Mouse Leydig MA-10 Cells Research Articles

Investigating the influence of XAV-939, a tankyrase inhibitor, on the density and gating of erg-mediated K+ currents in mouse MA-10 Leydig tumor cells

XAV-939(XAV) is a chemical compound that inhibits the activity of tankyrase. However, the precise way in which XAV alters membrane ionic currents is not well understood. In this study,our goal was to examine the impact of XAV on the ionic currents in mouse MA-10 Leydig cells, specifically focusing on the magnitude, gating properties,and voltage-dependent hysteresis of erg-mediated K+currents(IK(erg)). In our whole-cell current recordings we observed that the addition of XAV inhibited the density of IK(erg) in a concentration-dependent manner with an IC50 of 3.1 μM. Furthermore we found that continued exposure to XAV, further addition of neither liraglutide nor insulin-like growth factor-1 counteracted XAV-mediated inhibition of IK(erg). Additionally the presence of XAV suppressed the mean current versus voltage relationship of IK(erg) across the entire voltage-clamp step analyzed. This compound shifted the steady-state activation curve of IK(erg) to a less negative potential by approximately 12 mV. The presence of XAV increased the time constant of deactivating IK(erg) in MA-10 cells. The voltage-dependent clockwise hysteresis of IK(erg) responding to prolonged upright isosceles-triangular ramp voltage became diminished by adding XAV; moreover subsequent addition of NS3623 effectively reversed XAV-induced decrease of hysteretic area of IK(erg). XAV also inhibited the proliferation of this cell line and the IC50 value of XAV-induced inhibition of cell proliferation was 2.8M. Overall the suppression of IK(erg) by XAV may serve as a significant ionic mechanism that contribute to the functional properties of MA-10 cells. However, it is important to note that this effect cannot be attributed solely to the inhibition of tankyrase.

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.

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.

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. 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. 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. 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. 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.

The Effects of Organophosphate Esters Used as Flame Retardants and Plasticizers on Granulosa, Leydig, and Spermatogonial Cells Analyzed Using High-Content Imaging.

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making.

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.

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.

Steroidogenic acute regulatory protein transcription is regulated by estrogen receptor signaling in largemouth bass ovary

Estrogenic contaminants in the environment are linked to the occurrence of reproductive abnormalities in many aquatic species, including largemouth bass (Micropterus salmoides; LMB). Previous work has shown that many different types of xenoestrogens regulate expression of the Steroidogenic Acute Regulatory protein (StAR), a cholesterol-transporting protein vital to steroid hormone biosynthesis; however, the regulatory mechanisms of StAR are incompletely characterized in fish. To learn more about endogenous expression patterns of StAR in the ovary, LMB were collected from the St. John’s River (Florida, USA) over an entire breeding season to investigate StAR expression. Plasma 17β-estradiol (E2) and StAR mRNA levels were positively correlated in females, and StAR mRNA levels displayed ~ 100-fold increase between primary oocyte growth stages and final maturation. To further study the regulation of StAR, female LMB in the laboratory were fed at ≃2% of their weight on a diet laden with 17α-ethinylestradiol (EE2, 70 or 200 ng EE2 per gram feed). Diets were designed to achieve a physiologically-relevant exposure to EE2, and StAR expression was assessed in vivo. We observed a dose-dependent suppression of StAR mRNA levels, however both diets led to high, pharmacological levels in the blood and do not represent normal physiological ranges of estrogens. In the 200 ng EE2/gm feed group, ovarian StAR mRNA levels were suppressed to approximately 5% of that of the LMB control group. These investigations suggest that LMB StAR increases in expression during oocyte maturation and that it is suppressed by E2 feedback when estrogen levels are high, through the HPG axis. A 2.9 kb segment of the LMB StAR promoter was examined for putative E2 response elements using in silico software, and a putative estrogen receptor binding element (ERE/-1745) was predicted in the promoter. The functionality of the ERE was examined using MA-10 mouse Leydig cells transfected with the LMB StAR promoter. Estrogen receptor (ER) interaction with ERE/-1745 was evaluated under basal and human chorionic gonadotropin (hCG)-treated conditions in the presence and absence of E2. Chromatin immunoprecipitation (ChIP) experiments revealed that ESR1 binding to the promoter was enriched under basal conditions and E2 exposure elicited an increase in enrichment (4-fold) above that observed under basal conditions. ESR2 was not strongly enriched at the ERE/-1745 site, suggesting that StAR may be preferentially regulated by LMB estrogen receptor 1 (esr1). Taken together, these different experiments provide evidence that LMB StAR is under the control of estrogens and that ESR1 binds directly to the LMB StAR promoter in an E2-responsive manner.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function\u2014in vivo and in vitro evaluation

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 μg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17β-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and β; ERα, ERβ and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Bisphenol A disrupts steroidogenesis and induces a sex hormone imbalance through c-Jun phosphorylation in Leydig cells

Bisphenol A (BPA) is a well-known endocrine disrupting chemical (EDC) that is used to manufacture plastic consumer products. It is well known that exposure to BPA can induce defects in gonad development and negatively influences reproductive function in both males and females. In this study, we assessed the effects of BPA on hormone production in Leydig cells, which secrete hormones in the testes and support male fertility. We examined two steroidogenic enzymes, CYP11A1 and CYP19 that involved in sex hormone synthesis in mouse MA-10 Leydig cells. We found that BPA activated CYP gene in both mRNA and protein levels then resulted in alteration of the normal sex hormone ratio. Furthermore, we found that BPA induced c-Jun phosphorylation and contributed to CYP gene expression. Similar results were observed in an animal study. In conclusion, BPA disrupts the hormone environment in testis via steroidogenic gene activation through the JNK/c-Jun signaling pathway.

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.

MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells.

In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca(2+)/CAMK (Ca(2+)/calmodulin-dependent kinase)-myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at -232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action.

Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5'-flanking region of the StAR gene demonstrated the importance of the -254/-1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the -254/-1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells.

Deleterious Cholesterol Hydroperoxide Trafficking in Steroidogenic Acute Regulatory (StAR) Protein-expressing MA-10 Leydig Cells: IMPLICATIONS FOR OXIDATIVE STRESS-IMPAIRED STEROIDOGENESIS

Steroidogenic acute regulatory (StAR) proteins in steroidogenic cells are implicated in the delivery of cholesterol (Ch) from internal or external sources to mitochondria (Mito) for initiation of steroid hormone synthesis. In this study, we tested the hypothesis that under oxidative stress, StAR-mediated trafficking of redox-active cholesterol hydroperoxides (ChOOHs) can result in site-specific Mito damage and dysfunction. Steroidogenic stimulation of mouse MA-10 Leydig cells with dibutyryl-cAMP (Bt2cAMP) resulted in strong expression of StarD1 and StarD4 proteins over insignificant levels in nonstimulated controls. During incubation with the ChOOH 3β-hydroxycholest-5-ene-7α-hydroperoxide (7α-OOH) in liposomes, stimulated cells took up substantially more hydroperoxide in Mito than controls, with a resulting loss of membrane potential (ΔΨm) and ability to drive progesterone synthesis. 7α-OOH uptake and ΔΨm loss were greatly reduced by StarD1 knockdown, thus establishing the role of this protein in 7α-OOH delivery. Moreover, 7α-OOH was substantially more toxic to stimulated than nonstimulated cells, the former dying mainly by apoptosis and the latter dying by necrosis. Importantly, tert-butyl hydroperoxide, which is not a StAR protein ligand, was equally toxic to stimulated and nonstimulated cells. These findings support the notion that like Ch itself, 7α-OOH can be transported to/into Mito of steroidogenic cells by StAR proteins and therein induce free radical damage, which compromises steroid hormone synthesis.

Mechanisms of Action of Hormone-sensitive Lipase in Mouse Leydig Cells: ITS ROLE IN THE REGULATION OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

The Microsomal Enzyme 17β-Hydroxysteroid Dehydrogenase 3 Faces the Cytoplasm and Uses NADPH Generated by Glucose-6-Phosphate Dehydrogenase

Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism.

Estradiol-mediated suppression of CYP1B1 expression in mouse MA-10 Leydig cells is independent of protein kinase A and estrogen receptor

Estrogens have multifaceted roles in mammalian testis. In the present study, we focused on estradiol as a potential regulator of testicular cytochrome P450 1B1 (CYP1B1) expression and investigated the possible mechanisms involved in the estradiol-mediated suppression. CYP1B1 protein levels were measured in the testes of rats that were treated with 17β-estradiol benzoate (1.5 mg/kg) at different stages of development. In addition, CYP1B1 mRNA levels were measured in mouse MA-10 Leydig tumor cells treated with (a) various concentrations of 17β-estradiol benzoate, (b) 17β-estradiol benzoate in the presence of exogenous luteinizing hormone (LH), or (c) 17β-estradiol benzoate in the presence of ICI 182,780, a competitive steroidal antagonist of estrogen receptors (ERs). Treatment of neonatal, pubertal, or adult rats with 17β-estradiol benzoate was associated with a reduction of approximately 90% in testicular CYP1B1 protein content compared to age-matched controls. Treatment of MA-10 cells with 17β-estradiol benzoate (10-500 nM) produced a concentration- and time-dependent decrease in CYP1B1 mRNA levels, but had no effect on LH receptor mRNA levels or on protein kinase A (PKA) activity. However, 17β-estradiol benzoate (10-500 nM), regardless of the concentration tested, failed to attenuate the LH-elicited increase in CYP1B1 mRNA or PKA activity in MA-10 cells that were co-treated with LH and estradiol. Similarly, ICI 182,780 (10-1000 µM) did not reverse the suppressive effect of estradiol on CYP1B1 mRNA expression in MA-10 cells co-treated with estradiol and ICI 182,780. The results indicate that downregulation of testicular CYP1B1 by estradiol was independent of PKA activity and was not mediated by ERs in MA-10 cells.

CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists

Cytochrome P450 1B1 (CYP1B1) is highly expressed in testis, but there is conflicting information regarding the inducibility of testicular CYP1B1 by aryl hydrocarbon receptor (AhR) agonists.To assess AhR-mediated regulation, testicular CYP1B1 expression was measured following treatment of adult rats with 3-methylcholanthrene and various dosages of benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The effect of TCDD on CYP1B1 expression in R2C rat Leydig and MA-10 mouse Leydig cells in culture was also determined.Immunoblot analysis showed that treatment with benzo[a]pyrene at dosages up to 200 mg/kg/day and 3-methylcholanthrene at 25 mg/kg/day did not induce testicular CYP1B1 expression. Treatment with TCDD at dosages of 1, 5 or 100 μg/kg had no effect, but testicular CYP1B1 protein levels were increased by approximately 50% at dosages of 10 and 50 μg/kg.CYP1B1 mRNA levels in MA-10 and CYP1B1 protein levels in R2C cells were not induced by exposure to TCDD (10–1000 nM).Overall, the results indicate that rodent testicular CYP1B1 is not inducible by AhR agonists.

Blocking L-type calcium channels reduced the threshold of cAMP-induced steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells

Previous studies have reported the roles of Ca(2+) in steroidogenesis. The present study has investigated an inhibitory effect of Ca(2+) influx through L-type Ca(2+) channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca(2+) influx through L-type Ca(2+) channels using the selective Ca(2+) channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca(2+) channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca(2+) influx through L-type Ca(2+) channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca(2+) channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca(2+) channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca(2+) channel blockers are able to induce Star gene expression. These observations indicate that Ca(2+) influx through L-type Ca(2+) channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca(2+) channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.

Involvement of the Thromboxane A2 Receptor in the Regulation of Steroidogenic Acute Regulatory Gene Expression in Murine Leydig Cells

Recent studies suggested an involvement of thromboxane A2 in cyclooxygenase-2-dependent inhibition of steroidogenic acute regulatory (StAR) gene expression. The present study further investigated the role of thromboxane A2 receptor in StAR gene expression and steroidogenesis in testicular Leydig cells. The thromboxane A2 receptor was detected in several Leydig cell lines. Blocking thromboxane A2 binding to the receptor using specific antagonist SQ29548 or BM567 resulted in dose-dependent increases in StAR protein and steroid production in MA-10 mouse Leydig cells. The results were confirmed with Leydig cells isolated from rats. StAR promoter activity and StAR mRNA level in the cells were also increased after the treatments, suggesting an involvement of the thromboxane A2 receptor in StAR gene transcription. Furthermore study indicated that blocking the thromboxane A2 receptor reduced dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 protein, a transcriptional repressor of StAR gene expression. Specific binding of the antagonists to the receptors on cellular membrane was demonstrated by binding assays using (3)H-SQ29548 and binding competition between (3)H-SQ29548 and BM567. Whereas SQ29548 enhanced cAMP-induced StAR gene expression, in the absence of cAMP, it was unable to increase StAR protein and steroidogenesis. However, when the receptor was blocked by the antagonist, subthreshold levels of cAMP were able to induce maximal levels of StAR protein expression, suggesting that blocking the thromboxane A2 receptor increase sensitivity of MA-10 cells to cAMP stimulation. Taken together, the results from the present and previous studies suggest an autocrine loop, involving cyclooxygenase-2, thromboxane A synthase, and thromboxane A2 and its receptor, in cyclooxygenase-2-dependent inhibition of StAR gene expression.