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Department of Obstetrics and Gynecology (O.B., T.T.), Division of Reproductive Endocrinology and Infertility, University of Florida College of Medicine, Gainesville, Florida 32610-0294; Departments of Obstetrics and Gynecology (B.R.C., R.A.W., C.R.M.), Endocrinology and Metabolism (R.J.A.), and Biochemistry (D.B.H., C.R.M.), The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9032
Address all correspondence and requests for reprints to: Orhan Bukulmez, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, Florida 32610-0294. E-mail: obukulmez{at}obgyn.ufl.edu.
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Abstract |
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Objective: The objective of the study was to determine whether androstenedione (A4), the predominant sex steroid in peritoneal fluid, regulates endometrial aromatase expression.
Design: This was a cell/tissue culture study.
Setting: The study was conducted at an academic center.
Methods: Quantitative real-time PCR, HPLC, and chromatin immunoprecipitation were used in this study.
Results: Treatment of cultured human endometrial explants and stromal cells with A4 (10 nM) significantly up-regulated expression of aromatase mRNA transcripts containing exon IIa at their 5'-ends. In endometrial stromal cells and the human endometrial surface epithelial (HES) cell line, induction of aromatase mRNA by A4 was associated with increased expression of SF-1. In HES cells, tritiated A4 was metabolized to estradiol, testosterone (T), dihydrotestosterone, and androstanediol. Both estradiol and T, but not nonaromatizable androgens, up-regulated aromatase and SF-1 mRNA in HES cells. Chromatin immunoprecipitation revealed that A4 enhanced recruitment of SF-1 to its response element (–136 bp) upstream of CYP19 exon IIa. This, together with the findings that both estrogen receptor antagonist, ICI 182,780, and aromatase inhibitor, fadrozole, suppressed A4 and T induction of aromatase and SF-1 mRNA, indicates that the inductive effects of A4 and T are mediated by their conversion to estrogens.
Conclusions: Exposure of endometrial cells to A4 may enhance CYP19 gene expression through its aromatization to estrogens.
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Introduction |
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Both androstenedione [4-androsten 3,17-dione (A4)] and testosterone (T) serve as substrates for the aromatization reaction and are converted to estrone and E2, respectively. Further conversion of estrone to highly potent E2 is catalyzed by 17β-hydroxysteroid dehydrogenase type I. Type III 17β-hydroxysteroid dehydrogenase catalyzes the conversion of A4 to T (6). Female peritoneal fluid (PF) contains relatively high concentrations of A4 (
10 nM), compared with other steroids. In PF, E2 is present at 0.8–0.9 nM and T is found at 0.4–1.5 nM (7, 8), whereas PF levels of albumin and SHBG are lower than in plasma. Thus, concentrations of bioavailable steroid hormones are greater in PF than in the systemic circulation (9).
Human aromatase is transcribed from a single gene, CYP19 (10), localized on chromosome 15q21 (11). CYP19 contains a number of alternative first exons that encode the 5'-untranslated regions of aromatase mRNAs that are transcribed in a tissue-specific manner and alternatively spliced onto a common junction upstream of the ATG translation start site (4). Tissue-specific promoters lie upstream of these tissue-specific first exons. In the ovary and breast tumors, the start site of the transcription (in exon IIa) lies proximal to exon II, which contains the start site of translation (12). In breast tumors, aromatase transcripts also contain untranslated exon I.3, which lies just upstream of exon IIa promoter (13). We and others have demonstrated that aromatase transcripts in both eutopic endometrium and endometriosis implants predominantly contain exon IIa at their 5'ends (2, 3, 14, 15).
The conversion of A4 and T to estrone and E2, respectively, has been reported in normal and neoplastic endometrium (16, 17). However, to date, there is limited knowledge regarding the regulation of CYP19 expression in the endometrium. Promoter analysis has revealed that CYP19 exons IIa and I.3 share common regulatory elements (15), including those for steroidogenic factor-1 (SF-1) (2, 18). Whereas elevated expression of SF-1 has been associated with enhanced aromatase expression in endometriosis (2, 14), little is known about the hormones and factors that directly influence its transcriptional up-regulation in the endometrium itself.
The objective of this investigation was to test the hypothesis that endometrial aromatase expression is up-regulated by steroids within the peritoneal cavity, in particular by A4, the major aromatase substrate in PF. Enhanced estrogen synthesis within endometrial tissue disseminated into the peritoneal cavity by retrograde menstruation may be an important initial stimulus for survival and proliferation of these cells, which may play a role in the pathogenesis of endometriosis. To address this issue, we used human endometrial explants and stromal cells in primary culture to study the effects of A4 on aromatase expression. To assess the underlying mechanisms for A4-mediated induction of aromatase, we used the human endometrial surface epithelial (HES) cell line, which was demonstrated to serve as an excellent model for studying gene expression in human endometrium (19).
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Materials and Methods |
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Proliferative phase endometrial samples were collected from women of reproductive age with no evidence of endometriosis or submucosal fibroids, either undergoing hysterectomy for benign reasons (e.g. pelvic relaxation) or during diagnostic laparoscopy/hysteroscopy. The tissue collection protocol was approved by the Institutional Review Board at University of Texas Southwestern Medical Center. Phenol red-free, serum-free D-MEM/F12 medium (Sigma-Aldrich, St. Louis, MO) with 0.1% BSA, and antibiotic/antimycotic was used in all stages of tissue preparation and explant culture. The tissues obtained were first washed in medium and cut into uniform approximately 2-mm3 pieces with a sterile scalpel blade. Then eight to 10 tissue pieces were immediately transferred onto a square sieve covered with sterile filter paper in a 6-well plate. The explants were treated and incubated for 24 h at 37 C in a humidified 95% air-5% CO2 environment. At the end of the incubation, the explants were immediately placed into TRIzol reagent (Invitrogen, Carlsbad, CA) and homogenized for RNA extraction. Treatments included vehicle (absolute EtOH American Chemical Society/United States Pharmacopeia (ACS/USP) grade-200 proof; Pharmco/Aaper, Brookfield, CT) and A4 (Sigma-Aldrich; 10 nM, dissolved in vehicle) for 24 h. The final added volume of vehicle itself and all steroids dissolved in the vehicle were 1 µl/ml of serum-free medium (0.1% EtOH).
Isolation and culture of endometrial cells
Endometrial stromal cells were isolated from a small portion of tissues obtained from premenopausal women (28–39 yr old) using the selection criteria as mentioned above.
Briefly, endometrial tissue was minced with a sterile surgical blade and digested in Hank’s balanced salt solution (Sigma-Aldrich) containing collagenase B (1 mg/ml, 15 U/mg; Roche, Indianapolis, IN), deoxyribonuclease I (0.1 mg/ml, 1500 U/mg; Roche), penicillin (200 U/ml), and streptomycin (200 mg/ml) for 60 min at 37 C with agitation. The dispersed endometrial epithelial and stromal cells were separated by filtration through a 70-µm sterile cell strainer (BD Biosciences, Bedford, MA). The filtered stromal cells were cultured in D-MEM/F-12 (Life Technologies, Inc./Invitrogen) containing fetal bovine serum (10%; Invitrogen) and 1% antibiotic/antimycotic (Life Technologies, Inc./Invitrogen) in a 95% air-5% CO2 environment at 37 C. All experiments were performed in DMEM/F12 phenol red-free, serum-free medium (Sigma-Aldrich) with routine additions of 1% antibiotic/antimycotic and 1 g/l of BSA. The cells were cultured either with vehicle or A4 (10 nM), as described for explants. Time-course experiments were performed in triplicate for 12–72 h. Given that maximum up-regulation of aromatase and SF-1 mRNAs were detected after 24 h of incubation, the majority of experiments were carried out using this incubation time.
Human endometrial surface epithelial cell line
HES cells, kindly provided by Dr. A. T. Fazleabas (University of Illinois at Chicago, Chicago, IL), were used to examine some of the underlying mechanisms involved in the induction of aromatase mRNA expression in human endometrium. These cells, which were isolated from proliferative, noncancerous endometrium at hysterectomy and spontaneously immortalized during culture (20), have been reported to express ER (21). In our characterization studies, we observed that these cells expressed androgen receptor and ERβ (data not shown). HES cells also expressed aromatase predominantly via CYP19 promoter IIa. The cells were grown in DMEM/F12 with 10% fetal bovine serum and 1% antibiotic/antimycotic mixture. For experimentation, the cells were cultured at 60–70% confluence in either 6-well dishes or 10-cm tissue culture dishes in DMEM/F12 phenol red-free, serum-free medium (containing 0.1% BSA) and treated with vehicle (EtOH as described above), A4 (5–100 nM), T (10 nM), E2 (10 nM), 5
-dihydrotestosterone (DHT; 10 nM), or 5-androstane-3, 17β-diol (A-diol; 10 nM) for 24 h. HES cells incubated with A4, T, or E2 (10 nM) also were coincubated with the ER antagonist, ICI 182,780 (10 µM; Tocris Cookson Inc., Ballwin, MO), or the aromatase inhibitor, fadrozole (25 µM, formestane, F2552–16; Sigma).
HPLC
HES cells were cultured for 24 h in serum-free medium in 6-well plates in triplicate, in the presence of 10 nM tritiated A4 (NET-469; androst-4-ene-3, 17-dione, [1,2,6,7-3H]; PerkinElmer, Waltham, MA) or 10 nM cold A4 or vehicle, or no treatment. The medium from each well was analyzed to evaluate the metabolites of 10 nM of tritiated A4.
HPLC was performed by injecting aliquots into a Breeze model 1525 HPLC pump system equipped with model 717 plus autoinjector (Waters Corp., Milford, MA) and a Supelco 33 x 3 mm, 3 µm C8 reverse-phase column (Sigma). The column effluent was analyzed with a model 2487 dual-wavelength UV detector set to 280 nm and a β-RAM model 3 in-line radioactivity detector (IN/US Systems, Inc., Tampa FL). The samples were separated at 30 C and a flow rate of 1 ml/min with a program that involved a stepwise gradient of 30–60% methanol in water: 30% methanol for 8 min; linear gradient to 50% methanol over 8 min; and linear gradient to 60% methanol over 1 min. The retention times in minutes were as follows: E2 and A4, 7.1 min; T, 8.5 min; DHT, 10.2 min; A-diol, 12.4 min. E2 and A4 were separated by thin-layer chromatography using plastic-backed silica gel plates and a mobile phase of 3:1 chloroform-ethyl acetate, with A4 migrating faster.
Quantitative real-time RT-PCR
Total RNA from cell and explant cultures was extracted by the one-step method of Chomczynski and Sacchi (22) using TRIzol reagent (Invitrogen). RNA was treated with deoxyribonuclease to remove any contaminating DNA, and then 4 µg of RNA was reverse transcribed using random primers and Superscript II RNaseH-reverse transcriptase (Invitrogen). The relative abundance of each mRNA product in tissue samples was determined by quantitative PCR using a modification of previously published methods (23). Primer sets directed against human CYP19 exon IIa mRNA transcripts (forward, 5'-CAGGAGCTATAGATGAACCTTTTAGGG-3'; reverse, 5'-CTTGTGTTCCTTGAC CTCAGAGG-3'), SF-1 (forward, 5'-GCCCTGAAACAGCAGAAGAA-3'; reverse, 5'-GCCCTGTCTCCAGCTTGAA-3'), and h36B4 (forward, 5'-TGCATCAGTACCCCATTCTATCA-3'; reverse, 5'-AAGGTGTAATCCGTCTCCACAGA-3') were designed using Primer Express software (PE Applied Biosystems, Foster City, CA), based on published sequences for these mRNAs.
For the quantitative analysis of mRNA expression, the ABI Prism 7700 detection system (Applied Biosystems) was employed using the DNA binding dye SYBR Green (PE Applied Biosystems) for the detection of PCR products. Thermocycling was performed as reported previously (3). We calculated the relative fold changes using the comparative cycle times method with human ribosomal protein h36B4 mRNA as the reference guide.
Chromatin Immunoprecipitation (ChIP)
HES cells were cultured for 24 h in serum-free medium, in the absence or presence of A4 (10 nM) or vehicle (three dishes/treatment, 1 x 107 cells/dish). After culture, the cells were washed once with PBS and incubated with 1% formaldehyde for 10 min at room temperature to cross-link proteins and DNA. Cross-linking was terminated by the addition of glycine (0.125 M, final concentration). ChIP was performed using a modification (24) of previously published methods (25). Briefly, precleared chromatin was aliquoted into 300-µl amounts and incubated with antibodies for SF-1 [goat polyclonal SF-1 antibody (1:100), sc-10976X; Santa Cruz Biotechnology, Santa Cruz, CA] at 4 C overnight. Two aliquots were reserved as controls: one incubated without antibody and the other with nonimmune IgG. Protein A/G Plus agarose beads (60 µl) were added to each tube, the mixtures incubated for 2 h at 4 C, and the immune complexes were collected by centrifugation. The beads containing the immunoprecipitated complexes were washed and eluted as described previously (25). Cross-linking of the eluted immunoprecipitated chromatin complexes and input controls (10% of the total soluble chromatin) was reversed by heating the samples at 65 C for 4 h. The samples were treated with proteinase K, and the DNA was purified by phenol-chloroform extraction and precipitated in EtOH overnight at –20 C. Purified DNA from samples and input controls was diluted in 10–100 µl Tris/EDTA buffer just before PCR. Real-time PCR was employed using forward (5'-TCAAGGGCAAGATGATAAGGTTC-3') and reverse (5'-AACAAGGAAGCCCAAGAAAGATC-3') primers that amplify a approximately 100-bp region surrounding the proximal SF-1 response element (–136 bp) of the ovary-specific CYP19 IIa promoter (26).
Data analysis
The data were expressed as mean ± SEM. ANOVA with Bonferroni test or Kruskal-Wallis with Mann-Whitney U test was used. The two-tailed significance level was P < 0.05.
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Results |
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Explant cultures were treated either with vehicle or A4 at 10 nM for 24 h. Notably, A4 significantly increased expression of aromatase mRNA containing exon IIa (CYP19IIa), compared with vehicle-treated control explants (Fig. 1A
, vehicle vs. A4, P = 0.019).
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In consideration of the suggested role of SF-1 in up-regulation of aromatase in endometriotic stromal cells, compared with normal endometrium (2), we analyzed the effects of A4 on aromatase and SF-1 mRNA levels in primary cultures of endometrial stromal cells after 24 h of incubation. As can be seen in Fig. 1B
, A4 (10 nM) caused a pronounced up-regulation of aromatase mRNA transcripts containing exon IIa. Interestingly, A4 also had a comparable stimulatory effect on SF-1 mRNA levels, compared with the vehicle (Fig. 1B, for CYP19IIa mRNA P = 0.028 and for SF-1 mRNA P = 0.018).
Androstenedione enhances SF-1 and aromatase expression in HES cells at physiological concentrations
HES cells were cultured for 24 h with vehicle or with increasing concentrations of A4 (5–100 nM). Similar to the experiments on endometrial explants and primary cultures of endometrial stromal cells, the most pronounced effect of A4 on aromatase (CYP19IIa) and SF-1 mRNA expression was observed in cells treated with A4 at 10 nM (Fig. 1C). With regard to aromatase expression, the differences between vehicle vs. 10 nM A4 (P = 0.006), 10 nM A4 vs. 100 nM A4 (P = 0.008), and 10 nM A4 vs. 5 nM A4 (P = 0.029) were significant. Ten nM A4 significantly up-regulated SF-1 as compared with vehicle-treated cells (P = 0.013) and cells treated with 100 nM of A4 (P = 0.007).
HES cells metabolize A4 to E2, T, DHT, and A-diol
To begin to elucidate the mechanisms for A4 stimulation of SF-1 and aromatase expression in endometrial cells, it was of interest to analyze steroid metabolites formed from A4 in cultured HES cells. HES cells were cultured in serum-free medium with 10 nM tritiated A4 for 24 h. HPLC of the medium demonstrated the presence of a number of metabolites, including E2, T, DHT, and A-diol (Fig. 2).
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HES cells were cultured for 24 h with vehicle or 10 nM A4, T, DHT, A-diol, or E2, in the absence or presence of ICI 182,780 (10 µM) or fadrozole (25 µM). A4, T, and E2 markedly induced aromatase and SF-1 mRNA expression, whereas the nonaromatizable C19-steroids, DHT, and A-diol had no effect (Fig. 3, A and B, respectively). Furthermore, the ER antagonist, ICI 182,780 blocked the stimulatory effects of A4 and E2 on aromatase and SF-1 mRNA expression (Fig. 3, A and B, respectively). The aromatase inhibitor, fadrozole suppressed the stimulatory effects of A4, T, and E2 on aromatase and SF-1 expression, albeit with significantly less efficacy than ICI 182,780 (Fig. 3, A and B). Collectively, these findings suggest that the stimulatory effects of A4 and T are mediated via their metabolism to E2 and binding to ER.
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In consideration of the stimulatory effect of A4 on SF-1 expression and the known role of SF-1 in the induction of CYP19 expression via promoter IIa (27), it was of interest to use ChIP to analyze effects of A4 on binding of endogenous SF-1 to a genomic region upstream of CYP19 exon IIa containing the proximal SF-1 response element. HES cells were treated with 10 nM A4 or vehicle for 24 h and processed for ChIP. Treatment of HES cells with A4 significantly increased the recruitment of SF-1 to CYP19IIa promoter by about 3.5-fold, compared with vehicle-treated cells (Fig. 4, P = 0.043). Nonimmune IgG controls were no different from background, based on real-time PCR cycle times values (data not shown).
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Discussion |
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To our knowledge, this is the first report to demonstrate that aromatase in human endometrium is up-regulated by A4 via its conversion to estrogen. Our findings that the ER antagonist, ICI 182,780, significantly blocked and the aromatase inhibitor, fadrozole, suppressed the stimulatory effects of A4, T, and E2 on both aromatase and SF-1 mRNA expression suggest a novel positive feedback role of locally formed estrogen in the induction of aromatase via enhanced SF-1 binding to the CYP19IIa promoter.
C19-steroids also have been reported to induce aromatase in gonadal tissues. In bovine granulosa cells, A4 was found to induce aromatase mRNA expression. Although T manifested a similar effect, DHT failed to induce aromatase (28), suggesting that aromatization of the C19-steroids to estrogen is required. Interestingly, it has been reported that E2/ER may up-regulate aromatase expression in human breast cancer cell lines via CYP19 promoter I.1 by a nongenomic mechanism involving cross talk of ER with growth factor-mediated pathways (29). On the other hand, in cultured rat epididymal cells, addition of A4, T, and DHT increased aromatase activity (30). Because DHT was as effective as A4 and T, it was postulated that androgen induction of aromatase might be mediated by androgen receptor in the epididymis (30).
Induction of aromatase by A4 in human endometrial cells may be of significance in the pathogenesis of endometriosis because A4 is the predominant sex steroid in PF at all phases of the menstrual cycle (7, 8). Moreover, A4 has been shown to increase the proliferation and survival of endometrial cells and explants collected from early secretory phase (31). Thus, it is plausible that A4 along with E2 in the PF could play a major role in enhancing aromatase expression in the endometrial tissue disseminated into the peritoneal cavity, which may be important for their proliferation and survival. The findings in this study may be more relevant to endometrial tissue detached from the uterine lining and transported to the peritoneal cavity than to peritoneal implants of endometriosis where inflammatory response may play a more important role.
It should be noted that the endometrial explants and stromal cell cultures used in the present study were obtained from proliferative phase endometrium. Proliferative phase endometrial samples were used because proliferative and early secretory phase endometrium are readily maintained in culture, whereas endometrial cells from mid- to late-secretory phase manifest poor survival in culture (32). This may be due to the fact that terminally differentiated epithelial cells are more dominant in secretory phase specimens, whereas proliferative phase tissues contain increased numbers of cells with high proliferative potential, including stem cells that can give rise to epithelial, stromal, and smooth muscle components, typical of endometriosis implants (33). It could be argued that proliferative phase endometrium is not a relevant tissue to study because it does not give rise to endometriotic implants, which are believed to be derived from secretory/menstrual endometrium by retrograde menstruation (3). However, it has been suggested that endometriosis may result from the detachment and tubal transport of endometrial cells with a higher potential for proliferation and infiltrative growth (34) and that endometriotic lesions may be derived from the basalis layer of endometrium (34, 35, 36), which can give rise to all tissue components of endometriosis implants (33).
It has been suggested that up-regulation of aromatase in endometriosis is mediated by aberrant up-regulation of SF-1 and its binding to the CYP19IIa promoter (2, 14). In this study we have demonstrated for the first time that the steroid substrates of aromatase, A4, and T, as well as E2 itself, act to enhance expression of SF-1 in human endometrium. These effects were blocked by the ER antagonist ICI 182,780 and to some extent suppressed by the aromatase inhibitor, fadrozole, suggesting the importance of ER. Although endometriotic lesions are believed to originate from the reflux of secretory endometrium, which has lower levels of ER than endometrium from the proliferative phase (36), ER immunostaining has been shown to be maintained in the basalis layer of secretory phase endometrium (36).
To date, ligand-induced activation and expression of SF-1 remains controversial (37, 38). Recently structural studies have revealed that phosphatidylinositol second messengers are bound to SF-1 and are required for maximal activity (39, 40). On the other hand, ligand-independent activation of SF-1 also has been reported (41). In the present study, we observed that SF-1 mRNA expression was up-regulated in HES cells by A4, T, and E2. Furthermore, A4 enhanced recruitment of SF-1 to CYP19IIa promoter. The intriguing finding that up-regulation of SF-1 mRNA expression by A4, T, and E2 was suppressed by either ICI 182,780 or fadrozole suggests that E2 acting via ER may directly modulate SF-1 mRNA expression in human endometrium.
In conclusion, based on our collective findings, we suggest that exposure of endometrial cells to A4 within the peritoneal cavity may cause a marked up-regulation of CYP19 mRNA expression via its metabolism to E2. The estradiol formed acts via the ER to induce expression of SF-1, which in turn further activates CYP19 promoter IIa expression resulting in increased local estrogen production. This positive feed-forward mechanism may facilitate the survival and proliferation of endometrial cells within the peritoneal cavity and play a crucial role in the pathogenesis of endometriosis.
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Footnotes |
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O.B., D.B.H., B.R.C., R.J.A., T.T., and R.A.W. have nothing to declare. C.R.M. served on scientific advisory boards for AstraZeneca (estrogens and lung cancer), Wyeth (Frontiers in Nuclear Receptor Action), and Burroughs Wellcome (preterm birth) within the past 2 yr.
First Published Online June 17, 2008
Abbreviations: A4, Androstenedione; A-diol, 5-androstane-3, 17β-diol; ChIP, Chromatin Immunoprecipitation; DHT, 5-dihydrotestosterone; E2, estradiol; ER, estrogen receptor; HES, human endometrial surface epithelial; PF, peritoneal fluid; SF-1, steroidogenic factor-1; T, testosterone.
Received February 4, 2008.
Accepted June 11, 2008.
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B activation of cyclooxygenase 2 expression. Mol Endocrinol 20:2724–2733. Cancer Res 63:3546–3555This article has been cited by other articles:
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