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Departments of Obstetrics and Gynecology and Molecular Genetics (B.G., S.S., S.Y., J.Z., M.T., Z.F., S.E.B.), 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
The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms’ tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E2 stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.
ENDOMETRIOSIS IS A CHRONIC disease that is manifest by pelvic pain and infertility and defined as the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites. It is estimated to affect 2–10% of women in the reproductive age group (1, 2). Endometriosis is viewed to be a polygenically inherited disease of complex multifactorial etiology (3). Implantation of eutopic (intrauterine) endometrium on peritoneal surfaces in the abdomen via retrograde menstruation through the uterine tubes has been proposed to be the most common mechanism responsible for this disease.
Both circumstantial and laboratory evidence indicate a proliferative role played by estrogen in the establishment and maintenance of endometriosis (4). Aromatase P450 (P450arom) is the rate-limiting enzyme for the synthesis of estrogen and catalyzes the conversion of C19 steroids to estrogens. This conversion takes place in a number of human tissues such as the ovary, placenta, adipose tissue, skin, and the brain (5). We previously demonstrated significantly higher levels of P450arom mRNA and activity in the stromal cell component of endometriotic tissues compared with the eutopic endometrium in which P450arom mRNA or enzyme activity was either undetectable or barely detectable (6, 7). We have also shown that aromatase activity and P450arom mRNA levels can be induced by cAMP analogs or prostaglandin (PG) E2 in endometriotic stromal cells to levels comparable to those found in ovarian granulosa cells or the placental syncytiotrophoblast but not in eutopic endometrial stromal cells (7). The clinical significance of this local induction of aromatase activity in endometriotic tissue was exemplified recently by the successful use of an aromatase inhibitor to treat an unusually aggressive and resistant case of recurrent postmenopausal endometriosis (8). 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 observed in a number of women with endometriosis.
We reported previously that the high levels of baseline and cAMP-induced aromatase activity were partially mediated by SF-1 via binding of SF-1 to a cis-acting element at -136/-124 bp upstream of P450arom gene promoter II (9). We also found that the -517/-215-bp region was essential for the most striking increases in baseline activity and cAMP fold-induction. SF-1 belongs to the orphan nuclear receptor family and regulates the expression of many steroidogenic genes in the adrenal and gonads via binding to nuclear receptor half site (NRHS) in their promoter regions. A number of regulators [e.g. steroid receptor coactivator-1, cAMP response element-binding protein, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1), early growth response-1, sex-determining region Y-box 9, and Wilms’ tumor suppressor gene (WT1)] interact with SF-1 to modify its transactivating functions (10, 11, 12, 13, 14).
DAX-1 is another member of the orphan nuclear receptor family (15, 16). DAX-1 is also expressed in the adrenal and gonads and shows a strikingly similar expression pattern to that of SF-1 (17, 18). This colocalization of SF-1 and DAX-1 led to the suggestion of a functional interaction between these two orphan nuclear receptors (17, 18). In fact, DAX-1-deficient male mice are infertile and have small testes (19). P450arom mRNA levels in testes and circulating estrogen levels are significantly increased in DAX-1-deficient mice due to the lack of inhibition on SF-1-mediated P450arom expression (20). DAX-1 has also been shown to repress SF-1-mediated transactivation of other steroidogenic genes, including genes for steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase type II, Müllerian inhibiting substance (MIS), and P450c17 (Cyp17; Refs. 14 and 21, 22, 23).
The product of the WT1 contains a zinc finger domain and is essential for mammalian gonadal and kidney development (24). WT1 is highly conserved in mammalian species throughout the evolution. WT1 transcripts are present in a number of reproductive tissues including testicular Sertoli cells, ovarian granulosa cells, and endometrial stromal cells (25, 26, 27, 28). In vitro and in vivo studies suggest that WT1 can act as a transcriptional repressor or activator (29, 30). WT1 was reported to regulate the transcription of the MIS gene by virtue of a protein-protein interaction with SF-1 (31). Alternative splicing of the WT1 gene generates four isoforms, all of which are expressed in WT1-positive tissues; the fifth exon may or may not be present, and an alternative splice site in intron 9 allows the addition of three amino acids (Lys-Thr-Ser, or KTS) between the third and fourth zinc fingers of the WT1 protein (32). WT1(-KTS) isoform was reported to be more potent than the +KTS isoform in the stimulation of the MIS gene promoter (14).
In view of these observations, we have examined the modulation of cAMP/SF-1-mediated human P450arom gene expression by WT1 and DAX-1 in endometriotic and endometrial cells. We also studied differential expression and possible in vivo roles of WT1 and DAX-1 in endometriosis and normal endometrium. We should also point out that this is the first report demonstrating the regulation of aromatase expression by WT1 in any cell type or species.
Materials and Methods
Tissue acquisition and processing
Cyst walls of ovarian endometriomas (n = 8, 5 proliferative and 3 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. with minor modifications (33). Eight of the eutopic endometrial samples for stromal cell cultures were obtained at the time of surgical removal of the ovarian endometriomas. The remaining six endometrial samples were obtained from endometriosis-free women undergoing hysterectomy for cervical intraepithelial neoplasia (three proliferative and three luteal phase samples). Extraovarian 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 luteal phases, as confirmed via histological analysis. The age range of the patient population was 23–45 yr.
Stromal cell culture methodology was described previously (7, 33). 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 10% fetal bovine serum (FBS; Life Technologies, Inc./BRL, Grand Island, NY). 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. Medium was changed at 48 h intervals until the cells became confluent. Human tissues were collected as per a research protocol and informed consent approved by the Institutional Review Board of the University of Illinois at Chicago.
Plasmid constructs and site-directed mutagenesis
Human SF-1 cDNA (
3.1 kb) in the eukaryotic expression vector pcDNA2 was a generous gift from Drs. Meera Ramayya and Keith L. Parker (UT Southwestern Medical Center at Dallas, Dallas, TX). Human DAX-1 cDNA (1.5 kb) in the expression vector pBKCMV was a generous gift from Dr. J. Larry Jameson (Northwestern University, Chicago, IL). Mouse wt1 cDNA (with or without KTS domains) in pcDNA3.1 vectors were generous gifts from Dr. Daniel A. Haber (Harvard Medical School, Boston, MA). Preparation of the deletion mutants of the CYP19 (P450arom) gene 5'-flanking sequence in pGL3-basic luciferase vector has been described previously (34). Generation of the -517/-16-bp luciferase plasmid bearing a mutation in the -136/-124-bp SF-1 binding motif has also been described previously (Fig. 1
) (34). Briefly, the NRHS (-136/-124 bp) sequence was changed from 5'-ACCAGGTCAGAAA-3' to 5'-ACCcccTCAGAAA-3'; using the GeneEditor in vitro site-directed mutagenesis system (Promega Corp., Madison, WI), as per the manufacturer’s instructions. (The lowercase nucleotides indicate the mutations.) The mutations and the orientation of insert were confirmed by direct sequencing. Plasmids used in transfection experiments were purified using an EndoFree Plasmid Isolation Kit (QIAGEN, Valencia, CA), and their purity was verified by spectrophotometry and agarose gel electrophoresis.
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Primary endometrial and endometriotic stromal cells were transfected using Lipofectamine Plus (Life Technologies, Inc.-BRL) with the following plasmids: 1) 0.5 µg of deletion or mutant constructs of P450arom promoter II cloned into pGL3-basic luciferase reporter plasmids; 2) 0.5 µg of pcDNA3 or 0.3 µg pBKCMV expression plasmid (Invitrogen, Carlsbad, CA), containing the cDNAs encoding SF-1, and WT1 or DAX-1; and 3) Renilla luciferase (5 ng, Promega Corp.) employed as an internal control for transfection efficiency. Total DNA transfected per well was kept at a constant total of 2 µg/well, adding empty pcDNA3 vector if necessary. The day before transfection, primary stromal cells were seeded into 35-mm dishes at 2 x 105 cell/dish. At the time of transfection, stromal cells were 80% confluent. The transfection solution was made of 200 µl of Opti-MEM reduced-serum medium containing PLUS reagent (6 µl), precomplexed DNA, and 4 µl of 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 (Bt2cAMP) for 24 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, 2 mM ethylenediaminotetraacetic acid. Luciferase and Renilla luciferase readings were obtained using a dual-luciferase reporter assay system (Promega Corp.), and LUMAT LB9507 luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).
Results are presented as the mean ± SEM of data from triplicate replicates. Illustrated results are representative of five reproducible experiments using cells from five different patients.
Immunohistochemistry
Mouse monoclonal antibody against human WT1 (recognizing an amino-terminal epitope of human origin) and rabbit polyclonal antibody against DAX-1 (recognizing an amino-terminal epitope of DAX-1 of human origin) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The immunohistochemical procedures were performed, as previously described, using the biotin-streptavidin amplified technique with a histone immunostaining kit (Nichirei, Tokyo, Japan). Antigen retrieval was performed by heating the slides in an autoclave at 120 C for 5 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0). The dilutions of the primary antibodies used in this study were 1:1250 for WT1 and 1:500 for DAX-1. The antigen-antibody complex was visualized with 3.3'-diaminobenzidine solution (0.01 M 3.3'-diaminobenzidine in 0.05 M Tris-HCl buffer, pH 7.6; and 0.006% hydrogen peroxide) and counterstained with hematoxylin. Human tissues of kidney and adrenal gland were used as positive controls for WT1 and DAX-1 antibodies, respectively. As negative controls, normal mouse or rabbit IgG was used instead of the primary antibodies. No specific immunoreactivity was detected in these sections.
The immunoreactivity was quantified by an H-scoring system as originally described by McCarty et al. (35) with modifications. H-scores were generated by adding together 2x percentage of strongly stained nuclei in 10 high power fields and 1x percentage of weakly stained nuclei in 10 high power field giving a range from 0–200 (36). WT1 or DAX-1 staining was observed exclusively in nuclei, as expected.
Statistical analysis
A two-tailed t test and Bonferroni correction were used to compare the means of H-scores for WT1 and DAX-1 in stromal and epithelial cells of endometrium and endometriosis. Because we performed multiple t tests between these groups (Table 2), 
-value for each individual test after Bonferroni correction was 0.01274. This brought the overall -level to 0.05.
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Regulatory sequences in P450 promoter II that confer SF-1-mediated transcription in endometrial and endometriotic stromal cells
To identify regions of the P450arom promoter II that are responsible for SF-1-mediated transcription of the P450arom gene in endometrial and endometriotic stromal cells, we first transfected serial deletion mutants of P450arom promoter II into these cells. Plasmids containing -100, -214, -278, -517, and -694 bp regions of the promoter II fused to the luciferase reporter gene were transfected into endometrial and endometriotic stromal cells in culture. These cells were cultured in the absence of serum or hormone, with or without human SF-1 expression vector. SF-1 induced the luciferase activity of -214 bp (but not the -100 bp) promoter II plasmids by 1.4-fold in endometrial stromal cells. The addition of SF-1 to -278, -517, -694 bp luciferase plasmid in endometrial stromal cells gave rise to 2.3-, 4-, and 3.6-fold inductions, respectively (Fig. 2A). These results indicate that the proximal region between -214 and -100 bp conferred minimal responsiveness to SF-1 in endometrial stromal cells, and that this was markedly potentiated by the -517/-214-bp region. The NRHS (-136/-124 bp) and cAMP response element (CRE; -211/-197 bp) in the -214/-140-bp region have been shown to be functionally important previously (Fig. 1
) (9, 37, 38).
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). Fold-induction by SF-1 in endometriotic cells, however, was more pronounced (2-, 3.4-, 7-, and 4-fold for -214, -278, -517, and -694 bp construct, respectively) compared with those in endometrial cells (Fig. 2B). This was suggestive of a more efficient transcriptional machinery favoring binding of stimulatory factors and possible deficiency of factors that inhibit P450arom promoter II in endometriotic vs. endometrial cells. WT1 or DAX-1 down-regulate SF-1-induced transcriptional activation of P450arom promoter II gene in a dose-dependent manner in endometrial stromal cells
Previous studies have demonstrated the roles of SF-1 in transcriptional activation of human P450arom gene and DAX-1 in the transcriptional repression of murine Cyp19 gene (9, 20, 38). WT1 and DAX-1 have been shown to independently modify SF-1-mediated gene transcription of MIS via direct interaction with SF-1 (14). The role of DAX-1 in the regulation of the human P450arom gene or WT1 in the regulation of the P450arom gene in any species, however, has not been reported to date. We therefore studied the effects of WT1 and DAX-1 on SF-1-mediated stimulation of the human P450arom promoter II activity by transfecting mammalian expression plasmids.
The effects of WT1 and DAX-1 on P450arom promoter II activity were dose-dependent (Fig. 3). We first demonstrated that SF-1-induced promoter activity in a dose-dependent fashion, as expected (Fig. 3A). Then, we assessed the effect of WT1 isoforms with or without a KTS domain on SF-1-mediated P450arom promoter II activity (Fig. 3B). WT1(-KTS) is much more potent than WT1(+KTS) in the suppression of SF-1-induced P450arom promoter activity (Fig. 3B). The addition of WT1(-KTS) effectively decreased SF-1-induced promoter II activity in a dose-dependent manner (Fig. 3C). DAX-1 also inhibited SF-1-mediated promoter II activity in a dose-dependent fashion (Fig. 3D).
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After investigating the roles of SF-1, WT1, and DAX-1 in the regulation of P450arom promoter II in endometrial cell line, we next sought to determine the roles of these factors in the regulation of P450arom in endometriotic stromal cells. Figure 4, A and B, shows the effects of ectopically expressed SF-1, WT1, or DAX-1 on the activity of -517 bp promoter II construct in Bt2cAMP-treated endometriotic stromal cells. SF-1 significantly increased P450arom transcription in endometriotic stromal cell, whereas either WT1 or DAX-1 markedly reduced promoter II activity. Hence, our results suggest that WT1 or DAX-1 may play an important role in the regulation of P450arom expression in the human endometriotic stromal cells.
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To better understand the functions of the cis-acting element NRHS at -136/-124 bp, we introduced a disruptive mutation at this site in the -517-bp construct and determined its effects on promoter activity. First, we observed that Bt2cAMP potentiated promoter II activity in the presence or absence of SF-1 using the wild-type construct. The disruption of the -136/-124-bp NRHS significantly decreased both baseline and SF-1-induced promoter II activity in endometrial stromal cells (Fig. 5, A and B). In view of our previous data demonstrating dominant binding of SF-1 to the NRHS at -136/-124 bp, we suggest that the interaction of SF-1 with this site is critical for P450arom promoter II activity in endometrial stromal cells (9).
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, A and B). The addition of WT1 to endometrial stromal cells decreased the baseline and Bt2cAMP-induced promoter activity in the presence or absence of SF-1, suggesting that WT1 inhibits promoter II activity mediated by an interaction of SF-1 with the -136/-124-bp NRHS (Fig. 5A). On the other hand, disruption of the -136/-124-bp NRHS totally abolished promoter II activity regardless of the presence of SF-1, Bt2cAMP, or WT1 (Fig. 5A).
The addition of DAX-1 also inhibited SF-1 or Bt2cAMP-mediated induction of promoter II activity (Fig. 5B). As in the case of WT1, site-directed mutagenesis suggests that the stimulatory effect of SF-1 and the inhibitory effect of DAX-1 on P450arom promoter II activity were at least in part mediated by the -136/-124-bp NRHS (Fig. 5, A and B).
In vivo distribution of WT1 and DAX-1 in endometrial and endometriotic tissues
Cellular distribution and levels of WT1 and DAX-1 were evaluated by immunohistochemistry in endometrial and endometriotic tissue using the human anti-WT1 and DAX-1 antibodies (Santa Cruz Biotechnology, Inc.). In four luteal and two proliferative endometrial tissues and simultaneously obtained extraovarian endometriotic implants, WT1 and DAX-1 were detected in various cell types of the majority of the samples (Fig. 6).
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, A and B). Interestingly, endometrial or endometriotic epithelial cells contained strikingly less WT1 compared with stromal cells. DAX-1, on the other hand, was readily detected in both endometrial and endometriotic epithelial and stromal cells (Fig. 6, C and D).
We used a semiquantitative H-scoring system to compare WT1 and DAX-1 immunointensity between the cell types represented in Fig. 6. When endometrium and endometriosis were compared, a statistically significant difference was observed only between endometriotic and endometrial stromal cells with respect to WT1 (Tables 1 and 2). Endometriotic stromal WT1 levels were significantly lower than those in endometrial stromal cells (Table 2). Moreover, we found that WT1 is expressed in significantly higher levels in stromal (vs. epithelial) cells in endometrium. We also observed a similar trend in endometriosis, although this did not reach statistical significance (Table 2). No statistically significant differences between epithelial and stromal cells were observed with respect to DAX-1 expression, although a trend for preferential expression in the epithelium was observed (Table 2).
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This study was performed to determine molecular mechanisms responsible for the modulation of aromatase expression by SF-1, WT1, and DAX-1 in endometrial and endometriotic stromal cells. We previously showed that significantly increased SF-1 in endometriotic vs. endometrial stromal cells represents a critical mechanism for aromatase expression and estrogen production in endometriosis (9). Binding of SF-1 to the critical -136/-124-bp element (NRHS) in P450arom promoter II may be the key event that brings the entire transcriptional complex to initiate P450arom expression in endometriosis. Chicken ovalbumin upstream promoter-transcription factor (COUP-TF), on the other hand, is present in both endometriosis and endometrium and inhibits aromatase expression in the absence of SF-1 in endometrium (9). SF-1 serves as a dominant activator for P450arom promoter II via competing with COUP-TF for binding to the NRHS at -136/-124 bp in cultured endometriotic stromal cells (9). This competition between SF-1 and COUP-TF, however, is not sufficient to explain aberrant aromatase expression in endometriosis, because 20% of eutopic endometrial samples also expressed the dominant activator SF-1; yet these tissues did not contain aromatase in levels comparable to those in endometriosis. Thus, there may be other fail-safe mechanisms in addition to COUP-TF for ensuring the suppression of aromatase expression in endometrium.
Our present findings regarding the predominant expression of WT1 in endometrial vs. endometriotic cells and dominant repressor effect of WT1 on P450arom promoter II are strongly suggestive that WT1 serves as a physiologically important inhibitor of aromatase in the eutopic endometrium. Previously published data regarding stromal expression of WT1 in the endometrium are supportive of our findings (28).
Significantly decreased WT1 expression in endometriosis is interesting in view of recently implicated roles of other tumor suppressors in endometriosis. It was proposed that mutations of the tumor suppressor genes p53 and phosphatase and tensin homolog might play important roles in the etiology of endometriosis (39, 40). There are, however, conflicting data in the literature regarding the possible roles of these tumor suppressors in endometriosis (41, 42). To date, no definitive mechanisms have been shown for tumor suppressors in the pathogenesis of endometriosis. We demonstrate, for the first time, the down-regulation of a tumor suppressor, namely, WT1 in endometriosis. Moreover, we uncover one of the functions of WT1, namely suppression of estrogen production in endometrium. Studies are currently under way to understand whether aromatase inhibition is a general function of WT1 in other human tissues.
DAX-1 shows profiles of expression and function similar to those of COUP-TF. In other words, DAX-1 is ubiquitously expressed in both endometrium and endometriosis and down-regulates aromatase. DAX-1 may represent another fail-safe mechanism for the inhibition of aromatase in normal endometrium.
Acknowledgments
We thank Meera Ramayya, Keith L. Parker (UT Southwestern Medical Center at Dallas), J. Larry Jameson (Northwestern University), and Daniel A. Haber (Harvard Medial School) for the generous gifts of human SF-1 cDNA, human DAX-1 cDNA, and mouse wt1 cDNA, respectively.
Footnotes
This work was supported by the National Institutes of Health Grant HD38691 (to S.E.B.).
Abbreviations: Bt2cAMP, Dibutyryl cAMP; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; CRE, cAMP response element; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1; FBS, fetal bovine serum; KTS, Lys-Thr-Ser; MIS, Müllerian inhibiting substance; NRHS, nuclear receptor half site; P450arom, aromatase P450; PG, prostaglandin; SF-1, steroidogenic factor-1; WT1, Wilms’ tumor suppressor gene.
Received April 2, 2002.
Accepted May 23, 2002.
References
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-136/-124 bp promoter II construct. Ectopic expression of SF-1 and treatment with Bt2cAMP-stimulated promoter II activity of the wild-type -517-bp construct but not that of the -517