SF-1 (Steroidogenic Factor-1) and C/EBPß (CCAAT/Enhancer Binding Protein-ß) Cooperate to Regulate the Murine StAR (Steroidogenic Acute Regulatory) Promoter
Adam J. Reinhart,
Simon C. Williams,
Barbara J. Clark and
Douglas M. Stocco
Department of Cell Biology and Biochemistry (A.J.R., S.C.W.,
D.M.S.) and Southwest Cancer Center (S.C.W.) Texas Tech
University Health Science Center Lubbock, Texas 79430
Department of Biochemistry University of Louisville School of
Medicine (B.J.C) Louisville, Kentucky 40292
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ABSTRACT
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The steroidogenic acute regulatory (StAR) protein
mediates the rate-limiting step of steroidogenesis, which is the
transfer of cholesterol to the inner mitochondrial membrane. In
steroidogenic tissues, StAR expression is acutely regulated by trophic
hormones through a cAMP second messenger pathway, leading to increased
StAR mRNA levels within 30 min, reaching maximal levels
after 46 h of stimulation. The molecular mechanisms underlying such
regulation remain unknown. We have examined the StAR
promoter for putative transcription factor-binding sites that may
regulate transcription in a developmental and/or hormone-induced
context. Through sequence analysis, deoxyribonuclease I (DNAse I)
footprinting and electrophoretic mobility shift assays (EMSAs), we have
identified two putative CCAAT/enhancer binding protein (C/EBP) DNA
elements at -113 (C1) and -87 (C2) in the mouse StAR
promoter. Characterization of these sites by EMSA indicated that
C/EBPß bound with high affinity to C1 and C2 was a low-affinity C/EBP
site. Functional analysis of these sites in the murine StAR
promoter showed that mutation of one or both of these binding sites
decreases both basal and (Bu)2cAMP-stimulated
StAR promoter activity in MA-10 Leydig tumor cells, without
affecting the fold activation
[(Bu)2cAMP-stimulated/basal] of the promoter.
Furthermore, we have demonstrated that these two C/EBP binding sites
are required for steroidogenic factor-1 (SF-1)-dependent
transactivation of the StAR promoter in a nonsteroidogenic
cell line. These data indicate that in addition to SF-1, C/EBPß is
involved in the transcriptional regulation of the StAR gene
and may play an important role in developmental and hormone-responsive
regulation of steroidogenesis.
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INTRODUCTION
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The rate-limiting step of steroidogenesis is the delivery of
cholesterol from cellular stores to the inner mitochondrial membrane,
where it is converted to pregnenolone by the cytochrome P450
side-chain cleavage enzyme (P450scc; Refs. 1, 2). The
steroidogenic acute regulatory (StAR) protein mediates this transfer of
cholesterol to the inner mitochondrial membrane and thus, is required
for this regulatory step (reviewed in Refs. 3, 4, 5, 6). Expression of the
StAR protein is rapidly stimulated in steroidogenic tissues in response
to trophic hormone through a cAMP second messenger pathway (7, 8). At
the level of StAR gene transcription, it has been reported
that an increase in mRNA levels is detectable within 30 min after
stimulation of MA-10 cells with (Bu)2cAMP (9). It has also
been reported that the initial cAMP-stimulated induction of StAR mRNA
does not require de novo protein synthesis (10). Therefore,
the complement of transcription factors required to confer
hormone-responsive activation of the StAR promoter must be
present before stimulation. In addition, posttranslational modification
of one or more transcription factors in response to cAMP stimulation
may play a critical role in the activation of this promoter.
Recently it has been suggested that steroidogenic factor-1 (SF-1), an
orphan nuclear receptor, may play important roles in the regulation of
StAR transcription (10, 11, 12, 13, 14). SF-1 has been shown to play a
role in the transcriptional regulation of many genes involved in
steroidogenesis, including steroid hydroxylase genes (15, 16, 17), as well
as LHß (18), the ACTH receptor (19), and the GnRH receptor (20). SF-1
null mice have also revealed a role for SF-1 in the development of the
gonads and adrenal glands (11, 21). Combined, these reports suggest an
important role for SF-1 in the regulation of steroidogenesis at a
number of levels. However, there are many lines of evidence suggesting
that SF-1, although important for StAR transcription, may
not be a key transcription factor in the acute regulation of the
StAR gene in response to cAMP stimulation. For example,
transfection studies in nonsteroidogenic cell lines have shown that
SF-1 is capable of transactivating a StAR reporter (13, 14).
Yet, when multiple SF-1-binding sites were mutated in the mouse
StAR promoter and analyzed in MA-10 cells, the cAMP
responsiveness (fold activation) from the promoter was not disrupted
(10). These data indicate that SF-1 is required for proper activation
of the StAR promoter but may not confer cAMP responsiveness
in steroidogenic cells. These findings have led us to examine other
transcription factors, whose activity is acutely regulated in response
to trophic hormone in steroidogenic tissues, for their involvement in
the transcriptional regulation of the StAR gene.
Recent studies in our laboratory have suggested that the CCAAT/enhancer
binding protein (C/EBP) family of basic leucine zipper transcription
factors may be involved in the regulation of steroidogenesis in Leydig
cells. Thus far, six members of the C/EBP family have been identified:
C/EBP
, C/EBPß, C/EBP
, C/EBP
, C/EBP
(Ig/EBP), and C/EBP
(CHOP; Ref. 22). C/EBPß is the only member of the C/EBP family
expressed in unstimulated primary Leydig cell cultures and MA-10 cells
(A. J. Reinhart, D. Nalbant, S. C. Williams, and D. M.
Stocco, unpublished observation) and C/EBPß levels increase in MA-10
cells by 4.5-fold upon 4 h treatment with 1 mM
(Bu)2cAMP (23). It has also been shown that C/EBPß
activity can be altered, presumably by protein kinase A (PKA), upon
treatment with cAMP analogs (24, 25, 26, 27). Therefore, C/EBPß was examined
as a candidate transcription factor in the transcriptional regulation
of the StAR gene.
In the present study, we examined the StAR promoter for
potential binding sites for transcription factors that may be involved
in the regulation of StAR transcription. Two putative C/EBP
response elements were identified; one of these sites was shown to bind
to C/EBPß, and we determined that the other site was a low-affinity
protein-binding site. Functional analysis revealed that mutation of
these sites decreased basal and cAMP-stimulated activity from the
StAR promoter in MA-10 cells. Furthermore, we report that
SF-1-dependent transactivation of the StAR promoter in COS-1
cells required these putative C/EBP response elements, suggesting that
C/EBPß and SF-1 may interact to regulate StAR gene
transcription.
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RESULTS
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Characterization of the C/EBP-Binding Sites within the Mouse StAR
Promoter
Previous studies have established that the elements required to
confer cAMP responsiveness to the mouse StAR gene are
contained within the first 254 bp of the promoter (10). Therefore, we
examined this promoter region from mouse (10), rat (28), human (14),
ovine (J. L. Juengel and G. D. Niswender, personal
communication) and porcine (29), to identify protein-binding
sites that might mediate this response. The 5'-flanking regions of the
StAR gene from these five species were aligned (Fig. 1A
) and revealed that the greatest degree
of sequence similarity was found within 120 bases of the transcription
start site. Previously identified elements in this region included a
TATA box at nucleotide -35 (all coordinates are relative to the
transcriptional start site in mouse at +1) and three SF-1-binding sites
corresponding to -42, -91, and -135 in the mouse sequence (10, 12, 14, 28). Several additional blocks of homology were revealed by these
analyses. Two of these (named C1 and C2) displayed significant homology
to the consensus binding site for members of the CCAAT/enhancer binding
site family of transcription factors (ATTGCGCAAT; Ref. 22). Naturally
occurring C/EBP sites generally exhibit divergence from this consensus
sequence, but typically retain one well conserved half-site (22). The
C1 site is centered at -113 and contains one perfect half-site (GCAAT)
and an overall 7 of 10 match to the consensus sequence (Fig. 1B
). The
C1 site is almost completely conserved in all species (9 of 10
matches). The second potential C/EBP site (C2) also displays 7 of 10
matches to the consensus sequence in mouse (Fig. 1B
) and contains an
almost perfect half-site (ACAAT). However, this sequence is only
partially conserved in the five species shown here (Fig. 1A
).

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Figure 1. Identification of Two Putative C/EBP-Binding Sites
in the StAR Promoter
A, The sequences of the 5'-flanking regions of the mouse (M), rat (R),
ovine (O), porcine (P), and human (H) StAR genes were
aligned using the ClustalW (K. C. Worley, Human Genome Center,
Baylor College of Medicine, Houston, TX) sequence alignment program,
and identical bases in all five sequences are indicated with
asterisks. Binding sites for SF-1 are
shaded and labeled. Two putative C/EBP-binding sites
were identified based on their similarity to the consensus
C/EBP-binding site and are shaded and labeled. Other
highly conserved sequences discussed in the text are
boxed and labeled A and B. B, The two putative
C/EBP-binding sites from the mouse promoter were compared with the
consensus C/EBP-binding site determined by binding site selection (26 ).
Sequence identities are indicated with vertical bars
between the sequences. Both C1 and C2 share seven bases with the
consensus site.
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The sequence alignment in Fig. 1A
revealed the presence of two
additional well conserved sequence elements that have not yet been
characterized (Fig. 1A
, boxed regions A and B). The first is
a 10-bp element centered at -63 of the mouse promoter, which resembles
a binding site for members of the GATA family of transcription factors.
Although GATA-4 and a testis-specific version of GATA-1 are expressed
in testis and MA-10 Leydig cells, it is not clear whether they are
involved in regulating the StAR gene (30, 31). The second
putative element is a perfectly conserved 6-bp sequence (TGATGA)
centered at -53 of the mouse promoter, which does not match the
binding site of known transcription factors in the transfac matrix
table (release 3.2) when searched using TFSEARCH (version 1.3) (© 1995
Yutaka Akiyama, Kyoto University, Kyoto, Japan). Further
analyses are clearly required to address the possible roles of these
elements in regulating StAR gene expression.
DNAse I footprint analysis of the mouse StAR promoter from
-66 to -254 was performed to identify possible transcription
factor-binding sites in this region. A schematic diagram of the
radioactive probe used in the footprint analysis is presented in Fig. 2A
. Addition of 25 and 50 µg of nuclear
extract prepared from (Bu)2cAMP-stimulated MA-10 cells
revealed a broad region of protection interspersed with two DNAse
I-hypersensitive sites, indicated by arrows (Fig. 2B
). The
protected regions included the C1 site and the SF-1 element at -135
and are marked by vertical bars adjacent to the sequence
(Fig. 2B
).

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Figure 2. DNAse I Footprint Analysis of the
StAR Promoter Revealed a Protected Region Encompassing
the Putative C/EBP Site at -113
A double-stranded probe corresponding to the coding strand of the
StAR promoter spanning -254 to -66 was radiolabeled
and used in DNAse I footprinting assays to detect regions of the
StAR promoter that were protected from DNAse I digestion
by the presence of proteins bound to the promoter. A, Schematic
representation of the StAR promoter from -254 to -66
indicating the SF-1, C1, and C2 elements. The line
depicts the DNAse I-protected region that is flanked by the
hypersensitive sites. B, Radiolabeled probe was incubated for 30 min in
the absence (-) or presence of 25 µg or 50 µg of nuclear extract
purified from (Bu)2cAMP-treated MA-10 cells and then DNAse
I treatment was for 15 or 30 sec as indicated. The
arrows indicate the hypersensitive sites, and the
vertical lines indicate the protected regions. The StAR
promoter sequence is shown on the left with the
vertical lines again indicating the protected regions.
The GCAAT sequence is within the protected regions, and the C1 site
used for EMSA spans -124 to -101. The SF-1 element at -135,
CCACCTTGG, is shown in the sequence within a protected region. The C2
region begins at -95 and continues to the undigested probe. Four
separate DNAse I footprint analyses have qualitatively shown the same
results: the presence of two hypersensitive sites and protection of the
C1 region. The lowercase c indicates a base change in
the sequence due to the engineered BglII site.
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We next performed electrophoretic mobility shift assays (EMSAs) to
identify proteins in Leydig cells that bind the C1 and C2 sites.
Radioactively labeled oligonucleotides containing the C1 or C2 binding
sites were incubated with nuclear extracts prepared from resting and
(Bu)2cAMP-stimulated MA-10 cells. Protein-DNA complexes
with similar mobilities were formed on both the C1 and C2
oligonucleotides, although complex formation occurred more efficiently
on the C1 oligonucleotide (Fig. 3
).
Competition binding studies were carried out to determine whether the
protein-DNA complexes represented specific interactions and whether the
proteins binding to both sites were related (Fig. 3
). Addition of
100-fold molar excess of unlabeled C1 oligonucleotide abolished
formation of the protein-C1 complex (Fig. 3
, lane 2). However, addition
of a similar amount of a related oligonucleotide (C1m; Fig. 3
, lane 3),
in which residues within the predicted C/EBP recognition sequence had
been mutated, failed to prevent complex formation, indicating that
proteins were specifically binding to the potential C/EBP motif.
Cross-competition assays were also performed using the C1 and C2
oligonucleotides. Unlabeled C1 blocked complex formation on the C2
oligonucleotide (Fig. 3
, lane 7), whereas C1m did not (Fig. 3
, lane 8),
suggesting that proteins with similar DNA recognition specificities
bound these two sites. The unlabeled C2 oligonucleotide was incapable
of competing for complex formation with the C1 or the C2
oligonucleotide (Fig. 3
, lanes 4 and 9), probably reflecting the
apparent greater protein-binding affinity of the C1 oligonucleotide as
compared with C2. Collectively, these data indicate that the C1 and C2
motifs appear to be binding sites for identical or related proteins in
MA-10 nuclear extracts.

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Figure 3. The Putative C/EBP-Binding Sites Form Complexes
with Proteins Expressed in MA-10 Nuclear Extracts
Five micrograms of protein prepared from nuclear extracts of MA-10
cells stimulated for 6 h with (Bu)2cAMP were incubated
with 32P-labeled probes representing the putative C/EBP
sites at -113 (C1) or -87 (C2) in the presence or absence of 100-fold
molar excess of unlabeled competitor oligonucleotides. The competitor
oligonucleotides were either the unlabeled wild-type C/EBP binding
sites (C1, C2) or mutant versions of these sites (C1m, C2m).
DNA-protein complexes were subjected to electrophoresis through a 4%
nondenaturing polyacrylamide gel, and then dried gels were visualized
by phosphoimagery and autoradiography. The inset at the
bottom of the gel is a phosphoimage of the region of the gel
maked with an asterisk, which shows binding to the C2
oligonucleotide.
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Having established that the C1 site binds with high affinity to
proteins present in nuclear extracts from stimulated MA-10 cells, we
next sought to determine whether C/EBPß binds to C1, and whether this
binding was regulated by (Bu)2cAMP stimulation of the
cells. Supershift experiments were performed using an antiserum raised
against the amino terminus of C/EBPß (32). Initially recombinant
C/EBPß was produced in rabbit reticulocyte lysates and incubated with
radiolabeled C1 oligonucleotide in the absence and presence of the
C/EBPß antiserum. A specific DNA-protein complex was formed when the
C1 probe was mixed with recombinant C/EBPß that was absent in
unprogrammed lysates (compare Fig. 4
, lanes 1 and 2). This complex was completely supershifted by the
C/EBPß antiserum (lane 3). Nuclear extracts prepared from both
(Bu)2cAMP-stimulated and unstimulated MA-10 cells formed
complexes with the C1 probe, and further incubation with the C/EBPß
antiserum resulted in a supershifted complex in both extracts (Fig. 4
, lanes 5 and 7). The intensity of this complex was relatively unaffected
by (Bu)2cAMP stimulation (Fig. 4
, compare lanes 45 with
67 and 89 with 1011), suggesting that there are proteins other
than C/EBPß in these complexes, whose levels are not altered by
(Bu)2cAMP-stimulation, that may be rate-limiting in the
formation of the complex. The major shifted complex in MA-10 nuclear
extracts could be resolved into three distinct bands (Fig. 4
, right panel, labeled a, b, and c), and the supershifted
complex appeared to be derived primarily from the middle band. Multiple
C/EBP-like binding activities have been observed in nuclear extracts
from many cell types, which are unaffected by addition of currently
available antisera directed against known C/EBP family members. These
complexes may represent unrelated proteins with DNA-binding
specificities similar to C/EBP, or heterodimeric or posttranslationally
modified complexes that are not recognized by C/EBP antisera. The
C/EBPß-C1 complex (band b), which contributes to the supershifted
band does not migrate with the complex formed with recombinant
C/EBPß, indicating that C/EBPß may exist in a heterodimeric form in
MA-10 cells. At present, the putative partner(s) for C/EBPß are
unknown.

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Figure 4. C/EBPß Binds to the C1 Site
In vitro transcribed and translated C/EBPß or 5 µg
of protein isolated from nuclear extracts prepared from unstimulated or
(Bu)2cAMP-stimulated MA-10 cells were incubated with a
32P-labeled C1 oligonucleotide and then incubated in the
presence or absence of 1 µl of a C/EBPß-specific antiserum.
DNA-protein complexes were subjected to electrophoresis through a 4%
nondenaturing polyacrylamide gel for approximently 2 h at 200 V,
and then dried gels were visualized by autoradiography. SS indicates
the position of the supershifted band. The right panel
is a example of a 4% nondenaturing polyacrylamide gel that was run for
1.5 h at 200 V and was exposed to x-ray film for less time than
the example in (a) to achieve a higher resolution of the bands, which
shows that three bands, labeled a, b and c, resolve in the major
shifted complex.
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Since C/EBP
and C/EBPß share binding site preferences (22, 33),
and have both been shown to be expressed in reproductive tissues (34, 35), we examined their expression in MA-10 cells. By Western
analysis, we have determined that C/EBPß, but not C/EBP
, could be
detected in MA-10 cells, and that MA-10 nuclear extracts did not
contain C/EBP
-binding activity as evidenced by EMSA using the C1
oligo (data not shown).
C/EBP DNA Elements Are Required for Activation of the StAR
Promoter
To assess the role of the C1 and C2 sites in StAR
promoter function, we compared the activity in MA-10 cells of the
wild-type StAR promoter to mutants carrying changes in
either or both of these sites. The mutations were tested in the context
of a 966-bp fragment of the StAR gene that had previously
been shown to support basal and cAMP-inducible expression in MA-10
cells (10). The same mutations in the C1 and C2 sites used in the EMSAs
were introduced into the StAR promoter either alone or in
combination. Each construct was transfected into MA-10 cells, and
luciferase activities were measured in untreated cells and cells
incubated in the presence of (Bu)2cAMP for 6 h. The
wild-type promoter construct (-966 StAR Luc) displayed low basal
activity, which was stimulated 6.2-fold by (Bu)2cAMP (Fig. 5
). Mutation of either the C1 or C2 site
alone (-966 StAR C1m and -966 StAR C2m) resulted in significantly
lower basal activities, 20% and 15% of the wild-type value,
respectively. Mutation of both the C1 and C2 sites (-966 StAR C1m,
C2m) resulted in a further decrease in basal promoter activity to 10%
of the wild-type activity; however, the cAMP responsiveness of the
promoter was again relatively unchanged (Fig. 5
). Although the absolute
cAMP-induced activities of the mutated reporters was lower than the
wild-type promoter, the fold activation of all four reporters was
similar. These data indicate that the C1 and C2 sites are important for
high-level basal expression from the StAR promoter.

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Figure 5. C/EBP DNA Elements Are Required for Activation of
the StAR Promoter in MA-10 Cells
MA-10 cells were transfected with 2 µg of either StAR -966,
StAR -966C1m, StAR -966C2m, or StAR -966C1m,2 m, and then reporter
activity was measured from (Bu)2cAMP-stimulated or
unstimulated cells. In all cases, 75 ng of pRL-SV40 were also
transfected as a control for transfection efficiency. Data are
represented as reporter activity divided by the activity of SV40-RNU.
Fold activation represents the (Bu)2cAMP-stimulated
reporter activity divided by the unstimulated level. Data represent
averages ± SEM from three experiments including the
StAR -966C1m,2 m reporter, and two additional experiments not
including the StAR -966C1m,2 m reporter, all of which were normalized
to the activity of the StAR -966 reporter from unstimulated cells in
each experiment. Statistical analysis between control (StAR -966) and
mutant reporters revealed some significant differences as determined by
Students unpaired two-tailed t tests (#,
P < 0.05 between unstimulated reporters; *,
P < .05 between (Bu)2cAMP-stimulated
reporters).
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SF-1 Requires C/EBP DNA Elements to Activate the StAR Promoter
To better understand the role of C/EBPß in StAR gene
transcription, we conducted transactivation experiments in COS-1 cells,
a nonsteroidogenic cell line that does not express C/EBPß (S. C.
Williams, unpublished observation), or SF-1 (14). C/EBPß and SF-1
expression vectors were cotransfected, either alone or in combination,
with the wild-type and mutant promoter constructs mentioned above.
StAR promoter activity was stimulated approximately 2-fold
by C/EBPß, and this effect was lost or diminished when either the C1
or C2 sites were mutated (Fig. 6
; p-966
StAR C1m and C2m). A StAR promoter construct carrying
mutations in both sites (p-966 StAR C1m,C2m) was completely
unresponsive to C/EBPß. SF-1 stimulated StAR promoter
activity approximately 5-fold; however, mutating the C1 site, and
especially the C2 site, diminished this activity (Fig. 6
). In fact, the
promoter construct bearing mutations in the C2 site was totally
unresponsive to SF-1, despite the fact that the predicted SF-1 sites in
this promoter remain intact. Coexpression of C/EBPß and SF-1 did not
result in additive or synergistic activation of the StAR
promoter (Fig. 6
). This observation may be due to the high levels of
both C/EBPß and SF-1 protein in the transiently transfected cells,
resulting in squelching or titration of required cofactors, leading to
a decrease in reporter activity. These data indicate that C/EBPß can
stimulate the activity of the StAR promoter and that
efficient SF-1-dependent activation of this promoter requires intact C1
and C2 sites.

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Figure 6. C/EBP Sites Are Required for SF-1 to Transcativate
the StAR Promoter
COS-1 cells were transfected with 2 µg of either StAR -966,
StAR -966C1m, StAR -966C2m, or StAR -966C1m,2 m, and cotransfected
with 2 µg of either or both C/EBPß or SF-1 expression plasmids, and
reporter activity was measured. Data represent averages ±
SEM from three experiments that were normalized to the
activity of StAR -966 reporter alone. All values were compared with
the p-966 StAR Luc reporter cotransfected with the control vector by
Students one-tailed unpaired t test (*,
P < 0.05).
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C/EBPß and SF-1 Physically Interact in Vitro
The data presented above indicate a functional interaction between
SF-1 and proteins binding to the C1 and C2 sites. Due to the close
proximity of SF-1 and putative C/EBPß binding sites, we next examined
whether these proteins might physically associate. Bacterially
expressed glutathione S-transferase (GST) or a chimeric
GST-SF-1 protein was mixed with radiolabeled recombinant C/EBPß in
the presence of a portion of the StAR promoter (-5 to -158) and a
reversible protein cross-linking agent. After purification on
glutathione-agarose beads, the remaining proteins were resolved by
SDS-PAGE (Fig. 7
). Recombinant C/EBPß
was specifically retained by GST-SF-1, indicating that SF-1 and
C/EBPß are likely to physically associate, and that this association
may be necessary for efficient activation of the StAR promoter.

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Figure 7. SF-1 and C/EBPß Physically Interact in
Vitro
Bacterially expressed GST-SF-1 or GST was incubated with radiolabeled
recombinant C/EBPß in the presence of a fragment of the
StAR promoter spanning -5 to -158. Proteins were
reversibly cross-linked and purified using glutathione-agarose beads
and then subjected to SDS-PAGE.
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DISCUSSION
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The promoter regions of eukaryotic genes are generally composed of
multiple binding sites for transcriptional activators and repressors
that act in combination to regulate expression of a linked gene
(36, 37, 38). Comparison of the 5'-flanking sequence of the StAR
gene revealed the existence of several blocks of conserved sequences
within 120 bp of the transcription start site. Previous analyses had
revealed the existence of binding sites for the orphan receptor, SF-1,
and a variant TATA-like element located 2530 bp from the start site.
We report here the identification of two novel elements required for
high-level expression from the StAR promoter in Leydig
cells.
The upstream element, C1, is strongly bound by recombinant C/EBPß and
by C/EBPß in MA-10 nuclear extracts and appears to be the primary
site through which C/EBPß activates the StAR promoter. We
have considered two putative, nonexclusive functions for C/EBPß in
StAR gene regulation, namely, the activation of
StAR gene expression during development and the rapid
activation of StAR transcription in response to trophic
hormone. The observation that mutation of the C1 site did not abolish
cAMP induction (fold activation) of the StAR promoter
suggests that either C/EBPß does not mediate the cAMP-dependent
regulation of StAR gene expression, at least during the
acute phase of stimulation, or (Bu)2cAMP stimulation of
Leydig cells does not completely mimic all of the effects of hormone
stimulation. However, mutating the C1 site decreased basal level
activity from the StAR promoter to 20% of the wild-type
level. This finding, combined with our previous observation that both
C/EBPß and StAR protein levels increase during Leydig cell
development (23), indicates a role for C/EBPß in developmental
regulation of StAR gene expression. In support of a broad
role of C/EBPß in the developmental regulation of steroidogenesis,
analysis of the promoter regions of genes encoding steroidogenic
enzymes in Leydig cells, such as 3ß-hydroxysteroid dehydrogenase
(3ßHSD), cytochrome P450 side-chain cleavage
(P450scc), and 17,
-hydroxylase (CYP17), has
revealed the presence of putative C/EBP sites (our unpublished
observation), although functional studies of these sites are lacking at
present. Therefore, C/EBPß may participate in the regulation of
multiple Leydig cell genes during development.
Our mutational analysis also identified a second element (C2) that is
required for high-basal level expression of the StAR
promoter. Although the C2 site is not as highly conserved as the C1
site, it appears to be at least equally important for StAR
gene transcription as its mutation decreased promoter activity to 15%
of wild-type levels. We initially considered the C2 site to be a
binding site for C/EBPß based on the presence of an almost perfect
half-site in the mouse, porcine, and human genes, and a 7 of 10 match
to the consensus C/EBP binding site in the mouse gene. However, the C2
site complexed weakly with nuclear extract from MA-10 cells and was
unable to compete for C/EBP binding to the C1 element. In addition,
mutation of the C2 site in the StAR promoter had only a
slight negative effect on transactivation by C/EBPß in COS-1 cells.
These data could be interpreted in two ways. First, the C2 site may not
be a bona fide C/EBPß binding site in vivo,
instead serving as the binding site for another, as yet unidentified,
protein. Some candidate proteins might be members of the CCAAT
box-binding protein families, such as the constitutively expressed
nuclear factor-Y [NF-Y; a heterotrimer of NF-YA, NF-YB, and NF-YC (39, 40)]. Second, the C2 site may be a weak binding site for C/EBPß, and
efficient usage of this site by C/EBPß may require the presence of
cooperating factors such as SF-1. In support of this hypothesis, a
cryptic C/EBP-binding site is present in the promoter of the
liver-specific cytochrome P450 2D5 (2D5) gene as part of a
bi-partite binding site for C/EBPß and Sp1 (41). C/EBPß is unable
to bind to, or to activate transcription through, this element in the
absence of Sp1, and the selective interactions with Sp1 explain the
difference in the ability of C/EBPß and C/EBP
to activate the
2D5 promoter (41). Additional studies are required to
identify the proteins that bind the C2 site in vivo and
whether C/EBPß must interact with other proteins to bind to C2.
We (in this report) and others (14) have demonstrated that
exogenously expressed SF-1 is capable of transactivating the
StAR promoter in COS-1 cells. However, SF-1-dependent
activation was diminished or lost when either one or both the C1 or C2
sites were mutated, despite the fact that these mutations would not be
expected to significantly affect SF-1 binding to its cognate sites.
Furthermore, we have shown that SF-1 and C/EBPß associate in
vitro. We interpret these results to indicate that SF-1 physically
interacts with C/EBPß and possibly other proteins bound to these
sites and that these interactions are a prerequisite for SF-1 action.
These interactions could be direct protein-protein interactions or may
involve the recruitment of accessory factors such as coactivators to
the promoter. In regard to the former possibility, C/EBPß has been
shown to functionally and/or physically interact with numerous members
of the steroid hormone superfamily, including the estrogen receptor
(42), glucocorticoid receptor (43), and hepatocyte nuclear factor-4
[HNF4 (44)]. SF-1 has also been shown to interact with a number of
proteins that may be involved in the transcriptional regulation of the
StAR gene, including the steroid receptor coactivator-1
[SRC-1/NCoA-1 (45, 46)] and DAX-1 [for dosage-sensitive sex
reversal-adrenal hypoplasia congenita critical region on the X
chromosome, gene 1 (47)]. The requirement of intact C1 and C2 sites
for efficient SF-1-mediated transactivation of the StAR
promoter and the physical interaction observed between SF-1 and
C/EBPß distinguishes the StAR promoter as an important
model with which to investigate how transcription factors may cooperate
to regulate transcription.
A central unanswered question surrounding the StAR gene is
the mechanism by which the promoter responds acutely to trophic hormone
stimulation and, more specifically, how the interactions between
transcription factors bound to the StAR promoter affect
cAMP-dependent regulation of the StAR gene. We have shown in
this report that the complex of proteins bound to the C1 site is
relatively unaffected by (Bu)2cAMP stimulation. We have
also reported that disruption of the C1 and C2 sites interfered with
basal (unstimulated) transcription of the StAR gene, and
that these sites were required for SF-1-dependent transcription from
the StAR promoter. Additionally, it has been shown that
StAR transcriptional activation does not require de
novo protein synthesis (10). Collectively, these findings indicate
that C/EBPß (and/or other proteins) bound to the C1 and C2 sites
interacts with SF-1 regardless of cAMP stimulation, and that cAMP
stimulation regulates a step distal to the formation of this complex,
which, in turn, may activate transcription from the StAR
promoter. There are several nonexclusive mechanisms by which this may
transpire. For example, upon stimulation with trophic hormone,
increased cAMP levels cause the release of the catalytic subunit of
PKA, which can enter the nucleus (48). Posttranslational modifications
have been shown to activate C/EBPß independently from its ability to
bind DNA (25), and a specific target residue for PKA has been
identified in the C/EBPß basic region (27). This posttranslational
modification of C/EBPß may activate transcription either directly
through C/EBPß or through recruitment of coactivators to the
promoter. An alternative model involves the orphan nuclear receptor
DAX-1. DAX-1 has been shown to be a powerful repressor of
StAR promoter activity, through binding to a hairpin loop
structure located proximal to the C2 site (49). Our data demonstrate
that similar complexes, which include C/EBPß, are formed on the C1
site regardless of cAMP stimulation. SF-1 and other factors may also
bind to the promoter in the absence of cAMP stimulation, which should
result in a high level of basal transcription from the promoter. Since
DAX-1 has been shown to repress StAR, its presence on this
promoter could effectively inhibit transcription, even in the presence
of positive factors such as SF-1, Sp1, and C/EBPß. Upon cAMP
stimulation, DAX-1 may be displaced from the promoter, or disassociated
from corepressors, allowing high levels of transcription from the
StAR promoter. DAX-1 null mice have recently been described,
and steroidogenesis appears to be relatively normal (50). The
observed phenocopy appears to be less severe than expected, given that
mutations in the DAX-1 gene in humans results in X-linked, adrenal
hypoplasia congenita (AHC). The precise role of DAX-1 in the regulation
of the StAR gene remains a most interesting question, which
clearly requires additional study and a more detailed analysis of the
DAX-1 null mice. Work is currently underway to study in more detail the
direct interaction between C/EBPß and SF-1 and to study whether
transcriptional coactivators and repressors are involved in SF-1- or
C/EBPß-mediated regulation of the StAR gene.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The MA-10 mouse Leydig tumor cell line was a generous gift from
Dr. M. Ascoli (Department of Pharmacology, University of Iowa College
of Medicine, Iowa City, Iowa). The cells were grown in Waymouths
MB/752 medium containing 15% horse serum and 40 µg gentamycin
sulfate/ml (referred to as WAY+). COS-1 African green monkey kidney
cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in DMEM
supplemented with 10% FBS and 100 U of penicillin/ml and 10 U of
streptomycin sulfate/ml. All cells were grown at 37 C in a humid
atmosphere of 5% CO2. Media, additives, and serum were
purchased from Gibco BRL (Gaithersburg, MD).
Plasmids and Construction of StAR Promoter
Mutants
pMEX C/EBPß has been described previously (51), and the SF-1
expression plasmid was a generous gift of Dr. Keith Parker (University
of Texas, Southwestern Medical School, Dallas, TX).
Site-directed mutagenesis was performed to mutate both of the
C/EBP-binding sites in the StAR promoter. The Gene Editor
kit (Promega Corp., Madison, WI) was used to introduce
mutations into p-966 Luc (described in Ref. 10); referred to in this
study as Star -966) to eliminate the C/EBP elements such that the
mutated sequences would contain a novel SalI restriction
site. The oligonucleotides used to introduce the mutations were as
follows (mutations are underlined):
C1m: CACTGCAGGATGGTCGACTCATTCCATCCT
C2m:
CTTGACCCTCTGGTCGACGACTGATGACTT
C1,2m: GCACTGCAGGATGGTCGACTCATTCCATCCTT -
GACCCTCTGGTCGACGACGATGAC.
Resulting plasmids were partially sequenced to confirm that the
C/EBP-binding sites had been mutated as expected.
Transfections
MA-10 and COS-1 cells were transfected by electroporation.
Briefly, 350 µl of a suspension of cells (12.5 x
106 cells/ml) were mixed with various amounts of effector
and reporter plasmids along with sheared salmon sperm DNA (Sigma Chemical Co., St. Louis, MO.) as carrier DNA to equalize the
total amount of DNA electroporated to 70 µg in each electroporation.
For the transfection studies in MA-10 cells, 75 ng of pRL-SV40 vector
(a plasmid that constitutively expresses Renilla luciferase under the
control of the SV40 promoter; Promega Corp.) was also
transfected in all cases as a transfection control. Cells were
electroporated in cuvettes (Invitrogen, Carlsbad, CA) with
a gap width of 4 mm, using the electro cell manipulator 600 (BTX
Inc., San Diego, CA) using the following parameters: capacitance =
960 µFarads; voltage = 250 V; resistance = 129
,
yielding an electroporation time of 2030 msec. After electroporation,
1 ml normal growth medium was added to the cuvette, and the cells were
incubated for 15 min at room temperature (RT). The cells were then
brought to 12 ml in normal growth medium, and 2 ml were placed in each
well of a six-well (35-mm) dish. Twenty-four hours after
electroporation, the medium was replaced with fresh medium. Twenty-four
hours later, three wells of each six-well plate were treated for 6
h with 1 mM (Bu)2cAMP (Sigma Chemical Co., St. Louis MO), in 1 ml of WAY+, while the control wells
received only 1 ml of WAY+. After (Bu)2cAMP stimulation the
cells were harvested for luciferase assays as described below.
Luciferase Assays
Extracts for luciferase assays were prepared using luciferase
assay system reporter lysis buffer (Promega Corp.). At the
time of harvesting, medium was removed, and the cells were rinsed three
times with ice-cold PBS. Reporter lysis buffer (250 µl) was added to
the cells, and the cells were scraped into 1.5-ml centrifuge tubes. The
cellular debris was then pelleted by centrifugation at 13,800 x
g at 4 C, and the supernatant fluid was placed in a 1.5-ml
centrifuge tube and was either used immediately or stored at -80
C.
Luciferase assays were performed using luciferase or dual luciferase
assay kits (Promega Corp.) exactly as described in the
protocol provided with the kit. Relative light units were measured
using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Students unpaired one-tailed or
two-tailed t tests were performed using Statview SE+
graphics software (Abacus Concepts, Berkeley, CA).
EMSA
Nuclear extracts were prepared from confluent cell cultures as
described (52). Briefly, cell monolayers were rinsed three times with
ice-cold PBS and scraped in 1 ml of PBS into 1.5-ml centrifuge tubes.
The cells were pelleted by centrifugation at 1500 x g
for 3 min. The pellets were resuspended in 400 µl of buffer A (10
mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM
EDTA; 0.1 mM EGTA; 1 mM dithiothreitol;
1 mM phenylmethylsulfonyl fluoride). The cells were swelled
for 15 min at 4 C, and then 25 µl of 10% NP-40 were added and the
tubes were vortexed. The homogenates were centrifuged 30 sec at
13,800 x g in a microfuge to pellet the nuclei; then
50 µl of buffer C (20 mM HEPES, pH 7.9; 0.4 M
NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM
dithiothreitol; 1 mM phenylmethylsulfonyl fluoride) was
added, and the samples were vigorously rocked for 15 min at 4 C. The
nuclear lysate was then centrifuged for 5 min at 13,800 x
g in a microfuge at 4 C, and the supernatant fluid was
placed into a fresh microfuge tube and stored at -80 C or used
immediately. The pSVSportC/EBPß plasmid (provided by Dr. Elmus Beale,
Texas Tech University, Health Sciences Center, Lubbock, TX) was
transcribed using SP6 polymerase and was translated using the TNT kit
(Promega Corp.). The double-stranded DNA probes used were
C1 (C/EBP binding site at -113) and C2 (C/EBP binding site at -87),
and mutants of C1 and C2 in which underlined bases have been
mutated to disrupt the specific binding of C/EBP proteins:
C1: GGCTGCAGGATGAGGCAATCATTCCA
C1m: GGCTGCAGGATGGTCGACTCATTCCA
C2: GGGACCCTCTGCACAATGACTGATG
C2m: GGGACCCTCTGGTCGACGACTGATG
To generate radioactive probes, the sense and antisense
oligonucleotides were heated to 75 C for 5 min and then slowly cooled
over 2 h to room temperature in annealing buffer [10
mM Tris-HCl (pH 7.5), 100 mM NaCl, 1
mM EDTA]. 5'-GGG overhangs present in the double-stranded
oligonucleotides were filled in using
[32P] dCTP 3000
Ci/mmol (DuPont NEN, Boston, MA) and Klenow (Promega Corp.) at 37 C for 30 min. The 32P-labeled probes
were purified using Probe Quant spin columns (Pharmacia Biotech, Piscataway, NJ). Binding reactions were performed by
mixing 5 µg of nuclear extract with a binding cocktail containing 4%
Ficoll, 10 mM HEPES (pH 7.9), 1 mM EDTA (pH
8.0), and 1 µg poly (dI:dC) and the labeled probe at a final
concentration of 5 nM in 15 µl. Where noted, the protein
was first incubated 20 min at room temperature, in binding cocktail
with 100-fold molar excess of the unlabeled competitor DNA before
addition of the labeled DNA. For the supershift experiments, the
binding reaction was performed as described above for 20 min, after
which 1 µl of the C/EBPß antiserum was added and the reaction was
incubated an additional 20 min at room temperature. After the binding
reaction, the entire reaction was subjected to electrophoresis through
a 4% nondenaturing polyacrylamide gel. The gel was then dried and
autoradiography and phosphorimagery (Molecular Dynamics, Inc., Sunnyvale, CA) were performed.
DNAse I Footprint
To generate a radiolabeled DNA probe, the region spanning -254
to -35 of the StAR promoter was amplified using the
following oligonucleotide primers; 5'-primer is a 20 mer that spans
bases -254 to -235 and has additional bases at the 5'-end to generate
an MluI restriction endonuclease site, and the 3'-primer is
a 44 mer that spans -101 to -35 and; contains point mutations to
generate a BglII resriction endonuclease site centered at
base -63 and a XhoI resriction endonuclease site centered
at -95. The amplification product was cloned into the
MluI-SmaI sites of the pSport vector (Life Technologies, Gaithersburg, MD), and the sequence was verified
by the dideoxynucleotide sequencing method of Sanger using the T7
Sequenase Kit Version 2 (Amersham Pharmacia Biotech, Arlington Heights, IL). The StAR
promoter fragment was excised from pSport by MluI and
KpnI digestion and gel purified using QIAquick gel
extraction kit (Qiagen, Chatsworth, CA) and treated
with calf intestinal phosphatase (CIP, Promega Corp.). CIP
was inactivated by phenol-cholorform extraction, and the DNA was
precipitated with ethanol. Two picomoles of probe were radiolabed using
-[32P]ATP (DuPont NEN, Boston MA) and T4
polynucleotide kinase (Promega Corp.) followed by
inactivation of the kinase and digestion with BglII. The
resultant probe is labeled on the coding strand and spans -254 to -66
of the StAR promoter. The probe was purified by
phenol-chloroform extraction and ethanol precipitation, after which
DNAse I footprint analysis was performed using the Core Footprinting
System (Promega Corp.) with minor modifications. In brief,
2040 fmol of probe (50K-100K cpm) were added to the DNA
protein-binding reaction (10 mM Tris/Cl, pH 8.0, 150
mM KCl, 2.5 µg poly dI:dC, 4 µg/ml calf thymus DNA,
10% glycerol, and 2550 µg MA-10 nuclear extract. The reaction was
incubated on ice for 30 min and then transferred to 25 C, and
CaCl2 and MgCl2 were added to a final
concentration of 2.5 mM and 5 mM, respectively.
One unit of DNAse I (Promega Corp.) was added to the
reaction and was incubated for 15 sec in the presence of nuclear
extract or 30 sec in the presence or absence of nuclear extract. The
reactions were stopped by the addition of an equal volume of stop
buffer containing 200 mM NaCl, 30 mM EDTA, 1%
SDS, and 100 µg/ml yeast RNA, and the DNA was recovered by
phenol-chloroform extraction and ethanol precipitation. The reactions
were resuspended in formamide loading buffer, and the DNA was resolved
on a 6% polyacrylamide sequencing gel. The gel was dried and exposed
to x-ray film. Maxam and Gilbert (53) chemical sequencing reactions
were performed using 40 fmol of probe following standard protocols
(54).
GST Pull Down Assay
A GST-SF-1 fusion protein was prepared and isolated according to
standard protocols (52). The plasmid containing the SF-1 coding
sequence in the pGEX-1
T vector (Pharmacia Biotech,
Piscataway, NJ), was described previously (55), and the control GST
plasmid was pGEX4T-3 (Pharmacia Biotech). Both GST and
GST-SF-1 were subjected to PAGE and stained with Coomassie blue. The
appearance of a band at the correct molecular weight confirmed that
intact proteins were produced. In vitro transcribed and
translated C/EBPß was prepared as described above, except that the
STP3 kit (Novagen, Madison, WI) was used in the presence of
35[S]methionine according to protocols supplied by the
manufacturer. A binding reaction was prepared containing 10 µl of
in vitro transcribed and translated 35S-labeled
C/EBPß, 20 µg of the GST-SF-1 or GST protein bound to 70 µl of
glutathione linked to beaded agarose (Sigma Chemical Co.),
1 µg poly (dI:dC) in the binding cocktail described in the EMSA
methods above, with 50 ng of a PCR product spanning -5 to -158 of the
mouse StAR promoter (primer sequences avaliable upon
request). The binding reaction was incubated 20 min at 4 C,
DTSSP (Pierce Chemical Co., Rockford, IL), a
reversible cross-linking reagent, was added to a final concentration of
5 mM and incubation continued for an additional 20 min at 4
C. The binding reactions were then centrifuged at 13,000 x
g, the supernatant was removed, and the pellets were washed
four times in the binding cocktail. Pellets were resuspended in
denaturing sample buffer and incubated at 100 C for 10 min and
subjected to SDS-PAGE. Autoradiography and phosphorimagery were
performed on dried gels.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. Steven King for many helpful
discussions and Dr. Joseph Orly for sharing data before publication and
for helpful discussions. We acknowledge the technical assistance of
Deborah Alberts, Matthew Dyson, Rebecca Combs, Darrell Eubank, and
Demet Nalbant. We also thank Drs. Mark McLean, Holly LaVoie, and
Jennifer Juengel for providing us with StAR promoter
sequences before publication and Dr. Keith Parker for providing us with
plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
This research was supported by NIH Grants HD-17481 (D.M.S.) and
DK-51656 (B.J.C.) and a Scientist Development Grant from the American
Heart Association (S.C.W.).
Received for publication October 2, 1998.
Revision received February 5, 1999.
Accepted for publication February 17, 1999.
 |
REFERENCES
|
---|
-
Jefcoate CR, DiBartolomeis MJ, Williams CA, McNamara BC 1987 ACTH regulation of cholesterol movement in isolated adrenal cells.
J Steroid Biochem 27:721729[CrossRef][Medline]
-
Crivello JF, Jefcoate CR 1980 Intracellular movement of
cholesterol in rat adrenal cells. Kinetics and effects of inhibitors.
J Biol Chem 255:81448151[Free Full Text]
-
Stocco DM, Clark BJ 1996 Regulation of the acute production
of steroids in steroidogenic cells. Endocr Rev 17:221244[Medline]
-
Stocco DM 1997 A StAR search: implications in controlling
steroidgenesis. Biol Reprod 56:328336[Abstract]
-
Stocco DM, Clark BJ 1997 The role of the steroidogenic acute
regulatory protein in steroidogenesis. Steroids 62:2936[CrossRef][Medline]
-
Clark BJ, Stocco DM 1997 Steroidogenic acute regulatory
protein: the StAR still shines brightly. Mol Cell Endocrinol 134:18[CrossRef][Medline]
-
Clark BJ, Wells J, King SR, Stocco DM 1994 The purification,
cloning, and expression of a novel luteinizing hormone-induced
mitochondrial protein in MA-10 mouse Leydig tumor cells.
Characterization of the steroidogenic acute regulatory protein (StAR).
J Biol Chem 269:2831428322[Abstract/Free Full Text]
-
Clark BJ, Stocco DM 1995 Expression of the steroidogenic
acute regulatory (StAR) protein: a novel LH-induced mitochondrial
protein required for the acute regulation of steroidogenesis in mouse
Leydig tumor cells. Endocr Res 21:243257[Medline]
-
Clark BJ, Combs R, Hales KH, Hales DB, Stocco DM 1997 Inhibition of transcription affects synthesis of steroidogenic acute
regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor
cells. Endocrinology 138:48934901[Abstract/Free Full Text]
-
Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ 1997 Characterization of the promoter region of the mouse gene encoding the
steroidogenic acute regulatory protein. Mol Endocrinol 11:138147[Abstract/Free Full Text]
-
Caron KM, Clark BJ, Ikeda Y, Parker KL 1997 Steroidogenic
factor 1 acts at all levels of the reproductive axis. Steroids 62:5356[CrossRef][Medline]
-
Sugawara T, Kiriakidou M, McAllister JM, Kallen CB, Strauss
JF, 3rd 1997 Multiple steroidogenic factor 1 binding elements in the
human steroidogenic acute regulatory protein gene 5'-flanking region
are required for maximal promoter activity and cyclic AMP
responsiveness. Biochemistry 36:72497255[CrossRef][Medline]
-
Sugawara T, Kiriakidou M, McAllister JM, Holt JA, Arakane F,
Strauss JF, 3rd 1997 Regulation of expression of the steroidogenic
acute regulatory protein (StAR) gene: a central role for steroidogenic
factor 1. Steroids 62:59[CrossRef][Medline]
-
Sugawara T, Holt JA, Kiriakidou M, Strauss JF, 3rd 1996 Steroidogenic factor 1-dependent promoter activity of the human
steroidogenic acute regulatory protein (StAR) gene. Biochemistry 35:90529059[CrossRef][Medline]
-
Chau YM, Crawford PA, Woodson KG, Polish JA, Olson LM,
Sadovsky Y 1997 Role of steroidogenic-factor 1 in basal and
3',5'-cyclic adenosine monophosphate-mediated regulation of cytochrome
P450 side-chain cleavage enzyme in the mouse. Biol Reprod 57:765771[Abstract]
-
Zhang P, Mellon SH 1997 Multiple orphan nuclear receptors
converge to regulate rat P450c17 gene transcription: novel mechanisms
for orphan nuclear receptor action. Mol Endocrinol 11:891904[Abstract/Free Full Text]
-
Zhang P, Mellon SH 1996 The orphan nuclear receptor
steroidogenic factor-1 regulates the cyclic adenosine
3',5'-monophosphate-mediated transcriptional activation of rat
cytochrome P450c17 (17 alpha-hydroxylase/c1720 lyase). Mol Endocrinol 10:147158[Abstract]
-
Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site
is required for activity of the luteinizing hormone beta subunit
promoter in gonadotropes of transgenic mice. J Biol Chem 271:1078210785[Abstract/Free Full Text]
-
Cammas FM, Pullinger GD, Barker S, Clark AJ 1997 The mouse
adrenocorticotropin receptor gene: cloning and characterization of its
promoter and evidence for a role for the orphan nuclear receptor
steroidogenic factor 1. Mol Endocrinol 11:867876[Abstract/Free Full Text]
-
Duval DL, Nelson SE, Clay CM 1997 A binding site for
steroidogenic factor-1 is part of a complex enhancer that mediates
expression of the murine gonadotropin-releasing hormone receptor gene.
Biol Reprod 56:160168[Abstract]
-
Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA,
Tourtellotte LM, Simburger K, Milbrandt J 1995 Mice deficient in the
orphan receptor steroidogenic factor 1 lack adrenal glands and gonads
but express P450 side-chain-cleavage enzyme in the placenta and have
normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci
USA 92:1093910943[Abstract]
-
Johnson PF, Williams SCC 1994 CAAT/Enhancer Binding (C/ EBP)
Proteins. In: Tronch F, Yaniv M (eds) Liver Gene Expression. Landes Co,
Austin, TX, pp 231258
-
Nalbant D, Williams SC, Stocco DM, Khan SA 1998 Luteinizing
hormone-dependent gene regulation in Leydig cells may be mediated by
CCAAT/enhancer-binding protein-ß. Endocrinology 139:272279[Abstract/Free Full Text]
-
Niehof M, Manns MP, Trautwein C 1997 CREB controls LAP/C/EBP
beta transcription. Mol Cell Biol 17:36003613[Abstract]
-
Tae HJ, Zhang S, Kim KH 1995 cAMP activation of CAAT
enhancer-binding protein-beta gene expression and promoter I of
acetyl-CoA carboxylase. J Biol Chem 270:2148721494[Abstract/Free Full Text]
-
Metz R, Ziff E 1991 cAMP stimulates the C/EBP-related
transcription factor rNFIL-6 to trans-locate to the nucleus and induce
c-fos transcription. Genes Dev 5:17541766[Abstract]
-
Chinery R, Brockman JA, Dransfield DT, Coffey RJ 1997 Antioxidant-induced nuclear translocation of CCAAT/enhancer-binding
protein beta. A critical role for protein kinase A-mediated
phosphorylation of Ser299. J Biol Chem 272:3035630361[Abstract/Free Full Text]
-
Sandhoff TW, Hales DB, Hales KH, McLean MP 1998 Transcriptional regulation of the rat steroidogenic acute regulatory
protein gene by steroidogenic factor 1. Endocrinology 139:48204831[Abstract/Free Full Text]
-
LaVoie HA, Garmey JC, Veldhuis JD 1999 Mechanisms of IGF-I
augmentation of FSH-stimulated porcine StAR gene promoter activity in
granulosa cells. Endocrinology 140:146153[Abstract/Free Full Text]
-
Ito E, Toki T, Ishihara H, Ohtani H, Gu L, Yokoyama M, Engel
JD, Yamamoto M 1993 Erythroid transcription factor GATA-1 is abundantly
transcribed in mouse testis. Nature 362:466468[CrossRef][Medline]
-
Feng ZM, Wu AZ, Chen CL 1998 Testicular GATA-1 factor
up-regulates the promoter activity of rat inhibin
-subunit gene in
MA-10 Leydig tumor cells. Mol Endocrinol 12:378390[Abstract/Free Full Text]
-
Williams SC, Cantwell CA, Johnson PF 1991 A family of
C/EBP-related proteins capable of forming covalently linked leucine
zipper dimers in vitro. Genes Dev 5:15531567[Abstract]
-
Johnson PF 1993 Identification of C/EBP basic region residues
involved in DNA sequence recognition and half-site spacing preference.
Mol Cell Biol 13:69196930[Abstract]
-
Piontkewitz Y, Enerback S, Hedin L 1996 Expression of CCAAT
enhancer binding protein-alpha (C/EBP alpha) in the rat ovary:
implications for follicular development and ovulation. Dev Biol 179:288296[CrossRef][Medline]
-
Sirois J, Richards JS 1993 Transcriptional regulation of the
rat prostaglandin endoperoxide synthase 2 gene in granulosa cells.
Evidence for the role of a cis-acting C/EBP beta promoter element.
J Biol Chem 268:2193121938[Abstract/Free Full Text]
-
Roesler WJ, Park EA 1998 Hormone response units: one plus one
equals more than two. Mol Cell Biochem 178:18[CrossRef][Medline]
-
Ogbourne S, Antalis TM 1998 Transcriptional control and the
role of silencers in transcriptional regulation in eukaryotes. Biochem
J 331:114[Medline]
-
Nelson SB, Eraly SA, Mellon PL 1998 The GnRH promoter: target
of transcription factors, hormones, and signaling pathways. Mol Cell
Endocrinol 140:151155[CrossRef][Medline]
-
Dorn A, Bollekens J, Staub A, Benoist C, Mathis D 1987 A
multiplicity of CCAAT box-binding proteins. Cell 50:863872[Medline]
-
Sinha S, Maity SN, Lu J, de Crombrugghe B 1995 Recombinant rat
CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA
complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. Proc Natl
Acad Sci USA 92:16241628[Abstract]
-
Lee YH, Williams SC, Baer M, Sterneck E, Gonzalez FJ, Johnson
PF 1997 The ability of C/EBP beta but not C/EBP alpha to synergize with
an Sp1 protein is specified by the leucine zipper and activation
domain. Mol Cell Biol 17:20382047[Abstract]
-
Stein B, Yang MX 1995 Repression of the interleukin-6 promoter
by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Mol Cell
Biol 15:49714979[Abstract]
-
Nishio Y, Isshiki H, Kishimoto T, Akira S 1993 A nuclear
factor for interleukin-6 expression (NF-IL6) and the glucocorticoid
receptor synergistically activate transcription of the rat alpha 1-acid
glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:18541862[Abstract]
-
Stauffer DR, Chukwumezie BN, Wilberding JA, Rosen ED,
Castellino FJ 1998 Characterization of transcriptional regulatory
elements in the promoter region of the murine blood coagulation factor
VII gene. J Biol Chem 273:22772287[Abstract/Free Full Text]
-
Crawford PA, Polish JA, Ganpule G, Sadovsky Y 1997 The
activation function-2 hexamer of steroidogenic factor-1 is required,
but not sufficient for potentiation by SRC-1. Mol Endocrinol 11:16261635[Abstract/Free Full Text]
-
Ito M, Yu RN, Jameson JL 1998 Steroidogenic factor-1 contains
a carboxy-terminal transcriptional activation domain that interacts
with steroid receptor coactivator-1. Mol Endocrinol 12:290301[Abstract/Free Full Text]
-
Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated
transactivation via a carboxy-terminal domain that is deleted in
adrenal hypoplasia congenita. Mol Cell Biol 17:14761483[Abstract]
-
Nigg EA, Hilz H, Eppenberger HM, Dutly F 1985 Rapid and
reversible translocation of the catalytic subunit of cAMP-dependent
protein kinase type II from the Golgi complex to the nucleus. EMBO J 4:28012806[Abstract]
-
Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA
binding and transcriptional repression by DAX-1 blocks steroidogenesis.
Nature 390:311315[CrossRef][Medline]
-
Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL 1998 Role of
Ahch in gonadal development and gametogenesis. Nat Genet 20:353357[CrossRef][Medline]
-
Williams SC, Baer M, Dillner AJ, Johnson PF 1995 CRP2 (C/EBP
beta) contains a bipartite regulatory domain that controls
transcriptional activation, DNA binding and cell specificity. EMBO J 14:31703183[Abstract]
-
Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid
detection of octamer binding proteins with mini-extracts, prepared
from a small number of cells. Nucleic Acids Res 17:641955
-
Maxam AM, Gilbert W 1980 Sequencing end-labeled DNA with
base-specific chemical cleavages. Methods Enzymol 65:499599[Medline]
-
Ausubel FM, Brent R, Kingston RE, Moore ME, Smith JA, Seidman
JG, Struhl K (eds) 1998 Current Protocols in Molecular Biology. John
Wiley & Sons, Inc., New York
-
Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor I, a key
regulator of steroidogenic enzyme expression, is the mouse homolog of
fushi tarazu-factor I. Mol Endocrinol 6:12491258[Abstract]