ACTIVATION OF CYP11A AND CYP11B GENE PROMOTERS BY THE STEROIDOGENIC CELL-SPECIFIC TRANSCRIPTION FACTOR, AD4BP

We have examined the transcriptional activity of four cis-elements, Ad1(CRE), Ad2, Ad3, and Ad4, that are present in the promoter of the bovine CYP11B (11beta-hydroxylase P-450) gene using beta-globin reporter gene constructs and transient transfection into steroidogenic and nonsteroidogenic cell types. Only Ad1(CRE), a CRE homolog, showed forskolin-dependent transcriptional activity in adrenal tumor Y-1 cells, whereas the other elements were not able to stimulate transcription by themselves. As Ad3 and Ad4 had previously been identified as the cis-elements required for full cAMP-dependent transcription of this gene, we examined the effect of combinations of different cis-elements on the transcription of the reporter gene. In Y-1 cells, Ad1(CRE) and four tandem copies of any one of the other cis-elements substantially activated transcription in response to forskolin treatment. The template carrying Ad1(CRE) and Ad4 was also active in testicular Leydig cells, I-10, whereas it was inactive in nonsteroidogenic PC-12 cells. Transcriptional activation by the 4xAd4/Ad1(CRE) combination presumably depended on the presence of Ad4-binding protein (Ad4BP), which is absent in PC-12 cells, as shown by immunoblot analysis. This was confirmed by cotransfecting an expression vector for Ad4BP into PC-12 cells, which caused forskolin-dependent transcription to increase in proportion to the amount of expression vector. In Y-1 cells, transcriptional activation by forskolin was mimicked by cotransfection of an expression vector for the catalytic subunit of protein kinase-A. Finally, constructs in which the chloramphenicol acetyltransferase reporter gene is under the control of the native promoters of the CYP11A and CPY11B genes are inactive in nonsteroidogenic CV-1 cells, but could be activated by cotransfecting expression vectors for both Ad4BP and PKA.