E2β stimulates ovine uterine artery endothelial cell H2S production in vitro by estrogen receptor-dependent upregulation of cystathionine β-synthase and cystathionine γ-lyase expression†

Abstract

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17β (E2β) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2β was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2β at 48-72 h. E2β also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2β stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERβ stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2β. Specific antagonist of either ERα or ERβ blocked E2β-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERβ agonists or their antagonists had no additive effects. Thus, E2β stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERβ-dependent CBS and CTH transcription in UAEC in vitro.

Keywords: endothelium; estrogen; estrogen receptors; hydrogen sulfide; uterine artery; vasodilation.

Figure 1.

Effects of E₂β on H₂S production—role of CBS, CTH, and ER. (A) Primary uterine artery endothelial cells (UAEC) were treated with vehicle or estradiol-17β (E₂β, 10 nM) with or without the estrogen receptor (ER) antagonist ICI 182780 (ICI, 1 μM) for 48 h. Protein extracts (1 × 10⁶ cells) were used to determine hydrogen sulfide (H₂S) production. (B) UAEC were treated with vehicle or 10 nM E₂β for 48 h, and protein extracts were used to determine H₂S production in the presence or absence of an inhibitor of cystathionine β-synthase (CBS, cystathionine γ-lyase (CTH), or CHH and BCA, respectively. Data (means ± SEM) were collected from different cell preparations cells prepared from three to five ewes. *** P < 0.001 vs control.

Figure 2.

Time course and concentration response of E₂β on mRNA and protein expressions of CBS and CTH. Primary uterine artery endothelial cells (UAEC) were treated with 10 nM estradiol-17β (E₂β) for up to 3 days to assess cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA (A) and protein (B), or with increasing concentrations of E₂β (0–1 μM) for 48 h to assess CBS and CTH mRNA (C) and protein (D). Data (means ± SEM) were collected from different cell preparations cells prepared from three to five ewes. Bars with different letters differ significantly (P < 0.05); capital and lower case letters pertain to CBS and CTH, respectively.

Figure 3.

ER-dependency of E₂β effects on mRNA and protein expressions of CBS and CTH. Primary uterine artery endothelial cells (UAECs) were treated with vehicle, 10 nM estradiol-17β (E₂β), 1 μM ICI 182 780 (ICI), or both for 48 h. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA (A) and protein (B) were determined. (C) Immunofluorescence labeling of CBS and CTH proteins. Nuclei were stained with DAPI (blue). Protein expression was determined by relative green fluorescence intensity as fold change of control. Data (means ± SEM) were collected from different cell preparations from three to five ewes. *** P < 0.001 vs. Control. Scale bar = 50 μm.

Figure 4.

Specific role of ERα or ERβ in mediating E₂β stimulation of mRNA and protein expressions of CBS and CTH. (A, B) ERα or ERβ activation: primary uterine artery endothelial cells (UAECs) were treated with vehicle, 10 nM of estradiol-17β (E₂β), PPT (ERα agonist), DPN (ERβ agonist), or PPT + DPN for 48 h. (C, D) ERα or ERβ inhibition: primary uterine artery endothelial cells (UAECs) were treated with vehicle or estradiol-17β (E₂β) (10 nM) with or without 1 μM MPP (ERα antagonist), PHTPP (ERβ antagonist), or MPP + PHTPP. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA (A, C) and protein (B, D) were determined. Data (means ± SEM) were collected from different cell preparations from three to five ewes. Bars with different letters differ significantly; capital and lower case letters pertain to CBS and CTH, respectively. *** P < 0.001 vs Control.

Figure 5.

ER-dependency of E₂β activation of CBS and CTH promoters. Human cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) promoter luciferase-reporter constructs were transfected into primary uterine artery endothelial cells (UAECs). Twenty-four hours later, the cells were treated with vehicle or estradiol-17β (E₂β, 10 nM) with or without estrogen receptor (ER) antagonist ICI 182780 (ICI, 1 μM) for 24 h. Luciferase activity was determined as an index for promoter action. Data (means ± SEM) were collected from cells prepared from three to five ewes. *** P < 0.001 vs Control.