Molecular Background of Estrogen-Dependent and -Independent Gene Expressions in Endometriotic Lesions

Abstract

Background: Endometriosis is an estrogen-dependent disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. As a possible background, we hypothesized a role of the local environment with high estrogen depending on aromatase upregulation in endometriotic lesions. Objective: To test our hypothesis, we re-evaluated the expression profile of estrogen receptor (ER), and then searched for the estrogen-dependent gene expressions in endometriotic cells. Finally, we approached the epigenetic background of gene expressions in endometriotic cells. Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma. Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was evaluated using RT-PCR. Specific primer sets of unique 5’-UTR exons/exon 2 in ESR1 and specific primer sets of unique 5’-UTR exons/exon1 in ESR2 were used for the analysis of promoter usage. Primer sets of exon 7 and exon 8 in ESR2 were used to evaluate the expression of ERβisoform. Using SERM(PPT and DPN), ER-dependent gene expression was estimated. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis and eRNA expression. Results: 1) Relative expression of ERα mRNA in endometriotic cells was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is almost at a comparable level of the ERα. 3) In addition to ERβ1 mRNA, a splice variant ERβ2 was expressed at a comparable level of the ERβ1. 4) Top ten genes, up- or down-regulated in response to SERM, were extracted in endometriotic cells. 5) TGFA expression was upregulated at a comparable level in response to PPT and DPN. 6) A stretch of hypomethylated sequence, which includes an ERE at 50kb upstream from the TSS, was suggested as active enhancer. 7) ESR1 and ESR2showed a marginal response to SERM. 8) GATA6 and CYP19 were highly expressed in endometriotic cells, and hypomethylated sequences in these genes were suggested as active enhancer. Conclusion: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined ER-dependent and -independent gene expressions using endometriotic cells. The results suggest one aspect of gene expression in endometriosis lesions.