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Departments of Obstetrics and Gynecology (S.Y., Z.F., J.Z., B.G., M.T., S.B.), Molecular Genetics (S.Y., Z.F., J.Z., B.G., M.T., S.B.), and Pathology (K.F.), University of Illinois at Chicago, Chicago, Illinois 60612; and Department of Pathology (T.S., H.S.), Tohoku University, Sendai, 980-8575 Japan
Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Department of Obstetrics and Gynecology, University of Illinois at Chicago, 820 South Wood Street, M/C808, Chicago, Illinois 60612. E-mail: . sbulun{at}uic.edu
Abstract
In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE2 via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) ß knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBP
increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPß or C/EBP
abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBP, -ß, or -. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBP but not C/EBPß or C/EBP in endometriotic stromal cells. In contrast, C/EBPß and C/EBP but not C/EBP were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBP, -ß, and - in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPß was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBP up-regulates, whereas C/EBPß and C/EBP inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPß in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.
ENDOMETRIOSIS IS DEFINED as the presence of endometrium-like tissue outside the uterine cavity. It is a common chronic disorder affecting 1 in 10 women in the reproductive age group (1, 2). Implantation of eutopic (intrauterine) endometrium on peritoneal surfaces in the abdomen via retrograde menstruation through the uterine tubes is viewed to be the most common mechanism responsible for this disease.
Endometriosis tissue that grows in the ovaries was reported to be monoclonal and may arise through somatic mutations (3). Evidence of mutations in the tumor suppressor protein PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene in endometrioid subtype of epithelial ovarian cancer further suggests that somatic genetic alterations represent early events in the transformation of benign endometriotic cells (3, 4). These early mutations may give rise to overproduction of mitogens for endometriotic tissue. Estrogen is the best known mitogen for endometriosis.
Both circumstantial and laboratory evidence are indicative of a critical role of estrogen in the establishment and maintenance of this disease (5). The conversion of C19 steroids to estrogens by aromatase P450 (P450arom) takes place in a number of human tissues (e.g. the ovary, placenta, adipose tissue, skin, and brain) (6). We previously demonstrated significant levels of P450arom mRNA and activity in the stromal cell component of endometriotic tissues, whereas aromatase expression is either only barely detectable or most commonly absent in the eutopic endometrium (7, 8). Moreover, aromatase activity and P450arom mRNA levels in cultured endometriotic stromal cells could be induced by cAMP analogs or PGE2 to extremely high levels comparable with those found in ovarian granulosa cells or the placental syncytiotrophoblast, whereas significant levels of P450arom mRNA or enzyme activity could not be detected in eutopic endometrial stromal cells either before or after treatments with cAMP analogs or PGE2 (8). The clinical significance of local aromatase activity that is induced strikingly by PGE2 in endometriotic tissue was exemplified recently by the successful use of an aromatase inhibitor to treat an unusually aggressive case of recurrent postmenopausal endometriosis that was resistant to any other surgical or hormonal modalities of treatment (9). Therefore, aberrant P450arom expression in endometriotic tissue in contrast to eutopic endometrium accounts for local biosynthesis of estrogen that promotes the growth of these lesions and possibly mediates the resistance to conventional hormonal treatments, which is observed in a number of women with endometriosis. The molecular mechanisms that are responsible for aberrant P450arom expression may provide insights into the etiology of endometriosis and lead to identification of molecular targets for the development of novel treatment strategies. Thus, to determine the factors that stimulate P450arom expression in endometriotic stromal cells and its suppression in the eutopic endometrial stromal cells, we studied transcriptional regulation of the P450arom gene in these two cell types.
We reported previously that the high levels of baseline and cAMP-induced aromatase activity were partially mediated by steroidogenic factor-1 (SF-1) via binding of SF-1 to a cis-acting element at -136/-124 bp upstream of P450arom gene promoter II (10). We also found that the -517/-215-bp region is essential for the most striking increases in baseline activity and cAMP fold-induction. In this previous study, we have not used site-directed mutagenesis to ascertain the roles of individual cis-acting elements in promoter II. In this study, we used targeted mutations of four potential cis-acting elements to define their functions in P450arom expression. Moreover, we characterized the regulation of P50arom promoter II activity in endometriosis and endometrium by members of the CCAAT/enhancer binding protein (C/EBP) transcription factor subfamily.
Materials and Methods
Tissue acquisition and processing
Cyst walls of ovarian endometriomas (n = 8; five proliferative and three luteal phase samples) (dissected from surrounding tissue under microscope) and eutopic endometrial tissues (n = 14) were processed to culture stromal cells using a protocol previously described by Ryan et al. (11), with minor modifications (8). Eight of the eutopic endometrial samples for stromal cell cultures were obtained at the time of surgical removals of the ovarian endometriomas. The remaining six samples were obtained from endometriosis-free women undergoing hysterectomy for cervical intraepithelial neoplasia (three proliferative and three luteal phase samples). Extra-ovarian endometriosis and eutopic endometrial tissue biopsies for immunohistochemistry were obtained simultaneously from another group of women (n = 6) undergoing laparoscopy for endometriosis during both proliferative and luteral phases, as confirmed histologically. The age range of the patient population was 23–45 yr.
Stromal cell culture methodology was described previously (8, 11). Briefly, endometrial or endometriotic tissues were rinsed with sterile saline solution, minced finely, and digested with collagenase B (1 mg/ml) and deoxyribonuclease (0.1 mg/ml) at 37 C for 30–60 min. Stromal cells were then suspended in DMEM/F-12 (1:1; Life Technologies, Inc., Grand Island, NY) containing 10% FBS. Fresh suspensions of these cells were plated in 35-mm culture dishes at a cell density of 50,000/cm2 and kept in an incubator in a humidified atmosphere with 5% CO2 at 37 C. Twelve to 24 h after the attachment of stromal cells, culture medium was removed and cells were washed twice with DMEM/F-12. Then, cells were maintained in DMEM/F-12 containing 10% FBS. The medium was changed at 48-h intervals until the cells became confluent. Stromal cells were then placed in serum-free medium for 24 h. Then, cells were treated overnight in serum-free medium. Cells used for nuclear extract preparation were processed immediately. Tissues were collected as per a research protocol, and informed consent was approved by the Institutional Review Board of the University of Illinois at Chicago.
Transient transfections and luciferase assays
Endometrial and endometriotic stromal cells in primary culture were transfected using Lipofectamine Plus (Life Technologies, Inc.) with the following plasmids: 1) modified PGL3-basic luciferase reporter plasmid (1 µg) that contains serial deletion mutants of P450arom promoter II; 2) pcDNA3 expression plasmid (0.5 µg; Invitrogen, Carlsbad, CA), which contains the cDNA of either C/EBP (human), C/EBPß (human), or C/EBP (human); and 3) ß-galactosidase expression plasmid (0.2 µg; Promega Corp., Madison, WI) as an internal control for transfection efficiency. The day before transfection, stromal cells in primary culture were seeded into 35-mm dishes at 2 x 105 cells/dish. At the time of transfection, stromal cells were 80% confluent. The transfection solution was made of 200 µl OPTI-MEM I reduced serum medium containing PLUS reagent (6 µl), precomplexed DNA (1.2 µg), and 4 µl lipofectamine reagent. After transfection for 3 h in transfection solution at 37 C in 5% CO2, medium was changed to antibiotic-free DMEM/F-12 containing 10% FBS for overnight recovery. Cells were then kept in serum-free medium for 12 h. Thereafter, cells in serum-free medium were treated with 0.5 mM dibutyryl cAMP for 16 h. After treatment, transfected cells were washed twice in PBS and lysed in 250 µl luciferase lysis buffer [0.1 M potassium phosphate (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol, and 2 mM EDTA). Luciferase assays were performed in 20 µl cell lysates using a luciferase reporter assay system kit (Promega Corp.). ß-Galactosidase assays were performed on 10 µl cell lysate using the Galacto-light Chemiluminescent Reporter Assay (Tropix, Inc., Bedford, MA). Luminescence activities were measured using a LUMAT LB9507 luminometer (BERTHOLD GmbH & Co., KG, Bad Wildbad, Germany). Results are presented as the average of data from triplicate replicates. The empty luciferase vector PGL3-basic was arbitrarily assigned a unit of 1, and the rest of the results were expressed as multiples of the PGL3-basic vector. Illustrated results are representatives of five reproducible experiments on cells from five different patients.
Isolation of nuclear proteins from cells in culture
After cells were grown to confluence, media were aspirated and cells were rinsed with PBS and scraped from the dishes. All cells were combined in a 50-ml polypropylene tube and centrifuged for 3 min at 500 x g. The supernatant was removed by aspiration, and cellular pellet was resuspended in an ice-cold buffer (Cytoplasmic Extraction Reagent I, CER-I) provided by the manufacturer (Pierce Chemical Co., Rockford, IL). Resuspended cells were incubated on ice for 10 min. The cells were incubated on ice for 1 min after the addition of the second buffer (Cytoplasmic Extraction Reagent II, CER-II). Then, the third buffer (Nuclear Extraction Reagent, NER) was added to insoluble pellet fraction after being centrifuged for 5 min at 16,000 x g. The sample was then incubated on ice for 40 min and vortexed for 15 sec every 10 min. The supernatant (nuclear protein) was collected after centrifugation for 10 min at 16,000 x g. Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA), and nuclear extracts were kept in -80 C until use.
Site-directed mutagenesis
To generate serial plasmids bearing mutated consensus-binding sequences for transcription factors C/EBPs, SF-1, and cAMP response element binding protein (CREB), site-directed mutagenesis was performed using the GeneEditor in vitro site-directed mutagenesis system (Promega Corp.), as per the manufacturer’s instructions. A -517-bp promoter II/PGL3-basic construct containing wild-type -517/-16 bp of P450arom promoter II 5'-flanking DNA was used as a template for site-directed mutagenesis. Briefly, a DNA template (0.5 pmol) was denatured by 2 M NaOH and annealed with mutagenic and selection oligonucleotides in the annealing buffer. Mutant strand was synthesized in the reaction mixture containing 1x synthesis buffer, 5 U T4 DNA polymerase, and 2 U T4 DNA ligase at 37 C for 90 min. The mutagenesis reaction mixture was then used to transform BMH 71–18 mutS competent cells. These transformed competent cells were incubated in a medium containing GeneEditor antibiotic selection mix overnight to select the desired mutant plasmids. The plasmids isolated from the BMH 71-18 mutS were transformed into JM109 competent cells. The transformed JM109 competent cells were grown overnight on the LB plates containing ampicillin and GeneEditor antibiotic selection mix to further select the mutated plasmids. The disruption mutation of the binding consensus was confirmed by DNA sequencing. Consensus binding sequences for mutation and primers used were depicted in Table 1
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The nuclear extracts used for EMSA were prepared as described previously (10). Confluent cells were incubated in serum-free DMEM/F-12 for 24 h before they were processed for the extraction of nuclear proteins.
Double-stranded oligonucleotides were synthesized by Life Technologies, Inc. The double-stranded oligonucleotide probes were end-labeled with [
-32P]ATP using T4 kinase and used in EMSA after removal of unincorporated nucleotides by Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Inc. EMSAs were performed as described previously (10). Briefly, a 5-µg sample of nuclear extract was incubated with the radiolabeled double-stranded oligonucleotide probe (20,000 cpm/reaction) for 15 min at room temperature in a reaction buffer containing HEPES (20 mmol/liter, pH 7.6), KCl (75 mmol/liter), EDTA (0.2 mmol/liter), 20% glycerol, and 2 µg poly(dI-dC)-poly(dI-dC) as a nonspecific competitor. Protein-DNA complexes were resolved on 6% nondenaturing polyacrylamide gels with 0.25x Tris-borate-EDTA running buffer and were visualized by autoradiography. For DNA competition EMSA, a nonradiolabeled double-stranded oligonucleotide probe was added simultaneously with the labeled probe. EMSA was performed after the addition of 0.5 µl of an antibody against C/EBP, C/EBPß, C/EBP, CREB-binding protein (CBP), CREB-1, activating transcription factor-2 (ATF-2; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA), or phosphorylated CREB-1 (Sigma, St. Louis, MO) to the binding reaction, followed by a 30-min incubation on ice before electrophoresis. The phosphorylated CREB-1 antibody was reactive with serine 133 phosphorylated CREB-1. We used a C/EBP binding site probe (5'-GAAGAAGATTGCCTAAACAA-3') that represents an identical 20-bp long sequence -322/-303) in the promoter II regulatory region of the P450arom gene. This probe contains the -317/-304 C/EBP binding site, which shows 98% homology to the originally described consensus sequence (Fig. 1). The probe (5'-GAATGCACGTCACTCTACCCACT-3') represents an identical 23-bp long sequence (-214/-192) within the promoter II regulatory region of the P450arom gene and contained a critical imperfect cAMP-response element (CRE) (-211/-197) identified previously (10).
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The total protein used for Western blotting was prepared using M-PER mammalian protein extraction reagent (Pierce Chemical Co.). Cells were pelleted by centrifugation at 2,500 x g for 10 min. M-PER reagent was added to the cell pellet containing 1x protease inhibitor mixture and was gently mixed for 10 min. Cell debris was removed by centrifugation at 27,000 x g for 15 min. Protein concentration was determined by using BCA-200 protein assay reagents (Pierce Chemical Co.). Twenty-five micrograms of total protein were mixed 1:1 with Laemmli sample buffer (Bio-Rad Laboratories, Inc.) and heated at 95 C for 5 min. Samples were subjected to 12% SDS-PAGE electrophoresis. Equal protein loading was confirmed by staining in a parallel gel. Proteins were then transferred to nitrocellulose filters in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Filters were blocked with PBS containing 5% nonfat dried milk for overnight and incubated with a specific antibody at 1:500 dilution, for 1 h at room temperature. After washing with TTBS [0.1 M Tris.HCl (pH 7.5), 0.9% sodium chloride, and 0.05% Tween 20) for 4 x 15 min, filters were incubated with 1:10,000 horseradish peroxidase-labeled second antibody for 1 h at room temperature and washed with TTBS for 4 x 15 min. Immunodetection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Chemical Co.). Filters were stripped with 0.2 M NaOH for 5 min, rewashed with TTBS for 15 min each time for 1 h, and re-used for other antibody hybridization. Antibodies against C/EBP, C/EBPß, and C/EBP were purchased from Santa Cruz Biotechnologies, Inc.
Immunohistochemistry
Antihuman C/EBP, C/EBPß, and C/EBP antibodies were purchased from Santa Cruz Biotechnologies, Inc. The immunohistochemical procedures were performed, as described previously, on 2.5-µm thick sections mounted on poly-L-lysine-coated slides using the biotin-streptavidin amplified technique with a Histone immunostaining kit (Nichirei, Tokyo, Japan). Briefly, this staining procedure was performed as: 1) routine deparaffinization; 2) inactivation of endogenous peroxidase activity with 0.3% H2O2 in methyl alcohol for 20 min at 23 C; 3) blocking with 1% goat serum for 45 min at 23 C; 4) incubation with the primary antibody at 4 C for 18 h; 5) incubation with biotinylated goat antirabbit antibody for 30 min at 23 C; 6) incubation with peroxidase-conjugated streptavidin for 30 min at 23 C; 7) colorimetric reaction with a solution containing 0.05% Tris-HCI (pH 7.6), 0.66 M 3,3'-diaminobenzidine and 2 M H2O2; and 8) counterstaining with 1% methyl green.
The immunoreactivity was quantified by an H-scoring system as originally described by McCarty et al. (12) with modification (13). H-scores were generated by adding together twice the percentage of strongly stained nuclei in 10 high-power fields and once the percentage of weekly stained nuclei in 10 high-power fields giving a range from 0–100 (13).
Statistical analysis
A two-tailed t test was used to compare the means of H-scores for each C/EBP isoform in stromal and epithelial cells of endometrium and endometriosis. The significance level () was 0.05, and the power (1-ß) was 0.8.
Results
Three critical cis-acting elements in P450arom promoter II confer cAMP-induced transcription in endometriotic stromal cells
We identified four potential cis-acting elements in the 517 flanking region of the P450arom promoter II using the TFSEARCH database (http://www.blast.genome.ad.jp/sit/TFSEARCH) (Fig. 1
). First, we confirmed our previous observation (10) regarding the presence of two major regions in promoter II using serial deletion mutants of promoter in endometriotic stromal cells from eight patients. We obtained similar results. A representative experiment is shown in Fig. 2A. The major promoter regulatory regions were: 1) -214/-100-bp region that confers up to 3-fold cAMP induction, and 2) -517/-215-bp region responsible for a robust 7-fold induction by dibutyryl cAMP. In this study, we also demonstrated a similar pattern of promoter II regulation in eutopic endometrial stromal cells (n = 14), although these levels were seemingly reduced compared with those in endometriotic cells (Fig. 2B). We have not detected any significant differences between endometrial cells from patients with endometriosis (n = 8) and those without endometriosis (n = 6).
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). We proceeded by introducing disruption mutations of four potentially important cis-acting elements in the most active promoter II construct (i.e. the -517 bp/luciferase fusion gene) (Fig. 3). This experiment revealed three critical elements that are essential for promoter II activity in endometriotic stromal cells (n = 3 patients): 1) a SF-1 binding sequence (-136/-124 bp); 2) a CRE (-211/-197 bp); and 3) a C/EBP binding site (-317/-304 bp). Disruption mutation of another C/EBP binding site (-350/-337 bp) did not change the baseline activity and decreased cAMP fold-induction only modestly (Fig. 3). On the other hand, individual disruption mutations of the -136/-124-bp SF-1, -211/-197-bp CRE, or -317/-304-bp C/EBP binding sites completely abolished both baseline and cAMP-induced activity of promoter II (Fig. 3).
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Effects of C/EBP isoforms on P450arom promoter II activity
Overexpression of C/EBP increased baseline promoter II activity in endometriotic stromal cells by 3-fold (n = 8 patients; representative experiment is shown in Fig. 4A). Bt2cAMP-induced promoter activity was also increased, but this effect was more modest (1.5-fold induction). Consequently, cAMP fold-induction decreased in response to overexpression of C/EBP (Fig. 4A). In contrast, overexpression of C/EBPß or C/EBP nearly abolished promoter II activity both under baseline and cAMP-treated conditions (Fig. 4A). We observed comparable changes in promoter II activity on overexpression of C/EBP isoforms in eutopic endometrial stromal cells (n = 14 patients; representative experiment is shown in Fig. 4B). Although similar proportions were preserved, the absolute values of promoter activity in eutopic endometrial cells were consistently lower than those observed in endometriotic stromal cells (Fig. 4B).
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To explore the roles of C/EBPs in mediating the activity of promoter II in endometriotic stromal cells further, we evaluated the binding of these factors to a sequence (-322/-303 bp) in promoter II containing the -317/-304-bp C/EBP binding site, nuclear extracts from endometriotic and endometrial stromal cells, and antibodies against C/EBP, C/EBPß, and C/EBP (Fig. 5). This C/EBP binding site (-317/-304 bp) was chosen because disruption of this sequence completely abolished promoter II activity (Fig. 3). Using EMSA, we identified two specific DNA-protein complexes (l and 2) as verified by competition with the cold probe (Fig. 5). The probe containing the -317/-304-bp region formed a specific complex (complex 2) with nuclear proteins from endometriotic cells that migrated at a slower rate compared with the common complex (complex 1) formed with nuclear proteins from both endometriotic and eutopic endometrial cells (Fig. 5). The DNA-protein complex 2 formed specifically with endometriotic cell nuclear proteins was not altered significantly with the addition of antibodies against C/EBP, -ß, and -. An alternative second antibody that was raised against C/EBPß (C/EBP
l98) also did not produce a readily detectable alteration (Fig. 5). Although this sequence was originally identified as a C/EBP site based on the computer- assisted database search, our results were suggestive that nuclear proteins other than C/EBPs primarily contributed to the formation of these complexes in endometriotic and endometrial cells. This experiment is a representative of endometriotic (n = 6) and endometrial (n = 6) cells from nine patients.
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C/EBP isoforms were recently shown to bind various CREs in various promoters by a number of investigators (14, 15). Because C/EBP isoforms did not bind to the initially tested -317/-304-bp site, we directed our attention to another critical DNA motif responsible for P450arom promoter activity in endometriotic cells, that is -211/-197 bp CRE (Figs. 1 and 3). Use of nuclear extracts from endometrial cells showed three distinct DNA-protein complexes (Fig. 6A). All three complexes were diminished (complex 1) or abolished (complexes 2 and 3) using antibodies against C/EBPß or C/EBP but not C/EBP. In addition, antibodies against other transcription factors showed the presence of CREB-1, ATF-2, and CBP in these DNA-protein complexes, as expected (Fig. 6A) (16, 29).
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antibody but not anti-C/EBPß or -C/EBP antibodies (Fig. 6B). CBP, CREB-1, and ATF-2 were also present in these transcriptional complexes. Figure 6, A and B, is representative of reproducible experiments from endometrial and endometriotic cells from six patients.
We concluded that the stimulatory (for P450arom in endometriotic/endometrial stromal cells) transcription factor C/EBP binds to the -211/-197-bp CRE in endometriotic cells, whereas inhibitory factor C/EBPß and - bind to the same site in endometrial cells.
Protein levels of C/EBPs in endometriotic and endometrial stromal cells
We determined the levels of C/EBPs in endometriotic and endometrial stromal cells using Western analyses. C/EBP, -ß, and - were present in both cell types (Fig. 7). Treatment with Bt2cAMP (0.5 µM) gave rise to induction of C/EBP and C/EBPß in both cell types. C/EBPß levels were readily detectable in endometrial cells and extremely low in endometriotic cells (Fig. 7). In particular, the C/EBPß isoform liver inhibitory protein was abundant in endometrial cells and barely detectable in endometriotic cells. No differences were noted with respect C/EBP or C/EBP between two cell types in culture (Fig. 7). Fig. 7 shows representative experiments using endometrial and endometriotic cells from six patients.
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Finally, we determined by immunohistochemistry the cellular distribution of C/EBP isoforms using the same antibodies used for Western analyses. In four luteal and two proliferative endometrial tissues and simultaneously biopsied extra-ovarian endometriotic implants, all isoforms were detected in the majority of the samples (Fig. 8). We compared the number and intensity of immunoreactive nuclei for each cell type using an H-scoring system (Table 2a). A two-tailed t test comparing the means of scores for each isoform in specific cell types showed a statistically significant difference between endometrial and endometriotic stromal cells only with respect to C/EBPß levels. C/EBPß was readily detectable in endometrial stromal cells but not in significant number of endometriotic stromal cells (Table 2b). This result was consistent with diminished C/EBPß protein in endometriotic cells using Western analysis (Fig. 7) and the lack of C/EBPß binding activity to promoter II (Fig. 6B) in endometriotic stromal cells.
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Our conclusions from this study support the central hypothesis that molecular aberrations in endometriotic tissue favor elevated local concentrations of E2 either by enhancing production or inhibiting metabolism (8, 10). The production of estrogen in endometriotic tissue is enhanced specifically by the aberrant presence of P450arom expression and aromatase enzyme activity (8, 10). We previously reported that chicken ovalbumin upstream promoter-transcription factor (COUP-TF) binds to the -136/-124-bp element to accomplish suppression of aromatase in eutopic endometrium. On the other hand, aberrantly expressed stimulatory factor SF-1 competes by COUP-TF for binding, displaces COUP-TF, and activates aromatase expression in endometriosis (10). This mechanism, however, accounts for only part of aromatase regulation, because other cis-acting elements and transcription factors also mediate gene expression. This is consistent with the general cell biology concept that disease-free tissues such as eutopic endometrium include numerous overlapping mechanisms as a fail-safe system to prevent excessive production of mitogens such as estrogen, whereas pathological tissues such as endometriosis counteract with acquiring a number of backup systems to generate mitogens. In the present study, we uncovered that C/EBP isoforms differently play a stimulatory or inhibitory effect on the P450arom expression in endometriosis or normal endometrium. A DNA motif at -211/-197 bp, in P450arom promoter II and aberrant binding of C/EBP to this element are responsible, in part, for aberrant aromatase expression. cAMP is a common regulator for pathological expression of aromatase in endometriotic cells, and its stimulation was dose responsiveness. cAMP may mediate the binding activity of C/EBP to promoter II in endometriotic stromal cells. Furthermore, down-regulation of C/EBPß as confirmed by immunohistochemistry and Western analysis complemented by a decreased promoter binding activity (EMSA) in endometriotic stromal cells may represent the primary mechanism for the lack of P450arom suppression in this cell type.
C/EBPs belong to the basic leucine zipper class of transcription factors (17). This family consists of several related proteins encoded by distinct genes (18). Among the six C/EBP family members, C/EBP, -ß, and -, are activators of transcription of several genes (19, 20, 21). Negative regulation of gene expression by C/EBPß in the IL-6 promoter has also been described (22). C/EBP mainly regulates the IL-1ß-mediated induction of platelet derived growth factor (23). In addition, aromatase is overexpressed in the ovaries of C/EBPß knockout mice, indicative of an inhibitory role of C/EBPß for the P450arom gene (24). Contrary to our expectations, however, we recently found that ectopic expression of C/EBPß stimulates P450arom promoter II strikingly in adipose tissue fibroblasts (25). We also found that C/EBPß, in fact, binds to this promoter and mediates the estrogenic effects of malignant epithelial cells on adjacent adipose fibroblasts. Thus, it seems that the stimulatory vs. inhibitory effects of C/EBP isoforms on a specific gene promoter are modified extensively by the complement of available transcription factors within that particular cell type (26, 27, 28).
Here, we also demonstrated that a DNA motif at -317/-304 bp in promoter II is essential for aromatase expression in endometriotic cells (Fig. 3). Nuclear proteins from endometriotic cells gave rise to an aberrant DNA-protein complex at this site, which did not contain detectable amounts of C/EBPs. Efforts are underway to determine the nature of proteins in this complex. Finally, the partners of C/EBP isoforms in the transcriptional complex bound to the -211/-197-bp site included phosphorylated CREB and CBP (16). Cooperative binding of CREB and other ATF members with C/EBP isoforms to CREs have been reported for other promoters (29, 30, 31). Thus, in the case of P450arom promoter II in endometriotic cells, the complex containing C/EBP, CREB, and CBP acts as a stimulator whereas the replacement of C/EBP with -ß or - in this complex in endometrial cells silences promoter II.
In summary, this study is significant with respect to the characterization of three critical regulatory elements in the promoter region of the P450arom gene and its regulation in endometriosis by introducing selective disruption mutations. Furthermore, we defined a new -317/-304-bp cis-acting element, which confers both baseline activity and cAMP induction of P450arom promoter activity in endometriosis. Finally, we described a previously unknown function of C/EBPs in the regulation of aromatase expression in endometriosis. C/EBP isoforms confer their stimulatory or inhibitory effect on P450arom expression in part via the -211/-197-bp CRE in promoter II. In particular, significantly decreased expression of the inhibitory factor C/EBPß in endometriosis compared with endometrium may in part account for aberrant aromatase expression in this pathologic tissue.
Acknowledgments
Dee Alexander provided skilled editorial assistance.
Footnotes
This work was supported by Grant HD38691 from the National Institute of Child Health and Human Development and NIH Grant HD38691 (to S.E.B.).
Abbreviations: ATF, Activating transcription factor; Bt2cAMP, dibutyryl cAMP; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer binding protein; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; CRE, cAMP-response element; CREB, cAMP response element-binding protein; P450arom, aromatase P450; SF, steroidogenic factor.
Received June 14, 2001.
Accepted January 20, 2002.
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H. Ishikawa, V. Fencki, E. E. Marsh, P. Yin, D. Chen, Y.-H. Cheng, S. Reisterd, Z. Lin, and S. E. Bulun CCAAT/Enhancer Binding Protein {beta} Regulates Aromatase Expression via Multiple and Novel Cis-Regulatory Sequences in Uterine Leiomyoma J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 981 - 991. [Abstract] [Full Text] [PDF]
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 O. Bukulmez, D. B. Hardy, B. R. Carr, R. A. Word, and C. R. Mendelson Inflammatory Status Influences Aromatase and Steroid Receptor Expression in Endometriosis Endocrinology, March 1, 2008; 149(3): 1190 - 1204. [Abstract] [Full Text] [PDF]
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 J.J. Kim, H.S. Taylor, Z. Lu, O. Ladhani, J.M. Hastings, K.S. Jackson, Y. Wu, S.W. Guo, and A.T. Fazleabas Altered expression of HOXA10 in endometriosis: potential role in decidualization Mol. Hum. Reprod., May 1, 2007; 13(5): 323 - 332. [Abstract] [Full Text] [PDF]
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J. A. Riancho, C. Valero, A. Naranjo, D. J. Morales, C. Sanudo, and M. T. Zarrabeitia Identification of an Aromatase Haplotype That Is Associated with Gene Expression and Postmenopausal Osteoporosis J. Clin. Endocrinol. Metab., February 1, 2007; 92(2): 660 - 665. [Abstract] [Full Text] [PDF]
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 E. Attar and S.E. Bulun Aromatase and other steroidogenic genes in endometriosis: translational aspects Hum. Reprod. Update, January 1, 2006; 12(1): 49 - 56. [Abstract] [Full Text] [PDF]
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 S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383. [Abstract] [Full Text] [PDF]
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A. D. Burkart, A. Mukherjee, E. Sterneck, P. F. Johnson, and K. E. Mayo Repression of the Inhibin {alpha}-Subunit Gene by the Transcription Factor CCAAT/Enhancer-Binding Protein-{beta} Endocrinology, April 1, 2005; 146(4): 1909 - 1921. [Abstract] [Full Text] [PDF]
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H. S. Sun, K.-Y. Hsiao, C.-C. Hsu, M.-H. Wu, and S.-J. Tsai Transactivation of Steroidogenic Acute Regulatory Protein in Human Endometriotic Stromal Cells Is Mediated by the Prostaglandin EP2 Receptor Endocrinology, September 1, 2003; 144(9): 3934 - 3942. [Abstract] [Full Text] [PDF]
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 T. P. Connolly Cyclooxygenase-2 Inhibitors in Gynecologic Practice Clin. Med. Res., April 1, 2003; 1(2): 105 - 110. [Abstract] [Full Text] [PDF]
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