The Human 3?-Hydroxysteroid Dehydrogenase/5-4 Isomerase Type 2 Promoter Is a Novel Target for the Immediate Early Orphan Nuclear Receptor Nu
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内分泌学杂志 2005年第2期
Ontogeny-Reproduction Research Unit, Centre Hospitalier de l’Université Laval (CHUL) Research Center (L.J.M., J.J.T.), Ste-Foy, Québec, Canada; and Department of Obstetrics and Gynecology, Faculty of Medicine, Université Laval (J.J.T.), Ste-Foy, Québec, Canada
Address all correspondence and requests for reprints to: Dr. Jacques J. Tremblay, Ontogeny-Reproduction, Room T1-49, CHUL Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: jacques-j.tremblay@crchul.ulaval.ca.
Abstract
The human (h) 3?-hydroxysteroid dehydrogenase/5-4 isomerase type 2 (3?-HSD2) enzyme, encoded by the hHSD3B2 gene, is mainly found in gonads and adrenals. This enzyme catalyzes an essential early step in the biosynthesis of all classes of steroid hormones. The critical nature of the enzyme is supported by the occurrence of human syndromes that are associated with insufficient 3?-HSD2 expression and/or activity. Although the need for a functional 3?-HSD2 enzyme is indisputable, the molecular mechanisms that regulate HSD3B2 expression (both basal and hormone-induced) in steroidogenic cells remain poorly understood. A role for the Nur77 family of immediate-early orphan nuclear receptors in steroidogenesis has received recent interest. For example, Nur77 is present in gonads and adrenals, where its expression is robustly and rapidly induced by hormones that stimulate steroidogenic gene expression. Moreover, the expression patterns of Nur77 and at least one key steroidogenic gene (hHSD3B2) closely parallel one another. We now report that the hHSD3B2 promoter is indeed a novel target for Nur77 in both testicular Leydig cells and adrenal cells. We have mapped a novel response element located at –130 bp specific for Nur77 and not other orphan nuclear receptors (steroidogenic factor-1 and liver receptor homolog-1) previously shown to regulate hHSD3B2 promoter activity. This Nur77 element is essential and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter, and its mutation blunts basal and hormone-induced hHSD3B2 promoter activity in steroidogenic cells. We also show that Nur77 synergizes with all members of the steroid receptor coactivator family of coactivators on the hHSD3B2 promoter. Taken together, our identification of Nur77 as an important regulator of HSD3B2 promoter activity helps us to better define the tissue-specific and hormonal regulation of the HSD3B2 gene in steroidogenic cells.
Introduction
THE HUMAN 3?-hydroxysteroid dehydrogenase/5-4 isomerase type 2 (3?-HSD2) gene, HSD3B2, is expressed in both the gonads and the adrenals and encodes the 3?-HSD2 enzyme that is required for the initial steps of all classes of steroid hormone biosynthesis (reviewed in Ref.1). Its crucial role in steroidogenesis is underscored by the existence of human (h) HSD3B2 gene mutations that are responsible for 3?-HSD2 deficiency. Affected individuals have congenital adrenal hyperplasia accompanied by various degrees of salt-wasting in both sexes. In addition, because 3?-HSD2 is also required for testosterone synthesis in testicular Leydig cells, genetic males are pseudohermaphrodites with female external genitalia (1).
Despite its importance for normal steroidogenesis and proper male sex differentiation, very little is known about the mechanisms that regulate expression of the hHSD3B2 gene in steroidogenic cells. HSD3B2 gene expression, however, is known to be hormonally regulated by angiotensin II (AII) and ACTH, which acts on adrenal cells, and LH/hCG, which regulates steroidogenesis in testicular Leydig cells and ovarian thecal and luteinized granulosa cells (reviewed in Ref.1). AII acts through the diacylglycerol and inositol triphosphate pathways (2), whereas ACTH and LH/hCG mediate their effects through activation of the cAMP/protein kinase A (PKA) signaling pathway. This pathway triggers at least two distinct mechanisms that are both essential for an optimal response. One does not require de novo protein synthesis; rather, it relies on phosphorylation of transcription factors already present in the cell. The other mechanism requires protein synthesis, involving rapid induction of genes encoding transcription factors (3, 4, 5, 6, 7, 8). To date, only a few transcription factors have been shown to bind and activate the hHSD3B2 promoter. These include two members of the NR5A family of orphan nuclear receptors, steroidogenic factor-1 (SF-1) (9) and liver receptor homolog-1 (LRH-1) (10), as well as the signal transducer and activator of transcription proteins 5 and 6 (11, 12, 13, 14). These transcription factors are all found in steroidogenic tissues expressing hHSD3B2 (10, 11, 15, 16, 17, 18, 19, 20, 21). However, because they are also present in other tissues that do not express HSD3B2, such as pituitary gonadotrope cells, liver, pancreas, the zona reticularis of the adrenals, and testicular Sertoli cells (22, 23, 24), other transcription factors are probably involved in tissue-specific HSD3B2 expression. Moreover, the expression of SF-1 and LRH-1 is unchanged, decreased, or only slightly increased in steroidogenic cells in response to ACTH, LH/hCG, or cAMP analogs, which indicates that they are not likely to be the rapidly induced transcription factor required for maximal hormonal stimulation (22, 25). Thus, the newly synthesized transcription factor involved in the hormone-induced up-regulation of HSD3B2 expression has yet to be identified.
Nur77, also known as NGFI-B, TR3, or NR4A1, is a member of the NR4A family of orphan nuclear receptors, which also includes Nurr1 (NR4A2) and Nor1 (NR4A3). Members of this family are immediate-early response genes, and their expression, particularly that of Nur77, is strongly and rapidly induced after various stimuli in numerous tissues (26, 27), including hormonally stimulated steroidogenic cells (28, 29, 30, 31). Nur77 can bind to DNA in three different ways: as a monomer to a Nur77-binding response element (NBRE), which is very similar to SF-1-binding sites (32); as a homo- or heterodimer to an inverted repeat response element (NurRE) (33, 34); or to a DR5 element by heterodimerization with retinoid X receptor (35). Members of the Nur77 family have been identified as important regulators of hormonally regulated gene expression in several endocrine tissues, including the pituitary (proopiomelanocortin gene) (33), the ovary (20-HSD gene) (36), and, more recently, the adrenals (Cyp11B2 gene) (37) and testis (Cyp17 gene) (38).
We now report that the hHSD3B2 promoter is a novel target for Nur77 in both testicular Leydig cells and adrenal cells. Using detailed promoter analyses, we have identified a novel NBRE element located at –130 bp that is specifically recognized by Nur77, but not SF-1 or LRH-1. This novel NBRE element is also essential and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter. Moreover, mutation of the NBRE element blunts cAMP-mediated activation of the hHSD3B2 promoter in both Leydig and adrenal cells. Finally, we show that Nur77 synergizes with SRC coactivators (SRC-1/2/3) to further enhance hHSD3B2 promoter activity. Thus, our results provide critical new insights into the tissue-specific expression and hormonal regulation of hHSD3B2 gene transcription in steroidogenic cells.
Materials and Methods
Plasmids
The –1073 to +53 bp human HSD3B2 promoter fragment was provided by Dr. Jacques Simard (Centre Hospitalier de l’Université Laval Research Center, Université Laval, Québec, Canada). Deletions of the hHSD3B2 promoter to –842, –538, –495, –340, –224, –95, and –60 bp were obtained by PCR using the –1073 bp hHSD3B2 promoter as template, along with a common reverse primer containing a KpnI (underlined) cloning site (5'-GGGGTACCCGTAACTTAGATTGTTAAAAGCTGG-3') and the following forward primers: –842 bp, 5'-CGGGATCCCCTTGTAATGCCCAGATTACATC-3'; –538 bp, 5'-CGGGATCCTCCATTAGGAACCCAGAGCTCTCC-3'; –495 bp, 5'-CGGGATCCGGTTTTGGATATATTGGGTGGAAAAG-3'; –340 bp, 5'-CGGGATCCCCAGGTGGATTTACTGTACAAGGAC-3'; –224 bp, 5'-CGGGATCCCTGTTAAGGCTAAAGCCAAGAC-3'; –95 bp, 5'-CGGGATCCGAGGAGGGAGCAATGAGTATG –3'; and –60 bp, 5'-CGGGATCCGGTAATAAGGGCTGAGACACAAGC-3'. The –1073 bp reporters containing a mutation of the NBRE element at –130 and/or SF-1 element at –60 were obtained by site-directed mutagenesis using the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) and the following pair of oligos (the mutations are underlined): NBRE element at –130: sense, 5'-GTTCCTGGGAAGAATTAGAGATATAACCTAAATTTCACTATTATTCTGAG-3'; antisense, 5'-CTCAGAATAATAGTGAAATTTAGGTTATATCTCTAATTCTTCCCAGGAAC-3'; and SF-1 element at –60: sense, 5'-GAGTATGTGGCAGGAGTTCAATTTAATAAGGGCTGAGACACAAG-3'; antisense, 5'-CTTGTGTCTCAGCCCTTATTAAATTGAACTCCTGCCACATACTC-3'. All promoter fragments were cloned into a modified pXP1 luciferase reporter plasmid (39, 40) and subsequently verified by sequencing. The mouse SF-1 expression vector has been previously described (40). Expression vectors for full-length rat Nur77, NOR-1, and Nurr1 (33) were provided by Dr. Jacques Drouin (Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Canada). The human LRH-1 expression vector (41) was provided by Dr. Luc Bélanger (Centre de Recherche en Cancérologie, Centre de Recherche du CHUQ, Université Laval, Québec, Canada). A pcDNA3-derived expression vector for the PKA catalytic subunit was obtained by transferring the PKA cDNA from the MT-PKAc (42) obtained from Dr. Mark Montminy (The Salk Institute for Biological Studies, La Jolla, CA). The mouse SRC-1, SRC-2, and SRC-3 expression plasmids (43) were gifts from Dr. Joseph Torshia (University of Western Ontario, London, Canada).
Cell culture and transfections
Mouse Leydig MA-10 cells (44), provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA), were grown in Waymouth’s MB752/1 medium supplemented with 20 mM HEPES, 15% horse serum, and 50 mg/liter gentamicin, and streptomycin sulfates at 37 C in 5% CO2. Human adrenal H295R cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM/Ham’s F-12 (1:1) medium supplemented with 15 mM HEPES, 1.2 g/liter NaHCO3, 2.5% NuSerum, and 10 ml/liter of ITS+ Premix (BD Biosciences, San Jose, CA) at 37 C in 5% CO2. All transfections were performed in 12-well plates using the Lipofectamine 2000 method (Invitrogen Canada, Burlington, Canada) according to the manufacturer’s recommendations. Briefly, the day before transfection, MA-10 and H295R cells were plated at 200,000 and 300,000 cells/well, respectively. The next day, cells were transfected with 1.5 μg hHSD3B2 promoter construct fused to Firefly luciferase, 250 ng cytomegalovirus-driven expression vector, 10 ng phRL-TK Renilla luciferase as an internal control, and pSP64 as carrier DNA up to 2 μg/ well using 5 μl Lipofectamine 2000 and antibiotic- and serum-free medium. Five hours later, cells were provided with complete medium. The following day, the Dual Luciferase Assay System (Promega Corp., Madison, WI) was used to harvest the cells and measure luciferase activities using an EG&G Berthold LB 9507 luminometer. In experiments with cAMP stimulation, cells were treated with 0.5 mM dibutyryl cAMP [(Bu)2cAMP] for 6 h before harvesting. Several DNA preparations of the plasmids were used to ensure reproducibility of the results. The data reported represent the average of at least three experiments, each performed in duplicate.
EMSAs
Nuclear extracts were obtained from MA-10 cells treated with either vehicle or 0.5 mM (Bu)2cAMP for 4 h. Recombinant Nur77, SF-1, and LRH-1 proteins were in vitro translated using the T7 QuickCoupled TnT system (Promega Corp.). DNA binding assays were performed using either 10 μg nuclear extracts or 1–2 μl in vitro translated protein as described previously (45), with the exception of dI:dC, where 100 ng were used for EMSAs performed with in vitro translated proteins. The 32P-labeled double-stranded oligonucleotides used as probes were as follows: hHSD3B2 NBRE element (underlined) at –130 bp (sense, 5'-GATCCTAACCTAAAGGTCACTATTA-3'; antisense, 5'-GATCTAATAGTGACCTTTAGGTTAG-3'), and hHSD3B2 SF-1/LRH-1 element (underlined) at –60 bp (sense, 5'-GATCCGGAGTTCAAGGTAATAAGGA-3'; antisense, 5'-GATCTCCTTATTACCTTGAACTCCG-3'). For the competition experiments, double-stranded oligonucleotides corresponding to mutated versions of the NBRE and SF-1/LRH-1 elements were used. The sequences of the oligonucleotides are the same as those described above, except that the NBRE element (TAAAGGTCA) was mutated into TAAATTTCA, and the SF-1/LRH-1 element was changed from TCAAGGTAA to TCAATTTAA. For supershift experiments, 1.6 μg of a commercially available anti-Nur77 antiserum (M-210, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were also added to the binding reaction.
Results
Activation of hHSD3B2 promoter activity by Nur77 factors
Nur77 is known to be expressed in the adrenals and testes, where its expression is induced in response to ACTH and LH, two hormones that act mainly through the cAMP/PKA signaling pathway (28, 29). Because the H295R and MA-10 cell lines are commonly used as models for adrenal and testicular steroidogenic cells, respectively, we tested whether Nur77 expression could also be induced by cAMP in these cell lines. As shown in Fig. 1A, Nur77 protein levels were strongly induced in a time-dependent manner in response to (Bu)2cAMP in both MA-10 Leydig and H295R adrenal cells. These results thus raise the intriguing possibility that Nur77 could represent the hormonally induced transcription factor required for maximal stimulation of hHSD3B2 promoter in these cells. In support of this hypothesis, the hHSD3B2 promoter contains two regulatory elements (at –320 and –60 bp) for the binding of orphan nuclear receptors known to bind DNA as monomers, including SF-1 and LRH-1 (9, 10), and potentially Nur77, which we now propose (32, 41).
FIG. 1. Activation of the hHSD3B2 promoter by Nur77 family members. A, Induction of Nur77 after cAMP treatment. MA-10 mouse Leydig cells (left panel) and H295R human adrenal cells (right panel) were treated with 0.5 mM (Bu)2cAMP for the indicated times, and nuclear extracts were isolated. Twenty-microgram aliquots of extracts were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, and the Nur77 protein was revealed using a Nur77 polyclonal antibody. n.s., Nonspecific. B, MA-10 Leydig cells (left panel) and H295R adrenal cells (right panel) were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl; ) or expression vectors (250 ng) for Nur77 family members (), SF-1 (), and LRH-1 ( ) as indicated. The positions of the two previously characterized binding sites for orphan nuclear receptors within the –1073 to +53 bp hHSD3B2 promoter fragment (–320 and –60 bp) are represented by triangles. Results are shown as fold activation over the control value (±SEM).
To test whether Nur77 could participate in hHSD3B2 transcription in steroidogenic cells, we performed transient transfection assays in MA-10 Leydig and H295R adrenal cells, two homologous cell lines known to have endogenous 3?-HSD expression/activity. As shown in Fig. 1B, Nur77 as well as the other Nur77 family members, Nurr1 and Nor1, found in steroidogenic tissues (29, 31) could activate a –1073 bp hHSD3B2 reporter construct (3-fold in MA-10 cells and 1.7-fold in H295R cells), whereas SF-1 and LRH-1 had no statistically significant effect (P > 0.05, by t test). This is probably due to the fact that these cells already contain significant levels of SF-1 and LRH-1 (9, 15, 46).
Localization of the Nur77-responsive element
To locate the Nur77-responsive element, 5'-progressive deletions of the hHSD3B2 promoter were generated and tested for Nur77 responsiveness in MA-10 and H295R cells (Fig. 2A). A deletion construct to –340 bp that retained the two previously characterized SF-1/LRH-1 elements (–320 and –60 bp) was still activated by Nur77. Deletion to –224 bp that removed the distal (–320 bp) SF-1/LRH-1 element had no effect on Nur77-dependent activation of the hHSD3B2 promoter. Additional deletion to –95 bp while retaining the proximal (–60 bp) SF-1/LRH-1 element, however, completely abrogated transactivation of the hHSD3B2 promoter by Nur77. Taken together, these results indicate that the two SF-1/LRH-1 elements are not required for Nur77-dependent activation of the hHSD3B2 promoter and that a novel, as yet unidentified, element located between –224 and –95 bp is responsible for Nur77 responsiveness. Indeed, sequence analysis of this promoter region revealed the presence of a consensus NBRE located at position –130 bp (Fig. 2B). The importance of this novel NBRE element in Nur77-mediated hHSD3B2 promoter activation was further analyzed by mutagenesis in the context of the –1073 bp hHSD3B2 reporter. As shown in Fig. 3A, mutation of the –130 bp NBRE element (TAAAGGTCATAAATTTCA) completely abolished hHSD3B2 promoter activation by Nur77 in both MA-10 and H295R cells. Mutation of the –60 bp SF-1/LRH-1 element (TCAAGGTAATCAATTTAA), however, had no effect on Nur77-dependent hHSD3B2 promoter activation. Thus, the novel NBRE element (–130 bp) is both necessary and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter.
FIG. 2. Mapping of the Nur77-responsive element in the hHSD3B2 promoter. A, MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with various 5' deletion constructs of the hHSD3B2 promoter (the 5'-end point of each construct is indicated on the left of the graphs) with either an empty expression () or expression vectors for Nur77 (250 ng; ). The positions of the two orphan nuclear binding sites are indicated (triangles). Results are shown as fold activation over the control value (±SEM). B, Sequence of a novel NBRE element located at position –130 bp. The sequence is compared with the previously described sites at positions –320 and –60 bp.
FIG. 3. The novel NBRE element at –130 bp is necessary and sufficient for Nur77-dependent hHSD3B2 promoter activation. A, MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with either an empty expression vector () or expression vectors for Nur77 (250 ng; ) along with several –1073 to +53 bp hHSD3B2 reporters as indicated on the far left side of the figure: a wild-type reporter, a reporter harboring a point mutation (underlined) in the NBRE element at –130 bp (TAAAGGTCATAAATTTCA), a reporter with the –60 bp element mutated (TCAAGGTAATCAATTTAA), and a reporter with both the –130 and –60 bp elements mutated. The mutated elements are represented by a large X. All results are shown as fold activation over the control value (±SEM). B, Increased Nur77 DNA-binding activity on the novel –130 bp NBRE element after (Bu)2cAMP treatment of MA-10 Leydig cells. EMSA was used to determine the binding of the Nur77 protein present in MA-10 Leydig cells in the absence or after a 4-h stimulation with 0.5 mM (Bu)2cAMP. Nur77 binding was supershifted by a Nur77 antiserum (Nur77). P.I., Preimmune serum.
The presence of Nur77 DNA-binding activity in MA-10 Leydig cells was tested directly by EMSA (Fig. 3B). In unstimulated MA-10 cells, a weak band (Fig. 3B, lane 3) that comigrated with in vitro produced Nur77 (Fig. 3B, lane 2) was observed. This binding was strongly enhanced in nuclear extracts from (Bu)2cAMP-treated MA-10 cells (Fig. 3B, lane 4). Furthermore, this band was supershifted by the addition of an anti-Nur77 antiserum (Fig. 3B, lane 6). Thus, endogenous Nur77 DNA-binding activity on the novel NBRE element is strongly induced by cAMP analogs.
Specific binding of Nur77 to the NBRE element (–130 bp)
The DNA binding specificity of Nur77 to the novel NBRE element (–130 bp) was next assessed by EMSA. Consistent with the specific requirement for the NBRE element in Nur77-mediated activation of the hHSD3B2 promoter (Fig. 3A), in vitro translated Nur77 bound with high affinity to the NBRE element (Fig. 4A), but not to the proximal (–60 bp) SF-1/LRH-1 element (Fig. 4B). Nur77 binding was specifically competed by increasing doses of unlabeled oligonucleotides (Fig. 4A, lanes 7 and 8), but not by oligonucleotides harboring a mutation in the NBRE element (Fig 4A, lanes 9 and 10) or oligonucleotides corresponding to the proximal SF-1/LRH-1 element (Fig. 4A, lanes 3 and 4).
FIG. 4. The novel NBRE element is a Nur77-specific binding site. EMSA was used to assess the binding of in vitro produced Nur77 to a double-stranded 32P-labeled oligonucleotide corresponding to either the NBRE element at –130 bp (A) or the SF-1/LRH-1 site at –60 bp (B) of the hHSD3B2 promoter. Binding of in vitro synthesized SF-1 (C) and LRH-1 (D) was also assessed on the –60 bp element. Binding of the proteins was then challenged by increasing doses (; molar excesses of 2- and 5-fold) of unlabeled oligonucleotides corresponding to the wild-type –60 bp element (–60 wt), the –60 bp element mutated (underlined) from TCAAGGTAA to TCAATTTAA (–60 mut), the wild-type –130 bp element (–130 wt), and the –130 bp element containing a mutation (underlined) in the NBRE element (TAAAGGTCATAAATTTCA).
EMSA was also used to test the possibility that SF-1 and LRH-1 might bind to the NBRE element. On the –60 bp element, both SF-1 and LRH-1 could strongly bind, as expected (lanes 2 in Fig. 4, C and D). This binding was completely competed by self oligonucleotides (lanes 3 and 4 in Fig. 4, C and D), but only slightly by an oligonucleotide corresponding to the NBRE (–130 bp) element even at the highest dose (lanes 7 and 8 in Fig. 4, C and D). Thus, these results indicate that the Nur77 and SF-1/LRH-1 orphan nuclear receptors each have their specific binding sites within the hHSD3B2 promoter, raising the possibility that they might cooperate to regulate hHSD3B2 promoter activity. However, as shown in Fig. 5, no transcriptional cooperation was observed among the three orphan nuclear receptors. Rather, coexpression of Nur77 and SF-1 (but not LRH-1) resulted in a loss of Nur77-dependent hHSD3B2 promoter activation, specifically in MA-10 cells. Although the underlying reasons behind this effect remain unknown, this might be due to limiting amounts of an additional coactivator common to both SF-1 and Nur77 in MA-10, but not H295R, cells.
FIG. 5. Effects of orphan nuclear receptor combinations on hHSD3B2 promoter activity. MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl; ) or expression vectors (250 ng) for Nur77, SF-1, and LRH-1, alone or in the combinations indicated. The positions within the –1073 to +53 bp hHSD3B2 promoter fragment of the two previously characterized binding sites for orphan nuclear receptors (–320 and –60 bp) and the novel NBRE element identified in the present work (–130 bp) are represented by two triangles and a hatched circle, respectively. Results are shown as fold activation over the control value (±SEM).
NBRE is required for maximal basal and cAMP-induced hHSD3B2 promoter activities in steroidogenic cells
It is well established that HSD3B2 expression in steroidogenic tissues is regulated by several hormones, including ACTH and LH from the pituitary, two hormones that mediate their effects, at least in part, through the cAMP/PKA pathway (reviewed in Ref.1). Interestingly, these same trophic hormones are also known to induce Nur77 expression in steroidogenic tissues (28, 29, 30, 31). We therefore compared the activation of the –1073 bp hHSD3B2 reporter construct by Nur77 and 0.5 mM (Bu)2cAMP in MA-10 Leydig and H295R adrenal cells. As shown in Fig. 6A, activation of the hHSD3B2 promoter mediated by Nur77 overexpression was similar to the activation induced by (Bu)2cAMP. The combination of Nur77 and (Bu)2cAMP produced an additive effect (Fig. 6A). These results suggest that Nur77 and other cAMP-regulated transcription factors might function in a linear or parallel pathway to regulate hHSD3B2 promoter activity in steroidogenic cells.
FIG. 6. The NBRE element is required for maximal cAMP stimulation of the hHSD3B2 promoter. A, Additive effects of Nur77 and cAMP on hHSD3B2 promoter activity. MA-10 Leydig (left panel) and H295R adrenal cells (right panel) were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty (Ctl; ) or a Nur77 expression vector (250 ng; ). Transfected cells were then treated with or without 0.5 mM (Bu)2cAMP for 6 h before harvesting as indicated. Results are shown as fold activation over the control value (±SEM). B, MA-10 Leydig cells were transfected with the four –1073 to +53 bp hHSD3B2 reporter constructs shown on the left (described in Fig. 3) and treated with either vehicle (–, ) or 0.5 mM (Bu)2cAMP (+, ) 6 h before harvesting. Results are shown as the percent activity (±SEM) relative to the activity of the –1073 bp wild-type reporter in the absence of cAMP treatment (which was arbitrarily set at 100%).
To establish the significance of Nur77 and its NBRE element in cAMP-regulated hHSD3B2 transcription, hHSD3B2 reporter constructs harboring point mutations in the –130 bp NBRE and/or –60 bp SF-1/LRH-1 elements were transfected in MA-10 cells and tested for cAMP responsiveness. As shown in Fig. 6B, mutation of the –130 bp NBRE led to a 40% decrease in cAMP responsiveness compared with the wild-type promoter (from 3- to 1.8-fold stimulation), whereas a 24% decrease (from 3- to 2.3-fold stimulation) was observed when the –60 bp SF-1/LRH-1 element was mutated. Mutation of both elements led to no further decrease compared with the NBRE mutation alone. The results of the mutation experiments, summarized in Table 1, indicate that the Nur77/NBRE complex is a more important contributor to the cAMP responsiveness of the hHSD3B2 promoter than is the proximal SF-1/LRH-1 element. They also indicate that although an intact NBRE element is necessary for maximal stimulation, additional regulatory elements/transcription factors contribute to the cAMP regulation of the hHSD3B2 promoter.
TABLE 1. Contribution of the NBRE and SF-1/LRH-1 elements to basal and cAMP-induced hHSD3B2 promoter activity in MA-10 steroidogenic cells
As for several transcription factors, Nur77 would be expected to exert its effects through cooperations with coactivators. Indeed, Nur77 has been shown to cooperate with cAMP responsive element binding protein-binding protein and SRC family members (SRC-1/2/3). The SRC-1 data, however, are conflicting because some reports have described a transcriptional cooperation between Nur77 and SRC-1 (38, 47, 48), whereas others have not (49). This discrepancy could be explained by the fact that all these experiments were performed in heterologous cells using synthetic reporters consisting of multiple copies of a Nur77-binding site fused to a minimal promoter. To test whether Nur77 could transcriptionally cooperate with SRC coactivators on the natural hHSD3B2 promoter, cotransfections were performed in MA-10 Leydig cells. Although SRC-1, SRC-2, and SRC-3 alone had no effect (Fig. 7), combination of these coactivators with Nur77 resulted in a synergistic activation (average of 6-fold compared with 3.1 for Nur77 alone) of the –1073 bp hHSD3B2 reporter. In addition, PKA-mediated protein phosphorylation enhanced this synergy to nearly 9-fold (Fig. 7), which is consistent with previous findings that coactivator recruitment and interaction with Nur77 can be enhanced by cAMP/PKA (48). Thus, our results indicate that Nur77-dependent activation of hHSD3B2 promoter probably involves coactivator recruitment that is enhanced by the cAMP/PKA pathway.
FIG. 7. Transcriptional cooperation among Nur77, SRC, and PKA. MA-10 Leydig cells were transiently transfected with the –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl) or expression vectors for Nur77 (250 ng), SRC-1, SRC-2, or SRC-3 (500 ng), alone (empty bars) or in combination ( ) a), in the absence or presence of an expression vector encoding the PKA catalytic subunit (25 ng; ). Results are shown as fold activation over the control value (±SEM).
Discussion
The orphan nuclear receptor Nur77 belongs to a family of immediate early response transcription factors involved in the hormonal stimulation of gene expression in several tissues and cell types (26). Steroidogenic tissues are no exception to this, because Nur77 expression was shown to be strongly induced in vivo by LH/hCG in the testis (28) and ovary (30) as well as by ACTH and AII in the adrenal cortex (29, 50). These data suggested a role for Nur77 in steroidogenic gene expression in all three steroidogenic tissues. Indeed, Nur77 has been shown to activate the promoters of some steroidogenic genes (20-HSD, Cyp17, Cyp11B2, and Cyp21) (36, 37, 38, 51). In this study we have identified the promoter of the 3?-HSD gene, encoding a key enzyme essential for the synthesis of all classes of steroids, as a novel target for Nur77.
The hHSD3B2 promoter is a novel target for Nur77 in steroidogenic tissues
The HSD3B2 gene is expressed in the testis, ovary, and adrenals, and its expression within these tissues is cell and zone specific. Indeed, HSD3B2 is expressed in testicular Leydig cells, in ovarian thecal and luteinized granulosa cells, and in cells of the zona glomerulosa and fasciculata (but not reticularis) of the adrenals (reviewed in Ref.1). Therefore, transcription factors regulating the tissue-, cell-, and zone-specific expression of HSD3B2 are also likely to have a similar expression pattern. The transcription factors currently known to regulate hHSD3B2 promoter activity, however, cannot by themselves explain its highly specific expression pattern. In contrast, the Nur77 expression pattern does fulfill this criterion. Within the adrenals, Nur77 expression is zone specific like HSD3B2; it is predominantly expressed in the zona glomerulosa and fasciculata, but not in the zona reticularis (52). Nur77 is also coexpressed with hHSD3B2 in testicular Leydig cells (28) and in ovarian thecal and luteinized granulosa cells (30, 31). Taken together, these data point to a role for Nur77 in hHSD3B2 transcription. In agreement with this, we have now identified the hHSD3B2 promoter as a target for Nur77 in both Leydig and adrenal cells. Similar findings were recently reported by Bassett et al. (53). Furthermore, recent studies by Hong et al. revealed that Nur77 could activate the promoter of the mouse HSD3B1 gene, the mouse ortholog of the human HSD3B2 gene (47). The lack of a steroidogenic phenotype in Nur77–/– mice, however, would suggest otherwise (54). This can be explained by the fact that another Nur77 family member, Nurr1, was up-regulated in Nur77–/– mice, suggesting that the various Nur77 family members may compensate for one another in vivo (54). Consistent with this, Nurr1 is expressed in steroidogenic tissues (29, 31), and we have shown that it can activate the hHSD3B2 promoter as well as Nur77 (Fig. 1B).
Nur77 is known to bind as a monomer to DNA elements highly related to those recognized by the orphan nuclear receptors SF-1 and LRH-1 (32). Although the hHSD3B2 promoter contains two binding sites for SF-1 and LRH-1 located at –320 and –60 bp (9, 10), we found that Nur77-dependent activation of the hHSD3B2 promoter did not require any of these elements. Rather, Nur77 activates hHSD3B2 transcription through a novel element, located at –130 bp, that can be specifically bound by Nur77 and not SF-1 or LRH-1. This element is indeed necessary and sufficient to confer Nur77 responsivenes to the hHSD3B2 promoter in steroidogenic cells. Furthermore, mutation of this element led to a decrease of about 35% in basal hHSD3B2 promoter activity in Nur77-expressing MA-10 Leydig and H295R adrenal cells. Mutation of the –60 bp SF-1/LRH-1 element, however, resulted in a drop of more than 80% in basal hHSD3B2 promoter activity in these same cells, clearly indicating that SF-1 and/or LRH-1 are truly important regulators of basal HSD3B2 transcription. The fact that Nur77 and SF-1/LRH-1 mediate their effects through specific regulatory elements, such as on the hHSD3B2 promoter described in the present study, would be consistent with the nonredundant, yet complementary, roles played by these orphan nuclear receptors in endocrine development and function (22, 24, 26).
Roles of orphan nuclear receptors in basal and hormone-induced HSD3B2 expression
Of the three orphan nuclear receptors now known to be implicated in basal hHSD3B2 transcription (Nur77, SF-1, and LRH-1), Nur77 appears to be the most sensitive to hormonal regulation. Thus, although SF-1 and LRH-1 expression is either unaffected, decreased, or only slightly increased in response to hormones (LH/hCG, ACTH, and AII) that regulate steroidogenesis (18, 25, 50, 52, 55, 56), Nur77 expression, in contrast, is known to be strongly and rapidly induced (reviewed in Ref.26). This is not surprising given that Nur77 is an immediate-early response factor. Like 3?-HSD, Nur77 expression is robustly induced after hormonal stimulation in the three major steroidogenic tissues; it is strongly induced by LH/hCG in granulosa cells (30, 31), by LH/cAMP in testicular Leydig cells (28), and by both ACTH/cAMP and AII in adrenal cells (29, 50, 51, 52). Therefore, the Nur77 expression pattern and its hormonal regulation in steroidogenic tissues are consistent with a role for this transcription factor in hormone-induced 3?-HSD expression. Indeed, our cAMP regulation data and transcriptional cooperation with the SRC coactivators support such a role. By consolidating our results with those taken from the literature, we present a model (Fig. 8) in which the orphan nuclear receptors Nur77, SF-1, and LRH-1 can be integrated as nonredundant, yet complementary, regulators for the tissue-, zone-, and cell-specific and hormone-dependent expression of the hHSD3B2 gene.
FIG. 8. Proposed model for the implication of orphan nuclear receptors in basal and hormone-induced hHSD3B2 transcription. The binding of the pituitary trophic hormones LH or ACTH to their respective G protein-coupled receptors (left side) triggers activation of the cAMP intracellular signaling pathway, leading to activation of PKA. AII binds to its AT1 receptor (right side) triggering activation of phospholipase C (PLC) and production of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 increases intracellular calcium levels, which, in turn, activates calcium-dependent kinase (CamK), whereas DAG activates protein kinase C (PKC). These pathways all lead to increased Nur77 expression in steroidogenic cells (28 50 ) as well as phosphorylation of target proteins, including transcription factors such as orphan nuclear receptors Nur77, SF-1, and LRH-1. These DNA-bound transcription factors also directly interact with coactivators such as SRC-1/2/3, and this interaction is stronger when proteins are phosphorylated (48 57 ). The SRC coactivators then recruits the cointegrator cAMP responsive element binding protein-binding protein (58 ) into a transcriptionally active complex, leading to increased hHSD3B2 transcription.
Acknowledgments
We thank Drs. Jacques Simard, Jacques Drouin, Luc Bélanger, Marc Montminy, Joseph Torchia, and Mario Ascoli for generously providing the plasmids and cell lines used in this study.
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Address all correspondence and requests for reprints to: Dr. Jacques J. Tremblay, Ontogeny-Reproduction, Room T1-49, CHUL Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: jacques-j.tremblay@crchul.ulaval.ca.
Abstract
The human (h) 3?-hydroxysteroid dehydrogenase/5-4 isomerase type 2 (3?-HSD2) enzyme, encoded by the hHSD3B2 gene, is mainly found in gonads and adrenals. This enzyme catalyzes an essential early step in the biosynthesis of all classes of steroid hormones. The critical nature of the enzyme is supported by the occurrence of human syndromes that are associated with insufficient 3?-HSD2 expression and/or activity. Although the need for a functional 3?-HSD2 enzyme is indisputable, the molecular mechanisms that regulate HSD3B2 expression (both basal and hormone-induced) in steroidogenic cells remain poorly understood. A role for the Nur77 family of immediate-early orphan nuclear receptors in steroidogenesis has received recent interest. For example, Nur77 is present in gonads and adrenals, where its expression is robustly and rapidly induced by hormones that stimulate steroidogenic gene expression. Moreover, the expression patterns of Nur77 and at least one key steroidogenic gene (hHSD3B2) closely parallel one another. We now report that the hHSD3B2 promoter is indeed a novel target for Nur77 in both testicular Leydig cells and adrenal cells. We have mapped a novel response element located at –130 bp specific for Nur77 and not other orphan nuclear receptors (steroidogenic factor-1 and liver receptor homolog-1) previously shown to regulate hHSD3B2 promoter activity. This Nur77 element is essential and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter, and its mutation blunts basal and hormone-induced hHSD3B2 promoter activity in steroidogenic cells. We also show that Nur77 synergizes with all members of the steroid receptor coactivator family of coactivators on the hHSD3B2 promoter. Taken together, our identification of Nur77 as an important regulator of HSD3B2 promoter activity helps us to better define the tissue-specific and hormonal regulation of the HSD3B2 gene in steroidogenic cells.
Introduction
THE HUMAN 3?-hydroxysteroid dehydrogenase/5-4 isomerase type 2 (3?-HSD2) gene, HSD3B2, is expressed in both the gonads and the adrenals and encodes the 3?-HSD2 enzyme that is required for the initial steps of all classes of steroid hormone biosynthesis (reviewed in Ref.1). Its crucial role in steroidogenesis is underscored by the existence of human (h) HSD3B2 gene mutations that are responsible for 3?-HSD2 deficiency. Affected individuals have congenital adrenal hyperplasia accompanied by various degrees of salt-wasting in both sexes. In addition, because 3?-HSD2 is also required for testosterone synthesis in testicular Leydig cells, genetic males are pseudohermaphrodites with female external genitalia (1).
Despite its importance for normal steroidogenesis and proper male sex differentiation, very little is known about the mechanisms that regulate expression of the hHSD3B2 gene in steroidogenic cells. HSD3B2 gene expression, however, is known to be hormonally regulated by angiotensin II (AII) and ACTH, which acts on adrenal cells, and LH/hCG, which regulates steroidogenesis in testicular Leydig cells and ovarian thecal and luteinized granulosa cells (reviewed in Ref.1). AII acts through the diacylglycerol and inositol triphosphate pathways (2), whereas ACTH and LH/hCG mediate their effects through activation of the cAMP/protein kinase A (PKA) signaling pathway. This pathway triggers at least two distinct mechanisms that are both essential for an optimal response. One does not require de novo protein synthesis; rather, it relies on phosphorylation of transcription factors already present in the cell. The other mechanism requires protein synthesis, involving rapid induction of genes encoding transcription factors (3, 4, 5, 6, 7, 8). To date, only a few transcription factors have been shown to bind and activate the hHSD3B2 promoter. These include two members of the NR5A family of orphan nuclear receptors, steroidogenic factor-1 (SF-1) (9) and liver receptor homolog-1 (LRH-1) (10), as well as the signal transducer and activator of transcription proteins 5 and 6 (11, 12, 13, 14). These transcription factors are all found in steroidogenic tissues expressing hHSD3B2 (10, 11, 15, 16, 17, 18, 19, 20, 21). However, because they are also present in other tissues that do not express HSD3B2, such as pituitary gonadotrope cells, liver, pancreas, the zona reticularis of the adrenals, and testicular Sertoli cells (22, 23, 24), other transcription factors are probably involved in tissue-specific HSD3B2 expression. Moreover, the expression of SF-1 and LRH-1 is unchanged, decreased, or only slightly increased in steroidogenic cells in response to ACTH, LH/hCG, or cAMP analogs, which indicates that they are not likely to be the rapidly induced transcription factor required for maximal hormonal stimulation (22, 25). Thus, the newly synthesized transcription factor involved in the hormone-induced up-regulation of HSD3B2 expression has yet to be identified.
Nur77, also known as NGFI-B, TR3, or NR4A1, is a member of the NR4A family of orphan nuclear receptors, which also includes Nurr1 (NR4A2) and Nor1 (NR4A3). Members of this family are immediate-early response genes, and their expression, particularly that of Nur77, is strongly and rapidly induced after various stimuli in numerous tissues (26, 27), including hormonally stimulated steroidogenic cells (28, 29, 30, 31). Nur77 can bind to DNA in three different ways: as a monomer to a Nur77-binding response element (NBRE), which is very similar to SF-1-binding sites (32); as a homo- or heterodimer to an inverted repeat response element (NurRE) (33, 34); or to a DR5 element by heterodimerization with retinoid X receptor (35). Members of the Nur77 family have been identified as important regulators of hormonally regulated gene expression in several endocrine tissues, including the pituitary (proopiomelanocortin gene) (33), the ovary (20-HSD gene) (36), and, more recently, the adrenals (Cyp11B2 gene) (37) and testis (Cyp17 gene) (38).
We now report that the hHSD3B2 promoter is a novel target for Nur77 in both testicular Leydig cells and adrenal cells. Using detailed promoter analyses, we have identified a novel NBRE element located at –130 bp that is specifically recognized by Nur77, but not SF-1 or LRH-1. This novel NBRE element is also essential and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter. Moreover, mutation of the NBRE element blunts cAMP-mediated activation of the hHSD3B2 promoter in both Leydig and adrenal cells. Finally, we show that Nur77 synergizes with SRC coactivators (SRC-1/2/3) to further enhance hHSD3B2 promoter activity. Thus, our results provide critical new insights into the tissue-specific expression and hormonal regulation of hHSD3B2 gene transcription in steroidogenic cells.
Materials and Methods
Plasmids
The –1073 to +53 bp human HSD3B2 promoter fragment was provided by Dr. Jacques Simard (Centre Hospitalier de l’Université Laval Research Center, Université Laval, Québec, Canada). Deletions of the hHSD3B2 promoter to –842, –538, –495, –340, –224, –95, and –60 bp were obtained by PCR using the –1073 bp hHSD3B2 promoter as template, along with a common reverse primer containing a KpnI (underlined) cloning site (5'-GGGGTACCCGTAACTTAGATTGTTAAAAGCTGG-3') and the following forward primers: –842 bp, 5'-CGGGATCCCCTTGTAATGCCCAGATTACATC-3'; –538 bp, 5'-CGGGATCCTCCATTAGGAACCCAGAGCTCTCC-3'; –495 bp, 5'-CGGGATCCGGTTTTGGATATATTGGGTGGAAAAG-3'; –340 bp, 5'-CGGGATCCCCAGGTGGATTTACTGTACAAGGAC-3'; –224 bp, 5'-CGGGATCCCTGTTAAGGCTAAAGCCAAGAC-3'; –95 bp, 5'-CGGGATCCGAGGAGGGAGCAATGAGTATG –3'; and –60 bp, 5'-CGGGATCCGGTAATAAGGGCTGAGACACAAGC-3'. The –1073 bp reporters containing a mutation of the NBRE element at –130 and/or SF-1 element at –60 were obtained by site-directed mutagenesis using the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) and the following pair of oligos (the mutations are underlined): NBRE element at –130: sense, 5'-GTTCCTGGGAAGAATTAGAGATATAACCTAAATTTCACTATTATTCTGAG-3'; antisense, 5'-CTCAGAATAATAGTGAAATTTAGGTTATATCTCTAATTCTTCCCAGGAAC-3'; and SF-1 element at –60: sense, 5'-GAGTATGTGGCAGGAGTTCAATTTAATAAGGGCTGAGACACAAG-3'; antisense, 5'-CTTGTGTCTCAGCCCTTATTAAATTGAACTCCTGCCACATACTC-3'. All promoter fragments were cloned into a modified pXP1 luciferase reporter plasmid (39, 40) and subsequently verified by sequencing. The mouse SF-1 expression vector has been previously described (40). Expression vectors for full-length rat Nur77, NOR-1, and Nurr1 (33) were provided by Dr. Jacques Drouin (Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Canada). The human LRH-1 expression vector (41) was provided by Dr. Luc Bélanger (Centre de Recherche en Cancérologie, Centre de Recherche du CHUQ, Université Laval, Québec, Canada). A pcDNA3-derived expression vector for the PKA catalytic subunit was obtained by transferring the PKA cDNA from the MT-PKAc (42) obtained from Dr. Mark Montminy (The Salk Institute for Biological Studies, La Jolla, CA). The mouse SRC-1, SRC-2, and SRC-3 expression plasmids (43) were gifts from Dr. Joseph Torshia (University of Western Ontario, London, Canada).
Cell culture and transfections
Mouse Leydig MA-10 cells (44), provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA), were grown in Waymouth’s MB752/1 medium supplemented with 20 mM HEPES, 15% horse serum, and 50 mg/liter gentamicin, and streptomycin sulfates at 37 C in 5% CO2. Human adrenal H295R cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM/Ham’s F-12 (1:1) medium supplemented with 15 mM HEPES, 1.2 g/liter NaHCO3, 2.5% NuSerum, and 10 ml/liter of ITS+ Premix (BD Biosciences, San Jose, CA) at 37 C in 5% CO2. All transfections were performed in 12-well plates using the Lipofectamine 2000 method (Invitrogen Canada, Burlington, Canada) according to the manufacturer’s recommendations. Briefly, the day before transfection, MA-10 and H295R cells were plated at 200,000 and 300,000 cells/well, respectively. The next day, cells were transfected with 1.5 μg hHSD3B2 promoter construct fused to Firefly luciferase, 250 ng cytomegalovirus-driven expression vector, 10 ng phRL-TK Renilla luciferase as an internal control, and pSP64 as carrier DNA up to 2 μg/ well using 5 μl Lipofectamine 2000 and antibiotic- and serum-free medium. Five hours later, cells were provided with complete medium. The following day, the Dual Luciferase Assay System (Promega Corp., Madison, WI) was used to harvest the cells and measure luciferase activities using an EG&G Berthold LB 9507 luminometer. In experiments with cAMP stimulation, cells were treated with 0.5 mM dibutyryl cAMP [(Bu)2cAMP] for 6 h before harvesting. Several DNA preparations of the plasmids were used to ensure reproducibility of the results. The data reported represent the average of at least three experiments, each performed in duplicate.
EMSAs
Nuclear extracts were obtained from MA-10 cells treated with either vehicle or 0.5 mM (Bu)2cAMP for 4 h. Recombinant Nur77, SF-1, and LRH-1 proteins were in vitro translated using the T7 QuickCoupled TnT system (Promega Corp.). DNA binding assays were performed using either 10 μg nuclear extracts or 1–2 μl in vitro translated protein as described previously (45), with the exception of dI:dC, where 100 ng were used for EMSAs performed with in vitro translated proteins. The 32P-labeled double-stranded oligonucleotides used as probes were as follows: hHSD3B2 NBRE element (underlined) at –130 bp (sense, 5'-GATCCTAACCTAAAGGTCACTATTA-3'; antisense, 5'-GATCTAATAGTGACCTTTAGGTTAG-3'), and hHSD3B2 SF-1/LRH-1 element (underlined) at –60 bp (sense, 5'-GATCCGGAGTTCAAGGTAATAAGGA-3'; antisense, 5'-GATCTCCTTATTACCTTGAACTCCG-3'). For the competition experiments, double-stranded oligonucleotides corresponding to mutated versions of the NBRE and SF-1/LRH-1 elements were used. The sequences of the oligonucleotides are the same as those described above, except that the NBRE element (TAAAGGTCA) was mutated into TAAATTTCA, and the SF-1/LRH-1 element was changed from TCAAGGTAA to TCAATTTAA. For supershift experiments, 1.6 μg of a commercially available anti-Nur77 antiserum (M-210, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were also added to the binding reaction.
Results
Activation of hHSD3B2 promoter activity by Nur77 factors
Nur77 is known to be expressed in the adrenals and testes, where its expression is induced in response to ACTH and LH, two hormones that act mainly through the cAMP/PKA signaling pathway (28, 29). Because the H295R and MA-10 cell lines are commonly used as models for adrenal and testicular steroidogenic cells, respectively, we tested whether Nur77 expression could also be induced by cAMP in these cell lines. As shown in Fig. 1A, Nur77 protein levels were strongly induced in a time-dependent manner in response to (Bu)2cAMP in both MA-10 Leydig and H295R adrenal cells. These results thus raise the intriguing possibility that Nur77 could represent the hormonally induced transcription factor required for maximal stimulation of hHSD3B2 promoter in these cells. In support of this hypothesis, the hHSD3B2 promoter contains two regulatory elements (at –320 and –60 bp) for the binding of orphan nuclear receptors known to bind DNA as monomers, including SF-1 and LRH-1 (9, 10), and potentially Nur77, which we now propose (32, 41).
FIG. 1. Activation of the hHSD3B2 promoter by Nur77 family members. A, Induction of Nur77 after cAMP treatment. MA-10 mouse Leydig cells (left panel) and H295R human adrenal cells (right panel) were treated with 0.5 mM (Bu)2cAMP for the indicated times, and nuclear extracts were isolated. Twenty-microgram aliquots of extracts were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, and the Nur77 protein was revealed using a Nur77 polyclonal antibody. n.s., Nonspecific. B, MA-10 Leydig cells (left panel) and H295R adrenal cells (right panel) were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl; ) or expression vectors (250 ng) for Nur77 family members (), SF-1 (), and LRH-1 ( ) as indicated. The positions of the two previously characterized binding sites for orphan nuclear receptors within the –1073 to +53 bp hHSD3B2 promoter fragment (–320 and –60 bp) are represented by triangles. Results are shown as fold activation over the control value (±SEM).
To test whether Nur77 could participate in hHSD3B2 transcription in steroidogenic cells, we performed transient transfection assays in MA-10 Leydig and H295R adrenal cells, two homologous cell lines known to have endogenous 3?-HSD expression/activity. As shown in Fig. 1B, Nur77 as well as the other Nur77 family members, Nurr1 and Nor1, found in steroidogenic tissues (29, 31) could activate a –1073 bp hHSD3B2 reporter construct (3-fold in MA-10 cells and 1.7-fold in H295R cells), whereas SF-1 and LRH-1 had no statistically significant effect (P > 0.05, by t test). This is probably due to the fact that these cells already contain significant levels of SF-1 and LRH-1 (9, 15, 46).
Localization of the Nur77-responsive element
To locate the Nur77-responsive element, 5'-progressive deletions of the hHSD3B2 promoter were generated and tested for Nur77 responsiveness in MA-10 and H295R cells (Fig. 2A). A deletion construct to –340 bp that retained the two previously characterized SF-1/LRH-1 elements (–320 and –60 bp) was still activated by Nur77. Deletion to –224 bp that removed the distal (–320 bp) SF-1/LRH-1 element had no effect on Nur77-dependent activation of the hHSD3B2 promoter. Additional deletion to –95 bp while retaining the proximal (–60 bp) SF-1/LRH-1 element, however, completely abrogated transactivation of the hHSD3B2 promoter by Nur77. Taken together, these results indicate that the two SF-1/LRH-1 elements are not required for Nur77-dependent activation of the hHSD3B2 promoter and that a novel, as yet unidentified, element located between –224 and –95 bp is responsible for Nur77 responsiveness. Indeed, sequence analysis of this promoter region revealed the presence of a consensus NBRE located at position –130 bp (Fig. 2B). The importance of this novel NBRE element in Nur77-mediated hHSD3B2 promoter activation was further analyzed by mutagenesis in the context of the –1073 bp hHSD3B2 reporter. As shown in Fig. 3A, mutation of the –130 bp NBRE element (TAAAGGTCATAAATTTCA) completely abolished hHSD3B2 promoter activation by Nur77 in both MA-10 and H295R cells. Mutation of the –60 bp SF-1/LRH-1 element (TCAAGGTAATCAATTTAA), however, had no effect on Nur77-dependent hHSD3B2 promoter activation. Thus, the novel NBRE element (–130 bp) is both necessary and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter.
FIG. 2. Mapping of the Nur77-responsive element in the hHSD3B2 promoter. A, MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with various 5' deletion constructs of the hHSD3B2 promoter (the 5'-end point of each construct is indicated on the left of the graphs) with either an empty expression () or expression vectors for Nur77 (250 ng; ). The positions of the two orphan nuclear binding sites are indicated (triangles). Results are shown as fold activation over the control value (±SEM). B, Sequence of a novel NBRE element located at position –130 bp. The sequence is compared with the previously described sites at positions –320 and –60 bp.
FIG. 3. The novel NBRE element at –130 bp is necessary and sufficient for Nur77-dependent hHSD3B2 promoter activation. A, MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with either an empty expression vector () or expression vectors for Nur77 (250 ng; ) along with several –1073 to +53 bp hHSD3B2 reporters as indicated on the far left side of the figure: a wild-type reporter, a reporter harboring a point mutation (underlined) in the NBRE element at –130 bp (TAAAGGTCATAAATTTCA), a reporter with the –60 bp element mutated (TCAAGGTAATCAATTTAA), and a reporter with both the –130 and –60 bp elements mutated. The mutated elements are represented by a large X. All results are shown as fold activation over the control value (±SEM). B, Increased Nur77 DNA-binding activity on the novel –130 bp NBRE element after (Bu)2cAMP treatment of MA-10 Leydig cells. EMSA was used to determine the binding of the Nur77 protein present in MA-10 Leydig cells in the absence or after a 4-h stimulation with 0.5 mM (Bu)2cAMP. Nur77 binding was supershifted by a Nur77 antiserum (Nur77). P.I., Preimmune serum.
The presence of Nur77 DNA-binding activity in MA-10 Leydig cells was tested directly by EMSA (Fig. 3B). In unstimulated MA-10 cells, a weak band (Fig. 3B, lane 3) that comigrated with in vitro produced Nur77 (Fig. 3B, lane 2) was observed. This binding was strongly enhanced in nuclear extracts from (Bu)2cAMP-treated MA-10 cells (Fig. 3B, lane 4). Furthermore, this band was supershifted by the addition of an anti-Nur77 antiserum (Fig. 3B, lane 6). Thus, endogenous Nur77 DNA-binding activity on the novel NBRE element is strongly induced by cAMP analogs.
Specific binding of Nur77 to the NBRE element (–130 bp)
The DNA binding specificity of Nur77 to the novel NBRE element (–130 bp) was next assessed by EMSA. Consistent with the specific requirement for the NBRE element in Nur77-mediated activation of the hHSD3B2 promoter (Fig. 3A), in vitro translated Nur77 bound with high affinity to the NBRE element (Fig. 4A), but not to the proximal (–60 bp) SF-1/LRH-1 element (Fig. 4B). Nur77 binding was specifically competed by increasing doses of unlabeled oligonucleotides (Fig. 4A, lanes 7 and 8), but not by oligonucleotides harboring a mutation in the NBRE element (Fig 4A, lanes 9 and 10) or oligonucleotides corresponding to the proximal SF-1/LRH-1 element (Fig. 4A, lanes 3 and 4).
FIG. 4. The novel NBRE element is a Nur77-specific binding site. EMSA was used to assess the binding of in vitro produced Nur77 to a double-stranded 32P-labeled oligonucleotide corresponding to either the NBRE element at –130 bp (A) or the SF-1/LRH-1 site at –60 bp (B) of the hHSD3B2 promoter. Binding of in vitro synthesized SF-1 (C) and LRH-1 (D) was also assessed on the –60 bp element. Binding of the proteins was then challenged by increasing doses (; molar excesses of 2- and 5-fold) of unlabeled oligonucleotides corresponding to the wild-type –60 bp element (–60 wt), the –60 bp element mutated (underlined) from TCAAGGTAA to TCAATTTAA (–60 mut), the wild-type –130 bp element (–130 wt), and the –130 bp element containing a mutation (underlined) in the NBRE element (TAAAGGTCATAAATTTCA).
EMSA was also used to test the possibility that SF-1 and LRH-1 might bind to the NBRE element. On the –60 bp element, both SF-1 and LRH-1 could strongly bind, as expected (lanes 2 in Fig. 4, C and D). This binding was completely competed by self oligonucleotides (lanes 3 and 4 in Fig. 4, C and D), but only slightly by an oligonucleotide corresponding to the NBRE (–130 bp) element even at the highest dose (lanes 7 and 8 in Fig. 4, C and D). Thus, these results indicate that the Nur77 and SF-1/LRH-1 orphan nuclear receptors each have their specific binding sites within the hHSD3B2 promoter, raising the possibility that they might cooperate to regulate hHSD3B2 promoter activity. However, as shown in Fig. 5, no transcriptional cooperation was observed among the three orphan nuclear receptors. Rather, coexpression of Nur77 and SF-1 (but not LRH-1) resulted in a loss of Nur77-dependent hHSD3B2 promoter activation, specifically in MA-10 cells. Although the underlying reasons behind this effect remain unknown, this might be due to limiting amounts of an additional coactivator common to both SF-1 and Nur77 in MA-10, but not H295R, cells.
FIG. 5. Effects of orphan nuclear receptor combinations on hHSD3B2 promoter activity. MA-10 Leydig (left panel) and H295R adrenal (right panel) cells were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl; ) or expression vectors (250 ng) for Nur77, SF-1, and LRH-1, alone or in the combinations indicated. The positions within the –1073 to +53 bp hHSD3B2 promoter fragment of the two previously characterized binding sites for orphan nuclear receptors (–320 and –60 bp) and the novel NBRE element identified in the present work (–130 bp) are represented by two triangles and a hatched circle, respectively. Results are shown as fold activation over the control value (±SEM).
NBRE is required for maximal basal and cAMP-induced hHSD3B2 promoter activities in steroidogenic cells
It is well established that HSD3B2 expression in steroidogenic tissues is regulated by several hormones, including ACTH and LH from the pituitary, two hormones that mediate their effects, at least in part, through the cAMP/PKA pathway (reviewed in Ref.1). Interestingly, these same trophic hormones are also known to induce Nur77 expression in steroidogenic tissues (28, 29, 30, 31). We therefore compared the activation of the –1073 bp hHSD3B2 reporter construct by Nur77 and 0.5 mM (Bu)2cAMP in MA-10 Leydig and H295R adrenal cells. As shown in Fig. 6A, activation of the hHSD3B2 promoter mediated by Nur77 overexpression was similar to the activation induced by (Bu)2cAMP. The combination of Nur77 and (Bu)2cAMP produced an additive effect (Fig. 6A). These results suggest that Nur77 and other cAMP-regulated transcription factors might function in a linear or parallel pathway to regulate hHSD3B2 promoter activity in steroidogenic cells.
FIG. 6. The NBRE element is required for maximal cAMP stimulation of the hHSD3B2 promoter. A, Additive effects of Nur77 and cAMP on hHSD3B2 promoter activity. MA-10 Leydig (left panel) and H295R adrenal cells (right panel) were cotransfected with a –1073 to +53 bp hHSD3B2 promoter construct and either an empty (Ctl; ) or a Nur77 expression vector (250 ng; ). Transfected cells were then treated with or without 0.5 mM (Bu)2cAMP for 6 h before harvesting as indicated. Results are shown as fold activation over the control value (±SEM). B, MA-10 Leydig cells were transfected with the four –1073 to +53 bp hHSD3B2 reporter constructs shown on the left (described in Fig. 3) and treated with either vehicle (–, ) or 0.5 mM (Bu)2cAMP (+, ) 6 h before harvesting. Results are shown as the percent activity (±SEM) relative to the activity of the –1073 bp wild-type reporter in the absence of cAMP treatment (which was arbitrarily set at 100%).
To establish the significance of Nur77 and its NBRE element in cAMP-regulated hHSD3B2 transcription, hHSD3B2 reporter constructs harboring point mutations in the –130 bp NBRE and/or –60 bp SF-1/LRH-1 elements were transfected in MA-10 cells and tested for cAMP responsiveness. As shown in Fig. 6B, mutation of the –130 bp NBRE led to a 40% decrease in cAMP responsiveness compared with the wild-type promoter (from 3- to 1.8-fold stimulation), whereas a 24% decrease (from 3- to 2.3-fold stimulation) was observed when the –60 bp SF-1/LRH-1 element was mutated. Mutation of both elements led to no further decrease compared with the NBRE mutation alone. The results of the mutation experiments, summarized in Table 1, indicate that the Nur77/NBRE complex is a more important contributor to the cAMP responsiveness of the hHSD3B2 promoter than is the proximal SF-1/LRH-1 element. They also indicate that although an intact NBRE element is necessary for maximal stimulation, additional regulatory elements/transcription factors contribute to the cAMP regulation of the hHSD3B2 promoter.
TABLE 1. Contribution of the NBRE and SF-1/LRH-1 elements to basal and cAMP-induced hHSD3B2 promoter activity in MA-10 steroidogenic cells
As for several transcription factors, Nur77 would be expected to exert its effects through cooperations with coactivators. Indeed, Nur77 has been shown to cooperate with cAMP responsive element binding protein-binding protein and SRC family members (SRC-1/2/3). The SRC-1 data, however, are conflicting because some reports have described a transcriptional cooperation between Nur77 and SRC-1 (38, 47, 48), whereas others have not (49). This discrepancy could be explained by the fact that all these experiments were performed in heterologous cells using synthetic reporters consisting of multiple copies of a Nur77-binding site fused to a minimal promoter. To test whether Nur77 could transcriptionally cooperate with SRC coactivators on the natural hHSD3B2 promoter, cotransfections were performed in MA-10 Leydig cells. Although SRC-1, SRC-2, and SRC-3 alone had no effect (Fig. 7), combination of these coactivators with Nur77 resulted in a synergistic activation (average of 6-fold compared with 3.1 for Nur77 alone) of the –1073 bp hHSD3B2 reporter. In addition, PKA-mediated protein phosphorylation enhanced this synergy to nearly 9-fold (Fig. 7), which is consistent with previous findings that coactivator recruitment and interaction with Nur77 can be enhanced by cAMP/PKA (48). Thus, our results indicate that Nur77-dependent activation of hHSD3B2 promoter probably involves coactivator recruitment that is enhanced by the cAMP/PKA pathway.
FIG. 7. Transcriptional cooperation among Nur77, SRC, and PKA. MA-10 Leydig cells were transiently transfected with the –1073 to +53 bp hHSD3B2 promoter construct and either an empty expression vector (Ctl) or expression vectors for Nur77 (250 ng), SRC-1, SRC-2, or SRC-3 (500 ng), alone (empty bars) or in combination ( ) a), in the absence or presence of an expression vector encoding the PKA catalytic subunit (25 ng; ). Results are shown as fold activation over the control value (±SEM).
Discussion
The orphan nuclear receptor Nur77 belongs to a family of immediate early response transcription factors involved in the hormonal stimulation of gene expression in several tissues and cell types (26). Steroidogenic tissues are no exception to this, because Nur77 expression was shown to be strongly induced in vivo by LH/hCG in the testis (28) and ovary (30) as well as by ACTH and AII in the adrenal cortex (29, 50). These data suggested a role for Nur77 in steroidogenic gene expression in all three steroidogenic tissues. Indeed, Nur77 has been shown to activate the promoters of some steroidogenic genes (20-HSD, Cyp17, Cyp11B2, and Cyp21) (36, 37, 38, 51). In this study we have identified the promoter of the 3?-HSD gene, encoding a key enzyme essential for the synthesis of all classes of steroids, as a novel target for Nur77.
The hHSD3B2 promoter is a novel target for Nur77 in steroidogenic tissues
The HSD3B2 gene is expressed in the testis, ovary, and adrenals, and its expression within these tissues is cell and zone specific. Indeed, HSD3B2 is expressed in testicular Leydig cells, in ovarian thecal and luteinized granulosa cells, and in cells of the zona glomerulosa and fasciculata (but not reticularis) of the adrenals (reviewed in Ref.1). Therefore, transcription factors regulating the tissue-, cell-, and zone-specific expression of HSD3B2 are also likely to have a similar expression pattern. The transcription factors currently known to regulate hHSD3B2 promoter activity, however, cannot by themselves explain its highly specific expression pattern. In contrast, the Nur77 expression pattern does fulfill this criterion. Within the adrenals, Nur77 expression is zone specific like HSD3B2; it is predominantly expressed in the zona glomerulosa and fasciculata, but not in the zona reticularis (52). Nur77 is also coexpressed with hHSD3B2 in testicular Leydig cells (28) and in ovarian thecal and luteinized granulosa cells (30, 31). Taken together, these data point to a role for Nur77 in hHSD3B2 transcription. In agreement with this, we have now identified the hHSD3B2 promoter as a target for Nur77 in both Leydig and adrenal cells. Similar findings were recently reported by Bassett et al. (53). Furthermore, recent studies by Hong et al. revealed that Nur77 could activate the promoter of the mouse HSD3B1 gene, the mouse ortholog of the human HSD3B2 gene (47). The lack of a steroidogenic phenotype in Nur77–/– mice, however, would suggest otherwise (54). This can be explained by the fact that another Nur77 family member, Nurr1, was up-regulated in Nur77–/– mice, suggesting that the various Nur77 family members may compensate for one another in vivo (54). Consistent with this, Nurr1 is expressed in steroidogenic tissues (29, 31), and we have shown that it can activate the hHSD3B2 promoter as well as Nur77 (Fig. 1B).
Nur77 is known to bind as a monomer to DNA elements highly related to those recognized by the orphan nuclear receptors SF-1 and LRH-1 (32). Although the hHSD3B2 promoter contains two binding sites for SF-1 and LRH-1 located at –320 and –60 bp (9, 10), we found that Nur77-dependent activation of the hHSD3B2 promoter did not require any of these elements. Rather, Nur77 activates hHSD3B2 transcription through a novel element, located at –130 bp, that can be specifically bound by Nur77 and not SF-1 or LRH-1. This element is indeed necessary and sufficient to confer Nur77 responsivenes to the hHSD3B2 promoter in steroidogenic cells. Furthermore, mutation of this element led to a decrease of about 35% in basal hHSD3B2 promoter activity in Nur77-expressing MA-10 Leydig and H295R adrenal cells. Mutation of the –60 bp SF-1/LRH-1 element, however, resulted in a drop of more than 80% in basal hHSD3B2 promoter activity in these same cells, clearly indicating that SF-1 and/or LRH-1 are truly important regulators of basal HSD3B2 transcription. The fact that Nur77 and SF-1/LRH-1 mediate their effects through specific regulatory elements, such as on the hHSD3B2 promoter described in the present study, would be consistent with the nonredundant, yet complementary, roles played by these orphan nuclear receptors in endocrine development and function (22, 24, 26).
Roles of orphan nuclear receptors in basal and hormone-induced HSD3B2 expression
Of the three orphan nuclear receptors now known to be implicated in basal hHSD3B2 transcription (Nur77, SF-1, and LRH-1), Nur77 appears to be the most sensitive to hormonal regulation. Thus, although SF-1 and LRH-1 expression is either unaffected, decreased, or only slightly increased in response to hormones (LH/hCG, ACTH, and AII) that regulate steroidogenesis (18, 25, 50, 52, 55, 56), Nur77 expression, in contrast, is known to be strongly and rapidly induced (reviewed in Ref.26). This is not surprising given that Nur77 is an immediate-early response factor. Like 3?-HSD, Nur77 expression is robustly induced after hormonal stimulation in the three major steroidogenic tissues; it is strongly induced by LH/hCG in granulosa cells (30, 31), by LH/cAMP in testicular Leydig cells (28), and by both ACTH/cAMP and AII in adrenal cells (29, 50, 51, 52). Therefore, the Nur77 expression pattern and its hormonal regulation in steroidogenic tissues are consistent with a role for this transcription factor in hormone-induced 3?-HSD expression. Indeed, our cAMP regulation data and transcriptional cooperation with the SRC coactivators support such a role. By consolidating our results with those taken from the literature, we present a model (Fig. 8) in which the orphan nuclear receptors Nur77, SF-1, and LRH-1 can be integrated as nonredundant, yet complementary, regulators for the tissue-, zone-, and cell-specific and hormone-dependent expression of the hHSD3B2 gene.
FIG. 8. Proposed model for the implication of orphan nuclear receptors in basal and hormone-induced hHSD3B2 transcription. The binding of the pituitary trophic hormones LH or ACTH to their respective G protein-coupled receptors (left side) triggers activation of the cAMP intracellular signaling pathway, leading to activation of PKA. AII binds to its AT1 receptor (right side) triggering activation of phospholipase C (PLC) and production of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 increases intracellular calcium levels, which, in turn, activates calcium-dependent kinase (CamK), whereas DAG activates protein kinase C (PKC). These pathways all lead to increased Nur77 expression in steroidogenic cells (28 50 ) as well as phosphorylation of target proteins, including transcription factors such as orphan nuclear receptors Nur77, SF-1, and LRH-1. These DNA-bound transcription factors also directly interact with coactivators such as SRC-1/2/3, and this interaction is stronger when proteins are phosphorylated (48 57 ). The SRC coactivators then recruits the cointegrator cAMP responsive element binding protein-binding protein (58 ) into a transcriptionally active complex, leading to increased hHSD3B2 transcription.
Acknowledgments
We thank Drs. Jacques Simard, Jacques Drouin, Luc Bélanger, Marc Montminy, Joseph Torchia, and Mario Ascoli for generously providing the plasmids and cell lines used in this study.
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