From the Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen, Norway
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ABSTRACT |
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Steroidogenic factor-1 (SF-1) is a nuclear
receptor that is essential for the proper development and function of
steroid hormone-producing cells. The activation function-2 (AF-2)
domain in SF-1 is a short -helix in the C terminus that is conserved
with respect to other nuclear receptors and is important for
transactivation of target genes. In order to investigate the possible
role of the AF-2 domain of SF-1 in cAMP-dependent
transcriptional regulation of the bovine steroid hydroxylase gene
CYP17, mutations were introduced and the effects were characterized.
The mutant SF-1 proteins were expressed at comparable levels in
nonsteroidogenic Cos-1 cells that lack SF-1, and their abilities to
bind an SF-1 site from the CYP17 gene were not affected. Transient
transfections of wild-type and mutant SF-1 in Cos-1 cells showed that
the capacity to transactivate a reporter gene under the control of the
SF-1 site from CYP17 was reduced by the mutations in the AF-2 domain of
SF-1. A point mutation in the AF-2 region, E454A, resulted in a
relative reporter gene activity that was 21% of that observed with
wild-type SF-1. Co-transfections of adrenocortical Y-1 cells, which
express endogenous SF-1, with the catalytic subunit of
cAMP-dependent protein kinase (PKA-C) and the
SF-1-dependent reporter gene showed on average a 16-fold
increase in activity in the presence of PKA-C. Introduction of the AF-2
mutants of SF-1 into Y-1 cells completely abolished the PKA-C-mediated
stimulation of the reporter gene. The transdominant negative effect of
the mutant SF-1 proteins suggests that the AF-2 domain is essential for
the activation of SF-1 by the cAMP-dependent protein
kinase-dependent signaling pathway.
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INTRODUCTION |
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Steroid hormone biosynthesis in the adrenal cortex, testis, and ovary is under the control of the trophic hormones adrenocorticotropic hormone, luteinizing hormone, and follicle-stimulating hormone from the pituitary (1, 2). Through signaling via G-protein coupled cell surface receptors, these hormones increase intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA).1 As a consequence, steroidogenesis is stimulated through a rapid mobilization and movement of the substrate cholesterol to the enzymes, which convert it to biologically active hormones. In addition, cAMP and PKA increase the transcription of the genes encoding the biosynthetic enzymes, including the steroid hydroxylases which belong to the cytochrome P-450 superfamily of enzymes (2). Steroidogenic factor-1 (SF-1) or Ad4BP is a transcription factor belonging to the nuclear receptor superfamily, which appears to be crucial for not only the expression of essentially all components of the steroidogenic pathways but also for the development of the steroidogenic tissues per se (3-5). Thus, disruption of the SF-1 gene in mice leads to complete agenesis of the adrenals and gonads (6).
Several lines of evidence have suggested that SF-1 may be directly
involved in the cAMP-dependent regulation of steroid
hydroxylase gene expression. Often SF-1-binding sites in target genes
reside within regions that have been functionally assigned
cAMP-responsiveness and require an intact PKA to be active (7, 8).
Also, mutations that interfere with SF-1 binding often attenuate cAMP
responsiveness (9). Finally, SF-1 contains a putative PKA
phosphorylation site (10), can be phosphorylated by PKA in
vitro (11) and is immunoprecipitated as a phosphoprotein from
adrenocortical cells grown in the presence of
[32P]orthophosphate
(12).2 However, the exact
role of SF-1 in mediating the cAMP response is not well understood, and
results in the literature are in part conflicting. In the bovine CYP17
gene, which encodes the cytochrome P-450 17-hydroxylase, an SF-1
site is present within cAMP-responsive sequence 2 (CRS2) (13), and
mutations that interfere with SF-1 binding correlate with decreases in
cAMP-stimulated transcription of a linked reporter gene (9).
Furthermore, transfection of SF-1 into a nonsteroidogenic cell lacking
this transcription factor activates an otherwise inactive
CRS2-dependent reporter construct, and co-transfection of
the catalytic subunit of PKA (PKA-C) increases the activity
severalfold. In order to determine whether this response relies on
structures within SF-1 itself, amino acid substitutions and deletions
were introduced. Here we show that mutations within a conserved core
region of a putative C-terminal activation function-2 (AF-2) domain of
SF-1 compromises its transactivating ability. Furthermore, such mutant
SF-1 proteins are able to dominantly suppress the activity of wild-type
SF-1 in adrenocortical tumor cells transfected with PKA-C.
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EXPERIMENTAL PROCEDURES |
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Chemicals-- Cell culture media and sera were purchased from Life Technologies, Inc. 32 The ECL (chemiluminescence) kit for Western blotting was from Amersham Pharmacia Biotech, and the luciferase assay kit was from BIO Thema AB (Luciferase Assay kit, production number 484-001, Dalaro, Sweden). All other chemicals were from Sigma or Merck.
Cell Culture and Transient Transfection-- Y-1 cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum, 10 units penicillin/ml, and 10 µg streptomycin/ml. The day before transfection, Y-1 cells were plated in 6-well plates at a density of 1 × 106 cells/well. Before transfection, the standard growth medium was replaced by fresh medium, and the cells were incubated for a minimum of 2 h. The calcium phosphate co-precipitation method was used to transfect Y-1 cells as described previously (13), and 3 µg of total DNA was added per well. Cos-1 cells were cultured in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal calf serum, 10 units penicillin/ml, and 10 µg streptomycin/ml. The day before transfection, Cos-1 cells were plated in 6-well plates at a density of 1 × 106 cells/well. Cos-1 cells were transfected by the DEAE-dextran method according to standard procedures (14). Cells were incubated with transfection medium containing 3 µg of DNA for 1 h. After removing the transfection medium, 52 mg/liter of chloroquine was added in culture medium, and the cells were incubated for an additional 5 h. The culture medium containing chloroquine was removed, and the cells were incubated for another 18 h with culture medium before they were harvested.
Luciferase Assay-- After 24 h of transfection, cells were washed with cold phosphate-buffered saline, and 200 µl of lysis buffer was used to harvest the cells by scraping. 40 µl of the cell extracts were used for luciferase determinations on a LUCY-1 luminometer (Anthos, Austria). Preparation of lysis buffer and the luciferase enzyme assay was according to the procedures of a commercial kit (BIO Thema AB, Luciferase Assay kit, production number 484-001 Dalaro, Sweden).
Plasmid Constructions-- Constructions of the reporter plasmid pT81-4CRS2 and the expression plasmid pCMV5-SF-1 are as described (15). For mutagenesis of the AF-2 domain, the QuickChangeTM Site-directed Mutagenesis kit (Stratagene) was used. Briefly, the double-stranded plasmid pCMV5-SF-1 and two synthetic oligonucleotide primers with the desired mutations, each complementary to opposite strands, were annealed and extended by means of Pfu DNA polymerase. Following temperature cycling, the products were treated with DPN I in order to digest the parental plasmid. The DNA vectors with desired mutations were transformed into Escherichia coli (Epicurian Coli® XL1-supercompetent cells), and the mutations were confirmed by the dideoxynucleotide sequencing method (USBTM, Sequenase, version 2.0).
Western Blot Analysis-- To determine expression levels, Cos-1 cells were transfected with plasmids encoding wild-type SF-1 and AF-2 mutants. After 24 h, cells were lysed, and extracts were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Western blot analysis of SF-1 proteins was carried out with a polyclonal antiserum to Ad4BP/SF-1 (kindly provided by Dr. Ken Morohashi), and immunoreactive proteins were visualized by the enhanced chemiluminescence method (ECL, Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Analysis
(EMSA)--
Double-stranded synthetic oligonucleotides containing the
SF-1-binding site in CRS2 of the bovine CYP17 gene (15) was labeled with -32P and purified by native polyacrylamide gel
electrophoresis. 10 µg of nuclear extracts from transfected Cos-1
cells containing wild-type and mutant SF-1 protein were incubated with
the 32P-labeled probe. The reaction conditions were as
described previously (13). Protein-DNA complexes and unbound probe were
separated by nondenaturing electrophoresis on a 4% polyacrylamide gel
in 0.5 × TBE (0.045 M Tris borate, 0.001 M EDTA).
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RESULTS AND DISCUSSION |
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Several lines of evidence suggest that the nuclear receptor SF-1
may be directly involved in cAMP-dependent transcriptional regulation of steroid hydroxylase genes (see the Introduction). However, direct evidence of functionally important structures in SF-1
for the cAMP effect is lacking. The ligand-binding domain of nuclear
receptors is located in the C-terminal region and consists of
approximately 225 amino acids involved not only in ligand binding but
also in homo-heterodimerization, hormone-dependent
transcriptional activa-tion, as well as hormone-reversible
transcriptional repression (16, 17). The C-terminal activation function
domain (AF-2) is common to all ligand-activated members of the nuclear
receptor superfamily, and SF-1 has been shown to be activated by
25-hydroxy cholesterol in an AF-2-dependent manner (18).
The AF-2 region in SF-1 and the alignment with various nuclear
receptors are shown in Fig.
1A. The AF-2 sequence is
LLIEML represented as XE
(Fig. 1B),
where
represents hydrophobic residues and where E (glutamic acid)
is common to most of the nuclear receptors. In order to test the
possible importance of this region with respect to
cAMP-dependent transactivation via SF-1, a series of
mutations and deletions were introduced in the AF-2 domain.
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In mutants 1-3, the nonpolar Leu451 and Leu452 were changed either to the small nonpolar residue alanine or deleted. The conserved glutamic acid at position 454 was substituted for alanine in mutant 4. The entire AF-2 core was deleted in mutant 5, whereas in mutant 6, the arginine and the two asparagine residues N-terminal to the AF-2 core were also deleted (Fig. 1C). Wild-type and mutant plasmids were transfected into Cos-1 cells together with the reporter plasmid pT81-4CRS2Luc containing the SF-1-binding site from the proximal promoter region of the bovine CYP17 gene.
Proper expression from wild-type and mutant plasmids was analyzed by Western blot analysis and EMSA. Using a polyclonal antiserum to SF-1, whole cell extracts from transfected Cos-1 cells show the expression of both wild-type and mutant SF-1 proteins at comparable levels (Fig. 2A). 10 µg of nuclear extracts of transfected Cos-1 cells were also used in EMSA, and no significant differences in DNA binding were observed when comparing wild-type and mutant SF-1 proteins (Fig. 2B). It was therefore concluded that wild-type and mutant SF-1 proteins are expressed at comparable levels and that the proteins carrying mutations in their AF-2 core domain have retained their ability to bind DNA. To positively identify the mobility of SF-1, steroidogenic H295 cell extracts incubated in the presence or absence of antibodies against SF-1 before loading on the gel are also shown (Fig. 2B, right-hand two lanes).
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Transactivation by Wild-type and Mutant SF-1 in Nonsteroidogenic
Cos-1 Cells--
To determine the effect of mutations in the AF-2 core
domain of SF-1 with respect to its ability to stimulate the expression of pT81-4CRS2LUC, wild-type and mutant plasmids were transfected into
Cos-1 cells. After 24 h of transfection, cells were lysed, and the
luciferase activity was determined. No activity was observed in
extracts from cells transfected with the empty expression vector and
the reporter plasmid (data not shown). Significant differences in
transactivation were obtained when comparing wild-type and mutant SF-1
(Fig. 3). All the six mutants showed
decreased ability to transactivate pT81-4CRS2LUC. In mutants 4, 5, and
6, the relative luciferase activity obtained was 21, 32, and 14% of
that obtained with wild-type SF-1, respectively. The -helical region
of AF-2 is strongly hydrophobic and direct contact with ligand has been shown in the thyroid receptor (T3R
) and the retinoic acid receptor (RAR
) (19, 20). According to the crystal structures of T3R
and
RAR
, the leucine residues Leu451 and Leu452
as well as Glu454 in SF-1 are predicted to be surface
exposed in the helical core and form part of an interaction surface
recognized by cofactors or ligand. Leu451 and
Glu454 are among the most conserved residues in other
nuclear receptors, and Glu454 is not only surface exposed
but also extends into the solvent. The dramatic effect of mutating
Glu454 in SF-1 supports the notion that this is an
essential residue for transactivation by nuclear receptors.
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SF-1 Mutated in the AF-2 Domain Dominantly Suppresses cAMP-stimulated Transcription-- In Y-1 cells (mouse adrenocortical tumor cells), which express endogenous SF-1, transfection with the reporter plasmid pT81-4CRS2LUC resulted in clearly detectable levels of luciferase activity (Fig. 4). When the mutant SF-1 proteins were expressed, a significant reduction in the level of basal activity was observed. Transfection of the mutants 5 and 6, which carry deletions of the AF-2 domain, gave rise to relative luciferase activities that were 44 and 32%, respectively, of the activities obtained when the empty expression vector was used for transfection. These results show that the basal activity of the pT81-4CRS2LUC is attenuated through transfection with plasmids encoding SF-1 proteins deleted in the AF-2 core region.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. K. Morohashi for antibodies against SF-1/Ad4BP and Dr. K. Umesono for the plasmid encoding the hGR/green fluorescent protein fusion protein.
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FOOTNOTES |
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* This work was supported by grants from the Norwegian Cancer Society and the Norwegian Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Anatomy and
Cell Biology, University of Bergen, Årstadveien 19, N-5009 Bergen,
Norway. Tel.: 47-55-586361; Fax: 47-55-586360; E-mail: johan.lund{at}pki.uib.no.
1 The abbreviations used are: PKA, cAMP-dependent protein kinase; SF-1, steroidogenic factor-1; hGR, human glucocorticoid receptor; EMSA, electrophoretic mobility shift analysis; PKA-C, catalytic subunit of PKA; CRS2, cAMP-responsive sequence 2; AF-2, activation function-2; Ad4BP, adrenal 4-binding protein.
2 A. L. Jacob and J. Lund, unpublished observations.
3 M. Bakke and J. Lund, unpublished observations.
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REFERENCES |
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