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

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 alpha on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ER alpha on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38 alpha MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ER alpha phosphorylation and downstream activation of Akt, and eNOS. The key role of ER alpha Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ER alpha subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ER alpha with p38 MAPK as ER alpha 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 alpha and these data uncover a new mechanism of ER alpha activation that has broad implications for NO bioactivity and endothelial cell phenotype.