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Endocrinology Vol. 142, No. 12 5097-5106
Copyright © 2001 by The Endocrine Society

RECEPTORS

DAX-1 Represses the High-Density Lipoprotein Receptor Through Interaction with Positive Regulators Sterol Regulatory Element-Binding Protein-1a and Steroidogenic Factor-1

Dayami Lopez, Wendy Shea-Eaton, Mark D. Sanchez and Mark P. McLean

Departments of Obstetrics and Gynecology (D.L., W.S.-E., M.D.S., M.P.M.) and Biochemistry and Molecular Biology (M.P.M.), University of South Florida, 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}hsc.usf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The high-density lipoprotein receptor (HDL-R) mediates the selective uptake of high-density lipoprotein cholesterol in nonplacental steroidogenic tissues. We have previously demonstrated that sterol regulatory element-binding protein-1a (SREBP-1a) and steroidogenic factor-1 (SF-1) positively regulate HDL-R gene transcription. In the present study, we examined whether DAX-1 (dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1) could influence the expression of the HDL-R gene. Cotransfection studies demonstrated that DAX-1 was able to repress SREBP-1a and SF-1-dependent activation of the HDL-R promoter. Mammalian two-hybrid assays demonstrated that DAX-1 could interact with SREBP-1a. In addition, electrophoretic mobility shift assays demonstrated that initial incubation of DAX-1 with SREBP-1a protein in the absence of DNA prevented subsequent binding of SREBP-1a to the HDL-R sterol regulatory elements in a dose-dependent manner, whereas, in the case of SF-1, DAX-1 formed a complex with SF-1 protein on the DNA. These data suggest that DAX-1 inhibits SREBP-1a- and SF-1-dependent activation of the HDL-R promoter through different mechanisms. This investigation confirms that DAX-1 has an important role in regulating steroidogenesis by interfering with SREBP-1a and SF-1 induction of a gene involved in the transport of cholesterol, thereby limiting the amount of substrate available for steroid hormone production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HIGH-DENSITY lipoprotein receptor (HDL-R), or scavenger receptor class B type I, mediates the selective uptake of cholesterol and cholesteryl esters, substrates for steroidogenesis, in the adrenal gland and gonads (1, 2, 3, 4, 5). Similar to other genes involved in steroid hormone production, HDL-R gene expression is induced by ACTH (6, 7, 8, 9, 10), LH (5, 6, 7, 8, 9, 10), and human CG (5, 6, 7, 11). It is well known that the binding of ACTH, LH, or human CG to its receptor promotes a series of reactions that leads to a decrease in tissue cholesteryl ester content and an increase in cAMP levels (11, 12, 13, 14, 15, 16). Earlier studies in our laboratory have shown that the rat HDL-R is regulated at the transcriptional level by sterol regulatory element binding protein-1a (SREBP-1a) and steroidogenic factor-1 (SF-1) in response to the decrease in cholesterol and the increase in cAMP levels, respectively (12, 17). Transfection studies and gel shift assays revealed the presence of two sterol regulatory elements (SREs; proximal and distal) and one SF-1-binding motif (SFB) in the rat HDL-R promoter (12, 17). The human HDL-R promoter has also been shown to contain an SF-1-binding motif through which SF-1 binds and activates this gene in Y1 cells and an E-box previously shown to bind SREBP-1a (18). These data suggest that the rat and human HDL-R genes may be regulated by the same factors (SREBP-1a and SF-1).

It is well known that negative factors also play a critical role in gene regulation. One negative factor that appears to be important for the regulation of the HDL-R gene is the dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1 (DAX-1). DAX-1 is an orphan nuclear receptor that has been shown to be required for adrenal development (19, 20, 21, 22). This orphan nuclear receptor displays a novel DNA-binding domain that lacks the characteristic zinc finger motif that is highly conserved among true nuclear receptors (23, 24, 25). Instead, the amino terminus of DAX-1 consists of three and one-half repeats of an alanine- and glycine-rich 65–67 amino acid motif that has been proposed to serve as its DNA-binding domain (23, 24, 25). The findings that DAX-1 and SF-1 are colocalized to steroidogenic tissues (26) and that targeted mutations of the SF-1 gene result in phenotypic features similar to those seen with DAX-1 mutations (27) suggest that DAX-1 and SF-1 may function together or in sequential steps during embryonic development. In vitro studies indicate that DAX-1 can strongly repress steroidogenesis and antagonize SF-1-mediated stimulation of genes such as steroidogenic acute regulatory protein (StAR) (28, 29), P450 side-chain cleavage enzyme (29), 3ß-hydroxysteroid dehydrogenase (29), Müllerian-inhibiting substance (30), aldose reductase-like protein (31), and P450 17{alpha} hydroxylase (32). DAX-1 has also been shown to act as a corepressor of the estrogen receptors {alpha} and ß (33). Interestingly, ovarian DAX-1 protein levels have been shown to increase in response to PGF2{alpha} treatment (28), leading to a decrease in HDL-R mRNA levels (34). These data suggest that DAX-1 may be responsible for the reduction in HDL uptake following PGF2{alpha} treatment.

Two mechanisms have been proposed by which DAX-1 represses gene expression. The first mechanism involves the direct binding of DAX-1 to DNA or RNA at stem-loop structures preventing gene expression (25, 35). Stem-loop structures to which DAX-1 binds have been identified in the mouse DAX-1 and human StAR promoters (25). The second DAX-1 inhibitory mechanism suggests that DAX-1 represses transcription by directly interacting with other nuclear receptors such as SF-1 (36, 37). Furthermore, the efficiency of DAX-1’s inhibitory effect on the P450 17{alpha} hydroxylase gene depends on the number of SF-1-binding elements found in the promoter (32). DAX-1 also acts as an adapter molecule that recruits other corepressors to DNA-bound complexes like SF-1 (37) and in this way prevents the interaction between the trans-acting factor and the transcription initiation complex (38, 39). Corepressors reported to interact with DAX-1 include the nuclear receptor corepressor and Alien (37, 40).

In the present study, the effect of DAX-1 on the regulation of the HDL-R gene was examined. DAX-1 inhibition of the HDL-R gene was found to be coactivator (SREBP-1a or SF-1) dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All oligonucleotides and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The pGL3-basic luciferase vector, renilla luciferase vector, and the dual luciferase reporter assay system were obtained from Promega Corp. (Madison, WI). The murine SF-1 cDNA under the control of the cytomegalovirus (CMV) promoter (SF-1-pCMV) was obtained from Dr. Keith L. Parker (University of Texas, Southwestern Medical Center, Dallas, TX). The rabbit polyclonal IgG antimouse SF-1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The NH2-terminal segment (active fragment) of SREBP-1a under control of the CMV promoter (SREBP-1a-pCMV5) and SREBP-1a-polyhistidine-tagged in the pRSET B vector were kindly provided by Dr. T. Osborne (Department of Molecular Biology and Biochemistry, University of California, Irvine). The rabbit polyclonal SREBP-1 and DAX-1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The cDNA encoding the murine DAX-1 was obtained in pcDNA 3.1 (+) (Invitrogen, Carlsbad, CA) from Dr. E. R. B. McCabe (UCLA School of Medicine, Los Angeles, CA). The mouse adrenal Y1, human liver HepG2, and human bladder carcinoma HTB-9 cell lines were obtained from American Type Culture Collection (Manassas, VA). The QuickChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). [{alpha}32P]Deoxy (d)-CTP (3000 Ci/mmol), the T7 Sequenase DNA sequencing kit, polydeoxyinosinic-deoxycytidylic acid, and the glutathione S-transferase (GST)-fusion protein purification kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). [35S]dATP (1000–1500 Ci/mmol) was obtained from Dupont/NEN Life Science Products (Wilmington, DE). The Fugene 6 transfection reagent was obtained from Roche Molecular Biochemicals (Indianapolis, IN). DMEM: nutrient mixture F-12 (DMEM/F12) was obtained from Life Technologies, Inc./BRL (Grand Island, NY). FBS was purchased from Summit Biotechnology (Ft. Collins, CO). BioMax-MR films were obtained from Fisher Scientific (Norcross, GA). Rivastatin (Merck & Co., Inc., Rahway, NJ) was provided by Dr. G. C. Ness (Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, FL). All other chemicals were purchased from Fisher Scientific or Sigma (St. Louis, MO).

Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange site-directed mutagenesis kit (Stratagene) as previously described (17). Oligonucleotides used for site-directed mutagenesis to delete the hairpin in the p-2267 HDL-R promoter construct were 5'-GTGCCAGGGATATCTGCCCTCTGTG-3', 5'-CTAGGCCAGCGATATCAGCAGGAAG-3', and their respective complements. Following site-directed mutagenesis, the mutated p-2267 construct was digested with EcoRV and religated thereby generating the hairpin negative promoter construct (p-2267 HN-HDL-R). To prepare truncated DAX-1 (tDAX-1), oligonucleotides used were 5'-GATCCGCTGAATGATGAAGTGCCCTTTTCC-3' and its complement. This mutation introduces two premature stop codons resulting in a truncated protein that lacks 42 amino acids at the carboxy terminal. The nucleotides in boldface letters correspond to the mutated bases. Mutations were confirmed by sequencing using the T7 sequenase DNA sequencing kit and [35S]dATP.

Cell transfections and luciferase assays
The p-2267 and p-719 HDL-R promoter-luciferase gene constructs were prepared as previously described (12). The hairpin negative HDL-R promoter construct (p-2267 HN-HDL-R) was prepared as described above. Y1, HTB-9, and HepG2 cells were transfected with the specified HDL-R promoter-luciferase gene constructs either in the presence or absence of DAX-1-pcDNA3.1 ± SREBP-1a-pCMV5 or SF-1-pCMV using the Fugene 6 transfection method (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Cells were first plated in either 6-well tissue culture plates at a density of 3 x 106 cells/well or 12-well tissue culture plates at a density of 5 x 104 cells/well and incubated for 24 h at 37 C (5% CO2). Fresh DMEM/F12 medium + 10% FBS was added before transfections. With the exception of DAX-1, one (for 6-well plates) or 0.5 µg (for 12-well plates) of each plasmid to be transfected was incubated with Fugene 6 (2:1, Fugene 6:DNA) in 100 µl of medium for 15 min. DAX-1 expression plasmid concentrations used in these studies varied from 0.5 to 10 µg (see Figures). DNA-Fugene 6 complex was then added to the cells. Cells were allowed to incubate for 48 h at 37 C (5% CO2). Dibutyryl cAMP ([Bu]2AMP, 1 mM), 8-bromo cAMP (8-Br-cAMP, 1 mM), or Rivastatin (Riv, 50 µM) was added to plates 24 h before the end as per the study design.

Cell lysate preparation and luciferase assays were performed as previously described (17). Cotransfection of a plasmid containing the renilla luciferase gene (0.5 µg for 6-well plates or 0.25 µg for 12-well plates) under control of the SV40 early enhancer/promoter region was used as a control to correct for differences in transfection efficiencies. Luciferase data were expressed as the mean ± SEM, in which the value of luciferase activity for the pGL3-basic empty vector was set to 1.0, unless otherwise specified. 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 (41). Values of P < 0.05 was considered significant for all tests.

For Western blot analysis, cells were lysed in PBS by repetitive freezing and thawing, and proteins were then concentrated by adding 8 vol of ice-cold acetone. Electrophoresis and transfer was performed as previously described (12).

Recombinant protein preparation
GST-DAX-1 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 7700x 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 then added to a final concentration of 1% and the sample 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) as per the manufacturer’s recommendations. After washing the column with PBS, the fusion protein was digested with precision protease to remove the GST moiety and used in gel mobility shift assay. For histidine-tagged SREBP-1a protein purification, the cell pellet after the initial centrifugation was resuspended in guanidinium lysis buffer provided in the XPRESS system (Invitrogen, Carlsbad, CA). Purification was performed using an immobilized metal affinity column according to the manufacturer’s recommendations. Recombinant protein samples were concentrated 4- to 6-fold using Centricon-10 concentrators (Millipore Corp., Bedford, MA). Protein concentrations were determined using the protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Preparation of Y1 nuclear extracts
Adrenal Y1 cells (5 x 107) were treated with 0.25% trypsin in 1 mM EDTA and then centrifuged. Nuclear extracts were prepared from the cell pellets as previously described (12). Protein samples were concentrated 4- to 6-fold using Centricon-10 concentrators (Millipore Corp.), and concentrations were determined using the protein assay (Bio-Rad Laboratories, Inc.).

Gel mobility shift assay
Oligonucleotides corresponding to the HDL-R promoter region from -1966 to -1940 [distal SRE (dSRE): 5'-CTGCCCCCCTCACACCCTCCTCTGTAG-3'] and from -246 to -220 [proximal SRE (pSRE): 5'-CCATCAGAGCACCGCCCACTCCCCGCC-3'], or from -655 to -628 [SFB: 5'-GACAGTGCATCAAGGCCGCGAGGGACA-3'] 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 probe was then labeled using the Klenow fragment of DNA polymerase and [{alpha}32P]dCTP (3000 Ci/mmol). Unlabeled oligonucleotides were used as a competitor in some experiments. Five micrograms nuclear protein (or 50 ng recombinant protein, unless otherwise indicated) were incubated either 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 dithiothreitol, and 4 mM Tris-HCl (pH 8.0)], 2 µg polydeoxyinosinic-deoxycytidylic acid, and 1 µg BSA. After incubation, 50,000–100,000 cpm of the radiolabeled probe was added and the mixture incubated for 20 min at 15 C. Where indicated, SREBP-1a or SF-1 antibody was also added to the reaction for supershift analysis. The DNA protein complexes were resolved on a 4% (or 6% in case of the SF-1 protein) nondenaturing acrylamide gel at 4 C in 1x TGE (0.25 M Tris, 1.9 M glycine, and 10 mM EDTA). Gels were then vacuum dried and exposed to BioMax-MR films at -80 C for 12–24 h or developed using a Cyclone storage phosphor system (Packard Instrument Co., Meriden, CT). Densitometric quantitation of the autoradiograms was performed using the UVP imaging system and Labworks software (UVP Laboratory Products, Upland, CA).

For Western blot analysis, unlabeled mobility shift assay gels were electroblotted onto nitrocellulose membranes (0.2-µm pore) in buffer containing 0.25 M Tris-HCl (pH 8.3), 1.92 M glycine, and 20% methanol at 4 C overnight using 200 mA.

Western blot analysis
Western blot analysis was carried out with a 1:1000 dilution of the rabbit polyclonal antiserum to DAX-1, SREBP-1a, or SF-1 in 3% milk. Immunoreactive proteins were then visualized using a 1:6000 dilution of horseradish peroxidase-conjugated goat antirabbit antisera (Santa Cruz Biotechnology, Inc.) in 3% milk and the SuperSignal ULTRA chemiluminescent substrate method (Pierce Chemical Co., Rockford, IL).

Mammalian two-hybrid assay
The plasmids pBIND, pACT, and pG5-luciferase used in these assays were provided in the mammalian two-hybrid system (Promega Corp.). The plasmid SF-1-pBIND was constructed using full-length mouse SF-1 cDNA inserted into the BamHI and NotI sites. To construct the plasmid SREBP-1a-pBIND, the NH2-terminal active segment of SREBP-1a was inserted into the EcoRI and KpnI sites. The DAX-1-pACT plasmid was prepared using full-length mouse DAX-1 cDNA inserted into the XhoI/SalI and NotI sites. The tDAX-1-pACT plasmid was prepared by site-directed mutagenesis as described above. To examine luciferase activities, HTB-9 cells (plated in six-well plates) were cotransfected with the pG5-luciferase vector either in the presence or absence of SF-1-pBIND (or SREBP-1a-pBIND) ± DAX-1-pACT (or tDAX-1-pACT). For these experiments, transfections were performed with 2 µg of each plasmid using the Fugene 6 transfection reagent Roche Molecular Biochemicals according to the manufacturer’s instructions. After transfection, cells were incubated for 48 h at 37 C before harvesting. Transfection efficiencies were corrected using renilla activity expressed by the pBIND plasmid. Statistical analysis was performed as described in Cell transfections and luciferase assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the effects of DAX-1 on luciferase activity under control of the HDL-R promoter in the absence of any transcriptional activators, human bladder HTB-9 cells were transfected with the p-2267 (Fig. 1BGo) or the p-719 (Fig. 1CGo) HDL-R promoter construct in the presence or absence of DAX-1-pcDNA3.1 plasmid. Figure 1AGo shows a schematic representation of both promoter constructs. These studies were completed in HTB-9 cells because these cells have undetectable levels of HDL-R, SF-1, SREBP-1a, or DAX-1. DAX-1 reduced HDL-R promoter-driven luciferase activity of both promoter constructs by 50% and 70%, respectively (P < 0.01; Fig. 1Go, B and C). These data suggest that DAX-1 can inhibit basal HDL-R promoter activity. The p-2267 promoter construct consistently displayed basal activity levels similar to the pGL3-basic (control) plasmid alone, whereas the p-719 promoter construct induced luciferase activity 30-fold (P < 0.001) over pGL3-basic activity (Fig. 1Go, B and C). This could be owing to the presence of a negative regulatory region in the p-2267 promoter that prevents maximal activation of the HDL-R promoter in HTB-9 cells. It has been previously shown that this promoter region contains two Yin Yang 1 (YY1) binding sites (42) that may be responsible for decreased basal activity of the p-2267 HDL-R promoter construct in the HTB-9 cells.



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  		Figure 1. Effects of DAX-1 on the HDL-R promoter activity in the absence of either SF-1 or SREBP-1a in human bladder carcinoma HTB-9 cells. A, Schematic representation of the regulatory elements found in each HDL-R promoter construct. HP (hairpin), Putative DAX-1 binding motif. HTB-9 cells (6-well plates) were transfected with the specified promoter construct (p-2267 in B and p-719 in C) in the presence or absence of DAX-1-pcDNA3.1 plasmid. The amounts of plasmid DNA used per well in these transfection experiments were 0.5 µg for renilla and 1 µg each for the other plasmids. Variations in total DNA amount per well were corrected by adding enough pcDNA3.1 empty vector. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pGL3-basic empty plasmid was set to 1.0 and are from a typical experiment performed in triplicate. These experiments were repeated two times. *, P < 0.01, obtained by comparing with the HDL-R promoter activity in the absence of DAX-1.

 
To examine whether DAX-1 affects SF-1- or SREBP-1a-dependent HDL-R promoter activity, HTB-9 cells were cotransfected with the indicated construct in the presence of increasing amounts of DAX-1-pcDNA3.1 and SF-1-pCMV or SREBP-1a-pCMV5 plasmids. [Bu]2cAMP was added to the corresponding plates 24 h before lysing the cells. Because SF-1 has been shown to be the mediator of cAMP stimulation of the HDL-R gene (17), [Bu]2cAMP was added only to plates containing SF-1. As shown in Fig. 2Go, A and B, DAX-1 significantly reduced SF-1 + [Bu]2cAMP- or SREBP-1a-dependent activation of the HDL-R promoter in a dose-dependent manner. Ten micrograms DAX-1-pcDNA3.1 plasmid reduced SF-1 + [Bu]2cAMP- and SREBP-1a-dependent activation of the HDL-R promoter by 93% and 96% (P < 0.001), respectively (Fig. 2Go, A and B). The DAX-1’s effect on SF-1 and SREBP-1a-dependent activity of the HDL-R promoter were specific because the total amount of DNA plasmid transfected per well was corrected using pcDNA3.1 empty vector. In addition, Western blot analysis demonstrated that DAX-1 protein levels increased as increasing amounts of the DAX-1 expression plasmid were transfected into the cells, whereas SREBP-1a protein levels were not significantly changed (Fig. 2C).



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  		Figure 2. Effects of adding increasing amounts of DAX-1 on SF-1- or SREBP-1a-dependent HDL-R promoter activity in HTB-9 cells. Cells (six-well plates) were transfected with the indicated HDL-R promoter construct in the presence of increasing amounts (1–10 µg; 1x = 1 µg) of DAX-1-pcDNA3.1 plasmid ± SF-1-pCMV (A) or SREBP-1a-pCMV5 (B). The amounts of plasmid DNA used for the other plasmids were 1 µg (renilla 0.5 µg). Variations in total DNA amount per well were corrected by adding enough pcDNA3.1 empty vector. Dibutyryl cAMP ([Bu]2cAMP; 1 mM) was added to the corresponding plates 24 h before lysing the cells. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pGL3-basic empty plasmid was set to 1.0 and are from a typical experiment performed in triplicate. These experiments were repeated three times. *, P < 0.01 and **, P < 0.001 were obtained by comparing with the HDL-R promoter activity in the presence of SF-1 + [Bu]2cAMP (A) or SREBP-1a (B). C, Transfections were performed as described in B. Forty-eight hours later, cells were lysed in PBS by repetitive freezing and thawing, and proteins were concentrated by adding 8 vol ice cold acetone. Electrophoresis, transfer, and Western blot analysis were performed as described in Materials and Methods. Representative Western blots are shown.

 
Similar DAX-1 repression studies were performed using mouse adrenal Y1 (Fig. 3AGo) and human liver HepG2 cells (Fig. 3BGo). These cells endogenously express SF-1 and SREBP-1a, respectively. Rivastatin, a cholesterol biosynthesis inhibitor, causes a decrease in intracellular cholesterol levels with a corresponding increase in mature SREBP levels. As shown in Fig. 3Go (upper panels), DAX-1 was able to significantly repress basal, 8-Br-cAMP, and rivastatin-dependent activation of the HDL-R promoter. Likewise, DAX-1 was able to repress SF-1 (±8-BrcAMP) and SREBP-1a (±Riv)-dependent activation of the HDL-R promoter in Y1 and HepG2 cells, respectively (Fig. 3Go, lower panels).



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  		Figure 3. Effects of DAX-1 on HDL-R promoter activity in mouse adrenal Y1 (A) and human liver HepG2 (B) cells. Cells (plated in 12-well plates) were transfected with the p-2267 HDL-R promoter construct in the presence or absence of DAX-1-pcDNA3.1 plasmid ± SF-1-pCMV (A) or SREBP-1a-pCMV5 (B). The amounts of plasmid DNA used per well in these transfection experiments were 0.25 µg for renilla and 0.5 µg each for the other plasmids. A, 8-Bromo-cAMP (1 mM) was added to the corresponding plates 24 h before harvesting. B, Rivastatin (Riv; 50 µM) was added to some of the plates 24 h before lysing the cells. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pGL3-basic empty plasmid was set to 1.0 and are from a typical experiment performed in triplicate. These experiments were repeated three times in the Y1 cells and twice in the HepG2 cells. *, P < 0.001, **, P < 0.02, and ***, P < 0.05 were obtained by comparing with the HDL-R promoter activity in the absence of DAX-1.

 
It has been previously shown for the human StAR and mouse DAX-1 genes that DAX-1 can bind and repress their promoter activity through DNA hairpin structures (25). To examine whether a similar mechanism may occur within the HDL-R gene, sequence analysis of the rat HDL-R promoter (12) was performed. It was found that the rat HDL-R promoter contains a putative DAX-1-binding motif composed of 15-bp loops and 13-bp stems and is located between -404 and -353 of the HDL-R promoter (Fig. 4Go).



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  		Figure 4. Putative DAX-1-binding sites in the rat HDL-R gene promoter. This motif is composed of 15-bp loops and 13-bp stems and is located between -404 and -353 of the HDL-R promoter.

 
To determine whether the putative DAX-1 hairpin-binding motif (HP) identified in the HDL-R promoter (Fig. 4Go) was involved in DAX-1 inhibition of HDL-R gene expression, a mutated promoter construct was prepared as described in Materials and Methods. Figure 5Go, B and C, demonstrates that deleting the DAX-1-binding site (p-2267 HN-HDL-R) did not significantly affect basal or SF-1 ± [Bu]2cAMP or SREBP-1a-dependent activation of the HDL-R promoter in HTB-9 cells. The lack of the putative DAX-1 hairpin-binding structure did not prevent DAX-1 repression of the HDL-R promoter in the presence or absence of SF-1 or SREBP-1a (Fig. 5Go, B and C). Similar results were obtained in Y1 and HepG2 cells (data not shown). These results suggest that DAX-1 does not mediate repression of the HDL-R gene by binding to the hairpin structure found in this promoter. These results correlate with our findings that recombinant DAX-1 protein did not bind to the HDL-R promoter hairpin structure (data not shown) under conditions identical to those used for the human StAR and mouse DAX-1 hairpin structures (25).



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  		Figure 5. Effects of deleting the putative DAX-1 binding motif found in the HDL-R promoter on DAX-1 repression of this gene. A, Schematic representation of the wild-type (p-2267 HDL-R) and hairpin-negative (p-2267 HN-HDL-R) promoter constructs. HP refers to DAX-1 hairpin binding motif. HTB-9 cells (plated in 12-well plates) were transfected with the specified promoter construct in the presence or absence of DAX-1 ± SF-1 (B) or SREBP-1a (C) expression plasmids. The amounts of plasmid DNA used per well in these transfection experiments were 0.25 µg for renilla and 0.5 µg each for the other plasmids. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pGL3-basic empty plasmid was set to 1.0 and are from a typical experiment performed in triplicate. These experiments were repeated two times. B, [Bu]2cAMP (1 mM) was added to the corresponding plates 24 h before harvesting.

 
It has been previously shown using both two-hybrid (28, 30) and GST pull-down (30) assays that DAX-1 and SF-1 proteins interact. To examine whether DAX-1 and SF-1 protein-protein interaction influences SF-1 protein binding to the SFB found in the HDL-R promoter, mobility shift assays were performed. For this, nuclear extracts prepared from Y1 cells were incubated with 32P-labeled SFB oligonucleotide in the presence or absence of DAX-1 recombinant protein. As shown in Fig. 6AGo, 5Go µg Y1 adrenal cell nuclear proteins formed a major complex (lower arrow, lane 2) with radiolabeled SFB oligonucleotide. The addition of 125-fold excess of unlabeled SFB (lane 3) or SF-1 specific antibody (9.6 µg IgG, lane 4) reduced protein/DNA complex formation demonstrating the specificity of the complex formed and that SF-1 protein is part of that complex. Adding recombinant DAX-1 to the binding reaction shifted the SF-1/SFB complex to a higher molecular weight (upper arrow) in a dose-dependent manner (Fig. 6AGo, lanes 5–6 and 7–8). Apparently, DAX-1 and SF-1 formed a complex with the SFB motif causing the supershift. These effects appear to be specific for DAX-1 because the addition of recombinant GST to the binding reaction before or after the addition of DNA did not alter the protein/DNA complex (Fig. 6AGo, lanes 9 and 10). DAX-1 did not bind to the SFB motif in the absence of Y1 nuclear extracts (data not shown) suggesting that the supershifted complex resulted from direct protein-protein interaction between DAX-1 and SF-1.



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  		Figure 6. Recombinant DAX-1 forms a complex with SF-1 on the SFB found in the promoter of the HDL-R gene. A, Two concentrations of recombinant DAX-1 (50 and 150 ng) were allowed to bind to 5 µg Y1 nuclear extracts before (lanes 5 and 6) or after (lanes 7 and 8) the addition of 32P-labeled SFB oligonucleotide. A 125-fold excess of unlabeled SFB oligonucleotide was used as a competitor, where indicated. SF-1 antibody (SF-1 AB, 9.6 µg IgG) was added to lanes 4 and 11 to identify the presence of SF-1 in the DNA/protein complexes (arrows). Recombinant GST (negative control) was added to some of the lanes before (9 ) or after (10 ) the addition of 32P-labeled SFB oligonucleotide. The DNA-protein complexes were resolved on a 6% nondenaturing acrylamide gel at 4 C in 1x TGE. This experiment was repeated two times. B, A standard mobility shift assay with triplicate gels was run on the SFB oligonucleotide using Y1 nuclear extracts (5 µg) with increasing concentrations recombinant DAX-1 (50–300 ng). Recombinant GST (negative control) was added to one lane. The top gel represents the normal autoradiogram from the mobility shift assay, whereas the middle and bottom gels were transferred to nitrocellulose membrane, treated as a Western blot, and probed with SF-1 and DAX-1 specific antibodies, respectively.

 
The interaction between DAX-1 and SF-1 was not influenced by the order in which the proteins and the DNA were added to the binding reaction nor did DAX-1 prevent SF-1 from binding the SFB motif (Fig. 6AGo). The presence of DAX-1 in the supershifted complex was demonstrated using Western blot analysis (Fig. 6BGo, lanes 4–6), whereas the presence of SF-1 in the supershifted complex was demonstrated by abrogation (Fig. 6AGo, lane 11) and by Western blot analysis (Fig. 6BGo, lanes 4–6). Using our current conditions, SF-1 protein binding to the DNA prevents antibody recognition. Therefore, the presence of SF-1 in the SF-1/SFB complex when the binding reactions were performed in the absence of DAX-1 or in the presence of GST was not detected by Western blot analysis (Fig. 6BGo, lanes 2 and 7, respectively). It appeared that DAX-1 interaction with SF-1 induced a conformational change in the SF-1 protein that permitted DNA binding but also exposed that portion of the SF-1 protein necessary for antibody recognition.

To examine whether DAX-1 affected SREBP-1a binding to the HDL-R SREs in a similar fashion, recombinant SREBP-1a protein was incubated with DAX-1 before the addition of dSRE or pSRE, and the results are shown in Fig. 7AGo. In the absence of DAX-1, SREBP-1a formed a single DNA/protein complex with both SREs, which was specifically reduced by the addition of cold oligonucleotide (100x) and supershifted with an SREBP-1a specific antibody (2 µg IgG; Fig. 7AGo, lanes 2–4 and 9–11, respectively). Surprisingly, rather than supershifting the DNA/protein complex as in the case of SF-1 (Fig. 6Go), DAX-1 prevented SREBP-1a binding to the DNA in a dose-dependent manner (Fig. 7AGo). Recombinant DAX-1 protein (100 ng) reduced SREBP-1a binding to dSRE more efficiently than binding to pSRE, whereas higher amounts of DAX-1 (250–500 ng) reduced SREBP-1a binding to pSRE more efficiently than to dSRE (Fig. 7AGo). The effects of DAX-1 on SREBP-1a binding were not significantly influenced by the order in which the radiolabeled dSRE oligonucleotide was added (i.e. before and after adding the DNA to SREBP-1a containing binding reaction; Fig. 7BGo). Similar results were obtained in the case of the pSRE (data not shown). To rule out the possibility that a nuclear factor may be responsible for causing the supershift seen with SF-1, binding reactions were also performed using nuclear extracts containing SREBP-1a, and once again, DAX-1 prevented SREBP-1a binding to the dSRE (data not shown). These results suggested that the effects were factor dependent (i.e. SF-1 vs. SREBP-1a).



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  		Figure 7. Recombinant DAX-1 inhibits SREBP-1a binding to the SREs found in the HDL-R promoter. A, Increasing concentrations of recombinant DAX-1 (100–500 ng) were allowed to bind to recombinant SREBP-1a (400 ng) before the addition of 32P-labeled dSRE or pSRE oligonucleotide. A 100-fold excess of unlabeled dSRE or pSRE oligonucleotide was used as a competitor, where indicated. SREBP-1a antibody (SREBP-1 AB, 2 µg IgG) was added to some of the lanes (4 11 ) to identify the presence of SREBP-1a in the DNA-protein complexes (arrows). The DNA-protein complexes were resolved on a 4% nondenaturing acrylamide gel at 4 C in 1x TGE. Typical mobility shift assay autoradiograms are shown. Densitometric quantitation of the autoradiograms was performed using the UVP imaging system and Labworks software (UVP Laboratory Products). The percent remaining was calculated by comparing the density of the major protein-DNA band seen with SREBP-1a alone with the band formed in the presence of SREBP-1a/DAX-1. This experiment was repeated twice for both SREs. B, Increasing concentrations of recombinant DAX-1 (100–1000 ng) were allowed to bind to recombinant SREBP-1a (400 ng) after the addition of 32P-labeled distal SRE oligonucleotide. The percent remaining was calculated as described in A.

 
To determine whether SREBP-1a and DAX-1 physically interact with each other, mammalian two-hybrid assay was performed as described in Materials and Methods. As shown in Fig. 8Go, cotransfecting DAX-1 and SREBP-1a with the pG5-basic vector caused an 8-fold increase (P < 0.001) in luciferase activity, compared with cells cotransfected with SREBP-1a-pBIND and pACT empty vector. These results demonstrated that DAX-1 and SREBP-1a were able to interact in the mammalian two-hybrid assays.



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  		Figure 8. Interaction between DAX-1 and SREBP-1a using the mammalian two-hybrid assay. HTB-9 cells (plated in 6-well plates) were transfected with the pG5-luciferase vector in the presence of the indicated vectors (2 µg of each plasmid DNA per well). The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pG5-basic plasmid transfected in the presence of the pACT/pBIND negative controls was set to 1.0. The data are from a typical experiment performed in triplicate. These experiments were repeated four times. *, P < 0.001 was obtained by comparing with the pACT/SREBP-1a-pBIND control.

 
It has been previously shown that DAX-1 inhibition of SF-1-dependent transactivation (28, 36) is mediated by a carboxy-terminal transrepression domain. Thus, to examine whether this domain was involved in DAX-1 repression of the HDL-R promoter, a truncated DAX-1 form was generated as described in Materials and Methods and used in transfection studies. The results obtained using HTB-9 cells are shown in Fig. 9Go. The lack of the carboxy-terminal transrepression domain caused a loss of repression relative to the wild-type DAX-1 in the presence of SF-1 or SREBP-1a. Similar results were obtained in Y1 and HepG2 cells (data not shown). Mammalian two-hybrid assays were performed to examine whether tDAX-1 could still interact with SF-1 and SREBP-1a. The data in Fig. 10Go are represented as fold-induction ± SEM where the value of luciferase activity for the construct transfected in the presence of the pACT/pBIND negative controls was set to 1.0 (data not shown). As shown, the lack of the transrepression domain within the DAX-1 protein also resulted in a loss of interaction between DAX-1 and SF-1 or SREBP-1a. These results suggested that the DAX-1 transrepression domain was involved in the interaction of DAX-1 with proteins such as SF-1 and SREBP-1a.



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  		Figure 9. Effects of deleting the C-terminal transrepression domain of the DAX-1 protein (tDAX-1) on DAX-1 repression of the HDL-R promoter activity. HTB-9 cells (plated in 12-well plates) were transfected with the p-2267 HDL-R promoter construct in the presence or absence of tDAX-1 ± SF-1 (A) or SREBP-1a (B) expression plasmids. Control transfections were performed in the presence of wild-type DAX-1 expression plasmid (DAX-1). The amounts of plasmid DNA used per well in these transfection experiments were 0.25 µg for renilla and 0.5 µg each for the other plasmids. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the pGL3-basic empty plasmid was set to 1.0 and are from a typical experiment performed in triplicate. These experiments were repeated two times. A, [Bu]2cAMP (1 mM) was added to the corresponding plates 24 h before harvesting. *, P < 0.01 and **, P < 0.05 were obtained to the HDL-R promoter activity in the absence of DAX-1.

 


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  		Figure 10. An intact C-terminal transrepression domain is required for DAX-1 interaction with SF-1 or SREBP-1a as demonstrated using the mammalian two-hybrid assay. HTB-9 cells (plated in 6-well plates) were transfected with the pG5-luciferase vector in the presence of the indicated vectors (2 µg of each plasmid DNA per well). The data are represented as fold-induction ± SEM where the value of luciferase activity obtained from the pG5-basic plasmid transfected in the presence of the pACT/pBIND negative controls was set to 1.0 (data not shown). The data are from a typical experiment performed in triplicate. These experiments were repeated two times. *, P < 0.01 was obtained by comparing with the pACT/pBIND negative control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented in this study provide evidence that DAX-1 inhibits rat HDL-R gene transcription by directly interacting with the coactivators of this gene, SREBP-1a and SF-1. Although DAX-1 was able to inhibit HDL-R gene transcription through both cofactors (SREBP-1a or SF-1), the exact mechanisms for this inhibition appeared to differ between the coactivators. In the case of SF-1, DAX-1 did not affect SF-1 binding to the SF binding motif but rather formed a complex with SF-1 on the DNA that may possibly prevented SF-1 interaction with other coactivators or the transcription initiation complex. In the case of SREBP-1a, however, DAX-1 prevented SREBP-1a binding to the HDL-R SREs, resulting in a subsequent decrease in promoter activity.

The finding that DAX-1 does not interfere with SF-1 binding to the DNA has been previously reported (30, 36). However, these groups were not able to show heterodimer formation between DAX-1 and SF-1 in mobility shift assays (30, 36) perhaps owing to the close proximity between the two complexes (SF-1/DNA vs. SF-1/DAX-1/DNA). In the current study, we were able to separate the complexes using a higher percentage acrylamide gel (6% vs. 4%). One interesting fact, however, is the Western blot result showing that the SF-1 specific antibody was able to recognize the SF-1 protein in the protein-DNA complexes only when DAX-1 protein was present, perhaps owing to a conformation change in the SF-1 protein upon interaction with DAX-1. This conformation change in the SF-1 protein may prevent SF-1 interaction with its coactivator or allow the interaction of SF-1 with a corepressor. DAX-1 has also been shown to interact with estrogen receptors {alpha} or ß on the DNA, forming a higher molecular weight complex (33).

The most novel finding of the current study is that DAX-1 inhibited SREBP-1a-dependent activation of the HDL-R promoter by directly interacting with SREBP-1a to prevent SREBP-1a binding to the HDL-R SREs. DAX-1 was also able to reduce SREBP-1a/SRE binding even if SREBP-1a was allowed to bind the DNA first followed by DAX-1 addition, suggesting that the protein region of SREBP-1a that interacts with DAX-1 may be critical for SREBP-1a DNA binding. If the amount of SREBP-1a that was bound to the DNA was reduced owing to the presence of DAX-1, factors involved in the formation of the transcription initiation complex may not be properly recruited, causing a decrease in HDL-R gene transcription. Whether the recruitment of corepressors by DAX-1 is involved in DAX-1’s repression of SREBP-1a-dependent HDL-R promoter activity requires further study.

Another interesting finding was that DAX-1’s efficiency to prevent SREBP-1a binding to the DNA differs depending on the amount of DAX-1 present in the binding reaction and the type of SRE. Because both proteins used in these experiments, SREBP-1a and DAX-1, were purified to the homogeneity, the only other difference that could be responsible for this discrepancy would be the oligonucleotide sequence (both oligonucleotides are 30 bp long). The oligonucleotide containing the pSRE also contains a putative Sp1 site, whereas the oligonucleotide containing the dSRE does not contain any other known response elements. Whether this difference in DAX-1’s efficiency is critical to its action in vivo will require additional study.

We have recently reported that the repressor YY1 also inhibits HDL-R gene transcription by preventing SREBP-1a binding to the DNA (42). Interestingly, both DAX-1 (28) and YY1 (Nackley, A. C., W. Shea-Eaton, D. Lopez, and M. P. McLean, unpublished observations) protein levels increase significantly in rat ovary after PGF2{alpha} treatment, suggesting that these negative transcriptional regulators may act together to inhibit the expression of the HDL-R gene during luteolysis. The necessity for two repressors to downregulate the HDL-R gene under these conditions is still unknown.

Herein, it was also demonstrated that the hairpin structure found in the HDL-R promoter is not involved in DAX-1 inhibition of this gene. Similar results have been reported for the rat StAR gene (28). However, in the case of the human StAR and mouse DAX-1 genes, the hairpins were able to bind DAX-1, and deleting them from the promoter prevented DAX-1 inhibition (25). Further support for DAX-1 inhibition by mechanisms that do not require hairpin secondary structures comes from experiments with the human CYP17 gene that does not contain a hairpin structure, yet it is strongly inhibited by DAX-1 (32). It has been proposed that efficient repression of genes by DAX-1 may require multiple SF-1 elements (32). Genes such as the type II 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase promoter, which contains a single SF-1 binding site are not significantly repressed by DAX-1 (29, 43). In the case of the rat HDL-R gene, there is a single SF-1 binding site; however, the data indicate that DAX-1 strongly represses this gene perhaps owing to the presence of the SREs. The finding that deleting the HDL-R hairpin structure did not prevent DAX-1 inhibition of the HDL-R promoter in the absence of SF-1 and SREBP-1a suggests that DAX-1 may also inhibit this promoter through interaction with additional factors. In addition to its inhibitory effects on transcription, DAX-1 has also been found to inhibit translation by directly binding RNA hairpin structures (35). Whether DAX-1 is able to inhibit the translation of the HDL-R mRNA through RNA hairpin structures needs to be examined.

Because mutations of human DAX-1 cause X-linked adrenal hypoplasia congenita (20, 21, 23), extensive studies have been done to identify regions within the DAX-1 protein involved in DAX-1’s inhibitory effects. Zhang et al. (33) have shown that DAX-1 and estrogen receptor interaction is mediated by the DAX-1 N-terminal repeat domain. The C-terminal transrepression domain has been implicated in SF-1-mediated transactivation (28, 36). In the current study, we demonstrated that deleting the last 42 amino acids of the DAX-1 protein (transrepression domain) also eliminated DAX-1 inhibition of the HDL-R promoter. These results are consistent with the findings that the C-terminal DAX-1 protein region was involved in nuclear receptor corepressor recruitment (37). Interestingly, most naturally occurring DAX-1 deletions eliminate the carboxy-terminal inhibitory domain (36). In contrast to other studies (30, 36), we have demonstrated that deleting the transrepression domain of the DAX-1 protein prevented DAX-1 interaction with SF-1 and SREBP-1a. The differences in these results could be owing to the techniques used to examine protein-protein interactions (GST pull-down vs. mammalian two-hybrid) or to the exact deletion analyzed.

In conclusion, the data shown here demonstrate that DAX-1 may use different repressive mechanisms to inhibit SF-1- and SREBP-1a-activation of the HDL-R gene. This investigation confirms that DAX-1 has an important role in regulating steroidogenesis by interfering with SREBP-1a- and SF-1-induction of a gene involved in the transport of cholesterol thereby limiting the amount of substrate available for steroid hormone production.


   Acknowledgments
 
We thank Dr. Keith L. Parker, Dr. Tim Osborne, Dr. E. R. B. McCabe, and Dr. G. C. Ness for gifts of materials.


   Footnotes
 
This work was supported by grants from the National Institutes of Health Grants HD-31644 and HD-35163, and American Heart Association Florida Affiliate Grant-in-Aid 0150973B (to M.P.M.). D.L. was supported by National AHA Scientist Development Grant 0030172N.

Abbreviations: CMV, Cytomegalovirus; DAX-1, dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1; dSRE, distal SRE; GST, glutathione S-transferase; HDL-R, high-density lipoprotein receptor; HP, hairpin-binding motif; pSRE, proximal SRE; SF-1, steroidogenic factor-1; SFB, SF-1-binding motif; SF-1-pCMV, CMV promoter; SRE, sterol regulatory elements; SREBP-1a, sterol regulatory element-binding protein-1a; SREBP-1a-pCMV5, SREBP-1a under control of the CMV promoter; StAR, steroidogenic acute regulatory protein; tDAX-1, truncated DAX-1; TGE, 0.25 M Tris, 1.9 M glycine, and 10 mM EDTA.

Received April 2, 2001.

Accepted for publication August 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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