Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen
Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen
复制标题
DOI：
10.1074/jbc.m210828200
复制
发表时间：
2003-01-24
影响因子：
4.8
通讯作者：
Bender, JR
中科院分区：
文献类型：
--
作者：
Haynes, MP;Li, L;Bender, JR
17β-雌二醇激活内皮一氧化氮合酶 (eNOS)，通过磷脂酰肌醇 3-激酶 (PI3-激酶)/Akt 途径增强内皮细胞释放一氧化氮 (NO)。该途径的上游调节因子尚不清楚。我们现在证明 17β-雌二醇通过人内皮细胞中的 Src 激酶快速激活 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，但不会消除离子霉素刺激的 NO 释放。与这些结果一致，PP2 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 消除了 17β-雌二醇诱导的 PI3 激酶激活，表明 PP2 抑制激酶位于 PI3 激酶和 Akt 的上游。 17β-雌二醇诱导的雌激素受体/c-Src 关联与快速 c-Src 磷酸化相关。此外，转染激酶死亡的 c-Src 会抑制 17β-雌二醇诱导的 Akt 磷酸化，而组成型活性 c-Src 会增加基础 Akt 磷酸化。雌激素对 c-src、fyn 和 yes 基因纯合缺失的小鼠胚胎成纤维细胞的刺激未能诱导 Akt 磷酸化，而维持 c-Src 表达的细胞则显示出雌激素诱导的 Akt 激活。雌激素快速激活 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 的连续激活，从而增强 NO 释放，表明 c-Src 是雌激素刺激的 PI3 激酶/Akt/eNOS 途径的关键上游调节因子。
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.