Characterization of the hamster CYP11B2 gene regulatory regions

We have isolated a hamster CYP11B2 gene encoding the cytochrome P450 aldosterone synthase. In comparison with the CYP11B2 gene of other species, cis-elements named Ad1, Ad2, Ad3, and Ad4, were identified in the 5'-untranslated region of the hamster gene. Mouse adrenal tumor cells were transiently transfected with chimaeric reporter constructs, fused to the bacterial chloramphenicol acyltransferase (CAT) reporter gene,to study the regulation of expression of the hamster CYP11B2 gene. The highest basal expression was obtained with the -130 bp construct Decreasing the length of the regulatory region of the CYP11B2 gene beyond that of -130 bp, to exclude Ad2 and Ad1 elements, resulted in successive decreases in CAT activity. Increasing the length of the regulatory region beyond that of -130 bp also resulted in a reduction of CAT activity, indicating the presence of inhibitory cis-elements in this area of the gene. Forskolin stimulated the CAT activity of all constructs, the highest of which occurred with the -130 bp construct, indicating that the gene is controlled by the PKA signalling pathway. TPA, however, had no stimulatory effects on any of these constructs. Staurosporine, an inhibitor of the PKC pathway, stimulated cells transfected with the different constructs in a similar manner as forskolin, indicating that PKC might act, at least in Y-1 cells, as a negative regulator on the hamster CYP11B2 promoter.