Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells. Cells were stimulated with different steroidogenic compounds in the presence of [32P]orthophosphoric acid for 2 h and phosphoproteins analyzed by two-dimensional polyacrylamide gel-electrophoresis (PAGE). Results demonstrated an increase in the phosphorylation of four proteins (22 kDa, pI 5.9; 24 kDa, pI 6.7 and 30 kDa, pI 6.3 and 6.5) in response to 34 ng/ml hCG, 1 mM dbcAMP and 100 microM forskolin. Conversely, treatment of cells with PMA increased the phosphorylation of only one of these proteins (30 kDa, pI 6.3). At least two of these proteins (30 kDa, pI 6.5 and 6.3) appear to be identical to proteins which we and others have shown to be synthesized in response to trophic hormone stimulation in adrenal, luteal and Leydig cells. In addition, they also appear to be identical to adrenal cell mitochondrial proteins demonstrated to be phosphorylated in response to ACTH. These data indicate that proteins similar to those phosphorylated in adrenal cells in response to ACTH are phosphorylated in hormone stimulated testicular Leydig cells and that these proteins may be involved in steroidogenic regulation.     

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.     

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Differential regulation of steroid hormone biosynthesis in R2C and MA-10 Leydig tumor cells: role of SR-B1-mediated selective cholesteryl ester transport

The rat R2C Leydig tumor cell line is constitutively steroidogenic in nature, while the mouse MA-10 Leydig tumor cell line synthesizes large amounts of steroids only in response to hormonal stimulation. Earlier studies showed abundant cAMP-independent steroid production and constitutive expression of steroidogenic acute regulatory (StAR) protein in R2C cells. The objective of the current study was to identify possible genetic alterations in the R2C cell line responsible for rendering it a constitutively steroidogenic cell line, especially those that might have altered its cholesterol homeostatic mechanisms. Measurement of the levels of cholesterol esters and free cholesterol, precursors for steroidogenesis, indicated that R2C mitochondria were fourfold enriched in free cholesterol content compared with MA-10 mitochondria. In addition to the previously demonstrated increased expression of StAR protein, we show that R2C cells possess marginally enhanced protein kinase A activity, exhibit higher capacity to take up extracellular cholesterol esters, and express much higher levels of scavenger receptor-type B class 1 (SR-B1) and hormone sensitive lipase (HSL). These observations suggest that the high level of steroid biosynthesis in R2C cells is a result of the constitutive expression of the components involved in the uptake of cholesterol esters (SR-B1), their conversion to free cholesterol (HSL), and its mobilization to the inner mitochondrial membrane (StAR).     

Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole

In this study we attempted to examine the effects of ketoconazole on steroid biosynthesis and to determine which steps in the steroidogenic pathway were blocked using MA-10 Mouse Leydig tumor cells. This cloned cell line produces progesterone as the major steroid following stimulation by hCG or dbcAMP. At a concentration of 1 microM ketoconazole completely inhibited the hCG- and dbcAMP-stimulated progesterone synthesis in MA-10 Leydig cells. The conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone was also suppressed by this drug. The presence of ketoconazole inhibited mitochondrial steroid synthesis but required high concentrations of the drug as compared to inhibition in intact cells. No accumulation of pregnenolone was observed in the presence of ketoconazole indicating that the activity of 3 beta-hydroxysteroid dehydrogenase was not affected. We conclude that ketoconazole directly inhibits the activity of cholesterol side-chain cleavage enzyme (CSCC), a rate-determining enzymatic step in steroidogenesis, by interacting with cytochrome P-450scc.     

Stimulation of cholesterol side-chain cleavage enzyme activity by cAMP and hCG in MA-10 Leydig tumor cells

Using a cloned Leydig tumor cell line (designated MA-10), we have studied the activity of cholesterol side-chain (CSCC) enzyme, the rate-determining step in steroidogenesis, in mitochondria isolated from cells pretreated either with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Results showed a slight but significant increase in CSCC activity with treatment by cAMP (25% increase) and hCG (60% increase), as compared to mitochondria isolated from nontreated control cells. However, this stimulation of CSCC activity appears to be of limited significance when compared to the approximately 1000-fold or greater increase observed in progesterone production in the presence of hCG or dbcAMP. On the other hand, unstimulated MA-10 cells or isolated mitochondria efficiently converted 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone, and this conversion was not affected by cycloheximide. The addition of cholesterol to intact cells or to isolated mitochondria did not affect progesterone production. Our observations clearly indicate that given the proper hydroxy substrates (22R-hydroxycholesterol or 25-hydroxycholesterol), MA-10 Leydig cells are able to convert them into progesterone without any stimulation by steroidogenic stimuli, i.e. cAMP or hCG. Since MA-10 Leydig cells can efficiently convert 22R-hydroxycholesterol--an intermediate in CSCC reaction--into progesterone, these results suggest that the key regulatory step in the mechanism of trophic hormone-stimulated steroid production is the first hydroxylation step of the 3 sequential monooxygenation reactions involved in the conversion of cholesterol to pregnenolone.     

The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors

Acute regulation of steroidogenesis in steroidogenic tissue is controlled by the transfer of cholesterol from the outer to the inner mitochondrial membrane where cleavage to produce pregnenolone occurs. Hormonal stimulation of MA-10 mouse Leydig tumor cells results in a large increase in steroidogenesis and the concomitant appearance of a series of 30-kDa proteins which have been localized to the mitochondria. In the present study we have shown that the appearance of these proteins occurs in a dose-responsive manner with both human chorionic gonadotropin and cyclic AMP analog. We have also shown that while steroidogenesis is inhibited rapidly in response to a cessation of protein synthesis, the 30-kDa mitochondrial proteins remain in the mitochondria, posing a potential dilemma for arguments favoring their role in the acute regulation of steroidogenesis. We report that the 30-kDa mitochondrial proteins arise from two precursor proteins with molecular masses of 37 and 32 kDa which are also found to be associated with the mitochondria. The use of pulse-chase experiments and the inhibitors ortho-phenanthroline and carbonyl cyanide m-chlorophenylhydrazone demonstrated the precursor-product relationship between the 37-, 32-, and 30-kDa proteins. We have also demonstrated that, as shown for a number of other mitochondrial proteins, the 30-kDa proteins are transferred to the inner mitochondrial membrane by a process requiring both proteolytic removal of the targeting sequences and an electrical potential across the inner mitochondrial membrane. We propose that during this transfer contact sites form between the two mitochondrial membranes and may offer an ideal situation for the transfer of cholesterol from the outer membrane to the inner membrane by an as yet unknown mechanism. Following transfer, the 30-kDa proteins remain in the inner membrane no longer able to function in the further transfer of cholesterol, and it is the continuing synthesis and processing of more precursor proteins which provides additional substrate for steroidogenesis.     

Disrupting mitochondrial function with surfactants inhibits MA-10 Leydig cell steroidogenesis

It is well established that surfactants can elicit cytotoxic effects at threshold concentrations by changing the permeability and solubilizing components of cell membranes. The purpose of this study was to characterize the relationship between perturbation of the mitochondrial membrane resulting from treatment with representative cationic, nonionic, and anionic surfactants and the extent to which this perturbation affects steroid formation and StAR protein expression and activity in MA-10 Leydig cells. The StAR protein is synthesized as an active 37 kDa extramitochondrial form, which is processed into a 30 kDa intramitochondrial form after cholesterol transfer and mitochondrial import and processing. It has been shown in several in vitro studies that the mitochondrial electrochemical gradient is required for the StAR protein to transfer cholesterol to the inner mitochondrial membrane. Each substance that was tested produced a concentration-dependent decrease in steroid formation in hCG-stimulated MA-10 cells. Decreases in progesterone production were accompanied by loss of mitochondrial membrane potential and by a decrease in the levels of the 30 kDa form of the StAR protein. However, levels of the 37 kDa form of the StAR protein did not decrease, indicating no effect on StAR protein expression. These results demonstrate how perturbation of the mitochondrial membrane by surfactants inhibits import, processing, and cholesterol transfer activity and underscore the importance of including sensitive assays that evaluate mitochondrial function when screening for potential effects on steroidogenesis with in vitro test systems.     

Effects of lindane on steroidogenesis and steroidogenic acute regulatory protein expression

Lindane, the gamma isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use worldwide. Numerous reports have shown that this pesticide adversely affects reproductive function in animals. Although the pathogenesis of reproductive dysfunction is not yet fully understood, recent reports indicate that lindane can directly inhibit adrenal and gonadal steroidogenesis. Because Leydig cells play a pivotal role in male reproductive function through the production of testosterone, the mouse MA-10 Leydig tumor cell line was used to assess the potential effects of gamma-HCH and its isomers, alpha-HCH and delta-HCH, on steroid production, steroidogenic enzyme expression and activity, and steroidogenic acute regulatory (StAR) protein expression. StAR mediates the rate-limiting and acutely regulated step in hormone-stimulated steroidogenesis, the intramitochondrial transfer of cholesterol to the P450(scc) enzyme. Our studies demonstrate that alpha-, delta-, and gamma-HCH inhibited dibutyryl ([Bu](2)) cAMP-stimulated progesterone production in MA-10 cells in a dosage-dependent manner without affecting general protein synthesis; and protein kinase A or steroidogenic enzyme expression, activity, or both. In contrast, each of these isomers dramatically reduced (Bu)(2)cAMP-stimulated StAR protein levels. Therefore, our results are consistent with the hypothesis that alpha-, delta-, and gamma-HCH inhibited steroidogenesis by reducing StAR protein expression, an action that may contribute to the pathogenesis of lindane-induced reproductive dysfunction.     

The inhibitory effects of lead on steroidogenesis in MA-10 mouse Leydig tumor cells

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.     

Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.     

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.