Role of GPER in estrogen-dependent nitric oxide formation and vasodilation

Abstract

Estrogens are potent regulators of vasomotor tone, yet underlying receptor- and ligand-specific signaling pathways remain poorly characterized. The primary physiological estrogen 17β-estradiol (E2), a non-selective agonist of classical nuclear estrogen receptors (ERα and ERβ) as well as the G protein-coupled estrogen receptor (GPER), stimulates formation of the vasodilator nitric oxide (NO) in endothelial cells. Here, we studied the contribution of GPER signaling in E2-dependent activation of endothelial NO formation and subsequent vasodilation. Employing E2 and the GPER-selective agonist G-1, we investigated eNOS phosphorylation and NO formation in human endothelial cells, and endothelium-dependent vasodilation in the aortae of wild-type and Gper-deficient mice. Both E2 and G-1 induced phosphorylation of eNOS at the activation site Ser1177 to similar extents. Endothelial NO production to E2 was comparable to that of G-1, and was substantially reduced after pharmacological inhibition of GPER. Similarly, the clinically used ER-targeting drugs 4OH-tamoxifen, raloxifene, and ICI182,780 (faslodex, fulvestrant™) induced NO formation in part via GPER. We identified c-Src, EGFR, PI3K and ERK signaling pathways to be involved in GPER-dependent NO formation. In line with activation of NO formation in cells, E2 and G-1 induced equally potent vasodilation in the aorta of wild-type mice. Gper deletion completely abrogated the vasodilator response to G-1, while reducing the response to E2 by ∼50%. These findings indicate that a substantial portion of E2-induced endothelium-dependent vasodilation and NO formation is mediated by GPER. Thus, selective targeting of vascular GPER may be a suitable approach to activate the endothelial NO pathway, possibly leading to reduced vasomotor tone and inhibition of atherosclerotic vascular disease.

Keywords: Endothelium; Estrogen; GPER; GPR30; NO; SERD; SERM; Vascular; Vasodilation; eNOS.

Figure 1. GPER is expressed on intracellular membranes in human endothelial cells

The donor sex of TIVE cells was determined to be male by FISH analysis for X (red) and Y (green) chromosomes (A). GPER (white bars) and eNOS (black bars) expression in TIVE cells was determined by qPCR at the indicated passages (B). Gene expression of ERα and GPER was similar between TIVE and HUVEC at passage 3 (C). TIVE cells were stained by immunofluorescence for GPER (green) under non-permeabilizing (D) or permeabilizing (E) conditions demonstrating intracellular expression (staining only under permeabilizing conditions) of GPER. Cells were counterstained with nuclear DAPI (blue). Immunofluorescence of ERα (green) and GPER (red) indicate predominantly cytosolic localization for GPER and predominantly nuclear localization for ERα in both HUVEC (F) and TIVE (G) cells. Data were analyzed by one-way ANOVA with repeated measures followed by Bonferroni’s post-hoc test (B) or Student’s t-test (C) and graphed as mean±s.e.m.; *P<0.01 vs. passage 3.

Figure 2. GPER stimulates eNOS phosphorylation

Endothelial cells were treated with the GPER-selective agonist G-1 (1, 10, and 100 nM) or E2 (100 nM) and blotted for eNOS phosphorylation at activation residue Ser1177. Data (n=4–7) were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test and graphed as mean±s.e.m.; *P<0.05 vs. vehicle (Veh, DMSO 0.01%).

Figure 3. Selective and non-selective GPER activation mediates NO formation

Endothelial cells were treated with vehicle (Veh, DMSO 0.01%) or the GPER-selective antagonist G36 (1 μM) prior to stimulation with the GPER-selective agonist G-1, the non-selective ER agonist E2, the SERMs 4OH-tamoxifen (4-OHT) and raloxifene (Ralox), or the SERD ICI182,780 (ICI, 100 nM each). For comparison, the response to the muscarinic M₃ receptor agonist acetylcholine (ACh, 100 nM) is shown. NO formation was determined through the detection of stable NO metabolites NO₂⁻/NO₃⁻. Data (n=3–8) were analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test and graphed as mean±s.e.m.; *P<0.01 vs. vehicle, ^†P<0.05 vs. E2, ^‡P<0.05 vs. without G36.

Figure 4. GPER contributes to NO production via multiple signaling pathways

NO formation in endothelial cells was induced by the GPER-selective agonist G-1 or the non-selective ER agonist E2 (100 nM each). Cells were pretreated with multiple inhibitors of GPER signaling components that are known upstream activators of eNOS: PP2 for c-Src (A), AG1478 (AG) for EGFR (B), LY294002 (LY) for PI3K (C), and PD98059 (PD) for ERK1/2 (D). Data (n=4–6) were analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test and graphed as mean±s.e.m.; *P<0.01 vs. vehicle (Veh, DMSO 0.01%), ^†P<0.05 vs. E2, ^‡P<0.05 vs. without inhibitor.

Figure 5. GPER partially mediates endothelium-dependent vasodilation to E2

Direct vasodilator responses to the non-selective ER agonist E2 and the GPER-selective agonist G-1 (3 μM each) were induced in the aorta from wild-type (Gper^+/+) and GPER-deficient (Gper^−/−) mice (A). For comparison, ER-independent vasodilation to the muscarinic M₃ receptor agonist acetylcholine (ACh,1 μM) is shown (B). Data (n=3–8) were analyzed by two-way ANOVA with repeated measures followed by Bonferroni’s post-hoc test and graphed as mean±s.e.m.; *P<0.001 vs. vehicle (CTL, EtOH 0.1%), ^†P<0.001 vs. Gper^+/+. PGF_2α, prostaglandin F_2α; PE, phenylephrine.