Burgos-Trinidad M, Youngblood G L, Maroto M R, Scheller A, Robins D M, Payne A H
Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor 48109, USA.
Mol Endocrinol. 1997 Jan;11(1):87-96. doi: 10.1210/mend.11.1.9871.
In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked 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 expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected 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 -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting 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-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.
在小鼠睾丸间质细胞的原代培养中，睾酮通过雄激素受体（AR）介导的机制，抑制cAMP诱导的P450 17α-羟化酶/C17-20裂解酶（P450c17）蛋白的从头合成以及P450c17 mRNA的积累。为了研究雄激素抑制小鼠Cyp17基因cAMP诱导表达的机制，将含有与氯霉素乙酰转移酶（CAT）报告基因相连的小鼠Cyp17 5'-侧翼序列的构建体与小鼠AR表达质粒共转染到MA-10肿瘤睾丸间质细胞中。在二氢睾酮存在的情况下，含有-1021 bp Cyp17启动子序列的报告构建体的cAMP诱导表达受到抑制。相比之下，当-1021 bp Cyp17-CAT构建体与缺失DNA结合域第二个锌指的人AR表达质粒共转染时，未观察到二氢睾酮的抑制作用，这表明DNA结合参与了AR介导的Cyp17表达抑制。对小鼠Cyp17启动子5'-侧翼区域-346 bp缺失的分析表明，该区域足以赋予雄激素对Cyp17 cAMP诱导表达的抑制作用。脱氧核糖核酸酶I足迹分析表明，AR与Cyp17启动子-330至-278 bp之间的序列相互作用。该区域与先前鉴定的位于Cyp17启动子-346至-245 bp之间的cAMP反应区域重叠。这些结果表明，AR介导的抑制涉及AR与Cyp17启动子cAMP反应区域序列的结合，可能干扰介导Cyp17 cAMP诱导的蛋白质的结合。
