PARENT SESSION
PLATFORM SESSION 14. GENE REGULATION AND FUNCTION I

Monday, August 2, 2004
2:00 PM–4:00 PM
Buchanan A102
Chair: Colin Clay Co-Chair: Ted Chauvin

(328) SQUALENE EPOXIDASE/MONOXYGENASE ACTIVITY ASSOCIATED WITH THE CYTOCHROME P450 17-HYDROXYLASE ENZYME; ROLE IN LEYDIG CELL CHOLESTEROL BIOSYNTHESIS.

Papadopoulos, Vassilios¹, Yao, Zhi-xing¹, Liu, Ying¹, ¹ Department of Biochemistry and Molecular Biology, Washington, DC

ABSTRACT- Cytochrome P450 17-hydroxylase/17,20-lyase (P450c17; EC 1.14.99.9) is a microsomal monoxygenase essential for glucocorticoid and sex steroid biosynthesis. It has been suggested that P450c17 may have other catalytic properties distinct of its function in pregnenolone and progesterone metabolism. MA-10 Leydig tumor cells form mainly progesterone as the final steroid product, thus offering a cell model to investigate alternative function(s) of the enzyme. The MA-10 mouse P450c17 gene was cloned and P450c17 gene knockdown cells (MA-10^c17kd) were generated by homologous recombination. No differences in cell morphology, proliferation rates, cAMP-dependent protein kinase activity and progesterone formation in the presence of 22R-hydroxycholesterol were observed between MA-10^c17kd and MA-10^wild-type cells. However, a dramatic decrease in hCG-stimulated progesterone synthesis by MA-10^c17kd cells was observed. Metabolic study by radiolabeled mevanolactone confirmed the inhibition of steroid formation in MA-10^c17kd cells. This decrease in steroid synthesis was rescued upon transfection of the cells with the mouse P450c17 cDNA. Silencing P450c17 expression in MA-10^wild-type cells using RNA interference methods also resulted in the reduced ability of the cells to form progesterone in response to hCG treatment. These studies indicated that P450c17 might be involved in cholesterol biosynthesis. Treatment of MA-10^c17kd cells with the substrates squalene, squalene epoxide, lanosterol, zymosterol and desmosterol demonstrated that only the addition of all substrates with the exception of squalene could rescue the hCG-stimulated progesterone formation by MA-10^c17kd cells, suggesting that in these cells the squalene epoxidase/monoxygenase (EC 1.14.99.7) activity was reduced. Indeed, we found that cholesterol synthesis from ¹⁴C-squalene was reduced in MA-10^c17kd cells compared to MA-10^wild-type cells. Taken together these data indicate that the P450c17 enzyme, in addition to its steroid 17-monoxygenase activity also has squalene epoxidase/monoxygenase activity, critical in cholesterol biosynthesis. (Supported by NSF grant IBN-0110711).

KEY WORDS: squalene epoxidase/monoxygenase, P450c17 gene deletion, progesterone, cholesterol