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Endometrial Endothelial Cell Steroid Receptor Expression and Steroid Effects on Gene Expression

Department of Obstetrics, Gynecology, and Reproductive Sciences (G.K., F.S., J.H., C.J.L.), Yale University School of Medicine, New Haven, Connecticut 06520-8063; Center for Reproductive Sciences (R.T.), University of California, San Francisco, California 94143; Reproductive and Developmental Sciences (H.O.D.C.), Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom; and Centre for Women’s Health Research (P.A.W.R.), Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Graciela Krikun, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: graciela.krikun{at}yale.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Controversy exists regarding the expression of specific steroid receptor proteins and mRNA in human microvascular endometrial endothelial cells (HEECs). Thus, we studied steroid receptor expression in early passaged HEEC cultures and freshly isolated HEECs. Analysis of estrogen receptor (ER) and progesterone receptor (PR) mRNA levels was carried out with real-time quantitative RT-PCR, and the repertoire of genes activated by their respective steroid ligands was assessed by mRNA microarray analyses of 18,400 genes and expressed sequence tags. We observed that cultured and freshly isolated HEECs each express ER-ß mRNA but not ER-. In addition, PR mRNA was also detectable in both HEEC sources. Microarray analysis demonstrated that treatment of HEEC cultures with either estradiol or medroxyprogesterone acetate produced differential effects on a wide variety of genes, and cluster analysis demonstrated that many of the genes are involved in intracellular signaling and enzymatic pathways. Thus, quantitative RT-PCR and microarray analyses demonstrate that HEECs express ER-ß and PR mRNA and that gene expression by HEECs is differentially regulated by treatment with estrogen or progestin.

	Introduction

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LONG-TERM PROGESTIN-ONLY contraceptives such as Implanon (Organon, Oss, The Netherlands) and Depo-Provera (Depo Pharmacia, North Peapack, NJ) are extremely effective and safe (1, 2, 3). Unfortunately, continued compliance with these agents is limited by the high prevalence of unpredictable breakthrough uterine bleeding. Such bleeding arises from aberrant endometrial angiogenesis manifested by increased numbers of superficial, dilated, thinned walled vessels (4, 5, 6). However, it is uncertain whether this aberrant angiogenesis results from progestin effects mediated by its receptor on endothelial cells or by progestin effects on perivascular stromal cells, which in turn act in paracrine effectors on adjacent endothelial cells. Under normal circumstances, the human endometrium undergoes characteristic changes during the menstrual cycle under the strict regulation of steroid hormones acting via their cognate receptors. There are two dominant forms of the estrogen receptor (ER), and ß, and two dominant forms of the progesterone receptor (PR), A and B (7, 8, 9, 10, 11, 12). Although the expression of these receptors has been clearly identified in the endometrial glandular epithelium and stromal cells (7, 8, 9, 10, 11, 12, 13), evidence of their presence in the endometrial microvasculature has been the subject of conflicting reports in the literature. Critchley et al. (13) demonstrated immunoreactive ER-ß, but not ER-, in the nuclei of endothelial cells from most spiral arteries, capillaries, and veins of the human and nonhuman primate endometrium. Lecce et al. (11) also presented immunohistochemical evidence that ER-ß was present in endothelial cells, in addition to the nuclei of glandular epithelial and stromal cells. Iruela-Arispe et al. (14) reported positive ER and PR protein and mRNA in passaged (x5) endometrial endothelial cell cultures; however, these authors did not discriminate between ER isoforms. In contrast, Press et al. (15) reported that immunoreactive PR could not be detected in endometrial endothelial cells. Thus, we sought to confirm the nature of ovarian steroid receptor expression in human microvascular endometrial endothelial cells (HEECs) and to ascertain whether these steroids resulted in a differential gene expression profile.

	Subjects and Methods

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Human endometrial cell isolation

Endometrial tissue specimens were obtained from women of reproductive age undergoing hysterectomies for myomas who were not receiving hormonal therapy. Tissues obtained were either from the proliferative or the secretory phase of the menstrual cycle. All subjects provided written informed consent and institutional approval from the Human Investigation Committee of the Yale-New Haven Hospital (New Haven, CT). Endometrial tissue was transported to a sterile laminar flow hood, and isolation of the component cells was conducted as follows. HEECs from our laboratory were isolated as previously described (16) and either directly frozen for studies on noncultured cells or cultured and grown to confluence on flasks coated with 2% gelatin in EGM-2 MV Singlequot Medium with 5% stripped fetal calf serum (Cambrex Bio Science, Inc., Walkersville, MD). The cells were harvested by trypsin-EDTA and split 1:6 for three to five passages.

For the purposes of confirming the results obtained with HEECs isolated in our laboratory, we also obtained HEECs isolated in the laboratory of Dr. Peter Rogers that were sent for analysis to our laboratory in Yale University. Dr. Rogers prepared the cells as follows. Endometrial tissue was digested as described by Schatz et al. (16) and then trypsinized and pelleted. Red blood cells were removed by resuspension of the cells in 10 ml PBS/BSA solution and overlayed with Ficoll Paque (Amersham Biosciences, Buckinghamshire, UK). This suspension was centrifuged, and interface cells were carefully removed and washed twice with 30 ml PBS/BSA solution. Ulex europa lectin-specific-coated Dynabeads (Dynal Biotech Inc., Brown Deer, WI) were added at four beads per cell in a 100- to 200-µl vol in a 0.65-ml tube and rotated for 1 h at 4 C. Dynal-MPC (Dynal Biotech Inc.) was used to collect the bead-coated cells, washed, and microscopically checked for purity.

Human endometrial cell cultures

Passaged confluent cells were maintained in EGM-2MV medium to which was added vehicle control, estradiol (E₂; 10^–8 M), or medroxyprogesterone acetate (10^–7 M) for 48 h. Primary human endometrial stromal, glandular epithelial, and decidual cells (DCs) were obtained and grown to confluence and shown to be of high purity as previously described (17, 18).

mRNA analyses

Total RNA was isolated with Tri Reagent (Sigma-Aldrich, St. Louis, MO) as previously described (18). After optimization of all probes by semiquantitative RT-PCR, real time-quantitative RT-PCR was conducted as follows. RT was initially carried out with avian myeloblastosis virus reverse transcriptase (Invitrogen, Carlsbad, CA). A quantitative standard curve was then created using a range of 500 pg to 250 ng cDNA. The curve was created with the Roche Light Cycler (Roche, Indianapolis, IN) by monitoring the increasing fluorescence of PCR products during amplification. Once the standard curve was established, quantitation of the unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of ß-actin from these same unknowns. Melting curve analysis was conducted to determine the specificity of the amplified products and the absence of primer-dimer formation. All products obtained yielded the correct melting temperature.

The primers shown below were previously described (20, 21, 22), and the ER-ß primers were synthesized and gel purified at the Yale DNA Synthesis Laboratory, Critical Technologies.

Microarray analysis

Cells were harvested from a Falcon T-25 flask with QIAzol lysis reagent and total RNA isolated according to the manufacturer’s instruction (Qiagen, Valencia, CA). A total of 100 µg RNA was cleaned and precipitated using RNeasy Mini Kit (Qiagen) to prepare the template for cDNA synthesis. A T7-(dT)24 oligo-primer was used to synthesize double-stranded cDNA by the SuperScript Choice System (Invitrogen), which was subsequently cleaned up by Phase Lock Gels-Phenol/Chloroform extraction and ethanol precipitation. The ENZO BioArray High Yield RNA Transcript Labeling Kit (T7) (ENZO, Farmingdale, NY) was used to generate biotinylated cRNA. Additional cRNA clean-up was carried out by RNeasy Mini Kit before the fragmentation of biotinylated cRNAs with 5x fragmentation buffer (200 mM Tris, pH 8.1; 500 mM KOAc; 150 mM MgOAc). The chemically fragmented cRNAs were then hybridized on Affymetrix (Santa Clara, CA) HG-U133A 2.0 human chips, screening for 18,400 human genes and expressed sequence tags, followed by fluorescence labeling and optical scanning.

Raw data without normalization generated from Affymetrix GeneChip Operating Software Version 1.1.1 (Affymetrix) were analyzed by GeneSpring software 6.1 (Silicon Genetics, Redwood City, CA). The gene readouts were normalized to the 50th percentile of the distribution of all measurements in each chip. Per-gene normalization was performed using the median value of each gene throughout different chips in the same experimental condition. The normalized data were first filtered based on flag calls on each transcript to eliminate genes that were absent in all experimental conditions. Fold ratios were derived from comparing normalized data between control and treatment groups that were up- or down-regulated more than 4.0-fold by E₂ or medroxyprogesterone acetate (MPA). A Venn diagram was used to generate gene lists regulated by E₂ or MPA or both steroids.

Immunohistochemical analysis

Immunostaining for ER-, ER-ß, and PR was conducted as previously described in frozen endometrial tissue specimens (13). Endometrial tissue was collected from women undergoing hysterectomy as described above. All women reported regular menstrual cycles (25–35 d) and had not received exogenous hormones or used an intrauterine device in the 3 months before inclusion in the study. Samples were fixed overnight at 4 C in 4% paraformaldehyde, rinsed, and stored in 70% ethanol before routine processing into paraffin. Menstrual cycle phase was confirmed by histological dating and by serum samples taken at the time of hysterectomy for determination of circulating progesterone and E₂ levels by RIA.

Tissue sections were dewaxed, rehydrated, and subjected to antigen retrieval either in a microwave oven for ER- and PR or in a pressure cooker for ER-ß. For ER-, the sections were incubated at 37 C for 60 min with a 1:400 dilution of mouse monoclonal antibody clone 1D5 (Dako Corp., Cambridge, UK). Negative controls were performed by replacing the primary antibody with mouse IgG at a matched concentration. For ER-ß, the sections were incubated overnight at 4 C at a 1:800 dilution of anti-hER-ß antibody (anti-hER-ß1, MCA 1974S; Serotec Ltd., Oxford, UK). The negative control step involved incubation of prediluted antibody with an excess of the unconjugated form of the peptide used for immunization overnight at 4 C.

For PR, sections were incubated with primary antibody mouse monoclonal antibody (NCL-PgR, NovoCastra Laboratories Ltd., Newcastle, UK) at a dilution of 1:40 for 60 min at 37 C. This antibody recognizes both isoforms of PR (PR-A and PR-B). Negative controls were performed by replacing the primary antibody with mouse IgG at a matched antibody concentration. Visualization of epitopes was with chromagen 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, CA).

	Results

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Detection of steroid receptor mRNA

The presence of ER- was clearly demonstrated after quantitative RT-PCR from RNA extracted from three separate preparations of cultured endometrial stromal cells and endometrial glands. Figure 1, top, shows the melting curve analysis for ER- for the stromal cells and glands. Correct product formation was demonstrated from all preparations, and sequencing at the correct molecular weight band confirmed the identity of ER- after RT-PCR. In contrast, Fig. 1, bottom, shows that no ER- product is present in six different preparations of cultured endothelial cells as demonstrated by the lack of a specific melting curve. Because of the possibility that culturing and passaging cells may affect the levels of ER- expression, total RNA was isolated from uncultured and, hence, unpassaged HEECs and analyzed by quantitative RT-PCR. Again, no ER- mRNA was observed (data not shown).

View larger version (158K): [in this window] [in a new window]   FIG. 1. Melting curve after quantitative RT-PCR for ER-. Top, Melting curve for cultured HESCs, HEGEs, positive control (Pos Ctl; green line), and negative control in which the RT step was omitted (Bl, orange line). Bottom, Melting curve analysis for cultured HEECs. HEGE, Human endometrial glandular cells.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Real-time quantitative RT-PCR for ER-. The expression of ER- was compared in cultured and unpassaged/uncultured endometrial cell. The cultured HEECs were obtained from our laboratory as well as from Dr. Roger’s laboratory. The expression of the receptor is expressed as a ratio to ß-actin used as an internal control.

We next compared the expression of ER-ß in low-passage cultured and freshly isolated HEECs with that of cultured endometrial stromal and glandular epithelial cells by quantitative real- time RT-PCR. All cells demonstrated the presence of ER-ß (Fig. 3), and all were observed within the appropriate melt curve (data not shown).

View larger version (8K): [in this window] [in a new window]   FIG. 3. Real-time quantitative RT-PCR for ER-ß. The expression of ER-ß was compared in cultured and unpassaged/uncultured endometrial cells as in Fig. 2. The expression of the receptor is expressed as a ratio to ß-actin used as an internal control.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Real-time quantitative RT-PCR for PR. The expression of PR was compared in cultured and unpassaged/uncultured endometrial cells as in Fig. 2. The expression of the receptor is expressed as a ratio to ß-actin used as an internal control.

The finding that ER-ß and PR mRNA were expressed in cultured HEECs led us to analyze the expression of these proteins by immunohistochemistry of endometrial sections. In this case, we did not observe PR or ER- expression by the endothelial or glandular epithelial cells but did observe low levels in the surrounding stromal cells. In contrast, high levels of ER-ß were present in the glandular epithelium, the stromal cells, and the endometrial endothelial cells (Fig. 5).

View larger version (109K): [in this window] [in a new window]   FIG. 5. Expression of steroid receptors in HEECs in midsecretory endometrium. A, PR is present in the stromal cells (S) and absent in both the glandular epithelium (G) and the endometrial endothelial cells (arrows). B, ER- is present in some endometrial stromal cells and absent in the glandular epithelium and endometrial endothelial cells (arrows). C, ER-ß is present in the glandular epithelium, the stromal cells, and the endometrial endothelial cells (arrows). Magnification, x40; scale bar, 50 µm.

Given our findings demonstrating that cultured HEECs expressed PR and ER-ß mRNA, we investigated the differential effects of estrogen and progestin in these cells. Hence, cultured HEECs were treated for 2 d with vehicle control or with 10^–8 M E₂ or 10^–7 M MPA, and total RNA was extracted and analyzed by microarray analysis. Compared with controls, several differences were observed for the E₂- or MPA-treated HEECs. It was interesting to note that, although present, no steroidal regulation of the classic angiogenic factors was observed (Table 1). These factors, namely vascular endothelial growth factor, basic fibroblast growth factor, and angiopoietin-2, as well as their corresponding receptors, have been implicated in aberrant endometrial angiogenesis in patients receiving long-term progestin-only contraceptives (23, 24, 25, 26). Consistent with our previous findings on the protein expression of angiopoietins, both treatments with E₂ or MPA resulted in absent calls for angiopoietin-1 and present calls for angiopoietin-2 in HEECs (22). Surprisingly, however, the vehicle control HEECs received a present call for angiopoietin-1.

Finally, Table 2 limits the analysis to clusters of genes regulating specific pathways and enzyme systems that were either up- or down-regulated more than 4-fold by E₂ or MPA. It is interesting that many of these genes were reported to be similarly regulated by estrogen or progestin in other systems (Table 2), including the following: transformation gene ERBB-3, creatine kinase, cytochrome P450CYP3A4, thrombin-activatable fibrinolysis inhibitor, arylsulfatase D, arachidonate 12-lipoxygenase, tyrosine hydroxylase, manganese superoxide dismutase, and human placental alkaline phosphatase-like and estrogen sulfotransferase (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Additionally, when compared with isolated endometrial DCs, the HEECs showed classic expression of endothelial cell markers such as von Willebrand factor, platelet endothelial cell adhesion molecule (CD31), and Tie-1 and Tie-2 (Table 3). The latter was also expressed by DCs. In contrast, the HEECs did not express DC markers such as CXCL12, prolactin, or IGFBP-5 (Table 3). Quantitative RT-PCR analysis confirmed the presence or absence of several of these endpoints (data not shown). These findings demonstrate the purity of the isolated endothelial cells.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

We have demonstrated independent effects of estrogen and progestin on these cells using microarray assessments of mRNA expression. Interestingly, we found no effects of either steroid on endothelial expression of angiogenic factors (Table 1) whose dysregulation has been associated with the abnormal uterine bleeding found in patients receiving long-term progestin-only contraceptives as well as a variety of other uterine pathological states. Indeed, prior studies have shown that hypoxia and/or thrombin enhanced endometrial stromal and glandular epithelial expression of vascular endothelial growth factor and angiopoietin-2 while suppressing angiopoietin-1 (17, 19, 23).

In contrast, the finding that treatment of HEECs with estrogen or progestin resulted in differential expression on other genes suggests that the ER-ß and PR may indeed be functionally active. Analysis of gene expression by pathway-clustering suggests that steroid hormones exert potent effects on many unexpected endpoints including MAPK-kinase-kinase 8, suggesting that intermediate signaling pathways are activated by ovarian steroids in HEECs. Interestingly, previous experiments from our laboratory have shown that endometria from women treated with long-term progestin contraceptives displayed enhanced expression of stress-induced kinases (23).

Our findings regarding the expression of steroid receptors in endometrial endothelial cells have broad clinical implications. Although no changes were observed on classic angiogenic genes, the induction of intermediate signaling and enzymatic pathways suggests that steroids may act synergistically with paracrine mediators or act indirectly to mediate downstream angiogenic pathways.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants RO1HD33937-06 (to C.J.L.) and RO1 HL70004-01A1 (to C.J.L.), Medical Research Council Programme Grant 0000066 (to H.O.D.C.), project grants from the World Health Organization (Grant A05273 to P.A.W.R.), and the Australian National Health and Medical Research Council (Grant 143805 to P.A.W.R.).

First Published Online December 21, 2004

Abbreviations: DC, Decidual cell; E₂, estradiol; ER, estrogen receptor; HEEC, human microvascular endometrial endothelial cell; HESC, human endometrial stromal cells; MPA, medroxyprogesterone acetate; PR, progesterone receptor.

Received September 13, 2004.

Accepted December 2, 2004.

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