Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells
Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells
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DOI：
10.1128/mcb.22.24.8467-8477.2002
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发表时间：
2002-12-01
影响因子：
5.3
通讯作者：
Yang, ZH
中科院分区：
文献类型：
--
作者：
Ming, XF;Viswambharan, H;Yang, ZH
内皮一氧化氮合酶 (eNOS) 通过血管内皮细胞产生一氧化氮 (NO) 来调节心血管稳态。它可以通过 Ser-1177 磷酸化被蛋白激酶 B (PKB)/Akt 激活。我们对 Rho GTPase/Rho 激酶 (ROCK) 通路在 eNOS 表达和激活调节中的作用感兴趣。在人脐静脉内皮细胞 (HUVEC) 中使用腺病毒介导的基因转移，我们在此表明，活性 RhoA 和 ROCK 不仅如先前报道的那样下调 eNOS 基因表达，而且还抑制 eNOS Ser-1177 磷酸化和细胞 NO 产生，同时抑制 PKB 激活。此外，在活性 RhoA 存在的情况下，共表达 PKB 的组成型活性形式可以恢复 eNOS 的磷酸化，但不能恢复基因表达。此外，我们发现凝血酶抑制 eNOS 磷酸化以及通过 Rho/ROCK 途径的表达。活性 PKB 的表达可逆转 eNOS 磷酸化，但对凝血酶诱导的 eNOS 表达下调没有影响。综上所述，这些数据表明 Rho/ROCK 途径通过抑制 PKB 负向调节 eNOS 磷酸化，而它不依赖于 PKB 下调 eNOS 表达。
Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only down-regulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.