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Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-?.

ABSTRACT: Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17? (E2) rapidly stimulated protein SNO that maximized within 10-30?min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ER?, but not ER?, antagonists. A specific ER? but not ER? agonist was able to induce protein SNO. Overexpression of ER?, but not ER?, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ER? in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation.

SUBMITTER: Zhang HH

PROVIDER: S-EPMC3125455 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-β.

Zhang Hong-hai HH Feng Lin L Wang Wen W Magness Ronald R RR Chen Dong-bao DB

Journal of cellular physiology 20120101 1

Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-in ...[more]

PMID: 21374595

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Project description:Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5'-regulatory region of the sheep NOS3 gene.

Dataset Information

Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-?.

Publications

Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-β.

Similar Datasets

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