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Expression of the Mouse Gonadotropin-Releasing Hormone Receptor Gene in T3-1 Gonadotrope Cells Is Stimulated by Cyclic 3',5'-Adenosine Monophosphate and Protein Kinase A, and Is Modulated by Steroidogenic Factor-1 and Nur77

Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa

Address all correspondence and requests for reprints to: Dr. Janet Hapgood, Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa. E-mail: jhap{at}sun.ac.za.

	Abstract

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Regulation of GnRH receptor (GnRHR) expression levels in the pituitary is a crucial control point in reproduction. The promoter of the mouse GnRHR gene contains nuclear receptor half-sites (NRS) at –244/-236 and -15/-7 relative to the translation start site. Although binding of steroidogenic factor-1 (SF-1) to the –244/-236NRS is implicated in mediating basal and gonadotrope-specific expression, no function or protein-DNA interactions have previously been described for the –15/-7NRS. We report that levels of the endogenous GnRHR mRNA in T3-1 cells are stimulated by forskolin and 8-bromo-cAMP. We also show that the orphan nuclear receptor Nur77 is expressed in T3-1 cells, and that both SF-1 and Nur77 bind to the –15/-7NRS and –244/-236NRS in vitro. We show that the activity of the proximal (-579/+1) mouse GnRHR promoter is up-regulated by protein kinase A, via a mechanism that is modulated by SF-1, both positively and negatively, through binding to the –244/-236NRS or the –15/-7NRS, respectively. Nur77 appears to be capable of acting as a negative regulator of this response, via the –15/-7NRS. Furthermore, we show that forskolin up-regulates SF-1 mRNA levels in T3-1 cells, indicating that the levels of SF-1 play a role in modulating the protein kinase A response.

	Introduction

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REGULATION of GnRH receptor (GnRHR) gene expression in pituitary gonadotropes plays a central role in the control of mammalian reproductive function (1). The response of pituitary gonadotropes to GnRH correlates directly with the level of GnRHR on the cell surface, which in turn is regulated by several hormones, including 17ß-estradiol, progesterone, testosterone, inhibin, activin, and GnRH itself (2, 3, 4, 5). Although the molecular mechanisms by which tissue-specific and hormonal factors regulate transcription of this gene have not been fully elucidated, several recent reports using pituitary-derived mouse cell lines and GnRHR promoter-reporter constructs have begun to unravel some of these mechanisms. In T3-1 cells (6), gonadotrope-specific regulatory elements are present in a 1.2-kb 5'-flanking genomic fragment (-1226/+12 relative to the start codon) of the mouse GnRHR gene (7) (all numbering from here on will be relative to the translation start site). A tripartite basal gonadotropic-specific enhancer occurs within the same promoter fragment and includes a binding site for the orphan nuclear receptor, steroidogenic factor-1 (SF-1), at –244/-236, a consensus activator protein-1 (AP-1) site at –336/-330, and a noncanonical key element termed GnRHR-activating sequence at –391/-380 (8, 9, 10). Cell-specific and hormonal regulation of the mouse GnRHR gene in T3-1 cells partially converge at the tripartite enhancer element, with GnRH responsiveness primarily conferred through protein kinase C (PKC) activation of the AP-1 element (11), and activin responsiveness conferred through the GnRHR-activating sequence element (12).

There is evidence that both the protein kinase A (PKA) and PKC pathways are involved in stimulation of mouse GnRHR gene transcription. However, results obtained in different model cell lines appear to differ, most likely reflecting the different repertoires of G proteins and/or transcription factors. Using rat pituitary GGH₃ cells, a GH₃-derived cell line stably expressing the rat GnRHR (13), it was shown that the response of the mouse GnRHR gene to GnRH involves stimulation by cAMP and the PKA pathway (14, 15), stimulation by Ca²⁺-dependent pathways and the PKC pathway (16), as well as inhibition of GnRH-stimulated activity by the MAPK pathway (16). A cAMP response element (CRE) at –107/-100 mediates the response to both cAMP and GnRH in GGH₃ cells (15). In contrast, experiments in T3-1 cells have shown that GnRH stimulation of the expression of a –600 mouse GnRHR promoter-reporter construct is mediated in full by the PKC pathway and via the AP-1 site at –336/-330, but not via the PKA pathway (11). These results were supported by Norwitz et al. (17), who also identified another element at –354/-347 that is required for the full GnRH response. These results in T3-1 cells are consistent with the report that the endogenous receptor in T3-1 cells is coupled exclusively to G_q/11 proteins (18). In contrast, the endogenous GnRHR in LßT2 cells, another mouse gonadotrope cell line, is coupled to both G_q/11 and G_s proteins (19). Interestingly, cultured rat pituitary tissue responds to GnRH stimulation by accumulating cAMP intracellularly (20), indicating that in primary cells, the GnRHR is coupled to G_s proteins. Thus, although it has been consistently demonstrated that transcription of the mouse GnRHR gene in T3-1 cells is up-regulated by GnRH via a mechanism involving the PKC pathway, but not the PKA pathway, whether the mouse gene is regulated by the PKA pathway in response to other signals seems less clear. White et al. (11) found that activation of the PKA pathway for 4 h by forskolin had no effect on the basal activity of a -600 mouse GnRHR promoter fragment. In contrast, for the rat GnRHR gene, in which both the SF-1 site (-244/-237) and the imperfect CRE (-110/-103) are conserved (see Fig. 1), it has been shown that pituitary adenylate cyclase-activating polypeptide (PACAP) up-regulates promoter activity via PKA in T3-1 cells after 16 h via these elements (21). Very few studies have investigated the effects of activation of kinase pathways on endogenous GnRHR mRNA levels. Although Norwitz et al. (17) showed that the endogenous GnRHR mRNA levels in T3-1 cells are up-regulated by GnRH, this result is in contrast to the findings of Tsutsumi et al. (22), who reported that mRNA activity, but not mRNA levels, was up-regulated in response to GnRH in these cells. Whether cAMP regulates endogenous GnRHR mRNA levels in T3-1 cells has not been previously investigated to our knowledge.

View larger version (18K): [in this window] [in a new window]   Figure 1. Comparison of the proximal promoters of the rat, mouse, and human GnRHR genes. The boxed areas represent the 5'-untranslated region, and ATG denotes the translation start sites. Pit-1, Pituitary-specific response element-1; CAAT, CAAT box binding element; TATA, TATA box binding element; GRAS, GnRH receptor activating sequence; PEA-3, phorbol ester sensitive sequence. This figure is not drawn to scale, but relative sizes of the promoter regions are indicated below each representation. Compiled from information obtained from Refs.8 10 11 12 14 15 18 20 39 , and52 .

	Materials and Methods

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Materials
CCRF-CEM (a human T cell leukemia cell line) nuclear extracts (5 µg/µl) and purified anti-Nur77 antibody (for Western blots) were obtained from Geneka Biotechnology (Montréal, Canada), For electrophoretic mobility shift assays (EMSAs), anti-Nur77 antiserum was obtained from Dr. Perlmann (Karolinska Institute, Stockholm, Sweden). Rabbit preimmune serum was obtained from Dr. Bellstedt (University of Stellenbosch, Stellenbosch, South Africa). Antiserum against whole SF-1 protein was obtained from Dr. Morohashi (Kyushu University, Kyushu, Japan). Secondary goat antirabbit serum and rabbit peroxidase-antiperoxidase as well as all test compounds were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Plasmids
pGL2-basic promoterless luciferase expression vector, pGL2-basic vector-specific primers GL1 (sense) and GL2 (antisense), and pSV-ß-galactosidase expression vector were purchased from Promega Corp. (Madison, WI). The cytomegalovirus promoter (pCMV)-SF-1 expression construct containing the full-length (2-kb) human SF-1 cDNA was obtained from Dr. Parker (University of Texas, Dallas, TX) (23). For in vitro transcription-translation, the full-length cDNA was excised with EcoRI and cloned into pSPT19 (Roche, Indianapolis, IN). Mouse Nur77 cDNA (2 kb) in the pCMX-Nur77 expression vector was obtained from Dr. Drouin (Institut de Recherches Cliniques de Montréal, Montréal, Canada). The pFC-PKA catalytic subunit expression vector was a gift from Dr. Vanden Berghe (Royal University of Ghent, Ghent, Belgium). The full-length (2.1-kb) human fibroblast cytoplasmic ß-actin cDNA in the Okayama-Berg expression vector (24) was obtained from Dr. Parker (University of Cape Town, Cape Town, South Africa). Mouse GnRHR cDNA (2.1 kb) (25) in the pcDNAI/Amp vector (Invitrogen, San Diego, CA) as well as clone 111 containing 1.1 kb of the mouse GnRHR gene in pBluescript SK were obtained from Dr. Sealfon (Mt. Sinai Medical School, New York, NY). The ATG start codon of clone 111 in pBluescript SK was mutated to a BglII site using oligonucleotide 1S to yield plasmid pGB. Plasmid pGBM, where the two central Cs of the -15/-7NRS region were mutated to two Ts, as well as the ATG start codon mutated to a BglII site were also created from clone 111 in pBluescript SK by a similar strategy, using oligonucleotide 2S. Thereafter, 591-bp BglII/BamHI fragments were excised from pGB and pGBM and cloned into the BglII site of pGL2-basic to yield pLG and pLGM1, respectively. The sequence in the region of the -15/-7NRS for pLG is as follows: GCCTGTCCTTGGAGAAAATAgatctaag, where capital letters denote the wild-type GnRHR sequences, bold letters denote the original position of the ATG codon, and the underlined region denotes the wild-type -15/-7NRS region. The –244/-236NRS was mutated to a PstI site by PCR using overlapping primers –244/-236mps and –244/-236mpa, containing the modified –244/-236NRS sequence. Using pLG as template, two separate PCR reactions were performed, with either GL1 and –244/-236mpa or GL2 and –244/-236mps as primers, generating products of approximately 400 and 300 bp, respectively. Subsequently, these products were used in a PCR fusion reaction with primers GL1 and GL2, generating a product of approximately 700 bp containing the –579/+1 promoter sequence with the modified –244/-236NRS. The product was subsequently cloned back into SacI and HindIII sites of pGL2-basic, to yield pLGM2. All reporter constructs were sequenced for confirmation. For oligonucleotide sequences, refer to Table 1.

RNA isolation
T3-1 cells were plated in 100-mm petri dishes at a density of 2 x 10⁶ cells/dish. For Northern blotting, test compounds were added to fresh medium supplemented with 10% serum. Incubations were performed as described in the figure legends. Subsequently, the medium was removed, and the cells were harvested in 1 ml Tri-reagent (Sigma-Aldrich Corp.)/dish. Total RNA was isolated according to the Tri-reagent protocol (26).

Rapid amplification of 5' cDNA ends (5'RACE)
Total RNA from male mouse pituitaries or T3-1 cells was subjected to 5'RACE using a 5'RACE system (Life Technologies, Inc.) according to the protocol supplied (catalogue no. 18347-025). Briefly, this involved single-stranded cDNA synthesis using a gene-specific antisense primer (GSP2) and homopolymeric tailing, followed by amplification of the dC-tailed cDNA by PCR using an anchor primer and a nested GSP2. A second nested PCR was performed using a universal anchor primer and an internally nested GSP3. Thereafter, PCR products were analyzed by agarose gel electrophoresis, cloned, and subjected to DNA sequence analysis. For oligonucleotide sequences, refer to Table 1.

Northern blots
The cDNA probes (25 ng) were labeled with 50 µCi [-³²P]deoxy-CTP (AEC, Amersham Pharmacia Biotech, Arlington Heights, IL) using the Megaprime DNA Labeling System (Amersham Pharmacia Biotech). Total RNA (20 µg/well) was separated on a 1% denaturing formaldehyde agarose gel in 1x morpholinepropanesulfonic acid buffer, transferred overnight onto a Hybond N⁺ membrane (Amersham Pharmacia Biotech) in 20x standard saline citrate (SSC) buffer, and cross-linked to the membranes. The membranes were prehybridized in DIG EasyHyb solution (Roche) at 50 C for 30 min. Labeled DNA probes (10⁸–10⁹ dpm/µg DNA) were denatured, and hybridization (25 ng DNA/100 cm²) was performed overnight at 50 C. After hybridization, membranes were washed twice for 5 min each time at room temperature in a 2x SSC buffer containing 0.1% sodium dodecyl sulfate, followed by two washes at 50 C in a 0.1x SSC buffer containing 0.1% sodium dodecyl sulfate. The washed membranes were exposed to Hyperfilm (Amersham Pharmacia Biotech) at -80 C. Membranes were stripped, followed by probing for ß-actin. Quantification of signals was performed by densitometric analysis of the autoradiograms.

Transient transfections
T3-1 cells were plated in 12-well culture plates (Nunc, Naperville, IL) at 2 x 10⁵ cells/well in a volume of 1 ml DMEM containing 10% fetal calf serum and antibiotics as described above. Twenty-four hours after plating, medium was replaced with fresh antibiotics-free medium. Transfections were subsequently performed according to the Fugene 6 (Roche) product protocol. One microgram of luciferase reporter construct, 0.5 µg simian virus promoter-ß-galactosidase, and expression constructs, as indicated in relevant figure legends, together with Fugene 6 reagent at a ratio of 1 µg DNA/2 µl Fugene 6 in 100 µl serum-free, high glucose DMEM were added per well. Thirty hours after transfection, the medium was replaced with fresh antibiotic-free medium, and cells were incubated with various test compounds as indicated in the figure legends. Cells were harvested in 100 µl lysis buffer (Galacto-Star assay system, Tropix, Bedford, MA)/well. The ß-galactosidase assays (Galacto-Star assay system) and luciferase assays (Promega Corp. luciferase assay system) were performed with 20 µl cell extract in black 96-well plates in a Luminoskan RS luminometer (Labsytems, Chicago, IL). The luciferase assay values were divided by the ß-galactosidase assay values to correct for differences in transfection efficiency, and these values were then expressed relative to the pLG control value, which was taken as 1.

Nuclear extracts
Twenty 75-cm² flasks of T3-1 cells were grown to confluence and washed twice with PBS. Nuclear extracts were prepared at 4 C essentially as described previously (27). The cells were harvested in PBS, and a crude nuclear pellet was obtained as described. This was resuspended in 4 vol buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.05 mM phenylmethylsulfonylfluoride (PMSF)]. Nuclei were collected by centrifugation at 25,000 x g for 20 min and resuspended in 500 µl buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 1 mM EDTA, 25% (vol/vol) glycerol, 0.5 mM DTT, and 0.05 mM PMSF]. Nuclei were lysed in 250 µl 1 M KCl while gently rocking for 30 min. Subsequently, the insoluble debris was pelleted at 25,000 x g for 30 min. The supernatant was dialyzed three times for 2 h each time in 200 vol buffer D [10 mM HEPES (pH 7.9), 5 mM MgCl₂, 50 mM KCl, 1 mM EDTA, 10% (vol/vol) glycerol, 3 mM DTT, and 0.3 mM PMSF] and stored in aliquots at -80 C.

EMSAs
Probes (10 pmol double-stranded oligonucleotides) were end-labeled with 100 µCi [-³²P]ATP (Amersham Pharmacia Biotech) and 10 U Escherichia coli polynucleotide kinase (Roche). Protein-DNA incubations were performed for 10 min at room temperature in a final volume of 5 µl in assay buffer containing 100 mM NaCl, 7.5 mM HEPES (pH 7.9), 10 mM Tris-HCl, 8% (vol/vol) glycerol, 1.25 mM MgCl₂, 1 mM EDTA (pH 8.0), 0.25 mM EGTA (pH 7.0), 0.67 mM PMSF, and 3.5 mM DTT, containing 0.5 µg poly(dI-dC) (Roche) and 6 µg BSA (Roche, molecular biology grade). Labeled probe (0.1 pmol; 150,000–250,000 cpm) and 1.5 µl in vitro transcribed-translated protein (diluted 1:7 in 1x assay buffer), CCRF-CEM nuclear extracts (diluted 1:9 in 1x assay buffer, unless indicated otherwise in figure legends), or T3-1 nuclear extracts (1–2 µg protein) were used for each assay. In vitro transcription-translation of SF-1 cDNA (2 µg) was performed according to the Promega Corp. TnT Quick Coupled Transcription/Translation protocol. For assays containing anti-SF-1 (undiluted), anti-Nur77 (Perlmann; diluted 1:1.5 in 1x assay buffer), or preimmune antisera (diluted 1:29 in 1x assay buffer), reactions were performed as described above, followed by further incubation for 10 min at room temperature after the addition of 1 µl specific antibody or rabbit preimmune serum. For competition assays, double-stranded oligonucleotide competitor DNA, labeled probe, and proteins were added sequentially to the assay mix and incubated for 10 min at room temperature. EMSAs were performed using 4% polyacrylamide gels (29:1 acrylamide/bis-acrylamide) at 100 V in 1x Tris acetate EDTA buffer for 3.5 h at room temperature. The gels were transferred to blotting paper, dried, and exposed to Hyperfilm at -80 C. For oligonucleotide sequences, refer to Table 1.

SDS-PAGE and Western blots
Protein samples containing either 5 µl undiluted in vitro transcribed-translated protein or varying amounts of nuclear extracts were separated on an 8% SDS-PAGE gel for 5 h at 120 V in 25 mM Tris-HCl, 250 mM glycine (Merck \|[amp ]\| Co., Inc., Rahway, NJ; reagent grade), and 0.1% sodium dodecyl sulfate, pH 8.3 (28). Proteins were transferred onto a nitrocellulose membrane (BA 85, Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) by electroblotting for 16 h at 120 mA in 25 mM Tris-HCl, 192 mM glycine, and 20% (vol/vol) methanol, pH 8.3. Membranes were blocked with casein buffer (154 mM NaCl, 0.5% casein, 10 mM Tris base, and 0.02% thiomersol) at room temperature for 1 h and incubated overnight with anti-SF-1 antiserum (1:3000) at 4 C or blocked overnight in casein buffer at 4 C and incubated with anti-Nur77 antibody (1:2000) at 37 C for 2 h. Incubations with the secondary and peroxidase-antiperoxidase antibodies were each performed at 37 C for 90 min at 1:1000 and 1:5000 dilutions, respectively. All antibody dilutions were made in casein buffer. After each antibody incubation, the membrane was washed for 15 min in PBS and for 5 min in PBS containing 0.1% (vol/vol) Tween at room temperature. Protein-antibody complexes were visualized by enhanced chemiluminescence detection (RPN2106, Amersham Pharmacia Biotech).

Statistical analysis
Results were analyzed with PRISM software (version 3.1, GraphPad Software, Inc., San Diego, CA) using one-way ANOVA with either Dunnett’s posttest (when comparing all values to a single control) or Bonferroni’s posttest (when comparing all values to each other).

	Results

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Colocation of the GnRHR transcription start site in T3-1 cells and mouse pituitary tissue, upstream from the –15/-7NRS
Total RNA isolated from T3-1 cells and mouse pituitary tissue was analyzed by 5'RACE. The gene-specific primer used in the final PCR reaction (corresponding to position +32/+57) revealed a single distinct band at about 170 bp for both T3-1 and pituitary RNA upon electrophoretic analysis, corresponding to a start site around -60 relative to the start codon. No larger PCR products corresponding to further upstream sites were detected for several repeat experiments. Cloning of the 170-bp product and sequencing of several clones revealed that the major transcription start site for both T3-1 cells and mouse pituitary tissue occurs at the A residue at position -62 relative to the start codon.

Activators of the PKA pathway result in an increase in endogenous GnRHR mRNA
To determine whether the endogenous GnRHR gene in T3-1 cells is responsive to elevated levels of cAMP and forskolin, the cells were incubated for 16 h in the absence and presence of either forskolin or 8-bromo-cAMP (8-Br-cAMP). Total RNA was isolated and probed by Northern blotting with radiolabeled mouse GnRHR cDNA (Fig. 2A). The results presented in Fig. 2 show that both 8-Br-cAMP and forskolin resulted in a significant (2.5-fold) increase in GnRHR mRNA (4.5 kb) levels relative to levels in unstimulated cells. Furthermore, forskolin induced the expression of a second, smaller mRNA isoform (1.8 kb) in a concentration-dependent fashion, with 100 µM forskolin resulting in markedly increased expression of this isoform compared with expression at 10 µM.

View larger version (55K): [in this window] [in a new window]   Figure 2. Levels of endogenous GnRHR mRNA in T3-1 cells are increased by forskolin and 8-Br-cAMP. A, T3-1 cells were incubated for 16 h in the absence (ctrl; lanes 1 and 2) or presence of 1 mM 8-Br-cAMP (cAMP; lanes 3 and 4), 10 µM forskolin (10FSK; lanes 5 and 6), or 100 µM forskolin (100FSK; lanes 7 and 8). Total RNA was probed for GnRHR mRNA by Northern blotting. B, The membrane was stripped and reprobed for ß-actin mRNA. Lane numbers are as described in A. C, The combined results of five independent experiments, similar to and including the experiment shown in A and B, in which each incubation was performed in duplicate, were quantified by densitometric scanning. Note that the analysis was performed on the signal corresponding to the larger (4.5-kb) GnRHR mRNA isoform. Labels are as described for A. The intensity of the GnRHR 4.5-kb signal divided by that of the ß-actin signal, both in the absence of forskolin, was taken as 100%. *, P < 0.05.

SF-1 and Nur77 proteins are present in T3-1 nuclear extracts and recognize the -15/-7NRS in vitro

View larger version (111K): [in this window] [in a new window]   Figure 3. SF-1 and Nur77 proteins from T3-1 nuclear extracts recognize and bind to the –15/-7NRS. Autoradiographs of EMSAs showing DNA-protein complex formation between T3-1 nuclear extract proteins and the radiolabeled –15/-7NRS (A–C) and NBRE (D) probes. A, Lanes 1–3 contained in vitro translated SF-1, with anti-SF-1 antiserum (Ab) added in lane 2 and preimmune serum (s) added in lane 3. Lanes 4–7 contained T3-1 nuclear extract proteins, with anti-SF-1 antiserum (Ab) added in lanes 5 and 6 (a duplicate incubation), and preimmune serum (s) added in lane 7. Supershifted complex I is indicated by an arrow (ss). The positions of complex I (SF-1 with the –15/-7NRS probe) and complex II (Nur77 with the –15/-7NRS probe) are indicated with arrows. B, Lanes 1–3 contained CCRF-CEM nuclear extracts (positive control for Nur77), with anti-Nur77 antiserum (Ab) added in lane 2 and preimmune serum (s) added in lane 3. Lanes 5–8 contained T3-1 nuclear extract proteins, with anti-Nur77 antiserum (Ab) added in lanes 6 and 7 (a duplicate incubation), and preimmune serum (s) added in lane 8. Lane 4 contained anti-Nur77 antiserum in the absence of nuclear proteins. Complexes I and II are as described for A. Supershifted complex II is indicated by an arrow (ss). C, Lanes 1 and 3 contained CCRF-CEM nuclear extracts (diluted 1:4 in 1x assay buffer), and lane 2 contained CCRF-CEM nuclear extracts (diluted 1:1 in 1x assay buffer). In vitro translated SF-1 was added in lanes 2–4. Complexes containing SF-1 and Nur77 protein, respectively, are indicated by arrows labeled correspondingly. D, Lanes 1–3 contained CCRF-CEM nuclear extracts, with anti-Nur77 antiserum (NAb) added in lane 2, and preimmune serum (s) added in lane 3. Lanes 4–8 contained T3-1 nuclear extract proteins, with anti-Nur77 antiserum (NAb) added in lane 5, anti-SF-1 antiserum (SAb) added in lane 6, and preimmune serum (s) added in lane 7. Lane 8 contained a 25-fold molar excess of nonradiolabeled –15/-7NRS oligonucleotide (-15). The positions of complex III (SF-1 with the NBRE probe) and complex IV (Nur77 with the NBRE probe) are indicated with arrows. E, The presence of SF-1 and Nur77 proteins in T3-1 nuclear extracts was confirmed by Western blot analysis. The positive controls for SF-1 (lane 1) and Nur77 (lane 3) were 5 µl in vitro transcribed-translated SF-1 and 20 µg CCRF-CEM nuclear extracts, respectively. Thirty micrograms of T3-1 nuclear extract protein were loaded in lanes 2 and 4. Lanes 1 and 2 were probed with anti-SF-1 antiserum, and lanes 3 and 4 were probed with anti-Nur77 antibody.

To serve as further confirmation that the protein in complex II was indeed Nur77, the binding of T3-1 nuclear extract proteins to an established Nur77-binding site was investigated. As with the –15/-7NRS probe, several DNA-protein complexes were detected (Fig. 3D) when T3-1 nuclear extracts were incubated with a radiolabeled double-stranded probe containing the Nur77-binding site from the human proopiomelanocortin promoter (NBRE; Fig. 3D, lane 4) (33). One of these complexes, designated complex III, had similar mobility to that of one of the complexes formed between the NBRE probe and CCRF-CEM nuclear extracts (Fig. 3D, lane 1). The intensities of both of these complexes decreased in the presence of anti-Nur77 antiserum (Fig. 3D, lanes 2 and 5). Although a decrease in intensity of the CCRF-CEM nuclear protein complex was consistently observed in the presence of preimmune serum, this decrease was less pronounced than that seen for anti-Nur77 antiserum. The intensity of complex III was not influenced by anti-SF-1 antiserum (Fig. 3D, lane 6). In fact, it appears that complex IV, with higher mobility than complex III, contains SF-1 protein, as its intensity was diminished by the addition of anti-SF-1 antiserum. These results strongly indicate that complex III contains Nur77 protein from T3-1 nuclear extracts. Furthermore, binding of the protein in complex III was competed for by the addition of a 25-fold molar excess of nonradiolabeled –15/-7NRS oligonucleotide (Fig. 3D, lane 8), providing additional evidence that Nur77 protein from T3-1 nuclear extracts can recognize the –15/-7NRS.

The presence of both SF-1 and Nur77 proteins in T3-1 nuclear extracts was confirmed by Western blotting (Fig. 3E). Once again, no cross-reactivity was observed for the SF-1 antiserum with the Nur77 control protein or the Nur77 antiserum with the SF-1 control protein (not shown).

SF-1 and Nur77 proteins in T3-1 nuclear extracts differ in their sequence requirements for binding to the –15/-7NRS and –244/-236NRS
The sequence requirements for binding of SF-1 (complex I) and Nur77 (complex II) from T3-1 nuclear extracts to the –15/-7NRS were tested in an EMSA with radiolabeled –15/-7NRS oligonucleotide as probe (Fig. 4A) in the absence and presence of varying concentrations of nonradiolabeled competitor DNA containing either the wild-type –15/-7NRS sequence or a mutated version thereof. As the two adjacent cytosines within a NRS had previously been shown to be essential for binding of SF-1 protein (34), the corresponding bases in the –15/-7NRS were changed to two thymidines to yield the oligonucleotide –15/-7NRSm. Although a 5-fold excess of nonradiolabeled –15/-7NRS oligonucleotide can effectively compete for SF-1 binding to radiolabeled –15/-7NRS (Fig. 4A, lanes 2 and 3), the –15/-7NRSm oligonucleotide was unable to compete for SF-1 binding even at a 125-fold molar excess (lanes 4–6). In contrast, the –15/-7NRSm oligonucleotide was able to compete for Nur77 protein binding. However, Nur77 protein seemed to have a lower affinity for –15/-7NRSm than for wild-type –15/-7NRS, because complete competition for Nur77 binding by –15/-7NRSm was observed at a 25-fold molar excess (Fig. 4A, lane 5) compared with a 5-fold molar excess observed for wild-type –15/-7NRS (Fig. 4A, lane 2).

View larger version (60K): [in this window] [in a new window]   Figure 4. SF-1 and Nur77 proteins from T3-1 nuclear extracts bind to both the –15/-7NRS and the 244/-236NRS but have different sequence requirements for DNA binding. A and B, Autoradiographs of competition EMSAs, using radiolabeled –15/-7NRS as probe. A, Lanes 1 and 7 contained no competitor DNA. Lanes 2 and 3 contained 5- and 25-fold molar excesses of –15/-7NRS, respectively; lanes 4–6 contained 5-, 25-, and 125-fold molar excesses of –15/-7NRSm, respectively. B, Lanes 1 and 7 contained no competitor DNA. Lanes 2 and 3 contained 5- and 25-fold molar excesses of –244/-236NRS, respectively; lanes 4–6 contained 5-, 25-, and 125-fold molar excesses of –244/-236NRSm, respectively. C and D, Autoradiographs of competition EMSAs using radiolabeled –244/-236NRS as probe. Lane numbers for C are as described for A, and numbers for D are as described for B. E, Autoradiograph of competition EMSA using radiolabeled NBRE as probe. Lanes 1 and 6 contained no competitor DNA. Lanes 2 and 3 contained 25-fold molar excesses of –15/-7NRS (wt) and –15/-7NRSm (m), respectively, and lanes 4 and 5 contained 25-fold molar excesses of –244/-236NRS (wt) and –244/-236NRSm (m), respectively.

These experiments were subsequently repeated using the –244/-236NRS oligonucleotide as radiolabeled probe (Fig. 4, C and D). Complexes I and II, as formed by the –15/-7NRS probe, were also observed for the –244/-236NRS probe, and the proteins in the complexes were confirmed by specific antibodies to be SF-1 and Nur77, respectively (data not shown). Once again, a 5-fold molar excess of both –15/-7NRS (Fig. 4C, lane 2) and –244/-236NRS (Fig. 4D, lane 2) was able to completely compete for binding of both SF-1 and Nur77. As described previously, a 125-fold molar excess of either mutated competitor was unable to compete for binding of SF-1 (Fig. 4C, lane 6, and Fig. 4D, lane 6), whereas a 5-fold molar excess was sufficient to compete for Nur77 binding (Fig. 4C, lane 4, and Fig. 4D, lane 4). Finally, the competition results for Nur77 binding were confirmed using the NBRE oligonucleotide as probe (Fig. 4E) in the absence and presence of each of the four competitor oligonucleotides. Both the wild-type and mutated oligonucleotides competed for Nur77 binding (Fig. 4E, lanes 2–5).

The –579/+1 GnRHR promoter is responsive to stimulation by forskolin, PACAP, and GnRH
To investigate the responses of the proximal mouse GnRHR promoter to activators of the PKA pathway, we prepared a promoter construct, designated pLG, by inserting 580 bp of the mouse GnRHR gene, from position –579 to +1 relative to the translation start site, into the promoterless pGL2-basic luciferase reporter vector. T3-1 cells were transfected with pLG, followed by incubation for varying times with 10 µM forskolin (Fig. 5A). No significant effect was observed on promoter activity until 16 h of forskolin stimulation, at which time an approximately 2-fold increase in activity was obtained. To determine whether PACAP has a stimulatory effect on the mouse GnRHR proximal promoter similar to that shown for the rat promoter (21), a similar experiment was performed with PACAP on the mouse pLG construct (Fig. 5B). A significant increase in promoter activity of pLG was observed after only 30 min of stimulation with PACAP, with maximum activity (5-fold) observed after 8 h (Fig. 5B). After 16 h, the activity was still significantly higher (2-fold) than that of uninduced pLG (Fig. 5B). To compare the responses of the mouse promoter to forskolin and PACAP with the response to GnRH, a similar experiment was performed on pLG with GnRH (Fig. 5C). Although the promoter responded to GnRH with a maximal increase of about 1.5-fold after 4 h, no significant effect was observed after 8 or 16 h.

View larger version (12K): [in this window] [in a new window]   Figure 5. The –579/+1 GnRHR promoter responds to stimulation with forskolin, PACAP, and GnRH. T3-1 cells were transfected with 1 µg/well pLG and stimulated for varying times with either 10 µM forskolin (A), 20 nM PACAP (B), or 100 nM GnRH (C). Control incubations for each time point were performed with mock additions of test compounds in solvent only, i.e. DMSO (final concentration, 0.1%, vol/vol; A) or water (B and C). Cell extracts were harvested and assayed for luciferase and ß-galactosidase activity. Luciferase assay values were normalized by ß-galactosidase assay values to correct for differences in transfection efficiency between wells. Time points (in hours) are indicated below each bar. Values are expressed as the normalized induced value relative to the normalized control value for each time point, which was taken as equal to 1 for the zero time point. The results of a single typical experiment are shown, in which each time point was performed in duplicate. Each experiment was performed at least twice. *, P < 0.05; **, P < 0.01.

View larger version (20K): [in this window] [in a new window]   Figure 6. The response of the –579/+1 GnRHR promoter to forskolin stimulation is modulated by SF-1 binding to –15/-7NRS and –244/-236NRS. A, Diagrammatic representation of the luciferase expression constructs used in the transfection experiments. luc, Luciferase-coding sequence; -15NRS, wild-type -15/-7NRS; -15NRSm, mutated -15/-7NRS; -244NRS, wild-type -244/-236NRS; -244NRSm, mutated -244/-236NRS. B, T3-1 cells were transfected with 1 µg/well of each of the three GnRHR promoter reporter constructs, as indicated below the graph bars. Cells were stimulated overnight with 10 µM forskolin (FSK) in 0.1% DMSO (vol/vol; +) or with 0.1% DMSO (vol/vol) alone (-), as indicated. Cell extracts were harvested and analyzed as described in Fig. 5. The graph shows the combined results of four independent experiments, all performed in duplicate. Statistically significant differences (P < 0.05) are indicated by different lowercase letters above the graph bars.

To confirm that the forskolin-induced increase in transcriptional activity of the –579/+1 promoter fragment is the result of activation of the PKA pathway by elevated cAMP levels, the same constructs described above were successively transfected into T3-1 cells together with a construct expressing the PKA catalytic subunit. To investigate the role of SF-1 protein in the response of the –579/+1 promoter fragment to PKA, a construct expressing SF-1 cDNA was also cotransfected in some experiments. The wild-type –579/+1 GnRHR promoter fragment (pLG) responded strongly to PKA (Fig. 7A). Although overexpression of SF-1 had no effect on basal –579/+1 promoter activity, it substantially (2-fold) enhanced the PKA response of pLG. As already shown in Fig. 6B, mutating –15/-7NRS such that SF-1 could no longer bind there (pLGM1) had no effect on basal levels of transcriptional activity of the –579/+1 promoter fragment. Furthermore, overexpression of SF-1 had no effect on basal pLGM1 activity. Similar to the result obtained for forskolin, the PKA response of pLGM1 was substantially increased (Fig. 7B) compared with that of pLG, i.e. about 32-fold compared with about 13-fold. This is consistent with the result presented in Fig. 6B, indicating that binding of SF-1 at –15/-7NRS negatively modulates the PKA response. Overexpression of SF-1 increased the PKA response of pLGM1 to about 100-fold above basal pLG activity or about 3-fold above that seen in the absence of overexpressed SF-1, as found for pLG.

View larger version (12K): [in this window] [in a new window]   Figure 7. The –579/+1 GnRHR promoter responds to activation of the PKA pathway and is modulated by SF-1 and Nur77. T3-1 cells were transfected with 1 µg/well pLG (A), pLGM1 (B), or pLGM2 (C) together with 0.25 µg pFC-PKA (PKA) and 0.5 µg of either pCMV-SF-1 (SF-1) or pCMX-Nur77 (Nur77) as indicated below the graph bars. Cell extracts were harvested and analyzed as described in Fig. 5. The results of a single representative experiment are shown, in which each point was performed in duplicate. Each experiment was repeated at least three times. For each of the mutant constructs, a reference point (uninduced pLG) was taken and set as 1. Although the magnitudes of the responses differed between experiments, the general trend was well established. Statistically significant differences (P < 0.05) are indicated by different lowercase letters above the graph bars.

The PKA response of the –579/+1 GnRHR promoter can be repressed by Nur77
To investigate the role of Nur77 protein in basal activity and in the response of the –579/+1 promoter fragment to PKA, experiments similar to those described above were performed with a construct expressing the Nur77 cDNA (Fig. 7). Overexpression of Nur77 had no effect on the basal activity of all three of the promoter constructs. Unlike SF-1, Nur77 had no influence on the PKA response of pLG. In experiments performed with pLGM2, in which responsiveness to SF-1 was lost, Nur77 overexpression also had no effect on the PKA response (Fig. 7C). However, an unexpected and very interesting result was obtained for the pLGM1 construct, where it was found that overexpression of Nur77 resulted in a substantial (3-fold) attenuation of the PKA response (Fig. 7B). This result indicates that in the absence of SF-1 binding at –15/-7NRS, Nur77 is able to repress the PKA response.

The effect of overexpression of Nur77 in the presence of both overexpressed PKA and SF-1 was also investigated for pLG and pLGM1 (Fig. 8). As overexpressed SF-1 could not enhance the PKA response of pLGM2, this construct was not investigated. As shown previously in Fig. 7, the PKA response of both pLG and pLGM1 was enhanced by overexpressing SF-1 (Fig. 8). Overexpressed Nur77 completely abrogated the enhancing effect of SF-1 on the PKA response of pLG. A small reduction in the SF-1-enhanced PKA response of pLGM1 was also consistently observed in the presence of overexpressed Nur77, although this effect was not statistically significant.

View larger version (21K): [in this window] [in a new window]   Figure 8. Nur77 antagonizes the SF-1-enhanced PKA response of the –579/+1 GnRHR promoter. T3-1 cells were transfected with 1 µg/well of either pLG or pLGM1 together with 0.25 µg pFC-PKA (PKA) and 0.5 µg pCMV-SF-1 (SF-1) in the absence or presence of 0.5 µg pCMX-Nur77 (Nur77), as indicated below the graph bars. Cell extracts were harvested and analyzed as described in Fig. 5. The results of a single representative experiment are shown, in which each point was performed in duplicate. Each experiment was repeated at least three times. Although the magnitude of the responses differed between experiments, the general trend was well established. Statistically significant differences (P < 0.05) are indicated by different lowercase letters above the graph bars.

View larger version (56K): [in this window] [in a new window]   Figure 9. Levels of endogenous SF-1 mRNA in T3-1 cells are increased by forskolin. A, T3-1 cells were incubated for 16 h in the absence (ctrl; lanes 1 and 2) or presence of either 10 µM forskolin (10FSK; lanes 3 and 4) or 100 µM forskolin (100FSK; lanes 5 and 6). Total RNA was analyzed and probed for SF-1 mRNA by Northern blotting. B, The membrane was stripped and reprobed for ß-actin mRNA levels to serve as an internal control. Lane numbers are as described in A. C, Quantification by densitometric scanning of the results shown in A and B. Labels of bar graphs are as described for A. The intensity of the SF-1 mRNA signal, divided by that of the ß-actin signal, both in the absence of forskolin, was taken as 100%. **, P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although it has been established that SF-1 protein binds to the mouse GnRHR –244/-236NRS in vitro (9), the binding of T3-1 nuclear extract proteins to the -15/-7NRS has not been previously investigated. We report here for the first time that both SF-1 and Nur77 bind specifically to the –15/-7NRS. We also show that Nur77 protein binds to the –244/-236NRS. We report that SF-1 and Nur77 proteins have different DNA binding specificities, with Nur77, unlike SF-1, still being able to recognize the mutated NRS. The detection of Nur77 protein and mRNA has not previously been reported in T3-1 cells, although it is well known that Nur77 protein is expressed in pituitary corticotropes (33, 37). However, the presence of Nur77 transcripts and their up-regulation by GnRH have been reported in LßT2 cells, another gonadotrope cell line (38). This together with our findings suggests that Nur77 plays a role in regulation of gene expression via NRS sites in pituitary gonadotropes.

We report for the first time that in T3-1 cells the endogenous GnRHR gene is up-regulated by forskolin and 8-Br-cAMP. The major mRNA transcript (4.5 kb) is up-regulated by about 2.5-fold by both compounds. The more marked up-regulation of a smaller transcript of approximately 1.8 kb by forskolin is intriguing and may be due to the use of an alternative transcription start site or alternative RNA processing regulated by the PKA pathway. The up-regulation observed for the endogenous 4.5-kb transcript is likely to be mediated predominantly via transcriptional regulation, as indicated by the similar 2- to 2.5-fold increase in the transcriptional activity of the wild-type -579/+1 promoter fragment when stimulated with forskolin in transient transfection assays. Our promoter assays clearly show that the –579/+1 mouse GnRHR promoter is up-regulated by PKA, supporting the argument that the forskolin-induced increase in mRNA levels and promoter activity was due to activation of the PKA pathway. Our promoter time-course studies with forskolin clearly show that this regulation is only observed after more than 8 h, with a significant increase at 16 h. We did not observe any effect with forskolin after 4 h, indicating that the PKA effects are most likely acting indirectly on the GnRHR promoter and require prior regulation of another gene. In support of our results, the rat (21, 35) and human (39) GnRHR promoters have also been shown by others to be responsive to activators of the PKA pathway in T3-1 cells. Interestingly, Cheng et al. (39) found a time course for forskolin induction of the human promoter similar to our result with the mouse promoter, with maximal induction occurring about 12 h or later. Our results are consistent with those of White et al. (11), who found that a similar construct of the mouse GnRHR promoter was unaffected after 4 h of forskolin treatment in T3-1 cells. These researchers did not, however, investigate longer times of incubation.

Our transfection results show that the –15/-7NRS is not required for basal or PKA-stimulated transcription of the mouse GnRHR gene. We have also found that this site is not involved in gonadotrope-specific expression (not shown), in agreement with previously published results (8). However, our results suggest that the levels of SF-1 protein and the binding of SF-1 at the proximal and distal NRS sites play a modulatory role in regulating the extent of the PKA stimulatory response. SF-1 protein, and intact SF-1-binding sites at the –15/-7NRS and –244/-236NRS do not appear to be required for the PKA response. Nevertheless, our combined data strongly support a role for SF-1 protein in positive modulation of the PKA response via the –244/-236NRS and negative modulation via the –15/-7NRS. It is difficult to envisage at this stage how the same protein could have both positive and negative effects on PKA stimulation. It is possible that promoter context and architecture determine the outcome of the response. For example, PKA-activated SF-1 binding at the –15/-7NRS may recruit a corepressor, whereas SF-1 binding at the distal NRS may recruit a coactivator due to interaction with different factors at the proximal and distal sites. SF-1 has been shown to interact with both transcriptional repressors, such as N-CoR (via DAX-1) (40), and activators, such as Sp1 (41) and SRC-1 (42). The positioning of the –15/-7NRS in the 5'-untranslated region may also play a role in its actions as a negative modulator of the PKA response by interfering with components of the basal transcriptional machinery. The fact that endogenous SF-1 mRNA is up-regulated by forskolin, but that overexpression of SF-1, in the absence of overexpressed PKA, has no effect on promoter activity suggests that the modulatory effects of SF-1 on the PKA response require the direct or indirect enzymatic modification of SF-1 by PKA.

We have shown that Nur77 protein, like SF-1, binds to both the proximal and distal NRS sites. Furthermore, Nur77 has a different DNA binding specificity to SF-1, as indicated by its binding to the mutated NRS sites. Transfection experiments with overexpressed proteins clearly show that Nur77 does not regulate basal promoter activity. However, our results with the pLGM1 mutant strongly suggest that endogenous Nur77 could act as a repressor of the PKA response via the proximal NRS under conditions where SF-1 is not bound to the proximal NRS. In this experiment, Nur77 would most likely be bound at the proximal NRS. The result for the pLGM2 mutant construct, showing that overexpression of Nur77 does not prevent the PKA response, would indicate that Nur77 binding at the distal NRS does not inhibit the PKA response. The finding that overexpression of Nur77 can completely blunt enhancement of the PKA/SF-1-induced reporter activity on pLG is consistent with a negative modulatory role for Nur77. A similar trend, albeit not significant, is seen with pLGM1. Our combined results suggest that modulation of the relative levels and activity of SF-1 and Nur77 could play an important role in the response of the GnRHR promoter to activation of the PKA pathway.

Nur77 has also been implicated in playing a role in the response to the PKA pathway for other genes. Nur77 expression in corticotropes is up-regulated by CRH during the response of the hypothalamic-pituitary-adrenal axis to chronic stress (33), and this up-regulation can be mimicked by forskolin in the AtT20 corticotrope cell line (43). Similarly, ACTH stimulates Nur77 expression in Y1 adrenocortical cells via increased cAMP levels (44). Further experiments are necessary to determine the effect of activation of the PKA pathway on Nur77 levels and activity in T3-1 cells. Our results showing that SF-1 modulates up-regulation of the mouse GnRHR gene by PKA activators are consistent with the role established for SF-1 for several other genes. In the case of certain adrenal steroidogenic enzymes, SF-1 sites appear to act together with CREs to mediate a positive response to cAMP (45, 46), whereas in other cases, SF-1 appears to be the only direct (47, 48) or indirect (49, 50) target for the PKA pathway. Our results for the mouse GnRHR, showing up-regulation of SF-1 and positive modulation by SF-1 via the distal NRS, suggest that similar mechanisms are involved compared with some of these other genes.

Our finding that the proximal and distal NRS sites are not necessary for the PKA response raises the question of which cis-elements and transcription factor(s) are essential for this response. Both the rat (21) and the human (39) GnRHR promoters have been shown to respond to PKA pathway activators via similar imperfect cAMP response elements in T3-1 cells. Both the –15/-7NRS and –244/-236NRS as well as the imperfect CRE are conserved in rat and mouse GnRHR promoters. It thus seems likely that the imperfect cAMP response element in the mouse GnRHR promoter is essential for the PKA response in T3-1 cells, and that a factor(s) binding there interacts directly or indirectly with SF-1 at the proximal and distal NRS sites. Further experiments are in progress to test these hypotheses.

In T3-1 cells, the PKA response of the mouse GnRHR promoter is likely to be involved in the response to PACAP, but not GnRH. We show that the mouse GnRHR promoter is up-regulated by PACAP, similar to the results obtained for the rat (21, 35) and human (39) promoters in T3-1 cells. We detected a significant increase in promoter activity in response to both forskolin and PACAP after 16 h, consistent with a role for PACAP in indirect up-regulation of the GnRHR promoter via the PKA pathway in these cells. However, up-regulation by PACAP was also observed after only 30 min, unlike the case with forskolin, indicating that additional pathways may be involved in PACAP directly regulating the GnRHR promoter (51). The approximately 2-fold induction after 16 h for both forskolin and PACAP, as opposed to the about 5-fold increase after 8 h with PACAP, would also be consistent with a role for PKA in the later, but not the earlier, response to PACAP. Our finding that GnRH up-regulates the GnRHR promoter after 4 h, but that this effect is not seen after 16 h, indicates that PKA is not involved in the GnRH response in T3-1 cells, because no effect is seen after 4 h in our promoter studies with forskolin. In addition, this result is consistent with the Northern blot results reported by Norwitz et al. (17), showing that GnRHR mRNA levels in T3-1 cells are maximally induced after 4 h with GnRH. The lack of activation of the PKA pathway by GnRH in T3-1 cells is most likely due to the fact that GnRHR is not coupled to G_s proteins in these cells (18). In summary, we have shown that the mouse GnRHR promoter responds to activators of the PKA pathway in T3-1 cells. Given that there is strong evidence that both GnRH (20) and PACAP (51) activate this pathway in primary pituitary gonadotropes, our results most likely reflect a physiologically relevant mechanism for the effects of both of these hormones in vivo. In addition, our results suggest that other signals that activate the PKA pathway in pituitary gonadotropes in vivo regulate the expression of the GnRHR gene.

	Acknowledgments

 
Thanks to Carmen Pheiffer for preparation of the pLGM1 construct, and to Ellis Schipper for experimental contributions. We also thank Pamela Mellon and Shannon Snyder for their generous supply of T3-1 cells. Dr. Stuart Sealfon kindly supplied clone 111. Ken Morohashi and Thomas Perlmann very kindly supplied us with antibodies. We thank Johann Riedemann, Emerentia Hutchinson, and Donita Africander for expert technical assistance.

	Footnotes

 
This work was supported by grants from the Medical Research Council and National Research Foundation in South Africa and from Stellenbosch University (to J.H.).

Abbreviations: AP-1, Activator protein-1; 8-Br-cAMP, 8-bromo-cAMP; CRE, cAMP response element; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; GnRHR, GnRH receptor; GSP2, gene-specific antisense primer; NRS, nuclear receptor half-site; PACAP, pituitary adenylate cyclase-activating polypeptide; pCMV, cytomegalovirus promoter; PKA, protein kinase A; PKC, protein kinase C; PMSF, phenylmethylsulfonylfluoride; 5'RACE, rapid amplification of 5' cDNA ends; SF-1, steroidogenic factor-1; SSC, standard saline citrate.

Received August 21, 2002.

Accepted for publication January 29, 2003.

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