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.

Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.

The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.

The Delta-Opioid Receptor; a Target for the Treatment of Pain.

Nowadays, pain represents one of the most important societal burdens. Current treatments are, however, too often ineffective and/or accompanied by debilitating unwanted effects for patients dealing with chronic pain. Indeed, the prototypical opioid morphine, as many other strong analgesics, shows harmful unwanted effects including respiratory depression and constipation, and also produces tolerance, physical dependence, and addiction. The urgency to develop novel treatments against pain while minimizing adverse effects is therefore crucial. Over the years, the delta-opioid receptor (DOP) has emerged as a promising target for the development of new pain therapies. Indeed, targeting DOP to treat chronic pain represents a timely alternative to existing drugs, given the weak unwanted effects spectrum of DOP agonists. Here, we review the current knowledge supporting a role for DOP and its agonists for the treatment of pain. More specifically, we will focus on the cellular and subcellular localization of DOP in the nervous system. We will also discuss in further detail the molecular and cellular mechanisms involved in controlling the cellular trafficking of DOP, known to differ significantly from most G protein-coupled receptors. This review article will allow a better understanding of how DOP represents a promising target to develop new treatments for pain management as well as where we stand as of our ability to control its cellular trafficking and cell surface expression.

In vivo mapping of a GPCR interactome using knockin mice.

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

In Vivo Ablation of the Conserved GATA-Binding Motif in the Amh Promoter Impairs Amh Expression in the Male Mouse.

GATA4 is an essential transcriptional regulator required for gonadal development, differentiation, and function. In the developing testis, proposed GATA4-regulated genes include steroidogenic factor 1 (Nr5a1), SRY-related HMG box 9 (Sox9), and anti-Müllerian hormone (Amh). Although some of these genes have been validated as genuine GATA4 targets, it remains unclear whether GATA4 is a direct regulator of endogenous Amh transcription. We used a CRISPR/Cas9-based approach to specifically inactivate or delete the sole GATA-binding motif of the proximal mouse Amh promoter. AMH mRNA and protein levels were assessed at developmental time points corresponding to elevated AMH levels: fetal and neonate testes in males and adult ovaries in females. In males, loss of GATA binding to the Amh promoter significantly reduced Amh expression. Although the loss of GATA binding did not block the initiation of Amh transcription, AMH mRNA and protein levels failed to upregulate in the developing fetal and neonate testis. Interestingly, adult male mice presented no anatomical anomalies and had no evidence of retained Müllerian duct structures, suggesting that AMH levels, although markedly reduced, were sufficient to masculinize the male embryo. In contrast to males, GATA binding to the Amh promoter was dispensable for Amh expression in the adult ovary. These results provide conclusive evidence that in males, GATA4 is a positive modulator of Amh expression that works in concert with other key transcription factors to ensure that the Amh gene is sufficiently expressed in a correct spatiotemporal manner during fetal and prepubertal testis development.

Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents.

Genetically-modified animal models have significantly increased our understanding of the complex central nervous system circuits. Among these models, inducible transgenic mice whose specific gene expression can be modulated through a Cre recombinase/LoxP system are useful to study the role of specific peptides and proteins in a given population of cells. In the present study, we describe an efficient approach to selectively deliver a Cre-GFP to dorsal root ganglia (DRG) neurons. First, mice of different ages were injected in both hindpaws with a recombinant adeno-associated virus (rAAV2/9-CBA-Cre-GFP). Using this route of injection in mice at 5 days of age, we report that approximately 20% of all DRG neurons express GFP, 6 to 8 weeks after the infection. The level of infection was reduced by 50% when the virus was administered at 2 weeks of age. Additionally, the virus-mediated delivery of the Cre-GFP was also investigated via the intrathecal route. When injected intrathecally, the rAAV2/9-CBA-Cre-GFP virus infected a much higher proportion of DRG neurons than the intraplantar injection, with up to 51.6% of infected lumbar DRG neurons. Noteworthy, both routes of injection predominantly transduced DRG neurons over spinal and brain neurons.

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.

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.

The Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells.

Testosterone is essential for spermatogenesis and the development of male sexual characteristics. However, steroidogenesis produces a significant amount of reactive oxygen species (ROS), which can disrupt testosterone production. The myocyte enhancer factor 2 (MEF2) is an important regulator of organogenesis and cell differentiation in various tissues. In the testis, MEF2 is present in Sertoli and Leydig cells throughout fetal and adult life. MEF2-deficient MA-10 Leydig cells exhibit a significant decrease in steroidogenesis concomitant with a reduction in glutathione S-transferase (GST) activity and in the expression of the 4 Gsta members (GST) that encode ROS inactivating enzymes. Here, we report a novel role for MEF2 in ROS detoxification by directly regulating Gsta expression in Leydig cells. Endogenous Gsta1-4 mRNA levels were decreased in MEF2-deficient MA-10 Leydig cells. Conversely, overexpression of MEF2 increased endogenous Gsta1 levels. MEF2 recruitment to the proximal Gsta1 promoter and direct binding on the -506-bp MEF2 element were confirmed by chromatin immunoprecipitation and DNA precipitation assays. In MA-10 Leydig cells, MEF2 activates the Gsta1 promoter and cooperates with Ca(2+)/calmodulin-dependent kinases I to further enhance Gsta1 promoter activity. These effects were lost when the -506-bp MEF2 element was mutated or when a MEF2-Engrailed dominant negative protein was used. Similar results were obtained on the Gsta2, Gsta3, and Gsta4 promoters, suggesting a global role for MEF2 factors in the regulation of all 4 Gsta genes. Altogether, our results identify a novel role for MEF2 in the expression of genes involved in ROS detoxification, a process essential for adequate testosterone production in Leydig cells.

Novel Targets for the Transcription Factors MEF2 in MA-10 Leydig Cells.

Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) are transcription factors recently identified in somatic cells of the male gonad. In other tissues, MEF2 factors are essential regulators of organogenesis and cell differentiation. So far in the testis, MEF2 factors were found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MEF2-depleted MA-10 Leydig cells, and the results were analyzed using Partek and Ingenuity Pathway Analysis software. Several genes were differentially expressed in MEF2-depleted Leydig cells, and 16 were validated by quantitative RT-PCR. A large number of these genes are known to be involved in fertility, gonad morphology, and steroidogenesis. These include Ahr, Bmal1, Cyp1b1, Hsd3b1, Hsd17b7, Map2k1, Nr0b2, Pde8a, Por, Smad4, Star, and Tsc22d3, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2-binding sites within the first 2 kb upstream of the transcription start site of the Por, Bmal1, and Nr0b2 promoters, suggesting direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, small interfering RNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1, and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction.

GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program.

A cell-autonomous molecular cascade initiated by AMP-activated protein kinase represses steroidogenesis.

Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.

GATA4 autoregulates its own expression in mouse gonadal cells via its distal 1b promoter.

Transcription factor GATA4 is required for the development and function of the mammalian gonads. We first reported that the GATA4 gene in both human and rodents is expressed as two major alternative transcripts that differ solely in their first untranslated exon (exon 1a vs. exon 1b). We had also showed by quantitative PCR that in mouse tissues, both Gata4 exon 1a- and 1b-containing transcripts are present in all sites that are normally positive for GATA4 protein. In adult tissues, exon 1a-containing transcripts generally predominate. A notable exception, however, is the testis where the Gata4 exon 1a and 1b transcripts exhibit a similar level of expression. We now confirm by in situ hybridization analysis that each transcript is also strongly expressed during gonad differentiation in both sexes in the rat. To gain further insights into how Gata4 gene expression is controlled, we characterized the mouse Gata4 promoter sequence located upstream of exon 1b. In vitro studies revealed that the Gata4 1b promoter is less active than the 1a promoter in several gonadal cell lines tested. Whereas we have previously shown that endogenous Gata4 transcription driven by the 1a promoter is dependent on a proximally located Ebox motif, we now show using complementary in vitro and in vivo approaches that Gata4 promoter 1b-directed expression is regulated by GATA4 itself. Thus, Gata4 transcription in the gonads and other tissues is ensured by distinct promoters that are regulated differentially and independently.

Functional cooperation between GATA factors and cJUN on the star promoter in MA-10 Leydig cells.

Steroid hormone biosynthesis requires the steroidogenic acute regulatory protein (STAR). STAR is part of a protein complex that transports cholesterol through the mitochondrial membrane where steroidogenesis begins. Several transcription factors participate to direct the proper spatiotemporal and hormonal regulation of the Star gene in Leydig cells. Mechanistically, this is believed to involve the functional interplay between many of these factors. Here we report a novel transcriptional cooperation between GATA factors and cJUN on the mouse Star and human STAR promoters in MA-10 Leydig cells. This cooperation was observed with different GATA members (GATA1, 4, and 6), whereas only cJUN could cooperate with GATA factors. GATA/cJUN transcriptional cooperation on the Star promoter is mediated via closely juxtaposed GATA and AP-1 binding motifs. Mutation of all functional GATA and cJUN elements abolished GATA/cJUN cooperation, which is in agreement with previous data reporting a direct interaction between GATA4 and cJUN in a heterologous system. These data add valuable new insights that further define the molecular mechanisms that govern Star transcription in steroidogenic cells of the testis.

Transcription of platelet-derived growth factor receptor α in Leydig cells involves specificity protein 1 and 3.

Platelet-derived growth factor (PDGF) A is secreted by Sertoli cells and acts on Leydig precursor cells, which express the receptor PDGFRA, triggering their differentiation into steroidogenically active Leydig cells. There is, however, no information regarding the molecular mechanisms that govern Pdgfra expression in Leydig cells. In this study, we isolated and characterized a 2.2 kb fragment of the rat Pdgfra 5'-flanking sequence in the TM3 Leydig cell line, which endogenously expresses Pdgfra. A series of 5' progressive deletions of the Pdgfra promoter was generated and transfected in TM3 cells. Using this approach, two regions (-183/-154 and -154/-105), each conferring 46% of Pdgfra promoter activity, were identified. To better define the regulatory elements, trinucleotide mutations spanning the -154/-105 region were introduced by site-directed mutagenesis in the context of the -2.2  kb Pdgfra promoter. Mutations that altered the TCCGAGGGAAAC sequence at -138  bp significantly decreased Pdgfra promoter activity in TM3 cells. Several proteins from TM3 nuclear extracts were found to bind to this G(C/A) motif in electromobility shift assay. Two of the proteins were identified as the transcription factors SP1 and SP3. Using transient transfections of TM3 Leydig cells, SP1 and SP3 were found to activate the Pdgfra promoter by threefold. The SP1/SP3-dependent activation of the Pdgfra promoter was severely blunted when the G(C/A) motif was mutated. Our study provides new insights into the regulatory mechanisms of Pdgfra transcription in Leydig cells, which includes a role for the transcription factors SP1 and SP3.

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.