Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

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.     

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.     

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.     

Extracellular signal-regulated kinases (ERK) 1, 2 are required for luteinizing hormone (LH)-induced steroidogenesis in primary Leydig cells and control steroidogenic acute regulatory (StAR) expression

The luteinizing hormone (LH) plays a critical role in steroidogenesis, by stimulating cAMP-dependent protein kinase A (PKA) and phospholipase A2 activity, and by mobilizing calcium and chloride ions. In contrast, whether the ERK 1, 2 mitogen-activated protein (MAP) kinases are involved in LH-induced steroidogenesis is less obvious. Here, we sought to clarify this point in rat primary Leydig cells, naturally bearing the LH receptor (LH-R) in male, and in the mouse tumoral Leydig cell line (MLTC 1). Pre-incubation of both cell types with the mitogen-activated protein kinase kinase (MEK) inhibitors U0126 and PD98059 reduced LH-induced steroidogenesis, and tonically enhanced the expression of the StAR protein. Furthermore, ERK1, 2 were inducibly phosphorylated following LH exposure of MLTC 1 cells. Altogether, our results indicate that in primary as well as in tumoral Leydig cells, inhibiting MEK dampened LH-induced steroidogenesis but enhanced basal as well as LH-induced StAR expression, suggesting that ERK1,2 could be involved in these responses.     

Mechanisms of epidermal growth factor signaling: regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells

Steroid hormone biosynthesis in the adrenals and gonads is regulated by the steroidogenic acute regulatory (StAR) protein through its action in mediating the intramitochondrial transport of cholesterol. A role for epidermal growth factor (EGF) in modulating steroidogenesis has been previously determined, but the mechanism of its action remains unknown. The present investigation was designed to explore the potential mechanism of action of mouse EGF (mEGF) in the regulation of steroid biosynthesis and StAR protein expression in mLTC-1 mouse Leydig tumor cells. We show that treatment of mLTC-1 cells with mEGF significantly increased the levels of progesterone (P), StAR protein, and StAR mRNA in a time- and dose-dependent manner. The coordinate induction of P synthesis and StAR gene expression by mEGF was effectively inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. Also, longer exposure of mLTC-1 cells to mEGF produced a marked decrease in LH-receptor mRNA expression. These effects of mEGF were exerted through high-affinity binding sites (K(d) approximately 0.53 nmol/L) in these cells. It was also determined that the arachidonic acid (especially lipoxygenase metabolites) and mitogen-activated protein kinase pathways were also involved in the mEGF-induced steroidogenic response. However, involvement of the latter pathway was further assessed in nonsteroidogenic COS-1 cells transfected with the Elk1 trans-reporting plasmids and resulted in a significant increase in luciferase activity in response to mEGF. Furthermore, deletion and mutational analyses demonstrated a predominant involvement of activator protein-1 in addition to the multiple mEGF responsive elements found within the 5'-flanking region (-151/-1 base pairs) of the mouse StAR gene. These findings provide novel insights into the mEGF-induced regulatory cascades associated with steroid synthesis and StAR protein expression in mouse Leydig cells.     

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.     

Analysis of the developmental expression of small VCP-interacting protein and its interaction with steroidogenic acute regulatory protein in Leydig cells

Small VCP-interacting protein (SVIP) is a 9-kDa protein that is composed of 76 amino acids, and it plays a role in the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Recent studies have shown that SVIP is an androgen-responsive protein and its expression is regulated by androgens. Because no data are available regarding the cellular localization and expression of SVIP in the mouse testis, where androgens are highly expressed, immunohistochemistry and western blotting were performed. In the fetal testis, we found that moderate but consistent staining of SVIP is present in the cytoplasm of Leydig cells. In prepubertal and adult life, SVIP remains present in Leydig cells as well as in the cytoplasm of some peritubular and Sertoli cells. From postnatal day 15 onward, SVIP is strongly expressed in the cytoplasm of Leydig cells.Furthermore, TM3, MA-10 Leydig and Sertoli cell lines were also used to evaluate the expression of SVIP. To identify the interacting partners, such as steroidogenic acute regulatory (STAR) protein, colocalization studies were performed by fluorescence microscopy, showing that STAR colocalized with SVIP in the adult mouse testis. The expression changes of STAR were studied by using SVIP siRNAs in Leydig cell line cultures. Depletion of SVIP resulted in decreased expression of STAR. Additionally, the number and size of lipid droplets were significantly increased in SVIP-depleted Leydig cells. Taken together, our data identify SVIP as a marker of Leydig cell lineage and as a regulator of STAR protein expression and lipid droplet status in Leydig cells.     

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.