Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells, PKC alpha, -epsilon, and -zeta were detected in hamster adrenal tons glomerulosa and NCI-H295 cells, and PKC theta in NCI-H295 cells, 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt(2)cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKC alpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKC zeta. Dominant negative (DN) PKC alpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity, AII-, KCl-, and Bt(2)cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKC alpha, -epsilon or -theta. The effect of Bt(2)cAMP was abolished by CA-PKC zeta but AII and KCl were still able to enhance the promoter activity, DN-PKC alpha, -epsilon, -theta, or -zeta did not inhibit these effects, Go6976 enhanced promoter activity, providing further evidence that PKC alpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKC alpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt(2)cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.