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BOR - Papers in Press, published online ahead of print October 19, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044560
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BIOLOGY OF REPRODUCTION 74, 322–330 (2006)
DOI: 10.1095/biolreprod.105.044560
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

The Orphan Nuclear Receptor NR4A1 Regulates Insulin-Like 3 Gene Transcription in Leydig Cells1

Nicholas M. Robert 3, Luc J. Martin 3, and Jacques J. Tremblay 2 34 

Ontogeny-Reproduction Research Unit,3 CHUL Research Centre, Ste-Foy, Québec, Canada G1V 4G2 Centre for Research in Biology of Reproduction,4 Department of Obstetrics and Gynecology, Faculty of Medicine, Université Laval, Ste-Foy, Québec, Canada G1K 794

ABSTRACT

Insulin-like 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the testis and by theca and luteal cells of the adult ovary. In males, INSL3 regulates testicular descent during fetal life, whereas in adults, it acts as a germ cell survival factor. In the ovary, INSL3 regulates oocyte maturation. Despite its importance for male sex differentiation and reproductive function in both sexes, very little is known regarding the molecular mechanisms that regulate Insl3 expression. So far, the nuclear receptor NR5A1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. NR5A1 by itself, however, cannot explain the spatiotemporal expression pattern of the Insl3 gene. In the present study, we have identified the orphan nuclear receptor NR4A1 as a novel regulator of INSL3 transcription in Leydig cells. Using RT-PCR, we found that Nr4a1 is coexpressed with Insl3 in purified Leydig cells and in several Leydig cell lines. Through detailed analyses of the mouse and human INSL3 promoter in Leydig cells, we have mapped a novel regulatory element located at –100 bp that is essential and sufficient to confer NR4A1 responsiveness. Consistent with a role for NR4A1 in Insl3 transcription, chromatin immunoprecipitation assays revealed that endogenous NR4A1 binds to the proximal Insl3 promoter in vivo. Finally, we found that NR4A1 is also implicated in cAMP-induced Insl3 transcription in Leydig cells. Taken together, our identification of NR4A1 as an important regulator of mouse and human INSL3 promoter activity helps us to better define the tissue-specific regulation of the INSL3 gene in gonadal cells.

gene regulation, growth factors, Leydig cells, ovary, testis

INTRODUCTION

Insulin-like 3 (INSL3) or relaxin-like factor (RLF) belongs to the insulin-IGF-relaxin family of growth factors and hormones and is specifically expressed in testicular Leydig cells and in the ovary [1, 2]. In fetal Leydig cells, Insl3 is strongly expressed from Embryonic Day 13.5 (e13.5) in mice [3] and its expression decreases after birth, with the disappearance of the fetal Leydig cell population [3, 4]. Insl3 expression in the adult population of Leydig cells rises just prior to puberty, peaks in adults animals, and then declines in aging animals [2–6]. The second wave of Insl3 expression depends on a functional hypothalamo-pituitary-gonadal (HPG) axis as revealed by the lack of Insl3 expression in hpg mice [4] that have a deletion of the Gnrh1 gene and thus a nonfunctional HPG axis [7, 8]. Interestingly, Insl3 expression could be restored by administration of hCG to hpg mice [4]. Another study also confirmed that Insl3 expression in the gonads is upregulated in vivo by LH [9]. Insl3 expression, however, is not limited to testicular Leydig cells; it is also found in follicular theca cells and luteal cells of the adult ovary, albeit at a much lower level than in testicular Leydig cells [3, 4].

In males, INSL3 plays two distinct roles. First, during fetal life, INSL3 controls the initial phase of testicular descent, which constitutes a normal and obligatory step of the male sex differentiation process [10, 11]. Definitive proof that INSL3 was indeed responsible for the first phase of testicular descent came from gene inactivation experiments in the mouse. Two knockout alleles of the Insl3 gene, Insl3tm1Imad/Insl3tm1Imad and Insl3tm1Par/Insl3tm1Par, were generated by two independent laboratories [12, 13]. Insl3–/– mice had bilateral cryptorchid testes located high in the abdominal cavity close to the kidneys [12, 13]. Further support for INSL3 as a regulator of testicular descent came from elegant transgenic experiments where INSL3 was overexpressed in pancreatic ß cells of Insl3–/– mice [14]. In these animals, normal testis descent was restored. Even more convincing was the generation of female transgenic mice (TG(Ins2-Insl3)4Imad) overexpressing INSL3, which led to descended ovaries [14, 15]. The second role of INSL3 is in adult males, where it was recently shown to act as a germ cell survival factor [9]. In females, INSL3 plays a role in oocyte maturation [9].

Because INSL3 plays critical roles in male sex differentiation and in reproductive function of both sexes, understanding the mechanisms that regulate its expression is of paramount importance. Although the INSL3 promoter has been isolated from several species [3, 16–18], very little is known about its transcriptional regulation. So far, three consensus binding sites for nuclear receptors have been identified in the rodent proximal Insl3 promoter, suggesting a role for members of this family of transcription factors in the regulation of Insl3 expression. Consistent with this, two groups have reported that the nuclear receptor NR5A1, also known as SF-1 and Ad4BP, can bind to and activate the mouse Insl3 promoter [19, 20]. Other evidence, however, indicates that NR5A1 is, to a certain extent, dispensable for Insl3 transcription in vivo. For instance, there was a partial descent of the testes to the level of the bladder in Leydig cell-specific Nr5a1-null mice, suggesting that these animals retain some capacity to produce INSL3 [21]. Moreover, of the three NR5A1 binding sites found in the rodent Insl3 promoter, only one is conserved in the human and pig INSL3 promoter. Finally, Nr5a1 expression in the testis remains high throughout life (reviewed in [22]), whereas Insl3 expression declines after birth and increases prior to puberty [2–4]. Therefore, other transcription factors are required for the initiation and maintenance of Insl3 transcription.

NR4A1 (Nur77, NGFI-B, TR3) is the founding member of the NR4A family of orphan nuclear receptors, which also includes NR4A2 (Nurr1) and NR4A3 (Nor1). These transcription factors are known to be immediate-early response factors in several tissues, including steroidogenic gonadal cells [23, 24]. In these cells, NR4A1 is present at low levels and is strongly induced in response to LH and cAMP analogs [23, 24]. In recent years, members of the NR4A family, particularly NR4A1 and NR4A2, have received increased attention as novel regulators of basal and hormone-induced gene transcription in testicular Leydig cells. Interestingly, NR4A1 is known to bind as monomer to a regulatory element similar to that recognized by NR5A1, suggesting that these two nuclear receptors might regulate a common set of genes [25]. Indeed, NR4A1 stimulates the promoters of several genes involved in testosterone biosynthesis in Leydig cells that are also known to be activated by SF-1. These include the human HSD3B2, rat Cyp17a1, mouse Hsd3b1, and mouse Star promoters [23, 26, 27].

We now report that the mouse and human INSL3 promoter is a novel target for the orphan nuclear receptor NR4A1 (Nur77/NGFI-B) in testicular Leydig cells. Using detailed promoter analyses, we have identified a novel NR4A1 response element (NBRE) located at –100 bp that is recognized by NR4A1. This species-conserved NBRE element is also essential and sufficient to confer NR4A1 and cAMP responsiveness to the mouse and human INSL3 promoter. Thus, our results provide important new insights into the mechanisms of INSL3 gene transcription in testicular Leydig cells.

MATERIALS AND METHODS

Plasmids

The –1137-bp to +11-bp human INSL3 promoter (hINSL3) fragment was isolated by PCR using human genomic DNA as template and the following primers: forward: 5'-CTA GGA TCC TGC CAT TAC AGG CCA AC-3' (BamHI cloning site is underlined); reverse: 5'-GG GGT ACC GGT GGG TGG CGC CGG GGC CAA GC-3' (KpnI cloning site is underlined). Deletions of the hINSL3 promoter to –920 bp, –656 bp, –322 bp, and –93 bp were obtained by PCR using the –1137-bp hINSL3 promoter as template, along with the same reverse primer described above and the following forward primers (BamHI cloning site is underlined): –920 bp, 5'-CG GGA TCC TGT AGT CCC AGC TAC TCA GG-3'; –656 bp, 5'-CG GGA TCC AGC CTG GAA GAC AGA GCA AG-3'; –322 bp, 5'-CG GGA TCC AGG CTG AGG CAG GAG AAT CGC-3'; –93 bp, 5'-CG GGA TCC TGG GCG GTC CTG AAG AAT G-3'. The –1137-bp reporter containing a mutation of the NBRE element at –100 was obtained by site-directed mutagenesis using the QuickChange XL mutagenesis kit (Stratagene, La Jolla, CA) and the following pair of oligos (the mutations are in bold and underlined): sense: 5'-CTG GCA CTA ACC CCA CCC TGA TTT TTT TCC TGG GCG GTC CTG-3', antisense: 5'-CAG GAC CGC CCA GGA AAA AAA TCA GGG TGG GGT TAG TGC CAG-3'. The –189-bp mouse Insl3 (mInsl3) promoter fragment was isolated by PCR using mouse genomic DNA and the following primers: forward: 5'-CG GGA TCC AAT GTT GGG GAG CGG CTC CTG-3' (BamHI cloning site is underlined), reverse: 5'-GG GGT ACC GTG GCA GGA GGC AGT GGG CAG-3' (KpnI cloning site is underlined). All promoter fragments were cloned into a modified pXP1 luciferase reporter plasmid [28, 29], and subsequently verified by sequencing. A pcDNA3-based expression vector for the truncated NR4A1 protein (aa 252–601) was constructed by PCR using the following primers: forward, 5'-ATG GTG ACC TCC ACC AAG TCC CGG-3' and reverse, 5'-TCA GAA AGA CAA GGT GTC CAT-3'. The mouse NR5A1 expression vector has been previously described [29]. Expression vectors for full-length NR4A1, NR4A2, and NR4A3 [30] were kindly provided by Dr. Jacques Drouin (Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Canada). The human NR5A2 expression vector [31] was provided by Dr. Luc Bélanger (Centre de recherche en cancérologie, Centre de recherche du CHUQ, Université Laval, Québec, Canada).

Cell Culture and Transfections

The mouse Sertoli cell lines 15P-1 [32] and TM4 [33] and Leydig cell line TM3 [33] were obtained from American Type Culture Collection. The mouse Sertoli cell line MSC-1 [34] was obtained from Dr. Michael Griswold (Washington State University, Pullman, WA). The mouse Leydig cell line MA-10 [35] was provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA). The 15P-1, TM3, TM4, and MSC-1 cell lines were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. MA-10 Leydig cells were grown and transfected as previously described [23]. When indicated, MA-10 cells were treated with 0.5 mM (Bu)2cAMP 4 h prior to harvesting. Data reported represent the average of at least three experiments, each done in duplicate.

Electromobility Shift Assays

Recombinant NR4A1, NR4A2, NR4A3, NR5A1, and NR5A2 proteins were in vitro translated using the T7 QuickCoupled TNT system (Promega, Madison, WI). Nuclear extracts were obtained from MA-10 cells treated with 0.5 mM (Bu)2cAMP for 4 h. DNA binding assays were performed using either 10 µg of nuclear extracts or 1–2 µl of in vitro-translated protein as described in [23]. The 32P-labeled double-stranded oligonucleotides used as probes were as follow: human INSL3 NBRE (shown in bold), sense: 5'-ACC CTT GAC CTT TTT CCT G-3' and antisense: 5'-CAG GAA AAA GGT CAA GGG T-3'; mouse Insl3 NBRE (shown in bold), sense: 5'-CGA GCC TCG ACC TTT TGG GTG CT-3', antisense: 5'-AGC ACC CAA AAG GTC GAG GCT CG-3'. For the competition experiments, double-stranded oligonucleotides corresponding to mutated versions of the NBRE elements were used. The sequences of the oligonucleotides are the same as those described above except that the NBRE element (AAA AGG TCA) was mutated into AAA ATT TCA. For supershift experiments, 1.6 µg of a commercially available anti-NR4A1 antiserum (M-210; Santa Cruz Biotechnology, Santa Cruz, CA) was also added to the binding reaction.

Animals

CD-1 mice were obtained on site and maintained on a 12L:12D light cycle with water and food ad libitum. Adult mice (~90 day old) were killed by CO2 inhalation and the testes were harvested. All experiments were conducted according to the Canadian Council for Animal Care and have been approved by the Animal Care and Ethics Committee of Laval University (protocol # 2003–068).

RNA Isolation and RT-PCR

Total RNAs from purified Leydig cells isolated from rats at different developmental stages (progenitor Leydig cells, PLC; immature Leydig cells, ILC; mature Leydig cells, MLC) were kindly provided by Dr. Matthew Hardy (The Population Council, Rockefeller University, New York, NY). Total RNA was prepared from adult testes of CD-1 mice and the Leydig cell lines MA-10 and TM3 by the single-step acid guanidinium thiocyanate-phenol-chloroform method [36]. Different first-strand cDNAs were synthesized from a 5-µg aliquot of the various RNAs using the Superscript II Reverse Transcriptase System (Invitrogen Canada, Burlington, ON). One twentieth of the first-strand cDNA preparations (one 50th for Tubulin) were used as templates in the PCR reactions using VENT DNA polymerase (New England Biolabs, Mississauga, ON) and oligonucleotide primers specific for Insl3 (forward primer: 5'- CAT GCG CGC GCC GCT GCT AC-3', reverse primer: 5'-TCA GTG GGG ACA CAG ACC C-3'), Nr4a1 (forward primer: 5'-GCC AAG TAC ATC TGC CTG GC-3', reverse primer: 5'- CCA GGC CTG AGC AGA AGA TGA GC-3'), Nr5a1 (forward primer: 5'-CCG GAA CAA GTT TGG GCC CAT GT-3', reverse primer: 5'-CAG ACG AAC TCC TGG CGG TCC AG-3'), and Tubulin (forward primer: 5'-TCC ATC CAC GTC GGC CAG GCT-3', reverse primer: 5'- GTA GGG CTC AAC CAC AGC AGT-3'). The various PCRs were done simultaneously on a Tgradient thermocycler (Biometra) using the following conditions: 3 min at 94°C followed by 28 cycles of denaturation (50 sec at 94°C), annealing (1 min at various temperatures; see below), extension (1 min at 72°C), and a final extension of 5 min at 72°C. The annealing temperatures were 65°C for Insl3, 58°C for NR4A1, 62°C for NR5A1, and 60°C for Tubulin. The PCR products were subcloned in pBluescript (Stratagene) and sequenced on a ABI 3730/XL automated sequencer (Centre de génomique de Québec, Québec City, Canada) to confirm the nature of the amplified cDNAs.

Real-Time PCR

Real-time PCR was used to quantify Insl3 mRNA levels. The Insl3-specific primers are the same as those described above. A standard curve was first established using serial dilutions (500 pg to 5 fg) of a plasmid containing the Insl3 cDNA. The real-time PCRs were carried out using a LightCycler 1.5 instrument from Roche Diagnostics, Laval, Canada. Reactions were performed according to the manufacturer's recommendations using either 5 ng of cDNAs or plasmid standard. As an internal control, the 18S rRNA was amplified using the following primers: forward, 5'-GTA ACC CGT TGA ACC CCA TT-3' and reverse, 5'-CCA TCC AAT CGG TAG TAG CG-3'. The PCRs were performed using the following conditions: 10 min at 95°C followed by 35 cycles of denaturation (5 sec at 95°C), annealing (5 sec at 58°C for 18S rRNA and at 64°C for Insl3 mRNA), and extension (20 sec at 72°C) with single acquisition of fluorescence at the end of the extension step. After amplification, the samples were slowly heated at 0.2°C/sec from 75 to 95°C with continuous reading of fluorescence to obtain a melting curve. The specificity of each PCR product was then determined by using the melting-curve analysis program of the LightCycler software and confirmed by agarose gel electrophoresis. The Insl3 and 18S PCR products showed a single peak in the analysis. Quantitative analyses of the data were performed using the LightCycler analysis software package (Roche Diagnostics). Second-derivative maximum analysis, arithmetic base line adjustment, and polynomial calculation were used. Quantification of mRNA levels was calculated by reference to the respective standard curve. Relative gene expression was expressed as a ratio of Insl3 mRNA levels to 18S rRNA levels. Each amplification was performed in triplicate using at least two different preparations of first-strand cDNAs.

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation (ChIP) assays were performed according the technique described by Spencer et al. [37], with some modifications. Briefly, MA-10 cells were plated at 1.5 x 106 cells in 60-mm dishes. Forty-eight hours after plating, proteins were cross-linked to DNA by adding 1% formaldehyde directly to the media and incubating for 10 min at 37°C. Cells were washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A). Cells were collected, pelleted, and resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.0) containing appropriate protease inhibitors. Sonication was performed on ice using a Braun-Sonic 1510 Sonicator for 4 cycles of 15-sec pulses at six output control and 40% duty cycle in order to obtain DNA fragments between 200 bp and 1000 bp in size. Sheared chromatin was then diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl) containing appropriate protease inhibitors. A 1% aliquot was kept to quantitate the amount of DNA present in different samples before immunoprecipitation (input). Cross-linked DNA was immunoprecipitated with anti-NR4A1 (M-210, Santa Cruz Biotechnology) overnight with rotation at 4°C. Immunocomplexes were collected using a salmon sperm/protein G sepharose slurry and subsequently washed one time each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% IGEPAL, 1% SDS, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and twice with Tris-EDTA buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). Protein-DNA complexes were eluted from protein G sepharose beads by addition of elution buffer (1% SDS, 0.1 M NaHCO3) and rotation at room temperature. Cross-links were reversed by addition of 200 mM of NaCl and heating at 65°C for 4 h. Proteins were degraded using proteinase K treatment (1 h at 55°C) and the DNA fragments were purified by phenol/chloroform extraction and EtOH precipitation. PCRs were done using 0.5 µl of input chromatin sample and 5 µl of NR4A1-immunoprecipitated DNA sample with primers specific for the proximal region (–189 to +5 bp) of the mouse INSL3 promoter (forward: 5'-AAT GTT GGG GAG CGG CTC CTG-3'; reverse: 5'-GTG GCA GGA GGC AGT GGG CAG-3'). PCRs were carried out using Vent polymerase (New England Biolabs) at 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 30 sec, and completed by a final extension of 5 min at 72°C. The PCR products were analyzed by electrophoresis on a 2% ethidium bromide-stained agarose gel. ChIP results were confirmed by at least three separate experiments.

Statistical Analyses

Statistical analyses were done using the Kruskal-Wallis one-way analysis of variance followed by Mann-Whitney U-test to identify significant differences (for Figs. 2 and 8). For Figures 3, 4, and 7, individual comparisons were done using the Mann-Whitney U-test. For all statistical analyses, P < 0.05 was considered significant.


Figure 2
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  		FIG. 2. Activation of the mouse and human INSL3 promoter by NR4A1 family members. A) MA-10 mouse Leydig cells were cotransfected with a –1137 to +11-bp hINSL3 promoter construct and either an empty expression vector (Ctl, open bar) or increasing doses (20, 50, 100 ng; solid bars) of an expression vector for NR4A1. B) MA-10 Leydig cells were cotransfected with the hINSL3 promoter construct and either an empty expression vector (Ctl, open bar) or expression vectors (100 ng) for NR4A family members (solid bars), NR5A1 (hatched bar), and NR5A2 (stippled bar) as indicated. C) MA-10 Leydig cells were cotransfected with a –189-bp to +5-bp mouse Insl3-promoter construct along with an empty expression vector (Ctl, open bar) or expression vectors for NR4A1 (solid bar), NR5A1 (hatched bar), and NR5A2 (stippled bar) as indicated. The number of replicates is indicated for each experiment. Results are shown as fold activation over control (±SEM). Different letters indicate a statistically significant difference (P < 0.05)


Figure 8
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  		FIG. 8. A NR4A1 dominant negative protein blunts the cAMP-induced INSL3 promoter activity. MA-10 Leydig cells were cotransfected with either an empty expression vector (CTL), an expression vector for NR4A1 (100 ng), or treated with 0.5 mM (Bu)2cAMP for 4 h prior to harvesting (cAMP) in the absence (–) or presence of increasing doses (50, 100, 200 ng; solid triangle) of an expression vector for the NR4A1 dominant negative protein along with reporters as indicated on top of the figure: a wild-type reporter and a reporter harboring a point mutation in the NBRE at –100 bp (AAAGGTCA -> AAATTTCA) for each the human INSL3 (A) and mouse Insl3 (B) promoter. Different letters indicate a statistically significant difference (P < 0.05)


Figure 3
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  		FIG. 3. Mapping of the NR4A1-responsive element in the hINSL3 promoter. MA-10 Leydig cells were cotransfected with various 5' deletion constructs of the hINSL3 promoter (the 5'-end point of each construct is indicated on the left of the graphs) with either an empty expression (open bars) or expression vectors for NR4A1 (100 ng, solid bars). Results of five different experiments are shown as fold activation over control (±SEM). Activations statistically different (P < 0.05) from control are indicated by an asterisk (*)


Figure 4
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  		FIG. 4. The novel NBRE at –100 bp is necessary and sufficient for NR4A1-dependent activation of the mouse and human INSL3 promoter. A) Sequence of a novel NBRE element located at position –100 bp of the human INSL3 promoter. The sequence of the NBRE is conserved across species. The position of an NR5A1 (SF-1) element at –56 bp is indicated. B) MA-10 Leydig cells were cotransfected with either an empty expression (open bar), an expression vector for NR4A1 (100 ng, solid bars), along with reporters as indicated on top of the figure: a wild-type reporter and a reporter harboring a point mutation in the NBRE at –100 bp (AAAGGTCA -> AAATTTCA) for each the human (left panel) and mouse (right panel) INSL3 promoter. The number of replicates is indicated for each experiment. Results are shown as fold activation over control (±SEM). C) MA-10 Leydig cells were transfected with the same reporter constructs described in B. The number of replicates is indicated for each experiment. Results are shown as relative activity; the activity of the wild-type human (left panel) and mouse (right panel) INSL3 reporter was arbitrarily set at 100%. An asterisk (*) indicates an activation statistically different from control (P < 0.05)

RESULTS

Coexpression of NR4A1 and INSL3 in Leydig Cell Lines

NR4A1 is an orphan nuclear receptor expressed in Leydig cells [23, 24]. As a first step to assess the role of NR4A1 in Insl3 expression, we used RT-PCR to compare expression of Nr4a1, Nr5a1, and Insl3 in adult mouse testes, two Leydig cell lines (MA-10 and TM3), purified rat Leydig cells at different postnatal developmental stages (progenitor, immature, and mature Leydig cells), and three Sertoli cell lines (15P-1, MSC-1, TM4). As shown in Figure 1, we found that Nr4a1 is coexpressed with Insl3 in both Leydig cell lines as well as in purified Leydig cells at all developmental stages.


Figure 1
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  		FIG. 1. Nr4a1 is coexpressed with Insl3 in Leydig cells. RT-PCR analysis was used to detect Insl3, Nr4a1, and Nr5a1. Total RNA was isolated from adult mouse testes, Leydig cell lines MA-10 and TM3, Leydig cells purified from rat at different developmental stages (progenitor Leydig cells, PLC; immature Leydig cells, ILC; mature Leydig cells, MLC), and Sertoli cell lines 15P-1, MSC-1, and TM4. First-strand cDNAs were prepared as described in Materials and Methods. Omission of cDNAs was used as negative control (water). Amplification of Tubulin was performed to validate the quantity and integrity of the cDNAs. Shown is a representative experiment of at least three separate amplifications using different first-strand cDNA preparations

Activation of Mouse and Human INSL3 Promoter by NR4A1

The fact that Nr4a1 is coexpressed with Insl3 in Leydig cells suggests that this transcription factor could be involved in Insl3 transcription in these cells. To test this hypothesis, we isolated a fragment of the mouse and human INSL3 promoter and performed transient transfection assays in MA-10 Leydig cells. As shown in Figure 2, A and B, NR4A1, as well as the other NR4A family members NR4A2 and NR4A3, could activate a –1137-bp human INSL3 (hINSL3) reporter construct (3- to 4-fold), whereas NR5A1 and NR5A2 were weaker activators (1.8- and 1.6-fold, respectively). Similar results were obtained with the mouse Insl3 promoter (Fig. 2C).

Localization of the NR4A1-Responsive Element

To locate the NR4A1-responsive element, 5'-progressive deletions of the hINSL3 promoter were generated and tested for NR4A1 responsiveness in MA-10 cells (Fig. 3). A deletion construct to –322 bp was still activated by NR4A1. However, further deletion to –93 bp, that retains the NR5A1 element at –56 bp, completely abrogated transactivation of the hINSL3 promoter by NR4A1 (Fig. 3). Taken together, these results indicated that the NR5A1 element is not required for NR4A1-dependent activation of the hINSL3 promoter and that a novel, yet unidentified, element located between –322 and –93 bp is responsible for NR4A1 responsiveness. Indeed, sequence analysis of this promoter region revealed the presence of a consensus NR4A1 NBRE located at position –100 bp that is conserved across species (Fig. 4A). The importance of this novel NBRE was next assessed by site-directed mutagenesis. As shown in Figure 4B, mutation of the –100-bp NBRE (AAAGGTCA -> AAATTTCA) completely abolished NR4A1-dependent activation of the human (left panel) and mouse (right panel) INSL3 promoter. Mutation of the NBRE also led to a loss of ~20% in the basal activity of the human INSL3 promoter (Fig. 4C, left panel), whereas basal activity of the mouse Insl3 promoter was reduced by ~80% (Fig. 4C, right panel). The more dramatic effect seen on the mouse promoter could be due to the fact that MA-10 Leydig cells are of mouse origin and they would therefore correspond to a more natural and favorable environment for the mouse Insl3 promoter. Altogether, these results indicate that the NBRE at –100 bp is required for maximal INSL3-promoter activity in Leydig cells and that it is both necessary and sufficient to confer NR4A1-responsiveness to the mouse and human INSL3 promoter.

Specific Binding of NR4A1 to the NBRE (–100 bp)

DNA binding of NR4A1 to the novel NBRE (–100 bp) was first assessed by electromobility shift assays (EMSA). Consistent with the requirement for the NBRE in NR4A1-mediated activation of the mouse and human INSL3 promoter (Fig. 4B), in vitro translated NR4A family members (NR4A1, NR4A2, and NR4A3) bound to the NBRE (Fig. 5A, left panel). NR5A family members could also bind to this NBRE, although their binding appears weaker (Fig. 5A, right panel). Next, the DNA-binding specificity of NR4A1 was analyzed by competition experiments. As shown in Figure 5B, NR4A1 binding was specifically competed by increasing doses of unlabeled oligonucleotides (Fig. 5B, lanes 3 and 4) as well as by unlabeled oligonucleotides corresponding to the NBRE that we have recently identified in the human HSD3B2 promoter (Fig. 5B, lanes 7 and 8). NR4A1 binding, however, was not displaced by oligonucleotides harboring a mutation in the hINSL3 or the hHSD3B2 NBREs (Fig. 5B, lanes 5, 6, 9, and 10), known to abolish NR4A1-dependent activation (Fig. 4B and [23]).


Figure 5
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  		FIG. 5. NR4A1 binds to the novel NBRE. A) EMSA was used to assess the binding of in vitro-produced NR4A family members (NR4A1, NR4A2, NR4A3; left panel) and NR5A1 and NR5A2 (right panel) to a double-stranded 32P-labeled oligonucleotide corresponding to the NBRE at –100 bp of the hINSL3 promoter. The inset at the bottom of the right panel corresponds to an SDS-PAGE of the in vitro-produced proteins to validate the levels of the proteins. B) Competition experiments were used to assess the DNA-binding specificity of NR4A1 to a double-stranded 32P-labeled oligonucleotide corresponding to the NBRE at –100 bp. Binding of NR4A1 was challenged by increasing doses (black triangles; molar excesses of 2x and 5x) of unlabeled oligonucleotides corresponding to the wild-type –100-bp NBRE from the hINSL3 promoter (INSL3 wt), the –100-bp NBRE mutated from AAAGGTCA to AAATTTCA (INSL3 mut), the wild-type –130-bp NBRE from the human HSD3B2 promoter (HSD3B2 wt), and the –130-bp element containing a mutation (HSD3B2 mut) in the NBRE element (TAAAGGTCA -> TAAATTTCA)

NR4A1 DNA-binding activity was also tested by EMSA using nuclear extracts from MA-10 Leydig cells and the NBRE from the human (Fig. 6A, left panel) and mouse (Fig. 6A, right panel) INSL3 promoter. In both cases, a band (Fig. 6A, lanes 2 and 5) was detected. This band was mostly supershifted by the addition of an anti-NR4A1 antiserum (Fig. 6A, lanes 3 and 6). The remaining DNA-binding activity could be due to NR4A2, NR5A1, or NR5A2, which can bind to this element (Fig. 5A) but are not recognized by the anti-NR4A1 antiserum. Thus, endogenous NR4A1 can bind to the novel NBRE in the mouse and human INSL3 promoter.


Figure 6
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  		FIG. 6. Endogenous NR4A1 protein binds to the INSL3 promoter in vitro and in vivo. A) EMSA was used to determine the binding of the NR4A1 protein present in MA-10 Leydig cells to a double-stranded 32P-labeled oligonucleotide corresponding to the human (left panel) and mouse INSL3 (right panel) NBREs at –100 bp. The NR4A1 binding was supershifted by a NR4A1 antiserum ({alpha}NR4A1). P.I.: Preimmune serum. B) MA-10 cell lysates were prepared and in vivo NR4A1 interaction with the endogenous mouse Insl3 promoter was studied by ChIP. An aliquot of chromatin preparation before immunoprecipitation (input) was used as positive control. Chromatin was precipitated in duplicate with an anti-NR4A1 antiserum ({alpha}NR4A1) or incubated with water, which served as negative control. A 200-bp DNA fragment containing the NBRE present within the mouse Insl3 promoter was amplified by PCR. This DNA fragment comigrates with a fragment obtained by amplification of a plasmid containing the mouse Insl3 promoter (plasmid). Results are representative of three independent experiments

Next, ChIP assays were performed to detect in vivo binding of NR4A1 to the mouse Insl3 promoter in MA-10 cells. Proteins were cross-linked to the chromatin, which was then sheared and subjected to immunoprecipitation using an anti-NR4A1 antiserum ({alpha}NR4A1). By PCR, a band of ~200 bp was detected in the input sample (Fig. 6B, lane 3) as well as in the two samples immunoprecipitated with the anti-NR4A1 antiserum (Fig. 6B, lanes 4 and 5) but not from the control sample (Fig. 6B, lane 1). This band migrates at the same position as a band obtained with a plasmid containing the mouse Insl3 promoter (Fig. 6B, lane 2). NR4A1 is thus bound on the Insl3 promoter in vivo in MA-10 Leydig cells.

The NBRE is Required for the cAMP-Mediated INSL3 Promoter Activation

The fact that Nr4a1 and Insl3 are both upregulated in response to LH/hCG in vivo raises the possibility that NR4A1 might be involved in this upregulation of Insl3. We first tested whether cAMP treatment of MA-10 Leydig cells could stimulate Insl3 expression. Using real-time PCR, we found that Insl3 mRNA levels were increased ~2.2-fold following a 4-h treatment with 0.5 mM (Bu)2cAMP (Fig. 7A). Next, we tested whether cAMP could activate the mouse and human INSL3 promoter activity and if this required the NBRE at –100 bp. As shown in Figure 7B, a 2.5-fold cAMP-dependent activation of the human (left panel) and mouse (right panel) INSL3 promoter was observed (hatched bars). This stimulation was lost when the NBRE was mutated, suggesting that NR4A1, which binds to this element (Figs. 5 and 6), might act as a mediator of cAMP in the regulation of the mouse and human INSL3 promoter activity.


Figure 7
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  		FIG. 7. The NBRE is required for cAMP stimulation of the INSL3 promoter. A) Real-time PCR was used to quantify Insl3 mRNA levels in MA-10 cells treated with either vehicle (–) or 0.5mM (Bu)2cAMP for 4 h (+). Insl3 mRNA levels in each samples were compared with those of the 18S rRNA housekeeping gene. An asterisk (*) indicates a statistically significant difference (P < 0.05). B) MA-10 Leydig cells were transfected with the same reporters described in Figure 4B and treated with either vehicle (–) or 0.5mM (Bu)2cAMP for 4 h (+) prior to harvesting. The number of replicates is indicated for each experiment. Results are shown as fold activation over control (±SEM). An asterisk (*) indicates an activation statistically different from control (P < 0.05)

To assess whether endogenous NR4A family members are involved in the cAMP-induced Insl3 promoter activity, we generated a truncated NR4A1 protein (aa 252–601). Because this protein lacks its N-terminal activation domain but retains the DNA-binding domain, it could therefore bind to DNA but could no longer activate the mouse and human INSL3 promoter (CTL bars in the left panels of Fig. 8, A and B). In addition, the NR4A1 truncated protein could antagonize in a dose-dependent manner the activation of the mouse and human INSL3 mediated by the wild-type NR4A1 protein (NR4A1 bars in the left panels of Fig. 8, A and B). Thus, the truncated NR4A1 protein behaves as a dominant negative competitor. When overexpressed in MA-10 Leydig cells, the NR4A1 dominant negative protein decreased cAMP-induced activity of the mouse and human INSL3 promoter (cAMP bars in the left panels of Fig. 8, A and B). These effects were specific and mediated through the NBRE because reporters harboring a mutation of this element were unaffected by the NR4A1 dominant negative protein (right panels of Fig. 8, A and B). Altogether, these results indicate that endogenous NR4A proteins are involved in the cAMP stimulation of the INSL3 promoter.

DISCUSSION

Although the physiological roles of INSL3 in mammalian reproductive function are well established, very little is known regarding the molecular mechanisms that regulate its expression in gonadal cells. So far, NR5A1 is the only transcription factor known to regulate mouse Insl3 promoter activity; it alone, however, cannot explain the highly specific and dynamic Insl3 expression pattern. Due to its similarities with Insl3 in terms of expression pattern and hormonal regulation, the NR4A1 transcription factor is an interesting candidate regulator of the Insl3 gene. During gonadal development, Nr4a1 is 3–4 times more abundantly expressed in the fetal testis than in the fetal ovary [38]. Just prior to puberty and upon activation of the HPG axis, Nr4a1 expression levels are upregulated in the testis [24]. Nr4a1 expression is also known to be increased in response to LH/hCG/cAMP in Leydig cells as well as in the ovary [23, 24, 39]. Taken together, these observations argue in favor of a role for NR4A1 in Insl3 transcription. In agreement with this, we have now identified the mouse and human INSL3 promoter as a target for NR4A1 in Leydig cells.

Although NR4A1 and NR5A1 are nuclear receptors that bind as monomers to similar DNA regulatory sequences, they nonetheless have specific requirements for the 5' nucleotides of their binding site. Indeed, NR4A1 strongly binds to the sequence (T/A)AA AGGTCA, whereas NR5A1 prefers (C/T)CA AGGTCA [25, 40]. Thus, NR4A1 can only weakly bind to the NR5A1 element and NR5A1 poorly recognizes the NBRE [25, 40]. Our in vitro EMSA data are consistent with this because we found that, although NR4A and NR5A family members can all bind to the novel NBRE (AAA AGGTCA) that we have identified in the mouse and human INSL3 promoter, binding of NR4A family members was stronger than that of NR5A family members (Fig. 5A). In agreement with this, we also found that NR4A1 and NR5A1 regulate INSL3 promoter activity via their respective binding sites. This is supported by the fact that NR4A1-dependent activation of the mouse and human INSL3 promoter was lost when the NBRE at –100 bp was either deleted or mutated despite the presence of an intact NR5A1 element at –56 bp (Figs. 3 and 4B). Similarly, mutation of the NBRE did not impair the activation of the INSL3 promoter by NR5A1 (data not shown). Moreover, we found no transcriptional cooperation between these two transcription factors (data not shown). It is well known, however, that ubiquitously expressed and/or cell-specific transcription factors interact to form a unique combinatorial code to direct proper tissue-, cell-, and promoter-specific transcription. Thus, it is highly probable that other transcription factors are involved in regulating INSL3 expression. Supporting this is the fact that Sertoli cell lines do express Nr4a1 and Nr5a1 but do not express Insl3 (Fig. 1). Furthermore, we have found that the proximal mouse and human INSL3 promoter contains other species-conserved regulatory elements besides the NBRE and NR5A1 motifs (unpublished observations). The transcription factors binding to these elements, however, remain to be identified.

The lack of an overt reproductive phenotype in Nr4a1tm1Jmi/Nr4a1tm1Jmi mice, in which the Nr4a1 gene has been deleted by homologous recombination [41], would appear to argue against a role for NR4A1 in Insl3 transcription. A possible explanation is that other NR4A nuclear receptors can compensate for the absence of NR4A1. Indeed, Nr4a2 was shown to be upregulated in Nr4a1–/– mice [41]. Moreover, NR4A1 and NR4A3 have been shown to be functionally redundant in vivo in thymocyte gene expression [42]. Thus, there is strong evidence that the various NR4A family members can indeed compensate for one another in vivo. This likely includes the mammalian gonads, where two other NR4A family members, NR4A2 and NR4A3, are coexpressed with NR4A1 [24, 43, 44]. In support of this, we showed that NR4A2 and NR4A3 can activate the INSL3 promoter as well as NR4A1 (Fig. 2B).

Because LH action is required for proper Insl3 expression in postnatal Leydig cells [4], NR4A1 might be one of the downstream effectors of LH in the regulation of Insl3 expression. LH is known to mediate its effects on Leydig cell differentiation and function by binding to its G-protein-coupled receptor leading to activation of the cAMP/PKA pathway (reviewed in [45]). The downstream effectors of LH signaling in Leydig cells, however, remain poorly characterized. We and others have shown that Nr4a1 expression is robustly induced both at the mRNA and protein levels in response to LH/hCG/cAMP in Leydig cells in vitro and in vivo [23, 24]. Consistent with the fact that Insl3 expression is regulated by the chronic effects of LH (in contrast with its acute effects on steroidogenesis) [4, 46], NR4A1 levels remain high for more than 12 h following hCG treatment [24]. Our current data support a role for NR4A family members in the cAMP-induced Insl3 transcription in Leydig cells. Indeed, mutation of the NBRE in the mouse and human INSL3 promoter, which prevents binding and activation by NR4A1 (Figs. 4B and 5), abolished cAMP responsiveness of the promoter (Fig. 7B). Furthermore, we used a truncated NR4A1 protein that acts as a dominant negative competitor to confirm the functional implication of NR4A family members endogenously expressed in MA-10 Leydig cells in the cAMP-mediated increase in mouse and human INSL3 promoter activity. We found that overexpression of this dominant negative NR4A1 protein in MA-10 cells significantly blunted the response to cAMP (Fig. 8).

The identification of NR4A1 as a regulator of Insl3 expression might also help us better understand the mechanisms of action of certain endocrine-disrupting chemicals that have deleterious effects on the male reproductive system. Indeed, the Insl3 gene is a known target for certain classes of endocrine-disrupting chemicals, particularly phthalates and estrogenic compounds, all of which have been associated with cryptorchidism (undescended testis) in humans and rodents [47–58]. Using mice models, it was shown that Insl3 expression levels in male embryos were severely reduced following in utero exposure to estrogens and phthalates [53, 59, 60]. In both cases, insufficient INSL3 levels were associated with failure of testicular descent. These studies also revealed that Nr5a1 expression in fetal Leydig cells was not affected by maternal exposure to estrogens or phthalates [59, 61]. Thus, it is likely that repression of Insl3 by estrogens and phthalates involves additional transcription factors. NR4A1 might represent such a factor.

ACKNOWLEDGMENTS

We would like to thank Drs. Jacques Drouin, Luc Bélanger, Michael Griswold, Matthew Hardy, and Mario Ascoli for generously providing expression plasmids, RNA samples, and cell lines used in this study. We are also indebted to Christine Légaré and members of the laboratory of Dr. Jean-François Bilodeau for their assistance with the real-time PCR experiments.

FOOTNOTES

1 Supported by the Canadian Institutes of Health Research (CIHR) grant number MOP-62752 to J.J.T and a studentship from the Natural Sciences and Engineering Research Council of Canada to L.J.M. J.J.T. is the recipient of a CIHR New Investigator Award. Back

2 Correspondence: Jacques J. Tremblay, Ontogeny-Reproduction, Room T1–49, CHUL Research Centre, 2705 Laurier Blvd., Ste-Foy, Québec, Canada G1V 4G2. FAX: 418 654 2765; Jacques-J.Tremblay{at}crchul.ulaval.ca Back

Received: 9 June 2005.

First decision: 8 July 2005.

Accepted: 13 October 2005.

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Biol Reprod, December 1, 2007; 77(6): 978 - 989.
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