Ethylene dimethanesulfonate effects on gene promoter activities related to the endocrine function of immortalized Leydig cell lines R2C and MA-10.

Ethylene dimethanesulfonate (EDS) is a molecule with known selective cytotoxicity on adult Leydig cells. A single intraperitoneal injection in rats but not mice, leads to male androgen deprivation and infertility. In vitro studies using rat and mouse immortalized Leydig cell lines, showed similar effects of cell death promoted by EDS in rat cells as seen in vivo, and suggest that EDS affects gene transcription, which could firstly compromise steroidogenesis before the apoptosis process. Using gene reporter assay, this study aimed to investigate EDS effects on the promoter activity of genes important for endocrine function (Star, Insl3) and response to toxic agents (Gsta3) in immortalized Leydig cell lines (rat R2C and mouse MA-10 cells), as well as identify possible EDS-responsive elements in the Star gene promoter. EDS exposure of R2C and MA-10 Leydig cells increased Gsta3 promoter activity after 4 h of treatment and decreased Insl3 promoter activity only in R2C cells after 24 h of treatment. EDS also decreased Star promoter activity in both Leydig cell lines. Using R2C cells, the EDS-responsive region in the Star promoter was located between -400 and -195 bp. This suggests that this region and the associated transcription factors, which include MEF2, might be targeted by EDS. Additional somatic gonadal cell lines expressing Star were used and EDS did not affect Star promoter activity in DC3 granulosa cells while Star promoter activity was increased in MSC-1 Sertoli cells after 24 h of treatment. This study contributes to the knowledge regarding the mechanism of EDS action in Leydig cells, and in other gonadal cell lineages, and brings new light regarding the rats and mice differential susceptibility to EDS effects, in addition to providing new avenues for experimental approaches to better understand Leydig cell function and dynamics in different rodent species.

ERK5 Cooperates With MEF2C to Regulate Nr4a1 Transcription in MA-10 and MLTC-1 Leydig Cells.

Leydig cells produce hormones required for the development and maintenance of sex characteristics and fertility in males. MEF2 transcription factors are important regulators of Leydig cell gene expression and steroidogenesis. ERK5 is an atypical member of the MAP kinase family that modulates transcription factor activity, either by direct phosphorylation or by acting as a transcriptional coactivator. While MEF2 and ERK5 are known to cooperate transcriptionally, the presence and role of ERK5 in Leydig cells remained unknown. Our goal was to determine whether ERK5 is present in Leydig cells and whether it cooperates with MEF2 to regulate gene expression. We found that ERK5 is present in Leydig cells in testicular tissue and immortalized cell lines. ERK5 knockdown in human chorionic gonadotrophin-treated MA-10 Leydig cells reduced steroidogenesis and decreased Star and Nr4a1 expression. Luciferase assays using a synthetic reporter plasmid containing 3 MEF2 elements revealed that ERK5 enhances MEF2-dependent promoter activation. Although ERK5 did not cooperate with MEF2 on the Star promoter in Leydig cell lines, we found that ERK5 and MEF2C do cooperate on the Nr4a1 promoter, which contains 2 adjacent MEF2 elements. Mutation of each MEF2 element in a short version of the Nr4a1 promoter significantly decreased the ERK5/MEF2C cooperation, indicating that both MEF2 elements need to be intact. The ERK5/MEF2C cooperation did not require phosphorylation of MEF2C on Ser387. Taken together, our data identify ERK5 as a new regulator of MEF2 activity in Leydig cells and provide potential new insights into mechanisms that regulate Leydig cell gene expression and function.

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.

Comprehensive and Quantitative Analysis of the Changes in Proteomic and Phosphoproteomic Profiles during Stimulation and Repression of Steroidogenesis in MA-10 Leydig Cells.

Leydig cells produce testosterone, a hormone essential for male sex differentiation and spermatogenesis. The pituitary hormone, LH, stimulates testosterone production in Leydig cells by increasing the intracellular cAMP levels, which leads to the activation of various kinases and transcription factors, ultimately stimulating the expression of the genes involved in steroidogenesis. The second messenger, cAMP, is subsequently degraded to AMP, and the increase in the intracellular AMP levels activates AMP-dependent protein kinase (AMPK). Activated AMPK potently represses steroidogenesis. Despite the key roles played by the various stimulatory and inhibitory kinases, the proteins phosphorylated by these kinases during steroidogenesis remain poorly characterized. In the present study, we have used a quantitative LC-MS/MS approach, using total and phosphopeptide-enriched proteins to identify the global changes that occur in the proteome and phosphoproteome of MA-10 Leydig cells during both the stimulatory phase (Fsk/cAMP treatment) and inhibitory phase (AICAR-mediated activation of AMPK) of steroidogenesis. The phosphorylation levels of several proteins, including some never before described in Leydig cells, were significantly altered during the stimulation and inhibition of steroidogenesis. Our data also provide new key insights into the finely tuned and dynamic processes that ensure adequate steroid hormone production.

Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells.

Hormone-induced Leydig cell steroidogenesis requires rapid changes in gene expression in response to various hormones, cytokines, and growth factors. These proteins act by binding to their receptors on the surface of Leydig cells leading to activation of multiple intracellular signaling cascades, downstream of which are several kinases, including protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase I (CAMKI), and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). These kinases participate in hormone-induced steroidogenesis by phosphorylating numerous proteins including transcription factors leading to increased steroidogenic gene expression. How these various kinases and transcription factors come together to appropriately induce steroidogenic gene expression in response to specific stimuli remains poorly understood. In the present work, we compared the effect of PKA, CAMKI and ERK1/2 on the transactivation potential of 15 transcription factors belonging to 5 distinct families on the activity of the Star gene promoter. We not only validated known cooperation between kinases and transcription factors, but we also identified novel cooperations that have not yet been before reported. Some transcription factors were found to respond to all three kinases, whereas others were only activated by one specific kinase. Differential responses were also observed within a family of transcription factors. The diverse response to kinases provides flexibility to ensure proper genomic response of steroidogenic cells to different stimuli.

A Short Promoter Region Containing Conserved Regulatory Motifs Is Required for Steroidogenic Acute Regulatory Protein (Star) Gene Expression in the Mouse Testis.

In the testis, Leydig cells produce steroid hormones that are needed to masculinize typical genetic males during fetal development and to initiate and maintain spermatogenesis at puberty and adulthood, respectively. Steroidogenesis is initiated by the transfer of cholesterol from the outer to the inner mitochondrial membrane through the action of steroidogenic acute regulatory protein (STAR). Given its importance for the steroidogenic process, the regulation of STAR gene expression has been the subject of numerous studies. These studies have involved the characterization of key promoter sequences through the identification of relevant transcription factors and the nucleotide motifs (regulatory elements) that they bind. This work has traditionally relied on in vitro studies carried out in cell cultures along with reconstructed promoter sequences. While this approach has been useful for developing models of how a gene might be transcriptionally regulated, one must ultimately validate that these modes of regulation occur in an endogenous context. We have used CRISPR/Cas9 genome editing to modify a short region of the mouse Star promoter (containing a subset of regulatory elements, including conserved CRE, C/EBP, AP1, and GATA motifs) that has been proposed to be critical for Star transcription. Analysis of the resultant mutant mice showed that this short promoter region is indeed required for maximal STAR mRNA and protein levels in the testis. Analysis also showed that both basal and hormone-activated testosterone production in mature mice was unaffected despite significant changes in Star expression. Our results therefore provide the first in vivo validation of regulatory sequences required for Star gene expression.

Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti-Müllerian hormone receptor type 2 transcription in mouse MA-10 Leydig cells.

BACKGROUND: Leydig cells produce testosterone and insulin-like 3, two hormones essential for male sex differentiation and reproductive function. The orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor type II (COUP-TFII), and the zinc finger factor GATA4 are two transcription factors involved in Leydig cell differentiation, gene expression, and function. OBJECTIVES: Several Leydig cell gene promoters contain binding motifs for both GATA factors and nuclear receptors. The goal of the present study is to determine whether GATA4 and COUP-TFII cooperate to regulate gene expression in Leydig cells. MATERIALS AND METHODS: The transcriptomes from GATA4- and COUP-TFII-depleted MA-10 Leydig cells were analyzed using bioinformatic tools. Functional cooperation between GATA4 and COUP-TFII, and other related family members, was assessed by transient transfections in Leydig (MA-10 and MLTC-1) and fibroblast (CV-1) cell lines on several gene promoters. Recruitment of GATA4 and COUP-TFII to gene promoters was investigated by chromatin immunoprecipitation. Co-immunoprecipitation was used to determine whether GATA4 and COUP-TFII interact in MA-10 Leydig cells. RESULTS: Transcriptomic analyses of GATA4- and COUP-TFII-depleted MA-10 Leydig cells revealed 44 commonly regulated genes including the anti-Müllerian hormone receptor type (Amhr2) gene. GATA4 and COUP-TFII independently activated the Amhr2 promoter, and their combination led to a stronger activation. A GC-rich element, located in the proximal Amhr2 promoter was found to be essential for GATA4- and COUP-TFII-dependent activation as well as for the COUP-TFII/GATA4 cooperation. COUP-TFII and GATA4 directly interacted in MA-10 Leydig cell extracts. Chromatin immunoprecipitation revealed that GATA4 and COUP-TFII are recruited to the proximal Amhr2 promoter, which contains binding sites for both factors in addition to the GC-rich element. Cooperation between COUP-TFII and GATA6, but not GATA1 and GATA3, was also observed. DISCUSSION AND CONCLUSION: Our results establish the importance of physical and functional cooperation between COUP-TFII/GATA4 in the regulation of gene expression in MA-10 Leydig cells, and more specifically the Amhr2 gene.

Insights Into the Roles of GATA Factors in Mammalian Testis Development and the Control of Fetal Testis Gene Expression.

Defining how genes get turned on and off in a correct spatiotemporal manner is integral to our understanding of the development, differentiation, and function of different cell types in both health and disease. Testis development and subsequent male sex differentiation of the XY fetus are well-orchestrated processes that require an intricate network of cell-cell communication and hormonal signals that must be properly interpreted at the genomic level. Transcription factors are at the forefront for translating these signals into a coordinated genomic response. The GATA family of transcriptional regulators were first described as essential regulators of hematopoietic cell differentiation and heart morphogenesis but are now known to impact the development and function of a multitude of tissues and cell types. The mammalian testis is no exception where GATA factors play essential roles in directing the expression of genes crucial not only for testis differentiation but also testis function in the developing male fetus and later in adulthood. This minireview provides an overview of the current state of knowledge of GATA factors in the male gonad with a particular emphasis on their mechanisms of action in the control of testis development, gene expression in the fetal testis, testicular disease, and XY sex differentiation in humans.

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.

Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function.

Cell differentiation and acquisition of specialized functions are inherent steps in events that lead to normal tissue development and function. These processes require accurate temporal, tissue, and cell-specific activation or repression of gene transcription. This is achieved by complex interactions between transcription factors that form a unique combinatorial code in each specialized cell type and in response to different physiological signals. Transcription factors typically act by binding to short, nucleotide-specific DNA sequences located in the promoter region of target genes. In males, Leydig cells play a crucial role in sex differentiation, health, and reproductive function from embryonic life to adulthood. To better understand the molecular mechanisms regulating Leydig cell differentiation and function, several transcription factors important to Leydig cells have been identified, including some previously unknown to this specialized cell type. This mini review summarizes the current knowledge on transcription factors in fetal and adult Leydig cells, describing their roles and mechanisms of action.

Mechanism of Action of an Environmentally Relevant Organochlorine Mixture in Repressing Steroid Hormone Biosynthesis in Leydig Cells.

Within Leydig cells, steroidogenesis is induced by the pituitary luteinizing hormone (LH). The binding of LH to its receptor increases cAMP production, which then activates the expression of genes involved in testosterone biosynthesis. One of these genes codes for the steroidogenic acute regulatory (STAR) protein. STAR is part of a complex that shuttles cholesterol, the precursor of all steroid hormones, through the mitochondrial membrane where steroidogenesis is initiated. Organochlorine chemicals (OCs) are environmental persistent organic pollutants that are found at high concentrations in Arctic areas. OCs are known to affect male reproductive health by decreasing semen quality in different species, including humans. We previously showed that an environmentally relevant mixture of OCs found in Northern Quebec disrupts steroidogenesis by decreasing STAR protein levels without affecting the transcription of the gene. We hypothesized that OCs might affect STAR protein stability. To test this, MA-10 Leydig cell lines were incubated for 6 h with vehicle or the OCs mixture in the presence or absence of 8Br-cAMP with or without MG132, an inhibitor of protein degradation. We found that MG132 prevented the OC-mediated decrease in STAR protein levels following 8Br-cAMP stimulation. However, progesterone production was still decreased by the OC mixture, even in the presence of MG132. This suggested that proteins involved in steroid hormone production in addition to STAR are also affected by the OC mixture. To identify these proteins, a whole cell approach was used and total proteins from MA-10 Leydig cells exposed to the OC mixture with or without stimulation with 8Br-cAMP were analyzed by 2D SDS-PAGE and LC-MS/MS. Bioinformatics analyses revealed that several proteins involved in numerous biological processes are affected by the OC mixture, including proteins involved in mitochondrial transport, lipid metabolism, and steroidogenesis.

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.

Mechanisms of MEHP Inhibitory Action and Analysis of Potential Replacement Plasticizers on Leydig Cell Steroidogenesis.

Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.

Identification of novel genes and pathways regulated by the orphan nuclear receptor COUP-TFII in mouse MA-10 Leydig cells†.

In males, Leydig cells are the main producers of testosterone and insulin-like 3 (INSL3), two hormones essential for sex differentiation and reproductive functions. Chicken ovalbumin upstream promoter-transcription factors I (COUP-TFI/NR2F1) and COUP-TFII (NR2F2) belong to the steroid/thyroid hormone nuclear receptor superfamily of transcription factors. In the testis, COUP-TFII is expressed and plays a role in the differentiation of cells committed to give rise to fully functional steroidogenic adult Leydig cells. Steroid production has also been shown to be diminished in COUP-TFII-depleted Leydig cells, indicating an important functional role in steroidogenesis. Until now, only a handful of target genes have been identified for COUP-TFII in Leydig cells. To provide new information into the mechanism of action of COUP-TFII in Leydig cells, we performed microarray analyses of COUP-TFII-depleted MA-10 Leydig cells. We identified 262 differentially expressed genes in COUP-TFII-depleted MA-10 cells. Many of the differentially expressed genes are known to be involved in lipid biosynthesis, lipid metabolism, male gonad development, and steroidogenesis. We validated the microarray data for a subset of the modulated genes by RT-qPCR. Downregulated genes included hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), prolactin receptor (Prlr), nuclear receptor subfamily 0, group B, member 2 (Shp/Nr0b2), ferredoxin 1 (Fdx1), scavenger receptor class B, member 1 (Scarb1), inhibin alpha (Inha), and glutathione S-transferase, alpha 3 (Gsta3). Finally, analysis of the Gsta3 and Inha gene promoters showed that at least two of the downregulated genes are potentially new direct targets for COUP-TFII. These data provide new evidence that further strengthens the important nature of COUP-TFII in steroidogenesis, androgen homeostasis, cellular defense, and differentiation in mouse Leydig cells.

The Nuclear Receptor COUP-TFII Regulates Amhr2 Gene Transcription via a GC-Rich Promoter Element in Mouse Leydig Cells.

The nuclear receptor chicken ovalbumin upstream promoter-transcription factor type II (COUP-TFII)/NR2F2 is expressed in adult Leydig cells, and conditional deletion of the Coup-tfii/Nr2f2 gene impedes their differentiation. Steroid production is also reduced in COUP-TFII-depleted Leydig cells, supporting an additional role in steroidogenesis for this transcription factor. COUP-TFII action in Leydig cells remains to be fully characterized. In the present work, we report that COUP-TFII is an essential regulator of the gene encoding the anti-Müllerian hormone receptor type 2 (Amhr2), which participates in Leydig cell differentiation and steroidogenesis. We found that Amhr2 mRNA levels are reduced in COUP-TFII-depleted MA-10 Leydig cells. Consistent with this, COUP-TFII directly activates a -1486 bp fragment of the mouse Amhr2 promoter in transient transfection assays. The COUP-TFII responsive region was localized between -67 and -34 bp. Chromatin immunoprecipitation assay confirmed COUP-TFII recruitment to the proximal Amhr2 promoter whereas DNA precipitation assay revealed that COUP-TFII associates with the -67/-34 bp region in vitro. Even though the -67/-34 bp region contains an imperfect nuclear receptor element, COUP-TFII-mediated activation of the Amhr2 promoter requires a GC-rich sequence at -39 bp known to bind the specificity protein (SP)1 transcription factor. COUP-TFII transcriptionally cooperates with SP1 on the Amhr2 promoter. Mutations that altered the GCGGGGCGG sequence at -39 bp abolished COUP-TFII-mediated activation, COUP-TFII/SP1 cooperation, and reduced COUP-TFII binding to the proximal Amhr2 promoter. Our data provide a better understanding of the mechanism of COUP-TFII action in Leydig cells through the identification and regulation of the Amhr2 promoter as a novel target.

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.

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.

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.

Molecular regulation of steroidogenesis in endocrine Leydig cells.

Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. Consequently, the process of steroid hormone biosynthesis is finely regulated. In the testis, the main steroidogenic cells are the Leydig cells. There are two distinct populations of Leydig cells that arise during development: fetal and adult Leydig cells. Fetal Leydig cells are responsible for masculinizing the male urogenital tract and inducing testis descent. These cells atrophy shortly after birth and do not contribute to the adult Leydig cell population. Adult Leydig cells derive from undifferentiated precursors present after birth and become fully steroidogenic at puberty. The differentiation of both Leydig cell populations is controlled by locally produced paracrine factors and by endocrine hormones. In fully differentially and steroidogenically active Leydig cells, androgen production and hormone-responsiveness involve various signaling pathways and downstream transcription factors. This review article focuses on recent developments regarding the origin and function of Leydig cells, the regulation of their differentiation by signaling molecules, hormones, and structural changes, the signaling pathways, kinases, and transcription factors involved in their differentiation and in mediating LH-responsiveness, as well as the fine-tuning mechanisms that ensure adequate production steroid hormones.