Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells.
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 downregulate 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.
Ming
Xiu-Fen
XF
Vascular Biology, Institute of Physiology, University of Fribourg, CH-1700 Fribourg, Switzerland.
Viswambharan
Hema
H
Barandier
Christine
C
Ruffieux
Jean
J
Kaibuchi
Kozo
K
Rusconi
Sandro
S
Yang
Zhihong
Z
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000730:Androstadienes / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin / Q000378:metabolism; D000077191:Wortmannin; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases
D000730:Androstadienes; D047908:Intracellular Signaling Peptides and Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin; D000077191:Wortmannin; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; C496314:NOS3 protein, human; C494918:AKT1 protein, human