ERβ- and prostaglandin E2-regulated pathways integrate cell proliferation via Ras-like and estrogen-regulated growth inhibitor in endometriosis

Abstract

In endometriosis, stromal and epithelial cells from the endometrium form extrauterine lesions and persist in response to estrogen (E2) and prostaglandin E2 (PGE2). Stromal cells produce excessive quantities of estrogen and PGE2 in a feed-forward manner. However, it is unknown how estrogen stimulates cell proliferation and survival for the establishment and persistence of disease. Previous studies suggest that estrogen receptor-β (ERβ) is strikingly overexpressed in endometriotic stromal cells. Thus, we integrated genome-wide ERβ binding data from previously published studies in breast cells and gene expression profiles in human endometriosis and endometrial tissues (total sample number = 81) and identified Ras-like, estrogen-regulated, growth inhibitor (RERG) as an ERβ target. Estradiol potently induced RERG mRNA and protein levels in primary endometriotic stromal cells. Chromatin immunoprecipitation demonstrated E2-induced enrichment of ERβ at the RERG promoter region. PGE2 via protein kinase A phosphorylated RERG and enhanced the nuclear translocation of RERG. RERG induced the proliferation of primary endometriotic cells. Overall, we demonstrated that E2/ERβ and PGE2 integrate at RERG, leading to increased endometriotic cell proliferation and represents a novel candidate for therapeutic intervention.

Figure 1.

RERG is an ERβ target gene in endometriosis. A, Gene expression profiling identified 624 genes that are differentially regulated in stromal cells isolated from NoEM and E-Osis tissues. B, Seventy genes differentially expressed in NoEM and E-Osis also contained an ERβ-binding site; ERβ-binding data were obtained from previously published ERβ ChIP-on-ChIP and ChIP-Seq studies performed in MCF7 cells. C, Quantitative RT-PCR demonstrating that RERG is overexpressed in E-Osis (n = 8) compared with NoEM stromal cells (n = 8). D, Immunoblot showing overexpression of RERG in E-Osis (n = 8) compared with NoEM stromal cells (n = 8). E, Densitometry analysis indicating that RERG is overexpressed in E-Osis. F, Representative immunohistochemical analysis of human NoEM and E-Osis endometrium demonstrating higher expression of RERG (red) in endometriotic tissues. Nuclei were stained with DAPI (blue). Images show hematoxylin and eosin staining imaged at a magnification of ×10 (size bar, 100 μm); insets show immunofluorescence imaged at a magnification of ×40 (size bar, 40 μm). *, P < .05, **, P < .001, ***, P < .0001, Student's t test.

Figure 2.

Estradiol, via ERβ, regulates the transcription of RERG. A, RERG mRNA expression levels increased in response to E2 in E-Osis stromal cells. B, RERG protein levels increased in response to E2 treatment in a time-dependent manner in E-Osis stromal cells. C, Densitometry analysis of RERG (n = 5). D, Pretreatment of E-Osis stromal cells with the RNA polymerase II inhibitor, ActD, blocked the E2-mediated increase of RERG. Cells were treated with vehicle (Veh; DMSO), E2 + Veh, or E2 + ActD. E, Immunoblot of RERG in E-Osis cells treated with E2, DPN, and PPT for 2 hours. F, Densitometry analysis (n = 5) of immunoblots showing E2, DPN, and PPT induction of RERG protein. H, NoEM stromal cells treated with E2, DPN, or PPT. I, Densitometry analysis of RERG expression (n = 3). *, P < .05; **, P < .001; ***, P < .0001, two-way ANOVA with Tukey's multiple comparison posttest.

Figure 3.

ERβ binds the RERG promoter region. A, ERβ ChIP was performed in E-Osis stromal cells (n = 3) treated with vehicle (Veh) or 10⁻⁷ E2 for 45 minutes. ChIP-qPCR was performed using primers spanning 2.5 kb upstream and downstream of the RERG transcription start sites. B, ERβ knockdown by siRNA (siESR2) confirmed RERG regulation by ERβ in the absence of E2 treatment. Densitometry analysis (n = 3) showed a significant decrease in ERβ (C) and RERG (D) expression after siRNA-mediated ERβ knockdown compared with controls (siCTL). E, Lentiviral-mediated ERβ overexpression using Lenti-ERβ (or Lenti-GFP as a control) in NoEM stromal cells showed a concordant increase in RERG. Densitometry analysis (n = 4) showing a significant increase in ERβ (F) and RERG (G) after pLenti-ERβ overexpression. *, P < .05; **, P < .001; ***, P < .0001; Student's t test or two-way ANOVA with Tukey's multiple comparison posttest.

Figure 4.

E2 and PGE2 induce RERG expression nuclear translocation of RERG. A, RERG expression in E-Osis stromal cells treated with vehicle (Veh; DMSO), PGE2 + Veh, or PGE2 + ActD. ActD pretreatment did not block PGE2-induced expression of RERG. B, Densitometry analysis (n = 3) of PGE2 and ActD-treated E-Osis cells. C, Preincubation with H89 decreased PGE2-induced RERG-FLAG phosphorylation in HEK-293T-RERG-FLAG cells. D, RERG expression in E-Osis stromal cells treated with Veh, E2, PGE2, or E2+PGE2. E, Confocal microscopy of E-Osis stromal cells showing the nuclear localization of RERG after E2, PGE2, or E2+PGE2 treatment. Images are shown at a magnification of ×100 (size bar, 10 μm). *, P < .05; **, P < .001; ***, P < .0001, two-way ANOVA with Tukey's multiple comparison posttest.

Figure 5.

RERG regulates E-Osis stromal cell proliferation. A, RERG knockdown 24, 48, and 72 hours post-siRNA transfection. B, siRNA knockdown of RERG results in a significant decrease in E-Osis stromal cell numbers compared with controls (n = 3). C, BrdU incorporation assay showing a significant decrease in E-Osis stromal cell proliferation 72 hours after RERG knockdown (n = 6). D, RERG knockdown (72 hours) resulted in a statistically significant decrease in RERG and PCNA expression relative to siRNA-mediated ERβ knockdown compared with controls (siCTL) cells. E and F, Densitometry analysis performed after RERG knockdown showing a significant decrease in RERG and PCNA expression (n = 4). *, P < .05; **, P < .001; ***, P < .0001, Student's t test or two-way ANOVA with Tukey's multiple comparison posttest.

Figure 6.

Estrogenic and proinflammatory signals regulate RERG in endometriosis. In E-Osis, ERβ induces RERG mRNA expression, whereas PGE2, via PKA, phosphorylates RERG. RERG phosphorylation is associated with its nuclear translocation. E2- and PGE2-mediated activation of RERG regulates E-Osis cell proliferation.