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J. Biol. Chem. 278 (4): 2118-2123

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Src Kinase Mediates Phosphatidylinositol 3-Kinase/Akt-dependent Rapid Endothelial Nitric-oxide Synthase Activation by Estrogen*

M. Page HaynesDagger §, Lei LiDagger §, Diviya SinhaDagger §, Kerry S. RussellDagger §, Koji HisamotoDagger §, Roland Baron, Mark CollingeDagger §, William C. Sessa||§, and Jeffrey R. BenderDagger §**

From the Dagger  Sections of Cardiovascular Medicine and Immunobiology, Departments of || Pharmacology,  Cell Biology and Orthopedics, and the § Vascular Biology and Transplantation Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536

17beta -Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta -estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta -estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta -estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta -estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta -estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta -estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.

* This work was supported in part by the National Institute of Health Grant HL61782 (to J. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: 295 Congress Ave, New Haven, CT 06536. Tel.: 203-737-2223; Fax: 203-737-2293; E-mail:

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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