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Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292
Address all correspondence and requests for reprints to: Dr. Barbara J. Clark, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292. E-mail: bjclark{at}louisville.edu
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Introduction |
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Hormonal treatment of steroidogenic cells controls StAR gene expression. Our previous studies have shown that treatment of MA-10 mouse Leydig tumor cells with trophic hormones or the cAMP analog, (Bu)2cAMP, induces StAR message within 30 min, and maximal steady state levels are reached within 4 h (9). This induction of StAR gene transcription does not require de novo protein synthesis, suggesting that posttranslational mechanisms are involved in the acute induction of StAR gene in response to increases in intracellular levels of cAMP (10).
Several factors that participate in StAR gene activation have been reported. One factor is the orphan nuclear receptor steroidogenic factor-1 (SF-1). SF-1 has been shown to have a critical role in the regulation of many of the steroid hydroxylase genes, and gene knockout studies in mice have shown that SF-1 is critical for development of the gonad and adrenal glands (11, 12, 13, 14). Regulation of SF-1 may occur through direct phosphorylation by a protein kinase A (PKA)-dependent pathway. This proposal is supported by studies in which SF-1 trans-activation of the steroid hydroxylase genes was lost in PKA-deficient cell lines (15, 16). Furthermore, potential PKA phosphorylation sites are present in SF-1, and in vitro studies have directly demonstrated a PKA-dependent phosphorylation of SF-1 (17, 18, 19). In addition, activation of the mitogen-activated protein kinase pathway has been shown to phosphorylate and active SF-1, supporting a role for phosphorylation of SF-1 for its function (20). Our initial analysis of two SF-1 sites in the mouse promoter, located at -45 and -135 from the transcription start site, indicated that these elements contributed to basal promoter activity but were not essential for the cAMP-dependent induction (10). The involvement of a SF-1 site in StAR gene activation was more recently confirmed with the rat promoter (21). Mutation of the SF-1 elements at -135 and -95, either individually or in combination, decreased SF-1-dependent reporter gene expression in a nonsteroidogenic cell line cotransfected for SF-1 expression (21). In these studies the (Bu)2cAMP-mediated increase in promoter activity was also depressed, suggesting that SF-1 may mediate part of the cAMP response. However, it is not known what effect the -95 SF-1 mutation would have in a steroidogenic cell line, where factors other than SF-1 may be required for basal and cAMP-dependent StAR transcription. On the other hand, the SF-1 element located at -105/-95 in the human promoter was shown to be critical for basal and maximal cAMP responsiveness in human granulosa-luteal cells and in H295R adrenocortical cells (22, 23). Thus, it appears that the role of SF-1 in StAR expression may be promoter and/or tissue specific.
C/EBPß has also been shown to modulate StAR basal promoter activity. C/EBPß is a basic leucine zipper transcription factor that is expressed in liver, intestine, lung, and adipose tissues (24). More recently, C/EBPß has been identified as the only isoform of C/EBP present in unstimulated primary Leydig cell cultures and MA-10 Leydig cells (25). Two putative CCAAT sites located at -113 and -87 in the murine StAR promoter were identified, and C/EBPß in MA-10 nuclear extracts was shown to bind to the -113 element (26). A weak protein-DNA interaction was observed with the -87 region of the promoter, but the factors were not identified. Mutation of the -87, but not the -113, site abolished SF-1-dependent trans-activation of StAR-luciferase reporter gene expression in transfected COS-1 cells (26). However, transient transfection of MA-10 cells with a StAR-luciferase reporter gene construct containing mutations at either the -87 or -113 C/EBPß-binding site did not affect the cAMP-dependent response, but greatly reduced the basal activity of the promoter. A protein-protein interaction between C/EBPß and SF-1 was further demonstrated; thus, these studies suggested a possible C/EBPß-SF-1 interaction that is required for basal promoter activity.
Although SF-1 and C/EBPß participate in basal expression of StAR, the
mechanism for cAMP-dependent induction has not been elucidated.
Previously, we narrowed the cAMP-responsive region of murine StAR to
-254 bp of the start site of transcription (10). Sequence analysis of
this region identified potential binding sites for transcription
factors that have been shown to mediate cAMP-dependent responses in
other genes. Two elements that are part of the focus in the present
study are a C/EBPß/nonconsensus activating
protein-1/nuclear receptor half-site located between
-87/-79 (CAN region) and a putative binding site for members of the
GATA family of zinc finger transcription factors located between
-68/-40. Activating protein-1 is composed of either Fos-Jun or
Jun-Jun dimers that bind to the consensus sequence TGA(G/C)TCA and
regulates genes either constitutively or by cAMP- or
Ca2+-mediated mechanisms (27, 28, 29). The GATA
family of transcription factors regulates gene expression,
differentiation, and cell proliferation by binding to the consensus DNA
sequence (A/T)GATA(A/G) (30). More recently, three members of the GATA
transcription factor family were shown to be expressed in the
developing gonads: GATA-1, GATA-4, and GATA-6 (31, 32, 33). GATA-1 message
has been isolated in MA-10 cells, and GATA-1 was shown to up-regulate
the basal promoter activity of the rat inhibin 
-subunit gene (34).
GATA-4 and GATA-6 have also been identified in testicular tissue, and
transient expression of GATA-4 was shown to trans-activate
an inhibin -promoter/reporter construct in mouse Leydig and
granulosa tumor cell lines (35). Thus, a new role is emerging for GATA
transcription factors in Leydig cell function.
To clarify the role of SF-1 in StAR expression in MA-10 mouse Leydig tumor cells, we extended our previous studies and demonstrated that SF-1 binds to a third element in mouse promoter at -95. We show that this SF-1 site, denoted SF1–3, is required for full basal activity of the StAR promoter. Additionally, SF-1 functions in part to stabilize protein-DNA interactions at the C/EBPß/nonconsensus activating protein-1 (AP-1)/nuclear receptor half-site (CAN) region located at -79. We also demonstrate that GATA-4 binds to the StAR promoter at -68 bp and contributes to 20% of the cAMP-dependent induction. We conclude that the SF1–3, CAN, and GATA-4 DNA-binding sites serve critical roles in maintaining full basal promoter activity, which, in turn, is necessary for maximal cAMP induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP was obtained from NEN Life Science Products (Wilmington, DE). Custom oligonucleotides were
purchased from Genosys (The Woodlands, TX) and Genemed Synthesis, Inc.
(San Francisco, CA). Glutathione-S-transferase (GST)-SF-1
plasmid was donated by Dr. Keith Parker, University of Texas
Southwestern Medical Center (Dallas, TX). Antibodies were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and
Upstate Biotechnology, Inc. (Lake Placid, NY). X-ray film
was obtained from Eastman Kodak Co. (Rochester, NY) and
NEN Life Science Products (Boston, MA).
Cell culture
The MA-10 mouse Leydig tumor cell line was a gift from Dr. M.
Ascoli (Department of Pharmacology, University of Iowa of Medicine,
Iowa City, IA). The cells were grown in Waymouth’s MB/752 medium
containing 15% horse serum and 40 µg gentamicin sulfate/ml.
Transient transfections and reporter assays
MA-10 cells were plated at 300,000 cells/well in a 24-well plate
24 h before transfection. Two micrograms of StAR-luciferase
reporter gene plasmid and 2 µg pCMV-ß-galactosidase (CMV,
cytomegalovirus) expression vector plasmid were cotransfected into
cells using 15 µg/ml Lipofectamine (Life Technologies, Inc., Grand Island, NY) and Waymouth’s medium without
antibiotics and serum as described previously (10). Twenty-four hours
posttransfection, the cells were treated with 1 mM
(Bu)2cAMP for 16–19 h. The Promega Corp. Luciferase Assay System was used to prepare cell lysates
and measure luciferase activity (Lumat LB 9507 luminometer,
Wallac, Inc., Gaithersburg, MD). ß-Galactosidase
activity was assayed using ß-D-galactopyranosidase
(Roche Molecular Biochemicals, Indianapolis, IN) as the
substrate and measuring the absorbance at 595 nm. Relative light unit
(RLU) values were normalized to ß-galactosidase activity for each
sample. Each treatment was performed in triplicate, and the mean
± SEM were determined. The data were expressed as fold
induction relative to the -254/+35 luc control, which was set at 100.
The mean ± SEM for the fold induction in response to
(Bu)2cAMP for all independent transfection
experiments was calculated, and a pooled Student’s t test
was performed on the data. P < 0.05 was considered
statistically significant.
StAR-luciferase constructs
PCR was used to generate the 5'-deletion constructs for the StAR
promoter. Linearized -254/+35 StAR promoter-pGL2-luciferase (-254
StAR promoter construct) was used as the template. To amplify the
region -150 to +35 (-150/+35 luc), MluI -150 and
GL2-primers were used in the PCR reaction, and
the product was digested with MluI and HindIII
and cloned into the MluI and HindIII sites of
pGL2-basic. The regions -105 to +35 (-105/+35 luc) and -68 to +35
region (-68/+35 luc) were amplified using SF1–3 top primer and
GL2 primers, and -68/-44 top primer and
GL2 primers, respectively, and the PCR product
for each clone was Klenow treated, digested with HindIII,
and cloned into the SmaI-HindIII sites of
pGL2-basic. The site-specific mutants generated in the -254/+35 luc
plasmid were constructed by generating two overlapping PCR products
that share an engineered restriction site to alter the core sequences
of interest. To produce the 5'-PCR product, the
GL1 primer was used to amplify the top strand,
and the bottom strand was amplified with the site-specific primer. The
3'-PCR product was produced with the site-specific mutant primer (top
strand) and GL2 (bottom strand). The PCR products
were digested with the appropriate restriction enzymes and cloned into
the SmaI-KpnI restriction sites of pGL2-basic.
The sequences of oligonucleotides used in the reactions are listed
under Oligonucleotides used in EMSA and PCR. The
SF1–3mut/GATA-4mut-luc, GATAmut/AP-1mut-luc, and SF1–3mut/AP-1mut-luc
constructs were created in a similar manner; the mutant SF1–3 site was
engineered into GATAmut-luc using the SF1–3mut-luc primer to generate
SF1–3mut/GATAmut-luc, the AP-1mut site was engineered into GATAmut-luc
using the AP-1mut-luc primer to generate GATAmut/AP-1mut-luc, and the
AP-1mut site was engineered into SF1–3mut-luc using the AP-1mut-luc
construct to generate SF1–3mut/AP-1mut-luc. One exception for
SF1–3mut/AP-1mut-luc is the 5'-PCR product was digested with
MluI and BglII, the 3'-PCR product was digested
with BglII and XbaI, and the two products were
cloned into the MluI-NheI sites of pGL2 basic.
All sequences were confirmed by double stranded sequencing using the
Sequenase II kit (United States Biochemical Corp.,
Cleveland, OH).
Electrophoretic mobility shift assay (EMSA)
Double stranded oligonucleotides were generated by mixing equal
molar concentrations of top and bottom strand primers in a high salt
annealing buffer [10 mM Tris (pH 7.5), 50 mM
NaCl, and 1 mM EDTA] and heating for 2 min at 95 C
followed by slow equilibration to room temperature. The
oligonucleotides were end labeled with T4 DNA kinase and
[-32P]ATP (NEN Life Science Products). To generate a probe for the -105/-44 region of
StAR, linearized -254 StAR promoter construct was used as the DNA
template in a PCR reaction with SF1–3 (top) and -68/-44 (bottom).
The -105/-44 fragment containing site-specific mutations or
double mutations was generated as described above using the
corresponding mutated reporter gene constructs as the template and
appropriate oligonucleotides. Each PCR fragment was gel purified and
radiolabeled as described above. Restriction digestion analysis was
used to confirm the PCR-generated probe contained the desired
mutations. MA-10 nuclear extracts (2.5–15.0 µg) or 0.25 µg
GST-SF-1 were incubated with 50 fmol radiolabeled oligonucleotide for
30 min on ice. The binding buffer contained the following: 50
mM Tris-Cl (pH 8.0), 100 mM KCl, 12.5
mM MgCl2, 1 mM EDTA, 1
mM dithiothreitol, 20% glycerol, and 2.5 µg poly(dI:dC).
Cold competition assays were performed with a 20- to 100-fold molar
excess of oligonucleotides. Antibody supershift assays were performed
with 2.5 or 15 µg MA-10 nuclear extract proteins or 0.25 µg
GST-SF-1 plus 4 µg monoclonal GATA-1, polyclonal GATA-4, polyclonal
C/EBPß, or polyclonal SF-1 antibodies. For the supershift assays, all
components of the binding reaction, excluding the radiolabeled probe,
were preincubated on ice for 30 min. Radiolabeled probe was then added,
and the incubation was continued for 30 min. Binding reactions were
resolved on a 4% nondenaturing polyacrylamide gel. The dried gels were
exposed to x-ray film, and the radioactive bands were visualized by
autoradiography.
The following oligonucleotides were used in the EMSA binding reactions. Only the top strand is listed, and changed base pairs in the mutated probes are underlined.
Sequences of oligonucleotides used for PCR and EMSA probes
The following sequences were used: SF1–3,
5'-CATTCCATCCTTGACCCTCTGCA-3'; SF1–3mut,
5'-CATTCCATCCTCGAGCCTCTGCA-3'; -68/-44,
5'-TTTTTTATCTCAAGTGATGATGCAC-3'; -68/-44 GATAmut,
5'-TTTTTCCGGACAAGTGATGATGCAC-3'; -68/-44 SREBPmut
(SREBP, sterol regulatory binding protein),
5'-TTTTTTATCTCTCGAGATGATGCAC-3'; -87/-64,
5'-TGCACAATGACTGATGACTTTTT-3'; -87/-64 AP-1mut,
5'-TGCACAATAGATCTTGACTTTTT-3'; -87/-64 1/2
mut, 5'-TGCACAATGACTGAGATCTTTTT-3';
GL11, 5'-TGTATCTTATGGTACTGTAACTG-3';
GL21, 5'-CTTTATGTTTTTGGCGTCTTCCA-3'; and -150
Mlu, 5'-ACACACGCGTAGTCTGCTCCCTCCCACCTTGGCCA-3' (1 indicates
the primers used for pGL2-basic vector).
Nuclear extract preparation
Confluent MA-10 mouse Leydig tumor cell cultures were treated
with fresh Waymouth’s medium in the absence (control) or presence of
(Bu)2cAMP for 4 h, and nuclear extracts were
prepared following previously described protocols (36). The extracts
were flash-frozen in liquid nitrogen and stored at -80 C until
use.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
(Bu)2cAMP treatment of MA-10 cells resulted in an
8.7-fold increase in luciferase reporter gene activity for the -254
StAR promoter construct. Deletion to -150 caused both basal and
(Bu)2cAMP-stimulated promoter activity to
increase 4- and 6-fold, respectively, which represents a 70% increase
in the cAMP-dependent fold induction compared with the -254 StAR
construct. Further deletion to -105 caused a 46% decrease in basal
activity compared with -254 StAR, but the fold induction in response
to the cAMP stimulus was not diminished. However, a 5-fold decrease in
the cAMP-dependent induction was observed with the -68 StAR promoter
construct. These results suggest that multiple elements may be required
for the cAMP response of the mouse StAR promoter: a possible negative
regulatory region located between the -150/-105 and a positive
regulatory region located between the -105/-68 region of the
promoter. The -254/-65 region of the promoter was cloned upstream of
a heterologous promoter (-254/-66StAR-pGL3luc), but the single copy
of this region was not sufficient to confer cAMP-dependent
responsiveness (data not shown). Therefore, elements 5' and 3' of -68
appear to required for the induction of StAR gene by cAMP.
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is the sequence of the -150 region of
murine StAR, with the location of these elements indicated. An
alignment with the human and rat sequences is also shown to indicate
the high degree of sequence conservation within this region of the StAR
promoter. We previously characterized the SF-1–1 (-135), SF-1–2
(-45), and the C/EBPß (-113) elements (10, 26), whereas the SF-1
site located at -95 bp has been shown to bind SF-1 in the human and
rat StAR promoters (21, 37). The region between -87/-64 is not as
well conserved and unique to the mouse sequence are potential
C/EBPß, nonconsensus AP-1, and
nuclear-receptor half-site elements that we refer to as the
CAN region. Another highly conserved region, located between -68/-44,
is a putative binding site for GATA-1 and SREBP. This region is
adjacent to the proposed cAMP-responsive region (-105/-68) identified
by 5'-deletion analysis and was deleted in the nonresponsive
3'-deletion construct (-254/-66StAR-pGL3luc), indicating that this
region also may contribute to the cAMP response.
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. The SF1–3 DNA probe (-105/-83) formed a specific protein-DNA
complex that was observed using nuclear proteins isolated from either
untreated (lane 1) or (Bu)2cAMP-treated (lane 5)
MA-10 cells (Fig. 3). The specificity of
complex formation was shown by competition with unlabeled SF1–3 probe
(lanes 2 and 6), and the complex was not formed when an oligomer that
contained two point mutations in the CATCCTTG core sequence (SF1–3mut)
was used in the binding reaction (lanes 4 and 8). A protein-DNA complex
of similar mobility was observed when the probe was incubated with
purified GST-SF-1 (lane 9), and addition of an antibody specific for
SF-1 greatly diminished or abolished DNA-protein interaction (lanes 3
and 7). Thus, these data verify that SF-1 also binds to this region of
the mouse promoter. As the mobility and intensity of the SF-1 complex
are the same with nuclear proteins from either control or
(Bu)2cAMP-stimulated cells, these data indicate
that SF-1 binding is independent of the cAMP stimulus in MA-10 cells.
This element is referred to as SF1–3 to indicate the third
SF-1-binding site within 150 bp of the mouse promoter (Fig. 2A).
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, A and B). Complex A is a
doublet that is present in binding reactions with nuclear proteins from
both control and (Bu)2cAMP-treated MA-10 cells,
whereas complex B is a slower migrating band that is present only with
nuclear proteins from stimulated cells (Fig. 4A). As this region
contains a CAAT box, and C/EBPß has been proposed or shown to
interact with this region (26, 38), we used a C/EBPß antibody to test
for the presence of C/EBPß in complex A or B. As shown in Fig. 4B, addition of the antibody did not affect protein-DNA interactions with
the CAN probe and nuclear proteins from stimulated cells (lane 2). We
verified that a protein-DNA complex is formed with a consensus C/EBP
oligonucleotide (lane 3) and that this complex is supershifted by
antibody (lane 4), confirming that C/EBPß is present in the nuclear
extract (25, 26). Cold competition with the consensus C/EBP
oligonucleotide also failed to indicate any involvement of C/EBPß
with this element (lane 5). The specificity of complex formation was
demonstrated by cold competition with the CAN probe (lane 8) and a
mutation in the putative nuclear receptor half-site (1/2mut) was also
effective as a competitor probe (lane 6). Mutation in the putative AP-1
site (AP-1mut), on the other hand, did not compete with the CAN probe
for protein binding (lane 7). We next tested for Fos/Jun binding to
this region, but addition of antibodies that recognize all family
members did not affect complex formation (data not shown).
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). Complex formation was abolished
by cold competition with the GATA oligonucleotide (lane 2) or the
SREBPmut oligonucleotide that contains a mutation in the putative SREBP
site (lane 4). Conversely, using the SREBPmut as a probe did not affect
complex formation (lane 3), indicating that the SREBP element is not
involved in protein-DNA interactions at this region. On the other hand,
no complex was formed with a probe that had a mutation within the GATA
DNA-binding site (lane 5), and the mutant GATA probe (GATA mut) did not
compete for protein binding (lane 6). A complex of similar mobility was
observed using a probe with the consensus sequence for GATA binding
(lanes 7 and 8) and cold competition experiments using this consensus
GATA probe resulted in the lack of complex formation (lane 10).
Although this sequence was identified as a potential GATA-1-binding
site, GATA-1 antibodies did not affect complex formation (compare lanes
1 and 9). However, binding to the consensus GATA probe appeared to be
diminished by GATA-1 antibodies (compare lanes 7 and 8). Therefore, we
tested for other members of the GATA family, and GATA-4 antibodies
resulted in a supershift of the complex (lanes 11–13). The
supershifted complex was apparent using nuclear extracts from either
control (lane 11) or (Bu)2cAMP-treated (lanes 12
and 13) MA-10 cells with the StAR GATA probe (lanes 11 and 12) or the
consensus GATA probe (lane 13). A less prominent band appears with the
-68/-44 GATA probe; however, the significance of this interaction is
not known. At present, these results indicate that GATA-4 in MA-10
cells binds to the mouse StAR promoter.
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). Mutation of the SF1–3 site resulted
in a 43% reduction in basal luciferase activity compared with the
-254 StAR promoter construct (WT), but the fold induction in response
to (Bu)2cAMP stimulation was not significantly
different. Mutation of the GATA-4-binding site also reduced basal
promoter strength (
36%); however, the cAMP response was also
decreased from 8.7-fold (WT) to 6.9-fold (GATA). Lastly, mutation of
the putative AP-1 site (AP-1mut) or the nuclear receptor half-site
(1/2mut) resulted in decreased basal activity by 63% or 57%,
respectively, but, again, fold activation in response to
(Bu)2cAMP was unchanged compared with that with
the -254 StAR promoter construct (WT). Although the fold induction by
(Bu)2cAMP-treatment was not diminished with the
SF1–3 and CAN region mutations, overall promoter strength was reduced.
This decrease in response appeared to closely parallel the severity of
the mutation on basal promoter activity. These data indicate that the
SF1–3, CAN, and GATA sites all contribute to basal promoter strength.
However, neither the SF1–3 nor the CAN region is individually required
for the cAMP-dependent response of the StAR promoter. On the other
hand, GATA-4 may account for 20–30% of the cAMP response of the
murine StAR promoter.
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). The overall basal
activity of StAR promoter was dramatically decreased (88%) compared
with that of the -254 StAR promoter construct (WT), indicating that
mutation of both SF1–3 and GATA had an additive effect on promoter
strength. Despite minimal basal activity, the promoter caused a
4.8-fold increase in the cAMP-dependent response. However, this
represents only 45% of the cAMP response for the -254 StAR promoter
construct. Thus, the double mutation has a greater effect than the
single GATA mutation on the cAMP response. Therefore, we examined the
possibility that either the SF1–3 or GATA-4 site acts functionally
with the AP-1 site to promote the cAMP response (Fig. 6B). Double
mutations in the SF-1 and AP-1 elements (SF1–3/AP-1) or the GATA-4 and
AP-1 (GATA/AP-1) elements did not result in any greater effect on StAR
promoter activity compared with an additive effect of the single
mutations alone. The basal activities of SF1–3mut/AP-1mut and
GATAmut/AP-1mut were 16% (AP-1 plus SF-1) and 23% (SF-1 plus GATA)
that of the -254 StAR promoter construct, respectively, whereas the
(Bu)2cAMP-dependent increases were 90% (SF1–3
effect) and 64% (GATA-4 effect) that of the -254 StAR. Thus, it
appears that mutation of the AP-1 element has the single greatest
effect on StAR promoter activity, due mainly to decreased basal
expression, whereas the SF1–3 and GATA-4 double mutations had the most
severe effect on promoter function. One possibility is that SF1–3 and
GATA-4 function to stabilize a common factor(s) in the CAN (-87/-64)
region. To test this proposal, we examined the effects of abolishing
one DNA-binding site on protein-DNA interactions at other sites within
the -105/-44 bp of StAR promoter.
Protein-DNA interactions within the cAMP-responsive regions of the
StAR promoter
Four major protein-DNA complexes (I–IV) were detected by EMSA
analysis using a DNA probe that spans the -105 to -44 region
(-105/-44) of the StAR promoter (Fig. 7A). Complex formation appeared to be
independent of cAMP treatment in that similar complexes were formed
with nuclear extracts from both control and
(Bu)2cAMP-treated MA-10 cells. Antibodies to
SF-1, GATA-4, and C/EBPß were used to help identify the presence of
these proteins in the four complexes (Fig. 7B). The SF-1 antibody
abolished complex I and diminished complex III (lane 2), whereas GATA-4
antibodies caused the appearance of a supershifted complex concomitant
with diminished complex II (lane 3). C/EBPß antibodies had no effect
on protein-DNA interactions within this region of the StAR promoter
(lane 4). Cold competition experiments with the oligonucleotides
corresponding to the SF1–3 (-105/-44), CAN (-87/-64), and GATA
(-68/-44) regions of the promoter were performed to verify the
contribution of each site to complex formation. First, cold competition
with unlabeled -105/-44 probe greatly diminished complex formation
(lane 5). The SF1–3 probe eliminated complex I and diminished
complexes III and IV without affecting complex II formation (lane 6),
whereas the GATA probe competed for complex II formation without
affecting complex I, III, or IV (lane 9). The CAN probe eliminated
complex IV (lane 7), whereas the consensus C/EBPß oligonucleotide
(lane 8) had no effect on the protein-DNA interactions with the StAR
promoter.
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).
Complex I was abolished, and complexes III and IV were diminished by
mutation of the SF1–3 site (SF1–3mut, lane 2), which is consistent
with the results of the SF-1 antibody and cold competition experiments.
Complex II was not affected by the SF1–3 mutation, and addition of the
GATA-4 antibody caused a supershift of the remaining complex II (lane
3). Conversely, mutation of the GATA-4 site eliminated complex II
formation (GATAmut, lane 4), and the remaining complexes (I, III, and
IV) were greatly diminished by SF-1 antibodies (lane 5). The CAN probe
was an effective competitor for complexes III and IV that remained with
the GATAmut probe (lane 6) probe, but did not further reduce complex
formation with the AP-1mut probe (lane 7), suggesting that factors
binding to the CAN region contribute to formation of these complexes.
Indeed, mutation of the AP-1-like site eliminated complex IV and
surprisingly also reduced all other protein-DNA interactions (lane 10).
The remaining complexes I and III were eliminated by the addition of
SF-1 antibodies (lane 9), whereas GATA-4 antibodies resulted in a
supershift of complex II (lanes 8).
As StAR basal promoter activity was greatly reduced by mutations within
two of the three SF1–3, CAN, or GATA elements (Fig. 6B), we also
tested the effects of these mutations on protein-DNA interactions (Fig. 7D). A double mutation in SF-1 and GATA (SF1–3/GATA mut) resulted in
the loss of all complex formation (Fig. 7D, lane 2), whereas complex II
was still present with the probe containing a double mutation in SF1–3
and AP-1 (SF1–3/AP-1 mut, lane 3). Double mutations of GATA and AP-1
caused a marked decrease in protein-DNA interactions for complexes III,
and IV and eliminated complex II (lane 4). These data are consistent
with an additive effect of the single mutations on protein-DNA
interactions, with the exception that complex I did not appear affected
by the GATAmut/AP-1mut probe. This was not an expected result based on
the decrease in complex I with the AP-1 mut probe (Fig. 7C, compare
lanes 10 and 11). These results indicate that complexes III and IV are
dependent upon SF-1 and possibly, to a lesser extent, GATA-4.
In sum, the individual mutations in the SF1–3 or AP-1-like regions and the SF-1 antibodies do not affect GATA-4 binding; therefore, we conclude that GATA-4 binding within the -105/-44 region is independent of other factors. Further, the GATA-4 mutation does not prevent SF-1-dependent complex formation (I, III, and IV), indicating that SF-1 binding is not dependent on GATA-4 binding. CAN factors appear to contribute to complex IV formation, as mutation of the AP-1-like element or cold competition with the CAN region eliminated this complex. However, protein binding within the CAN region in the context of the longer probe appears to be dependent on SF-1 binding to SF1–3 and may be influenced by GATA-4 binding. Conversely, an intact AP-1 element appears to be required for efficient SF-1 binding, indicating that protein binding to SF1–3 and CAN regions may be interdependent. Together, these data clearly demonstrate that complex I is due to SF-1 binding, and complex II is due to GATA-4 binding. The data are consistent with complexes III and IV being dependent upon SF-1, with complex III most likely containing SF-1, whereas CAN factor binding contributes to complex IV.
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Discussion |
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Identification of the -105 to -95 region as a SF-1-binding site was an expected result, as this element was previously shown to bind SF-1 in the human and rat StAR promoters (21, 37). However, these are the first studies to show a distinction between regulations of the human and mouse StAR promoter by cAMP in steroidogenic cell lines. Mutation of the SF-1 site (-95) in the human promoter demonstrated 90% and 80% decreases in basal and forskolin-stimulated promoter activity in human granulosa-lutein cells (37). Our recent studies in the human H295R adrenocortical cells also have shown that the SF-1 element at -95 is required for cAMP and angiotensin II induction of human StAR promoter-luciferase reporter gene expression (23). Transient transfection of COS-1 cells with the rat StAR promoter confirmed a SF-1-dependent activation of the StAR promoter that required the SF-1 site at -95 and that mutation of this site resulted in a decrease in the cAMP response that was due to lowered basal promoter activity (21). Our results demonstrate that the mouse and rat promoters are functionally more similar to each other than to the human StAR promoter. This apparent promoter-specific difference may be reflective of a divergence within the StAR promoter sequences between the human and the mouse. The human sequence has an additional five bases downstream of the SF-1 site and does not contain the CAAT box, the the AP-1-like site, or the nuclear receptor half-site that have been shown to be important for the murine promoter in MA-10 cells (26, 38). Thus, it is apparent that SF-1 is important in StAR gene regulation but is not sufficient for the cAMP-dependent response in MA-10 cells. Possibly, the sequence divergence reflects greater interactions of SF-1 with other factors binding in this region of the mouse StAR promoter.
We have confirmed the functional importance of the AP-1-like element within the -87/-68 (CAN) region of mouse StAR promoter for basal transcriptional activity (26, 38). In addition, this study provides the first evidence that (Bu)2cAMP treatment of MA-10 cells results in the formation of a unique protein-DNA complex at this region of the StAR promoter (complex B). Identification of the CAN factor(s) should provide an important link to elucidating the mechanism of action for cAMP-dependent regulation of StAR. Presently, our data suggest that multiple elements may contribute to activation of the StAR promoter. Double mutations in either the SF1–3 and AP-1 or GATA and AP-1 elements have a greater effect on basal promoter activity that represents the combined effects of the single mutations. The GATAmut/AP-1 mut also resulted in decreased cAMP response that is attributed to the GATA element. On the other hand, the effect of the SF1–3 and GATA double mutation on basal promoter activity was greater than the combined effects of the single mutations. As mutations that disrupted the AP-1-like site (AP-1mut, SF1–3mut/AP-1mut, and GATAmut/AP-1mut) have similar effects on basal activity as the SF1–3mut/GATAmut, these data suggest that all three elements work together to promote full basal activity of the StAR promoter that, in turn, is required for a maximal cAMP response. What is not clear at the time is why the cAMP-dependent response, although reduced, remains intact despite apparent elimination of protein-DNA binding interactions within the -105/-44 region of StAR promoter with the SF1–3mut/GATAmut. As we did not observe a cAMP-dependent complex with the -105/-44 probe, the cAMP-dependent CAN-binding factor(s) may not have been detected in this analysis. Indeed, the cAMP-dependent complex was observed only with increased concentrations of nuclear extract, suggesting that this factor(s) may be expressed in lesser abundance. Thus, it is possible that the AP-1 region is still functional in the SF1–3mut/GATAmut double mutation.
Previously, we demonstrated that a protein(s) in MA-10 nuclear extracts binds to the CAAT box located in the -93/-71 region, but with lower affinity compared with the C/EBPß site located at -113 (26). Antibody supershift EMSA experiments showed that C/EBPß binds to the -113 site, but binding to the proximal site was not tested. We now have directly tested C/EBPß binding to the CAN region and verify that C/EBPß does not bind to this region of the StAR promoter. It is possible that our previous studies did not detect the factor(s) we now observe because the probe used previously (-93/-71) did not contain the entire CAN region (-87/-64) binding sites, which may have affected protein binding to this region. Indeed, complexes A and B appear to be dependent upon an intact AP-1-like element and the flanking TGATGA sequence. Functionally, mutation of the putative AP-1 site resulted in approximately a 63% decrease in the basal promoter activity of StAR without affecting the cAMP response, a result very similar to the effects seen by mutating the CAAT box at -87 (26). Therefore, mutation of the CAAT box most likely affects a factor(s) binding to the AP-1-like element in the CAN region or vice versa.
Our results in MA-10 cells are distinct from the recent report of murine StAR promoter activity in rat ovarian cells, which demonstrated that C/EBPß is a prominent component in a DNA-protein complex formed with the -87/-64 region (38). Interestingly, the binding site recognized by C/EBPß in ovarian cell extracts is not the CAAT box core, but is the AP-1-like element. One possibility for this apparent tissue-specific difference in C/EBPß binding is that different factors bind to the CAN region. This tissue-specific difference may reflect the cycloheximide sensitivity of the cAMP response for StAR. Previously, StAR gene expression was reported to be mediated by a cycloheximide-sensitive mechanism in human granulosa-lutein cells (40). C/EBPß expression has been shown to be increased by FSH in ovarian granulosa cells and by (Bu)2cAMP in MA-10 cells (25, 38). Subsequent to submission of this paper, it was reported that C/EBPß is induced in human granulosa cells by 8-bromo-cAMP (41). The data supported C/EBPß in StAR basal promoter activity as well as the proposal that the cycloheximide-sensitive, cAMP-dependent response may be linked to the induction of C/EBPß. Thus, it is probable that a SF-1/C/EBPß or a GATA-4/C/EBPß interaction mediates the observed cycloheximide-sensitive response in rat ovarian granulosa cells. Consistent with this proposal, C/EBPß and SF-1 were shown to interact directly in a GST pull-down assay (26). However, StAR gene expression in MA-10 cells and Y1 mouse adrenocortical cells is not dependent upon de novo protein synthesis (10). Therefore, another factor(s) that is insensitive to cycloheximide may be used in MA-10 Leydig cells and Y1 cells to bind to the CAN region and promote maximal basal activation of StAR. A difference in tissue-specific transcription factor expression may thus account for differences in the acute regulation of StAR in granulosa vs. Leydig cell cultures.
We identified GATA-4 as the factor that binds to the highly conserved -68/-44 region of the mouse StAR promoter. Functionally, GATA-4 contributes to 20% of the cAMP response. Independently, GATA-4 was recently reported to bind to the same region and was shown to be required for maximal cAMP response in rat granulosa cells (38). Mutation of both the GATA-4 and AP-1-like elements decreased StAR basal promoter activity 97% and dramatically decreased the FSH-dependent increase in reporter gene expression in luteal-granulosa cells. In contrast, we demonstrate that a similar double mutation (GATAmut/AP1mut) had no greater effect on StAR promoter activity in MA-10 cells than the combined effects of the individual mutations within these elements. Again, this discrepancy between our results and those reported for rat granulosa cells indicates that the murine StAR gene may be regulated in a tissue-specific manner. In MA-10 cells, we propose the GATA-4 transcription factor has a role in stabilizing the factor(s) bound to the putative AP-1 site and that GATA-4 and SF-1 work together to stabilize this common factor(s). However, these putative CAN-binding proteins and SF-1 can function in the absence of GATA-4.
In sum, SF-1 binding is important for the basal activity of the StAR gene, most likely by affecting the interactions among the other proteins within the CAN region of the promoter. GATA-4 binding is independent of SF-1 binding and has an apparently minimal effect on SF-1- and CAN-protein binding, but may influence factor binding. Interaction between SF-1 and GATA-4 has been shown for regulation of the Müllerian-inhibiting substance gene; therefore, one model for SF-1 and GATA-4 is that they interact and function to stabilize binding of the CAN-binding factor(s) (42). Alternatively, it is possible that there is a redundancy of function for SF-1, CAN-binding protein, and GATA-4. This proposal would be consistent with our mutational analysis that shows that elimination of one binding factor does not compromise the cAMP response of the StAR promoter. Thus, each of the DNA-binding factors may work together to stabilize the unknown cAMP-mediated transcription factors and/or coactivators, but each factor alone may be sufficient to mediate part of the response.
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Received August 9, 1999.
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158-subunit
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