ISOLATION AND CHARACTERIZATION OF THE MOUSE P450 17 ALPHA-HYDROXYLASE C-17-20-LYASE GENE (CYP17) - TRANSCRIPTIONAL REGULATION OF THE GENE BY CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE IN MA-10 LEYDIG-CELLS

The biosynthesis in Leydig cells of the C19 steroid testosterone from the C21 precursor progesterone requires the activities of the enzyme cytochrome P450 17-alpha-hydroxylase/C-17-20 lyase (P450(17-alpha)). Previous studies from this laboratory demonstrated that the de novo synthesis of the P450(17-alpha) protein and the accumulation of P450(17-alpha) mRNA in mouse Leydig cell cultures is absolutely dependent on cAMP stimulation. To investigate further the cAMP regulation of P450(17-alpha) expression in Leydig cells, the structural gene encoding P450(17-alpha) (Cyp17) was isolated from a mouse genomic library using a full-length mouse P450(17-alpha) cDNA. Two overlapping genomic clones were isolated and characterized by restriction mapping and partial sequencing. The two clones together contain the entire coding region and approximately 10 kilobases of 5'-flanking sequences of Cyp17. To identify regions necessary for cAMP-induced transcription, 5'-flanking regions of Cyp17 were fused with the chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into MA-10 tumor Leydig cells. Studies localized the cAMP-responsive region of the gene to a region between -346 and -245 basepairs relative to the transcription initiation site. Transient transfections of MA-10 cells with a construct consisting of the -346/-245 sequences fused to a heterologous promoter, thymidine kinase, and the CAT reporter gene demonstrated a marked increase in cAMP stimulation of CAT expression, providing additional evidence that the -346/-245 sequences of the Cyp17 5'-flanking region confer cAMP-induced expression of Cyp17. This cAMP-responsive region of mouse Cyp17 bears no apparent homology to the cAMP-responsive regions identified in the human and bovine Cyp17 genes.