Haynes M Page, Li Lei, Sinha Diviya, Russell Kerry S, Hisamoto Koji, Baron Roland, Collinge Mark, Sessa William C, Bender Jeffrey R
Section of Cardiovascular Medicine, Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
J Biol Chem. 2003 Jan 24;278(4):2118-23. doi: 10.1074/jbc.M210828200. Epub 2002 Nov 12.
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.
17β-雌二醇可激活内皮型一氧化氮合酶（eNOS），通过磷脂酰肌醇3激酶（PI3激酶）/Akt信号通路增强内皮细胞释放一氧化氮（NO）。该信号通路的上游调节因子尚不清楚。我们现在证明，17β-雌二醇可通过Src激酶在人内皮细胞中快速激活eNOS。Src家族激酶特异性抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP2）可消除17β-雌二醇刺激而非离子霉素刺激引起的NO释放。与这些结果一致，PP2可阻断17β-雌二醇诱导的Akt磷酸化，但不抑制用组成型活性Akt转导的细胞释放NO。PP2可消除17β-雌二醇诱导的PI3激酶激活，表明PP2可抑制的激酶位于PI3激酶和Akt的上游。17β-雌二醇诱导的雌激素受体/c-Src结合与c-Src的快速磷酸化相关。此外，转染激酶失活的c-Src可抑制17β-雌二醇诱导的Akt磷酸化，而组成型活性c-Src可增加基础Akt磷酸化。用c-src、fyn和yes基因纯合缺失的小鼠胚胎成纤维细胞进行雌激素刺激未能诱导Akt磷酸化，而维持c-Src表达的细胞则表现出雌激素诱导的Akt激活。雌激素可快速激活c-Src，诱导雌激素受体、c-Src和P85（PI3激酶调节亚基）复合物形成。这种复合物的形成导致PI3激酶、Akt和eNOS的相继激活，从而增强NO释放，表明c-Src是雌激素刺激的PI3激酶/Akt/eNOS信号通路的关键上游调节因子。
