In vivo and in vitro evidence for ERβ regulation of G protein-coupled estrogen receptor (GPER) expression in normal women and those with endometriosis

Abstract

OBJECTIVE: GPER, also known as GPR30, is a membrane-bound estrogen receptor that stimulates multiple signaling pathways. Previous work in our laboratory has demonstrated expression of GPER in normal human endometrial epithelium, with highest expression in the proliferative phase, suggesting regulation by estradiol in vivo. We also observed estradiol induction of GPER in vitro. The aims of this study were to evaluate the receptors conferring estrogen regulation as well as study patterns of expression in women with endometriosis. DESIGN: Laboratory study. MATERIALS AND METHODS: Urinary LH-surge timed endometrial specimens were obtained from healthy volunteers. Matched samples of eutopic and ectopic endometrium from women with endometriosis were collected and menstrual cycle stage was assigned using Noyes criteria. Real time quantitative RT-PCR analysis of GPER mRNA expression was performed using Taqman® probes for GPER and a constitutive control (PPIA). Estrogen receptor (ER) selective agonists were utilized to determine the role of each ER on GPER mRNA expression. Immunohistochemistry utilized a GPER C-terminal rabbit polyclonal antibody (Eric Prossnitz, U. New Mexico) and a mouse monoclonal ERβ antibody (NovoCastra). ECC-1 endometrial epithelial cell line was used for in vitro studies. RESULTS: In ECC-1 cells, GPER mRNA expression was stimulated by treatment with estradiol and an ERβ-selective ligand, but not by agonists selective for ERα or GPER. In normal endometrium from the proliferative phase, strong immunostaining for GPER was seen in the epithelium and stroma. In the early secretory phase, the staining intensity in the epithelium was decreased, but the stromal staining remained strong. In the mid-secretory phase, stromal staining persisted, but staining was largely absent in the glandular epithelium. In the late secretory phase, minimal epithelial staining and moderate stromal staining was seen. In eutopic and ectopic endometrium from patients with endometriosis, immunostaining for GPER and ERβ closely correlated. Unlike normal endometrium, proliferative eutopic and ectopic endometriosis samples exhibited largely absent epithelial staining for both GPER and ERβ, but endothelium remained positive for both GPER and ERβ. CONCLUSIONS: 1. Estradiol induces GPER mRNA expression in ECC-1 cells and this effect is mediated through ERβ. 2. ERβ staining is strongly correlated with GPER staining. 3. GPER expression appears to be aberrant in patients with endometriosis.