Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

Abstract

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.Endothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial nitric oxide synthase (eNOS), plays a critical role in the regulation of vascular tone and the maintenance of vascular integrity by modulating various processes. It promotes vasodilatation, inhibits platelet activation and prevents vascular smooth muscle cell proliferation and/or migration (4). Abnormalities in eNOS gene expression or activation, which result in decreased NO production, are thought to contribute to the pathogenesis of various cardiovascular disorders such as atherosclerosis and hypertension (49).eNOS-mediated NO generation is a highly regulated cellular event. The mechanisms controlling eNOS activity involve multiple regulatory steps including gene expression, co-and posttranslational modification, intracellular localization, cofactors and phosphorylation (20). Although eNOS was originally described as a constitutive enzyme with enzyme activation achieved via calmodulin (CaM) binding in response to increased Ca 2ϩ , recent studies indicate that a variety of stimuli can modulate its expression at the transcriptional (15) and/or posttranscriptional level (6). Moreover, it is evidenced that phosphorylation of eNOS also modulates eNOS activity independent of Ca 2ϩ /CaM. Studies showed that Ser-1177 of human eNOS (Ser-1179 of bovine sequence) is phosphorylated directly by protein kinase B (PKB)/Akt (10, 18, 37), which results in an increase in electron flux through the reductase domain and increase in NO production (36). Shear stress and insulin have been shown to activate eNOS through the activation of PKB (14,41). eNOS phosphorylation at Ser-1177 by PKB therefore represents another important regulatory mechanism of eNOS activation in addition to Ca 2ϩ /CaM-depe...

Increased expression of the nitric oxide synthase gene and protein in corpus cavernosum by repeated dosing of udenafil in a rat model of chemical diabetogenesis

“…Interestingly, glucose-induced elevation of protein kinase C seems to be mediated by oxidative stress in rabbit CC smooth muscle cells [21]. Furthermore, Rho-kinase protein expression is increased in diabetic corporal tissue, and the RhoA/Rho-kinase signalling pathway plays an important role in the reduction of NO synthesis through eNOS suppression [22,23]. In the diabetic rat penile crura, ICP is markedly decreased, and the gene expression levels of apoptotic factors, such as Bak and Bax, are increased [24].…”

Endothelial dysfunction and hypertension in aging

“…105 Activation of the RhoA/ROCK pathway impairs NO bioavailability through inhibition of eNOS mRNA stability, eNOS posphorylation at Ser 1177 and the Akt/PI3K pathway and enhancement of eNOS phosphorylation at Thr495. [106][107][108] Endothelial function and hypertension in aging Y Higashi et al Several investigators have shown an interaction between the RhoA/ ROCk pathway and ROS. 109,110 Indeed, ROS induced by hyperglycemia enhances ROCK activity, leading to atherothrombogenesis through an increase in expression of plasminogen activator inhibitor-1 in vascular endothelial cells.…”

Nitric Oxide: Considerations for the Treatment of Ischemic Stroke

“…Rho kinase is implicated in the pathophysiology of atherosclerosis (Miyata et al, 2000), myocardial infarction, and hypertension (Uehata et al, 1997). Activation of Rho kinase in endothelial cells under hypoxic conditions (Wolfrum et al, 2004) has been related to the mechanism responsible for the downregulation of eNOS during and after ischemia (Takemoto et al, 2002;Ming et al, 2002;Wolfrum et al, 2004;Jin et al, 2006).…”