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Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells
Xiu-Fen Ming,1,2 Hema Viswambharan,1 Christine Barandier,1 Jean Ruffieux,1 Kozo Kaibuchi,3 Sandro Rusconi,2 and Zhihong Yang1*
Vascular Biology, Institute of Physiology,1
Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland,2
Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya, Aichi 466-8500, Japan3
Received for publication 27 February 2002.
Accepted for publication 11 September 2002.
Abstract:
Endothelial nitric oxide synthase (eNOS) is an important regulatorof cardiovascular homeostasis by production of nitric oxide(NO) from vascular endothelial cells. It can be activated byprotein 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. Usingadenovirus-mediated gene transfer in human umbilical vein endothelialcells (HUVECs), we show here that both active RhoA and ROCKnot only downregulate eNOS gene expression as reported previouslybut also inhibit eNOS phosphorylation at Ser-1177 and cellularNO production with concomitant suppression of PKB activation.Moreover, coexpression of a constitutive active form of PKBrestores the phosphorylation but not gene expression of eNOSin the presence of active RhoA. Furthermore, we show that thrombininhibits eNOS phosphorylation, as well as expression via Rho/ROCKpathway. Expression of the active PKB reverses eNOS phosphorylationbut has no effect on downregulation of eNOS expression inducedby thrombin. Taken together, these data demonstrate that Rho/ROCKpathway negatively regulates eNOS phosphorylation through inhibitionof PKB, whereas it downregulates eNOS expression independentof PKB.
* Corresponding author. Mailing address: Vascular Biology, Institute of Physiology, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland. Phone: 41-26-300-85-93. Fax: 41-26-300-96-36. E-mail: zhihong.yang{at}unifr.ch.
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