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Original Article |
Obstetrics and Gynecology Section, Department of Reproductive and Developmental Sciences, University of Edinburgh (H.O.D.C., T.A.H.), and Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (R.W.K., G.S.S., P.T.K.S.), Edinburgh, United Kingdom EH16 4SB; and School of Biological and Molecular Sciences, Oxford Brookes University (L.R.E., N.P.G.), Headington, Oxford, United Kingdom OX3 0PB
Address all correspondence and requests for reprints to: Prof. Hilary Critchley, Obstetrics and Gynecology Section, Center for Reproductive Biology, University of Edinburgh Medical School, Little France Cresent Edinburgh, United Kingdom EH16 4SB. E-mail: hilary.critchley{at}ed.ac.uk.
Abstract
Estrogen action is mediated via two subtypes of the estrogen receptor (ER), usually referred to as ER
and ERß. We have previously compared the spatial and temporal expressions of ER and ERß proteins in human endometrium and reported that endothelial cells exclusively express ERß. In the present study we have extended our investigations to compare the pattern of expression of wild-type (ERß1) and a newly identified ERß variant isoform (ERßcx/ß2) that lacks the ability to bind steroids.
mRNAs encoding both ERß1 and ERßcx/ß2 receptors were identified in human endometrial extracts by RT-PCR. Quantitative TaqMan R-TPCR demonstrated that levels of total mRNAs were increased significantly premenstrually as circulating progesterone levels declined. ERß1 and ERßcx/ß2 proteins were identified within multiple cell types within the endometrium using isotype-specific monoclonal antibodies; immunoexpression of ERßcx/ß2 appeared less intense than that of ERß1 in endometrial glandular epithelium and endothelial cells. Immunoexpression of ERß1 appeared unchanged throughout the menstrual cycle. In contrast, levels of ERßcx/ß2-specific immunoreactivity were specifically reduced in gland cells within the functional layer, but not in those of the basal layer, in the midsecretory phase. It is possible that coexpression of ERßcx/ß2 in cells containing ERß1 and/or ER may modulate the effects of estrogens on the endometrium.
STEROID HORMONES ARE the systemic factors that drive the endometrium through the characteristic sequential phases of the menstrual cycle and necessarily act via their cognate receptors. The consequential initiation of gene transcription and cascade of downstream local events is responsible for the key functions of the endometrium, these being implantation or, in the absence of pregnancy, menstruation and endometrial repair. The sex steroid receptors for estrogen (ER and ERß), androgen, and progesterone (PR) are expressed in the nuclei of endometrial glands and stroma (1, 2, 3, 4, 5, 6, 7).
Like other members of the steroid receptor superfamily, ER and ERß share a common arrangement of 5 structure-function domains, denoted A–F (8). ER and ERß are both encoded by 8 exons, but are the products of 2 genes located on different chromosomes (9). Polymorphic sites have been identified within both the human ER and ERß genes, and an association between particular polymorphisms and disturbances in reproductive function have been demonstrated. For example, Sundarrajan et al. (10) reported that the frequency of RasI and AluI ERß gene polymorphisms in 98 Chinese women with ovulatory or menstrual disorders was significantly higher than that in 150 controls with normal ovulatory menstrual cycles. The same group reported an association between a PvuII polymorphism in the ER gene and the outcome of in vitro fertilization treatment (11). The length of a dinucleotide (CA) repeat polymorphism in the flanking region of the ERß gene has been correlated with variations in blood pressure (12) and in levels of androgen and steroid hormone-binding globulin in blood (13). The PuvII polymorphism in the N-terminal region of ER is associated with the risk of development of benign uterine disease (14), but not with the development of preeclampsia (15).
In vitro studies have demonstrated that homodimers (ER-ER or ERß-ERß) or heterodimers (ER-ERß) can be formed when both isoforms are expressed in the same cell (16, 17). Both receptor homodimers are reported to induce similar trans-activation profiles in vitro using a luciferase reporter gene linked to an estrogen response element (ERE) when they were activated with estradiol or diethylstilbestrol, but to signal in opposite ways at an activating protein-1 site (18). Studies by Hall and McDonnell (19) have indicated that one role played by ERß may be to modulate ER transcriptional activity. For example, at subsaturating levels of ligand (10 pM estradiol) ERß was able to act as a dominant inhibitor of ER. A novel human ERß variant named hERßcx, formed by alternative splicing of the eight exon of ERß, was first identified by screening a human testis cDNA library (accession no. AB006589) (20). The same isoform, called hERßcx/ß2, and an additional three splice variants (hERß3–5) were independently identified by Moore et al. (21). Moore et al. (21) detected differential expression of mRNAs encoding these variants by RT-PCR in a wide range of human tissues and cell extracts, including the human uterus and Ishikawa cells. Furthermore, they demonstrated that the DNA and dimerization domains within ERßcx/ß2 remain functional and that the protein can bind to DNA containing a consensus ERE both as a homodimer and as a heterodimer with either ER or ERß1 (21). Within this report the hERß protein identified initially as the homolog to rat ERß (9, 22, 23) will be referred to as ERß1, and the ERßcx/ERß2 variant as ERßcx/ß2, to distinguish them from each other. The human ERßcx/ß2 cloned by Lu et al. (accession no. AF124790) is not the same variant as the ERßcx/ß2 described above, but is a form of ERß that lacks exon 5 (24).
The structure of an estrogenic ligand, its concentration, the presence of coregulators, and the subtype(s) of ER expressed in each cell will determine the pattern of estrogen-induced gene expression (25). Therefore, the cellular distributions of ERß1 and ERßcx/ß2 may have important implications for the regulation of normal reproductive processes within the uterus (implantation and menstruation) and also when these events are dysfunctional. There are relatively few data addressing the cell-specific sites of expression of ERß in the human uterus. An antibody used in previous studies (1, 5) was directed against a peptide common to the hinge domain of hERß1 and ERßcx/ß2, and therefore immunopositive cells identified may contain one or both receptor isoforms. The aim of the present study was therefore to extend our previous investigations by using RT-PCR with ERß1- and ERßcx/ß2-specific primers and immunohistochemistry with isotype-specific monoclonal antibodies to determine whether ERß1 and ERßcx/ß2 mRNA and protein are both expressed in human endometrium during the normal menstrual cycle.
Materials and Methods
Tissue collection
Full thickness endometrial tissue (lumen to the muscular myometrial layer, thereby including superficial and basal tissues) was collected from a total of 53 women undergoing hysterectomy or endometrial investigation for benign gynecological indications. From these, 33 were used for measurement of ERß mRNAs, and 24 were used for immunohistochemical analysis. All women described regular menstrual cycles (25–35 d) and had not received exogenous hormones or used an intrauterine contraceptive device in the 3 months preceding inclusion in the study. Written informed consent was provided by all subjects, and ethical approval for tissue collection was granted by the local research ethics committee. Tissue samples collected for immunohistochemistry were fixed overnight at 4 C in 4% paraformaldehyde, rinsed, and stored in 70% ethanol before routine processing into paraffin wax using a 18-h cycle on a TP1050 machine (Leica Corp., Knowlhill, Milton Keynes, UK). Endometrial tissue was also frozen in liquid nitrogen and stored at -70 C for subsequent RNA extraction for RT-PCR and real-time PCR analysis.
For all endometrial biopsies analyzed in the study, the stage of the menstrual cycle was consistent with the patient’s reported last menstrual period and histological dating using the criteria of Noyes et al. (26). Samples were classified as early, mid, or late proliferative and early, mid, or late secretory phase with at least four per group for measurement of ERß mRNAs. For immunohistochemical studies, samples were split into proliferative, early, mid, and late secretory phases, with at least four per group. Any cases with severe uterine pathology, for example, polyps or large fibroids, were excluded. All subjects had a serum sample collected at the time of surgery for the determination of circulating estradiol and progesterone levels by RIA as described previously (27). All samples were consistent with the designated cycle stage based on morphological criteria and last menstrual period (see Table 1
). Progesterone concentrations were significantly lower in the late secretory phase than in the midsecretory phase (P < 0.01). Samples were split into proliferative, early, mid, and late secretory phases for immunohistochemical studies and early, mid, and late proliferative phases and early, mid, and late secretory phases for TaqMan RT-PCR, with at least four in each group.
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Peptides P7 (specific for ERß1, wild type; accession no. AB006590) (23) and P8 (specific for hERßcx/ß2; accession no. AB006589) (20, 21) were synthesized at the Center for Proteins and Peptides, Oxford Brookes University (Oxford, UK). Monoclonal antibodies were prepared using standard methods and were screened against recombinant protein or peptide (28, 29). The specificity for the ERß isotype to which they were directed has been confirmed on Western blots using recombinant proteins (see Fig. 2a in Ref. 29). Neither antibody showed any cross-reactivity against ER (29); a positive control section of human adult testis was included in all immunohistochemical experiments, and in all cases the expected staining pattern was observed (not shown).
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The immunohistochemical protocol has been described previously in detail (1). Anti-hER was a mouse monoclonal antibody (clone 1D5) obtained from DAKO Corp. (Cambridge, UK). Briefly, 5-µm tissue sections were dewaxed in Histoclear (National Diagnostics, Atlanta, GA) and rehydrated in descending grades of alcohol to dH20. Antigen retrieval was carried out by pressure cooking in 0.05 M glycine/0.01% EDTA (pH 8) for 7 min at setting 2 (Tefal, Nottingham, UK). After cooling for 20 min, slides were washed in 0.05 M Tris-buffered saline (TBS), and endogenous peroxidase was quenched. Nonspecific binding of the primary antibody was blocked by incubating the slides in a 1:5 dilution of nonimmune rabbit serum (NRS; Diagnostics Scotland, Carluke, UK) in TBS containing 5% BSA (NRS/TBS/BSA; Sigma, Poole, UK). Sections were incubated at 4 C overnight in either a 1:100 dilution of anti-hERß1 antibody or a 1:50 dilution of anti-hERßcx/ß2 antibody made up in NRS/TBS/BSA. Antibody binding was detected by applying a 1:500 dilution of biotinylated rabbit antimouse antibody (DAKO Corp.) in NRS/TBS/BSA, followed by an avidin-biotin peroxidase solution (DAKO Corp.), both for 60 min at room temperature. Slides were then incubated in 3,3'-diaminobenzidine (DAKO Corp.) before counterstaining in Harris’s hematoxylin (Pioneer Research Chemicals Ltd., Colchester, UK), dehydrating, and mounting with Pertex (Cellpath plc, Hemel Hempstead, UK). On negative controls the primary antibody was replaced with NRS/TBS/BSA.
Scoring of immunoreactivity
The immunostaining intensity of ERß1 and ERßcx/ß2 proteins in human endometrium was assessed in a semiquantitative manner on a four-point scale. All tissue sections were scored blind by two observers: 0 = no staining, 1 = mild staining, 2 = moderate immunostaining, and 3 = intense immunostaining. We have previously validated this scoring system (30) in a subset of tissue sections in which immunoreactivity was measured with a computerized image analysis system, and a strong correlation between quantitative data derived from image analysis and subjective scores by a trained observer was obtained. Nonparametric statistical analysis was conducted using the Kruskal-Wallis nonparametric test (Instat, GraphPad Software, Inc., San Diego, CA), and Dunn’s test was used for post hoc comparisons.
Semiquantitative analysis of ERß mRNAs by RT-PCR
RNA was extracted from endometrial tissues using TRIzol RNA isolation reagent (Invitrogen, Paisley, UK) according to the manufacturer’s instructions and dissolved in ribonuclease-free water, and 5 µg total RNA were subjected to RT using Expand Reverse Transcriptase (Roche, Lewes, UK) and an oligo(deoxythymidine). cDNA was purified by heating to 100 C for 5 min, ribonuclease treatment at 37 C for 15 min, and finally by passing through a High Pure PCR Purification column (Roche). Purified cDNAs were quantified on Pharmacia Biotech Genequant (St. Albans, UK) and adjusted to a final concentration of 5 ng/µl in TE buffer [10 mM Tris-HCl (pH 7.5) and 1 mM EDTA]. PCRs were performed using AGS Gold Taq (Hybaid, Ashford, UK) with the following primers: ERß 5' common forward primer (5'-AGGAGTTGGTACACATGATCAG; exon 4) used with either ERß1-specific (exon 8; 5'-CACTGAGACTGTGGGTTCTGGGA) or ERßcx/ß2-specific (exon 8v; 5'-CACTGCTCCATCGTTGCTTC) reverse primers. The expected sizes of the amplified products from full-length transcripts were: ERß1 682 bp; and ERßcx/ß2, 577 bp. All samples were tested using primers directed against glyceraldehyde-3-phosphate dehydrogenase (5'-GAAC GGGAAGCTCACTGGCAT and 5'-GTCCACCACCCTGTTGCTGTAG; 240 bp). PCR conditions were as follows: 1 cycle of 94 C for 2 min, followed by 30 cycles of 94 C for 30 sec, 58 C for 30 sec, and 72 C for 45 sec, with a final cycle of 72 C for 10 min, in a 0.2-ml Sprint thermal cycler (Thermo Hybaid, Middlesex, UK). PCR products were separated on 2% agarose gels using 100-bp markers (Promega Corp., Madison, WI), stained with ethidium bromide, and photographed.
Quantitative RT-PCR
Real-time quantitative PCR was used to determine the amounts of ERß1 and ERßcx/ß2 mRNA. This PCR method monitors progress of the PCR via detection of a fluorescent signal released, by the action of Taq polymerase, from a specific probe that contains both fluorescent dye and quencher. In the present experiments the amount of specific amplicon present was related to ribosomal 18S and subsequently to an internal control.
Endometrial tissue samples were immersed in TRIzol RNA isolation reagent (Invitrogen) and homogenized, and RNA was extracted according to the manufacturer’s instructions. RNA was subjected to deoxyribonuclease (DNase) treatment using 1 U DNase 1 (amp grade)/µg RNA in DNase reaction buffer for 15 min at room temperature (Invitrogen) to remove genomic DNA contamination. The reaction was stopped by the addition of a final concentration of 2.5 mM EDTA, followed by heating to 99 C for 5 min.
Using random hexamers, 200 ng RNA were then reverse transcribed in a buffered solution containing 5.5 mM MgCl2, 2.5 µM random hexamers, 500 µM of each deoxy-NTP, 0.4 U/µl ribonuclease inhibitor, and 1.25 U/µl Multiscribe (all from PE Biosystems, Warrington, Cheshire, UK). The samples were then incubated for 60 min at 25 C, for 45 min at 48 C, and for 5 min at 95 C. Negative controls were included in every run. An RT-negative control had template RNA but no Multiscribe enzyme included, and an H2O RT had template RNA replaced by nuclease-free water.
A TaqMan real-time PCR reaction mix was then made up containing final concentrations of TaqMan universal PCR master mix (1x), ribosomal 18S forward and reverse primers and probe (50 nM; PE Biosystems), ERß1 or ERßcx/ß2 forward and reverse primers (300 nM), and ERß1 or ERß2 probe (200 nM; BioSource International, Inc., Camarillo, CA). One microliter of cDNA was added per 25 µl reaction volume, and each sample was tested in triplicate. A no template control (where water replaced cDNA) was included in every run, and the controls from the RT step were also run at least once for each set of primers and probes. Wells were sealed with optical caps, and the PCR reaction was run on ABI PRISM 7700 (PE Biosystems) using standard conditions.
Specific primers and probes for ERß1 and ERßcx/ß2 were designed using the Primer express program (PE Biosystems); in all cases they were chosen to span an intron to further reduce the chance of spurious readings due to genomic DNA contamination (Table 2). The 18S primers and probe were purchased from PE Biosystems. The linearity of the response of the primers and probe to specific cDNA was validated using serial dilutions of a cDNA sample, and within-assay variation of the PCR measurement of ERß in cDNA was calculated from six replicates. Significant differences were determined by one-way ANOVA, and individual differences were described using the least squares differences post hoc multiple comparison (SPSS, Inc., Chicago, IL).
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Expression of ERß1 and ERßcx/ß2 mRNAs in human endometrium
Analysis of cDNAs prepared from extracts of endometrium recovered at different stages of the menstrual cycle revealed that mRNAs encoding ERß1 and ERßcx/ß2 were both present in all samples examined (Fig. 1). Preliminary analysis using a semiquantitative approach suggested that levels of expression of ERßcx/ß2 were similar in all samples, but those of ERß1 appeared to vary with less mRNA during the early secretory phase.
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; levels of both ERß1 and ERßcx/ß2 mRNAs were significantly increased in samples obtained during the late secretory phase compared with other stages of the cycle (P < 0.05). For example, ERß1 mRNA was 1.73 times higher, and ERßcx/ß2 1.52 times higher than levels in the early proliferative phase (Fig. 2, A and B, respectively). The overall pattern of change in the levels of mRNAs for both ERß1 and ERßcx/ß2 followed a similar pattern, with lowest levels during the late proliferative/early secretory phases of the cycle. Immunoexpression of ERß1 and ERßcx/ß2 in human endometrium throughout the normal menstrual cycle
To determine spatial and temporal expression of ERß1 and ERßcx/ß2 proteins isotype-specific monoclonal antibodies were used to perform immunohistochemistry. Both isoforms were immunolocalized exclusively to cell nuclei and were expressed in both cells lining the glands (G) and in some, but not all, cells in the stroma within both the functional (Fig. 3) and basal layers (Fig. 4). Within the functional layer the immunointensity of staining for ERß1 appeared higher than ERßcx/ß2 in the glandular epithelium (compare Fig. 3, a with c) in all samples and was particularly marked in the mid secretory phase (Fig. 3, b compared with d). Differences between the intensity of staining of individual nuclei within the stroma of the same samples was not apparent. Whereas levels of ERß1 protein in the glandular epithelium appeared to vary little between samples obtained at different stages of the cycle (Fig. 3, b compared with a), the amount of ERßcx/ß2 protein detected in glandular epithelial cells was reduced in the glandular epithelium of the functional layer during the midsecretory phase (Fig. 3, d compared with c). Within the basal layer (Fig. 4) immunoexpression of ERß1 was generally more intense than that of ERßcx/ß2, and this difference was most striking within the glandular epithelium. Levels of expression of ERß1 and ERßcx/ß2 proteins within the basal region did not appear to vary across the cycle.
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) revealed that immunostaining for ERß1 was intense in endometrial endothelial cells across the cycle in both the functional and basal layers (Fig. 4a). In contrast, endometrial endothelial cells displayed a heterogeneous pattern of ERßcx/ß2 immunostaining ranging from immunonegative (indent arrowheads) to immunopositive (arrowheads and arrows; Fig. 5b). In agreement with previous data (1) we did not detect immunoexpression of ER (Fig. 5c).
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). In the functional layer immunostaining scores for ERßcx/ß2 were significantly reduced in the glands in the midsecretory phase (Fig. 6B), but remained at a persistent intensity in the endothelial (Fig. 6C) and stromal compartment (not shown). No significant pattern of change was noted in levels of expression of ERß1 protein in the functional layer of the same tissue set (Fig. 6, A and C). In the basal layer of endometrium, no significant pattern of change in intensity of immunoexpression of ERß1 or ERßcx/ß2 was noted (not shown).
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The endometrium is a target organ for estrogen action. The characteristic morphological and functional changes that are a feature of the normal menstrual cycle are the consequence of the sequential action of estrogen and progesterone on endometrial cellular components: epithelium, stroma, endothelium, and bone marrow-derived cells in the stroma. Several of the fundamental processes involved in normal endometrial function, for example proliferation, are regulated by estrogen, and estrogen also plays an important role in the vascularization of the endometrium.
We (1) and others (5) have reported that both ERß and ER are expressed in human endometrial tissues. One finding of our own studies was that there was a striking contrast between the pattern of expression of ER and ERß in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERß was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We have previously suggested that endometrial endothelial cells may be targets for agonists or antagonists that selectively target the ß form of ER (31). However, the studies we undertook employed a polyclonal antibody directed against a peptide within the hinge domain of ERß (P4) (28) and subsequent alignment of ERß1 and ERßcx/ß2 peptide sequences has revealed that this peptide is present in both isoforms. Therefore, we cannot rule out the possibility that some of the ERß protein previously detected could be due to expression of the truncated ERßcx/ß2 variant isoforms and not to full-length ERß1, and we have therefore undertaken a new study to examine whether ERßcx/ß2 as well as ERß1 mRNA and protein are expressed in human endometrium.
In agreement with a single sample analyzed by RTPCR and shown in the paper by Moore et al. (21), we found that mRNAs corresponding to both ERß1 and ERßcx/ß2 could be detected in extracts of human endometrial tissue. Using a semiquantitative approach ERßcx/ß2 was detected in all samples regardless of the stage of the cycle, and a small proportion of the mRNA appeared to lack exon 5 sequences; a similar exon 5-deleted form has been reported for ERß1 (24). Using quantitative TaqMan RT-PCR, we found that although the amounts of both mRNAs in total endometrial extracts were low, the highest levels of both mRNAs were present in the late secretory phase. The pattern of expression of mRNAs for ERß1 and ERßcx/ß2 appeared to parallel each other. Previous studies have used both semiquantitative and quantitative RT-PCR (32, 33) or in situ hybridization (34) approaches to determine whether ERß mRNA is expressed in the human endometrium. Investigators have generally reported that amounts of ERß mRNA in endometrium are lower than those of ER (33, 34, 35), and where the stage of the cycle has been included in the analysis the researchers have suggested that ERß mRNAs are lower in the secretory compared with the proliferative phase. In all these reports the probes or primers used would not have discriminated between the ERß1 and ERßcx/ß2 isoforms.
The reason for the increased levels of mRNA observed in the extracts at the end of the secretory phase is not known. However, we do know that there is an influx of bone marrow-derived cells into endometrium during the mid to late luteal phase and the numbers of uterine natural killer cells increase (36). Recently, Stygar et al. (37) have described the coexpression of ERß with CD45 leukocyte common antigen and CD68 macrophage-specific antigen in blood cells infiltrating the cervix during pregnancy. In the current study we observed that in tissue sections of superficial endometrium, particularly in samples collected in the late secretory phase, there was strong immunoreactivity for ERßcx/ß2 in selected cells within the epithelium of the glands and in the stromal compartment. Further studies are now underway to establish whether these cells are bone marrow derived.
In previous studies on the immunoexpression of ERß we have been able to detect significant amounts of nuclear protein even in tissues such as the human and rodent endometrium (1) where levels of mRNA are reported to be low (35). Here we have demonstrated that both ERß1 and ERßcx/ß2 proteins can be detected in cell nuclei within the human endometrium. We found that the immunointensity of staining for ERß1 is generally higher than that of ERßcx/ß2, and this difference was consistently observed in glandular epithelial and endothelial cells. Although it can be dangerous to make direct comparisons due to differences in the relative affinities of different antibodies, we have found that in other tissues, such as the testes (29), the reverse is true. There was no significant difference in the intensity of staining in the functional and basal layers, and there was no consistent change in the level of expression of ERß1 protein at different stages of the cycle. In contrast, ERßcx/ß2 immunoreactivity was significantly reduced in gland cells of the functional layer during the midsecretory phase. Analysis of antibody specificity has confirmed that the polyclonal antibody used in our previous studies (1, 38) is able to bind equally efficiently to recombinant ERß1 and ERßcx/ß2 when tested on Western blots (our unpublished observations). Thus, the reduced expression of ERß proteins previously reported to occur in glands within the functional layer (1) may be due to reduced expression of ERßcx/ß2, as the levels of ERß1 appeared unchanged. The rise in mRNAs encoding ERß1 and ERßcx/ß2 in total endometrial extracts obtained in the late secretory phase did not appear to be reflected in the amount of protein detected by immunohistochemistry in the cellular compartments examined. We do not have an explanation for the apparent discrepancy in the findings, although it is possible that some of the mRNAs are not translated, or a small increase in intensity was not detected.
Studies in vitro have demonstrated that ER and ERß homodimers can bind estrogenic ligands and activate reporter constructs; however, ERß appears to have a 4-fold lower binding affinity for estradiol than does ER (39). Ligand binding to ERs induces a conformational change in the structure of the protein, resulting in recruitment of interacting proteins (coactivators or corepressors) that can have a profound effect on ER-mediated gene transcription (reviewed in Ref. 40). ERßcx/ß2 is formed by alternative splicing of the ERß gene, resulting in loss of 61 amino acids, including those encoding the activating factor-2 domain present in ERß1 (20). As a result when expressed in vitro the ERßcx/ß2 does not bind estradiol (20), and when coexpressed with ER it exhibited dominant negative activity (20). However, using a gel-shift assay Moore et al. (21) have shown that not only can recombinant ERßcx/ß2 protein bind to the ERE DNA sequence, but that it can also form heterodimers with ER or ERß1 proteins when binding to the same sequence. These findings suggest that the expression of ERßcx/ß2 in cells containing ERß1 or ER might result in binding of heterodimeric receptor complexes on the response elements of estrogen-responsive genes and, taking into account the data in the study by Ogawa et al. (20), might reduce gene activation by the wild-type receptors. The relative role(s) played at a cellular level by the different ER subtypes (ER, ERß1, and ERßcx/ß2) in regulation of endometrial function during the normal menstrual cycle remains to be elucidated and may depend upon local metabolism of estrogenic ligands as well as the relative abundance of the receptor subtypes.
Estradiol is involved in the regulation of a number of genes within the human endometrium including PR, vascular endothelial growth factor (VEGF), and lactoferrin (41, 42, 43). Up-regulation of PR during the proliferative phase is consistent with the identification of EREs within the regulatory region of the PR gene (44, 45) and is good evidence of a functional ER-mediated pathway at this time of the cycle. It is notable that VEGF is a key mediator of the cyclical neovascularization that occurs within the functional layer of the primate endometrium; VEGF mRNA has been also been reported to increase in the midproliferative phase (46). Studies in vitro using transient transfection with a human VEGF promoter construct have demonstrated that estradiol bound to either ER or ERß can induce gene expression via a variant ERE (47). The activity of heterodimeric ERs containing combinations of ER, ERß, and ERßcx/ß2 has not yet been tested using endometrial cells. In the present study detailed analysis has identified two cellular types in which estrogens may act directly via ERß1 homodimers: firstly, within the majority of endothelial cells, and secondly, in the glandular epithelium during the mid/late secretory phase. The identification of ERß1-specific selective estrogen receptor modulators has been reported, and these cells would be potential targets for their actions (31, 48).
In summary, we have demonstrated that in addition to ER (1), both ERß1, the functional wild-type receptor, and ERßcx/ß2, a splice variant receptor isoform that is devoid of the ability to bind estradiol, are expressed in human endometrium. It is therefore possible that the expression of ERßcx/ß2 may influence the ability of some endometrial cells to respond to either endogenous or exogenous estrogenic ligands.
Acknowledgments
We thank Mike Millar and Sheila Macpherson for technical advice on immunohistochemistry, and Prof. David Baird for incisive comments on the manuscript.
Footnotes
This work was supported by Medical Research Council Programme Grant G0000066.
Abbreviations: DNase, Deoxyribonuclease; ER, estrogen receptor; ERE, estrogen response element; NRS, nonimmune rabbit serum; PR, progesterone receptor; TBS, Tris-buffered saline; VEGF, vascular endothelium growth factor.
Received March 29, 2002.
Accepted August 5, 2002.
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 S Hombach-Klonisch, A Kehlen, P A Fowler, B Huppertz, J F Jugert, G Bischoff, E Schluter, J Buchmann, and T Klonisch Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells J. Mol. Endocrinol., April 1, 2005; 34(2): 517 - 534. [Abstract] [Full Text] [PDF]
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G. Krikun, F. Schatz, R. Taylor, H. O. D. Critchley, P. A. W. Rogers, J. Huang, and C. J. Lockwood Endometrial Endothelial Cell Steroid Receptor Expression and Steroid Effects on Gene Expression J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1812 - 1818. [Abstract] [Full Text] [PDF]
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M. H. Herynk and S. A. W. Fuqua Estrogen Receptor Mutations in Human Disease Endocr. Rev., December 1, 2004; 25(6): 869 - 898. [Abstract] [Full Text] [PDF]
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U. A. Kayisli, J. Luk, O. Guzeloglu-Kayisli, Y. Seval, R. Demir, and A. Arici Regulation of Angiogenic Activity of Human Endometrial Endothelial Cells in Culture by Ovarian Steroids J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5794 - 5802. [Abstract] [Full Text] [PDF]
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T. A. Henderson, P. T. K. Saunders, A. Moffett-King, N. P. Groome, and H. O. D. Critchley Steroid Receptor Expression in Uterine Natural Killer Cells J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 440 - 449. [Abstract] [Full Text] [PDF]
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