Propranolol inhibits catecholestrogen-induced human endometrial stromal cell survival mediated by p38 MAPK signaling: potential therapy for endometriosis

Abstract

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Studies indicate that local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance local generation of CCEs in women with endometriosis. CCEs have low binding affinity to estrogen receptors whereas CCEs display high binding affinity to β2-adrenergic receptor (AR), which induce uterine endothelial cell proliferation during gestation. Our recent data demonstrated β2-AR expression in eutopic and ectopic endometrial tissue. In addition, binding of CCEs to β-AR enhances human endometrial stromal cell (HESC) viability, suggesting contribution of CCEs to the pathogenesis of endometriosis. Thus, we tested the hypothesis that the mechanism of CCE-enhanced HESC viability involves alterations in either proliferation or apoptosis mediated by β-AR-induced common intracellular signaling pathways, i.e. AKT, MAPK and/or NFκB. BrdU, Apoptotic Cell Detection ELISA, q-PCR, Western blot and XTT analyses were performed on cultured HESCs derived from endometrial biopsies. Cultured HESCs treated with 10-8 M E2 or 2-OHE2 or 4-OHE2 were measured by BrdU for proliferation and ELISA for apoptosis (n=3 with quadruplicate). Total RNA from cultured HESCs was isolated and pro-apoptotic, anti-apoptotic, and proliferation markers were evaluated by q-PCR (n=5 with duplicate). Total and phosphorylated AKT, p38 and ERK1/2 MAPKs, and NFκB levels were detected in lysates of cultured HESCs (n=3 with triplicate) treated for 10 min with vehicle (control) or 10-8 M E2 or 2-OHE2 or 4-OHE2 ± 2x10-5 M non-specific β-antagonist (propranolol). Subsequently, XTT assays were conducted with p38 MAPK inhibitor to assess the role of p38 MAPK on CCE-induced HESC viability (n=4 with triplicate). Results were analyzed by One-way ANOVA and post hoc Tukey test. An increased HESC proliferation index by E2 and 4-OHE2 (P<0.05 and P<0.05, respectively) and decreased apoptosis were detected in HESCs treated with 2-OHE2 and 4-OHE2 vs. control (P<0.01 and P<0.01, respectively). Analysis of apoptotic markers by q-PCR revealed a significant decrease in Bax mRNA expression in response to 2-OHE2 treatment vs. control (P<0.01). Among the several intracellular signaling cascades analyzed, only phosphorylation levels of p38 MAPK were increased by either treatment with 2-OHE2 or 4-OHE2 (P<0.05 and P<0.05 vs. control, respectively), but not with E2. β-AR antagonism with propranolol mitigated this increased phosphorylation in p38 MAPK levels (P<0.05 and P<0.01, respectively) and inhibition of p38 MAPK by SB203580 blocked CCE-induced HESC survival (P<0.05 and P<0.001, respectively). These data indicate that induction of endometrial stromal cell viability by CCE-β-AR interactions results from an imbalance in both proliferation and anti-apoptotic mechanisms and is specifically mediated by the activation of p38 MAPK signaling. These data also suggest that inhibition of β2-ARs with propranolol may be a novel treatment option in endometriosis.