TRANSCRIPTIONAL ACTIVITY OF THE HAMSTER CYP11B2 PROMOTER IN NCI-H295 CELLS STIMULATED BY ANGIOTENSIN-II, POTASSIUM, FORSKOLIN AND BISINDOLYLMALEIMIDE

We studied the regulation of the hamster CYP11B2 gene in the NCI-H295 cell line, which is known to produce aldosterone in response to stimul ation by angiotensin II (AII) and KCl. Ten deletion plasmids harboring the 5'-untranslated region of the CYP11B2 gene were used for chloramp henicol acetyltransferase (CAT) assays. Transient transfections showed progressively increasing basal promoter activity by constructs beyond the TATA bos, with a peak occurring with the -167 bp construct which contains putative Ad1, Ad2, Ad5 and the newly reported -143/-161 cis-e lement sequences. The promoter activity was lower with the construct c ontaining the putative Ad3 cis-element and increased with longer const ructs. This indicates the presence of both inhibitory and stimulatory cis-elements in this area of the gene. Expression of the reporter gene of all constructs was stimulated by AII and KCl, with the exception o f the construct containing only the TATA bos, which showed 6-fold and 10-fold increases occurring with the -167 bp deletion plasmid. The pat terns of increase in CAT activity with AII and KCl treatment were simi lar, showing that these two regulators can stimulate hamster CYP11B2 p romoter activity through common cis-elements. The calcium channel anta gonist nifedipine blocked the stimulatory effects of KCl on CAT activi ty, showing the involvement of calcium channels in the regulation of C YP11B2 gene transcription by KCl. 12-O-Tetradecanoylphorbol 13-acetate , a known stimulator of the protein kinase C (PKC) signaling pathway, was without significant effect on CAT activity. Bisindolylmaleimide, a specific inhibitor of PKC, had a significant enhancing effect (3.4- t o 6-fold), indicating that PKC may negatively regulate the expression of the hamster CYP11B2 gene in NCI-H295 cells. A mutation was induced in the sequence -143/-161 of the -350 bp construct in order to determi ne its importance in the regulation of hamster CYP11B2 promoter activi ty. The stimulatory effects of AII, KCI, forskolin and bisindolylmalei mide on CAT activity were significantly less in the mutant than in the wild type. These results confirm that this cis-element is necessary i n maintaining a high level of transcriptional activity in stimulated N CI-295H cells. In conclusion, using NCI-295H transfected cells, we hav e found that the 5'-untranslated region of the hamster CYP11B2 gene po ssesses transcriptional activity with stimulatory and also inhibitory cis-elements; CYP11B2 promoter activity can be stimulated by AII, KCl, forskolin, dibutyryl cAMP and bisindolylmaleimide. Our results sugges t that this gene is positively regulated through the protein kinase A signaling pathway and through calcium channels, whereas PKC may have a negative regulatory effect upon the transcription of the CYP11B2 gene . Furthermore, we have shown that the cis-element -143/-161 in the 5'- untranslated region of the hamster CYP11B2 gene is important in mainta ining a high level of promoter activity in stimulated NCI-295H cells.