Molecular Human Reproduction, Vol. 6, No. 8, 671-676,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Potential regulation of GnRH gene by a steroidogenic factor-1-like protein
1 Division of Basic Science Research, Department of Obstetrics and Gynecology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292, USA
Abstract
Steroidogenic factor-1 (SF-1) is a member of an orphan nuclear hormone receptor superfamily. It plays a critical role in the development and function of the hypothalamic–pituitary–gonadal and adrenal axis. However, whether SF-1 can regulate transcription of gonadotrophin-releasing hormone (GnRH) gene is not known. To examine this possibility, we first over-expressed SF-1 and found that it not only decreased steady state GnRH messenger ribonucleic acid (mRNA) levels but also reduced its promoter activity in GT1-7 neurons. The inhibitory effect of SF-1 was lost when the 5'-flanking region of GnRH gene containing two distal (-1479 to –1474 bp and –1059 to –1054 bp) hexamers was deleted. Gel mobility shift assays showed that GT1-7 cell nuclear extracts contained a protein that formed a specific complex with synthetic oligonucleotides containing the two distal hexamers or a consensus SF-1 binding sequence. The migration of this complex was, however, slower than the complex formed with MA-10 cell nuclear extracts which were shown to contain a 53 kDa SF-1 protein. The addition of anti-SF-1 antibody supershifted the complex formed with MA-10, but not with GT1-7 cell nuclear extracts. The same antibody, however, detected a 60 kDa protein and immunostained nuclei of GT1-7 neurons. These results are consistent with GT1-7 neurons containing an SF-1-like protein that can bind to the distal hexamer sequences in the 5'-flanking region of rat GnRH gene to inhibit its transcription.
gene transcription/GnRH/GT1-7 neurons/MA-10 cells/SF-1
Introduction
The mammalian homologue of the Drosophila Ftz-F1 gene encodes steroidogenic factor-1 (SF-1), also called Ad4BP, as well as embryonal long terminal repeat binding protein (ELP) (Tsukiyama et al., 1992
). Both are members of an orphan nuclear hormone receptor superfamily of transcription factors (Lala et al., 1992; Tsukiyama et al., 1992). The protein sequence of SF-1 is highly homologous to a subset of nuclear receptors, including ELP, nerve growth factor inducible gene (NGFI-B), retinoic acid receptor-like orphan receptor (ROR), oestrogen-related receptor (ERR) 1, ERR2 and mouse liver receptor homologue 1 (LRH1), which interact as monomers with TGACCT recognition motif (Galarneau et al., 1996; Parker and Schimmer, 1997). These orphan nuclear receptors differ in their expression profiles and may serve distinct functional roles (Parker and Schimmer, 1997). SF-1 regulates the expression of genes for steroidogenic enzyme, gonadotrophin subunit, anti-Müllerian hormone and gonadotrophin-releasing hormone (GnRH) receptor (Barnhart and Mellon, 1994; Halvorson et al., 1996). Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene show severe defects which include the absence of the adrenal cortex, ovary, testes, ventromedial hypothalamic nucleus and a markedly decreased expression of gonadotrophin subunit genes in the anterior pituitary gland (Parker and Schimmer, 1997). However, gonadotrophin subunit gene expression can be restored by treating animals with GnRH (Ikeda et al., 1995).
GnRH is a hypothalamic decapeptide that plays an important role in controlling the biosynthesis and secretion of gonadotrophins and, ultimately, reproductive function. The expression of the GnRH gene is tightly regulated by transcriptional activators and repressors which respond to various cellular signals (Wierman et al., 1997). Several transcription factors of the homeodomain and helix–loop–helix (HLH) domain families, e.g. octamer binding factor 1 (Oct-1) (Eraly et al., 1998) and GATA activate (Lawson et al., 1998), whereas rat transcriptional repressor of myelin-specific gene (SCIP)/Oct-6/Tst-1 (Wierman et al., 1997) and G-Prox-1 (Lei and Rao, 1998) inhibit the transcription of rat GnRH gene in immortalized hypothalamic GnRH-producing GT1-7 neurons. However, little is known about whether transcription factors of the orphan nuclear hormone receptor superfamily can directly regulate the expression of the rat GnRH gene. Sequence analysis of the 3 kb 5'-flanking region of the rat GnRH gene has revealed the presence of three hexamer copies which are similar to the recognition motif for SF-1 and several other members of the same family of nuclear receptors (Kepa et al., 1992). Two copies with a sequence of 5'-TGACCT-3' are located at the –1479 to –1474 bp and –1059 to –1054 bp regions and one copy with a sequence of 5'-AGGTCA-3' is present at the –493 to –488 bp region upstream of the transcription start site of the rat GnRH gene. This raised the possibility that SF-1 or a related protein may regulate transcription of the GnRH gene. This possibility was tested in the present study.
Materials and methods
Materials
The following reagents were purchased from commercial sources: DNA random priming and end labeling reagents, the promoterless luciferase reporter vector pGL2 basic DNA, ß-galactosidase (ß-gal) and luciferase assay systems from Promega Corp. (Madison, WI); [
-32P-dCTP] and [
-32P-ATP] from New England Nuclear Corporation (Boston, MA, USA); lipofectin reagent and all cell culture reagents from Gibco BRL Laboratories (Grand Island, NY); avidin–biotin–peroxidase complex (ABC) immunostaining kit from Vector Laboratories (Burlingame, CA, USA); enhanced chemiluminescent (ECL) Western blot detection kit from Amersham Life Science Inc (Arlington Heights, IL, USA); rabbit polyclonal antibody to mouse SF-1 DNA binding domain from Upstate Biotechnology (Lake Placid, NY, USA); BandShift kit from Pharmacia Biotech (Piscataway, NJ, USA) and oligonucleotides containing consensus SF-1 binding sequence of the steroid hydroxylase genes (5'-CATTTCTGACCTTGGTAGAGTG-3') and oligonucleotides corresponding to bases –1488 to –1467 (5'-GACTCTGTGTGACC TAAGACAA-3') and –1068 to –1047 (5'-GAGCACAGATGACCTGGGAAGC-3') of rat GnRH gene from Operon Technologies Inc. (Alameda, CA, USA). The following items were obtained as gifts: immortalized mouse GnRH producing GT1-7 neurons from Dr Pamela Mellon at the University of California San Diego (La Jolla, CA, USA); mouse Leydig tumour MA-10 cells from Dr Mario Ascoli at the University of Iowa College of Medicine (Iowa City, IA, USA); pGEM7-GnRHP plasmid containing –3026 to +116 bp of rat GnRH 5'-flanking region from Dr Margaret Wierman at the University of Colorado Health Science Center (Denver, CO, USA); human GnRH cDNA, cloned by Dr Peter Seeburg, from Dr Wolfgang Merz at the University of Heidelberg (Heidelberg, Germany); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA from Dr Russell Prough and pCMV ß-gal expression vector from Dr Thomas Geoghea, University of Louisville, KY, USA; pCMV vector from Dr Cameron Scarlett and SF-1 cDNA expression plasmid in the pCMV vector from Dr Keith Parker at the University of Texas Southwestern Medical School (Dallas, TX, USA).
Cell culture
GT1-7 cells are immortalized hypothalamic GnRH-containing neurons which are morphologically and functionally similar to their in-vivo counterparts (Mellon et al., 1990). These neurons were grown and maintained in a humid atmosphere of 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 4.5 g/l glucose. MA-10 cells are a mouse Leydig tumour cell line which respond to LH stimulation by an increase in progesterone which is the major steroid produced by the cells (Ascoli, 1981). These cells were grown in a humidified atmosphere of 2.5% CO2 at 37°C in Waymouth medium modified to contain 20 mmol/l HEPES, 1.1 g/l NaHCO3 and 15% horse serum.
Promoter reporter fusion constructs
Rat GnRH reporter fusion constructs were prepared as previously described (Lei and Rao, 1997). We excised a HindIII fragment of the 5'-flanking region of GnRH gene containing –3026 to +116 from pGEM7-GnRHP plasmid and sub-cloned into the HindIII site of PGL2-basic expression vector upstream of the coding region of the luciferase reporter gene. The sequential 5' deletion constructs were prepared from the –3026 to +116 bp fragment using the convenient restriction sites. All the constructs have the same 3' end of +116 bp. The sequence of deletion constructs was confirmed by multiple endonuclease enzyme analysis.
DNA transfections and assay for reporter activity
GT1-7 neurons were plated at a density of 5x105 cells per well in 6-well culture clusters and grown to 80% confluence. Then the serum containing DMEM was changed to serum-free DMEM and transfected with 25 µg of lipofectin reagent containing 10 µg of prostaglandin (PG)L2–GnRH promoter–luciferase fusion construct, 2 µg of pCMV-ß-gal expression vector and either 2 µg of pCMV vector or 2 µg of pCMV-SF-1 expression plasmid DNA. The cells were cultured for 12 h and then the culture medium was changed to DMEM supplemented with 10% fetal calf serum. After 72 h of transfection, the cells were lysed in 400 µl of reporter buffer from the Promega luciferase assay kit.
For luciferase assays, 20 µl cell lysates were mixed with 100 µl assay mixture [270 µmol/l Coenzyme A, 470 µmol/l Luciferin and 530 µmol/l ATP in 20 mmol/l Tricine, 1.07 mmol/l (MgCO3)4Mg(OH)2.5H2O, 2.67 mmol/l MgSO4, 0.1 mmol/l ethylenediaminetetracetic acid (EDTA) and 33.3 mmol/l dithiothreitol (DTT), (pH 7.8)] and the enzyme activity was immediately measured at room temperature using a luminometer.
For measurements of ß-gal activity, 10 µl of cell lysates were incubated for 30 min at 37°C with 150 µl of 2x assay mixture (120 mmol/l Na2HPO4, 80 mmol/l NaH2PO4, 2 mmol/l MgCl2, 100 mmol/l ß-mercaptoethanol, 1.33 mg/ml O-nitrophenyl--D-galactopyranoside), and the absorbance at 420 nm was determined using a multiplate reader. The measurement of ß-gal activity served to monitor transfection efficiencies and also to normalize luciferase data for ß-gal activity.
Northern blotting
Total RNA was isolated from the cells by a one-step guanidinium method as described previously (Lei and Rao, 1994). Aliquots (30 µg) were denatured and separated on formaldehyde–agarose gels. After transferring RNA to nylon membranes, hybridizations were performed overnight at 42°C with 2x106 c.p.m./ml of [32P-dCTP] GnRH cDNA labelled by the random priming method. The membranes were sequentially washed at 42°C twice with 2x SSC containing 0.1% sodium dodecyl sulphate (SDS) and then with 1x SSC for 20 min each time (1x SSC = 0.15 mol/l NaCl and 0.015 mol/l sodium citrate, pH 7.0). Washed membranes were exposed to X-ray film for 3 days at –80°C with intensifying screens. The membranes were stripped and rehybridized with [32P]-labelled GAPDH cDNA under the same conditions as used for hybridization of GnRH mRNA. Relative optical densities of autoradiographic bands were qualified by scanning the X-ray film using a Z-scan densitometer. Any variations in the amount of total RNA loaded was corrected by expressing values as ratios with the GAPDH signal.
Preparation of nuclear extracts and gel mobility shift assays
Nuclear extracts from GT1-7 and MA-10 cells were prepared as previously described (Lei and Rao, 1997). Briefly, cells were homogenized in a buffer containing 0.5 mmol/l DTT, 0.5 mmol/l phenylmethanesulphonyl fluoride (PMSF), 10 mmol/l HEPES, 1 mmol/l EDTA, 2 mol/l sucrose and 10% glycerol, pH 7.6 and nuclei were isolated. The crude nuclear pellets were lysed in buffer (1 mmol/l DTT, 0.1 mmol/l PMSF, 10 mmol/l HEPES, 100 mmol/l KCl, 0.1 mmol/l MgCl2, 0.1 mmol/l EDTA and 10% glycerol, pH 7.6). After centrifugation, 0.3 g/ml ammonium sulphate was added to precipitate nuclear proteins which were redissolved, and then dialysed overnight at 4°C.
Annealed oligonucleotides containing the SF-1 consensus binding sequence and putative SF-1 binding sequences of the rat GnRH promoter were end-labelled with [32P-ATP] and T4 DNA kinase. The labelled probes were purified by polyacrylamide gel electrophoresis (PAGE). Gel mobility shift assays were performed as described in the BandShift kit from Pharmacia Biotech. Briefly, 5 µg aliquots of nuclear extracts from GT1-7 and MA-10 cells were incubated for 20 min at room temperature with binding mixture [10 mmol/l Tris–HCl, pH 7.5, 100 mmol/l NaCl, 0.5 mmol/l DTT, 1% glycerol, 0.05% Nonidet P-40, 5 mmol/l MgCl2, 2 µg of poly (dI-dC), 0.1 mmol/l EDTA and 0.5 ng of labelled probe (30 000 c.p.m./reaction)]. For competitive studies, a 100-fold excess of unlabelled probe was added to the binding mixture. For gel mobility supershift experiments, 1 µl of either polyclonal anti-SF-1 antibody or non-specific rabbit immunoglobulin G (IgG) was added to the binding mixture and incubated for 30 min at 4°C and the labelled probe was then added. After incubation, DNA–protein complexes were resolved by 4% native PAGE in a buffer containing 7 mmol/l Tris–HCl, pH 7.5, 3 mmol/l sodium acetate and 1 mmol/l EDTA at 4°C. Gels were dried and exposed overnight at –80°C to X-ray films with intensifying screens.
Immunocytochemistry
The monolayer of GT1-7 neurons cultured on coverslips was fixed for 5 min with Bouin's solution and then permeabilized for 10 min at room temperature by incubating with 0.1% Triton X-100. Immunocytochemistry was performed by an ABC method using 1:25 dilution of polyclonal anti-SF-1 antibody (Lei and Rao, 1994). The 3,3'-diaminobenzidine was used as the substrate with nickel enhancement which gave a blue/black coloured product. Non-specific rabbit IgG was used for the procedural control.
Western immunoblotting
GT1-7 and MA-10 cells were homogenized in 50 mmol/l Tris–HCl buffer, pH 7.4, containing protease inhibitors. Aliquots (50 µg) of protein were separated by discontinuous 8% SDS–PAGE under reducing conditions and electroblotted to immobilon P-membranes. After blocking non-specific binding sites with non-fat dry milk, the membranes were incubated with 1:100 dilution of polyclonal anti SF-1 antibody and then with 1:1000 dilution of horseradish peroxidase-labelled secondary antibody. Immunoreactive proteins were detected by an ECL method (Lei and Rao, 1994). Non-specific rabbit IgG was used for the procedural control. The molecular sizes of the immunoreactive proteins were determined by running standard molecular weight marker proteins in an adjacent lane.
Statistical analysis
Each experiment was repeated three times in duplicate or triplicate. All values in the case of Figures 1 and 2 were pooled for calculation of means and SE and for paired t-test.
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Results
Transient transfection of GT1-7 neurons with SF-1 inhibits the transcription of GnRH gene
The effect of over-expression of SF-1 on steady state GnRH mRNA levels in GT1-7 neurons was determined by Northern blotting. The results showed that over-expression indeed decreased GnRH mRNA levels by ~70% (Figure 1
). This decrease was specific because GAPDH mRNA levels were not affected (Figure 1). We made deletion constructs of 5'-flanking region of GnRH gene fused to promoterless luciferase reporter gene (Figure 2). Co-transfection of GT1-7 neurons with 10 µg of –3026 to +116 bp construct and 2 µg of SF-1 expression vector resulted in a 70% decrease in luciferase activity (Figure 3). This decrease was maximal with 2 µg of SF-1 with no squelching effect observed on the pCMV promoter (data not shown). Deletion of the region upstream of –1031 bp, which contains two distal hexamers, resulted in a loss of inhibitory effect, suggesting that proximal hexamer is not required for the SF-1 effect (Figure 3).
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Nuclear extracts of GT1-7 neurons contain SF-1-like binding protein
Since the transient transfection experiments demonstrated that only two distal hexamers are required for the SF-1 action, we next examined whether nuclear extracts of GT1-7 neurons contained SF-1 binding protein. Gel mobility shift assays revealed that GT1-7 nuclear extracts formed specific complexes with the synthetic oligonucleotides synthesized from rat GnRH gene which contained distal hexamers (data not shown). These nuclear extracts also formed complexes with the synthetic oligonucleotide synthesized from steroid hydroxylase gene which contains consensus SF-1 binding sequence (Figure 4
). However, migration of DNA–protein complex formed with GT1-7 cell nuclear extracts was slower than that formed with the nuclear extracts of MA-10 cells (Figure 4). The addition of 100-fold excess unlabelled corresponding oligonucleotide inhibited the formation of DNA–protein complex. The competition among oligonucleotides containing two distal hexamers and consensus SF-1 binding sequence was not tested. However, from above data, they are expected to compete for nuclear protein binding.
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The addition of anti-SF-1 antibody could not induce a supershift of the binding complex in GT1-7 neurons (Figure 4
). This lack of supershift could be because GT1-7 neurons contain SF-1-like, rather than classical SF-1, protein and antibody binding to SF-1-like protein does not induce a supershift. There are examples of antibodies binding not inducing the supershift of transcription factors (Marshall-Heyman et al., 1994; Jaakkola et al., 1998). As expected, MA-10 cells showed higher levels of DNA–protein complex than GT1-7 neurons and the addition of excess unlabeled corresponding oligonucleotide inhibited formation of the complex and anti- SF-1 antibody supershifted the complex (Figure 4).
GT1-7 neurons contain a SF-1-like immunoreactive nuclear protein
Results of the gel shift experiments suggested that GT1-7 neurons probably contained a SF-1-like protein which could bind to the hexamers as well as the consensus SF-1 binding sequence, but anti-SF-1 antibody binding could not induce supershift. To further characterize this protein, we performed Western blotting with the same antibody used in gel shift experiments. Results showed that while nuclear extracts of GT1-7 neurons contained a 60 kDa protein (Figure 5), the nuclear extracts of MA-10 cells contained an expected 53 kDa protein. When a non-specific rabbit IgG was used in the control experiment, the 60 kDa protein in GT1-7 cells (Figure 5) and 53 kDa protein in MA-10 cells (data not shown) were not detected.
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Immunocytochemistry with the same antibody again demonstrated the immunostaining in GT1-7 neurons. Most nuclei were intensely immunostained with a small number showing faint staining (Figure 6A
). The immunostaining was specific as non-specific IgG used for a control showed no immunostaining (Figure 6B).
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Discussion
Transcription factor SF-1 is important for the development and function of the hypothalamus-pituitary–gonadal and adrenal axis. While its role in the expression of genes for gonadotrophin subunit in anterior pituitary and the steroidogenic enzymes in gonads and adrenal has been well established, its role in the expression of the GnRH gene is not clear.
Over-expression of SF-1 resulted in a 70% decrease in the steady state GnRH mRNA levels as well as GnRH promoter activity in GT1-7 neurons. For SF-1 to work, it must bind to its sites in the 5'-flanking region of GnRH gene. To determine their presence, we searched the 5'-flanking region of GnRH gene and found that the region downstream of 3056 bp did not contain any classical SF-1 binding sites. However, this region contained a TGACCT hexamer sequence at –1479 to –1474 bp and at –1059 to –1054 bp and another AGGTCA hexamer at–493 to –488 bp. These hexamers differ from the classical SF-1 binding sites by only two nucleotides at the 5'-end (Rice, 1991; Lala et al., 1992). Because of this close structural similarity, these hexamers may bind SF-1 or a related protein. Deletion analysis revealed that only two distal hexamers are required for the SF-1 action.
If this finding is to have any functional relevance, then GT1-7 neurons should contain SF-1 protein. Gel shift assays revealed that GT1-7 cell nuclear extracts do indeed contain protein that can bind to synthetic oligonucleotides containing the distal hexamers of the GnRH gene and the classical SF-1 DNA binding sequence. The mobility of the binding complex in GT1-7 neurons was slower, indicating that its molecular size might be higher compared with the classical SF-1 protein in MA-10 cells. Western blot analysis confirmed that while GT1-7 neurons contained a 60 kDa protein, MA-10 cells contained an expected 53 kDa SF-1 protein. The 60 kDa protein in GT1-7 neurons is probably a related member of the SF-1 family because it can bind to the consensus labelled SF-1 binding sequence and excess corresponding oligonucleotide could inhibit its binding.
The hexamer TGACCT represents a half-site of oestradiol receptor response element (Beato, 1989). GT1-7 neurons and a sub-population of GnRH neurons in the hypothalamus contain oestradiol receptors (Ahima and Harlan, 1992; Watson et al., 1992; King, 1995; Herbison et al., 1996). Therefore, it may have been of interest to know whether this hexamer could possibly bind oestradiol receptors and whether SF-1-like protein is related to oestradiol receptors. However, neither of these possibilities is likely considering that rat GnRH gene contains the hexamer that does not respond to oestradiol treatment. Moreover, oestradiol receptors, unlike SF-1 family members, bind as dimers to palindromic repeats with an appropriate spacing of these half-sites (Umesono and Evans, 1989).
SF-1 belongs to a subset of the nuclear receptor family which includes BGFI-B, ROR, ERR1, ERR2, COUP and LHR1 (Barnhart and Mellon, 1994; Galarneau et al., 1996; Parker and Schimmer, 1997). Common characteristics of this family are a great degree of sequence homology and the ability to bind as monomers to TGACCT motif (Parker and Schimmer, 1997). Whether the SF-1-like protein found in the present study is any of these other members of this superfamily is not known.
The SF-1-like protein may be antigenically related, but would probably be translated from a distinct mRNA from the classical SF-1 mRNA. This possibility is consistent with previous reports which could not detect classical SF-1 mRNA in GT1-7 neurons by Northern blotting and, in the preoptic area of rat hypothalamus, by ribonuclease protection assay.
We do not know the sequence of SF-1-like protein. Because it can bind to the hexamers in 5'-flanking region of the GnRH gene, it probably can negatively regulate GnRH gene transcription in GT1-7 neurons. Since neither its sequence nor identity are known, we could not perform the kind of experiments that were done for SF-1 itself.
In summary, immortalized GT1-7 neurons contain an SF-1-like protein which could bind to two distal hexamers in the promoter region of rat GnRH gene. Based on studies with SF-1, the binding of the SF-1-like protein is predicted to inhibit the transcription of GnRH. Further studies are required to identify this protein.
Notes
2 Present address: 14822 W. 71st Terrace, Shawnee, KS 66216, USA 
3 To whom correspondence should be addressed at: Department of Ob/Gyn, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, KY 40292, USA. E-mail: cvrao001{at}gwise.louisville.edu
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Submitted on March 1, 2000; accepted on June 2, 2000.
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