p38 Mitogen-Activated Protein Kinase Activates eNOS in Endothelial Cells by an Estrogen Receptor -Dependent Pathway in Response to Black Tea
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循环研究杂志 2005年第5期
The Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute (E.A., K.C., O.M.S., J.F.K.), Boston University School of Medicine, Boston, Mass
Molecular Cardiology Research Institute (R.H.K.), Tufts University School of Medicine, Boston, Mass.
Abstract
Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Because the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780, and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ER on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ER on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38 MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ER phosphorylation and downstream activation of Akt, and eNOS. The key role of ER Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ER subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ER with p38 MAPK as ER with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ER and these data uncover a new mechanism of ER activation that has broad implications for NO bioactivity and endothelial cell phenotype.
Key Words: antioxidants p38 endothelial dysfunction eNOS estrogen receptor
Introduction
Recent evidence indicate that flavonoids, a group of polyphenolic substances found in tea, fruit, vegetables, and wine favorably impact the endothelium.1,2 Normal endothelial function is critical for regulation of vasomotor tone, platelet activity, leukocyte adhesion, and vascular smooth muscle proliferation.3 These actions of the endothelium are mediated via release of several paracrine factors, including nitric oxide (NO).3
These normal functions of the endothelium are impaired in the setting of atherosclerosis and its associated vascular conditions such as hypertension, hypercholesterolemia, and diabetes.3 Impaired endothelial function has important consequences as individuals with the poorest endothelial function are at increased risk of cardiovascular events.4,5 Thus, reversing endothelial dysfunction has become a topic of intense investigation. In this regard, endothelial NO bioactivity is enhanced in patients with atherosclerosis by either the acute or chronic administration of black tea.6 In cultured cells, the black tea polyphenolic fraction promotes both eNOS catalytic activity and NO bioactivity.7 This effect is because of activation of phosphoinositol 3-kinase (PI 3-K) and Akt via a p38 MAPK-dependent mechanism.7
Despite observations that black tea polyphenols enhance endothelial cell NO bioactivity, important questions remain. The upstream components of polyphenol-mediated eNOS activation are not well described and the precise signals linking p38 MAPK to eNOS activation are largely unknown. The purpose of this study, therefore, was to investigate the mechanism of p38 MAPK-mediated eNOS activation by black tea polyphenols.
Materials and Methods
Materials
We obtained ICI 182,780 from Tocris (Ellisville, Mo). Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059) and JNK (SP600125) were obtained from Calbiochem (San Diego, Calif). Polyclonal antibodies directed against p38 MAPK, hemagluttinin (HA), ERK1/2, and the phosphorylated forms of p38 MAPK (Thr-180, Tyr-182), ERK1/2 (Thr-202, Tyr-204), JNK (Thr-183, Tyr-185), MAPKAPK-2 (Thr-222), GSK-3/ (Ser-21/9), and estrogen receptor-alpha (ER; Ser-118) were obtained from Cell Signaling Technology (Beverly, Mass). Polyclonal antibodies directed against phospho-eNOS (Ser1177) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-eNOS monoclonal antibody was obtained from Transduction Laboratories (Lexington, Ky). Antibody against ER (AER 320) was obtained from Laboratory Vision (Fremont, Calif). L-[3H]arginine (1 mCi, 53 mCi/mmol) was obtained from PerkinElmer Life Sciences (Wellesley, Mass) and cGMP assay kits were from Cayman (Ann Arbor, Mich). Black tea polyphenols, and black tea fractions were provided by Unilever, Inc. All other reagents were obtained from Sigma.
Cell Culture
Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics and grown on endothelial growth medium (Clonetics, Inc, San Diego, Calif). COS-7 cells (American Type Culture Collection, Rockville, Md) were grown in DMEM supplemented with 10% heat inactivated FBS, 50 e/mL heparin sulfate, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 e/mL streptomycin as described.8 For experiments, confluent endothelial cells were used between passages 4 and 8 and treated with physiologically relevant concentrations of black tea polyphenols (100 ng/mL) that we previously demonstrated activate eNOS.7 Before all experiments, cells were placed in serum- and phenol-free media (opti-MEM, Gibco, NY) for at least 16 hours, washed twice in HEPES-buffered physiologic salt solution (PSS), and experiments performed in HEPES-buffered PSS as described.7
eNOS Activity Assay
The catalytic activity of eNOS was estimated by the conversion of L-[3H]arginine to L-[3H]citrulline that was sensitive to inhibition by L-nitro-arginine methyl ester (L-NAME). Confluent cells in 100 mm culture dishes were washed and incubated in HEPES-buffered PSS for 30 minutes followed by the addition of 5 eol/L L-arginine plus 3.3 e藽i of L-[3H]arginine. Cells were then treated with the agonist of interest or vehicle for 15 minutes and cells were then lysed and L-[3H]citrulline determined as described.7
Immunoprecipitation and Western Blotting
Cells were washed with PSS and incubated in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L -glycerophosphate, 1 mmol/L Na3VO4, 1 e/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride for 30 minutes on ice as described.7 For ER immunoprecipitation, lysates were incubated with ER monoclonal antibody (4 e/mL) rotating for 16 hour at 4°C followed by a 1-hour incubation with protein A/G-agarose. Following centrifugation, the immunoprecipitates were washed, resuspended in loading buffer, and resolved by SDS-PAGE as described.7 Resolved proteins were transferred to a nitrocellulose membrane (Hybond; Amersham Biosciences, Inc.) and immunoblotting performed as previously described.7 Densitometric analysis of immunoblots was performed using PDI Imageware System.
Recombinant Adenoviral Vectors and Transfection
Recombinant adenovirus expressing a HA-tagged constitutively active MKK6 mutant (MKK6bE) was a kind gift from Dr. Jiahuia Han, Scripps Institute.9 Adenoviral construct encoding myristoylated, constitutively active Akt (myr-Akt) was a kind gift of Dr. Kenneth Walsh, Boston University. BAECs were infected in medium without FBS for 2 hours, washed, and incubated in fresh medium with FBS for 12 to 24 hours before experimentation.
Plasmid Constructs and Transfection
The construct for human eNOS cDNA was obtained from Dr. Richard Venema, Medical College of Georgia. Expression plasmids for ER and ERs118a have been described previously.10 Transfection of COS-7 cells was performed essentially as previously described.8,11 After transfection, cells were cultured in a phenol red- and estrogen-free media and either vehicle or estrogen was added to effect stimulation of the receptor.
Statistical Analysis
All numerical data are presented as means±SE. Western blots shown are representative of 3 or more independent experiments. For parametric data, comparisons among treatment groups were performed with one-way analysis of variance and an appropriate post hoc comparison. Instances involving only 2 comparisons were evaluated with a Student’s t test. Statistical significance was accepted if the null hypothesis was rejected with a P<0.05.
Results
Activation of eNOS by Black Tea Polyphenols Involves Estrogen Receptors
To probe pathways involved in black tea polyphenol-induced eNOS activation, we used a pharmacological approach that included the ER antagonist ICI 182,780. As shown in Figure 1A and B, polyphenol-induced eNOS phosphorylation and catalytic activation were both attenuated by ER antagonism. To confirm this finding, we used siRNA to silence ER expression and found it limited black tea polyphenol-induced eNOS phosphorylation on Ser-1177 (human sequence; Figure 1C) and eNOS activation (Figure 1D). Thus, black tea polyphenol-induced eNOS activation involves estrogen receptors.
Black Tea Polyphenols Induce ER Phosphorylation
Phosphorylation of ER occurs on multiple residues, including Ser-118.12 To determine whether black tea polyphenols induce ER Ser-118 phosphorylation, BAECs were treated with black tea polyphenols and probed with a phosphorylation-specific antibody. Under resting conditions, BAECs exhibit little ER Ser-118 phosphorylation, however, exposure to black tea polyphenols resulted in a time-dependent increase in ER Ser-118 phosphorylation (Figure 2A). Pharmacological ER inhibition by ICI 182,780 attenuated ER phosphorylation in response to black tea polyphenols (Figure 2B). These data indicate polyphenols induce ER phosphorylation in a manner that is inhibited by ICI 182,780.
Black Tea Polyphenol-Induced ER Phosphorylation Involves p38 MAPK
Phosphorylation of ER on Ser-118 has been linked to activation of extracellular signal-regulated kinase (ERK)12 and we have demonstrated p38 MAPK (p38 MAPK) is required for eNOS activation in response to black tea polyphenols.7 Therefore, to determine the involvement of MAPKs in our system, lysates from black tea polyphenol-treated BAECs were probed for activation of ERK, p38 MAPK, or c-Jun, N-terminal kinase (JNK). As shown in Figure 3A, p38 MAPK was activated in response to black tea polyphenols with no activation of ERK or JNK. Consistent with this observation, only inhibition of p38 MAPK had a material impact on the polyphenol-induced eNOS stimulation (Figure 3B).
We next investigated the role of p38 MAPK in ER phosphorylation. Pharmacological inhibition of p38 MAPK blunted black tea polyphenol-induced ER Ser-118 phosphorylation and phosphorylation of the p38 MAPK target, MAPKAPK2 (Figure 4A). We confirmed that ER phosphorylation was downstream of p38MAPK as adenoviral transfection with a dominant-negative p38 MAPK mutant prevented black tea polyphenol-induced ER Ser-118 phosphorylation (Figure 4B). Activation of p38 MAPK proved to be a proximal component of the black tea polyphenol response as dominant-negative p38 MAPK also prevented Akt phosphorylation, eNOS phosphorylation (Figure 4B), and eNOS activation (Figure 4C) in response to polyphenols. Consistent with this observation, constitutive activation of p38 MAPK with the MKK6bE adenovirus produced the same response as black tea polyphenols with regards to phosphorylation of ER, Akt, and eNOS as well as eNOS activation (Figure 4B and 4C). Combined treatment of BAECs with MKK6bE overexpression and black tea polyphenols was synergistic with regards to eNOS activation (Figure 4C).
We next investigated the relation between p38 MAPK, ER, and Akt in this signaling response. BAECs overexpressing MKK6bE exhibited phosphorylation of p38 MAPK, ER, Akt, and eNOS as well as eNOS catalytic activation (Figure 5A and B). Treating MKK6bE overexpressing cells with ICI 182,780 had no impact on p38MAPK activation, but this treatment prevented ER phosphorylation and downstream signaling to Akt and eNOS. Consistent with this observation, BAECs harboring a constitutively active Akt mutant (Myr-Akt) demonstrated eNOS phosphorylation and activation without any effect on ER or p38 MAPK (Figure 5A and B).
To confirm that p38 MAPK is upstream of ER, we used siRNA to inhibit ER expression and found it inhibited black tea polyphenol-induced eNOS Ser-1177 phosphorylation (human sequence) without any effect on p38 MAPK activation (Figure 5C). Collectively, these data establish a linear signaling cascade from p38 MAPK to ER, PI 3-K/Akt, and eNOS. Thus, ER Ser-118 phosphorylation appears critical for signal transduction from p38 MAPK to Akt.
P38 MAPK-Mediated eNOS Activation Requires ER Ser-118
To establish that black tea polyphenol-induced eNOS activation requires ER, we conducted experiments in COS-7 cells that lack ERs. In COS-7 cells transfected with eNOS alone, neither estradiol nor black tea polyphenols stimulated eNOS activity, whereas A23187 enhanced eNOS activity by 3-fold (Figure 6A). However, in COS-7 cells cotransfected with eNOS and ER, both estradiol and black tea polyphenols significantly increased eNOS activity and the response to A23187 was not altered (Figure 6A). Black tea polyphenol-mediated p38 MAPK activation was observed both in the presence and absence of ER transfection in COS-7 cells (Figure 6A).
To further define the role of ER Ser-118 in black tea polyphenol-induced eNOS activation, we used an ER mutant (ERs118a) containing an alanine in place of serine at position 118 (ERs118a) that is not subject to phosphorylation.10 As shown in Figure 6B, COS-7 cells transfected with ERs118a and eNOS showed significant attenuation of eNOS stimulation in response to black tea polyphenols compared with cells harboring the wild-type ER. These data indicate that ER Ser-118 is required for black tea polyphenol-induced eNOS stimulation.
ER and p38 MAPK Are Functionally Associated in Response to Black Tea Polyphenols
To probe for the existence of a functional complex including both p38 MAPK and ER, COS-7 cells cotransfected with eNOS and wild-type ER, were treated with black tea polyphenols and the lysates were immunoprecipitated with antibodies directed against ER. We then probed the precipitates for p38 MAPK and found a time-dependent increase in the pellet p38 MAPK with a corresponding decrease in supernatant p38MAPK. (Figure 7A). Similarly, the converse experiment involving immunoprecipitation of p38 MAPK from black tea polyphenol exposed COS-7 cells demonstrated evidence for ER in the pellet and a corresponding decrease in the supernatant (Figure 7B). This functional association between p38 MAPK and ER could not be detected in cells cotransfected with eNOS and ER(s118a) (Figure 7C and D). Immunoprecipitation of ER from BAECs also demonstrated polyphenol-induced complex formation between ER and p38 MAPK (data not shown). Collectively, these data support the notion that ER is an upstream mediator of Akt and eNOS activation by black tea polyphenols. Moreover, the data also suggest that p38 MAPK activates ER in response to black tea polyphenols.
Discussion
In this study we found that ER plays a key role in mediating the activation of eNOS in response to black tea polyphenols. In particular, we found that ER is phosphorylated on Ser-118 in response to black tea polyphenol-mediated p38 MAPK activation. This ER phosphorylation is required for p38 MAPK downstream signaling to Akt and eNOS that mediates black tea polyphenol-induced eNOS activation. We were able to implicate Ser-118 as a critical residue in this process as we observed ER Ser-118 phosphorylation in response to black tea polyphenols and mutation of this residue abrogated polyphenol-induced eNOS activation in COS cells. This residue also appeared important for the functional association of p38 MAPK with ER as mutants lacking this site did not associate with p38 MAPK in response to black tea polyphenols. These data suggest a novel adaptor function of ER that integrates p38 MAPK signaling with downstream targets.
The rapidity of the response to black tea polyphenols suggests a nongenomic role for ER in our system. These findings are in agreement with previous studies indicating that ER mediates nongenomic eNOS activation in response to estradiol.13eC15 Ligand engagement of ER induces its association with the p85-subunit of phosphoinositol 3-kinase (PI 3-K) and c-Src leading to Akt activation.15,16 This event promotes eNOS phosphorylation at Ser-1177,17 a key event that stimulates enzyme activation18,19 and enhances its sensitivity to calcium.20 The data presented here add to this body of literature by identifying p38 MAPK as an upstream effector of ER that mediates rapid eNOS activation. Moreover, we have strong evidence that p38 MAPK induces ER phosphorylation in a ligand-independent manner as molecular activation of p38 MAPK with MKK6bE recapitulated both ER phosphorylation and eNOS activation in the absence of polyphenols. To our knowledge, this is the first demonstration of rapid, nongenomic effects of ER stimulation that is ligand-independent. Typically, ligand-independent ER activation has been restricted to the transcriptional functions of this nuclear hormone receptor in response to stimuli such as epidermal growth factor,21 insulin-like growth factor,21 and the cyclins A22 and D1.23 Thus, the data provided here indicate a novel ER function as a consequence of ligand-independent activation.
Despite recent reports demonstrating polyphenol-induced ERK activation,24 we did not find a role for ERK in our system (Figure 3). Possible explanations for this observation might include the fact we used a tea polyphenol extract rather than a red wine extract or authentic resveratrol.24 Moreover, our data fit best with a ligand-independent mechanism rather than polyphenol binding to the estrogen receptor. In this regard, previous data depict ligand-independent ER activation as an ERK-mediated phenomenon that involves the ER Ser-118 residue.21 The data presented here add a new facet to this body of literature in that ER is a target of the p38 MAPK in endothelial cells. There is one prior report of p38 MAPK-mediated ER phosphorylation in a study that involved endometrial carcinoma cells and Thr-311 as the p38 MAPK target.25 In contrast, our report involves endothelial cells and Ser-118 appears to be the site of phosphorylation in response to black tea polyphenols. This discrepancy could be a function of the different cell types between these studies. Alternatively, it is possible that our study also involved p38 MAPK phosphorylation of ER on Thr-311 that facilitated ER Ser-118 phosphorylation by some other, as yet unrecognized, kinase. However, we did exclude ERK as mediating Ser-118 phosphorylation in this study, but other kinases do target this site including cdk7.26 Thus, determining the precise molecular events surrounding black tea polyphenol-mediated ER phosphorylation will require further study.
Evidence from this work does distinguish estradiol-mediated eNOS activation from that observed with black tea polyphenols. Although both agents induce ER Ser-118 phosphorylation, this event was only critical for polyphenol-induced eNOS activation. In COS-7 cells transfected with ER harboring an alanine at position 118 (ERs118a), we observed intact eNOS activation in response to authentic estradiol, whereas the black tea polyphenol response was attenuated compared with wild-type ER. These data are in keeping with the known role of Ser-118 in mediating ligand-independent ER function with little role in ligand-dependent responses.10 The results of our study may also be related to the presence of other phosphorylation sites on ER such as Ser-167, Thr-311, and Ser-522 that were not the subject of this investigation. In this regard, Ser-522 has been proven critical for G-protein-coupled ER responses whereas Thr-311 appears important for cancer cell-mediated metastasis.25
The data presented here indicate that p38 MAPK has a role in regulating the activity of a nuclear hormone receptor, ER. Although not described for ER, there is some precedent for nuclear hormone receptors to be modulated by p38 MAPK. For example, in the setting of cytokine stimulation, PPAR coactivator-1 is phosphorylated by p38 MAPK and this event regulates the control of genes involved in energy expenditure.27 Phosphorylation of the glucocorticoid receptor by p38 MAPK appears to reduce receptor activity and this may be involved in feedback regulation of the receptor in the setting of inflammation.28 The precise nature of how p38 MAPK interacts with ER is not clear from this work, but our experiments do suggest these 2 proteins occur in a common complex in response to polyphenol stimulation. Further investigation will be required to determine the molecular determinants of this event.
In summary, the data presented here indicate that black tea polyphenols stimulate eNOS activity largely through nongenomic ligand-independent activation of ER in vascular endothelial cells. The mechanisms involve a p38 MAPK-induced phosphorylation of ER on Ser-118, which in turn leads to the activation of the PI 3-K/Akt pathway and eNOS. Along with the implications regarding estrogen and vascular endothelial function, the present findings are important to the mechanisms underlying the rapid activation of estrogen receptors in vascular endothelium.
Acknowledgments
This is supported by grants from the National Institutes of Health (DK55656, HL60886, HL67206, HL68758). We thank Nikheil Rau for technical assistance.
References
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Anter E, Thomas SR, Schulz E, Shapira OM, Vita JA, Keaney JF Jr. Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols. J Biol Chem. 2004; 279 (45): 46637eC46643.
Chen K, Vita JA, Berk BC, Keaney JF Jr. c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves src-dependent EGF receptor transactivation. J Biol Chem. 2001; 276: 16045eC16050.
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Chen K, Albano A, Ho A, Keaney JF Jr. Activation of p53 by Oxidative Stress Involves Platelet-derived Growth Factor-{beta} Receptor-mediated Ataxia Telangiectasia Mutated (ATM) Kinase Activation. J Biol Chem. 2003; 278 (41): 39527eC39533.
Joel PB, Traish AM, Lannigan DA. Estradiol and phorbol ester cause phosphorylation of serine 118 in the human estrogen receptor. Mol Endocrinol. 1995; 9 (8): 1041eC1052.
Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW. Estrogen receptor mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest. 1999; 103 (3): 401eC406.
Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol. 2002; 64: 749eC774.
Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature. 2000; 407 (6803): 538eC541.
Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge M, Sessa WC, Bender JR. Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. J Biol Chem. 2003; 278 (4): 2118eC2123.
Haynes MP, Sinha D, Russell KS, Collinge M, Fulton D, Morales-Ruiz M, Sessa WC, Bender JR. Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells. Circ Res. 2000; 87 (8): 677eC682.
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Molecular Cardiology Research Institute (R.H.K.), Tufts University School of Medicine, Boston, Mass.
Abstract
Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Because the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780, and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ER on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ER on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38 MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ER phosphorylation and downstream activation of Akt, and eNOS. The key role of ER Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ER subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ER with p38 MAPK as ER with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ER and these data uncover a new mechanism of ER activation that has broad implications for NO bioactivity and endothelial cell phenotype.
Key Words: antioxidants p38 endothelial dysfunction eNOS estrogen receptor
Introduction
Recent evidence indicate that flavonoids, a group of polyphenolic substances found in tea, fruit, vegetables, and wine favorably impact the endothelium.1,2 Normal endothelial function is critical for regulation of vasomotor tone, platelet activity, leukocyte adhesion, and vascular smooth muscle proliferation.3 These actions of the endothelium are mediated via release of several paracrine factors, including nitric oxide (NO).3
These normal functions of the endothelium are impaired in the setting of atherosclerosis and its associated vascular conditions such as hypertension, hypercholesterolemia, and diabetes.3 Impaired endothelial function has important consequences as individuals with the poorest endothelial function are at increased risk of cardiovascular events.4,5 Thus, reversing endothelial dysfunction has become a topic of intense investigation. In this regard, endothelial NO bioactivity is enhanced in patients with atherosclerosis by either the acute or chronic administration of black tea.6 In cultured cells, the black tea polyphenolic fraction promotes both eNOS catalytic activity and NO bioactivity.7 This effect is because of activation of phosphoinositol 3-kinase (PI 3-K) and Akt via a p38 MAPK-dependent mechanism.7
Despite observations that black tea polyphenols enhance endothelial cell NO bioactivity, important questions remain. The upstream components of polyphenol-mediated eNOS activation are not well described and the precise signals linking p38 MAPK to eNOS activation are largely unknown. The purpose of this study, therefore, was to investigate the mechanism of p38 MAPK-mediated eNOS activation by black tea polyphenols.
Materials and Methods
Materials
We obtained ICI 182,780 from Tocris (Ellisville, Mo). Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059) and JNK (SP600125) were obtained from Calbiochem (San Diego, Calif). Polyclonal antibodies directed against p38 MAPK, hemagluttinin (HA), ERK1/2, and the phosphorylated forms of p38 MAPK (Thr-180, Tyr-182), ERK1/2 (Thr-202, Tyr-204), JNK (Thr-183, Tyr-185), MAPKAPK-2 (Thr-222), GSK-3/ (Ser-21/9), and estrogen receptor-alpha (ER; Ser-118) were obtained from Cell Signaling Technology (Beverly, Mass). Polyclonal antibodies directed against phospho-eNOS (Ser1177) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-eNOS monoclonal antibody was obtained from Transduction Laboratories (Lexington, Ky). Antibody against ER (AER 320) was obtained from Laboratory Vision (Fremont, Calif). L-[3H]arginine (1 mCi, 53 mCi/mmol) was obtained from PerkinElmer Life Sciences (Wellesley, Mass) and cGMP assay kits were from Cayman (Ann Arbor, Mich). Black tea polyphenols, and black tea fractions were provided by Unilever, Inc. All other reagents were obtained from Sigma.
Cell Culture
Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics and grown on endothelial growth medium (Clonetics, Inc, San Diego, Calif). COS-7 cells (American Type Culture Collection, Rockville, Md) were grown in DMEM supplemented with 10% heat inactivated FBS, 50 e/mL heparin sulfate, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 e/mL streptomycin as described.8 For experiments, confluent endothelial cells were used between passages 4 and 8 and treated with physiologically relevant concentrations of black tea polyphenols (100 ng/mL) that we previously demonstrated activate eNOS.7 Before all experiments, cells were placed in serum- and phenol-free media (opti-MEM, Gibco, NY) for at least 16 hours, washed twice in HEPES-buffered physiologic salt solution (PSS), and experiments performed in HEPES-buffered PSS as described.7
eNOS Activity Assay
The catalytic activity of eNOS was estimated by the conversion of L-[3H]arginine to L-[3H]citrulline that was sensitive to inhibition by L-nitro-arginine methyl ester (L-NAME). Confluent cells in 100 mm culture dishes were washed and incubated in HEPES-buffered PSS for 30 minutes followed by the addition of 5 eol/L L-arginine plus 3.3 e藽i of L-[3H]arginine. Cells were then treated with the agonist of interest or vehicle for 15 minutes and cells were then lysed and L-[3H]citrulline determined as described.7
Immunoprecipitation and Western Blotting
Cells were washed with PSS and incubated in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L -glycerophosphate, 1 mmol/L Na3VO4, 1 e/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride for 30 minutes on ice as described.7 For ER immunoprecipitation, lysates were incubated with ER monoclonal antibody (4 e/mL) rotating for 16 hour at 4°C followed by a 1-hour incubation with protein A/G-agarose. Following centrifugation, the immunoprecipitates were washed, resuspended in loading buffer, and resolved by SDS-PAGE as described.7 Resolved proteins were transferred to a nitrocellulose membrane (Hybond; Amersham Biosciences, Inc.) and immunoblotting performed as previously described.7 Densitometric analysis of immunoblots was performed using PDI Imageware System.
Recombinant Adenoviral Vectors and Transfection
Recombinant adenovirus expressing a HA-tagged constitutively active MKK6 mutant (MKK6bE) was a kind gift from Dr. Jiahuia Han, Scripps Institute.9 Adenoviral construct encoding myristoylated, constitutively active Akt (myr-Akt) was a kind gift of Dr. Kenneth Walsh, Boston University. BAECs were infected in medium without FBS for 2 hours, washed, and incubated in fresh medium with FBS for 12 to 24 hours before experimentation.
Plasmid Constructs and Transfection
The construct for human eNOS cDNA was obtained from Dr. Richard Venema, Medical College of Georgia. Expression plasmids for ER and ERs118a have been described previously.10 Transfection of COS-7 cells was performed essentially as previously described.8,11 After transfection, cells were cultured in a phenol red- and estrogen-free media and either vehicle or estrogen was added to effect stimulation of the receptor.
Statistical Analysis
All numerical data are presented as means±SE. Western blots shown are representative of 3 or more independent experiments. For parametric data, comparisons among treatment groups were performed with one-way analysis of variance and an appropriate post hoc comparison. Instances involving only 2 comparisons were evaluated with a Student’s t test. Statistical significance was accepted if the null hypothesis was rejected with a P<0.05.
Results
Activation of eNOS by Black Tea Polyphenols Involves Estrogen Receptors
To probe pathways involved in black tea polyphenol-induced eNOS activation, we used a pharmacological approach that included the ER antagonist ICI 182,780. As shown in Figure 1A and B, polyphenol-induced eNOS phosphorylation and catalytic activation were both attenuated by ER antagonism. To confirm this finding, we used siRNA to silence ER expression and found it limited black tea polyphenol-induced eNOS phosphorylation on Ser-1177 (human sequence; Figure 1C) and eNOS activation (Figure 1D). Thus, black tea polyphenol-induced eNOS activation involves estrogen receptors.
Black Tea Polyphenols Induce ER Phosphorylation
Phosphorylation of ER occurs on multiple residues, including Ser-118.12 To determine whether black tea polyphenols induce ER Ser-118 phosphorylation, BAECs were treated with black tea polyphenols and probed with a phosphorylation-specific antibody. Under resting conditions, BAECs exhibit little ER Ser-118 phosphorylation, however, exposure to black tea polyphenols resulted in a time-dependent increase in ER Ser-118 phosphorylation (Figure 2A). Pharmacological ER inhibition by ICI 182,780 attenuated ER phosphorylation in response to black tea polyphenols (Figure 2B). These data indicate polyphenols induce ER phosphorylation in a manner that is inhibited by ICI 182,780.
Black Tea Polyphenol-Induced ER Phosphorylation Involves p38 MAPK
Phosphorylation of ER on Ser-118 has been linked to activation of extracellular signal-regulated kinase (ERK)12 and we have demonstrated p38 MAPK (p38 MAPK) is required for eNOS activation in response to black tea polyphenols.7 Therefore, to determine the involvement of MAPKs in our system, lysates from black tea polyphenol-treated BAECs were probed for activation of ERK, p38 MAPK, or c-Jun, N-terminal kinase (JNK). As shown in Figure 3A, p38 MAPK was activated in response to black tea polyphenols with no activation of ERK or JNK. Consistent with this observation, only inhibition of p38 MAPK had a material impact on the polyphenol-induced eNOS stimulation (Figure 3B).
We next investigated the role of p38 MAPK in ER phosphorylation. Pharmacological inhibition of p38 MAPK blunted black tea polyphenol-induced ER Ser-118 phosphorylation and phosphorylation of the p38 MAPK target, MAPKAPK2 (Figure 4A). We confirmed that ER phosphorylation was downstream of p38MAPK as adenoviral transfection with a dominant-negative p38 MAPK mutant prevented black tea polyphenol-induced ER Ser-118 phosphorylation (Figure 4B). Activation of p38 MAPK proved to be a proximal component of the black tea polyphenol response as dominant-negative p38 MAPK also prevented Akt phosphorylation, eNOS phosphorylation (Figure 4B), and eNOS activation (Figure 4C) in response to polyphenols. Consistent with this observation, constitutive activation of p38 MAPK with the MKK6bE adenovirus produced the same response as black tea polyphenols with regards to phosphorylation of ER, Akt, and eNOS as well as eNOS activation (Figure 4B and 4C). Combined treatment of BAECs with MKK6bE overexpression and black tea polyphenols was synergistic with regards to eNOS activation (Figure 4C).
We next investigated the relation between p38 MAPK, ER, and Akt in this signaling response. BAECs overexpressing MKK6bE exhibited phosphorylation of p38 MAPK, ER, Akt, and eNOS as well as eNOS catalytic activation (Figure 5A and B). Treating MKK6bE overexpressing cells with ICI 182,780 had no impact on p38MAPK activation, but this treatment prevented ER phosphorylation and downstream signaling to Akt and eNOS. Consistent with this observation, BAECs harboring a constitutively active Akt mutant (Myr-Akt) demonstrated eNOS phosphorylation and activation without any effect on ER or p38 MAPK (Figure 5A and B).
To confirm that p38 MAPK is upstream of ER, we used siRNA to inhibit ER expression and found it inhibited black tea polyphenol-induced eNOS Ser-1177 phosphorylation (human sequence) without any effect on p38 MAPK activation (Figure 5C). Collectively, these data establish a linear signaling cascade from p38 MAPK to ER, PI 3-K/Akt, and eNOS. Thus, ER Ser-118 phosphorylation appears critical for signal transduction from p38 MAPK to Akt.
P38 MAPK-Mediated eNOS Activation Requires ER Ser-118
To establish that black tea polyphenol-induced eNOS activation requires ER, we conducted experiments in COS-7 cells that lack ERs. In COS-7 cells transfected with eNOS alone, neither estradiol nor black tea polyphenols stimulated eNOS activity, whereas A23187 enhanced eNOS activity by 3-fold (Figure 6A). However, in COS-7 cells cotransfected with eNOS and ER, both estradiol and black tea polyphenols significantly increased eNOS activity and the response to A23187 was not altered (Figure 6A). Black tea polyphenol-mediated p38 MAPK activation was observed both in the presence and absence of ER transfection in COS-7 cells (Figure 6A).
To further define the role of ER Ser-118 in black tea polyphenol-induced eNOS activation, we used an ER mutant (ERs118a) containing an alanine in place of serine at position 118 (ERs118a) that is not subject to phosphorylation.10 As shown in Figure 6B, COS-7 cells transfected with ERs118a and eNOS showed significant attenuation of eNOS stimulation in response to black tea polyphenols compared with cells harboring the wild-type ER. These data indicate that ER Ser-118 is required for black tea polyphenol-induced eNOS stimulation.
ER and p38 MAPK Are Functionally Associated in Response to Black Tea Polyphenols
To probe for the existence of a functional complex including both p38 MAPK and ER, COS-7 cells cotransfected with eNOS and wild-type ER, were treated with black tea polyphenols and the lysates were immunoprecipitated with antibodies directed against ER. We then probed the precipitates for p38 MAPK and found a time-dependent increase in the pellet p38 MAPK with a corresponding decrease in supernatant p38MAPK. (Figure 7A). Similarly, the converse experiment involving immunoprecipitation of p38 MAPK from black tea polyphenol exposed COS-7 cells demonstrated evidence for ER in the pellet and a corresponding decrease in the supernatant (Figure 7B). This functional association between p38 MAPK and ER could not be detected in cells cotransfected with eNOS and ER(s118a) (Figure 7C and D). Immunoprecipitation of ER from BAECs also demonstrated polyphenol-induced complex formation between ER and p38 MAPK (data not shown). Collectively, these data support the notion that ER is an upstream mediator of Akt and eNOS activation by black tea polyphenols. Moreover, the data also suggest that p38 MAPK activates ER in response to black tea polyphenols.
Discussion
In this study we found that ER plays a key role in mediating the activation of eNOS in response to black tea polyphenols. In particular, we found that ER is phosphorylated on Ser-118 in response to black tea polyphenol-mediated p38 MAPK activation. This ER phosphorylation is required for p38 MAPK downstream signaling to Akt and eNOS that mediates black tea polyphenol-induced eNOS activation. We were able to implicate Ser-118 as a critical residue in this process as we observed ER Ser-118 phosphorylation in response to black tea polyphenols and mutation of this residue abrogated polyphenol-induced eNOS activation in COS cells. This residue also appeared important for the functional association of p38 MAPK with ER as mutants lacking this site did not associate with p38 MAPK in response to black tea polyphenols. These data suggest a novel adaptor function of ER that integrates p38 MAPK signaling with downstream targets.
The rapidity of the response to black tea polyphenols suggests a nongenomic role for ER in our system. These findings are in agreement with previous studies indicating that ER mediates nongenomic eNOS activation in response to estradiol.13eC15 Ligand engagement of ER induces its association with the p85-subunit of phosphoinositol 3-kinase (PI 3-K) and c-Src leading to Akt activation.15,16 This event promotes eNOS phosphorylation at Ser-1177,17 a key event that stimulates enzyme activation18,19 and enhances its sensitivity to calcium.20 The data presented here add to this body of literature by identifying p38 MAPK as an upstream effector of ER that mediates rapid eNOS activation. Moreover, we have strong evidence that p38 MAPK induces ER phosphorylation in a ligand-independent manner as molecular activation of p38 MAPK with MKK6bE recapitulated both ER phosphorylation and eNOS activation in the absence of polyphenols. To our knowledge, this is the first demonstration of rapid, nongenomic effects of ER stimulation that is ligand-independent. Typically, ligand-independent ER activation has been restricted to the transcriptional functions of this nuclear hormone receptor in response to stimuli such as epidermal growth factor,21 insulin-like growth factor,21 and the cyclins A22 and D1.23 Thus, the data provided here indicate a novel ER function as a consequence of ligand-independent activation.
Despite recent reports demonstrating polyphenol-induced ERK activation,24 we did not find a role for ERK in our system (Figure 3). Possible explanations for this observation might include the fact we used a tea polyphenol extract rather than a red wine extract or authentic resveratrol.24 Moreover, our data fit best with a ligand-independent mechanism rather than polyphenol binding to the estrogen receptor. In this regard, previous data depict ligand-independent ER activation as an ERK-mediated phenomenon that involves the ER Ser-118 residue.21 The data presented here add a new facet to this body of literature in that ER is a target of the p38 MAPK in endothelial cells. There is one prior report of p38 MAPK-mediated ER phosphorylation in a study that involved endometrial carcinoma cells and Thr-311 as the p38 MAPK target.25 In contrast, our report involves endothelial cells and Ser-118 appears to be the site of phosphorylation in response to black tea polyphenols. This discrepancy could be a function of the different cell types between these studies. Alternatively, it is possible that our study also involved p38 MAPK phosphorylation of ER on Thr-311 that facilitated ER Ser-118 phosphorylation by some other, as yet unrecognized, kinase. However, we did exclude ERK as mediating Ser-118 phosphorylation in this study, but other kinases do target this site including cdk7.26 Thus, determining the precise molecular events surrounding black tea polyphenol-mediated ER phosphorylation will require further study.
Evidence from this work does distinguish estradiol-mediated eNOS activation from that observed with black tea polyphenols. Although both agents induce ER Ser-118 phosphorylation, this event was only critical for polyphenol-induced eNOS activation. In COS-7 cells transfected with ER harboring an alanine at position 118 (ERs118a), we observed intact eNOS activation in response to authentic estradiol, whereas the black tea polyphenol response was attenuated compared with wild-type ER. These data are in keeping with the known role of Ser-118 in mediating ligand-independent ER function with little role in ligand-dependent responses.10 The results of our study may also be related to the presence of other phosphorylation sites on ER such as Ser-167, Thr-311, and Ser-522 that were not the subject of this investigation. In this regard, Ser-522 has been proven critical for G-protein-coupled ER responses whereas Thr-311 appears important for cancer cell-mediated metastasis.25
The data presented here indicate that p38 MAPK has a role in regulating the activity of a nuclear hormone receptor, ER. Although not described for ER, there is some precedent for nuclear hormone receptors to be modulated by p38 MAPK. For example, in the setting of cytokine stimulation, PPAR coactivator-1 is phosphorylated by p38 MAPK and this event regulates the control of genes involved in energy expenditure.27 Phosphorylation of the glucocorticoid receptor by p38 MAPK appears to reduce receptor activity and this may be involved in feedback regulation of the receptor in the setting of inflammation.28 The precise nature of how p38 MAPK interacts with ER is not clear from this work, but our experiments do suggest these 2 proteins occur in a common complex in response to polyphenol stimulation. Further investigation will be required to determine the molecular determinants of this event.
In summary, the data presented here indicate that black tea polyphenols stimulate eNOS activity largely through nongenomic ligand-independent activation of ER in vascular endothelial cells. The mechanisms involve a p38 MAPK-induced phosphorylation of ER on Ser-118, which in turn leads to the activation of the PI 3-K/Akt pathway and eNOS. Along with the implications regarding estrogen and vascular endothelial function, the present findings are important to the mechanisms underlying the rapid activation of estrogen receptors in vascular endothelium.
Acknowledgments
This is supported by grants from the National Institutes of Health (DK55656, HL60886, HL67206, HL68758). We thank Nikheil Rau for technical assistance.
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