Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.
17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.
Haynes
M P
MP
Division of Cardiovascular Medicine and Molecular Cardiobiology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812, USA.
Sinha
D
D
Russell
K S
KS
Collinge
M
M
Fulton
D
D
Morales-Ruiz
M
M
Sessa
W C
WC
Bender
J R
JR
D000256:Adenoviridae / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D014161:Transduction, Genetic
D002867:Chromones; D004791:Enzyme Inhibitors; D004958:Estradiol; D009025:Morpholines; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011960:Receptors, Estrogen; D012710:Serum Albumin, Bovine; C043090:estradiol-bovine serum albumin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496314:NOS3 protein, human; C494918:AKT1 protein, human