Mol. Hum. Reprod. Advance Access originally published online on December 22, 2004
Molecular Human Reproduction 2005 11(2):79-85; doi:10.1093/molehr/gah139
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The NGFI-B family of transcription factors regulates expression of 3ß-hydroxysteroid dehydrogenase type 2 in the human ovary
1Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390 USA and 2Department of Obstetrics and Gynaecology, University of Adelaide, SA 5005, Australia
3 To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9032, USA
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Abstract |
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The nerve growth factor-induced clone B (NGFI-B) family of transcription factors are orphan members of the steroid hormone receptor superfamily. The NGFI-B expression was recently shown in the rat ovarian tissue and appears to be regulated by gonadotrophins. The purpose of our study was to investigate the role of the three members of this family [NGFI-B, Nur-related factor 1 (NURR1) and neuron derived orphan receptor 1 (NOR-1)] in the transcription of genes that encode key steroidogenic enzymes and examine expression in the human ovary. Real-time RT–PCR was used to quantify mRNA expression levels of the NGFI-B family members in human ovarian follicles, corpora lutea and in human granulosa cells after FSH, phorbol ester (TPA) and forskolin treatment. NGFI-B was expressed at higher levels than both NURR1 and NOR-1 in both ovarian follicles and corpora lutea. In human granulosa tumour (HGT) cells, the NGFI-B expression increased after TPA, and to a lesser extent, after forskolin treatment. Treatment of primary cultures of human granulosa cells with forskolin and FSH rapidly increased the NGFI-B mRNA levels followed by an increase in 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2). Transcription of HSD3B2 was studied by transfecting NGFI-B and steroidogenic factor 1 (SF1) expression vectors with reporter constructs prepared with human steroidogenic acute regulatory protein, cholesterol side-chain cleavage, and HSD3B2 genes. NGFI-B increased the transcription of HSD3B2 in HGT cells which is significantly more than SF1. Mutation or deletion of the NGFI-B response element in the HSD3B2 promoter significantly reduced the NGFI-B-mediated transcription of HSD3B2. Therefore, our data suggest that the NGFI-B may play a significant role in up-regulation of HSD3B2 that leads to the increase in progesterone production that is seen in granulosa cells at ovulation.
Key words: corpus luteum/HSD3B2/NGFI-B/Nur77/ovary/progesterone
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Introduction |
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The ovary functions to produce oocytes and secrete steroid hormones that orchestrate the reproductive process. The profile of the steroid hormone production changes considerably during the menstrual cycle and pregnancy. This variability is brought about by the changes in the development and maturation of the ovarian follicle and corpus luteum, as well as the cells that comprise them. The theca cells of the ovarian follicle produce androgens that are subsequently converted to estrogens by the granulosa cells (Ryan and Petro, 1966
). While the cells of the corpus luteum also make estradiol (E2), they primarily secrete and synthesize progesterone. The synthesis of these hormones is achieved by the actions of a number of steroidogenic enzymes, which change in response to alterations in gonadotrophin stimulation and responsiveness. A key area of investigation concerns the increase in progesterone production that occurs at the time of ovulation when granulosa cells of the large follicles are converted into granulosa-lutein cells of the corpus luteum. This process necessitates the induction of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A) and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2).
One of the most important of these regulatory enzymes is HSD3B2, which converts pregnenolone and 17-hydroxypregnenolone to progesterone and 17-hydroxyprogesterone, respectively. Its activity appears to be regulated at the level of transcription. In humans, two genes encode two very similar forms of 3ß-hydroxysteroid dehydrogenase, type 1 (HSD3B1) and type 2 (HSD3B2), which are 93% homologous at the amino acid level (Rheaume et al., 1991). While type 1 is predominantly found in the skin, placenta and breast tissue, type 2 is rather specific for human gonads and the human adrenal gland and is thought to be important in the regulation of ovarian steroid hormone production (Lachance et al., 1991).
Ovarian steroidogenesis is predominantly regulated by the steroidogenic enzyme expression. Current research in the area of ovarian steroidogenesis focuses on the transcriptional regulation of the genes encoding steroidogenic enzymes. The transcription factor, steroidogenic factor 1 (SF1) was found to be crucial for development of both ovary and adrenal gland and is regarded as a permissive factor for the expression of a number of steroidogenic enzymes (Leers-Sucheta et al., 1997; Sirianni et al., 2002). Dosage sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1) was also recently identified as an inhibitory factor in the control of several steroidogenic enzymes (Lalli et al., 1998; Sato et al., 2003). Liver receptor homologue-1 (LRH-1) has been shown to alter the expression of steroidogenic enzymes in the corpus luteum (Peng et al., 2003; Kim et al., 2004).
Nerve growth factor-induced clone B (NGFI-B), also called as Nur77, is an immediate early response gene that is known to be rapidly induced by a variety of extracellular stimuli. NGFI-B and its family members, Nur-related factor 1 (NURR1) and neuron derived orphan receptor 1 (NOR-1), are orphan receptors that bind as homodimers, heterodimers or monomers to specific binding elements containing the 5'-extended core motif (AAAGGTCA) (Wilson et al., 1991; Giguere, 1999). These transcription factors are highly expressed in the brain and pituitary and were originally described as neuronal transcription factors. Recently, however, their prominence in steroidogenesis was further elucidated (Maruyama et al., 1998). NGFI-B was found to be expressed in the adrenal, testis and ovaries and is known to activate HSD3B2 in adrenal cells (Wilson et al., 1993; Song et al., 2001; Bassett et al., 2004). NGFI-B has been shown to increase in the rat corpus luteum in response to prostaglandin F2
with subsequent induction of progesterone metabolizing enzymes in response to NGFI-B (Stocco et al., 2000, 2001). Furthermore, NGFI-B has been shown to be rapidly and transiently induced in in vitro cultured pre-ovulatory follicles in rats and in cultured human luteinized granulosa (HLG) cells (Park et al., 2003). Taken together, these data suggest that NGFI-B is important in the regulation of ovarian steroidogenesis.
In this manuscript, we focus on the role of NGFI-B family of transcription factors in the ovary. We examine their levels of expression in human follicles and corpora lutea and their regulation in granulosa cells in monolayer culture. In addition, we study the capacity of NGFI-B to up-regulate the transcription of HSD3B2 in human granulosa cells.
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Materials and methods |
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Ovarian tissue and cells
Ovarian follicles (n=5) and corpora lutea (n=10) were isolated from the human ovarian tissues. Human ovaries were obtained from premenopausal women at the time of elective hysterectomy and the ovaries were determined to be non-cancerous and otherwise normal at the time of examination of the pathology. All follicles were taken from ovaries in the follicular phase and corpora lutea from ovaries in the luteal phase of the menstrual cycle. The use of these tissues was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas.
Human granulosa tumour (HGT) cells were derived from granulosa cell tumour (Peng et al., 2003). This granulosa cell model reproduces many functions of the granulosa cells. Specifically, the cells retain the ability to produce progesterone and convert androstenedione to E2. This cell model also responds to forskolin and dbcAMP by increasing the production of progesterone and E2 (Peng et al., 2003).
HLG cells (n=2) were obtained by follicular aspiration from women of reproductive age (aged 25–35 year) undergoing oocyte retrieval for IVF. Briefly, women were treated with gonadotrophin-releasing hormone antagonist before and during controlled ovarian stimulation using recombinant human gonadotrophins. After follicular aspiration, HLG cells were washed twice with Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F12) (GIBCO BRL, Gaithersburg, MD) and then incubated for 30 min at 37°C in DME/F-12 containing 0.1% hyaluronidase to disperse them. The dispersed cells were resuspended in 20 ml medium and transferred to 50-ml tubes containing 3.5 ml Histopaque 1077 (Sigma Chemical Co., St Louis, MO). HLG cells were separated from the red blood cells by centrifugation at 600 g for 15 min. HLG cells formed a thin layer between the Histopaque and the medium. The cells were removed and then washed three times using DME/F-12 containing 5% Nu-Serum (BD Biosciences, Bedford, MA), 1% ITS Plus (BD Biosciences, Bedford, MA) and 1% antibiotics. In order to separate the granulosa cells from macrophages, the cells were resuspended in 25 ml medium and placed on 100 mm Petri dishes for incubation at 37°C for 15 min (Beckmann et al., 1991).
Cell culture
HGT cells were cultured in DME/F-12 medium supplemented with 2% Ultroser G (BioSepra SA, Villeneuvue la Garenne Cedex, France), 2% Nu-Serum, 1% ITS Plus and antibiotics. HLG cells were cultured in DME/F12 containing 2% Ultroser G, 1% ITS Plus and antibiotics/antimycotics. The cells were plated in 12-well culture dishes at a density of 20 000 cells/cm2 and allowed to reach near confluence. The cell culture medium was changed on days 3 and 5 of culture. On culture day 7, the cells were changed to DME/F12 medium containing 1% ITS Plus and antibiotics. On day 8, the medium was replaced with DME/F12 medium containing 1% ITS Plus and antibiotics and incubations and experimental conditions were carried out as described.
Reporter constructs and expression vectors
A transient expression system using a luciferase reporter gene was used to characterize the effects of NGFI-B, NURR1, NOR-1 and SF1 on transcription of HSD3B2, CYP11A and StAR. The coding sequences from human SF1, rat NGFI-B, mouse NURR1 and rat NOR-1 were excised from their vectors and subcloned into the pRC/RSV expression plasmid (Invitrogen, Carlsbad, CA). The HSD3B2, CYP11A and StAR constructs used in our study contained 963, 4400 and 1300 bp of DNA isolated from the upstream regulatory regions of the genes, respectively, and cloned into the promoterless pGL3-Basic luciferase reporter plasmid (Promega, Madison, WI), as previously described (Bassett et al., 2004). All three constructs were derived from the human DNA. The –963 HSD3B2 construct was prepared by using the available restriction site, XhoI. The deletion construct (–52 bp) was prepared by PCR through introduction of a unique HindIII 5' restriction site. All constructs were numbered relative to the transcriptional start site. For preparation of the consensus NGFI-B binding element (NBRE) mutant construct, the sequence 5'-AAAGGTCA-3' (–131/–124 bp) was mutated to 5'-AgAatTCA-3', which included an EcoRI restriction enzyme site, as previously described (Bassett et al., 2004).
Transfection assay
For transfection experiments, the HGT cells were subcultured into 12-well culture dishes at a density of 100 000 cells per well and incubated for 36 h. Transfection was carried out using 2 µl Fugene (Roche, Indianapolis, IN) and 1 µg reporter plasmid DNA in DME/F12 medium (1 ml) for 6 h at 37°C. For dose–response analyses, various amounts of expression plasmids were included in the transfection reaction and the total amount of DNA was kept constant by addition of carrier DNA (empty expression vector). Following transfection, cells were incubated with 2 ml low-serum medium (DME/F12 medium containing 0.1% Ultroser G and antibiotics) for 18–24 h to allow time for cell recovery and expression of foreign DNA. Where noted, the transfected cells were treated with agonists for various times. The cells were then lysed and assayed for activity using the luciferase assay system (Promega, Madison, WI). Data were normalized to ß-galactosidase expression vectors that were included in the transfection. All transfection experiments were performed in triplicate or quadruplicate.
Expression experiments, RNA extraction, and real-time RT–PCR
HGT cells were cultured in the same media as above. For the expression experiments, the cells were subcultured into 100-mm dishes and treated with either phorbol ester (TPA), a protein kinase C agonist (20 nM) or forskolin, an activator of adenylyl cyclase (10 µM) for 2 h. HLG cells were cultured as described above. For the agonist experiments, the cells were treated with FSH (50 ng/ml) or forskolin (10 µM). The cells were treated for 1, 3, 6, 12 or 24 h. Forskolin, TPA and FSH were purchased from Sigma Chemical (St Louis, MO).
Total RNA was extracted from the cells using the Ultraspec RNA isolation system (Biotecx Laboratories Inc., Houston, TX) and from ovarian tissue using the caesium chloride gradient method (Chirgwin et al., 1979). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis.
Real-time RT–PCR was used to quantify the expression of NGFI-B family members in the ovarian tissue or after addition of the agonists to the HGT cells. Four microgram of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Primers and probes for real-time RT–PCR were designed by using the Primer Express computer program (Applied Biosystems), as previously described (Bassett et al., 2004). For NGFI-B, NURR1 or NOR-1 quantification, a double-stranded DNA dye, SYBR Green I (Molecular Probes Inc., Eugene, OR) was used in conjunction with 15 µl 2 x SYBR Green Universal PCR Master Mix (Applied Biosystems) and 0.1 µM of each primer. Quantification of 18S ribosomal RNA and HSD3B2 mRNA was performed using a TaqMan Ribosomal RNA Reagent kit (Applied Biosystems) and 10 µl of primer/probe mix. For 18S, the final concentrations of primer and probe were 0.05 µM and 0.1 µM, respectively. All real-time RT–PCR reactions were carried out, in two steps, using the ABI Prism 7000 Sequence Detection System (Applied Biosystems) and the dissociation protocol. Step 1: 50°C for 2 min followed by 95°C for 10 min, one cycle. Step 2: 95°C for 15 s followed by 60°C for 60 s, 40 cycles. Plasmids containing cDNA for NGFI-B, NURR1 or NOR-1 were used to construct standard curves to quantify mRNA transcript levels. As an internal standard, each individual sample was normalized to its own 18S ribosomal RNA content. Messenger RNA levels were expressed as attomoles/µg of 18S rRNA. Alternatively, in the HLG cell experiments, relative gene expression was calculated by the 
Ct method. Briefly, the mRNA was normalized to a calibrator and in each case, the calibrator chosen was the basal sample. Final results were expressed as n-fold difference in gene expression relative to 18S rRNA and calibrator as follows: n-fold=2–(Ct sample–Ct calibrator), where Ct values of the sample and calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the 18S rRNA gene for each sample.
Statistical analyses
Data were analysed by analysis of variance using a SigmaStat software (SPSS Inc., Chicago, IL). Treatments were considered significantly different when P value was <0.05.
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Results |
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Expression of NGFI-B in human ovarian tissue
Expression of NGFI-B, NURR1 and NOR-1 was quantified in fresh tissue samples of ovarian follicles and corpora lutea using real-time RT–PCR (Figure 1). Both NGFI-B and NURR1 were found in both tissues, but NGFI-B mRNA levels were present in 18-fold higher than the amount of NURR1 in the follicle and 31-fold higher than NURR1 in the corpus luteum. NOR-1 was only weakly detectable in either tissue. Therefore, the remainder of the study focused primarily on the role of the predominant transcript, NGFI-B, in the human ovary.
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The effect of forskolin and TPA on NGFI-B expression
To determine whether NGFI-B is regulated by the same intracellular pathways that regulate steroidogenesis, the HGT cells were treated with an agent that increases the cellular cAMP production (forskolin) as well as an agonist of the protein kinase C signalling pathway TPA (Figure 2). RNA was isolated and real-time RT–PCR was performed to quantify expression of NGFI-B, NURR1 and NOR-1. NGFI-B expression increased in response to forskolin and TPA treatments by 58 and 140-fold, respectively. Expression of NURR1 and NOR-1 mRNA was extremely low in both basal and stimulated cells (data not shown).
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The effect of NGFI-B on HSD3B2 transcription
Reporter constructs prepared with the 5'-flanking region of the StAR, CYP11A and HSD3B2 genes were co-transfected with SF1 or NGFI-B expression vectors (Figure 3). Both StAR and CYP11A reporter constructs were more responsive to co-transfection with the SF1 expression vector, which increased the activity to 6-fold above that seen under basal conditions in the StAR promoter and 10-fold in the CYP11A promoter. In contrast, activity of the StAR and CYP11A promoters increased only 1.3 and 1.7-fold in response to the stimulation by NGFI-B. However, the HSD3B2 promoter construct was much more responsive to stimulation by NGFI-B than by SF1, showing a 17-fold increase in activity with NGFI-B as compared to only a 5-fold increase with SF1. This demonstrates that, although SF1 activates transcription of all three genes, NGFI-B differentially regulates transcription of HSD3B2.
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The dose–response of NGFI-B on HSD3B2 gene expression
Granulosa cells were co-transfected with HSD3B2 promoter constructs and varying concentrations of NGFI-B expression vector. Transcription of HSD3B2 increased in a linear fashion with increasing doses of the transcription factor (0.1, 0.3 and 1.0 µg/well) (Figure 4).
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The effect of NBRE alteration on NGFI-B-stimulated transcription of HSD3B2
To further elucidate the role of the suspected NBRE (5'-AAAGGTCA-3') in transcription of HSD3B2, the site was either deleted or mutated within the HSD3B2 promoter constructs (Figure 5A). These altered promoters were co-transfected in parallel with wild-type (963 bp) HSD3B2 promoter and the NGFI-B expression vector. The 963 bp HSD3B2 promoter showed full activity when co-transfected with NGFI-B (100%), while the mutated and deleted promoters showed no induction above basal levels (Figure 5B). The results of the mutation/deletion analysis demonstrate that NGFI-B requires the intact NBRE site for transcriptional regulation of HSD3B2.
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The effect of SF1 on NGFI-B-mediated transcriptional regulation of HSD3B2
To determine the effect of SF1 on NGFI-B-mediated transcription of HSD3B2, the HGT cells were co-transfected with HSD3B2 promoter constructs and NGFI-B expression vector, SF1 expression vector or both expression vectors. Maximal activity of the HSD3B2 promoter was seen with NGFI-B, which increased 11-fold from basal (Figure 6). The effect of SF1 was much less and only increased the activity to 5-fold. The combination of SF1 and NGFI-B produced results of intermediate values.
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The time course expression of NGFI-B and HSD3B2 in HLG cells
To further validate the findings of NGFI-B and HSD3B2 expression in our granulosa cell tumour model, we performed a time course analysis to determine the temporal relationship between the NGFI-B gene expression and the HSD3B2 gene expression, in response to FSH (50 ng/ml) and forskolin (10 µM) in primary HLG cells. RNA was isolated from HLG cells obtained from two patients. The HLG cells were subsequently treated with FSH or forskolin for various time intervals (1, 3, 6, 12 and 24 h). Transcription of NGFI-B increased dramatically at 1 h in response to FSH (25-fold), with a subsequent decrease to baseline expression levels by 6 h (Figure 7A and B). In contrast, induction of HSD3B2 gene expression was delayed compared to that of NGFI-B, with maximal stimulation of HSD3B2 mRNA levels at 24 h (3–7-fold). A similar pattern of NGFI-B and HSD3B2 gene expression was seen with the protein kinase A pathway agonist, forskolin (Figure 7C and D).
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Discussion |
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The present study demonstrates that NGFI-B and, to a lesser extent, NURR1 are present in ovarian follicles and corpora lutea and are induced by activation of the intracellular pathways that promote steroidogenesis. We show that NGFI-B stimulates transcription of HSD3B2 in the ovary at the transcriptional level through action at its NBRE site on the promoter and that it is only minimally affected by SF1. From these data, we suggest that NGFI-B may play an important role in the qualitative changes in ovarian steroidogenesis that occurs late in the follicular phase and throughout the luteal phase as granulosa cells shift from an estrogen producing follicle to a progesterone producing corpus luteum.
NGFI-B is a known participant in various endocrine systems. It is widely expressed in endocrine tissues, such as the pituitary and the adrenal glands under basal conditions and may be induced in these tissues by external stressors or signals (Maruyama et al., 1997). This orphan receptor is also known to activate transcription of steroidogenic enzymes in various tissues (Davis and Lau, 1994; Murphy and Conneely, 1997; Philips et al., 1997). The role of NGFI-B in steroidogenesis has been examined in both ovary and adrenal. Recent microarray analysis showed a positive association of expression levels of NGFI-B with HSD3B2 in the human adrenal gland. Furthermore, this study demonstrated an increased expression of HSD3B2 in response to NGFI-B in adrenal cells that secrete mainly cortisol (Bassett et al., 2004). Recent microarray analysis has demonstrated a reduced NGFI-B expression in polycystic ovary syndrome ovaries compared to normal ovaries (Diao et al., 2004; Jansen et al., 2004).
In the ovary, we found that NGFI-B is expressed in high levels in the corpus luteum. The evidence, NGFI-B expression in mature rat follicles increased markedly 1 h after LH treatment suggests that NGFI-B regulates transcription differentially in follicles versus corpora lutea (Park et al., 2001). In this study, we have used FSH to stimulate our primary granulosa cells, as we have found these cells to require significantly greater doses of LH to produce an equivalent steroidogenic response. This is likely due to desensitization from prolonged gonadotrophin exposure during controlled ovarian stimulation, which can be partially overcome with prolonged culture in vitro (Breckwoldt et al., 1996). Unfortunately, the dose of LH required to stimulate a significant steroidogenic response (3000 mIU/ml) is at a supraphysiologic level (Sasson et al., 2004). The use of both granulosa cell tumour model and primary human granulosa cells in this study allowed us to evaluate the role of NGFI-B in an ovary specific cell model and to confirm these findings in primary human granulosa cells.
Our findings of a rapid induction of NGFI-B with subsequent return to basal transcription by 6 h suggests that NGFI-B may be responsible for the rapid, acute changes in HSD3B2 expression during luteinization, while other factors may be responsible for chronic changes in HSD3B2 gene expression. Although NGFI-B protein half-life is approximately 30–40 min (Hazel et al., 1991), stimulated protein levels persist for at least 6 h in Leydig and adrenal cells, in response to a cAMP analogue (Martin and Tremblay, 2004). Interestingly, in the human Leydig cell, both LH and NGFI-B-mediated stimulation of the NBRE resulted in active and rapid progesterone production (Song et al., 2001). This expression pattern relates with that of human HSD3B2, which is only minimally expressed when the demand for progesterone is low, as in the early sfollicle, but it is maximally expressed in the corpus luteum, which produces enormous amounts of progesterone (Doody et al., 1990; Suzuki et al., 1993). Its ability to be rapidly induced, easily modulated and to activate HSD3B2 transcription make NGFI-B a likely candidate for influencing the rapid luteinization process (Maruyama et al., 1998; Kovalovsky et al., 2002).
The results of this study suggest that NGFI-B specifically binds the NBRE to activate transcription of HSD3B2. The NBRE is a well-characterized binding site for NGFI-B that is very specific for this transcription factor (Wilson et al., 1991) and the NBRE has also been observed in the promoter of 21-hydroxylase, the steroidogenic enzyme largely responsible for glucocorticoid and mineralocorticoid production from the adrenal (Wilson et al., 1993). In contrast to SF1, which binds sites that are similar to its own in sequence (including the NBRE), NGFI-B is site-specific (Crawford et al., 1995). The Nur77 response element (NurRE) is a recently identified palindromic sequence that binds NGFI-B homodimers and was discovered to activate corticotrophin releasing hormone mediated pro-opiomelanocort (CRH-mediated POMC) transcription with much higher potency than the NBRE (Philips et al., 1997). We were unable to locate a NurRE site in the promoter region of HSD3B2 suggesting that the NBRE at –124 (5'-AAAGGTCA-3') is likely the key cis-element for NGFI-B action.
Because human HSD3B1 is only expressed to 5% of the levels of HSD3B2 in the human ovary (Rheaume et al., 1991), its regulation by NGFI-B was not studied for this manuscript. Like HSD3B2, the HSD3B1 promoter does contain an NBRE, and NGFI-B may indeed influence transcription of HSD3B1 in the ovary or other tissues. Although their coding regions share considerable sequence homology, the promoter regions of HSD3B1 and HSD3B2 only share 78% sequence similarity (Lachance et al., 1991), potentially rendering these genes responsive to different tissue-specific regulatory factors.
The other orphan receptor family members, NURR1 and NOR-1, have also not been previously studied in the ovary. These transcription factors exhibit similar expression patterns and transcriptional roles as for NGFI-B, but unique roles for NURR1 and NOR-1 have been documented in other tissues (Fernandez et al., 2000). However, we found that their expression levels were very low in the ovary, suggesting that they would have minimal if any role in regulating ovarian steroidogenesis in human mature follicles and corpora lutea. Other major transcription factors regulating ovarian steroidogenesis include SF1 and LRH-1. SF1 is constitutively expressed in the ovary and activates HSD3B2 in granulosa and theca cells (Leers-Sucheta et al., 1997), but our data show that SF1 has minimal effect on action of NGFI-B on HSD3B2 transcription. SF1 can loosely bind certain NBRE cis-elements and, therefore, may have the propensity to displace NGFI-B from its own binding site (Crawford et al., 1995). However, our results indicate that no such significant displacement occurs for the NBRE seen in the HSD3B2 promoter. Similarly, LRH-1 activates HSD3B2 in the corpus luteum and may contribute to luteinization of granulosa cells (Peng et al., 2003). Due to the intricacy of ovarian steroidogenic enzyme transcriptional regulation, it is unlikely that NGFI-B acts alone, though factors modifying NGFI-B action in the ovary are yet to be determined.
Although many of the specific mechanisms for the acute and differential regulation of HSD3B2 remain to be elucidated, data suggest that NGFI-B plays a significant role in the post-ovulation increase in HSD3B2. We suspect that NGFI-B accomplishes a role in human ovarian luteinization similar to its role in the rat ovary. As an immediate-early gene, NGFI-B has the ability to be rapidly induced in response to an acute signal, and its transcriptional response to the LH surge suggests that NGFI-B levels rise immediately after ovulation to activate HSD3B2-mediated production of progesterone. Thus, NGFI-B may represent one of the key molecules regulating the rise in progesterone during luteinization of the granulosa cell.
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Acknowledgements |
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The authors would like to thank Bobbie Mayhew for her technical support. This work was supported by National Institutes of Health grant T32-HD007190 (BRC).
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References |
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Submitted on November 3, 2004; resubmitted on November 16, 2004; accepted on November 20, 2004.
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