Molecular Endocrinology, Vol 6, 927-934, Copyright © 1992 by Endocrine Society

ARTICLES

Isolation and characterization of the mouse P450 17 alpha- hydroxylase/C17-20-lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells

GL Youngblood and AH Payne
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.

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/C17-20 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.

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