Stimulation of Aromatase P450 Promoter (II) Activity in Endometriosis and Its Inhibition in Endometrium Are Regulated by Competitive Binding of Steroidogenic Factor-1 and Chicken Ovalbumin Upstream Promoter Transcription Factor to the Same cis-Acting Element

Khaled Zeitoun, Kazuto Takayama, M. Dod Michael and Serdar E. Bulun

Cecil H. Ida Green Center for Reproductive Biology Sciences Department of Obstetrics and Gynecology The University of Texas Southwestern Medical Center Dallas, Texas 75235-9051


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In stromal cells of endometriosis, marked levels of aromatase P450 (P450arom) mRNA and activity are present and can be vigorously stimulated by (Bu)2cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. Since eutopic endometrial tissue or stromal cells lack P450arom expression, we studied the molecular basis for differential P450arom expression in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometriotic lesions is regulated almost exclusively via the alternative promoter II. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic stromal cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a -214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/-214 distal region responsible for potentiation of cAMP response up to 13-fold. In the -214/-100 region, we studied eutopic endometrial and endometriotic nuclear protein binding to a nuclear receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response element-binding activity in nuclear proteins from both endometriotic and eutopic endometrial cells gave rise to formation of identical DNA-protein complexes. The NRHS probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells. Employing recombinant proteins and antibodies against steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS in eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding protein in endometriotic cells. In fact, COUP-TF transcripts were present in both eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but in only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dose-dependent direct competition between SF-1 and COUP-TF for occupancy of the NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentiated baseline and cAMP-induced activities of -517 promoter II construct, whereas overexpression of COUP-TF almost completely abolished these activities. In conclusion, COUP-TF might be one of the factors responsible for the inhibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, aberrant SF-1 expression in endometriotic stromal cells can override this inhibition by competing for the same DNA-binding site, which is likely to account for high levels of baseline and cAMP-induced aromatase activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endometriosis is the presence of endometrium-like tissue outside the uterine cavity and is most commonly found in the pelvic peritoneum and ovaries. It becomes manifest most commonly as pelvic pain and infertility in women of reproductive age (1, 2, 3, 4). Although the etiology and mechanism for the development of endometriosis are not well understood, both circumstantial and laboratory evidence are indicative of a critical role of estrogen in the growth of endometriotic tissue (5). In fact, all current medical treatments for endometriosis block either estrogen production or its action (1, 2). Estrogen is produced in the human ovary, placenta, adipose tissue, skin, and brain by the aromatization of C19 steroids, a reaction catalyzed by aromatase P450 (P450arom) (6). P450arom expression in these tissues is, in part, regulated by the alternative use of tissue-specific promoters in the placenta (distally located promoter I.1), ovary (classically located proximal promoter II), and adipose tissue (promoters I.4, I.3, and II) (6). Until recently, estrogen production was presumed to take place primarily in the ovaries and peripheral tissues (e.g., adipose tissue and skin) in women with endometriosis. We recently demonstrated, however, 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 to those found in ovarian granulosa cells or the placental syncytiotrophoblast, whereas significant levels of P450arom mRNA or activity could not be detected in eutopic endometrial stromal cells either before or after treatments with cAMP analogs or PGE2 (8). It should be pointed out here that the most abundant substrate for aromatase in endometriosis is circulating androstenedione of adrenal and ovarian origins, which is aromatized to estrone that is only weakly estrogenic and must be converted further to estradiol for full estrogenic action. In endometriotic tissues, we recently demonstrated, by Northern blotting mRNA of 17ß-hydroxysteroid dehydrogenase type 1, an enzyme that catalyzes the conversion of estrone to estradiol (9).

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 progestins (10). 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 also provide insights into the etiology of endometriosis and lead to identification of molecular targets for the development of novel treatment strategies. Thus, we sought to determine the factors that stimulate P450arom expression in endometriotic stromal cells and its inhibition in the eutopic endometrial stromal cells, and studied transcriptional regulation of the P450arom gene in these two cell types in a comparative manner. We previously demonstrated by exon-specific RT-PCR that the classically located promoter II is almost exclusively used to regulate P450arom expression in cultured endometriotic stromal cells incubated with a cAMP analog or PGE2 (8). In the current study, we initially used an alternative and independent method, i.e. rapid amplification of cDNA 5'-ends (5'-RACE) to determine the promoter-specific sequences in P450arom mRNA isolated from the extraovarian endometriotic tissues (11). This confirmed the almost exclusive use of promoter II for P450arom expression in vivo in endometriotic tissue. It was shown previously that two transcription factors, i.e. steroidogenic factor-1 (SF-1), which is an orphan nuclear receptor expressed in steroidogenic tissues in a specific manner, and cAMP response element (CRE) binding protein (CREB), which is expressed ubiquitously in many tissues, bind to separate regulatory sequences upstream of promoter II in bovine and murine ovarian granulosa cells to activate transcription (12, 13, 14). We report herein that the nuclear receptor half-site and CRE in promoter II are also important for aromatase expression in endometriotic stromal cells. Additionally, we discovered that an inhibitory type orphan nuclear receptor, i.e. chick ovalbumin upstream promoter factor (COUP-TF), mediates the inhibition of P450arom gene transcription in eutopic endometrial stromal cells (15, 16, 17, 18, 19). Competitive binding of these stimulatory (SF-1) and inhibitory (COUP-TF) transcription factors to the nuclear receptor half-site in promoter II was found to determine the presence or absence of P450arom promoter activity in these two homologous cell types. We also find that the ubiquitous expression of COUP-TF in both endometriotic and eutopic endometrial tissues and the expression of SF-1 in a more specific manner in endometriotic tissue constitute the basis for this complex mechanism.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aromatase Expression in Endometriotic Tissues Is Regulated by Promoter II
Aromatase expression in human tissues is under the control of alternatively used and partially tissue-specific promoters such as the classically located proximal promoter II in the ovary, distal promoter I.1 in the placenta, promoter 1.4 in the skin, and promoters I.4, I.3, and II in the adipose tissue. 5'-Untranslated regions of P450arom transcripts in these tissues contain first exons that are specific for each promoter. Therefore, we amplified and sequenced the 5'-ends of P450 transcripts in total RNA samples from extraovarian endometriotic tissues from three patients using the 5'-RACE procedure to determine the promoter(s) that are used for aromatase expression in endometriosis. Of 57 RACE clones isolated from endometriotic tissues of these three patients, 53 clones were found to contain the promoter II-specific sequence (Table 1Go). Three clones were the result of transcription via promoter I.3 and one clone was the result of transcription via promoter I.4. We have previously demonstrated that P450arom expression stimulated by cAMP analogs or PGE2 in endometriotic stromal cells was regulated almost exclusively by promoter II (8). Therefore, these 5'-RACE results complement our previous in vitro findings and demonstrate that P450arom expression in endometriotic tissue is also regulated in vivo almost exclusively via promoter II.


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Table 1. P450arom 5'-RACE Clones Detected in Total RNA from Extraovarian Endometriotic Tissues

 
Regions of Promoter II That Confer cAMP-Induced Transcription in Endometriotic Stromal Cells
To identify regions of the P450arom promoter II that are responsible for cAMP- (or PGE2)-induced transcription of the P450arom gene in endometriotic stromal cells, plasmids containing -100, -140, -214, -517, and -694 bp of the promoter II ligated to the luciferase reporter gene were transfected into endometriotic stromal cells that had been cultured in the absence of serum or hormone (Fig. 1Go). After transfection, the cells were stimulated with (Bu)2cAMP or were left untreated. Compared with the -100 promoter II plasmid, baseline luciferase activity increased slightly by 1.5-fold and 1.7-fold after transfections with the -214 and -694 promoter II plasmids, respectively. Transfection with the -517 promoter II plasmid resulted in the highest baseline luciferase activity (2.7-fold) compared with the -100 plasmid. (Bu)2cAMP induced luciferase activities of -140 and -214 promoter II plasmids by 1.9- and 3.7-fold, respectively. On the other hand, 10- and 13-fold inductions were achieved by the -694 and -517 promoter II plasmids, respectively. These results indicated that the proximal region between -214 and -100 bp conferred responsiveness to cAMP in endometriotic stromal cells, and that this was markedly potentiated by the -517/-214 bp region, whereas there might be inhibitory elements in the -694/-517 region. Next, we searched for potential regulatory elements in the -214/-140 bp region that might be responsible for the inhibition and stimulation of aromatase expression in eutopic endometrial and endometriotic stromal cells. First, we identified a consensus nuclear receptor half-site in the -140/-100 bp region that is known to bind SF-1 in ovarian granulosa cells (13) (Fig. 1Go). When this sequence is present in duplicate in promoters of other genes (e.g. CYP17), it was shown to bind COUP-TF as a dimer (20). There is also an imperfect CRE in this region that is necessary for cAMP induction of promoter II through binding of CREB in ovarian granulosa cells (12) (Fig. 1Go). Thus, we directed our attention to these two regulatory elements, i.e. nuclear receptor half-site and CRE, in the following nuclear protein-DNA-binding studies.



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Figure 1. Identification of Regulatory Regions Responsible for cAMP-Inducible P450arom Promoter II Activity in Human Endometriotic Stromal Cells

Reporter plasmids containing the 5'-flanking region of the human P450arom gene with deletion mutations are represented on the left. Relative positions and sequences of the imperfect CRE (-211/-203 bp) and the nuclear receptor half-site (-132/-125 bp) are indicated. Cells were transfected with each reporter plasmid together with pCMV-ßgal used as an internal control for transfections. Results are represented as luciferase activities expressed relative to the -100-bp construct, which was given the arbitrary value of 1, and are presented as the average of data from three replicate experiments ± SEM.

 
Differential Binding of Nuclear Proteins from Endometriotic and Eutopic Endometrial Stromal Cells to Nuclear Receptor Half-Site
Using bovine luteal cell nuclear proteins, we previously demonstrated binding of specific trans-activating factors to two consensus sequences upstream of P450arom promoter II: CREB to an imperfect CRE (-211/-203 bp) and SF-1 to a nuclear receptor half-site (-132/-125 bp) (12, 13). In an attempt to determine the basis for the presence of aromatase expression in endometriotic stromal cells and its absence in eutopic endometrial stromal cells, binding activities for CRE and the nuclear receptor half-site in nuclear extracts from these two cell types were demonstrated in a comparative manner. Nuclear proteins from both eutopic endometrial and endometriotic stromal cells demonstrated CRE binding activity and formed complexes of identical size (Fig. 2AGo). Although CREB does bind to CRE in active or inactive forms, which cannot be differentiated by electrophoretic mobility shift assay (EMSA), identical CRE-binding activities in both cell types were suggestive that CRE did not account for the differential aromatase expression in these two cell types. Nuclear receptor half-site probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells (cx2), which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells (cx1) (Fig. 2AGo). This endometriotic cell-specific complex (cx2) was identical to that formed with nuclear proteins of human granulosa cells (Fig. 2AGo). Formation of complex 1 (although of much lesser intensity in comparison with complex 2) was also observed upon binding of endometriotic stromal cell nuclear proteins to nuclear receptor half-site probe. These results were consistently reproduced in eutopic endometrial (n = 7) and endometriotic cells (n = 8) from 10 other women (Fig. 2BGo). No differences in nuclear receptor half-site binding activity were detected between eutopic endometrial cell extracts from patients with endometriosis and those from disease-free women. In addition, no differences were observed between proliferative and luteal phase endometrial or endometriotic tissues.



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Figure 2. Differential Binding of Nuclear Proteins to Promoter II Regulatory Sequences in Endometriosis and Eutopic Endometrial Stromal Cells

A, Nuclear proteins from eutopic endometrial (endometrium) and endometriotic (endometriosis) stromal cells bound to CRE upstream of the P450arom promoter II to form three identical complexes (left). These specific complexes could be competed away by the addition of excessive amounts of the cold probe. On the other hand, nuclear proteins from these two cell types formed two specific and distinct complexes with the nuclear receptor half-site (right). Nuclear proteins from eutopic endometrial stromal cells gave rise to a slow-migrating single complex, complex 1 (cx 1), whereas nuclear proteins from endometriotic stromal cells also formed an additional faster-migrating complex 2 (cx 2) that is identical to that formed by ovarian granulosa cells. Since promoter II is responsible for cAMP induction of aromatase expression in ovarian granulosa cells, we conclude that similar mechanisms are responsible for aromatase expression in the ovary and endometriosis. B, Differential binding of nuclear proteins to the nuclear receptor half-site in endometriosis and eutopic endometrium is confirmed in stromal cells from five other patients. Please note that complex 1 is formed by both eutopic endometrial and endometriotic cell nuclear proteins, although its intensity is considerably less in the case of endometriotic cell nuclear proteins. On the other hand, complex 2 formation is specific for endometriotic cell nuclear proteins. A total of seven eutopic endometrial and eight endometriotic cell cultures from 10 patients were tested with consistent results.

 
Only COUP-TF Binds to Nuclear Receptor Half-Site in Eutopic Endometrium, Whereas Both COUP-TF and SF-1 Bind to This Site in Endometriosis
Thus far, our results suggested that different trans-activating factors bind to the nuclear receptor half-site in eutopic endometrial and endometriotic stromal cells. As illustrated in Fig. 3Go, we demonstrated that this was the case. First, it was shown that in vitro transcribed/translated orphan nuclear receptor COUP-TF (both I and II isoforms) forms a complex with nuclear receptor half-site, which migrates at an identical rate to that of complex 1. On the other hand, the size of complex 2 is identical to a complex formed by another orphan nuclear receptor, SF-1 (Fig. 3AGo). Next, antibodies were used to verify the presence of COUP-TF and SF-1 in these complexes: in the case of both eutopic endometrial and endometriotic nuclear proteins, complex 1 formation with the nuclear receptor half-site probe was eliminated by COUP-TF antibody (recognizes both isoforms I and II) (Fig. 3BGo). Complex 1 formation with these nuclear proteins was also diminished by SF-1 antibody. Complex 2 formation by the endometriotic and granulosa cell nuclear proteins, on the other hand, was eliminated by SF-1 antibody but not by COUP-TF antibody (Fig. 3BGo). These results indicated that complex 2 represented SF-1 binding, whereas it was not clear whether complex 1 contained some SF-1 in addition to COUP-TF. Figure 3CGo illustrates that the complex formed by in vitro transcribed/translated mixture of COUP-TFI and II (corresponding to complex 1) diminished after incubation with the anti-SF-1 antibody, thus indicating that the interaction of SF-1 antibody with complex 1 was a consequence of cross-reaction of this antibody with COUP-TF but not due to the actual presence of SF-1 in this complex. In summary, COUP-TF bound to the nuclear receptor half-site in the eutopic endometrium to form complex 1, whereas both COUP-TF and SF-1 bound to this identical site in endometriosis to form two distinct complexes (1, 2). Complex 2 formed by endometriotic stromal cell nuclear proteins appeared much more intense than complex 1, suggesting that the most important portion of nuclear receptor half-site binding activity in endometriosis was accounted for by SF-1.



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Figure 3. Identification of Nuclear Proteins in Complex 1 and 2

A, In vitro synthesized COUP-TF and SF-1 bind to nuclear receptor half-site. EMSA that compares the sizes of protein-DNA complexes formed by binding of in vitro synthesized COUP-TF (I and II) and SF-1 to the nuclear receptor half-site with complexes 1 and 2 formed by eutopic endometrial and endometriotic nuclear proteins. The size of complex 1 is identical to that formed by COUP-TF, whereas the size of complex 2 is equal to that formed by SF-1. B, Differential binding of COUP-TF and SF-1 to nuclear receptor half-site in eutopic endometrium and endometriosis. Using a probe for nuclear receptor half-site, complex 1, (cx 1), which is the DNA-protein complex formed with eutopic endometrial cell nuclear proteins (endometrium), was eliminated with the addition of anti-COUP-TF antibody, indicating that this complex contained COUP-TF. Since complex 1 was also diminished by anti-SF-1 antibody, the question remained whether SF-1 was also present in complex 1. On the other hand, only anti-SF-1 antibody (but not anti-COUP-TF antibody) blocked the formation of complex 2 (cx 2) by endometriotic stromal cell nuclear proteins indicating the presence of SF-1 in this endometriosis-specific complex. Nuclear proteins from human ovarian granulosa cells were used as positive controls for SF-1 binding. Thus, cx 2 formation was clearly accounted for by SF-1 in endometriotic samples, whereas it was not clear whether cx 1 contained SF-1 in addition to COUP-TF. C, Cross-reaction of anti-SF-1 antibody with in vitro synthesized COUP-TF. To clarify whether SF-1 exists in complex 1 (cx 1) in addition to COUP-TF, we incubated in vitro synthesized COUP-TF and the nuclear receptor half-site probe with anti-SF-1 antibody. Anti-SF-1 antibody diminished the specific complex formation by COUP-TF, which is indicative of a cross-reaction between anti-SF-1 antibody and COUP-TF. Preimmune serum did not alter the formation of DNA-protein complexes, as expected. Thus, these data are collectively suggestive that COUP-TF binding to nuclear receptor half-site accounts for the formation of complex 1 by eutopic endometrial and endometriotic nuclear proteins, whereas SF-1 binding is responsible for the endometriosis-specific complex 2 formation.

 
SF-1 Binds to Nuclear Receptor Half-Site with a Higher Affinity Than That of COUP-TF
The nuclear receptor half-site in promoter II of the CYP19 (P450arom) gene is a classic SF-1 binding site, and SF-1 seemed to bind to this probe with a relatively high affinity (complex 2) in comparison with COUP-TF (complex 1) in endometriotic cells (Figs. 2Go and 3Go). Previous studies suggested that SF-1 binds to DNA as a monomer, whereas COUP-TF binds preferentially as a dimer to repeats of nuclear binding half-site variants (14, 21). To demonstrate relative binding affinities of SF-1 and COUP-TF to the nuclear receptor half-site in CYP19 (P450arom) gene promoter II, we compared binding activities of recombinant SF-1 and COUP-TF and endometriotic and eutopic endometrial nuclear extracts to the half-site in the CYP19 gene and also to the -129/-114 region in the CYP11B2 gene promoter, which contains an inverse repeat of the half-site with a two-nucleotide spacer (Fig. 4Go). As expected, recombinant COUP-TF and eutopic endometrial nuclear proteins (cx1) bound to the -129/-114 element (with half-sites) more avidly compared with the single nuclear receptor half-site in the CYP19 gene (Fig. 4Go), whereas SF-1 binding activity to either probe seemed to be the same. Additionally, a striking change was noted in binding activities of endometriotic nuclear proteins to each probe: complex 2 (SF-1) accounted for the primary binding activity to the half-site in the CYP19 gene, whereas complex 1 (COUP-TF) represented the primary binding activity in endometriotic nuclear proteins to a repeat of this half-site (-129/-114 element) in the CYP11B2 gene. This is suggestive that a single nuclear receptor half-site favors SF-1 binding (over COUP-TF) as in the CYP19 (P450arom) gene, whereas a repeat of this half-site gives rise to a higher binding activity of COUP-TF. Thus, the regulation of P450arom promoter II activity in endometriosis by SF-1, despite the presence of COUP-TF in the same tissue, might be, in part, due to relatively lower binding activity of COUP-TF to a single nuclear receptor half-site in this particular promoter.



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Figure 4. Relative Nuclear Receptor Half-Site Binding Activities of SF-1 and COUP-TF Are Determined by the Presence or Absence of a Repeat of This Sequence

Panel A depicts binding activities of COUP-TF (I and II) and SF-1 to the nuclear receptor half-site in CYP19 (P450arom) gene promoter II. In this case, SF-1 binding activity appears to be higher than that of COUP-TF. Panel B, To demonstrate the relative affinities of SF-1 and COUP-TF, another regulatory element (-129/-114 bp site) in the CYP11B2 gene that contains a repeat half-site was used. Please note that the binding activity of SF-1 did not change (in comparison with the single half-site in CYP19 gene promoter), whereas COUP-TF bound to the probe with double half-site much more avidly. Comparable changes were noted in binding activities of endometriotic and endometrial nuclear proteins as evident from the intensities of complex 1 (COUP-TF) and complex 2 (SF-1).

 
COUP-TF and SF-1 Compete for Binding to Nuclear Receptor Half-Site
As illustrated in Fig. 5Go, increasing quantities (1-, 5-, and 10-fold) of in vitro transcribed/translated SF-1 diminished the binding activity of a mixture of in vitro transcribed/translated COUP-TFI and II to the nuclear receptor half-site in a dose-dependent fashion. The size of the COUP-TF-DNA complex was identical to that of complex 1 formed by eutopic endometrial or endometriotic nuclear proteins, whereas the size of the SF-1-DNA complex corresponded to complex 2 formed by endometriotic nuclear proteins (Fig. 5Go).



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Figure 5. SF-1 Displaces COUP-TF from the Nuclear Receptor Half-Site

Using competition EMSA employing in vitro synthesized proteins, increasing amounts of SF-1 (1-, 5-, and 10-fold) were mixed with a constant amount of COUP-TF to demonstrate the competition for binding to the labeled nuclear receptor half-site probe. Increasing amounts of SF-1 diminished COUP-TF binding to nuclear receptor half-site in a dose-dependent fashion. Nuclear proteins extracted from endometriotic and endometrial stromal cells were used in the last two lanes to demonstrate the identity between complex 1 and COUP-TF binding and also between complex 2 and SF-1 binding.

 
Effects of COUP-TF and SF-1 on P450arom Promoter II Activity
In both eutopic endometrial and endometriotic cells, overexpression of SF-1 significantly increased both baseline and (Bu)2cAMP-induced luciferase activities of -517 promoter II plasmid, whereas overexpression of COUP-TF nearly abolished these activities (Figs. 6Go and 7Go). It would be noted that cAMP fold-induction of baseline promoter activity in eutopic endometrial cells (8-fold) was much lower than that of endometriotic cells (20-fold). The overexpression of SF-1 in endometriotic cells gave rise to an increase of activity in both untreated and cAMP-treated cells (Fig. 6Go). In eutopic endometrial cells in which SF-1 is not normally expressed, SF-1 overexpression gave rise to an increase of baseline luciferase activity by 40-fold, and cAMP treatment of these cells further increased activity by 11.5-fold to 460-fold the baseline level (Fig. 7Go). We should also note that, after the addition of SF-1, cAMP fold-induction values were preserved in both eutopic endometrial cells (11.5-fold) and endometriotic cells (21.5-fold). Addition of COUP-TF to both cell types strikingly abolished the promoter activity.



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Figure 6. Effects of COUP-TF and SF-1 on P450arom Promoter II Activity in a Primary Culture of Endometriotic Stromal Cells

Overexpression of SF-1 caused a marked increase in both baseline and (Bu)2cAMP-induced activities of the -517 promoter II construct, whereas overexpression of COUP-TFI and II markedly decreased these activities. Results are represented as luciferase activities expressed relative to the -100-bp construct, which was given the arbitrary value of 1, and are presented as the average of data from three replicate experiments ± SEM.

 


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Figure 7. Effects of COUP-TF and SF-1 on P450arom Promoter II Activity in a Primary Culture of Endometrial Stromal Cells

Overexpression of SF-1 caused a 40-fold increase in the basal luciferase activity, and treatment with (Bu)2cAMP further increased activities of the -517 promoter II construct by 11.5-fold to 460-fold the baseline level. Overexpression of COUP-TFI and II nearly abolished the cAMP-stimulated activity in this construct. Results are represented as luciferase activities expressed relative to the -100 bp construct, which was given the arbitrary value of 1, and are presented as the average of data from three replicate experiments ± SEM.

 
The presence of -517 promoter II activity in eutopic endometrial cells, despite the absence of aromatase transcripts or enzyme activity, may be explicable by the construct length. Longer constructs may contain other inhibitory elements that block transcription. We arbitrarily chose the -517 deletion construct, since it had the highest activity (Fig. 1Go), and the purpose of these experiments was to demonstrate stimulation by SF-1 and inhibition by COUP-TF in both cell types. All cultures were routinely subjected to aromatase activity assays. No baseline or cAMP-induced aromatase activity could be demonstrated in eutopic endometrial cells in culture, whereas aromatase activity in endometriotic stromal cells showed a baseline activity of 0.6–1.0 pmol/mg/protein/4 h. This activity was increased by (Bu)2cAMP stimulation by 25 to 30 times. These results are indicative of a critical inhibitory role of COUP-TF and stimulatory action of SF-1 for aromatase expression in endometriosis.

Detection of COUP-TF and SF-1 Transcripts in Eutopic Endometrial and Endometriotic Tissues
COUP-TFI and II transcripts were detected in total RNA samples of both eutopic endometrium (n = 12) and extraovarian endometriotic tissues (n = 8) using Northern blotting (Fig. 8Go). On the other hand, SF-1 transcripts were readily detectable by RT-PCR/Southern hybridization in all extraovarian endometriotic tissue samples (n = 12), whereas in only 3 of 15 eutopic endometrial tissue samples were SF-1 transcripts detected (Fig. 9Go). In 2 of these 3 positive eutopic endometrial samples, SF-1 transcripts were barely detectable (data not shown). No differences in COUP-TF or SF-1 expression were detected either between proliferative and luteal samples or between eutopic endometrial samples from patients with endometriosis and those from disease-free women.



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Figure 8. Northern Blot Analysis for COUP-TF

Northern blot analysis of total RNA (20 µg) from four secretory phase endometriotic (lanes 1–4) and four simultaneously biopsied (secretory phase) eutopic endometrial samples (lanes 5- 8), and disease-free normal peritoneum (per, lane 9). The following total RNA samples (3 µg) were used as controls: placenta (plc, lane 10) and testis (tes, lane 11). Using a COUP-TFII cDNA probe, transcripts of two sizes (1.3 kb and 1.5 kb) were detected. Since the 1.3-kb and 1.5-kb bands correspond to COUP-TFII and COUP-TFI transcripts, respectively, the membrane was not rehybridized with a COUP-TFI cDNA probe (COUP-TFI and II cDNAs display a homology of >80%). There does not appear to be a difference in the levels of COUP-TF transcripts between endometriosis and eutopic endometrium. Lower panel, Transcripts of ß-actin, a housekeeping gene.

 


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Figure 9. Differential SF-1 Expression in Eutopic Endometrium and Extraovarian Endometriosis

Southern blot analysis of RT-PCR products after amplification of SF-1 transcripts is shown in the upper panel. A positive signal was detected in only one of six eutopic endometrial RNA samples (1 µg), whereas all five endometriotic RNA samples (1 µg) were positive. RNA from normal-appearing peritoneum (0.5 µg) from a patient with endometriosis was used as a negative control, while testicular RNA (0.2 µg) was used as a positive control for SF-1. The lower panel shows amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts to control for the integrity of RNA used for RT-PCR.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study was performed to determine molecular mechanisms responsible for cAMP- or PGE2-induced aromatase expression in endometriotic stromal cells and its absence in stromal cells derived from eutopic endometrium. One mechanism appears to be the competition of stimulatory (SF-1) and inhibitory (COUP-TF) factors for occupancy of a specific binding site upstream of the P450arom promoter II to initiate or inhibit transcription (Fig. 10Go). SF-1 is an orphan nuclear receptor and is a key regulator of endocrine function within the hypothalamus, pituitary, gonads, and adrenal cortex and an essential factor in sex differentiation (14). In particular, SF-1 is a transcription factor with limited tissue distribution, which recognizes a conserved regulatory consensus site in the proximal promoter regions of genes encoding steroidogenic P450s. This conserved nuclear receptor half-site (AGGTCA) is also present in the proximal promoter (promoter II) of the CYP19 (P450arom) gene, and we demonstrated previously that SF-1 binding to this sequence in bovine ovarian granulosa cells enhanced transcription in response to cAMP analogs or FSH (13). Another critical regulatory element in promoter II for aromatase expression in ovarian granulosa cells was previously shown to be an imperfect CRE with which CREB was demonstrated to interact (12). This CRE did not seem to have a critical role in differential expression of aromatase in the eutopic endometrial and endometriotic cells, since CRE binding activity with nuclear proteins from both cell types gave rise to identical DNA-protein complexes. This, however, did not rule out binding of active and inactive forms of CREB to this CRE in these two cell types; also, no mutagenesis was preformed to evaluate the relative importance of CRE. In contrast to CRE, SF-1 and COUP-TF were shown to compete for binding to the nuclear receptor half-site and mediate, at least in part, cAMP-stimulated P450arom gene transcription in endometriosis and inhibition of transcription in the eutopic endometrium.



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Figure 10. Proposed Mechanism for the Regulation of P450arom Expression by SF-1 and COUP-TF in Eutopic Endometrium and Endometriosis

Panel A represents binding of COUP-TF readily to the nuclear receptor half-site in promoter II in the absence of SF-1 in eutopic endometrial stromal cells. Thus, COUP-TF exerts its inhibitory effect on the complex of general transcription factors (GTFs) that bind to the TATA box. Panel B, In endometriotic stromal cells that contain both SF-1 and COUP-TF, however, SF-1 binds to the nuclear receptor half-site with a higher affinity than that of COUP-TF and synergizes with CREB and other transcription factors to activate the transcription of the CYP19 (P450arom) gene in response to cAMP.

 
COUP-TF is another orphan nuclear receptor, which is primarily of inhibitory nature and binds promiscuously to a variety of repeats of AGGTCA (nuclear receptor half-site) or related sequences. Two isoforms of COUP-TF (I and II) are expressed in many human tissues and display 98% and 96% homology (in amino acid sequences) in the DNA-binding and ligand-binding domains, respectively (21). Through direct competition with vitamin D receptor, thyroid receptor, and retionic acid receptor for the available binding sites, COUP-TFs have been shown to repress hormonal induction of target genes (21). Additionally, COUP-TFs were previously demonstrated to inhibit the transactivation by SF-1 due to mutually exclusive binding to a direct repeat of nuclear receptor half-site in CYP17 (P45017{alpha}) gene promoter (20). A similar competitive model was proposed by Yu et al. for the DAX-1 gene. In this model, as in our study, SF1 stimulated while COUP-TF inhibited promoter activity. Both transcription factors acted via the same binding site in the DAX-1 gene promoter (22). We have shown herein that COUP-TFs bind to an inverted repeat of the half-site in CYP11B2 gene promoter much more avidly than to the single half-site in P450arom (CYP19) promoter II. Thus, it is conceivable that the binding affinity of SF-1, which binds to DNA as a monomer, for the single nuclear receptor half-site in P450arom promoter II is higher than that of COUP-TFs. In fact, this concept constitutes the basis for the model in Fig. 10Go: COUP-TFs are ubiquitously expressed in both eutopic endometrium and endometriosis, whereas SF-1 expression is limited to endometriosis. In the absence of SF-1, COUP-TFs bind to the nuclear receptor half-site in eutopic endometrium, albeit with a relaxed affinity. In endometriosis, however, SF-1 readily displaces COUP-TF and binds to this site with a higher affinity to activate promoter II.

As demonstrated in Fig. 1Go, the -214/-100 bp region of promoter II, which contains the nuclear receptor half-site and CRE, accounts for 30% of fold-induction in promoter activity in response to (Bu)2cAMP. Characterization of other regulatory elements in the -517/-214 bp region that mediate the potentiation of cAMP response is under way. The critical roles of COUP-TFs and SF-1 in differential aromatase expression, however, is evident by the near-complete abolishment of cAMP-induced promoter II activity of the -517-bp reporter construct upon overexpression of COUP-TFs in endometriotic cells (Fig. 6Go). Moreover, longer constructs may contain inhibitory regulatory element. The lack of such silencer regions in the -517 luciferase construct may explain the presence of -517 promoter II activity in eutopic endometrial cells despite the absence of aromatase expression in these cells (Fig. 7Go).

Finally, these orphan nuclear receptors may have other functions in the endometrium and endometriosis other than the regulation of aromatase expression. For example, COUP-TF has been shown to antagonize estrogen receptor activation of the lactoferrin and oxytocin promoters by binding to a DNA site that overlaps the estrogen response element (23). Thus, one can envision COUP-TF as a factor that protects the endometrium from estrogen action. Loss of such mechanisms in endometriosis (e.g. aberrant SF-1 expression) may promote the growth and development of endometriotic tissues. The presence of SF-1 expression in all endometriotic tissues that were tested, but only in a very small percentage of eutopic endometrial samples, is a very exciting finding. SF-1 in endometriosis may regulate the expression of other genes that are important for the growth of this tissue. Little is currently known with respect to regulation of SF-1 expression. Michael and co-workers (13) previously demonstrated a significant increase in SF-1 transcript levels in ovarian granulosa cells in response to forskolin. Thus, aberrant expression of aromatase in endometriotic stromal cells may be mediated, in part, by increased SF-1 levels due to exposure of these ectopically located cells to factors (e.g. increased levels of PGE2) that stimulate cAMP formation. Another possibility is the interaction of the cAMP-dependent signal transduction pathway with the COUP-TF corepressor complex. COUP-TF repressor properties are at least partially mediated by carrying the transcriptional corepressor N-CoR to target gene promoters (24, 25, 26). Some nuclear receptor-corepressor interactions were shown to be disrupted by cAMP in recent reports (27, 28). If this mechanism is important in the case of endometriosis, increased PGE2 may chronically stimulate the cAMP-dependent pathway and disrupt the protective action(s) of COUP-TF in normal endometrium, leading to aromatase expression (7, 8).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue Acquisition and Processing and RNA Isolation
At the time of laparoscopy or laparotomy, the following types of samples were obtained: 1) extraovarian endometriotic implants from patients with endometriosis, 2) ovarian endometriomas, 3) normal appearing peritoneum from patients with endometriosis, and 4) eutopic endometrial tissues from women with endometriosis and disease-free women. All samples were histologically confirmed, and the phase of the menstrual cycle was determined by histological examination. Tissue samples were frozen instantly in liquid nitrogen in the operating room and stored at -80 C. Total RNA was isolated from tissues by the guanidium thiocyanate-cesium chloride method (29). Samples for histology were transported in 10% formalin and embedded in paraffin. Samples used for culture were transported in HBSS with HEPES and 2% antibiotic/antimycotic solution for cell culture and were immediately processed. Written informed consent was obtained before surgical procedures, including a consent form and protocol approved by the Institutional Review Board for Human Research of the University of Texas Southwestern Medical Center.

Cell Cultures and Aromatase Activity
Endometriomas (dissected from surrounding tissue under microscope) and eutopic endometrial tissues were cultured using a protocol previously reported by Ryan et al. (30) with minor modifications (8). Tissues were rinsed with sterile saline solution, minced finely, and digested with collagenase B (1 mg/ml) and deoxyribonuclease I (0.1 mg/ml) at 37 C for 30–60 min. Epithelial cells were separated from stromal cells by filtration through a 75-µm sieve. Stromal cells were then suspended in DMEM/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% FBS. Fresh suspensions of stromal cells were plated in culture dishes and kept in an incubator in a humidified atmosphere with 5% CO2 at 37 C. Media were changed at 48-h intervals until the cells became 75% confluent. Stromal cells were then placed in serum-free medium for 16–24 h. Then the cells were treated overnight in serum-free medium. Cells used for RNA preparation were rinsed and then frozen with liquid nitrogen and kept at -80 C. Cells used for nuclear extract preparation were processed immediately. Aromatase activity in cells was assayed by measuring the rate of incorporation of [3H] from [3H]androstenedione to [3H]water, as previously described (31).

5'-RACE of P450arom
P450arom cDNA libraries were constructed from extraovarian endometriotic tissue samples from three women. Construction of RACE cDNAs was performed with modifications as described by Frohman and co-workers (32, 33). The first step, first-strand synthesis, was performed using 2 µg of total RNA, 10 pmol of an antisense primer (located in coding exon III, 5'-ACTTGCTGATAATGAGTGTT-3'), and Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD). The primer extension was carried out at 42 C for 1.5 h. RNA was degraded with RNAse Mix (GIBCO BRL). Before the tailing step, the single-stranded cDNA was denatured at 65 C for 5 min, placed on ice, and then tailed at the 3'-end with poly(C) using the enzyme terminal transferase. The amplifications were performed using the Perkin-Elmer Cetus (Norwalk, CT) buffer system, a nested P450arom antisense primer (located in coding exon II, 5'-CTGGTATTGAGGATATGCCCTCAT-AAT-3'), a poly(G) anchor primer, and Taq polymerase. An aliquot of the amplified product was run on a 1.8% agarose gel to visually estimate DNA concentrations. The cDNA was then ligated into the pAMP1 plasmid and transformed into DH5{alpha}-competent cells using the cloneAmp pAMP1 system (GIBCO BRL). Positive colonies were screened using the tetramethyl ammonium chloride method as described (34). Positive clones were sequenced using the dsDNA Cycle Sequencing System (GIBCO BRL).

Deletion Mutations
The 5'-deletion mutations of promoter II regulatory region of the P450arom gene were amplified from a subcloned promoter II genomic fragment using PCR and single-stranded oligonucleotides synthesized with sequence complementary to the promoter II sequence of interest plus nonannealing ends for the restriction sites SalI (5'-primer) and PstI (3'-primer). The PCR reactions were performed in 50 µl [150 ng plasmid template, 300 ng forward and reverse primers, 200 µM deoxynucleoside triphosphates, 5 U AmpliTaq polymerase in 1x AmpliTaq buffer (Perkin-Elmer Cetus)] with the following reaction conditions: a single denaturation cycle at 93 C for 3 min, 30 cycles of 93 C for 30 sec, melting temperature (Tm)-5 C for 1 min, and 72 C for 1.5–2 min, and a single cycle of 72 C for 7 min to polish any prematurely terminated products. PCR products were directly subcloned into pCR2.1 using the TA Cloning System as described in the manufacturer’s protocol (Invitrogen, Carlsbad, CA). To screen for clones carrying the appropriate inserts, the resultant bacterial colonies were transferred to Colony/Plaque Screen Hybridization Transfer Membrane (DuPont NEN, Boston, MA) and were lysed/denatured according to the recommendations of the manufacturer. Plasmid DNA was isolated from positive clones, and inserts were sequenced to ensure fidelity of the amplified sequences.

The promoter II fragments were released from the pCR 2.1 vector by restriction digest with SalI and PstI and were subcloned into SalI and PstI sites of the reporter vectors. The pGL3-Basic vector was modified by destroying the endogenous SalI restriction site in the original vector (Promega, Madison, WI) by digesting with SalI, blunting the ends with Klenow, and ligating the ends to restore the plasmid. This vector, pGL3-BX-Sal, was digested with XhoI and HindIII, and a synthetic, double-stranded oligonucleotide with XhoI and HindIII cohesive ends and internal sites for SalI, PvuII, PstI, and BglII (5'-TCGAGTCGACAGCTGTACTGCAGAGATC-TCCA-3') was ligated into the multiple cloning site of the vector, thereby generating the promoterless, enhancerless modified pGL3-B vector. pGL3-Bmod/promoter II constructs containing -694/-16, -517/-16, -278/-16, -140/-16, or -100/-16 bp of P450arom promoter II 5'-flanking DNA and the pGL3-Bmod plasmid without a promoter were transfected along with ß-galactosidase reference plasmid into primary endometriotic stromal cell cultures.

Transient Transfections and Luciferase Assays
Transient transfection of endometriotic stromal cells in culture were carried out in 35-mm dishes using the Lipofectamine reagent (GIBCO BRL) with the following plasmids: 1) 2 µg of the modified pGL3-Basic (PGL3-Bmod) luciferase reporter plasmid that contains serial deletion mutants of P450arom promoter II; 2) 2 µg of the pcDNA3 expression plasmid (Invitrogen) that contains either SF-1, COUP-TFI, or COUP-TFII cDNAs, and 3) ß-galactosidase expression plasmid (0.3 µg). After transfection for 6 h in serum-free and antibiotic-free media, medium was changed to DMEM/F12 (with antibiotics and 10% FBS). After an overnight recovery in the serum-containing medium, cells were kept in serum-free medium for 12 h. Thereafter, cells in serum-free medium were treated with 0.5 mM (Bu)2cAMP for 16 h.

After treatment, transfected cells were washed twice in PBS and lysed in 125 µl of a buffer [0.1 M potassium phosphate, pH 7.8; 1% Triton X-100; 1 mM dithiothreitol (DTT); 2 mM EDTA]. Luciferase assays were performed on 40 µl of cell lysate using an enhanced luciferase assay kit (Analytical Luminescence Laboratory, Sparks, MD). ß-Galactosidase assays were performed on 10 µl of cell lysate using the Galacto-Light Chemiluminescent Reporter Assay (Tropix, Inc., Bedford, MA). Luminescent activities were measured using a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). Results are presented as the average of data from three replicate experiments ± SEM.

Isolation of Nuclear Proteins from Cell Cultures and in Vitro Transcription and Translation of SF-1 and COUP-TF
The method described by Dignam et al. (35) was followed to isolate nuclear proteins from cultured cells (35). Briefly, 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 2 min at 700 x g. The supernatant was removed by aspiration, and the cellular pellet was resuspended in 10 cell-pellet volumes of cold buffer A (10 mM HEPES, pH 7.4; 1.5 mM MgCl2; 10 mM KC1; 9.5 mM DTT; 10 µg/ml leupeptin; 100 µg/ml pepstain; 2 µg aprotonin; 0.5 mM phenylmethylsulfonyl fluoride). The cells were homogenized in a 7- or 15 ml Dounce homogenizer on ice with 10–20 strokes by hand turning. Once greater than 90% of the cell membranes were broken (as determined by the failure to exclude trypan blue from the nucleus), the lysate was transferred to a 15-ml polypropylene tube and centrifuged for 2 min at 700 x g. After the supernatant was removed, the pellet was resuspended in 1 cell-pellet volume of buffer C (20 mM HEPES, pH 7.4; 420 mM NaC1; 1.5 mM MgC12; 0.2 mM EDTA; 0.5 mM DTT; 20% glycerol) and incubated on ice for 30 min with intermittent mixing. After centrifugation at 60,000 rpm for 5 min at 4 C, the supernatant was aliquoted and snap frozen in liquid nitrogen. Protein concentrations were determined by a modified Bradford assay (Bio-Rad, Hercules, CA), and nuclear proteins were stored at -80 C.

In vitro transcription and translation were performed using the TNT-coupled Reticulocyte Lysate System (Promega). SF-1, COUP-TFI, and COUP-TFII were transcribed from their cDNAs inserted in a PGEM-7Zf plasmid (Promega), as per manufacturer’s instructions. The following ingredients were assembled in a 1.5-ml microcentrifuge tube for the transcription and translation: TNT Rabbit Reticulocyte Lysate, TNT Reaction buffer, SP6 TNT RNA polymerase for COUP-TFI and SP7 TNT RNA polymerase for COUP-TFII and SF-1, amino acid mixture without methionine, RNAsin ribonuclease inhibitor, DNA template, and ribonuclease-free water. Reaction volume was 50 µl. The reaction mix was incubated for 90 min at 30 C. Proteins were then snap frozen and stored at -80 C.

EMSAs
Double-stranded oligonucleotide probes (10 pmol) were labeled in a reaction containing 2.5 µM dATP, dGTP, and dTTP, 1x buffer H (Boehringer Mannheim, Mannheim, Germany), 50 µCi of [{alpha}-32P]dCTP, and 3.75 U Klenow fragment (Boehringer Mannheim). The labeling reaction mix was incubated at 37 C for 15 min, and unincorporated nucleotides were removed from the labeled DNA by Bio-Spin 6 Chromatography Columns (Bio-Rad, Hercules, CA). Nuclear proteins were incubated with the radiolabeled, double-stranded DNA probes (10,000–15,000 cpm/reaction) for 10 min at room temperature in a reaction buffer (20 mM HEPES, pH 7.6; 75 mM KCl; 0.2 mM EDTA; 20% glycerol) and 2 µg poly(dI-dC)-poly(dI-dC) (Pharmacia, Uppsala, Sweden) as nonspecific competitor. Protein-DNA complexes were resolved on 6% nondenaturing polyacrylamide gels with 0.5x Tris-borate-EDTA running buffer and were visualized by autoradiography. For DNA competition EMSA, a nonradiolabeled double-stranded oligonucleotide was added simultaneously with labeled probe. Supershift EMSAs were performed by adding 1 µl of antiserum to the binding reaction, followed by a 30-min incubation on ice before electrophoresis (SF-1/Ad4BP antiserum was provided by Dr. Ken-ichiro Morohashi and COUP-TF antiserum was provided by Dr. Ming-Jer Tsai). We used the following double-stranded DNA probes: the imperfect CRE (5'-CGCGTGGGAATGCACGTCACTCTAG-3') and the nuclear receptor half-site (5'-CACTCTACCAAGGTCAGAAATG-3') represent identical sequences in the promoter II regulatory region of the (CYP19) P450arom gene. We also used a probe (5'-CGCGAAGGTCAAGGCTGGAGGCCC-3') that represents the -129/-114 bp regulatory region in the CYP11B2 gene promoter and contains an imperfect inverse repeat of the nuclear receptor half-site. This element was previously reported to bind both SF-1 and COUP-TF (36).

RT-PCR/Southern Hybridization
PCR amplification of sequences in the coding regions of SF-1 transcripts were performed using specific oligonucleotides that were designed to flank three exons. This procedure involved synthesizing initially a cDNA template by reverse transcription of 1 µg of total RNA from endometrial and endometriotic tissues using Superscript II reverse transcriptase (GIBCO BRL) and random primers. A specific region in the coding sequence of SF-1 cDNA was then amplified by PCR. The reaction was carried out in a 50-µl volume using Amplitaq DNA polymerase (Perkin-Elmer Cetus), 1 mM of deoxynucleoside triphosphates, 10 µM sense (5'-AGAATGGCCGACCAGACCTTC-3'; exon 3) and antisense (5'-CCAGGCTGAAGAGGATGATGAAC-3'; exon 5) primers and PCR buffer (Perkin-Elmer Cetus, Norwalk, CT) for 30 cycles. Denaturing was performed at 94 C for 40 sec, annealing at 51.5 C for 40 sec, and extension at 72 C for 60 sec. PCR products were then fractionated on 1.8% agarose gel to visualize the 340-bp band of expected size and transferred to nylon membranes for Southern blot analysis. The membranes were hybridized for 16 h with a 32P-labeled oligonucleotide probe (5'-GGTCGAACACCAGCAGCTCGCTCC-3', exon 4). Autoradiographs were then exposed to blotting membranes. Transcripts of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified and probed in a similar fashion to verify the integrity of comparable amounts of total RNA in each sample. This has been described previously (37).

Northern Blotting
Total RNA was size fractionated by electrophoresis on 1% formaldehyde-agarose gels (20 µg/lane) and transferred electrophoretically to a nylon membrane. The RNA was cross-linked to the nylon membranes by UV. The membranes were prehybridized for 24 h at 42 C in prehybridization buffer comprised of formamide (50% by volume), NaH2PO4 (250 mM, pH 7.2), NaCl (250 mM), SDS (7%, wt/vol), and denatured sheared salmon sperm DNA (100 µg/ml). Hybridizations were conducted for 16 h at 42 C in the same buffer after the addition of COUP-TFII cDNA probe (106 cpm/ml) radiolabeled with [{alpha}-32P]dCTP using random hexanucleotide primers and Klenow. After hybridization, the blots were washed with varying concentrations of saline sodium citrate and SDS at varying temperatures. The membranes were exposed to film with intensifying screens at -80 C for varying lengths of time. The presence of comparable amounts of total RNA in each lane was verified by hybridization of membranes with a ß-actin cDNA probe (CLONTECH Laboratories, Inc., Palo Alto, CA). Since the cDNAs of COUP-TFI and COUP-TFII are highly homologous (>80%) (38), and we detected two bands that corresponded to COUP-TFI (1.5 kb) and COUP-TFII (1.3 kb) mRNAs using the COUP-TFII probe, rehybridization of the membrane with the COUP-TFI probe was not performed (17, 18, 19).


    ACKNOWLEDGMENTS
 
Rosemary Bell provided skilled editorial assistance.

This work was supported by an unrestricted research grant from the American Society for Reproductive Medicine-Organon, by National Cancer Institute Grant CA-67167, and by American Association of Obstetricians and Gynecologists Postdoctoral Fellowship Award (K.Z.).


    FOOTNOTES
 
Address requests for reprints to: Serdar E. Bulun, M.D., Green Center for Reproductive Biology Sciences, Department of Obstetrics-Gynecology, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: Bulun{at}grnctr.swmed.edu

Received for publication June 3, 1998. Revision received October 14, 1998. Accepted for publication October 16, 1998.


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