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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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 1
). 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.
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. 1
). 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. 1
). 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. 1
). 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.
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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. 2A
). 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. 2A
). This
endometriotic cell-specific complex (cx2) was identical to that formed
with nuclear proteins of human granulosa cells (Fig. 2A
). 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. 2B
). 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.
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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. 3
, 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. 3A
). 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. 3B
). 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. 3B
). 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 3C
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.
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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. 2
and 3
). 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. 4
). 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. 4
), 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).
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COUP-TF and SF-1 Compete for Binding to Nuclear Receptor
Half-Site
As illustrated in Fig. 5
, 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. 5
).

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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.
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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. 6
and 7
). 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. 6
). 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. 7
). 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.
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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. 1
), 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
0.61.0 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. 8
). 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. 9
). 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 14) 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.
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|
 |
DISCUSSION
|
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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. 10
). 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
) 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. 10
: 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. 1
, 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. 6
). 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. 7
).
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
|
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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 3060 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 1624 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
-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 1.52 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 manufacturers 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 1020
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 manufacturers 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
[
-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 (10,00015,000 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
[
-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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