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.     

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 mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

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.     

Cytochrome P450 17alpha hydroxylase/17,20 lyase (CYP17) function in cholesterol biosynthesis: identification of squalene monooxygenase (epoxidase) activity associated with CYP17 in Leydig cells

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is a microsomal enzyme catalyzing two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the biosynthesis of adrenal and gonadal steroids. CYP17 is a potent oxidant, it is present in liver and nonsteroidogenic tissues, and it has been suggested to have catalytic properties distinct to its function in steroid metabolism. To identify CYP17 functions distinct of its 17alpha-hydroxylase/17,20-lyase activity, we used MA-10 mouse tumor Leydig cells known to be defective in 17alpha-hydroxylase/17,20-lyase activity. A CYP17 knocked down MA-10 clone (MA-10(CYP17KD)) was generated by homologous recombination and its steroidogenic capacity was compared with wild-type cells (MA-10(wt)). Although no differences in cell morphology and proliferation rates were observed between these cells, the human chorionic gonadotropin-induced progesterone formation and de novo synthesis of steroids were dramatically reduced in MA-10(CYP17KD) cells; their steroidogenic ability could be rescued in part by transfecting CYP17 DNA into the cells. Knocking down CYP17 mRNA by RNA interference yielded similar results. However, no significant difference was observed in the steroidogenic ability of cells treated with 22R-hydroxycholesterol, which suggested a defect in cholesterol biosynthesis. Incubation of MA-10(CYP17KD) cells with (14)C-labeled squalene resulted in the formation of reduced amounts of radiolabeled cholesterol compared with MA-10(wt) cells. In addition, treatment of MA-10(CYP17KD) cells with various cholesterol substrates indicated that unlike squalene, addition of squalene epoxide, lanosterol, zymosterol, and desmosterol could rescue the hormone-induced progesterone formation. Further in vitro studies demonstrated that expression of mouse CYP17 in bacteria resulted in the expression of squalene monooxygenase activity. In conclusion, these studies suggest that CYP17, in addition to its 17alpha-hydroxylase/17,20-lyase activity, critical in androgen formation, also expresses a secondary activity, squalene monooxygenase (epoxidase), of a well-established enzyme involved in cholesterol biosynthesis, which may become critical under certain conditions.