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).     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

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.     

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.     

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.     

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T₃) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T₃ response requires both on-going mRNA and protein synthesis. In addition, the effects of T₃ were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T₃. Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T₃-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5′-flanking region of the mouse StAR gene, which identified a region between −254 and −110 bp that was essential for T₃ function. Importantly, it was found that the SF-1 binding site at position −135 bp of the 5′-flanking region was greatly involved in T₃-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T₃ function. The relevance of T₃-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.     

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.     

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.     

Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis.

In previous studies we and others have described several mitochondrial proteins which are synthesized in response to acute hormone stimulation in several steroidogenic tissues. In both MA-10 mouse Leydig tumor cells and primary cultures of rat adrenal cortex cells, these proteins consist of a family of 37 kilodalton (kDa) and 32 kDa precursor forms and fully processed forms which are 30 kDa in molecular weight. The nature of the appearance of these proteins and their subcellular localization to the mitochondria, the site of the rate limiting step in steroidogenesis, has led to the speculation that they may be involved in the acute regulation of steroidogenesis. In the present study we have taken advantage of another steroidogenic cell, the R2C rat Leydig tumor cell, to perform studies which further indicate that these mitochondrial proteins are involved in the regulation of steroidogenesis. Unlike the MA-10 cell which requires hormone stimulation for steroid production, the R2C cell is a constitutive progesterone producer whose steroid production cannot be further increased with hormone stimulation. We have shown that the R2C cell line is less sensitive to the inhibition of steroid production by the metal chelator orthophenanthroline (OP) than is the MA-10 cell. We have demonstrated that progesterone production and the 30 kDa mitochondrial proteins remain present in the R2C cells at a concentration of OP which completely inhibits progesterone production and totally eliminates the 30 kDa proteins in MA-10 cells. As further evidence for the role of these proteins in steroidogenic regulation, we have isolated several revertants of the R2C parent (P) cell line which have lost the ability to synthesize progesterone constitutively, but which can be stimulated to synthesize this steroid by trophic hormone and cAMP analog. In these revertants, designated (R), the normally constitutively present 30 kDa proteins are greatly decreased compared to controls, but reappear in large amounts following hormone stimulation. Taken together, these data provide further evidence that the 30 kDa mitochondrial proteins are involved in the acute regulation of steroidogenesis in Leydig cells.     

Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress

The StarD4 and StarD5 proteins share approximately 30% identity, and each is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. We previously showed StarD4 expression is sterol-repressed, consistent with regulation by sterol regulatory element-binding proteins (SREBPs), whereas StarD5 is not sterol-regulated. Here we further address the regulation and function of StarD4 and StarD5. Unlike StAR, the START family prototype, StarD4 and StarD5 were not induced by steroidogenic stimuli in Leydig cells. However, StarD4 and StarD5 showed StAR-like activity in a cell culture steroidogenesis assay, indicating cholesterol transfer. In transgenic mice expressing active SREBPs, StarD4 was predominantly activated by SREBP-2 rather than SREBP-1a. The mouse and human StarD4 proximal promoters share approximately 70% identity, including several potential sterol regulatory elements (SREs). Reporters driven by the StarD4 promoter from either species were transfected into NIH-3T3 cells, and reporter activity was highly repressed by sterols. Site-directed mutagenesis of potential SREs identified a conserved functional SRE in the mouse (TCGGTCCAT) and human (TCATTCCAT) promoters. StarD5 was not sterol-repressed via SREBPs nor was it sterol-activated via liver X receptors (LXRs). Even though StarD4 and StarD5 were not LXR targets, their overexpression stimulated LXR reporter activity, suggesting roles in cholesterol metabolism. StarD5 expression increased 3-fold in free cholesterol-loaded macrophages, which activate the endoplasmic reticulum (ER) stress response. When NIH-3T3 cells were treated with agents to induce ER stress, StarD5 expression increased 6-8-fold. Because StarD4 is regulated by sterols via SREBP-2, whereas StarD5 is activated by ER stress, they likely serve distinct functions in cholesterol metabolism.