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Department of Obstetrics and Gynecology and Molecular Biology and Biochemistry University of South Florida, College of Medicine, Tampa, Florida 33606
Address all correspondence and requests for reprints to: Dr. Mark P. McLean, University of South Florida, Department of Obstetrics and Gynecology, 4 Columbia Drive, Suite 529, Tampa, Florida 33606. E-mail: mmclean{at}com1.med.usf.edu
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Introduction |
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Transcriptional regulation by cholesterol has been reported for numerous genes (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In particular, cell-specific regulation of the steroidogenic factor-1 (SF-1)-dependent human StAR promoter by certain oxysterols was shown to increase StAR protein levels in granulosa and theca interna cells without changing StAR messenger RNA (mRNA) levels, possibly through up-regulation of StAR protein translation or a reduction in protein degradation (7). However, earlier studies from this laboratory (9, 10) found that ovarian tissue from rats treated with tropic hormones had decreased levels of cholesterol, with a subsequent increase in mature sterol regulatory element (SRE)-binding protein (SREBP)-1a and StAR mRNA. Sterol-sensitive genes like StAR contain at least one SRE; and SREBP-1a, -1c, and -2 have been shown to bind to SREs and activate transcription. SREBPs are synthesized as 125-kDa precursors that are attached to the endoplasmic reticulum membrane and nuclear envelope (20, 21, 22, 23). In sterol-depleted cells, a two-step proteolytic process cleaves the 65-kDa NH2-terminal segment of the SREBP, which is then free to enter the nucleus, bind the SRE, and activate transcription of the target gene (20, 21, 22, 23). The importance of SREBP in activating transcription of several SRE-containing genes has been clearly demonstrated in experiments using transgenic mice overexpressing the NH2-terminal segment of SREBP-1a (24). In these mice, sterol-sensitive genes were increased several fold over control levels. SREBPs are known to be weak transcriptional activators and require the presence of ubiquitous transcription coactivators, like Sp1 and nuclear factor-Y (NF-Y), to elicit maximum activation. Sp1 was first identified as the transcription factor required for expression of the simian virus (SV40) early promoter (25). Cooperation between SREBP-1a and Sp1 was found to enhance sterol regulation of the low-density lipoprotein receptor (LDL-R) gene (26). NF-Y binds as a heterotrimer (NF-YA/NF-YB/NF-YC) to CCAAT or an inverted CCAAT box (ATTGG) (27). The combined actions of NF-Y and SREBP-1a resulted in enhanced binding and synergistic activation of the rat farnesyl diphosphate synthase gene (28). Recently, sterol regulation of the human fatty acid synthase promoter I was found to be dependent on both NF-Y and Sp1 (29).
In an earlier study from this laboratory (30), the orphan nuclear receptor SF-1 was shown to mediate cAMP-dependent responsiveness of the rat StAR promoter in rat luteal cells. Furthermore, a separate study by Lopez et al. (10) found that SF-1 and SREBP-1 were able to synergistically activate the high-density lipoprotein receptor (HDL-R) gene, demonstrating the combined actions of two regulatory pathways to provide cholesterol as substrate for steroidogenesis. Tropic hormones stimulate the biosynthesis of steroid hormones via a cAMP-dependent second-messenger system. Steroid hormone synthesis could then result in the depletion of intracellular cholesterol, which triggers the release of mature SREBP and the subsequent activation of genes involved in the synthesis and transport of cholesterol.
In the present study, cooperation between SREBP-1a and NF-Y, SF-1, or Sp1 in regulation of the rat StAR promoter is examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T vector, along with full-length
SF-1cDNA in the pCMV expression plasmid. The rat LDL-R promoter was
prepared by PCR from a genomic library and linked to the pGL3 basic
reporter construct. The rat HDL-R promoter was linked to the pGL3 basic
reporter construct as described elsewhere (10). The
plasmid pPacSp1 that contains the Drosophila actin 5C promoter upstream
of the coding sequence for human Sp1 was a gift from Al Courey
(Department of Chemistry & Biochemistry, University of
California at Los Angeles, Los Angeles, CA). Additional pAC5.1 vectors,
used in transfection studies, and Drosophila SL2 cells were obtained as
part of the Drosophila Expression System from Invitrogen
(Carlsbad, CA). NF-Y A, B, and C in pCITE were obtained from Dr. Sankar
Maity (M. D. Anderson Cancer Center, Houston, TX). After
introduction of an EcoRI site into NF-Y B and C, all three
complementary DNAs were transferred into pcDNA 3.1 using
EcoRI and XhoI. The QuickChange Site-Directed
Mutagenesis Kit was purchased from Stratagene (La Jolla,
CA). [
32P]-deoxycytidine 5'-triphosphate
(3000 Ci/mmol) and the T7 Sequenase DNA Sequencing Kit were purchased
from Amersham Pharmacia Biotech (Arlington Heights, IL).
[35S] deoxy-ATP (1000–1500 Ci/mmol) was
obtained from DuPont/NEN Life Science Products
(Wilmington, DE). [(Bu)2cAMP] was purchased
from Sigma (St. Louis, MO). All restriction enzymes,
Fugene 6 Transfection reagent, and the Calpain inhibitor I (ALLN:
N-acetyl-leucyl-leucyl-norleucinal) were obtained
from Roche Molecular Biochemicals (Indianapolis, IN).
DMEM: nutrient mixture F-12 was obtained from Life Technologies, Inc./BRL (Grand Island, NY). FBS was purchased from Summit Biotechnology (Ft. Collins, CO). Poly dI-dC and the Sephaglas
DNA Purification Kit were obtained from Pharmacia Biotech
(Piscataway, NJ). Dr. Gene C. Ness (Department of Biochemistry and
Molecular Biology, University of South Florida) kindly provided
Rivastatin from Merck & Co., Inc. (Rahway, NJ). Biomax-MR
films were obtained from Fisher Scientific (Norcross, GA).
All other chemicals were purchased from Fisher Scientific
or Sigma.
Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange
Site-Directed Mutagenesis Kit (Stratagene) according to
the manufacturer’s protocol. Briefly, 10 ng plasmid was incubated with
125 ng of the appropriate complementary oligonucleotides (see below)
and 1 µl deoxynucleotide triphosphates in 50 µl reaction buffer
(100 mM KCl, 100 mM (NH
4)2SO4,
200 mM Tris-HCl, pH8.8, 20 mM
MgSO4, 1% Triton X-100, and 1 µg/ml
nuclease-free BSA). One microliter of Pfu DNA polymerase
(2.5U/ul) was added to the reaction, and each reaction was heated to 95
C for 30 sec, followed by 35 cycles of denaturation at 95 C for 30 sec,
annealing at 55 C for 1 min, and extension at 68 C for 12 min. After
the cycling reaction, samples were subjected to digestion with
DpnI for 1 h at 37 C to remove the parental DNA
template. The mutations were confirmed by sequencing using the T7
Sequenase DNA Sequencing Kit and
[35S]-deoxy-ATP. Complementary oligonucleotides
(with mutations underlined) used to mutate each E-box were
as follows: E-box1
(5'-CTTTTTTATCTCCCGCGATGATGCAC-3'), E-box2
(5'-CATTTAAGGCAGAGCCCCCGCTTTGAGCC-3'), E-box3
(5'-GCTTTGAGCCCG- CCGCAGGACTCAG-3'), and E-box4
(5'-CAGCAGCAGAAATTT-
CAGCAGTAC-3'), respectively. The primer
(5'-CAGTTACTGGGTACCTAAGTGAATG-3') and its
complement were used to introduce a KpnI restriction site in
the StAR promoter in the preparation of the p-291 construct. The
complementary oligonucleotide used to mutate the Sp1 site was
(5'-TTTGGTTCTTTAGCTCTGG-3'). SREBP-1a mutations
were prepared as described previously (30).
Cell transfection
Mammalian cells were transfected with the specified StAR
promoter gene construct, either in the presence or absence of SREBP-1a
(or -1c) -pCMV5, SREBP-2 -pcDNA 3.1, Sp1-pCMV5 or NF-Y (-A, -B, and -C)
-pcDNA 3.1 using the Fugene 6 method according to the manufacturer’s
instructions. Insect cells were transfected with the same
promoter constructs but used pAC expression vectors containing
SREBP-1a, Sp1, or Renilla. Cells were first plated in 6-well
tissue-culture plates at a density of 5 x
105 cells per well (6-well plate) and incubated
for 24 h at 37 C (5% CO2). The media was
changed, and 2 µg of each DNA (and 1 µg Renilla) and Fugene 6 was
added to the culture plates, and the cells were allowed to incubate for
48 h. Cotransfection of the Renilla luciferase gene under control
of the SV40 early enhancer/promoter region was used as a control to
correct for differences in transfection efficiencies. Then, 50
µM of Rivastatin (Riv), 50 µg/ml of ALLN,
(Bu)2cAMP (1 mM), or PBS as a vehicle
control were added to the appropriate plates, 24 h before the cell
harvest, as indicated. Cells were washed twice with PBS and treated
with a passive lysis buffer for 20 min. Lysates were transferred to a
microcentrifuge tube and store at -80 C until the determination of
luciferase activity.
Luciferase assays
Luciferase assays were performed using the Dual Luciferase
Reporter Assay System according to the manufacturer’s instructions
(Promega Corp.). Briefly, 100 µl luciferase
substrate was added to 20 µl lysate, and luciferase activity was
measured using a 20/20 luminometer (Turner Designs, Sunnyvale, CA).
Luciferase data were expressed as the mean ± SEM.
Each luciferase assay experiment was performed in triplicate and
repeated for the number of times indicated in the figure legends. Data
from the individual parameters were compared by ANOVA followed by
Student’s-Newman-Keuls multiple-comparison test where applicable
(31). P < 0.05 was considered significant
for all tests.
Preparation of NF-Y or Sp1 nuclear extracts (NEs)
HTB-9 cells (5 x 107) were
transfected with 50 µg NF-Y (A, B, and C) in pcDNA 3.1 plasmids, or
Drosophila SL2 cells (5 x 107) were
transfected with 50 µg Sp1 in the pPacSp1 plasmid, using Fugene 6,
for 48 h. SL2 cells were collected by centrifugation, and the
HTB-9 cells were treated with 0.25% trypsin in 1 mM EDTA;
and then, after centrifugation, NEs were prepared from the cell pellets
as described previously (10).
Recombinant protein production
Glutathione S-transferase-Sp1 fusion proteins or
histidine-tagged SREBP-1a proteins were overexpressed in
Escherichia coli by induction of midlogarithmic-phase
cultures with 1 mM
isopropyl-ß-D-thiogalactopyranoside. After
incubating for 6 h at 27 C, cells were sedimented by centrifuging
at 7,700 x g for 10 min at 4 C. For GST-protein
purification, the cell pellet was resuspended in PBS and lysed by
sonication, Triton X-100 was added to a final concentration of 1%, and
the sample was incubated for 30 min at 4 C. The suspension was
centrifuged at 12,000 x g for 10 min at 4 C. Affinity
purification of the fusion protein was performed using the GST-fusion
Purification Kit (Pharmacia Biotech) as per the
manufacturer’s recommendations. Briefly, cleared lysates were passed
through the glutathione Sepharose 4B column. After washing with PBS,
the fusion protein was digested with precision protease to remove the
GST moiety and was used in gel mobility shift assays. For
histidine-tagged SREBP-1a protein purification, the cell pellet after
the initial centrifugation was resuspended in guanidium lysis buffer
provided in the XPRESS System (Invitrogen) and eventually
eluted off an immobilized metal affinity column according to the
manufacturer’s recommendations. Recombinant protein samples were
concentrated approximately 4- to 6-fold using Centricon-10
concentrators (Millipore Corp., Bedford, MA). Protein
concentrations were determined using the Bio-Rad Laboratories, Inc. protein assay (Bio-Rad Laboratories, Inc.,
Hercules, CA).
Electrophoretic mobility shift assay
Double-stranded oligonucleotides corresponding to SRE binding
sites in the rat StAR promoter were synthesized and annealed in 10
mM Tris-HCl (pH 7.5), 1 mM EDTA, 25
mM NaCl, 10 mM MgCl2 and
1 mM dithiothreitol. The oligonucleotide probes were
labeled using Klenow (Promega Corp.) and
[32P] ATP (NEN Life Science Products, Boston, MA). Unlabeled oligonucleotides (50-fold
excess) were used as competitors in some experiments, and antibodies
against SREBP-1a and Sp1 (Santa Cruz Biotechnologics Inc., Santa Cruz, CA) or NF-Y A and B (Chemicon
International, Temecula, CA) were used for supershift analysis. A total
of 200–500 ng histidine-tagged recombinant SREBP-1 fusion proteins
(rSREBP-1) were incubated in the presence or absence of
competitor, recombinant Sp1, recombinant SF-1 (rSF-1), or NE prepared
from HTB-9 cells overexpressing NF-Y (ABC) or Drosophila SL2 cells
overexpressing Sp1, for 20 min at 10 C, in binding buffer. The
[32P] ATP-labeled probe was added, and the
incubation was continued for an additional 20 min. The DNA/protein
complexes were resolved on a 5% nondenaturing acrylamide gel in TGE
buffer (0.25 M Tris, 1.9 M glycine, 10
mM EDTA final), which was subsequently dried and exposed to
BioMax-MR films at -80 C for 4–12 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Rivastatin is a cholesterol biosynthesis inhibitor that causes a
decrease in intracellular cholesterol levels, with a corresponding
increase in levels of mature SREBP-1a, resulting in an induction of
SREBP-regulated genes. The increase in endogenous SREBP-1a resulted in
a significant increase in luciferase activity under the control of the
StAR promoter. A similar increase in StAR promoter-driven activity was
seen in Fig. 1B when the p-1862 StAR construct was cotransfected with
the SREBP-1a -pCMV expression plasmid in the presence or absence of
ALLN, a Calpain inhibitor which prevents SREBP-1a degradation, thereby
increasing the accumulation of SREBP-1a protein and prolonging
activation of SREBP-regulated genes (21). These latter
studies were done in HTB-9 cells, which lack (or have undetectable
levels of) StAR and SREBP-1a.
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), SRE 2, 3, and 5 had the highest
affinity for rSREBP-1a.
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). Unexpectedly, elimination of all five
SRE (p-150) did not significantly decrease the levels of basal or
SREBP-1a-induced activation. In addition, as illustrated in the table
included in Fig. 2B
, there was no major difference in the fold
induction between any of the constructs. The p-150 StAR promoter
construct does contain a sequence from -85 to -76
(5'-AGTGACTGATGG-3'), which displayed 83% homology to a putative
SREBP/Yin Yang 1 binding site identified in the human StAR promoter
(34). However, although an oligonucleotide containing this
sequence did show low levels of binding with recombinant Yin Yang 1,
rSREBP-1a did not bind with high affinity to this site (data not
shown). These results suggested that SREBP-1a could be working through
one or more of the multiple E-boxes in the StAR promoter. Specific
binding of rSREBP-1a to an E-box was found (Fig. 4A), although the binding affinity was
several fold less than binding observed between SREBP-1a and SRE no. 3.
To identify which E-box was important in SREBP-1a induction of the StAR
promoter, site-directed mutagenesis was used to introduce a mutation in
each of the E-boxes in the p-545 StAR promoter construct (unpublished
observations). Only one E-box mutation was able to affect
SREBP-1a-induced activation, and this specific E-box mutation (denoted
by an arrow) in the mp-150, mp-291, and mp-545 StAR
constructs is shown in Fig. 4, B and C. When there was no consensus SRE
present in the promoter construct (mp-150), the mutation in the E-box
resulted in the complete loss of SREBP-1a induction (Fig. 4B). This is
in contrast with the results seen with the mutated StAR constructs
(mp-545 and mp-291) still possessing at least one SRE (Fig. 4C).
Mutation of the E-box under these conditions resulted in an increase in
SREBP-1a induced activation of the StAR promoter over basal levels. To
further examine the role of E-boxes in SREBP-1a-induced activation of
the StAR promoter, two SREBP-1a mutants were prepared by site-
directed mutagenesis, according to information obtained from an
earlier study (31). The SREBP-1a-Y320R mutant (which can
bind to an E-box but not an SRE) and the SREBP-1a-Y320A mutant (which
has lost all DNA binding ability) both failed to activate the rat StAR
promoter, suggesting that the E-box does not play a major role in
transcription activation of the StAR promoter in the presence of a
functional SRE.
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). The LDL-R
promoter was used as a positive control in these studies.
Overexpression of SREBP-1a, SREBP-1c, and SREBP-2 were all effective at
increasing StAR promoter-driven luciferase activity in HTB-9 cells,
although SREBP-1a and–1c were consistently more effective than
SREBP-2.
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), NF-Y in
HTB-9 cells (Fig. 7), and SF-1 in HTB-9
cells (Fig. 8) were investigated as
potential enhancers of SREBP-1a binding and possible coactivators of
StAR promoter-driven luciferase activity. Figure 6A is a schematic of
the rat p-1862 StAR promoter, showing the relative positions of the SRE
and Sp1 binding sites. The EMSA depicted in Fig. 6B shows a
dose-dependent increase in SREBP-1a binding to SRE no. 3 with
increasing amounts of Sp1. Maximal enhancement occurred with 2–3 µg
of Sp1 resulting in a 20- to 21-fold enhancement, respectively. There
was no enhancement of SREBP-1a binding with the addition of an equal
amount of GST or BSA and only a minor enhancement attributable to the
addition of NE from SL2 cells not overexpressing Sp1 (3.8-fold). Sp1
antibody was able to cause a supershift of the complex (Fig. 6B, right arrow), suggesting that Sp1 is a component of the
SREBP-1a/SRE complex. The Sp1 enhancement of SREBP-1a binding did not
correspond to an increase in StAR promoter-driven luciferase activity
in the presence of Sp1 and SREBP-1a (Fig. 6C). By contrast, the
combined action of Sp1 and SREBP-1a was able to cause synergistic
activation of the HDL-R promoter, which was included as a positive
control (Fig. 6C). The use of recombinant Sp1, instead of nuclear
extracted proteins, also enhanced SREBP-1a binding to SRE no. 3 2-fold,
even when 500 ng rSREBP-1a protein (optimal concentration) was used in
the binding reactions (data not shown). The EMSA data suggested that
Sp1 could interact with SREBP-1a in solution and somehow encourage or
stabilize SREBP-1a/SRE binding. The lack of a detectable supershift in
the presence of SREBP-1a and Sp1 (in the absence of any antibody) may
be the result of a loose association of factors like Sp1 with
SREBP-1a, which ultimately dissociates under the harsh electrophoresis
running conditions. Site-directed mutation of the Sp1 site resulted
in a reduction (32.7%) in SREBP-1a-induced activation of the p-1413
StAR promoter construct in HTB-9 cells (Fig. 6D). However, the same
mutation resulted in a significant reduction (43%; P
< 0.001) in SREBP-1a-induced activation of the p-545 StAR promoter
construct that lacks SREs 3–5 (Fig. 6D). This experiment was repeated
in Drosophila SL2 insect cells with exogenously added Sp1, with similar
results (data not shown).
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is a schematic of the rat p-1862 StAR promoter, showing the
relative positions of the SRE and NF-Y binding sites. Addition of NE
from HTB-9 cells overexpressing NF-Y (A, B, and C) in the EMSA-binding
reactions (Fig. 7B) was able to enhance SREBP-1a binding to SRE no. 3
(up to 3.7-fold), and the combined actions of NF-Y and SREBP-1a
resulted in a significant (4-fold; P < 0.001) increase
in the amount of StAR promoter-driven luciferase activity (Fig. 7C).
Addition of a mixture of NF-Y A and B antibodies to the binding
reaction resulted in a decrease in the major SREBP-1a/SRE complex,
suggesting the presence of NF-Y in this complex (Fig. 7B, right
arrow).
To determine whether a transcription factor that is involved in the
cAMP-dependent second-messenger system could influence SREBP-1a binding
or SREBP-1a-induced activation of the StAR promoter, EMSA and
luciferase assays in the presence of SF-1 were performed (Fig 8).
Figure 8A is a schematic of the rat p-1862 StAR promoter, depicting the
relative positions of the SRE and SF-1 binding sites. Addition of rSF-1
protein (DNA-binding domain only) enhanced SREBP-1a binding (up to
2-fold) to SRE no. 3 (Fig. 8B), and the combined actions of SREBP-1a
and SF-1 resulted in an increase in luciferase activity under control
of the p-1862 StAR promoter construct under both basal (1.6-fold) and
SREBP-1a induced (3.5-fold) conditions (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normally, release of mature SREBP protein occurs under conditions of sterol depletion; but when the mature form of SREBP is overexpressed in cultured cells using an SREBP-1a expression plasmid, the StAR and HDL-R promoter (current study) and the LDL-R promoter (36) are activated, even in the presence of sufficient amounts of cholesterol. Up-regulation of endogenous SREBP-1a, overexpression of SREBP-1a protein, or inhibition of SREBP-1a degradation all resulted in an increase in the rat StAR promoter-driven luciferase activity. Although SREBP-1a demonstrated specific binding to all five SRE’s in the rat StAR promoter, results with the deletion constructs demonstrated that activation of the rat StAR promoter could occur in the absence of any known SRE. This confirms a previous study demonstrating SREBP activation of the LDL-R promoter through an E-box, although sterol responsiveness was lost under these conditions (37). Although SREBPs were shown to bind to an E-box and cause activation of the LDL-R promoter through an E-box in this artificial system, sterol regulation of promoter action was dependent on an SRE and not an E-box. This type of complex control mechanism prevents a loss of specificity in sterol responsiveness, which would occur if other nonsterol- sensitive helix-loop-helix proteins had the potential to activate genes involved in cholesterol homeostasis. Our transfection studies, using mutant SREBP-1a proteins defined in an earlier study by Kim et al. (31), confirm the minor role of the E-box in SREBP-1a-induced activation. The SREBP family of proteins is composed of two isoforms of SREBP-1 (1a and 1c, which mainly regulate genes involved with fatty acid biosynthesis) and SREBP-2 (which is more specific for genes involved in the cholesterol biosynthetic pathway) (20, 21, 22, 23). In the current study, all the SREBP isoforms used were able to activate rat StAR promoter-driven luciferase activity. This is in contrast to results of a recent study using the human StAR promoter, where SREBP-1a was able to increase StAR reporter activity but SREBP-2 had no effect (7). The apparent contradictory results could be attributable to differences in the two promoters (rat vs. human), differences in endogenous factors in the host cells, or differences in the absolute amounts of DNA transfected.
SREBP proteins require the presence of coactivators to achieve maximal activation. The exact nature of the cooperative mechanism of SREBP-1a and coactivators has been the subject of numerous studies over the past few years. Sterol-regulated activation of the farnesyldiphosphate synthase gene was shown to be dependent on an NF-Y binding site and an SRE (27). SREBP stimulates the binding of Sp1 to its recognition site in the LDL-R promoter and subsequently enhances transcription after both proteins are bound to DNA (28). Protein-protein interaction between the trimeric NF-Y protein and the basic helix-loop-helix/leucine zipper of SREBP in the absence of DNA has been demonstrated in vitro (20). In the present study, Drosophila SL2 cells that lack endogenous Sp1 were used to determine whether these cofactors could synergistically activate transcription of the rat StAR promoter. The combined actions of Sp1 and SREBP-1a did not synergistically activate the rat StAR promoter, suggesting that an additional cofactor like NF-Y or SF-1 may be required. Sp1 was able to enhance SREBP-1a binding to an SRE in the rat StAR promoter, demonstrating that enhancement of binding and the ability to act as a coactivator of SREBP-1a-induced transcription are two distinct regulatory events. Sp1 and SREBP-1a did synergistically activate the HDL-R promoter under similar conditions, and Sp1 was able to enhance rSREBP-1a binding to an SRE in the rat HDL-R promoter (38), demonstrating the promoter specificity of this interaction. In the latter studies, Sp1 was able to increase SREBP-1a binding to the HDL-R SRE and to cause a supershift of the SREBP-1a/SRE complex, suggesting a different type of binding complex that may account for the HDL-R promoter-specific effects. Although the combined actions of SREBP-1a and NF-Y were able to synergistically activate the rat StAR gene when tested in HTB-9 cells, these cells contain endogenous Sp1; and even though Sp1 and SREBP-1a did not result in StAR activation, it is possible that the combination of all three transcription factors enhanced activation of the rat StAR gene. Whether Sp1 plays a ro1e in NF-Y/SREBP-1a synergistic activation of StAR still needs to be determined. EMSA results suggest that Sp1 enhances SREBP-1a/SRE binding through protein/protein interactions, because there is no Sp1 binding site in the oligonucleotide containing SRE no. 3, and the possibility exists that Sp1 can only act as a coactivator of the rat StAR gene in the presence of multiple additional cofactors like SREBP-1a, SF-1, and NF-Y. Protein/protein interactions of NF-Y-A with Sp1 have been previously demonstrated using the yeast two-hybrid system and GST pull-down assays (39). The differential efficacy of Sp1 in SREBP-1a-induced activation could also be a result of the availability of distinct transcriptional regulatory sites in specific promoters, which could bring Sp1 protein in contact with an interacting protein. In the rat p-1413 StAR promoter (with three consensus NF-Y sites and one Sp1 site), SREBP-1a could use NF-Y primarily and only use Sp1 secondarily, which could account for the small reduction in promoter activity after mutation of the Sp1 site. Mutation of the Sp1 site in a StAR promoter construct lacking all consensus NF-Y sites (p-545) resulted in a large decrease in SREBP-1a- induced activation, suggesting that Sp1 assumes a more indispensable role in this promoter fragment. The rat StAR promoter does contain a GTTGG site at -80, which specifically binds NF-Y (data not shown), and studies to determine the role of this site on StAR activation are currently underway. Overall, cofactor regulation of the rat StAR promoter seems different from that of the rat HDL-R promoter that has multiple Sp1 sites and a single NF-Y site, given that Sp1, not NF-Y, acts as the major cofactor necessary for SREBP-1a induction.
The current results with the rat StAR promoter confirm similar findings with the human FAS promoter I (29) using wild-type or 5'deletion constructs to demonstrate that Sp1 recruits SREBP to the SRE and that NF-Y is required to activate the cholesterol response. However, previous studies with the rat FAS promoter demonstrated that SREBP binding to two SRE and Sp1 binding to its binding site were sufficient for the sterol regulation (40). Because the rat StAR promoter contains multiple NF-Y binding sites, the individual and combined requirements of each of these sites will need to be determined through deletion and mutation analysis.
The results of this study confirm synergism between SREBP-1a and SF-1 that was previously demonstrated for the rat HDL-R promoter by this laboratory (10). Tropic hormones have been shown to exert their steroidogenic effects by binding to specific G protein-coupled receptors, which subsequently activates adenylate cyclase and increases intracellular cAMP levels. SF-1 (a tissue-specific orphan nuclear receptor) was shown to mediate cAMP-induced transcriptional activation of the rat StAR gene (30). Numerous studies demonstrate that SF-1-dependent regulation is mediated through interactions with additional transcriptional cofactors. The C/EBP response element of the human StAR promoter was shown to enhance cAMP responsiveness in the presence of SF-1 (34). Coexpression of SF-1 and C/EBPß was shown to be required for transcriptional activation of the mouse StAR promoter (41). Furthermore, SF-1 and Sp1 were found to physically interact and enhance regulation of the human StAR promoter (42). SF-1 binding was shown to be required for protein/DNA interactions in a highly conserved C/EBPß/AP-1 nuclear receptor half-site and for maximal activation of the mouse StAR promoter (43). Additionally, a recent paper by Reyland et al. (44) demonstrated that lipoproteins, which provide cholesterol as a substrate for steroidogenesis, regulate StAR mRNA and protein expression in Y1 adrenocortical cells. It seems likely that multifaceted transcriptional coactivators like Sp1, SF-1, and NF-Y provide an additional level of regulatory control in modulation of SREBP-mediated sterol responsiveness. These results provide the first evidence of SREBP-1a binding to SRE in the rat StAR promoter and demonstrate the potential convergence of two distinct regulatory pathways to participate in maximal activation of a steroidogenic gene.
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Footnotes |
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2 Supported by an American Heart Association-Florida Affiliate
Post-Doctoral Fellowship (9703004).
Received September 13, 2000.
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 S. H. Mellon, S. R. Bair, C. Depoix, J.-L. Vigne, N. B. Hecht, and P. B. Brake Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription Mol. Endocrinol., January 1, 2007; 21(1): 89 - 105. [Abstract] [Full Text] [PDF]
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T. Ozbay, A. Rowan, A. Leon, P. Patel, and M. B. Sewer Cyclic Adenosine 5'-Monophosphate-Dependent Sphingosine-1-Phosphate Biosynthesis Induces Human CYP17 Gene Transcription by Activating Cleavage of Sterol Regulatory Element Binding Protein 1 Endocrinology, March 1, 2006; 147(3): 1427 - 1437. [Abstract] [Full Text] [PDF]
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B. F. Clem and B. J. Clark Association of the mSin3A-Histone Deacetylase 1/2 Corepressor Complex with the Mouse Steroidogenic Acute Regulatory Protein Gene Mol. Endocrinol., January 1, 2006; 20(1): 100 - 113. [Abstract] [Full Text] [PDF]
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T. Ishikawa, K. Hwang, D. Lazzarino, and P. L. Morris Sertoli Cell Expression of Steroidogenic Acute Regulatory Protein-Related Lipid Transfer 1 and 5 Domain-Containing Proteins and Sterol Regulatory Element Binding Protein-1 Are Interleukin-1{beta} Regulated by Activation of c-Jun N-Terminal Kinase and Cyclooxygenase-2 and Cytokine Induction Endocrinology, December 1, 2005; 146(12): 5100 - 5111. [Abstract] [Full Text] [PDF]
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M. D. Pisarska, J. Bae, C. Klein, and A. J. W. Hsueh Forkhead L2 Is Expressed in the Ovary and Represses the Promoter Activity of the Steroidogenic Acute Regulatory Gene Endocrinology, July 1, 2004; 145(7): 3424 - 3433. [Abstract] [Full Text] [PDF]
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 N. Sekar and J. D. Veldhuis Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells Am J Physiol Endocrinol Metab, July 1, 2004; 287(1): E128 - E135. [Abstract] [Full Text] [PDF]
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 K. Tajima, A. Dantes, Z. Yao, K. Sorokina, F. Kotsuji, R. Seger, and A. Amsterdam Down-Regulation of Steroidogenic Response to Gonadotropins in Human and Rat Preovulatory Granulosa Cells Involves Mitogen-Activated Protein Kinase Activation and Modulation of DAX-1 and Steroidogenic Factor-1 J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2288 - 2299. [Abstract] [Full Text] [PDF]
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 Z. T. Ruiz-Cortes, Y. Martel-Kennes, N. Y. Gevry, B. R. Downey, M.-F. Palin, and B. D. Murphy Biphasic Effects of Leptin in Porcine Granulosa Cells Biol Reprod, March 1,&nbs |
