Nuclear Receptor SF-1 Research Articles

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

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. 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.

Lrh1 can help reprogram sexual cell fate and is required for Sertoli cell development and spermatogenesis in the mouse testis.

The mammalian nuclear hormone receptors LRH1 (NR5A2) and SF1 (NR5A1) are close paralogs that can bind the same DNA motif and play crucial roles in gonadal development and function. Lrh1 is essential for follicle development in the ovary and has been proposed to regulate steroidogenesis in the testis. Lrh1 expression in the testis is highly elevated by loss of the sex regulator Dmrt1, which triggers male-to-female transdifferentiation of Sertoli cells. While Sf1 has a well-defined and crucial role in testis development, no function for Lrh1 in the male gonad has been reported. Here we use conditional genetics to examine Lrh1 requirements both in gonadal cell fate reprogramming and in normal development of the three major cell lineages of the mouse testis. We find that loss of Lrh1 suppresses sexual transdifferentiation, confirming that Lrh1 can act as a key driver in reprogramming sexual cell fate. In otherwise wild-type testes, we find that Lrh1 is dispensable in Leydig cells but is required in Sertoli cells for their proliferation, for seminiferous tubule morphogenesis, for maintenance of the blood-testis barrier, for feedback regulation of androgen production, and for support of spermatogenesis. Expression profiling identified misexpressed genes likely underlying most aspects of the Sertoli cell phenotype. In the germ line we found that Lrh1 is required for maintenance of functional spermatogonia, and hence mutants progressively lose spermatogenesis. Reduced expression of the RNA binding factor Nxf2 likely contributes to the SSC defect. Unexpectedly, however, over time the Lrh1 mutant germ line recovered abundant spermatogenesis and fertility. This finding indicates that severe germ line depletion triggers a response allowing mutant spermatogonia to recover the ability to undergo complete spermatogenesis. Our results demonstrate that Lrh1, like Sf1, is an essential regulator of testis development and function but has a very distinct repertoire of functions.

Inhibition of Luman/CREB3 expression leads to the upregulation of testosterone synthesis in mouse Leydig cells.

Luman, also known as cAMP-response element-binding protein 3, is an endoplasmic reticulum stress-related protein that has been identified as a novel transcriptional coregulator of a variety of nuclear receptors. Herein, immunohistochemistry results showed that Luman was specifically expressed in mouse Leydig cells in an age-dependent increase manner, from prepuberty to sexual maturation. Luman was not detected in Sertoli cells within the seminiferous tubules at any developmental period. The immunofluorescent experiment indicated that Luman was mainly located within the cytoplasm of murine Leydig tumor cells (MLTC-1) and primary Leydig cells (PLCs). To investigate the physiological function of Luman, experiments were conducted to examine the consequences of short hairpin RNA- and small interfering RNA-mediated Luman knock-down in MLTC-1 and PLCs, respectively. Luman knock-down significantly upregulated the expression of steroidogenic acute regulatory, cytochrome P450 cholesterol side-chain cleavage enzymes, 3β-hydroxysteroid dehydrogenase, and 17-α-hydroxylase/C17-20 lyase in MLTC-1 cells and PLCs. Luman knock-down caused an increase in human chorionic gonadotropin-stimulated testosterone production in vitro and in vivo. The nuclear receptors SF-1 and Nur-77 were significantly increased upon Luman knock-down in MLTC-1. By contrast, the level of the nuclear receptor SHP decreased. Luciferase reporter assay results demonstrated that Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters. These data suggested that Luman expressed in mouse Leydig cells in an age-dependent increase manner. Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters, which lead to the increase of testosterone synthesis and steroidogenesis genes expression. In conclusion, these findings provide us with new insights into the role Luman played in male reproduction.

Theca Cell INSL3 and Steroids Together Orchestrate the Growing Bovine Antral Follicle.

Insulin-like peptide 3 (INSL3) and its specific receptor RXFP2 are both expressed by theca interna cells of the growing antral follicle where they form an essential regulatory element in the production of the steroid precursor androstenedione. Using primary cultures of bovine theca cells from the mid follicular phase together with steroid agonists and antagonists we have examined how ovarian steroids modulate INSL3 expression. Transcript analysis shows that these cells express estrogen receptors α and β, androgen and progesterone receptors, besides the orphan nuclear receptors SF1 and nur77. Whereas, exogenous androgens have little or no effect, the androgen antagonist bicalutamide stimulates INSL3 production. In contrast, estrogen receptor agonists, as also progesterone, are stimulatory. Importantly, estrogen receptor signaling is convergent with the protein kinase A signaling pathway activated by LH, such that the estrogen receptor antagonist can inhibit the mild stimulatory effect of LH, and vice versa the PKA antagonist H89 blocks stimulation by estradiol. A significant finding is that the major steroid metabolite androstenedione appears to act predominantly as an estrogen and not an androgen in this system. Transfection of INSL3 gene promoter-reporter constructs together with various steroid receptor expression plasmids supports these findings and shows that steroid action uses non-classical pathways not requiring canonical steroid-responsive elements in the proximal promoter region. Together, the results indicate that increasing estrogens in the follicular phase stimulate a feedforward loop driving INSL3 signaling and thereby promoting steroidogenesis in the growing antral follicle until the LH surge which effectively switches off INSL3 expression.

Transcription factors SF1 and cJUN cooperate to activate the Fdx1 promoter in MA-10 Leydig cells

The Ferredoxin 1 (FDX1) protein supports steroid biosynthesis in steroidogenic cells through electron transfer to the rate-limiting steroidogenic enzyme, CYP11A1. The latter catalyzes the conversion of cholesterol to pregnenolone through side chain cleavage inside the mitochondria. Thus far, only several transcription factors have been implicated in the regulation of mouse Fdx1 promoter activity in Leydig cells. These include the nuclear receptor SF1 and SP1. Since two conserved regulatory elements for AP1 transcription factors have been located at −764 and −617bp of the Fdx1 promoter, we hypothesized that cJUN may cooperate with other partners to regulate Fdx1 in Leydig cells. Indeed, we report that SF1 and cJUN interact and cooperate to activate the Fdx1 promoter in MA-10 and TM3 Leydig cells. Furthermore, we found that such activation requires different regulatory elements located between −124 and −306bp of the Fdx1 promoter and involves recruitment of SF1 to this region. Using RNA interference, the importance of SF1 in transcriptional regulation of Fdx1 was confirmed, whereas cJUN was dispensable even though it cooperated with SF1 to upregulate Fdx1 expression in MA-10 cells. Thus, our data provides new insights in the molecular mechanisms that control mouse Fdx1 transcription, possibly leading to regulation of CYP11A1 enzyme activation, in Leydig cells.

Molecular mechanisms regulated by the histone methyltransferase EZH2 in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a highly aggressive cancer. Although surgical resection remains the treatment of choice in localized ACC, clinical management of metastatic disease is ineffective. It is thus essential to identify the actors of malignant ACC progression to allow development of more effective therapeutic strategies. Deregulation of epigenetic factors is frequently involved in tumor progression. We have recently shown that the histone methyltransferase EZH2 is the most deregulated epigenetic histone modifier in ACC. EZH2 is over expressed downstream of TP53/RB1/E2F1 and is associated with tumor proliferation and poor prognosis. Pharmacological inhibition of EZH2 inhibits H295R cells growth and aggressive behaviour and induces apoptosis. This suggests that EZH2 is involved in malignant progression and could represent an interesting therapeutic target. Expression of the pro-apoptotic factor NOV/CCN3 is decreased in ACC, which is correlated with poor prognosis. Our molecular analyses show that EZH2 inhibition by siRNA or pharmacological treatment with DZNep increases expression of NOV/CCN3, suggesting that EZH2 over expression may favour malignant progression in ACC by inhibition of apoptosis stimulators. NOV has previously been identified as a negative target of the nuclear receptor SF1 in ACC cells, although the molecular mechanisms underlying this inhibition were unidentified. Interestingly, in prostate cancer, NOV expression is inhibited by the androgen receptor, through recruitment of EZH2 and deposition of the H3K27me3 mark. We are now conducting a combination of transfection and ChIP experiments to evaluate a similar cooperation between SF1 and EZH2 to repress NOV expression and block apoptosis in ACC.

Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis.

Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10μM GEN+10μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN+MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10μM combined GEN+MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN+MEHP. We propose a working model for GEN+MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally relevant doses.

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.

Molecular Characterization of dax1 and SF-1 and Their Expression Analysis During Sex Reversal in Spotted Scat, Scatophagus argus

The nuclear receptors dax1 and SF-1 play important and antagonistic roles in gonad differentiation in mammals. To study their potential functions in teleost fish, the full-length cDNAs of dax1 and SF-1 were cloned and characterized from spotted scat. Their expression patterns in adult tissues and juveniles treated with 17α-methyltestosterone (MT) and their gonadal histological changes were investigated, respectively. The tissue-specific expression results showed that dax1 was abundantly expressed in the gonads, spleen, brain, and intestine. SF-1 was preferentially expressed in the gonad and spleen. Histological results showed that sex reversal could be induced by MT, and the degenerating oocytes and proliferations of spermatogonia were observed in gonad. At the beginning of sex reversal (40 d after treatment), the mRNA level of dax1 significantly increased, and the transcript level of SF-1 mRNA was inhibited at the point of 20 d following treatment with MT. Our results suggested that masculinization could be induced by MT treatment in spotted scat, and MT could upregulate the dax1 expression, whereas it downregulates the expression of SF-1, which implied that dax1 and SF-1 might be involved in the gonad development from female to male and there was an antagonistic effect between dax1 and SF-1.

The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1.

The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.

Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours

Granulosa cell tumours of the ovary (GCT) express aromatase and produce oestrogens. The ovarian-specific aromatase promoter (pII) is regulated by members of the group 5A nuclear receptor family, SF-1 and LRH-1. Since both SF-1 and LRH-1 are implicated in proliferation and cancer, we hypothesised that alteration in the expression of either or both receptors may be associated with GCT. We therefore determined the expression of LRH-1, SF-1 and aromatase in a cohort of GCT, mucinous and serous cystadenocarcinomas, and normal ovaries. LRH-1 mRNA was present at low level in normal ovary and serous cystadenocarcinoma, but was elevated approximately 30-fold in GCT, and 8-fold in mucinous cystadenocarcinoma, compared to normal ovary. LRH-1 protein expression was confirmed in GCT by immunohistochemistry. SF-1 mRNA was significantly lower that of LRH-1 in all samples and not significantly altered in GCT, compared to normal ovary. Aromatase mRNA was present at low level in normal ovary and serous and mucinous cystadenocarcinoma, and significantly elevated (18-fold) in GCT compared to normal ovary. Despite the coordinate over-expression of both LRH-1 and aromatase in GCT versus normal ovary, their levels did not correlate in individual patients; rather, aromatase expression correlated with that of SF-1. Finally, although both LRH-1 and SF-1 activated aromatase promoter activity in transient transfection studies, gel-shift and chromatin immunoprecipitation data indicated that SF-1, but not LRH-1, bound to the aromatase promoter. We conclude that SF-1 regulates aromatase expression in GCT; over-expression of LRH-1 suggests that this receptor may be involved in the pathogenesis of GCT by mechanisms other than the regulation of aromatase. Its role in this disease therefore warrants further investigation.

In vivo evidence for the crucial role of SF1 in steroid-producing cells of the testis, ovary and adrenal gland

Adrenal and gonadal steroids are essential for life and reproduction. The orphan nuclear receptor SF1 (NR5A1) has been shown to regulate the expression of enzymes involved in steroid production in vitro. However, the in vivo role of this transcription factor in steroidogenesis has not been elucidated. In this study, we have generated steroidogenic-specific Cre-expressing mice to lineage mark and delete Sf1 in differentiated steroid-producing cells of the testis, the ovary and the adrenal gland. Our data show that SF1 is a regulator of the expression of steroidogenic genes in all three organs. In addition, Sf1 deletion leads to a radical change in cell morphology and loss of identity. Surprisingly, sexual development and reproduction in mutant animals were not compromised owing, in part, to the presence of a small proportion of SF1-positive cells. In contrast to the testis and ovary, the mutant adult adrenal gland showed a lack of Sf1-deleted cells and our studies suggest that steroidogenic adrenal cells during foetal stages require Sf1 to give rise to the adult adrenal population. This study is the first to show the in vivo requirements of SF1 in steroidogenesis and provides novel data on the cellular consequences of the loss of this protein specifically within steroid-producing cells.

The AP-1 family member FOS blocks transcriptional activity of the nuclear receptor steroidogenic factor 1

Steroid production in the adrenal zona glomerulosa is under the control of angiotensin II (Ang II), which, upon binding to its receptor, activates protein kinase C (PKC) within these cells. PKC is a potent inhibitor of the steroidogenic enzyme CYP17. We have demonstrated that, in the ovary, PKC activates expression of FOS, a member of the AP-1 family, and increased expression of this gene is linked to CYP17 downregulation. However, the pathway and the molecular mechanism responsible for the inhibitory effect of PKC on CYP17 expression are not defined. Herein, we demonstrated that Ang II inhibited CYP17 through PKC and ERK1/2-activated FOS and that blocking FOS expression decreased PKC-mediated inhibition. Although CYP17 transcription was activated by the nuclear receptor SF-1, expression of FOS resulted in a decrease in SF-1-mediated gene transcription. FOS physically interacted with the hinge region of SF-1 and modulated its transactivity, thus preventing binding of cofactors such as SRC1 and CBP, which were necessary to fully activate CYP17 transcription. Collectively, these results indicate a new regulatory mechanism for SF-1 transcriptional activity that might influence adrenal zone-specific expression of CYP17, a mechanism that can potentially be applied to other steroidogenic tissues.

Stimulating the GPR30 Estrogen Receptor with a Novel Tamoxifen Analogue Activates SF-1 and Promotes Endometrial Cell Proliferation

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.

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.

Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

There are increasing evidence in the literature reporting the detrimental effects of endocrine disruptors on the development and function of the male reproductive system. One example is cryptorchidism, or undescended testis, caused by exposure to excessive estrogens. Estrogens, acting through the estrogen receptor α (ERα), have been shown to repress expression of the gene encoding insulin-like 3 (INSL3), a small peptide produced by testicular Leydig cells that is essential for normal testis descent. The molecular mechanism of estrogen/ER action on Insl3 expression, however, remains poorly understood. Here we report estradiol (E 2) represses Insl3 mRNA levels in MA-10 cells, a Leydig cell line model. We also found that E 2 represses the activity of the human and mouse Insl3 promoter in these cells. The E 2-responsive region of the human INSL3 promoter was located to the proximal INSL3 promoter. This region does not contain a consensus estrogen response element indicating an indirect mechanism of action. In agreement with this, we found that E 2-responsiveness was lost when two previously characterized binding sites for the nuclear receptors NUR77 and SF1 were mutated. Finally we show that the E 2 repressive effect could be overcome by cotreatment with testosterone, a positive regulator of Insl3 transcription. Collectively our data provide important new insights into the molecular mechanism of estrogen action in Insl3 transcription in Leydig cells.

Steroidogenic factor-1 and endometriosis

Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE(2) induces the expression of the genes that encode these enzymes. Upon PGE(2) treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE(2) by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women.

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 nuclear receptors NUR77 and SF1 play additive roles with c-JUN through distinct elements on the mouse Star promoter

The steroidogenic acute regulatory protein plays an essential role in steroid biosynthesis in steroidogenic cells. It is involved in the transport of cholesterol through the mitochondrial membrane where the first step of steroidogenesis occurs. Star gene expression in testicular Leydig cells is regulated by the pituitary LH through the cAMP signaling pathway. So far, several transcription factors have been implicated in the regulation of Star promoter activity in these cells. These include the nuclear receptors NUR77 and SF1, AP-1 family members (particularly c-JUN), GATA4, C/EBPbeta, DLX5/6, and CREB. Some of these factors were also shown to act in a cooperative manner to further enhance Star promoter activity. Here, we report that NUR77 and c-JUN have additive effects on the Star promoter. These effects were abolished only when both elements, NUR77 at -95 bp and AP-1 at -78 bp, were mutated. Consistent with this, in vitro co-immunoprecipitation revealed that NUR77 and c-JUN interact and that this interaction is mediated through part of the ligand binding domain of NUR77. Furthermore, we found that SF1 could cooperate with c-JUN on the mouse Star promoter but this cooperation involved different regulatory elements. Collectively, our data not only provide new insights into the molecular mechanisms that control mouse Star transcription in Leydig cells but also reveal a novel mechanism for the regulation of NR4A1-dependent genes in tissues where NUR77 and c-JUN factors are co-expressed.