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Division of Reproductive Biology Research,3 and Division of Reproductive Endocrinology and infertility,4 Department of Obsterics and Gynecology, and Department of Preventative Medicine,5 Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
Department of Obstetrics and Gynecology,6 First Hospital of Peking University, Beijing, China
ABSTRACT
Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (–197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (–197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.
CpG island, DNA methylation, endometriosis, endometrium, ESR2, estradiol receptor, ovary, uterus
Endometriosis is defined as the presence of endometriumlike tissue outside of the uterine cavity. It is a common gynecologic condition, affecting 1 in 10 women in the reproductive age group [1]. Endometriosis is associated with severely painful menstruation, chronic pelvic pain, and infertility [1, 2]. Although the etiology and exact mechanism for the development of endometriosis is unclear, there is a large body of laboratory and circumstantial evidence that suggests a crucial role for estrogen in the establishment and maintenance of this disease [3–5].
Despite 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, a number of investigators reported markedly higher levels of estrogen receptor 2 (ESR2) and lower levels of estrogen receptor 1 (ESR1) in human endometriotic tissues and primary stromal cells compared with eutopic endometrial tissues and cells [6, 7]. Moreover, the levels of both isoforms of progesterone receptor (PGR), particularly progesterone receptor B (PGR B), are significantly lower in endometriosis compared with eutopic endometrium [8, 9]. The classical human ESR1 was cloned in 1986, and a second estrogen receptor, ESR2, was cloned from rat prostate and human testis in 1996 [10–12]. Both ESRs act as transcription factors and are believed to play a key role in endometrial and endometriosis growth regulation.
Hypermethylation of a CpG island has been associated with the transcriptional inactivation of genes. Recently, key nuclear receptor genes, such as ESR1, ESR2, and PGR, were shown to be regulated by methylation of their promoter regions in breast, prostate, and endometrial cancer tissues [13–15]. To assess the relative expression levels of these nuclear receptors and the DNA methylation mechanism in endometrium and endometriosis, an in vitro model of primary stromal cells from these two tissue sources was developed.
Currently, the biologic roles of ESR2 in endometrium and endometriosis are not well understood. We chose to investigate the molecular mechanism responsible for differential expression of ESR2 for two reasons. First, the most striking difference between endometriosis and endometrium was observed with respect to ESR2 levels compared with other steroid receptors, and ESR2 mRNA levels were very low or nearly undetectable in the endometrial stromal cells. Second, an ESR2-selective compound was shown to be therapeutic in a rodent endometriosis model [16, 17]. At present, no evidence has been provided to indicate whether DNA methylation is causally linked to differential ESR2 expression in endometriotic stromal cells and endometrial stromal cells. Direct evidence in support of the cytosine methylation of specific 5' CpGs that leads to transcriptional inactivation has not been reported.
Subjects and Primary Cell Culture
Eutopic endometrium from disease-free subjects (n = 8) and ectopic endometrium from the cyst walls of ovarian endometriomas (defined as a cystic ovarian lesion composed of endometrium-like tissue in its cyst wall and bloody fluid filled in this cyst; n = 8) were obtained immediately after surgery. The mean ages of subjects in each group were 42 ± 3 to 39 ± 3 years, respectively, and there were no significant differences between the two groups with respect to age or cycle phase. Moreover, we obtained three paired samples of ovarian endometriomas and eutopic endometrium from the same subjects. None of the patients had received any preoperative hormonal therapy. All samples were histologically confirmed, and the phase of the menstrual cycle was determined by preoperative history and histologic examination. Written informed consent was obtained before surgical procedures, including a consent form and protocol approved by the Institutional Review Board of Northwestern University. Stromal cells were isolated from these two types of tissues using a protocol previously reported by Ryan et al. with minor modifications [18, 19]. Briefly, tissues were rinsed with sterile PBS, minced finely, and digested with collagenase (Sigma, St. Louis, MO) and DNase (Sigma) at 37°C for 60 min. Stromal cells were separated from epithelial cells by filtration through a 70- and a 20-µm sieve, then they were suspended in Dulbecco modified Eagle medium (DMEM)/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% FBS and in a humidified atmosphere with 5% CO2 at 37°C.
RNA Extraction and Quantitative Analysis by Real-Time RT-PCR
Total RNA was isolated from stromal cells using TRIzol reagent (Sigma) and following the manufacturer's protocol. Total RNA was first treated with DNaseI (Ambion, Austin, TX) to remove contaminating genomic DNA from the RNA samples, then 1 µg RNA was used to generate cDNA with the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-time quantitative PCR was performed with the ABI 7900 Sequence Detection System and the ABI Taqman Gene Expression system (purchased from Applied Biosystems, Foster City, CA) for ESR1, ESR2, and eukaryotic 18S rRNA (18S). The SYBR Green assay was used for total PGR, PGR B, and 18S. 18S values were used for normalization. Primers used for SYBR Green assay were: total PGR, forward: 5'-GTCCTTACCTGTGGGAGCTG-3'; reverse: 5'-CAACAGCATCCAGTGCTCTC-3'. PGR B, forward: 5'-GTACGGAGCCAGCAGAAGTC-3'; reverse, 5'-TCTCTGGCATCAAACTCGTG-3'. 18S, forward: 5'-AGGAATTCCCAGTAAGTGCG-3'; reverse: 5'-GCCTCACTAAACCATCCAA-3'. Relative quantification of mRNA species was performed using the comparative threshold cycles (CT) method. In brief, CT was used to determine the mRNA level normalized to the average mRNA level in endometrial stromal cells. Thus, mRNA levels were expressed as an n-fold difference. For each sample, the gene CT value was normalized using the formula: 
CT = CT gene – CT 18S. To determine relative expression levels, the following formula was used: CT = CT sample – CT calibrator. This value was used to plot the gene expression employing the formula 2– CT.
Bisulfite Modification and Sequencing Analysis
Genomic DNA was extracted from the primary stromal cells using the DNeasy Tissue kit (Qiagen, Valencia, CA). DNA (500 ng) was treated with sodium bisulfite following the manufacturer's protocol (Zymo Research, Orange, CA). Purified DNA was dissolved in 10 µl M-Elution Buffer (Zymo Research). For PCR amplification, 3 µl bisulfite-modified DNA was added to a final volume of 20 µl. AmpliTaq Gold PCR Master Mix (Applied Biosystems) was used for all PCR amplifications. PCR amplifications were performed using the following primers for ESR2: forward: 5'-ATTATTTTTGTGGGTGGATTAGGAG-3', and reverse: 5'-AACCCCTTCTTCC-TTTTAAAAACC-3'. The thermal cycle conditions were as follows: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 2 min, and elongation at 72°C for 2 min, then followed by an incubation at 72°C for 7 min. PCR products (166 bp) were gel purified and cloned into the pGEM-Teasy vector (Promega, Madison, WI). Following transformation, six to eight clones with the correct insert were randomly picked for each PCR and were sequenced using an Applied Biosystems 377 instrument.
5-Aza-2'-deoxycytidine (5-aza-dC) Treatments
At approximately 40% confluence, endometrial stromal cells were placed in serum-free DMEM/F12 for 24 h and then treated with 20 µM DNA methyltransferase inhibitor, 5-aza-dC (Sigma) for 5 days, and medium was changed each day. Total RNA was isolated from the treated cells using TRIzol reagent. All experiments were conducted in triplicate and repeated three times in primary cultured cells from at least three different subjects.
Reporter plasmid vectors containing the ESR2 promoter sequences were constructed by PCR cloning. Genomic DNA from endometriotic stromal cells was used as the template for amplification. The primers were: reverse primer 5'-GATATCTTAGCACAATCAACCCAGAG-C-3' (position +564 relative to the transcription start site); forward primers 5'-GGTACCTTCCC-AGTGACCTCTTGA-3' (–525), 5'-GGTACCTGTGCGCCACTATCCTTG-3' (–197), and 5'-GGT-ACCTGTTTGAAATCCTGCGGTGAG-3' (+372). Restriction sites (Kpn2 site for forward primers and EcoRV site for reverse primers) were added to the 5'-end of primers, and promoter sequences were amplified using TAKARA LA Taq with GC buffer (TaKaRa, Otsu, Japan). PCR products were cloned into a modified pGL4 vector-SV40. The SV40 minimal promoter was digested with BglII and HindIII from pGL2-promoter vector and cloned into BglII- and HindIII-digested pGL4.10 vector (Promega). The final plasmids containing ESR2 promoter sequences were –525/+564, –197/+564, and +372/+564.
Transfection and Luciferase Reporter Gene Assay
Transfection experiments of endometrial and endometriotic stromal cells were performed using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. Briefly, the cells were grown in 24-well tissue culture plates so that the cell layer was 50%–60% confluent on the day of transfection. For each well, OPTI-MEM I containing 1.5 µl FuGENE 6 was mixed with 240 ng reporter plasmid and 60 or 80 ng pSV-ß-galactosidase vector (Promega) for endometrial or endometriotic stromal cells. The cells were harvested 48 h after transfection, and the luciferase activity was measured using a luciferase assay system (Promega). Beta-galactosidase activity was used to normalize transfection efficiency. All of the experiments were repeated three times in triplicate.
In Vitro Methylation of Reporter Plasmids
In vitro methylation assays were carried out according to the methods described by Robertson and Ambinder [20] and Singal et al. [21]. Briefly, region-specific methylation was carried out on the ESR2 promoter fragments of –525/+564 and –197/+564 after excision and isolation. DNA was incubated with SssI CpG methylase (New England Biolabs, Ipswich, MA) in the presence (methylated) or absence (mock-methylated) of S-adenosylmethionine, as recommended by the manufacturer, for 2 h. Methylated and mock-methylated fragments were re-ligated into their respectively unmethylated vectors. All constructs were sequenced to confirm the correct region of the ESR2 gene, and the efficiency of the methylation was determined through methylation-sensitive and methylation-insensitive restriction enzyme digestion with HpaII and MspI.
Cells were washed with ice-cold PBS and suspended in the protein extraction reagent (Pierce, Rockford, IL). Lysates were cleared by centrifugation at 15 700 x g for 10 min. Equal amounts of protein (15 µg) were resolved on 4%–15% Tris-HCL gels, transferred onto nitrocellulose membranes, and incubated with anti-human ESR1 or ESR2 antibodies diluted 1:100 or 1:2000 (purchased from Calbiochem, Darmstadt, Germany and Upstate, Chicago, IL). Anti-ACTB antibody was used as a loading control. Detection was performed using a supersignal west femto maximum sensitivity substrate system (Pierce). Band intensity of protein expression was quantified using the Quantity One Analysis Software (Bio-Rad Laboratories, Los Angeles, CA).
For mRNA levels and luciferase assays, the values are expressed as means ± SEM of measurements for primary cells cultured in triplicate. The results were representative of at least three independent experiments. Percent methylation of each clone obtained from each of the eight patients in each group was treated as a single value for the statistical analysis of bisulfite sequencing. The data were analyzed using Student's t-test with statistical significance at the level of P < 0.05. Spearman's rank correlation coefficient was calculated for the correlation between ESR2 mRNA levels and percent methylation, and a permutation test was used to assess its statistical significance.
ESR1, ESR2, Total PGR, and PGR B mRNA Levels in Endometrial and Endometriotic Stromal Cells
Real-time RT-PCR was used to quantify the mRNA levels of nuclear receptors in endometrial (n = 8 subjects) and endometriotic (n = 8 subjects) stromal cells. ESR1 mRNA levels were somewhat lower (7-fold; P = 0.037) in endometriotic stromal cells compared with endometrial stromal cells. ESR2 mRNA was strikingly higher (approximately 34-fold; P = 0.015) in endometriotic stromal cells, whereas it was much lower or nearly absent in endometrial stromal cells. Thus, the ratios of ESR1 to ESR2 were, on average, 841 and 21 in endometrial and endometriotic stromal cells, respectively (P < 0.001). Total PGR and PGR B mRNA levels in endometriotic stromal cells were also significantly lower than those in endometrial stromal cells (P = 0.027 and P = 0.029; Fig. 1). Western blot showed that ESR2 protein levels in endometriotic cells (n = 8 subjects) were significantly higher compared with endometrial cells (n = 8 subjects), whereas ESR1 protein levels in endometriotic cells were significantly lower compared with endometrial cells (P < 0.05; Fig. 1F). We also compared ESR1 and ESR2 expression in matched endometrial versus endometriotic stromal cells obtained simultaneously from separate groups of three subjects (P < 0.05; Fig. 2). Both mRNA and protein levels of ESR1 and ESR2 were significantly different in these two groups similar to the findings illustrated in Figure 1.
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DNA Methylation Profile of the ESR2 Promoter Region
Among the four steroid receptors that we examined, ESR2 mRNA levels displayed the highest and most strikingly differential expression between the two homologous cell types. Since this observation made promoter methylation a likely mechanism for the regulation of ESR2 in endometriosis versus endometrium, we pursued this line of investigation. We identified an approximately 550-bp classic CpG island (–197/+359) within the promoter and its downstream untranslated exon 0N region of the ESR2 gene. Methylation status of ESR2 promoter region was determined by bisulfite genomic sequencing. The detailed CpG methylation status of endometrial and endometriotic stromal cells was shown in Figure 3. There was a statistically significant difference in the methylation status within this region (–189/–24; P < 0.0001). It was heavily methylated in the majority of endometrial stromal cells (n = 8) that expressed lower levels of ESR2 and largely unmethylated in endometriotic stromal cells (n = 8) that expressed higher levels of ESR2 mRNA. Also, significant negative correlation was found between percentage of methylation of ESR2 promoter region and ESR2 mRNA expression (in logarithmc scale) among eight endometrial stromal cells and eight endometriotic stromal cells (Fig. 3D, Spearman's rank correlation coefficient –0.89, permutation test; P < 0.001).
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Induction of ESR2 mRNA Expression by 5-aza-dC
To determine the correlation between DNA methylation and downregulation of the ESR2 gene or its near-silencing, the endometrial stromal cells (with hypermethylation of ESR2 promoter) were treated with demethylating agent 5-aza-dC. The level of ESR2 mRNA was measured using real-time RT-PCR. As shown in Figure 4, the treatment in endometrial stromal cells with 5-aza-dC significantly increased ESR2 mRNA levels (P = 0.025).
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Regulation of ESR2 Promoter Activity by Methylation of ESR2
To elucidate the critical region in the ESR2 gene 5'-flanking sequence, which regulates promoter activity, we transfected serial deletion mutants (–525/+564, –197/+564, +372/+564) of the ESR2 promoter region fused to the luciferase reporter gene into endometriotic and endometrial stromal cells. The relative luciferase activities of the reporter gene constructs were determined in triplicate. We did not detect a significant difference in luciferase activity between –525/+564 and –197/+564 constructs, whereas the +372/+564 construct exhibited significant decreases (60.1% and 48.6%) in ESR2 promoter activity compared with the –197/+564 construct in endometriotic or endometrial cells (Fig. 5, A and B). This indicated that the –197/+372 bp region containing the CpG island is critical for baseline promoter activity in both endometriotic and endometrial cells (P < 0.01).
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Next, in vitro methylation analysis was performed to determine whether ESR2 promoter activity was regulated by the methylation of the ESR2 CpG island. Figure 5, C and D, showed that in vitro methylation of the CpG island in the –525/+564 or –197/+564 luciferase constructs significantly reduced ESR2 promoter activity in both cell types (Student's t-test, P < 0.001, and P < 0.01).
Development and progression of endometriosis depends on the presence of estrogen [22, 23]. However, the biologic influence of estrogen on target organs is modulated by changes in tissue hormone levels and the local distribution of its receptors ESR1 and ESR2. Studies in knockout mice and its nonidentical tissue distribution compared with ESR1 would suggest that ESR2 has a biologic function distinct from that of ESR1 [24]. We demonstrated that ESR1 expression was downregulated and ESR2 was upregulated in endometriotic stromal cells compared with endometrial stromal cells, which confirmed previous reports [7, 25]. This raises the possibility that at least some of the critical functions of estradiol are mediated by ESR2. Recently uncovered biologic roles of ESR2 regulating inflammatory processes in autoimmune diseases and endometriosis lend further credence to these findings [16, 17]. In fact, an ESR2-selective drug has been shown to treat endometriosis in a rodent model. This highly selective ESR2 agonist, ERB-041, is found to be inactive on classic estrogenic targets, such as the uterus, mammary gland, and bone. However, it has potent anti-inflammatory activity in two in vivo models: the HLA-B27 transgenic rat and Lewis rat adjuvant-induced arthritis. In a rodent model of endometriosis, the beneficial actions of this compound were interpreted to be independent of ESR2 in the lesion [16]. ESR2 has also been shown to induce cell proliferation and may cause growth of endometriosis via this mechanism [26].
ESR2 is regulated by two alternatively used promoters (exons 0N and 0K) upstream of a common coding region. Both promoter regions contain the CpG islands. We elected to evaluate the DNA methylation status of the CpG island, located at the proximal promoter-exon 0N, because this region was shown to be differentially methylated previously in normal versus malignant breast lesions [27, 28].
Differences in the ESR1 to ESR2 ratio between endometriotic and endometrial stromal cells could have important functional implications, since these ESRs have different ligand-binding characteristics [29, 30]. It also has been proposed that heterodimers of ESR1 and ESR2 can associate with estrogen-responsive elements in vitro [31]. Because it was reported that one possible role of ESR2 is to modulate ESR1 activity, the relative expression levels of two ESR subtypes are an important determinant of target genes regulated by estrogens and antiestrogens [32]. Therefore, it is conceivable that the set of estrogen target genes vary significantly in endometriotic versus endometrial stromal cells.
We observed a clear inverse relationship between the extent of methylation in the ESR2 promoter CpG island and its mRNA levels in endometrial and endometriotic stromal cells. This was verified mechanistically using treatments with demethylating agent and isolation of the regulatory region subject to methylation by assaying promoter activity. This is consistent with a large body of literature showing that DNA methylation at the transcription regulatory region is generally associated with gene silencing or downregulation [33–35]. In general, DNA methylation-mediated control of gene expression may be a major mechanism for the regulation of steroid receptor mRNA levels in various tissues in view of accumulating published evidence [13–15].
It has been demonstrated that DNA methylation can interfere with protein-DNA interaction, recruitment of histone deacetylases, and the induction of chromatin condensation necessary for gene inactivation [36, 37]. Methylation can directly interfere with the DNA binding of certain transcriptional factors. Also, some methyl-CpG binding proteins are shown to bind to methylated DNA and alter its DNA conformation, thus affecting the binding of various transcriptional regulators [38, 39]. These molecular alterations associated with the methylation of the ESR2 promoter may be responsible for its repression in endometrial stromal cells. Also, the expression of ESR2 in the stromal cells of endometriosis may be regulated by factors other than methylation. For example, sequence analysis of the 5'-flanking region of the ESR2 promoter 0N has shown the presence of several consensus transcriptional factor binding sites and cis-regulatory elements [40].
This is the first demonstration of a methylation-dependent mechanism responsible for strikingly elevated levels of ESR2 in endometriosis. This finding may have several clinical applications. Because the methylation of a specific gene can be detected in DNA from diagnosis biopsies [41], ESR2 methylation status could be a potentially helpful adjunct to morphologic criteria for the diagnosis of endometriosis. Moreover, testing for ESR2 promoter methylation in endometriotic lesions may identify patients who are candidates for treatment with ESR2-selective compounds. Finally, new drugs that regulate methylation may be used as potential therapeutics for endometriosis.
FOOTNOTES
1Supported by National Institutes of Health/National Institute of Child Health & Human Development grant U54-HD40093. 
Correspondence: 2Serdar E. Bulun, Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, 303 East Superior Street, Suite 4–250, Chicago, IL 60611. FAX: 312 503 0095; e-mail: s-bulun{at}northwestern.edu
Received: 26 March 2007.
First decision: 9 April 2007.
Accepted: 9 July 2007.
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