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.