Α1d-Adrenergic Receptor Expression in Human Endometrial Stromal Cells Contributes to Catecholestradiol-Induced Cell Survival: Implications for Endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance generation of CCEs in women with endometriosis. During gestation, CCEs bind adrenergic receptors (ARs) to mediate uterine endothelial cell proliferation. Our recent data demonstrated that compared to the basalis layer of the endometrium, the functionalis layer displayed increased β2-AR expression in women with endometriosis. Moreover, binding of CCEs to β-ARs enhanced human endometrial stromal cell (HESC) viability in culture, suggesting a modulatory role for CCE-β-AR binding in retrograde menstruation that contributes to endometriosis development. It is unknown if, in addition to β-ARs, α-ARs also have a role in this process. We, therefore, tested the hypothesis that ectopic endometrial tissue expresses α1D-AR and that 2-OHE2 and 4-OHE2 will potentiate HESC viability via α-ARs. Immunohistochemistry was performed on paired eutopic/ectopic endometriotic tissue and XTT analysis was conducted on cultured CCE-treated HESCs derived from endometrial biopsies. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained using a1D-AR antibody and evaluated semi-quantitatively by HSCORE. Confluent HESCs derived from endometrial biopsies at time of surgery for benign reasons were cultured in 96-well plates (5x103 cells/well) and treated with vehicle (control) or with 10-8 M E2 or 2-OHE2 or 4-OHE2 ± nonspecific α-AR inhibitor (phentolamine) for 48h. XTT assays measured cell viability. Experiments (n=4) were performed in duplicate and results were analyzed by One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed overall weak to moderate α1D-AR staining in endometrial epithelial cells and weak staining in endometrial stromal cells with no significant difference between eutopic and ectopic endometrial tissues in either phase. Compared to the basalis layer, both stromal and epithelial cells in the functionalis layer of eutopic endometrium displayed stronger α1D-AR staining. In vitro XTT analyses revealed phentolamine partially inhibited (P<0.05 and P<0.001, respectively) 2-OHE2 and 4-OHE2-enhanced HESC survival versus control (P<0.05 and P<0.05, respectively). This inhibitory effect of phentolamine was specific to CCEs since E2-enhanced HESC survival was not inhibited by phentolamine. Immunostaining with α1D-AR showed similar expression patterns as β2-AR in that there was increased staining to the functionalis layer of the endometrium; albeit, α1D-AR staining was overall weaker in both ectopic and eutopic tissue. Our in vivo and in vitro results indicate that induction of HESC viability by CCE-α-AR binding may contribute to the propagation of endometriosis. Future studies into different α-AR isoform expressions in endometriotic lesions are warranted.