{"paper_id":"1480b851-aef9-40c7-9d8e-58736ae2eca3","body_text":"In this review, the terms  endometriotic tissue  and  endometriosis  refer to the pathological state in which ectopic endometrium-like tissues grow within the pelvic peritoneum or ovaries. The terms  endometrial tissue  and  endometrium  refer to normal eutopic or intrauterine endometrial tissue. A biological distinction is also made between endometrium from disease-free women versus women with endometriosis. Sampson proposed the most widely accepted mechanism for the development of endometriosis on pelvic peritoneal surfaces as the implantation of endometrial tissue on the peritoneum through retrograde menstruation. Because retrograde menstruation occurs in >90% of all women, endometriosis is believed to be caused by molecular defects that favor survival and establishment of endometrial tissue in menstrual debris on the peritoneum. 10 – 12  Gene expression profiles characterized by microarray in the endometrium of women with or without endometriosis showed that several progesterone target genes were dysregulated during the window of implantation, at which time endometrium is exposed to the highest levels of progesterone. 13  For example, the prototype progesterone-responsive gene, glycodelin, was downregulated strikingly in endometrium of women with endometriosis compared with women without endometriosis. These findings suggested that the eutopic endometrium of women with endometriosis exhibit the pathology found within endometriotic tissue. 13 , 14\n\nIn a postmenopausal women undergoing in vitro fertilization with a donor egg, the exogenous administration of only estradiol (E2) and progesterone is sufficient to prepare the endometrium for implantation in the absence of ovarian function. This observation underscores the essential roles of these steroids in uterine physiology. Indeed, both E2 and progesterone are master regulators of endometrial tissue. Each hormone is estimated to regulate expression of hundreds of genes during various phases of the menstrual cycle. 15  Endometriotic and eutopic endometrial tissues respond to E2 and progesterone with apparently similar histological changes, and both tissues contain immunoreactive ERs and PRs. The eutopic endometrium predictably becomes atrophic in response to prolonged progestin therapy or oral contraceptives that contain progestins. Treatment with these agents, however, does not predictably suppress endometriotic tissue growth. Endometriotic tissue in ectopic locations, such as the peritoneum or ovary, is fundamentally different from eutopic endometrium within the uterus in terms of production of cytokines and prostaglandins, estrogen biosynthesis and metabolism, and clinical response to progestins. 8 , 16 , 17  There are substantial molecular differences with regard to progesterone response between normal endometrium and eutopic and ectopic tissues from women with endometriosis. 13 , 18 , 19\n\nBoth circumstantial and laboratory evidence strongly support the notion that estrogen is an extremely strong mitogen for endometriotic tissue. The biologically active estrogen E2, which is secreted by the ovary or locally produced by endometriotic tissue, acts as a classical steroid hormone to regulate growth of endometrial or endometriotic tissue. E2 enters cells and binds to the ER in estrogen-responsive cells. ER subtypes α and β are proteins with high affinity for E2 and are encoded by separate genes. The classical human ERα was cloned in 1986, and a second estrogen receptor, ERβ, was cloned from rat prostate and human testis in 1996. 20 – 22  Although both ERα and ERβ are present in the endometrium, ERα seems to be the primary mediator of the estrogenic action in this tissue. 23\nDespite its sensitivity to estrogen, endometriosis appears to contain a unique complement of steroid hormone receptors compared with that of its normal tissue counterpart, the eutopic endometrium. For example, several investigators reported markedly higher levels of ERβ and lower levels of ERα in human endometriotic tissues and primary stromal cells compared with eutopic endometrial tissues and cells. 24 , 25  Moreover, the levels of both isoforms of PR, particularly PR-B, are significantly lower in endometriosis compared with eutopic endometrium. 5 , 26\nThe E2-receptor complex acts as a transcription factor that becomes associated with the promoters of E2-responsive genes via direct DNA binding or binding to other docking transcription factors at basal promoter regions. 27  This interaction brings about ER-specific initiation of gene transcription, which promotes the synthesis of specific mRNAs and proteins. 27  PR is one of many E2-responsive genes, and E2 acts in eutopic endometrial tissues and stromal cells to promote endometrial responsiveness to progesterone. 28  In contrast, PR mRNA and protein levels are not elevated in biopsied endometriotic tissues exposed to high E2 levels during late proliferative phase or in endometriotic cells treated with E2, indicating that E2-induction PR expression in endometriosis is markedly blunted. 26\n\nBecause the response of endometriotic tissue to sex steroids is markedly different from that of endometrium, we and others determined the expression of steroid receptors in cells from these tissues. Our recent findings regarding the levels of steroid receptors in endometrial and endometriotic cells are consistent with previously published data. 24 , 26  Real-time polymerase chain reaction was used to quantify the mRNA levels of nuclear receptors in primary endometrial and endometriotic stromal cells. ERα mRNA levels were significantly lower (sevenfold;  Fig. 1 ) in endometriotic stromal cells compared with endometrial stromal cells. ERβ mRNA was strikingly higher (~34-fold;  Fig. 1 ) in endometriotic stromal cells, whereas it was much lower or nearly absent in endometrial stromal cells. 29  The ratios of ERα to ERβ were, on average, 841 and 21 in endometrial and endometriotic stromal cells, respectively. 29  Total PR and PR-B mRNA levels in endometriotic stromal cells were significantly lower than those in endometrial stromal cells. 29  Protein levels of ERα and ERβ were significantly different in these two groups similar to the findings regarding mRNA levels. 29  In conclusion, ERα, ERβ, and PR levels were markedly different in endometrium versus endometriosis-derived stromal cells. Endometriotic stromal cells contained extraordinarily higher ERβ and significantly lower ERα and PR levels compared with endometrial stromal cells.\n\nBecause of the extreme differential expression of ERβ between endometriotic and endometrial cells, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ERβ mRNA levels in endometriotic cells. 29  We identified a CpG island occupying the promoter region of the ERβ gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells versus endometriotic cells. Treatment with a demethylating agent significantly increased ERβ mRNA levels in endometrial cells. The critical region that confers promoter activity also bears the CpG island. 29  The activity of the ERβ promoter was strongly inactivated by in vitro methylation. Therefore, methylation of a CpG island at the ERβ promoter region is a primary mechanism responsible for differential expression of ERβ in endometriosis and endometrium. 29  Therefore, high ERβ mRNA and protein expression in endometriotic cells were mediated by an epigenetic defect involving hypomethylation of a CpG island occupying its promoter ( Fig. 2 ).\n\nERα deficiency in endometriosis may be responsible for failure of E2 to induce PR expression, thus contributing to secondary PR deficiency and progesterone resistance in women with this disease. In vivo observations strongly suggest that E2 induces ERα expression in mouse uterine tissue. 30  It is quite likely that E2 also plays a key role in regulating ERα expression in human endometrial stromal cells. However, strikingly high quantities of E2 produced via local aromatase activity in addition to high ERβ levels in stromal cells of endometriosis may perturb this regulation and may suppress ERα expression. 29 , 31  We tested the hypothesis whether ERβ is responsible for suppressing ERα promoter activity and mRNA and protein expression in endometriotic cells. The human ERα gene is regulated via multiple promoters; the three major promoters are A, B, and C and are used alternatively in various tissues. 32 – 34  Promoters A and B are located within the 2-kb region proximal to the translation start site, whereas promoter C lies some 101 kb upstream of this site. 32 , 35\nWe determined the role of ERβ in E2-dependent regulation of the ERα gene in endometrial and endometriotic stromal cells. 36  ERβ knockdown significantly increased ERα mRNA and protein levels in endometriotic stromal cells. 36  Conversely, ERβ overexpression in endometrial stromal cells decreased ERα mRNA and protein levels. ERβ knockdown significantly decreased proliferation of endometriotic stromal cells. 36  ERβ knockdown or overexpression primarily regulated ERα mRNA species arising from the far distal promoter C.\nWe screened the three ERα promoter regions using serial chromatin immunoprecipitation assays for binding of ERβ or ERα itself ( Fig. 3 ). E2 enhanced binding of both ERα and ERβ to a region containing a nonclassic activator protein 1 (AP1) motif in promoter A in endometriotic cells ( Fig. 3 ). We investigated several regions of ERα promoter C, which lies some 101 kb upstream of the common splice junction. In the presence of E2, ERβ bound to a genomic region flanking an AP1 site upstream of promoter C and a region bearing a specificity protein 1 (Sp1) motif immediately downstream of promoter C in endometriotic stromal cells. In contrast, ERα bound to neither promoter C sequences. Taken together, these findings suggest that ERβ-mediated inhibition of ERα expression may be mediated primarily by promoters A and C.\nIn summary, high levels of ERβ suppress ERα expression and response to estradiol in endometriotic stromal cells via binding to nonclassic DNA motifs in alternatively used ERα promoters. ERβ also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells.\n\nPR is a prototype target gene of ERα in several cell types including breast malignant epithelial cells. ERα mediates E2 induction of PR. It has been reported that two distinct E2-regulated promoters generate transcripts encoding the two functionally different human PR isoforms, PR-A and PR-B. 37  Previous studies have demonstrated that maximal PR mRNA and protein levels are reached after human breast cancer cells have been exposed to E2 for 3 days. 38 – 40  Two major transcriptional start sites have been identified. The upstream transcription start site gives rise to a full-length mRNA species that encodes the PR-B protein. Another mRNA species with a further downstream transcription initiation site gives rise to the truncated PR-A form. Despite the fact that both proposed PR promoter sequences are E2 responsive, neither contains a classical palindromic ERE sequence. 41  Several nonclassic regulatory elements (e.g., AP1, Sp1) in the human PR promoter have been reported. These sites have been shown to bind ERα and on one occasion ERβ. 28 , 41 – 49\nWe speculate that a critical level of ERα may be necessary for E2-dependent induction of PR in endometrial stromal cells. The occupancy of the PR promoter regions with varying ratios of ERα-to-ERβ may be critical in determining the effect of E2 on PR expression. A strikingly lower ERα-to-ERβ ratio in endometriotic stromal cells may be responsible for a shift from E2 stimulation to E2 inhibition of PR expression in endometriotic stromal cells ( Fig. 4 ).\n\nComplex mechanisms involving promoter regulation may be responsible for the observed aberrations in ERα, ERβ, and PR expression in endometriosis. The stromal cell component of endometriotic tissue may be the primary site of these abnormalities. In endometriotic stromal cells, ERβ promoter is pathologically hypomethylated and therefore hyperactive, leading to very high ERβ levels. ERβ suppresses ERα expression and results in strikingly high ERβ-to-ERα ratios in endometriotic cells. We speculate that a strikingly lower ERα-to-ERβ ratio in endometriotic stromal cells may cause a shift from E2 stimulation to E2 inhibition of PR expression in endometriotic stromal cells under in vivo circumstances ( Fig. 4 ). This hypothetical mechanism may be the answer for a critically important clinical question regarding the etiology of severely deficient PR-B in endometriotic stromal cells, which contributes to progesterone resistance in women with endometriosis. Lack of PR-B in the endometriotic stroma perturbs normal epithelial-stromal interactions, leading to epithelial cell defects, 17β-hydroxysteroid dehydrogenase-2 deficiency, and progesterone resistance in endometriosis. It is possible that this consequence of PR-B deficiency is only the tip of the iceberg with regard to pathogenesis of endometriosis, and that numerous other molecular aberrations may also contribute to the development of resistance to hormone treatments in women with endometriosis. Nevertheless, an understanding of the molecular mechanisms underlying PR-B deficiency may lead to the development of new treatments that target defective pathways. This concept supports the use of SPRMs with mixed agonist/antagonist properties and possibly higher affinity to PR to overcome PR deficiency in endometriotic stromal cells. Moreover, selective ERβ ligands that target high levels of ERβ in endometriotic tissue may be clinically beneficial via disrupting this mechanism.","source_license":"CC0","license_restricted":false}