p38 mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols

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 ERalpha on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ERalpha on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38alpha MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ERalpha phosphorylation and downstream activation of Akt, and eNOS. The key role of ERalpha Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ERalpha subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ERalpha with p38 MAPK as ERalpha with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ERalpha and these data uncover a new mechanism of ERalpha activation that has broad implications for NO bioactivity and endothelial cell phenotype.

Figure 1

Activation of eNOS by black tea polyphenols involves estrogen receptors. (A) BAECs in HEPES-buffered PSS were incubated with or without the estrogen receptor antagonist ICI 182,780 (100 nM) followed by incubation with 100ng/ml black tea polyphenols (BTP) for 15 min. Cells were then lysed, eNOS immunoprecipitated, and its phosphorylation status at Ser-1179 determined by immunoblotting with antibodies against phosphorylated or total eNOS as indicated. (B) BAECs were incubated as in (A) and eNOS catalytic activity estimated by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” (C) HUVECs were incubated without (CTL) or with siRNA directed against human ERα/ERβ (ER siRNA) or its scrambled control (CTL siRNA) for 72 hours before treatment with BTP as in (A). The expression of ERα and eNOS phosphorylation were determined by immunoblotting with the indicated antibodies. (D) HUVECs treated as in (C) were assessed for eNOS catalytic activity by the conversion of L[³H]arginine to L-[³H]citrulline as in “Methods.” All immunoblots are representative of 3 independent experiments and composite data are mean ± S.E of 4–8 independent experiments. Legend: *p<0.05 vs no additions, †p<0.05 for the effect of BTP by paired t-test.

Figure 1

Activation of eNOS by black tea polyphenols involves estrogen receptors. (A) BAECs in HEPES-buffered PSS were incubated with or without the estrogen receptor antagonist ICI 182,780 (100 nM) followed by incubation with 100ng/ml black tea polyphenols (BTP) for 15 min. Cells were then lysed, eNOS immunoprecipitated, and its phosphorylation status at Ser-1179 determined by immunoblotting with antibodies against phosphorylated or total eNOS as indicated. (B) BAECs were incubated as in (A) and eNOS catalytic activity estimated by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” (C) HUVECs were incubated without (CTL) or with siRNA directed against human ERα/ERβ (ER siRNA) or its scrambled control (CTL siRNA) for 72 hours before treatment with BTP as in (A). The expression of ERα and eNOS phosphorylation were determined by immunoblotting with the indicated antibodies. (D) HUVECs treated as in (C) were assessed for eNOS catalytic activity by the conversion of L[³H]arginine to L-[³H]citrulline as in “Methods.” All immunoblots are representative of 3 independent experiments and composite data are mean ± S.E of 4–8 independent experiments. Legend: *p<0.05 vs no additions, †p<0.05 for the effect of BTP by paired t-test.

Figure 2

Black tea polyphenols induce ERα phosphorylation. (A) BAECs in HEPES-buffered PSS were incubated with 100 ng/ml black tea polyphenols for the indicated time, lysed, and ERα Ser-118 phosphorylation determined by immunoblotting with antibodies specific for the indicated epitopes. (B) BAECs were incubated with or without ICI 182,780 or BTP as in Fig. 1A. Cells were then lysed and ERα Ser-118 phosphorylation determined by immunoblotting with antibodies against the indicated epitopes. All immunoblots are representative of 3 independent experiments.

Figure 3

Black tea polyphenol-induced eNOS activation involves p38 MAPK. (A) BAECs in HEPES-buffered PSS were incubated with 100 ng/ml black tea polyphenols (BTP) for the indicated time. Cells were then lysed and immunoblotted with antibodies against the phosphorylated (activated) forms of p38 MAPK, ERK1/2 and JNK as indicated. Loading was assessed by immunoblots for total p38 MAPK. (B) BAECs were incubated for 30 min without (CTL) or with inhibitors for MEK1 (PD98059; 20μM), p38 MAPK (SB203580; 5μM), or JNK (SP600125; 10μM) as indicated. Cells were then treated with BTP (100ng/ml; 15min) and eNOS catalytic activity determined as the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” Immunoblots are representative of 3 independent experiments and composite data are mean ± S.E. Legend: *p<0.05 vs corresponding treatment without BTP.

Figure 4

Black tea polyphenol-induced ERα phosphorylation involves p38 MAPK. (A) BAECs were incubated for 30 min with or without the p38 MAPK inhibitor SB203580 (5μM). Cells were then treated with black tea polyphenols (100ng/ml; 15min) or vehicle (CTL), lysed and both Ser-118 phosphorylation and expression of ERα determined with antibodies directed against the indicated epitopes. Activity of p38 MAPK was determined by immunoblotting with antibodies for the phosphorylated (activated) form of the p38 MAPK downstream target, mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK-2). (B) BAECs were transfected with adenoviral vectors expressing LacZ, a dominant-negative p38α mutant (dn p38α), or a constitutively-active MKK6 mutant (MKK6bE) for 24h. Cells were then equilibrated in HEPES-PSS (30 min) followed by treatment with black tea polyphenols (BTP; 100ng/ml) or its vehicle for 15 min. Cells were then lysed and the lysates either collected or immunoprecipitated with ERα or total eNOS antibody. Lysates were probed with antibodies against the indicated epitopes. Cell lysates were also immunoblotted for Flag and hemagluttinin (HA) to assess expression of dn p38α and MKK6bE, respectively. (C) BAECs treated as in B underwent assessment of eNOS catalytic activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” All blots are representative of 3 independent experiments and composite data are mean ± S.E derived from of 4–8 independent experiments. Legend: *p<0.05 vs LacZ without BTP treatment.

Figure 4

Black tea polyphenol-induced ERα phosphorylation involves p38 MAPK. (A) BAECs were incubated for 30 min with or without the p38 MAPK inhibitor SB203580 (5μM). Cells were then treated with black tea polyphenols (100ng/ml; 15min) or vehicle (CTL), lysed and both Ser-118 phosphorylation and expression of ERα determined with antibodies directed against the indicated epitopes. Activity of p38 MAPK was determined by immunoblotting with antibodies for the phosphorylated (activated) form of the p38 MAPK downstream target, mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK-2). (B) BAECs were transfected with adenoviral vectors expressing LacZ, a dominant-negative p38α mutant (dn p38α), or a constitutively-active MKK6 mutant (MKK6bE) for 24h. Cells were then equilibrated in HEPES-PSS (30 min) followed by treatment with black tea polyphenols (BTP; 100ng/ml) or its vehicle for 15 min. Cells were then lysed and the lysates either collected or immunoprecipitated with ERα or total eNOS antibody. Lysates were probed with antibodies against the indicated epitopes. Cell lysates were also immunoblotted for Flag and hemagluttinin (HA) to assess expression of dn p38α and MKK6bE, respectively. (C) BAECs treated as in B underwent assessment of eNOS catalytic activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” All blots are representative of 3 independent experiments and composite data are mean ± S.E derived from of 4–8 independent experiments. Legend: *p<0.05 vs LacZ without BTP treatment.

Figure 5

ERα Ser-118 phosphorylation links p38 MAPK to Akt activation. (A) BAECs were transfected with adenoviral vectors expressing LacZ, a constitutively-active MKK6 mutant (MKK6bE) or constitutively-active Akt (Myr-Akt) for 24h. Cells were then equilibrated in HEPES-PSS (30 min) in the presence or absence of ICI 172,780 (100nM) as indicated. Cells were then lysed and the lysates either collected or immunoprecipitated with ERα or eNOS antibody. The phosphorylation status of eNOS or ERα on were assessed in their respective immunoprecipitates by immunoblotting for the indicated epitopes. Lysates were probed for phosphorylation of p38MAPK and Akt as indicated and the expression of MKK6bE or Myr-Akt was estimated by the presence of an HA tag or phosphorylated glycogen synthase kinase 3 (P-GSK3). (B) BAECs treated as in (A) underwent assessment of eNOS catalytic activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” (C) HUVECS were subjected to siRNA-mediated ER gene silencing prior to treatment with 100 ng/ml black tea polyphenols (BTP) and then assessed for phosphorylation of p38 MAPK and eNOS as in “Methods.” All blots are representative of 3 independent experiments and composite data are mean ± S.E derived from of 3 independent experiments. Legend: *p<0.05 vs LacZ; ^†P<0.05 vs MKK6bE alone .

Figure 5

ERα Ser-118 phosphorylation links p38 MAPK to Akt activation. (A) BAECs were transfected with adenoviral vectors expressing LacZ, a constitutively-active MKK6 mutant (MKK6bE) or constitutively-active Akt (Myr-Akt) for 24h. Cells were then equilibrated in HEPES-PSS (30 min) in the presence or absence of ICI 172,780 (100nM) as indicated. Cells were then lysed and the lysates either collected or immunoprecipitated with ERα or eNOS antibody. The phosphorylation status of eNOS or ERα on were assessed in their respective immunoprecipitates by immunoblotting for the indicated epitopes. Lysates were probed for phosphorylation of p38MAPK and Akt as indicated and the expression of MKK6bE or Myr-Akt was estimated by the presence of an HA tag or phosphorylated glycogen synthase kinase 3 (P-GSK3). (B) BAECs treated as in (A) underwent assessment of eNOS catalytic activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” (C) HUVECS were subjected to siRNA-mediated ER gene silencing prior to treatment with 100 ng/ml black tea polyphenols (BTP) and then assessed for phosphorylation of p38 MAPK and eNOS as in “Methods.” All blots are representative of 3 independent experiments and composite data are mean ± S.E derived from of 3 independent experiments. Legend: *p<0.05 vs LacZ; ^†P<0.05 vs MKK6bE alone .

Figure 5

ERα Ser-118 phosphorylation links p38 MAPK to Akt activation. (A) BAECs were transfected with adenoviral vectors expressing LacZ, a constitutively-active MKK6 mutant (MKK6bE) or constitutively-active Akt (Myr-Akt) for 24h. Cells were then equilibrated in HEPES-PSS (30 min) in the presence or absence of ICI 172,780 (100nM) as indicated. Cells were then lysed and the lysates either collected or immunoprecipitated with ERα or eNOS antibody. The phosphorylation status of eNOS or ERα on were assessed in their respective immunoprecipitates by immunoblotting for the indicated epitopes. Lysates were probed for phosphorylation of p38MAPK and Akt as indicated and the expression of MKK6bE or Myr-Akt was estimated by the presence of an HA tag or phosphorylated glycogen synthase kinase 3 (P-GSK3). (B) BAECs treated as in (A) underwent assessment of eNOS catalytic activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline as in “Methods.” (C) HUVECS were subjected to siRNA-mediated ER gene silencing prior to treatment with 100 ng/ml black tea polyphenols (BTP) and then assessed for phosphorylation of p38 MAPK and eNOS as in “Methods.” All blots are representative of 3 independent experiments and composite data are mean ± S.E derived from of 3 independent experiments. Legend: *p<0.05 vs LacZ; ^†P<0.05 vs MKK6bE alone .

Figure 6

ERα mediates black tea polyphenol-induced eNOS activation in COS-7cells. (A) COS-7 cells were transfected with plasmids expressing eNOS and ERα either alone or in combination. After 36hr, cells were equilibrated in HEPES-PSS (30 min) followed by incubation with buffer alone, black tea polyphenols (BTP; 100ng/ml), estradiol (100nM) or A23187 (1μM) for 15 min. Cells were then assessed for eNOS catalytic activity as described in “Methods.” Cell lysates were probed for eNOS and ERα expression by immunoblotting. (B) COS-7 cells were co-transfected with eNOS and either wild-type (ERwt) or s118a mutant ERα (ERs118a) as in (A). After 36hr, cells were assessed for eNOS catalytic activity as in (A). Blots are representative of 3 independent experiments and composite data are the mean ± S.E of 6–8 independent experiments expressed as the fold-increase compared to vehicle alone. Legend: *p<0.05 vs no additions, †p<0.05 vs ERwt treated with BTP.

Figure 6

ERα mediates black tea polyphenol-induced eNOS activation in COS-7cells. (A) COS-7 cells were transfected with plasmids expressing eNOS and ERα either alone or in combination. After 36hr, cells were equilibrated in HEPES-PSS (30 min) followed by incubation with buffer alone, black tea polyphenols (BTP; 100ng/ml), estradiol (100nM) or A23187 (1μM) for 15 min. Cells were then assessed for eNOS catalytic activity as described in “Methods.” Cell lysates were probed for eNOS and ERα expression by immunoblotting. (B) COS-7 cells were co-transfected with eNOS and either wild-type (ERwt) or s118a mutant ERα (ERs118a) as in (A). After 36hr, cells were assessed for eNOS catalytic activity as in (A). Blots are representative of 3 independent experiments and composite data are the mean ± S.E of 6–8 independent experiments expressed as the fold-increase compared to vehicle alone. Legend: *p<0.05 vs no additions, †p<0.05 vs ERwt treated with BTP.

Figure 7

Functional association of p38 MAPK and ERα. (A) COS-7 cells co-transfected with eNOS and ERα were treated with black tea polyphenols (BTP; 100ng/ml) for the indicated times. After treatment, cells were lysed, ERα immunoprecipitated, and both the pellet (PLT) and supernatant (SNT) subjected to immunoblotting as indicated. (B) Cells were treated as in (A) except p38 MAPK and both the pellet and supernatant immunoblotted with antibodies to the indicated epitopes. (C) COS-7 cells were co-transfected with eNOS and mutant ERα (ERαs118a), treated with BTP (100mg/ml) for the indicated times, and subjected to immunoprecipitation and immunoblotting as in (A). (D) COS-7 cells were co-transfected and treated with BTP as in (C) followed by immunoprecipitation and immunoblotting as in (B). All blots are representative of 3 independent experiments.

Figure 7

Functional association of p38 MAPK and ERα. (A) COS-7 cells co-transfected with eNOS and ERα were treated with black tea polyphenols (BTP; 100ng/ml) for the indicated times. After treatment, cells were lysed, ERα immunoprecipitated, and both the pellet (PLT) and supernatant (SNT) subjected to immunoblotting as indicated. (B) Cells were treated as in (A) except p38 MAPK and both the pellet and supernatant immunoblotted with antibodies to the indicated epitopes. (C) COS-7 cells were co-transfected with eNOS and mutant ERα (ERαs118a), treated with BTP (100mg/ml) for the indicated times, and subjected to immunoprecipitation and immunoblotting as in (A). (D) COS-7 cells were co-transfected and treated with BTP as in (C) followed by immunoprecipitation and immunoblotting as in (B). All blots are representative of 3 independent experiments.