ERβ regulates ERα expression and response to estradiol via specific promoters in endometrium and endometriosis

Abstract

Gene expression in endometrium and its pathological counterpart endometriosis is highly regulated by estradiol (E2) and its nuclear receptors ERα and ERβ. The human ERα gene is regulated by a number of alternative promoters including 3 major ones - A, B and C, which are used in part in a tissue-specific manner. Although E2 regulates ERα expression in endometrium and endometriosis the underlying mechanism is unknown. ERα to ERβ ratio is strikingly higher in endometrial vs. endometriotic stromal cells due to elevated ERβ and lower ERα levels in endometriotic cells. We investigated how E2 and ERβ regulate ERα expression in endometrial and endometriotic stromal cells. We used primary stromal cells in culture from ovarian endometrioma walls (n = 3) and eutopic endometrium from disease-free woman (n = 3) to conduct experiments. Isolated endometrial and endometriotic stromal cells in primary culture were treated with E2 (10−7M) or vehicle for 15 min, 30 min, 1, 3, 6, 12 or 24 h. Real-time PCR employing exon-specific primers was used to quantify total and promoter (A, B, or C) - specific ERα mRNA levels. ERβ was selectively knocked down by siRNA. Westen analyses were used to determine ERα and ERβ protein levels. Real-time PCR employing exon-specific primers was used to quantify total and promoter (A, B, or C) – specific ERα mRNA levels in the presence of ERβ knockdown. Chromatin immunoprecipitation (ChIP) assays were performed to determine binding of ERβ to specific ERα promoter regions. Treatment with E2 regulated total ERα mRNA levels in a time-dependent manner in both endometrial and endometriotic stromal cells. Levels of ERα promoter A, B, or C-specific mRNA also showed comparable alterations in response to E2. ERβ knockdown gave rise to an induction of ERα mRNA by E2 in endometriosis that has high baseline ERβ levels. This was mediated primarily through promoters A and C of ERα gene. ChIP assays demonstrated ERβ binding to the promoters A and C but not promoter B of the ERα gene. E2 regulates ERα gene expression via its alternative promoters A, B and C in endometrial and endometriotic stromal cells. High ERβ levels in endometriotic cells might be responsible for the lack of E2-mediated induction of ERα expression via its promoters A and C. Based on the above results ERβ may serve as a potential target for medical treatment of endometriosis.