Dataset Information

Glucocorticoid response elements and 11 beta-hydroxysteroid dehydrogenases in the regulation of endothelial nitric oxide synthase expression.

ABSTRACT: Hypertensive and other effects of excess glucocorticoids might be in part mediated by the suppression of endothelial nitric oxide synthase (eNOS) expression. We studied the transcriptional and biochemical mechanisms that mediate or modulate the suppression of eNOS expression by glucocorticoids.We found that a mere three-fold increase in the concentration of the natural glucocorticoid cortisol (from 30 to 100 nmol/L) significantly decreased the expression level of eNOS in human endothelial cells. Deletion analysis of the eNOS promoter indicated that the segment within -119 bp upstream from the transcription start site was significantly involved in the effect of cortisol. Site-directed mutagenesis and chromatin immunoprecipitation analyses demonstrated the presence of a suppressive glucocorticoid response element (GRE) at -111 to -105 bp. 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) catalyse the interconversion of active and inactive glucocorticoids. The suppression of 11 beta-HSD2 using small interfering RNA markedly exacerbated the inhibition of eNOS by cortisol. The suppression of 11 beta-HSD1 abolished the inhibition of eNOS expression by cortisol.We identified the first GRE in the eNOS promoter region and demonstrated that endogenous 11 beta-HSD1 and 11 beta-HSD2 play significant and distinct roles in modulating the effect of glucocorticoids on eNOS expression.

SUBMITTER: Liu Y

PROVIDER: S-EPMC2639091 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Previous studies have demonstrated that G protein-coupled receptor kinase interacting-1 protein (GIT1) associates with endothelial nitric oxide synthase (eNOS) to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly associated binding partner, β-PIX (p21-activated kinase-interacting exchange factor β, ARHGEF7) is specifically important in the regulation of eNOS activity. We examined β-PIX expression in normal rat liver by immunohistochemistry and explored β-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of β-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and β-PIX required to regulate eNOS activity were mapped. β-PIX was expressed primarily in SECs in normal liver and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, and hepatocytes). β-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs and was important in stimulating eNOS activity. Of note, GIT1-β-PIX interaction led to synergistic enhancement of eNOS activity, and β-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of β-PIX or GIT1 in normal SECs using β-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains [Spa2 homology domain (SHD) and synaptic localization domain (SLD), aa 331-596] and the β-PIX COOH terminal (aa 496-555) appeared to be critical in the regulation eNOS activity. The data indicate that β-PIX regulates eNOS phosphorylation and function in normal SECs and highlight the importance of the GIT1/β-PIX/eNOS trimolecular complex in normal liver SEC function.NEW & NOTEWORTHY β-PIX is a multidomain protein known to be a GIT1 binding partner. We report here that in the normal liver, the distribution and cellular localization of β-PIX are restricted largely to sinusoidal endothelial cells. Furthermore, β-PIX interacts with eNOS and GIT1 promotes eNOS activity and NO production and therefore exerts a novel posttranslational regulatory function on eNOS activity in sinusoidal endothelial cells. We also have identified specific molecular domains important in GIT1 and β-PIX's interaction with eNOS, which may represent novel therapeutic targets in the control of sinusoidal blood flow and intrahepatic resistance.

Dataset Information

Glucocorticoid response elements and 11 beta-hydroxysteroid dehydrogenases in the regulation of endothelial nitric oxide synthase expression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets