The nuclear receptors SF1 and COUP-TFII cooperate on the Insl3 promoter in Leydig cells.

In brief: The insulin-like 3 (INSL3) hormone produced by Leydig cells is essential for proper male sex differentiation, but the regulation of Insl3 expression remains poorly understood. This study describes a new physical and functional cooperation between the nuclear receptors SF1 and COUP-TFII in Insl3 expression. Abstract: INSL3, a hormone abundantly produced by Leydig cells, is essential for testis descent during fetal life and bone metabolism in adults. The mechanisms regulating Insl3 expression in Leydig cells have been studied in several species but remain poorly understood. To date, only a handful of transcription factors are known to activate the Insl3 promoter and include the nuclear receptors AR, NUR77, COUP-TFII, and SF1, as well as the Krüppel-like factor KLF6. Some of these transcription factors are known to transcriptionally cooperate on the Insl3 promoter, but the mechanisms at play remain unknown. Here, we report that COUP-TFII and SF1 functionally cooperate on the Insl3 promoter from various species but not on the Inha, Akr1c14, Cyp17a1, Hsd3b1, Star, Gsta3, and Amhr2 promoters that are known to be regulated by COUP-TFII and/or SF1. The Insl3 promoter contains species-conserved binding sites for COUP-TFII (-91 bp) and SF1 (-134 bp). Mutation of either the COUP-TFII or the SF1 sequence had no impact on the COUP-TFII/SF1 cooperation, but the mutation of both binding sites abolished the cooperation. In agreement with this, we found that COUP-TFII and SF1 physically interact in Leydig cells. Finally, we report that the transcriptional cooperation is not limited to COUP-TFII and SF1 as it also occurred between all NR2F and NR5A family members. Our data provide new mechanistic insights into the cooperation between the orphan nuclear receptors COUP-TFII and SF1 in the regulation of Insl3 gene expression in Leydig cells.

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells.

The peptide hormone insulin-like 3 (INSL3) is produced almost exclusively by Leydig cells of the male gonad. INSL3 has several functions such as fetal testis descent and bone metabolism in adults. Insl3 gene expression in Leydig cells is not hormonally regulated but rather is constitutively expressed. The regulatory region of the Insl3 gene has been described in various species; moreover, functional studies have revealed that the Insl3 promoter is regulated by various transcription factors that include the nuclear receptors AR, NUR77, COUP-TFII, LRH1, and SF1, as well as the Krüppel-like factor KLF6. However, these transcription factors are also found in several tissues that do not express Insl3, indicating that other, yet unidentified factors, must be involved to drive Insl3 expression specifically in Leydig cells. Through a fine functional promoter analysis, we have identified a 35-bp region that is responsible for conferring 70% of the activity of the mouse Insl3 promoter in Leydig cells. All tri- and dinucleotide mutations introduced dramatically reduced Insl3 promoter activity, indicating that the entire 35-bp sequence is required. Nuclear proteins from MA-10 Leydig cells bound specifically to the 35-bp region. The 35-bp sequence contains GC- and GA-rich motifs as well as potential binding elements for members of the CREB, C/EBP, AP1, AP2, and NF-κB families. The Insl3 promoter was indeed activated 2-fold by NF-κB p50 but not by other transcription factors tested. These results help to further define the regulation of Insl3 gene transcription in Leydig cells.

The nuclear receptor NR2F2 activates star expression and steroidogenesis in mouse MA-10 and MLTC-1 Leydig cells.

Testosterone production is dependent on cholesterol transport within the mitochondrial matrix, an essential step mediated by a protein complex containing the steroidogenic acute regulatory (STAR) protein. In steroidogenic Leydig cells, Star expression is hormonally regulated and involves several transcription factors. NR2F2 (COUP-TFII) is an orphan nuclear receptor that plays critical roles in cell differentiation and lineage determination. Conditional NR2F2 knockout prior to puberty leads to male infertility due to insufficient testosterone production, suggesting that NR2F2 could positively regulate steroidogenesis and Star expression. In this study we found that NR2F2 is expressed in the nucleus of some peritubular myoid cells and in interstitial cells, mainly in steroidogenically active adult Leydig cells. In MA-10 and MLTC-1 Leydig cells, small interfering RNA (siRNA)-mediated NR2F2 knockdown reduces basal steroid production without affecting hormone responsiveness. Consistent with this, we found that STAR mRNA and protein levels were reduced in NR2F2-depleted MA-10 and MLTC-1 cells. Transient transfections of Leydig cells revealed that a -986 bp mouse Star promoter construct was activated 3-fold by NR2F2. Using 5' progressive deletion constructs, we mapped the NR2F2-responsive element between -131 and -95 bp. This proximal promoter region contains a previously uncharacterized direct repeat 1 (DR1)-like element to which NR2F2 is recruited and directly binds. Mutations in the DR1-like element that prevent NR2F2 binding severely blunted NR2F2-mediated Star promoter activation. These data identify an essential role for the nuclear receptor NR2F2 as a direct activator of Star gene expression in Leydig cells, and thus in the control of steroid hormone biosynthesis.

Growth Hormone-induced STAT5B Regulates Star Gene Expression Through a Cooperation With cJUN in Mouse MA-10 Leydig Cells.

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star messenger RNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (electrophoretic mobility shift assay and supershift) and in vivo (chromatin immunoprecipitation) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

FOXA3 Is Expressed in Multiple Cell Lineages in the Mouse Testis and Regulates Pdgfra Expression in Leydig Cells.

The three FOXA transcription factors are mainly known for their roles in the liver. However, Foxa3-deficient mice become progressively sub/infertile due to germ cell loss. Because no data were available regarding the localization of the FOXA3 protein in the testis, immunohistochemistry was performed on mouse testis sections. In the fetal testis, a weak but consistent staining for FOXA3 is detected in the nucleus of Sertoli cells. In prepubertal and adult life, FOXA3 remains present in Sertoli cells of some but not all seminiferous tubules. FOXA3 is also detected in the nucleus of some peritubular cells. From postnatal day 20 onward, FOXA3 is strongly expressed in the nucleus of Leydig cells. To identify FOXA3 target genes in Leydig cells, MLTC-1 Leydig cells were transfected with a series of Leydig cell gene reporters in the presence of a FOXA3 expression vector. The platelet-derived growth factor receptor α (Pdgfra) promoter was significantly activated by FOXA3. The Pdgfra promoter contains three potential FOX elements and progressive 5' deletions and site-directed mutagenesis revealed that the most proximal element at -78 bp was sufficient to confer FOXA3 responsiveness. FOXA3 from Leydig cells could bind to this element in vitro (electrophoretic mobility shift assay) and was recruited to the proximal Pdgfra promoter in vivo (chromatin immunoprecipitation). Finally, endogenous Pdgfra messenger RNA levels were reduced in FOXA3-deficient MLTC-1 Leydig cells. Taken together, our data identify FOXA3 as a marker of the Sertoli cell lineage and of the adult Leydig cell population, and as a regulator of Pdgfra transcription in Leydig cells.

LRH-1/NR5A2 cooperates with GATA factors to regulate inhibin alpha-subunit promoter activity.

Inhibin alpha is the common subunit of the dimeric inhibin proteins known for their role in suppressing pituitary FSH secretion. In this study, we have examined the role of GATA factors and the nuclear receptor, LRH-1/NR5A2, in the regulation of inhibin alpha-subunit promoter activity. The inhibin alpha promoter contains two GATA-binding motifs that can be activated by GATA4 or GATA6. The GATA-dependence of the promoter was demonstrated by downregulating GATA expression in MA-10 cells using siRNA technology. We next examined whether GATA factors could cooperate with LRH-1, a factor recently proposed to be an important regulator of inhibin alpha-subunit transcription. Both GATA4 and GATA6 strongly synergized with LRH-1. Consistent with the cAMP-dependence of the inhibin alpha-subunit promoter, GATA/LRH-1 synergism was markedly enhanced by PKA and the co-activator protein CBP. Thus, our results identify LRH-1 as a new transcriptional partner for GATA factors in the regulation of inhibin alpha-subunit gene expression.

GATA factors and the nuclear receptors, steroidogenic factor 1/liver receptor homolog 1, are key mutual partners in the regulation of the human 3beta-hydroxysteroid dehydrogenase type 2 promoter.

The human HSD3B2 gene encodes the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase type 2 (3beta-HSD2) enzyme that is required for steroid hormone biosynthesis. Mutations in the hHSD3B2 gene are responsible for a form of congenital adrenal hyperplasia and male pseudohermaphroditism whereas overexpression of hHSD3B2 has been recently associated with polycystic ovarian syndrome. Despite the importance of the hHSD3B2 gene, the molecular mechanisms that regulate its expression remain poorly understood. Transcription factors belonging to the GATA family are emerging as novel regulators of steroidogenesis. Indeed, GATA-4 and GATA-6 are abundantly expressed in steroidogenic cells of the gonads and adrenals. We now report that the human HSD3B2 promoter (hHSD3B2), which contains four consensus GATA elements, constitutes an important target for GATA factors. GATA-4 and GATA-6 by themselves are sufficient to activate transcription (up to 15-fold) from a -1073 bp hHSD3B2 promoter fragment and blockade of endogenous GATA expression and/or activity blunts hHSD3B2 promoter activity in steroidogenic cells. Deletion studies showed that the proximal GATA element located at -196 bp is sufficient to confer GATA responsiveness of the hHSD3B2 promoter and is required for full hHSD3B2 promoter activity in steroidogenic cells. Moreover, we report that GATA-4 and GATA-6 can physically interact with the nuclear receptors, steroidogenic factor 1 and liver receptor homolog 1, to synergistically activate hHSD3B2 promoter activity in both homologous and heterologous cells. Aberrant expression of transcription factors essential for hHSD3B2 expression might also be involved in some pathologies/syndromes associated with deregulated hHSD3B2 expression.

Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements.

The nuclear receptor NR4A1 is expressed in steroidogenic Leydig cells where it plays pivotal roles by regulating the expression of several genes involved in steroidogenesis and male sex differentiation including Star, HSD3B2, and Insl3 Activation of the cAMP and Ca(2+) signaling pathways in response to LH stimulation leads to a rapid and robust activation of Nr4a1 gene expression that requires the Ca(2+)/CAMKI pathway. However, the downstream transcription factor(s) have yet to be characterized. To identify potential Ca(2+)/CaM effectors responsible for hormone-induced Nr4a1 expression, MA-10 Leydig cells were treated with forskolin to increase endogenous cAMP levels, dantrolene to inhibit endoplasmic reticulum Ca(2+) release, and W7 to inhibit CaM activity. We identified Ca(2+)-responsive elements located in the discrete regions of the Nr4a1 promoter, which contain binding sites for several transcription factors such as AP1, CREB, and MEF2. We found that one of the three AP1/CRE sites located at -255 bp is the most responsive to the Ca(2+) signaling pathway as are the two MEF2 binding sites at -315 and -285 bp. Furthermore, we found that the hormone-induced recruitment of phospho-CREB and of the co-activator p300 to the Nr4a1 promoter requires the Ca(2+) pathway. Lastly, siRNA-mediated knockdown of CREB impaired NR4A1 expression and steroidogenesis. Together, our data indicate that the Ca(2+) signaling pathway increases Nr4a1 expression in MA-10 Leydig cells, at least in part, by enhancing the recruitment of coactivator most likely through the MEF2, AP1, and CREB transcription factors thus demonstrating an important interplay between the Ca(2+) and cAMP pathways in regulating Nr4a1 expression.

KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells.

Insulin-like 3 (INSL3), a Leydig cell-specific hormone, is essential for testis descent during foetal life and bone metabolism in adults. Despite its essential roles in male reproductive and bone health, very little is known regarding its transcriptional regulation in Leydig cells. To date, few transcription factors have been shown to activate INSL3 promoter activity: the nuclear receptors AR, NUR77, COUP-TFII and SF1. To identify additional regulators, we have isolated and performed a detailed analysis of a 1.1 kb human INSL3 promoter fragment. Through 5' progressive deletions and site-directed mutagenesis, we have mapped a 10 bp element responsible for about 80% of INSL3 promoter activity in Leydig cells. This element is identical to the CPE element of the placental-specific glycoprotein-5 (PSG5) promoter that is recognized by the developmental regulator Krüppel-like factor 6 (KLF6). Using PCR and western blotting, we found that KLF6 is expressed in several Leydig and Sertoli cell lines. Furthermore, immunohistochemistry on adult mouse testis revealed the presence of KLF6 in the nuclei of both Leydig and Sertoli cells. KLF6 binds to the 10 bp KLF element at -108 bp and activates the -1.1 kb human, but not the mouse, INSL3 promoter. KLF6-mediated activation of the human INSL3 promoter required an intact KLF element as well as Leydig/Sertoli-enriched factors because KLF6 did not stimulate the human INSL3 promoter activity in CV-1 fibroblast cells. Consistent with this, we found that KLF6 transcriptionally cooperates with NUR77 and SF1. Collectively, our results identify KLF6 as a regulator of human INSL3 transcription.

Role of nuclear receptors in INSL3 gene transcription in Leydig cells.

Insulin-like 3 (INSL3) is a hormone produced by testicular Leydig cells throughout life. During embryonic life it regulates an essential step of testicular descent, whereas in adults it acts as a male germ cell survival factor. Despite the importance of INSL3 for male sex differentiation and function, very little is known regarding the molecular mechanisms that regulate its expression. So far, the nuclear receptor SF-1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. In order to further our understanding of the transcriptional regulation of INSL3 expression, we have isolated the human INSL3 promoter and tested the effects of the nuclear receptors SF-1, LRH-1, and Nur77 on its activity in Leydig cells. In transfections assays, all three nuclear receptors activated the human INSL3 promoter but especially Nur77, which acted through a novel regulatory element. Thus, the human INSL3 promoter constitutes a novel target for the orphan nuclear receptor Nur77.

Friend of GATA (FOG)-1 and FOG-2 differentially repress the GATA-dependent activity of multiple gonadal promoters.

The GATA transcription factors are crucial regulators of cell-specific gene expression in many tissues. GATA proteins are abundantly expressed in gonads of several species. In vertebrates, GATA factors are expressed from the onset of gonadal development and are later found in multiple cell lineages of both the testis and ovary. GATA factors activate transcription of several gonadal genes including the hormone-encoding genes Müllerian inhibiting substance (MIS) and inhibin alpha and genes involved in steroidogenesis like P450 aromatase (Cyp 19) and steroidogenic acute regulatory protein. GATA factors also contribute to cell-specific gonadal gene expression through cooperative interactions with other transcription factors such as the orphan nuclear receptor steroidogenic factor-1. GATA transcriptional activity is also modulated by two multitype zinc finger proteins called the Friend of GATA (FOG) proteins, which were cloned as GATA-specific cofactors. The FOG proteins (FOG-1 and FOG-2) can act as either enhancers or repressors of GATA transcriptional activity, depending on the cell and promoter context. We now report that the FOG proteins are coexpressed with GATA factors in testicular cells in which they differentially repress the promoter activities of several GATA-dependent target genes. These findings implicate the FOG proteins in the regulation of GATA-dependent gene transcription in the gonads.

Expression of ladybird-like homeobox 2 (LBX2) during ovarian development and folliculogenesis in the mouse.

The Ladybird-like homeobox gene 2 (Lbx2) belongs to the homeodomain-containing family of transcription factor that are known to play crucial role in various developmental processes. During early mouse embryogenesis, Lbx2 was shown to be expressed in the developing eye, brain and urogenital system. Although Lbx2 was detected in the testis and epididymis throughout development, no data was available regarding its expression in the female gonad. Here we have determined Lbx2 expression throughout mouse ovarian development by in situ hybridization. In contrast to the strong expression in the male fetal gonad, no Lbx2 signal could be detected in the fetal ovary. Soon after birth, however, Lbx2 expression was detected at different levels in various ovarian compartments (oocyte, granulosa cells, theca cells) where its expression was highly dynamic depending on the stage of follicular maturation. Our data would be consistent with a role for LBX2 in ovarian maturation and folliculogenesis.

Nuclear receptors, testosterone, and posttranslational modifications in human INSL3 promoter activity in testicular Leydig cells.

Insulin-like peptide 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the mammalian testis. During embryonic life INSL3 is required for testicular descent, whereas in adults it is involved in bone metabolism and male germ cell survival. Despite these important roles, the molecular mechanisms regulating INSL3 expression remain poorly understood. So far, two transcription factors have been implicated in INSL3 transcription: the nuclear receptors SF1 and NUR77. Circumstantial evidence also points to a role for androgens. Using transient transfections in MA-10 Leydig cells, we found that testosterone regulates in a time- and dose-dependent manner the human INSL3 promoter. The INSL3 promoter, however, does not contain a classical androgen-responsive element. Testosterone responsiveness was found to be mediated through an element located in the proximal INSL3 promoter, which also contains a NUR77-SF1-binding site. Furthermore, we found that posttranslational modifications, such as phosphorylation and acetylation, modulate transcription factor activity and therefore also contribute to INSL3 promoter activity in Leydig cells. All together, these data provide new insights into the molecular mechanisms regulating INSL3 expression in the mammalian testis.

The nuclear receptors SF1 and LRH1 are expressed in endometrial cancer cells and regulate steroidogenic gene transcription by cooperating with AP-1 factors.

Excessive exposure to estradiol represents the main risk factor for endometrial cancer. The abnormally high estradiol levels in the endometrium of women with endometrial cancer are most likely due to overproduction by the tumour itself. Endometrial cancer cells express the genes encoding the steroidogenic enzymes involved in estradiol synthesis. Here we used RT-PCR and Western blot to show that the nuclear receptors SF1 and LRH1, two well-known regulators of steroidogenic gene expression in gonadal and adrenal cells, are also expressed in endometrial cancer cell lines. By transient transfections, we found that SF1 and LRH1, but not the related nuclear receptor NUR77, can activate the promoters of three human steroidogenic genes: STAR, HSD3B2, and CYP19A1 PII. Similarly, forskolin but not PMA, could activate all three promoters. In addition, we found that both SF1 and LRH1 can transcriptionally cooperate with the AP-1 family members c-JUN and c-FOS, known to be associated with enhanced proliferation of endometrial carcinoma cells, to further enhance activation of the STAR, HSD3B2, and CYP19A1 PII promoters. All together, our data provide novel insights into the mechanisms of steroidogenic gene expression in endometrial cancer cells and thus in the regulation of estradiol biosynthesis by tumour cells.

The orphan nuclear receptor NR4A1 regulates insulin-like 3 gene transcription in Leydig cells.

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.