Estrogen signaling in the cardiovascular system. Nucl. Recept. Signal 4, e013 DOI: 10.1621/ nrs.04013 Received for publication December 4, 2007. Accepted for publication January 24, 2008.
2197
