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Departments of Obstetrics and Gynecology and Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida 33606
Address all correspondence and requests for reprints to: Dr. Mark P. McLean, Departments of Obstetrics and Gynecology, 4 Columbia Drive, Room 529, Tampa, Florida 33606. E-mail: mmclean{at}com1.med.usf.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although cAMP stimulation is a common pathway in regulating
steroidogenic genes, each gene appears to be regulated by a different
mechanism (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). However, recent studies have shown that independent
of the mechanism involved, a common factor in this pathway is the
orphan nuclear receptor (18), steroidogenic factor-1 (SF-1) (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
SF-1 has been shown to regulate many steroidogenic genes, including the
human (3, 4), bovine (5, 6), rat (7), and mouse (8) cholesterol
side-chain cleavage cytochrome P450 gene (CYP11A), the human (9) and
rat (10, 11) aromatase cytochrome P450 gene (CYP19), the rat
17
-hydroxylase/c17,20 lyase gene (CYP17) (12), the human
3ß-hydroxysteroid dehydrogenase 
5
4-isomerase type
II (13), and the human (14, 15), mouse (16), and rat (17) steroidogenic
acute regulatory protein (StAR). All of these studies have clearly
shown that SF-1 can mediate cAMP responsiveness (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). SF-1 also
regulates the expression of genes encoding the peptide hormones
involved in regulating steroidogenesis, such as the fish gonadotropin
II ß-subunit (19); the bovine (20), rat (21), and mouse (22, 23) LH
ß-subunit; and the Mullerian inhibiting substance genes (24). The
mouse ACTH (25), GnRH (26), human anti-Mullerian hormone (27), and PRL
(28) receptors are also regulated by SF-1. In addition, DAX-1
(dosage-sensitive sex reversal adrenal hypoplasia congenita critical
region on the X-chromosome, gene-1), an orphan nuclear receptor that
inhibits SF-1 protein’s function, is regulated by SF-1 in humans (29)
and mice (30). These studies demonstrate the critical role that SF-1
plays in regulating genes within the reproductive axis.
The recently isolated and cloned high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), has been shown to play an important role in steroid hormone production (31, 32). This receptor mediates the uptake of cholesterol and cholesteryl esters, substrates for steroidogenesis, in the adrenal gland and gonads (31, 32). Similar to that of other genes involved in steroid hormone synthesis, the expression of the HDL receptor gene is induced by hCG (32, 33, 34, 35), ACTH (33, 35, 36, 37, 38), and LH (32, 33, 35, 36, 37, 38). A recent report has also shown that the human HDL receptor promoter contains a SF-1-binding site to which SF-1 protein binds in vitro and activates transcription of this gene in Y1 adrenal cells (39). These results suggested that the HDL receptor gene may be regulated by SF-1 similar to the other genes involved in steroidogenesis.
In the present study, we investigated the effects of cAMP on the regulation of the HDL receptor gene. We found that SF-1 is required for both basal and cAMP-induced regulation of the HDL receptor gene. Transcriptional regulation of HDL receptor gene expression by SF-1 in response to cAMP provides a locus for control of the uptake of cholesterol, the required substrate for the synthesis of all steroid hormones.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T vector
[glutathione-S-transferase (GST)-SF-1] were obtained from
Dr. Kieth L. Parker (University of Texas Southwestern, Dallas, TX). The
rabbit polyclonal IgG antimouse SF-1 (2.4 µg/µl) was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). The mouse
Y1 adrenal and human HTB-9 bladder carcinoma cell lines were obtained
from American Type Culture Collection (Manassas, VA). The
mouse Kin 8 adrenal cell line was provided by Dr. Bernard P. Schimmer
(University of Toronto, Toronto, Canada). The QuickChange Site-Directed
Mutagenesis Kit was purchased from Stratagene (La Jolla,
CA). Tri-Reagent, Background Quencher, Formazol, and High Efficiency
Hybridization Solution were obtained from Molecular Research Center, Inc. (Cincinnati, OH). [-32P]Deoxy
(d)-CTP (3000 Ci/mmol) and the T7 Sequenase DNA Sequencing Kit were
purchased from Amersham (Arlington Heights, IL).
[35S]dATP (1000–1500 Ci/mmol) was obtained from
DuPont/NEN (Wilmington, DE). All restriction enzymes and the nick
translation kit were obtained from Boehringer Mannheim
(Indianapolis, IN). Nitrocellulose membrane was purchased from
Schleicher & Schuell, Inc. (Keene, NH). The SuperSignal
ULTRA chemiluminescent substrate was obtained from Pierce Chemical Co. (Rockford, IL). DMEM-nutrient mixture F-12
(DMEM/F12) was obtained from Life Technologies (Grand
Island, NY). FBS was purchased from Summit Biotechnology
(Ft. Collins, CO). Poly(dI-dC) and the GST fusion protein purification
kit were obtained from Pharmacia Biotech (Piscataway, NJ).
BioMax-MR films were obtained from Fisher Scientific
(Norcross, GA). All other chemicals were purchased from Fisher Scientific or Sigma Chemical Co. (St. Louis,
MO).
Animals
Twenty-eight-day-old Sprague Dawley rats were purchased from
Harlan Industries (Madison, WI). All protocols to treat animals were
approved by the University of South Florida animal care committee.
Throughout the experiment, animals had free access to food and water
and were housed under a 12-h dark, 12-h light cycle. Follicular
development and ovulation were induced in rats by injection of 8 IU
PMSG. By this method, rats ovulate approximately 72 h after
treatment with PMSG (40).
Luteal cell dispersion
For luteal cell dispersions, ovaries were collected 10 days
postovulation. Ovaries from 10 animals were collected and dispersed in
10 ml of a solution containing 0.23 mg/ml collagenase, 0.03 mg/ml
deoxyribonuclease, and 1.5 mg/ml dispase and incubated on a Biostir
plate for 30 min with gentle stirring. After the incubation, the
collagenase-deoxyribonuclease-dispase solution was changed, and the
ovaries were incubated for another 30 min. This process was repeated
three times. Cells were then centrifuged and resuspended in dispersion
medium for trypan blue exclusion cell viability analysis.
Cell culture
Luteal cells were plated in six-well plates (
106
cells/well) and incubated with DMEM/F12 and 10% FBS for 36 h at
37 C (5% CO2). The medium was changed 2 h before
adding (Bu)2cAMP. After incubating the cells with
the specified dose of (Bu)2cAMP for the indicated
time, the medium was removed, and the cells were washed twice with PBS.
Cells were then used for RNA isolation.
RNA isolation and Northern blot analysis
Total RNA was isolated using the acid guanidinium
thiocyanate-phenol-chloroform extraction method (41) employing
Tri-Reagent. This method consistently yields 5–8 µg RNA/mg tissue.
Cells were scraped with a rubber policeman into 2 ml Tri-Reagent and
then transferred to a ribonuclease-free centrifuge tube. Cells were
homogenized with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) and centrifuged at 11,000 x
g for 15 min at 4 C. RNA was precipitated from the aqueous
phase with isopropanol. The RNA pellet was washed in 75% ethanol and
then resuspended in formazol. Electrophoresis and Northern blot
analysis to determine HDL receptor messenger RNA (mRNA) levels were
carried out as previously described (42). The rat HDL receptor cDNA was
obtained by PCR as previously described (43). The rat HDL receptor cDNA
insert (1579 bp) was then labeled with [-32P]dCTP
using the random primed DNA labeling method (44) or nick translation.
Blots were stripped and reprobed with the internal control
ß-actin.
Plasmids for luciferase assays
All HDL receptor promoter-luciferase gene constructs were
derivatives of the pGL3-basic luciferase vector. Standard molecular
biology techniques were used in all cloning procedures (45). The HDL
receptor promoter used in these experiments was obtained using the
PromoterFinder DNA Walking PCR kit (Lopez, D. and M. P. McLean,
unpublished) from CLONTECH Laboratories, Inc. (Palo Alto,
CA). To make the p-2267 plasmid, the entire HDL receptor promoter
obtained by this method was used. This promoter fragment was subcloned
into pGL3-basic luciferase vector using MluI and
HindIII sites. Nested deletions of the HDL receptor promoter
were carried out using restriction enzymes. The restriction enzymes
used were KpnI and HindIII for the p-1742
promoter fragment, BglII and HindIII for the
p-1421 fragment, XhoI and HindIII for the p-719
fragment, and SmaI and HindIII for the p-170
fragment. All promoter fragments were then subcloned into the
pGL3-basic luciferase vector.
Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)
according to the manufacturer’s protocol. Briefly, 10 ng plasmid were
incubated with 125 ng of the appropriate complementary oligonucleotides
(see below) and 1 µl dNTPs in 50 µl reaction buffer [100
mM KCl, 100 mM
(NH4)2SO4, 200 mM
Tris-HCl (pH 8.8), 20 mM MgSO4, 1% Triton
X-100, and 1 mg/ml nuclease-free BSA]. One microliter of
Pfu DNA polymerase (2.5 U/µl) 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. One microliter of the
mutant samples was used to transform XL-1 blue bacterial cells. The
mutations were confirmed by sequencing using the T7 sequenase DNA
sequencing kit and [35S]dATP.
Oligonucleotides used for site-directed mutagenesis to mutate the distal SF-1 binding site (SFBd) in the p-719 plasmid were SFBd-Mut1 (5'-GACAGTGCATCTCGGCCGCGAGGGACA-3') and its complement. To introduce a premature termination codon instead of Arg at position 448 in the activation function-2 domain (AF-2) and to substitute Ser430 by Ile, both in the SF-1-pCMV plasmid, the complementary oligonucleotides used were SF-1-Mut1 (5'-CGAGATGCCCTGAAACAACCTTCTC-3') and SF-1-Mut2 (5'-GCGGGCCCTGATCATGCAGGCCAAG-3'), respectively. The underlined nucleotides correspond to the mutated bases.
Cell transfections
Cells were transfected with the specified HDL receptor
promoter-luciferase gene construct in the presence or absence of
SF-1-pCMV using the calcium phosphate method (46). Cells were first
plated in six-well tissue culture plates at a density of 3.3 x
106 cells/well and incubated for 24 h at 37 C (5%
CO2). Fresh DMEM/F12 medium and 10% FBS were added 2
h before transfections. The DNA to be transfected was purified by
ethanol precipitation, resuspended in 0.25 M
CaCl2, and reprecipitated in an equal volume of 2 x
HEPES buffer for 20 min at room temperature. Precipitated DNA was then
added to the culture plates. After incubating for 4 h, the medium
was replaced, and the cells were allowed to incubate for 48 h at
37 C (5% CO2). Either (Bu)2cAMP or
8-bromo-cAMP (8-Br-cAMP; 1 mM) was added to some plates
24 h before the end of the incubation period. Cells were washed
twice with PBS and treated with a passive lysis buffer for 20 min.
Lysates were transferred to a microcentrifuge tube and stored at -80 C
until the determination of luciferase activity. Cotransfection of a
plasmid containing the renilla luciferase gene under control of the
simian virus 40 early enhancer/promoter region was used as a control to
correct for differences in transfection efficiencies.
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
were added to 20 µl lysate, and luciferase activity was measured
using a Turner Designs-20/20 luminometer (Sunnyvale, CA).
Fusion protein production
GST-SF-1 fusion protein was 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 7700 x g for 10 min at 4 C. The cell pellet was
resuspended in PBS and sonicated using a Sonic Dimembrator 60
(Fisher Scientific) with a 0.25-in. tip at full strength
in 10-sec bursts until cells were lysed. Triton X-100 was then 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, Piscataway, NJ) according to the manufacturer’s
recommendations. Briefly, cleared lysate was passed through the
glutathione-Sepharose 4B column. After washing the column with PBS, the
fusion protein was eluted with 10 mM reduced glutathione in
50 mM Tris-HCl, pH 8.0. Protein concentrations were
determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Purified GST fusion protein was used in
mobility shift assays.
Gel mobility shift assay
Complementary oligonucleotides corresponding to the HDL receptor
promoter regions from -655 to -628 (SFBd;
5'-GACAGTGCATCAAGGCCGCGAGGGACA-3') and from -110 to -83 (SFBp;
5'-CAGGCACACACCTTGCTGCTCGGTTTC-3') with GGG overhangs at the 5'-ends
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 (DTT). The
oligonucleotide probe was then labeled using the Klenow fragment of DNA
polymerase and [-32P]dCTP (3000 Ci/mmol). Unlabeled
SFBd oligonucleotide was used as a competitor in some experiments.
Mutated-SFBd (5'-GACAGTGCATCTCGGCCGCGAGGGACA-3') is
identical to SFBd except for two mutated bases (underlined
letters). In some experiments, mutated-SFBd was also 32P
labeled and used as a probe. Purified GST-SF-1 fusion protein (2.5
µg) was incubated in the presence or absence of competitor for 30 min
at room temperature in binding buffer [12 mM HEPES (pH
7.9), 12% glycerol, 60 mM KCl, 1 mM EDTA, 1
mM DTT, 4 mM Tris-HCl (pH 8.0), 2 µg
poly(dI-dC), and 0.4 µg BSA]. After incubation, 50,000 cpm of the
radiolabeled probe were added, and the mixture was incubated for 15 min
at 30 C. Where indicated, SF-1 antibody was also added to the reaction
for supershift analysis. The DNA-protein complexes were resolved on a
4% nondenaturing acrylamide gel at 4 C in 1 x TBE (0.05
M Tris, 0.05 M boric acid, and 0.001
M EDTA). Gels were then vacuum-dried and exposed to
BioMax-MR films at -80 C for 12–24 h.
Nuclear extracts
Nuclear extracts were prepared from COS-7 cells overexpressing
either wild-type or mutated SF-1 protein. COS-7 cells at a density of
108 cells/flask were transiently transfected with 50
µg/flask of the corresponding plasmid using the calcium phosphate
method (47) as described above. After transfections, cells were
incubated for 48 h at 37 C (5% CO2). Cells were then
washed twice with PBS and treated with 10 ml 0.25% trypsin-1
mM EDTA for 10 min. The cell pellet was recovered by
centrifugation at 500 x g for 5 min. Cell nuclei were
prepared as previously described (48). The nuclear pellet was
resuspended in 2.5 vol extract buffer [15 mM Tris-HCl (pH
7.5), 15 mM NaCl, 60 mM KCl, 0.34 M
sucrose, 15 mM ß-mercaptoethanol, 0.15 mM
spermine, 0.5 mM spermidine, 5% glycerol, and 0.2
mM phenylmethylsulfonylfluoride]. The suspension was
gently agitated for 30 min at 4 C. KCl was then added to a final
concentration of 300 mM. After incubating for another 30
min at 4 C, the samples were centrifuged in a microcentrifuge at top
speed for 30 min. Nuclear extracts were stored at -80 C until used for
Western blot analysis. Protein concentrations were determined using the
Bio-Rad protein assay (Bio-Rad Laboratories, Inc.).
Western blot analysis
Nuclear extracts (100 µg) were denatured at 100 C in loading
buffer (49) for 10 min and subjected to electrophoresis on a 7.5–18%
gradient SDS-PAGE according to the method of Laemmli (50). After
electrophoresis, samples were electroblotted onto nitrocellulose
membranes (0.2 µm pore size) in buffer containing 0.25 M
Tris-HCl (pH 8.3) and 1.92 M glycine for 16 h at 4 C.
To verify equal protein loading, nitrocellulose membranes were stained
with 0.1% Ponceau S (in 5% acetic acid) and destained in water.
Western blot analysis of SF-1 protein was carried out with a 1:2500
dilution in 3% milk of the rabbit polyclonal antiserum to mouse SF-1
(Upstate Biotechnology, Inc., Lake Placid, NY).
Immunoreactive proteins were then visualized using a 1:10,000 dilution
in 3% horseradish peroxidase-conjugated goat antirabbit antisera
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the
SuperSignal Chemiluminescent Substrate method (Pierce Chemical Co., Rockford, IL).
Data analysis
Northern blot results were analyzed using a Hoefer scanning
densitometer (Hoefer Scientific, San Francisco, CA). Equal
RNA loading was verified by ethidium bromide staining of the agarose
gel. Luciferase data were expressed as the mean ±
SEM. Each luciferase assay experiment was performed in
triplicate and repeated for the indicated number of times in the figure
legends. Data from the individual parameters were compared by ANOVA,
followed by Student-Newman-Keuls multiple comparison test when
applicable (51).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 1 mM
(Bu)2cAMP dramatically increased HDL receptor
mRNA levels by 118-fold at 24 h after treatment (P
< 0.01). Increases in mRNA levels began as soon as 3 h after
adding (Bu)2cAMP to the medium of the cells and
reached maximal levels by 24 h. A typical dose-response curve is
presented in Fig. 1B. As shown, treatment with a
(Bu)2cAMP dose as low as 0.001 mM for
24 h caused a 2-fold increase in HDL receptor mRNA levels. Maximum
levels were obtained with 1 mM
(Bu)2cAMP (P < 0.001). These
results suggest that cAMP may regulate HDL receptor gene expression at
the transcriptional level.
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. Briefly, five separate
adaptor-ligated rat genomic libraries were screened by PCR. For this,
two nested gene-specific primers were designed to the 5'-flanking
region of the rat HDL receptor cDNA (43). These primers were then used
with adaptor specific primers in nested-PCR reactions. A 0.8-kb DNA
fragment was obtained in the PvuII library (Fig. 2A). Two
new nested gene-specific primers were then designed to the 5'-flanking
region of the 0.8-kb DNA fragment and used in new nested PCR reactions
to obtain sequence further upstream. With these new primers, a 1.5-kb
promoter fragment was obtained in the EcoRV library (Fig. 2A). Both DNA promoter fragments were ligated together using a shared
XhoI site, which resulted in a 2.2-kb DNA fragment (Fig. 2A). The entire sequence (Lopez, D. and M. P. McLean, unpublished) was
then searched for any putative cAMP response element (CRE).
Surprisingly, no putative CRE was found in this region. Instead, two
putative SF-1-binding sites at positions -645 [SFB-distal (SFBd);
5'-TCAAGGCC-3'] and -102 [SFB-proximal (SFBp); 5'-CACCTTGC-3']
relative to the translation start site were found. The SFBd had 88%
identity to the SF-1-binding site reported for the human CYP11A gene
(5'-TCAAGGCA-3') (4) and 75% identity to the SF-1 site reported for
the human HDL receptor gene (5'-CCAAGGCT-3') (39). The SFBp had 87%
identity to the SF-1-binding site reported for the rat StAR gene
(5'-CACCTTGG-3') (17) and 75% identity to the SF-1-binding sites
reported for the bovine (20) and rat (21) LH ß-subunit gene
(5'-GACCTTGT-3') and the human StAR gene (5'-GACCTTGA-3') (15).
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, all HDL receptor promoter
constructs, except p-170, showed a 2- to 3-fold induction
(P < 0.05) in luciferase activity when cotransfected
with cSF-1 plasmid. This activity was enhanced by
(Bu)2cAMP treatment. Stimulation with
(Bu)2cAMP of HTB9 cells transfected with the
p-719 construct in the presence of cSF-1 produced the highest levels of
luciferase activity (27-fold; P < 0.01). None of the
constructs were induced by (Bu)2cAMP in the
absence of cSF-1 (data not shown). In addition, p-719 construct
cotransfected with rSF-1, a plasmid containing SF-1 cDNA in the reverse
orientation, showed no induction by (Bu)2cAMP
(Fig. 3). These results suggest that SF-1
directly mediates cAMP activation of the HDL receptor promoter.
Surprisingly, the p-170 construct showed no induction of luciferase
activity by SF-1 and (Bu)2cAMP, even though this
construct contained one putative SF-1-binding site, SFBp (Fig. 2A).
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, the luciferase activity of constructs
containing both SFBs was induced 2- to 5-fold compared with that of the
empty vector, an effect that was enhanced by 1 mM
8-Br-cAMP. Once again, the activity of the p-170 construct, which only
contains SFBp, was not significantly higher than that of the empty
vector (Fig. 4).
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, incubation of radiolabeled SFBd with purified GST-SF-1 fusion protein
resulted in the production of a slower migrating DNA-protein complex
(lane 2). When SF-1 antibody was included in the binding reaction (lane
3), a supershifted complex was formed (lane 3). No binding of SF-1
protein to SFBp was observed (Fig. 5C, lanes 6–8). This correlated
with the luciferase assay showing no activation through this motif
(Figs. 2 and 4). A known SF-1-binding site from the rat StAR promoter
(17) was used as a positive control (Fig. 5C, lanes 1–4).
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, lanes 2–6). As shown, the addition of
increasing levels of unlabeled SFBd gradually diminished the
DNA-protein complex. A 100-fold molar excess of unlabeled SFBd was able
to completely eliminate the SFBd-SF-1 complex. Likewise, the addition
of increasing amounts of SF-1 antibody gradually reduced the SFBd-SF-1
complex, whereas the supershifted complex increased (lanes 7–10).
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, SF-1 protein did not bind to the
mutated probe. The same mutation was then tested in cotransfection
studies. SF-1 and (Bu)2cAMP were unable to induce
luciferase activity of mutated p-719 construct in HTB9 cells (Fig. 7B).
However, when the mutated construct was transfected into Y1 cells, both
basal and 8-Br-cAMP-induced luciferase were reduced about 60% compared
with that of the wild-type promoter (Fig 8).
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).
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, both Kin 8 and Y1 cells express
similar very low constitutive levels of HDL receptor mRNA. Treatment
with 8-Br-cAMP increased HDL receptor mRNA levels 15-fold in Y1 cells
(Fig. 10). In Kin 8 cells, however, the increase in endogenous HDL
receptor mRNA levels by 8-Br-cAMP was only 2-fold (Fig. 10). These
results suggest that a functional PKA is required for SF-1 mediation of
cAMP regulation of the HDL receptor gene.
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). These mutations were prepared
using site-directed mutagenesis as described in Materials and
Methods. As shown in Fig. 11B, both wild-type and mutant SF-1
proteins were expressed at comparable levels as determined by Western
blot analysis.
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shows
that when the p-719 plasmid was cotransfected with either mutation, no
significant induction of luciferase activity was observed even in the
presence (Bu)2cAMP. Both mutations diminished
basal and cAMP-induced activity to the same extent (Fig. 11B). These
data suggest that both regions of the SF-1 protein are important for
basal and cAMP-induced regulation of the HDL receptor gene. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SF-1 mediates cAMP regulation of many components of the steroidogenic
pathway by different mechanisms. In some genes, the simple binding and
activation of gene transcription by SF-1 are insufficient. They require
interaction with other factors. For example, in the case of the rat
CYP19 gene, cAMP regulation of this gene requires both SF-1 and cAMP
response element-binding protein (11). The regulation of the fish
gonadotropin II ß-subunit gene requires SF-1 and estrogen receptor
(19), whereas the regulation of the bovine CYP11A gene requires SF-1
and Sp1 (6). The mouse LH ß-subunit gene activation requires the
cooperative interaction of SF-1 and early growth response protein-1
(22, 23), whereas the StAR gene requires multiple SF-1-binding sites
for maximal activation (15, 16, 17). For genes such as the human
3ß-hydroxysteroid dehydrogenase 54-isomerase type
II (13), 17-hydroxylase/c17,20 lyase (CYP17) (12), ACTH receptor
(25), PRL receptor (28), and DAX-1 (29, 30), no other factors
interacting with SF-1 have been reported. Whether cAMP regulation of
the rat HDL receptor gene requires other factors in addition to SF-1
remains to be determined. The finding that SFBd mutation only caused a
60% reduction in Y1 cells, even though SF-1 protein did not bind to
the mutated probe, suggests that cAMP regulation of the HDL receptor
gene requires other factors in addition to SF-1. Besides SFBd, the HDL
receptor promoter contains four estrogen receptor half-sites, two
sterol regulatory elements, and seven Sp1-binding sites (Lopez, D. and
M. P. McLean, unpublished). Whether any of these regulatory motifs
cooperates with SF-1 to mediate cAMP induction of the HDL receptor gene
remains to be determinated. It appears that the factor that cooperates
with SF-1 is not present in HTB9 cells because the same mutation
completely abolished SF-1 and cAMP effects on the HDL receptor promoter
activity in this cell type.
The second messenger cAMP mediates the intracellular responses to a wide variety of hormones in many cell types (58). These include glucagon in the liver and adipose tissues; FSH and LH in ovarian follicles; ACTH in adipose tissue and adrenal cortex; TSH in thyroid cells; PTH in bone cells; epinephrine in adipose, liver, intestine, cardiac, and skeletal muscle cells; vasopressin in the kidney; and PG I in blood platelets (58). In all of these cases, cAMP activates PKA (58). The difference in response according to the specific hormone and cell type depends on which factors are activated or inhibited by PKA (58). In steroidogenic tissues, SF-1 appears to be that specific factor (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). This correlates with the finding that SF-1 is mainly expressed in steroidogenic tissues (59, 60), and that cotransfection of the PKA catalytic subunit has been shown to mimic forskolin’s effects on SF-1-dependent reporter gene activation (5, 54).
The availability of steroidogenic Y1 mutant clones (Kin) resistant to activation by cAMP due to mutations in the regulatory subunit of PKA (52, 53) has provided an experimental model to specifically evaluate the role of this kinase in cAMP regulation of steroidogenic genes (12, 61, 62, 63, 64). Several mutants have been identified according to their degree of resistance to 8-Br-cAMP activation (52, 53). The Kin 8 cell line shows the highest degree of impairment in both PKA and cAMP-binding activities (52, 53). In the present report, we have found that transcriptional activation of the HDL receptor promoter in response to cAMP is reduced in Kin 8 cells, and that this reduction results in decreased receptor mRNA levels. Similar effects have been seen in the P450c17 (12), CYP21 (61), cholesterol side chain cleavage (62, 64), and steroid 11ß-hydroxylase (63, 64) genes. Although all of these genes show diminished gene expression in Kin 8 cells (12, 61, 62, 63, 64), some differences in their absolute requirement for PKA activity are seen (64). In the case of the SCC, both basal and 8-Br-cAMP-induced mRNA levels were markedly reduced but still detectable in the absence of a functional PKA (64). However, in the case of steroid 11ß-hydroxylase, both basal and 8-Br-cAMP-induced mRNA levels completely disappeared as a result of the PKA mutation (64). PKA does not appear to significantly affect basal HDL receptor mRNA levels, but it does affect 8-Br-cAMP-induced HDL receptor mRNA levels.
Although the current data, including this report, show a requirement for PKA in the SF-1 mediation of the cAMP responsiveness of genes, the exact mechanism for the functional role of SF-1 in the PKA signal transduction pathway is not totally clear. One possibility is that SF-1 could be phosphorylated by PKA. It has been previously shown that PKA is able to phosphorylate SF-1 in vitro (12). In addition, a phosphorylated form of SF-1 can be detected in rat granulosa cells after treatment with FSH (11). However, there is no evidence that this phosphorylated SF-1 form was produced by PKA (11). Whether PKA can directly phosphorylate SF-1 in vivo and whether phosphorylation directly affects trans-activation of the SF-1 protein remain to be determined. Furthermore, the possibility that phosphorylation of SF-1’s coactivators, instead of or in addition to SF-1 phosphorylation may be crucial in this regulatory process and needs to be examinated. Consistent with this possibility, it was reported that phosphorylation of CRE-binding protein, a coactivator of SF-1, is required in the case of CYP19 gene activation (11).
In the present report, we examined the roles of two regions of the SF-1 protein in the PKA-dependent trans-activation of the HDL receptor gene. The first region examined was the AF-2 domain of SF-1. Three main functions have been ascribed to this domain. First, the AF-2 domain is required for interaction with the steroid receptor coactivator-1, and its removal diminishes SF-1 activation capacity by 75% (56, 65). Second, this domain also appears to be involved in the activation of SF-1 by oxysterols, which are proposed ligands for SF-1 (66). Recently, however, it was shown that 25-hydroxycholesterol does not act as a ligand for SF-1 in mouse MA-10 cells (67), and there are still some questions about this function of the AF-2 domain. The third function assigned to this domain is that it is essential for the PKA activation of SF-1 (55). Although SF-1’s AF-2 domain does not contain any potential PKA phosphorylation site, it does contain a conserved threonine residue that has been proposed as a potential protein kinase C phosphorylation site (54). The second region of the SF-1 protein examined was a potential consensus site for PKA phosphorylation at Ser430 (54). This correlates with the finding that Ser is one of the residues that can be phosphorylated by PKA (12). Thus, this region was a good candidate for this type of study. Our results indicate that both mutations diminish basal and cAMP-induced activity of the HDL receptor promoter construct to the same extent. Whether phosphorylation of SF-1 actually occurs at Ser430 and whether deleting the AF-2 domain influences phosphorylation remain to be determined. It has been previously shown that deletion of SF-1’s AF-2 domain does not affect its binding to DNA (55). This suggests that the decrease in basal activity of the HDL receptor promoter results from SF-1’s reduced transactivation potential when the AF-2 domain is altered. The finding that both mutations decreased basal activity of the HDL receptor further supports the possibility that these regions may also be involved in other functions, such as cofactor interaction.
In summary, the rat HDL receptor promoter contains an SF-1-binding site (SFBd) through which SF-1 protein binds and activates transcription of the luciferase reporter gene. This motif is required for both basal and cAMP-induced regulation of the HDL receptor gene, similar to other genes involved in steroidogenesis (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). We also demonstrated that regulation of the HDL receptor by cAMP requires a functional PKA. Two regions of the SF-1 protein, the AF-2 domain and a serine residue within a potential consensus phosphorylation site for PKA, appear to be required for both basal and cAMP-induced regulation of the HDL receptor gene. The mediation of cAMP responsiveness of the HDL receptor gene by SF-1 suggests how important this trans-acting factor is in steroid hormone synthesis by assuring that all required elements (substrates and enzymes) are present when they are needed for maximal steroid production.
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Received August 3, 1998.
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158
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