From the Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
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ABSTRACT |
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Steroidogenic acute regulatory protein (StAR) is
a vital accessory protein required for biosynthesis of steroid hormones
from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are
acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the The first and key reaction in the enzymatic cascade of steroid
hormone biosynthesis is catalyzed in the mitochondria by cholesterol side chain cleavage cytochrome P450
(P450scc)1 (1-3). In the
presence of atmospheric oxygen and reducing power provided by
associated proteins, P450scc converts cholesterol substrate to the
first steroid prototype molecule, pregnenolone (1). In order to do so,
a supply of cholesterol is required to be transferred from cytosolic
pools into the inner membranes of the mitochondrion, where P450scc
resides (4-7). Recently, it was found that cholesterol delivery into
the mitochondria is enhanced by a novel protein (8, 9) designated
steroidogenic acute regulatory (StAR) protein (reviewed in Refs.
10-12). More studies have established the fact that StAR is a vital
protein essential for steroidogenesis in the adrenal cortex and the
gonads (13, 14). In rodents, StAR is also expressed in steroidogenic brain cells (15) and
placenta.2 Interestingly,
StAR is not expressed in human placenta, where its role is probably
assumed by a less efficient StAR substitute called MLN64 (16). Perhaps
the most compelling evidence for the critical role of StAR in
steroidogenesis was the discovery that various mutations of the StAR
gene encoding a functionally impaired protein (14) cause a syndrome
known as lipoid congenital adrenal hyperplasia (17). Affected
individuals die shortly after birth in the absence of adrenal steroids,
unless treated with steroid hormone replacement therapy. Similar
patterns were also observed in StAR null mice (14).
Trophic hormones, such as gonadotropins and ACTH, trigger up-regulation
of StAR expression by cAMP signaling (18-20). Additionally, Ca2+ changes evoke StAR expression in glomerulosa cells of
the adrenal cortex (21, 22). Very little is known about the factors
controlling StAR expression at the transcriptional level, downstream to
the signal transduction pathways. Special attention has been devoted to
examine the potential involvement of the steroidogenic factor-1 (SF-1,
or Ad4BP), which is a pivotal tissue-specific orphan nuclear receptor
essential for regulation of many steroid hydroxylases in
steroid-producing tissues (23, 24). In light of the fact that StAR
promoter includes several putative recognition sites for SF-1 binding,
several attempts have be made to determine if the latter factor is
involved in StAR regulation. At present, the available results are
somewhat inconsistent. Using the human, mouse, and rat promoters, an
apparent up-regulation of StAR transcription by SF-1 could be
demonstrated upon co-transfection of SF-1 cDNA and
promoter-reporter plasmids in non-steroidogenic cells (25-29). However, other studies analyzing the activity of StAR promoter in
SF-1-expressing cells did not support a role for SF-1 in a cAMP-inducible fashion (30). These data suggested that SF-1 may not
confer cAMP responsiveness in authentic steroidogenic cells and,
therefore, cannot be an exclusive transcription factor controlling the
acute regulation of StAR in such cells.
In search for alternative regulatory elements that can mediate the
acute response of StAR to hormones, we undertook to study the
inducibility of the mouse promoter in ovarian granulosa cells from
prepubertal rats. Earlier studies have unambiguously shown that
endogenous SF-1 in these cells is critical for the induction of P450scc
and P450aromatase by follicle-stimulating hormone (FSH) (31-34). In
contrast, the present study suggests that SF-1 is probably not involved
in hormonal activation of StAR promoter. We also demonstrate that
promoter regions capable of C/EBP Materials--
Ovine FSH (NIDDK-oFSH-20) was kindly provided by
the National Institute of Health NIAMD (Bethesda, MD.). Acetyl-CoA,
poly(dI-dC), RNase A, indomethacin, proteinase K, sodium
orthovanadate, aprotinin, NaF, pepstatin,
phenylmethylsulfonyl fluoride, peroxidase-conjugated goat anti-rabbit
and peroxidase-conjugated rabbit anti-goat sera were obtained from
Sigma. Dulbecco's modified Eagle's medium and Ham's F-12 medium were
from Grand Island Biological, New York. Polyclonal antisera to C/EBP Animals--
Intact, immature female Sprague-Dawley rats (21 days old) were obtained from Harlan (Jerusalem, Israel) and maintained
under 16:8 light:dark schedule with food and water ad
libitum. Animals were treated in accordance with the NIH Guide for
the Care and Use of Laboratory Animals. All protocols had the approval
of the Institutional Committee on Animal Care and Use, Alexander
Silberman Institute of Life Sciences, Hebrew University of Jerusalem.
Naive granulosa cells for CAT assays were expressed form
E2-primed rats (35). Preovulatory PMSG-hCG-treated ovaries
were prepared by administration of 15 IU of PMSG (PMSG 600, Intervet,
Angers, France) to 25-day-old rats, which were further treated with 4 IU of human chorionic gonadotropin (hCG, Organon Special Chemicals,
West Orange, NJ) administered (subcutaneously) 50 h later. The
animals were sacrificed at 8 h after hCG, and ovaries were
retrieved for protein extraction. Also, post-ovulatory ovaries enriched
with corpora lutea were similarly harvested 72 h after onset of
PMSG-hCG treatment.
Cell Cultures--
To obtain granulosa cells expressing low
basal levels of CAT, we used a modification of a previously described
method (35). Briefly, after incubation in hypertonic
sucrose/EGTA-containing medium, the ovaries were incubated for
additional 45 min in Dulbecco's modified Eagle's medium/F-12 medium
containing 10 µM indomethacin. The same medium also
served for further procedures, including needle pricking of the ovaries
and post-electroporation treatments. After electroporation the cells
were plated onto serum-coated wells (35) (24-well plated; Nunc,
Copenhagen, Denmark) and incubated at 37 °C in 95% air and 5%
CO2.
Electroporation and CAT Assay--
Estradiol-primed granulosa
cells (4 × 105) obtained from 3-4 ovaries were
electroporated in the presence of 20 µg of DNA as described before
(35). Cells from each cuvette were seeded into four wells and hormonal
treatments were initiated after a 3-h recovery period. Following a 6-h
treatment with FSH (100 ng/ml), cell lysates were prepared and CAT
activity was analyzed as described previously (35). Quantitation of the
CAT assay was performed using a Fuji Bio-Imaging analyzer (BAS-1000,
Fuji Photo Film, Tokyo, Japan). Protein was determined by a modified
method of Bradford (36) using the Bio-Rad protein assay. Data are
presented as percent of [14C]chloramphenicol (Amersham
International, Little Chalfont, United Kingdom) converted to its
acetylated products (per protein and time of assay) and the -fold
induction of CAT activity over basal values measured in the absence of
hormone. Data are presented as the mean ± S.E. of several
independent transfections as indicated in each figure.
Whole Tissue Extracts for EMSA--
Whole ovarian extracts for
EMSA was performed as described before (37). Briefly, ovaries were
homogenized in a Dounce homogenizer using 2-3 volumes of buffer A
containing 400 mM KCl, 10 mM
NaH2PO4, (pH 7.4), 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 5 µM NaF, 1 mM sodium orthovanadate, 5 µg/ml
aprotinin, 2 µM pepstatin, and 1 mM
phenylmethylsulfonyl fluoride. Following homogenization, the protein
slurry was freeze-thawed three times in liquid nitrogen and finally
centrifuged for 2 min at 14,000 × g. After
determination of the protein content, the supernatant was aliquoted and
kept at Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay (EMSA) was performed as described before (37).
Briefly, whole cell extracts (3-15 µg) were incubated with 2 ng of
double-stranded DNA, previously labeled by a fill-in reaction using
Klenow fragment (Promega, Madison, WI) and [ Promoter Constructs--
A previously published sequence of the
5'-flanking region of the mouse StAR gene (30), was used to clone most
of the StAR promoter constructs by a PCR-based approach.
5'-HindIII and 3'-XbaI cloning sites were
included in all forward and reverse primers, respectively. To generate
the
Further progressive deletions of the promoter constructs were prepared
using the
To generate constructs with point mutations, oligonucleotides
containing the point mutations of choice were used for the PCR reaction
as the forward primers, using the
The double mutant construct Western Blot and RT-PCR Analyses--
At the indicated time
points, granulosa cells were extracted by lysis buffer (RIPA) and
analyzed by SDS-PAGE and electro-blotting procedures as described
previously (40). After a 1-h incubation with anti-C/EBP
Total RNA was extracted by dissolving the granulosa cells in 0.5 ml of
RNAzol B (Tel-Test, Inc., Friendwood, TX) added to each culture well
(16 mm). Further steps followed the manufacturer's instructions.
Semiquantitative RT-PCR analysis of total RNA extracts from granulosa
cells was performed exactly as described previously (40).
Statistical Analysis--
Student's unpaired two-tailed
t test was performed using Microsoft Excel 97 statistical
analysis functions. Differences between the activities of the indicated
constructs were considered statistically significant at
p < 0.05.
Is SF-1 Involved in Regulation of StAR Promoter?--
Aiming to
identify the regulatory elements controlling StAR expression, we have
applied transient expression assays of the mouse StAR promoter by use
of granulosa cells from prepubertal rat ovary. To this end, a
At large, the activity values obtained by transfecting a series of
progressive deletions of the promoter showed that hormone inducibility
remained high in all constructs pruned down to
The apparent cooperativity of Sp1 and SF-1 in mediating cAMP-driven
expression of steroidogenic genes (34, 42) could have suggested that a
similar concept might be functionally relevant for FSH induction of
StAR. Therefore, CAT reporter transgenes containing point mutations in
the FSH Responsiveness Is Determined by the
Sequence analysis of the
Despite the fact that the sequence of C/EBP The Role of GATA-4--
However, the moderate attenuating effect
caused by mutating the C/EBP
Fig. 10 shows a single protein complex
labeled by either of the two DNA probes, Granulosa Cell Expression of C/EBP
By contrast to the profile of C/EBP The objective of the present study was to reveal the principal
sequence elements that render the promoter of StAR gene
responsive to FSH in cells of the rodent ovary. Based on previously
published sequence (30), we cloned a High responsiveness to FSH still remained if the promoter was pruned
down to position Upstream of position Indeed, deletion mutants and site-directed mutation within the The present study shows that the prepubertal granulosa cells already
express GATA-4 in vivo, and the level of this protein remained unaffected under any culture manipulations. By contrast, the
C/EBP Our EMSAs showed that the C/EBP Like C/EBP1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to
freshly prepared cells. FSH inducibility determined over a 6-h
incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were
sufficient for transcription of the StAR promoter constructs: a
non-consensus binding sequence (
81/
72) for CCAAT enhancer-binding protein
(C/EBP
) and a consensus motif for GATA-4 binding
(
61/
66). Western analyses showed that GATA-4 is constitutively
expressed in the granulosa cells, while all isoforms of C/EBP
were
markedly inducible by FSH. Site-directed mutations of both binding
sequences practically ablated both basal and hormone-driven
chloramphenicol acetyltransferase activities to less than 5% of the
parental
96/+6 construct. Unlike earlier notions, elimination of
potential binding sites for steroidogenic factor-1, a well known
tissue-specific transcription factor, did not impair StAR
transcription. Consequently, we propose that C/EBP
and GATA-4
represent a novel combination of transcription factors capable of
conferring an acute response to hormones upon their concomitant binding
to the StAR promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and GATA-4 binding are required
for activation of StAR transcription in FSH-treated cells. Thus, StAR
provides the first example of a steroidogenesis-associated protein that
is transcriptionally controlled by C/EBP
and/or GATA-4.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(sc-150x), C/EBP
(sc-61x), Sp1 (sc-059x), GATA-4 (sc-1237x), GATA-6
(sc-7244x), c-Fos (sc-253x), and c-Jun/AP-1 (sc-44x) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). SF-1 antibody was
purchased from Upstate Biotechnology Inc. (Lake Placid, NY).
70 °C until use.
-32P]-dCTP
(Amersham International, Little Chalfont, United Kingdom). Incubation
was performed using a final volume of 30 µl of buffer containing 100 mM KCl, 15 mM Tris-HCl (pH 7.5), 10 mM dithiothreitol, 1 mM EDTA, 5 mM
MgCl2, 12% glycerol, and 4.5 µg of poly(dI-dC). After
incubation for 35 min at room temperature, the binding reactions were
resolved on pre-run 5% acrylamide gel as described previously for
quantitative RT-PCR analyses (38). When competition experiments were
conducted in the presence of molar excess of cold probe, the protein
extracts were added last to the reaction mixture. When antibodies were
used for supershift (or ablation) of a given protein-DNA complex, the
protein extracts were preincubated for 25 min at room temperature with
2-8 µg of the antiserum, prior to the addition of the DNA-labeled
probe. The following oligonucleotide probes used for EMSA included
overhanging restriction site sequences: SCC1(SF-1) (32) (upper strand,
5'-GATCGCCCTCTCTTAGCCTTGAGCTA GTTA); consensus Sp1 (upper
strand, 5'-GATCCGATCGGGGCGGGGCGAGC);
148/
127 StAR (upper strand,
5'-TGCTCCCTCCCACCTTGGCCAG);
148/
127mut2Sp1 (upper strand,
5'-TGCTCCCTCtgACCTTGGCCAG);
87/
70 StAR (upper strand,
5'-GGCCAAGCTTGCACAATGACTGATGACT); and
73/42 StAR (upper strand,
5'-GGCCAAGCTTGACTTTTTTATCTCAAGTGATGATGCACAGCC).
1002/+6 DNA fragment, the oligonucleotide sequence
1002/
982
(5'-GGCCAAGCTTTTCTAAGGTTCCCTGGATCT) and
14/+6
(5'-GGCCTCTAGAAGCTGTGGCGCAGATCAAGT) were included in the PCR reaction
(38) using mouse genomic DNA (prepared as described in Ref. 39), as
template. The PCR product was digested with HindIII and
XbaI (New England Biolabs) and ligated (T4-DNA ligase; Roche
Molecular Biochemicals, Mannheim, Germany) into the HindIII and XbaI sites of a promoterless pCAT-Basic vector (Promega,
Madison, WI). The resulting construct was designated
1002StAR. Two
deletion constructs were generated from
1002StAR by StuI
and AccI (New England Biolabs, Beverly, MA) to generate
the
823/+6 (
823StAR) and
257/+6 (
257StAR) constructs, respectively.
14/+6 reverse primer and the appropriate 5'- forward
primers:
152StAR
(5'-GGCCAAGCTTAGTCTGCTCCCTCCCACCTTGGCCAGCACT);
123StAR
(5'-GGCCAAGCTTTGCAGGATGAGGCAATCATTCCAT);
96StAR
(5'-GGCCAAGCTTTGACCCTCTGCACAATGACTGA);
73StAR (the same
oligonucleotide sequence used for the EMSA probe,
73/
42StAR);
51StAR (5'-GGCCAAGCTTATGCACAGCCTTCCACGG).
14/+6 as the reverse primer (unless
stated otherwise):
152mutSF-1 (5'-
GGCCAAGCTTAGTCTGCTCCCTCCCAtaTTGGCCAGCACT);
152mut1"Sp-1"
(5'-GGCCAAGCTTAGTCTGCTCCCTCtgACCTTGGCCAGCACT);
152mut2"Sp-1"
(5'-GGCCAAGCTTAGTCTGCTCCCTggCACCTTGGCCAGCACT);
123mutC/EBP
-2 (5'-GGCCAAGCTTTGCAGGATGAGtcccaCATTCCAT);
96mut
2(5'-GGCCAAGCTTTGACCCTCTCtcccaGACTGAT);
96mut
-3
(5'-GGCCAAGCTTTGACCCTCTGCACAATGAtctgTGACTT;
96mutGATA
(5'-GGCCAAGCTTTGACCCTCTGCACAATGACTGATGACTTTTTTAagTCAAGTG);
73mutGATA (5'-
GGCCAAGCTTGACTTTTTTAagTCAAGTGATGATGCACAGCC); The
96rStAR
construct was built using the
96StAR primer as the forward primer,
and the
53/+6mutStAR
(5'-GGCCTCTAGATGATctcgacgtccaggacgcAAGCATTTAAGGCAGAGCACTTGATCTGCGCCACAGCT) as the reverse primer.
96doublemut was generated
using the
96mut
-3 as the forward primer, the
14/+6
oligonucleotide as the reverse primer, and the construct
96mutGATA as
the template. PCR reaction (total volume of 100 µl) consisted of 30 cycles at 94 °C (1 min), 60-65 °C (2 min), and 72 °C (3 min)
(38).
(1:2000) or
anti-GATA-4 (1:4000), the nitrocellulose membranes were washed and
further incubated for 1 h with the appropriate peroxidase-conjugated antibodies (1:10,000 dilution). Specific signals
were detected by chemiluminescence utilizing the LumiGlo substrate (New
England Biolabs). Quantitation of chemiluminescence signals on x-ray
films was performed as described previously (40).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1002 to
+6 fragment of the StAR gene was cloned by PCR and ligated to a
promoterless pCAT-Basic plasmid. We reasoned that the expression of the
mouse promoter in rat cells is justified by the fact that the proximal
regions of the rat and mouse promoters are almost identical, in
particular through the first 150 base pairs upstream to the
transcription start site (Fig. 1).
Testing the hormonal inducibility of the promoter constructs was
performed following a 6-h incubation with FSH added shortly after
transfection by electroporation. Semiquantitative RT-PCR and Western
blot analyses confirmed that under similar experimental conditions the
levels of StAR mRNA and protein rise acutely upon the addition of
FSH (Fig. 2).
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Fig. 1.
Putative binding sites for
trans-acting proteins potentially involved in
regulation of the mouse and rat StAR promoter. The 178/+6 region
in the murine StAR promoter (30) harbors the following potential
recognition sites (boxed) for trans-acting
proteins, which were examined in this study: Sp1 (
146/
137), SF-1
(
139/
132, SF-1, Ad4BP), C/EBP
-1 (
117/
108), C/EBP
-3
(
81/
72), and GATA (
66/
61). Additional SF-1 and C/EBP
sites
(broken line boxes) were proposed by
other investigators: SF-1-a (
102/
95); C/EBP
-2 (
90/
81) and
SF-1b (
46/
39). A TATA-like element is underlined.
Mismatched nucleotides of the rat promoter (72) are indicated by
highlighted superscripts.
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Fig. 2.
Time-dependent rise of StAR
mRNA and protein induced by FSH. Freshly prepared granulosa
cells were seeded to culture and 3 h later, FSH was added (100 ng/ml). At the indicated time points, duplicate wells, were harvested
with either RNAzol B or lysis buffer (see "Experimental
Procedures"). A, RT-PCR was performed to determine the
levels of StAR mRNA as described under "Experimental
Procedures." The presented autoradiogram depicts the amplified PCR
signals obtained for StAR and the ribosomal protein L19 mRNAs.
B, Western blot analysis was performed, and the enhanced
chemiluminescence reaction depicts the mitochondrial 30-kDa StAR
protein. The lower panel presents the time dependent
increase of StAR transcript and protein. Quantitation of StAR mRNA
and protein was performed as described under "Experimental
Procedures."
96/+6 (Fig.
3). The latter region exhibited the
highest -fold induction by FSH (44-fold), suggesting that two potential
upstream binding sites for SF-1 (
139/
132 and
102/
95), are not
necessarily required for the FSH activation of the promoter. These
results did not agree with earlier reports, which strongly advocated
the notion that SF-1 is implicated in regulation of StAR expression
(26-28, 30, 41). This inconsistency, together with the fact that
deletion of the
139/
132 SF-1 site significantly reduced the basal
activity of
152StAR (Fig. 3), urged us to cautiously reassess the
importance of this element by site-directed mutations and EMSAs. To our
surprise, SF-1 did not bind to a
148/
127 probe (Fig.
4), previously shown to be capable of
SF-1 binding using extracts of Y-1 adrenocortical cell line (30).
Instead, the rat cell extracts generated a slower migrating protein
complex, which was not affected by antiserum to SF-1 (Fig.
4A, lanes 2 and 4). A
closer examination of this sequence revealed a potential Sp1 site,
which is overlapping the SF-1 binding element to create an
"Sp1"/SF-1 motif (see Fig.
5B, probe
2). This G-rich element (
146, 5'-TGGGAGGGAG, lower strand) is nearly identical to an Sp1-like binding sequence previously reported
to be involved in cAMP-dependent regulation of the bovine P450scc transcription (34, 42). In StAR promoter, this Sp1-like site,
termed "Sp1," binds a protein that is antigenically cross-reactive with Sp1 antiserum (Fig. 4B, lanes 6 and 8). Moreover, molar excess of Sp1 consensus DNA can
compete for the binding of "Sp1" to its site in StAR promoter (Fig.
5A, lanes 4 and 5).
Finally, a site-directed mutation replacing GG with ca (Fig.
5B, lane 16) resulted in the loss of
"Sp1" band shift and rendered the SF-1 site available for a typical
SF-1 binding (Fig. 5B, lane 16).
Noteworthy, the "Sp1"/SF-1 element could bind both proteins,
providing the extracts were prepared from the mouse MA-10 cells (Fig.
5B, lane 14), which are highly
enriched with SF-1 content. These results suggest that the
148/
127
region has a dual capacity to bind both "Sp1"and SF-1, which
compete with each other depending on their relative content in a given
cell type.
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Fig. 3.
5'-Deletion analysis of the 1002/
96 StAR
promoter region. Progressive deletions of StAR promoter are
schematically illustrated in the left panel. Each
vector was transfected by electroporation into E2-primed
rat granulosa cells. Cells without hormone treatment (dotted
bars) and those treated with 100 ng/ml FSH (solid
bars) were incubated for 6 h before extracts were
prepared for CAT analysis as described under "Experimental
Procedures." CAT activity was determined using 5 µg of protein for
a 5-h assay. The results are presented as the mean ± S.E. of
percent of [14C]chloramphenicol converted to the
acetylated products. Hatched bars represent the
FSH -fold induction above basal activity. Multiple independent
transfections (n) were performed for each construct.
Activity levels were statistically significant when compared with the
respective values obtained for the
1002StAR construct: a,
p < 0.05; b, p < 0.001; b*
denotes statistically different values when compared with the
corresponding activities of the
152StAR construct (p < 0.001).
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Fig. 4.
SF-1 does not bind the 148/
127 StAR
promoter region. Extracts prepared from PMSG/hCG-treated rats (see
"Experimental Procedures") were used for the following
electrophoretic mobility shift assays (EMSAs): A, antiserum
to SF-1 (4 µg) was added to protein extracts for a 25 min
preincubation period prior to addition of either a
32P-labeled
148/
127 probe (lane
2), or a positive control SF-1 probe, designated SCC1
(lane 4) (32). Further EMSA procedure was
performed according to the protocol described under "Experimental
Procedures." B, antiserum to Sp1 was preincubated with the
protein extract prior to assay with labeled
148/
127 probe
(lane 6) or a consensus Sp1 (Cons.
Sp1) probe (lane 8). The corresponding
DNA-protein complexes formed in the absence of the antisera are
depicted in lanes 1, 3, 5,
and 7.
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Fig. 5.
148/
127 StAR promoter region binds an
Sp1-like ("Sp1") protein. Panels A and
B include EMSA performed as described in Fig. 4 by use of
the following 32P-labeled probes: consensus Sp1 binding
site (Cons. Sp1 (1);
148/
127
(2); an SF-1 binding site, SCC1(SF-1) (3); a
mutated
148/
127 probe designated mut2"Sp1" (4).
A, an ovarian extract from PMSG/hCG-treated rats was used as
a source for DNA-binding proteins. Lanes 1-3
depict a competition experiment using unlabeled self-competitor
148/
127 DNA, lanes 4 and 5 examine
the ability of molar excess of unlabeled consensus Sp1 DNA
(Cons. Sp1) to compete for the labeled
148/
127 probe, and lanes 6-10 examine
reciprocal competitions using a labeled consensus Sp1 binding site
probe. B, lane 11 depicts an Sp1
binding to a consensus Sp1 probe. Lanes 12 and
13 show the formation of an "Sp1" complex when a
148/
127 probe was incubated with either a rat, or a mouse ovarian
extract, respectively. The same
148/
127 probe yielded both SF-1 and
"Sp1" bands when tested with an extract of mouse MA-10 Leydig cell
line (lane 14). Lane 15 demonstrates a typical SF-1 binding to its consensus motif (SCC1).
Lane 16 depicts the effect of mutating the
148/
127 probe to mut2"Sp1" (probe 4).
132/
146 region were created in the context of
152StAR, as an
alternative approach for the deletion strategy described before. The
results in Fig. 6 show that a 5'-CAAGGTGG
mutation to 5'-CAAtaTGG (
152mutSF-1), did not affect the response of
StAR promoter to hormones, but instead improved it. The same mutation
was previously shown to ablate SF-1 binding and transcriptional
activation of the P450scc and P450aromatase promoters (31, 32). The
notion that binding of SF-1 is irrelevant for transcriptional
activation of StAR was further strengthened by the fact that a mutant
of the "Sp1" site (
152mut2"Sp1"), which capacitated SF-1
binding (Fig. 5, lane 16), did not improve any of
the construct performances (Fig. 6). Finally, when the "Sp1" site
was mutated (
152mut1"Sp1"), as previously done to critically
impaired its activity when harboring the bovine P450scc promoter (42),
no significant loss of hormonal inducibility or basal activity were
noticed (Fig. 6).
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Fig. 6.
Site-directed mutations of the "Sp1"/SF-1
site do not affect the hormonal activation of StAR-CAT. PCR-based
mutations were created within the context of 152/+6 of
StAR, and ligated to pCAT-Basic (
152StAR). Mutated
sequences of the "Sp1" (superscript dotted line) and the
SF-1 (underline) motifs are indicated by
lowercase letters. Transient expression was
conducted in the absence or the presence of FSH as described in Fig. 3.
Hatched bars represent the FSH -fold induction
above basal activity. CAT activity was determined using 5 µg of
protein for a 2-h assay. The results are presented as the mean ± S.E. of percentage of [14C]chloramphenicol converted to
its acetylated products. Three independent electroporations were
conducted with these constructs. a, p < 0.05 when compared with the basal activity value of
152StAR. The rest
of the data were not statistically different.
93/
51 Region: A Role
for C/EBP
--
Further 5' deletions of StAR promoter finally
resulted in a severe loss of FSH responsiveness. Fig.
7 shows that the
73/+6 and
51/+6 CAT
constructs retained only 12% and 0.3% of the FSH-driven activity
exhibited by the
96/+6 construct. To verify that no additional
sequences located downstream to
51 are potentially involved in
hormonal regulation of this promoter, we have randomized 17 base pairs
constituting the
49/
33 region. Clearly, this mutation performed
within the context of the
96/+6 construct did not affect its activity
(Fig. 7), suggesting that no important elements reside immediately
upstream to the TATA box.
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Fig. 7.
Functional analysis of the 96/
51 StAR
promoter region. Progressive deletions of
96StAR promoter
sequence are schematically illustrated in the left
panel. The bottom construct depicts the mutated
96rStAR,
in which the 17 bases
49/
33 were randomized. Transient expression
of the constructs in cells treated with or without FSH were performed
as described in Fig. 3. CAT activities were determined using 5 µg of
protein for a 5-h assay. The results are presented as the mean ± S.E. of percentage of [14C]chloramphenicol converted to
the acetylated products. Hatched bars represent
the FSH -fold induction above basal activity. Multiple independent
transfections (n) were performed for each construct.
Activity levels were statistically significant when compared with the
respective values obtained for the
96StAR construct: a,
p < 0.05; b, p < 0.005; c,
p < 0.001.
96/
51 region depicts a putative
near-consensus C/EBP
site (C/EBP
-2), residing upstream to a
conserved consensus GATA binding motif (Fig. 1). To test if the
C/EBP
-2 site may have a regulatory role in the rat granulosa cells,
we mutated this site in the context of
96StAR, as shown in Fig. 8. However, the mutated construct
(
96mut
-2) did not affect its 55-fold response to FSH, which was
not much different from the parental plasmid (Fig. 8). Further EMSA
studies provided an explanation for this observation by showing that a
specific C/EBP
-2 probe (
96/
75) did not bind to any protein (data
not shown). Instead, a downstream adjacent element reminiscent of an
AP-1 site, if anything else (5'-TGACTGA), was found capable of C/EBP
binding. We designated this non-consensus element C/EBP
-3 (see Fig.
1). EMSAs showed that a C/EBP
-3 oligonucleotide probe (
87/
70)
binds C/EBP
in a specific fashion; antiserum to C/EBP
caused a
supershift and ablation of the typical triplet bands bound to C/EBP
DNA (Fig. 9, lanes
1 and 3), and a specific antiserum to C/EBP
supershifted the upper band, suggesting that it consists of
C/EBP
/C/EBP
heterodimer (Fig. 9, lane 2).
Finally, three sera served for negative controls, including antiserum
to sterol-responsive element-binding protein (SREBP, Fig. 9,
lanes 5 and 6), previously shown to
activate StAR transcription in human granulosa cells (29), and
ineffective antisera to c-Fos and c-Jun (Fig. 9, lanes
8 and 9).
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Fig. 8.
Recognition elements for
C/EBP and GATA-4 binding confer
transcriptional activation to StAR promoter. The left
panel illustrates a series of
96StAR constructs
including mutated sequences (lowercase letters)
for binding of either C/EBP proteins (
96mut
-2,
96mut
-3),
or the GATA factors (
96mutGATA). Additionally,
double-mutated constructs were prepared as either
96double mut, or
73mutGATA. Transfection and
activity assays were conducted as described in Fig. 3. The results are
presented as the mean ± S.E. of percentage of
[14C]chloramphenicol converted to the acetylated
products. Hatched bars represent the FSH -fold
induction above basal activity. Multiple independent transfections
(n) were performed for each construct. Activity levels were
statistically significant when compared with the respective values
obtained for the
96StAR construct: a, p < 0.05; b, p < 0.01; c, p < 0.001.
View larger version (63K):
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Fig. 9.
The 87/
70 StAR promoter region binds
C/EBP
proteins. A,
electrophoretic mobility shift assays using PMSG/hCG ovary extract were
performed as described in Fig. 4 using a
87/
70
32P-labeled probe (lanes 1-15). The
formation of the three DNA-protein complexes (arrows,
lanes 1 and 4) was examined in the
presence of the following antisera: C/EBP
(lane
2), C/EBP
(lanes 3 and
7), sterol-responsive element-binding protein, SREBP
(lane 5 and 6), c-Fos (lane
8), and c-Jun (lane 9). B,
competition studies using the 32P-
87/
70 as a probe
(lane 11) and molar excess of unlabeled
self-competitor DNA (lanes 12 and 13),
or unlabeled C/EBP
-1 DNA (lanes 14 and
15). Lane 16 demonstrates lack of
binding to a mutated C/EBP
-3 labeled probe. C,
characterization of C/EBP
binding to a C/EBP
-1 probe
(
125/
100) was performed in the presence of either an antiserum to
C/EBP
(2 µg, lane 19), or anti-SF-1 serum as
a negative control (4 µg, lane 18).
-3 site
(TGACTGATGA) is so remotely different from a consensus
C/EBP
motif, it is absolutely required for C/EBP
binding, which
was lost upon a TGAtctgTGA mutation of the probe (Fig.
9B, lane 16). Yet, as could be
expected, the C/EBP
-3 motif does not necessarily exhibit the best
affinity for C/EBP
binding, as we learn from competitive EMSAs
mixing a 32P-C/EBP
-3 probe (
87/
70) with a
10-100-fold molar excess of the near-consensus C/EBP
-1 sequence
(
125/
100); in doing so, a 10-fold excess of C/EBP
-1 was enough
to displace 90% of the labeled C/EBP
-3 probe (Fig. 9,
lane 14). The reason for using the C/EBP
-1
sequence for competitor DNA lies in the curious fact that its motif
(ATGAGGCAAT) specifically binds immuno-cross-reactive C/EBP
(Fig.
9C). However, mutating or deleting the C/EBP
-1 site does
not impair transcriptional activation of the StAR promoter (Fig. 3,
bottom two constructs). By contrast,
functional analysis of the C/EBP
-3 sequence by site-directed
mutagenesis (Fig. 8,
96mut
-3) suggested that an intact
C/EBP
-3 site is, indeed, required for the activity of StAR promoter.
-3 site could not account for the
severely impaired activity of the
51StAR construct (Fig. 3).
Therefore, the involvement of an additional regulatory element
downstream of C/EBP
-3 was likely to be found. To test this
possibility, we examined the candidacy of a perfect GATA binding site
located at
61/
66 (Fig. 1). Like
96mut
-3, modification at this
GATA site (
96mutGATA) resulted in no more than a partial attenuation
of the construct activity (Fig. 8). However, double mutation of both
C/EBP
-3 and GATA sites (
96doublemut) resulted in a
nearly complete loss of CAT activity (Fig. 8). A similar marked
impairment of the FSH responsiveness was also observed when the GATA
site was mutated in the context of
73mutGATA, from which the
C/EBP
-3 motif was deleted. In our view, the residual but not
negligible FSH -fold induction observed in cells transfected with the
double-mutated construct (
96double mut) is probably
meaningless and reflects a pleiotropic effect FSH has on the basal
activity by acting as a trophic hormone. In fact, the activity
performances of the double mutant were similar to those obtained for
the maximally trimmed promoter construct
51StAR, which also exhibited
a significant 5-fold induction of CAT activity by FSH (Fig. 7).
However, this trophic improvement of the basal activity was no higher
than 3% of the FSH activity measured for the parental
96StAR
construct (Fig. 7) and, therefore, could not represent other than a
misleading basal activity
73/
42, or
73/+6 (Fig.
10, A and B, respectively). These results
convince that no other protein complexes can bind to the entire region
residing between
73 and +6. The binding specificity was demonstrated
by use of antiserum to GATA-4, which ablated and supershifted the
DNA-protein complex. The latter remained unaffected in the presence of
anti-GATA-6 serum (Fig. 10A, lanes 1 and 2). Additionally, mutating the GATA site in the context of the
73/+6 probe (5'-TTATCT
5'-TTAagT) associated with loss of
binding capacities, as evident by competition (Fig. 10B,
lanes 7 and 8) and direct binding
studies using radiolabeled probe (Fig. 10B, lane
9).
View larger version (74K):
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Fig. 10.
The 66/
61 GATA element binds a GATA-4
protein. Electrophoretic mobility shift assays using a PMSG/hCG
ovary extract were performed as described in Fig. 4 using a
73/
42,
73/+6, or a
73/+6mutGATA as labeled DNA probes. A,
antiserum to either GATA-4 (2 µg, lane 2) or
GATA-6 (2 µg, lane 3) was added to the protein
extracts 25 min prior to assay. B, competition for GATA-4
binding (lane 4) was assessed in the presence of
molar excess of unlabeled self competitor DNA (lanes
5 and 6), or an unlabeled
73/+6mutGATA probe
(lanes 7 and 8). Lane
9 tests the capacity of the latter mutated probe to complex
with proteins of the ovarian extract.
and GATA-4--
We also
aimed to study by Western blot analysis the levels of C/EBP
and
GATA-4 proteins in our tissue and cell preparations. First, we tested
granulosa cell extracts prepared at identical time points previously
used for determination of CAT activity. Fig.
11A shows that the C/EBP
antiserum cross-reacted with three protein bands of 45, 39, and 22 kDa,
known as C/EBP
isoforms (43). The levels of those proteins were
barely detectable in vivo (t0), but
seeding the cells for a few hours in culture substantially elevated the
levels of the higher molecular weight forms. The 22-kDa isoform of
C/EBP
was not affected by seeding to culture (Fig. 11A).
A 6-h treatment with FSH generated an increase of all the isoforms of
C/EBP
up to 7-fold. In agreement with our EMSAs (Fig.
9A), Western blot analysis of the ovarian cells detected C/EBP
protein bands (data not shown), which were also reported by
earlier studies of these proteins during follicular development (44).
View larger version (46K):
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Fig. 11.
Effect of seeding and FSH treatment on
expression of C/EBP and GATA-4 proteins.
Except for electroporation, granulosa cells from E2-primed
immature rats were prepared for culture exactly as described in Fig. 3.
Protein extracts were prepared from cells prior to seeding
(t0), or after a 9-h incubation (post-seeding)
in the absence of FSH. FSH treatment commenced 3 h after seeding
and cells were harvested 6 h later. SDS-PAGE and Western blot
analysis were performed as described under "Experimental
Procedures" using 40 µg of protein/lane. Chemiluminescent detection
of the reactive proteins was performed using antisera to C/EBP
(A) or GATA-4 (B). The antiserum to C/EBP
detects three major isoforms, designated LAP-I, LAP-II, and LIP (43).
The lower panel presents a semiquantitative analysis of the
data shown in A and B. C,
demonstration of C/EBP
and GATA-4 proteins in ovarian extracts
prepared from the PMSG/hCG-treated rats.
expression, neither seeding nor
FSH treatment had any effect on the high level of the GATA-4 content,
which is probably constitutively expressed in the rat granulosa cells
(Fig. 11B). It should be noted that, in correlation to our
EMSA data, we could not detect the GATA-6 protein by this Western blot
analysis (data not shown). Fig. 11C shows that the C/EBP
and GATA-4 proteins also exist at high levels in the ovarian extracts
we selected, for practical reasons, as a source for our EMSAs. Those
extracts were expected to express the necessary factors since StAR
expression during the post-hCG period is in its prime response
(40).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1002 to +6 fragment of the
5'-flanking region of the mouse StAR gene and placed it
upstream of CAT gene in a promoterless pCAT-Basic reporter plasmid.
Then, by contrast to most of the previous StAR studies expressing
promoter-reporter plasmids in established cell lines (26-30, 41), we
conducted our functional assays using primary naive granulosa cells
from prepubertal rats. Following electroporation of these cells in the
presence of plasmid DNA, a 6-h incubation in culture resulted in a
robust activation (up to 50-fold) of the promoter by FSH. Having such a
sensitive assay in hand, we combined a progressive deletion strategy
with site directed mutations aiming to validate, or eliminate, the
potential involvement of candidate cis-acting regulatory
elements in StAR expression. This approach led us to identification of
two novel elements, C/EBP
and GATA-4, which were never known before
to be required for transcriptional activation of genes encoding
steroidogenic proteins.
96 from the transcription start site. However, we
were interested in performing a limited analysis upstream of the
96
regions, in particular addressing those sites that were previously
suggested to be functionally involved in regulation of StAR expression.
For example, between
100 and
1000 of the StAR promoter reside at
least two putative binding sites for the tissue-specific orphan
receptor SF-1 (45), also known as Ad4BP (24). This factor is required
for activation of many genes expressed in steroidogenic tissues (23).
Therefore, a host of studies have recently proposed that SF-1 is also
essential for expression of StAR gene (26-30, 41). Using the naive
granulosa cell model, our findings do not support this notion since the
removal of all putative SF-1 sites (28, 30) hardly affected the
promoter performance (
96StAR and
96rStAR, Figs. 2 and 7,
respectively). A possible explanation for the apparent discrepancy
between the present findings and earlier works may reflect
species-dependent differences between the human (26) and
the murine (30) promoters. However, one cannot exclude the possibility
that the StAR promoter was never examined in steroidogenically
committed primary cells which contain normal levels of SF-1. Instead,
the role of SF-1 was demonstrated in luteal cells (27, 46) and
steroidogenic cell lines expressing exceptionally high endogenous
levels of the orphan receptor (30, 41), or by use of non-steroidogenic cells overexpressing transfected SF-1 plasmids (26-29, 41). Therefore, the role of SF-1 was always examined under conditions favoring its
interaction with StAR promoter, but did not necessarily simulate the
physiologically compatible requirements for regulation of StAR in
normal cells.
96 in the mouse promoter resides an
additional element of considerable interest, i.e. a
non-consensus putative binding site for Sp1 (
137/
146). A similar
sequence for binding of an Sp1-like protein ("Sp1") was found
important for cAMP induced transcription of the bovine P450scc gene
(42). Moreover, an analogous Sp1 site seemed to be important for
activation of the human StAR promoter (26). In rodents, however, this
"Sp1" element is overlapping a well studied SF-1 binding motif,
thus creating a mixed "Sp1"/SF-1 site. This composite sequence can bind SF-1 from Y-1 (30) or MA-10 cells (Fig. 4B), but is
apparently incapable of doing so when tested with granulosa cell
extracts. We may, therefore, conclude that "Sp1" and SF-1 compete
for binding to this site, so that SF-1 band shift in EMSAs is
demonstrable only in cell lines expressing extremely high levels of
SF-1, as discussed above. However, at present it is not clear if
activation of the StAR promoter in granulosa cells involves "Sp1"
action, since removal of the "Sp1"/SF-1 site down to position
123
attenuated the basal activity, but retained the hormonal responsiveness
intact. Interestingly, further pruning of the promoter down to
96
kept suppressing the basal activity of the promoter by removing a
near-consensus C/EBP
(C/EBP
-1) binding moiety at
116/
107.
Thus, the C/EBP
-1 site is irrelevant for activation of the promoter
despite its apparent excellence in binding typical C/EBP
proteins
(Fig. 9). Collectively, these results have suggested that the critical
element(s) controlling the inducibility of StAR promoter
must have resided further downstream, within the first 80-90 base
pairs of the promoter.
87 to
51 region revealed the involvement of trans-acting proteins that bind two sites located 10 base pairs apart: an upstream sequence interacting with C/EBP
(C/EBP
-3) and a consensus site specifically binding a GATA-4 protein. Contrary to our earlier expectations, the sequence core subserving for C/EBP
binding (TGACTGA) seemed more like an AP-1 site, so that a typical c-Jun/c-Fos binding was demonstrable upon a single base substitution to TGACTcA (data not shown). Even more confusing was the fact that overlapping with this odd C/EBP
-3 site resides another putative recognition element for C/EBP
, designated C/EBP
-2. However, mutating the core
sequence of C/EBP
-2 (ACAAT to tccca) did not affect the inducibility
of the
96StAR construct, while a four-base mutation of C/EBP
-3
reduced the basal and FSH-responsive activity by 50%. This shy effect
of the mutated C/EBP
-3 did not impress much until after the
elucidation of the GATA binding site at
66/
61. Mutation of the GATA
site resulted in a 45% drop of the promoter activity, but a double
mutation of both GATA-4 and C/EBP
-3 resulted in a dramatic 97% loss
of the construct activity. These results suggest that a concurrent
binding of the two regulatory proteins is required for activation of
StAR promoter.
proteins are absent in vivo, but their expression
in culture was markedly promoted by FSH. A similar induction of
C/EBP
expression was recently documented in testicular Leydig cells responding to cAMP (47). These results suggest that the level of
C/EBP
might determine the rate-limiting regulatory switch of StAR
transcription, while the constitutively expressed GATA-4 plays an
essential, yet more permissive role for that matter. It is noteworthy
that, other than StAR, members of the C/EBP family have been described
as regulators of acute responses, such as the control of certain
inflammatory functions (48). Moreover, the suggested involvement of
C/EBP proteins in the acute response of StAR to cAMP (8, 20) and
trophic hormones (40, 49, 50) is also well accepted in light of a well
studied example where C/EBP plays a critical role in transcriptional
regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene (51). Similar to the emerging scenario in StAR promoter, it has been shown
that cAMP and probably protein kinase A drive PEPCK transcription via a
complex interactions of C/EBP with other activators and co-activators
(51); also, the binding of C/EBP
to a non-consensus recognition
motif in the StAR promoter is reminiscent of the C/EBP binding to
non-dyad-symmetric sequences in the PEPCK promoter, a cyclic
AMP-responsive element-like site (52) and a site termed P3(I) (53).
-1 and C/EBP
-3 probes formed three
DNA-protein complexes, which were identical to those obtained when
binding capacities of rat granulosa cell extracts were previously examined with a C/EBP
motif located in the hormone-inducible prostaglandin synthase-2 promoter (54). The alternative approach testing the potential cross-reactivity of the C/EBP
proteins with
specific antibodies tested by Western blot analysis revealed that the
rat granulosa cells express three isoforms, previously identified as
liver-enriched activating proteins (LAPs) and liver-enriched inhibiting
protein (LIP) (43, 55). Clearly, LIP and LAP-I were markedly elevated
as a result of FSH treatment, while LAP-II level rose by merely seeding
the cells into culture. Therefore, it is conceivable to propose that
the onset of StAR transcription is controlled by a two-step mechanism:
first, the seeding-induced rise of LAP-II capacitates the immediate
response of the granulosa cells to FSH, and a follow-up activation of
the promoter may proceed thanks to the hormone-elevated levels of
LAP-I. The likelihood of such a mechanism is not too speculative since
the granulosa cells express high levels of FSH-activated cyclic
AMP-responsive element-binding protein (56, 57), which in turn, is
known to up-regulate LAP transcription (58, 59). Further studies should
address this hypothesis and reveal even more about the potential
involvement of the C/EBP
isoforms in regulation of StAR expression.
Also, it is not unlikely that other C/EBP proteins, such as C/EBP
,
can interact with the
87/
70StAR site. If truly so, C/EBP
should
be considered as potential substitute for C/EBP
in the
C/EBP
-deficient mouse ovary (60-63). It has been shown that the ovaries of the latter null mice do not ovulate (60, 64)
despite normal production of gonadal steroids and expression of
steroidogenic enzymes, probably including
StAR.3
, GATA-4 had not been described previously as a potential
regulator of genes encoding steroidogenic enzymes, or their accessory
proteins. Gata4-null mice die in utero by 9.5 days postcoitum (65, 66). However, recent studies have suggested that
this transcription factor is involved in control of gonadal development
and sex differentiation in rodents (67-69). Moreover, in adult mouse
tissues, high levels of GATA-4 are observed in the ovary, testis, and
heart. Interestingly, little or no GATA-4 protein was found in the
cells of the mouse corpus luteum (68), which do express record levels
of StAR (20, 40, 70, 71). One way to reconcile this apparent
inconstancy is provided by assuming that the role of GATA-4 during
follicular phase could be fulfilled by another member of this family,
like GATA-6, which is highly abundant in the corpus luteum (68).
Alternatively, we may propose that the mode of StAR expression in the
corpus luteum may not necessarily resemble its regulatory pattern in granulosa cells of the follicular phase. Accordingly, SF-1, which is
extremely high in the corpus luteum may control the gland expression of
StAR expression after all. This tempting speculation implies that
activation of StAR transcription may be achieved by more than one set
of cis-acting proteins, depending on the origin of the cells
and tissue under study. In this regard, the present findings raise the
question: what tissue-specific factor(s) can potentially replace SF-1
in our steroidogenically committed, but yet undifferentiated cell
model? Alternative experimental approaches addressing this challenging
question are currently under study.
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ACKNOWLEDGEMENT |
---|
We thank Jonathan Arensburg for excellent technical assistance and many helpful discussions.
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FOOTNOTES |
---|
* This work was supported by Israel Science Foundation Grant 547/97.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
orly{at}vms.huji.ac.il.
2 Y. Arensburg and J. Orly, unpublished data.
3 E. Sterneck, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: P450scc, cholesterol side chain cleavage cytochrome P450; StAR, steroidogenic acute regulatory protein; SF-1, steroidogenic factor-1; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; PEPCK, phosphoenolpyruvate carboxykinase; PAGE, polyacrylamide gel electrophoresis; hCG, human chorionic gonadotropin; PMSG, pregnant mare serum gonadotropin; FSH, follitropin.
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REFERENCES |
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