REPRESSION OF CAMP-INDUCED EXPRESSION OF THE MOUSE P450 17-ALPHA-HYDROXYLASE C17-20 LYASE GENE (CYP17) BY ANDROGENS/

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lya se (P450c17) protein and the accumulation of P450c17 mRNA, via an andr ogen receptor (AR)-mediated mechanism. To examine the mechanism by whi ch androgens repress the cAMP-induced expression of the mouse Cyp17 ge ne, constructs containing 5'-flanking sequences of the mouse Cyp17 lin ked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced exp ression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestoste rone was observed when the -1021 bp Cyp17-CAT construct was cotransfec ted with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions of t he -1021 bp fragment demonstrated that -346 bp of 5'-flanking region o f the mouse Cyp17 promoter are sufficient to confer androgen repressio n of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footpri nting analysis indicated that the AR interacts with sequences between -330 and -278 bp of the Cyp17 promoter, This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-respons ive region of the Cyp17 promoter, possibly interfering with the bindin g of the protein(s) that mediate cAMP induction of Cyp17.