CHARACTERIZATION OF THE HAMSTER CYP11B2 GENE ENCODING ADRENAL CYTOCHROME-P450 ALDOSTERONE SYNTHASE

A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo ) was isolated from a hamster genomic library. The gene, which contain ed 9 exons, was composed of 9,045 bp, of which 3,722 bp were located i n the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) an d other putative cis elements, previously named Ad1 to Ad6, were ident ified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nucl ear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a n ew protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis elemen t was specifically retarded by factors in the nuclear extracts of hams ter adrenal ZG. We then examined the transcriptional activity of the 5 ' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activ ity was obtained with the construct containing up to -134 bp. Increasi ng the length of the regulatory region of CYP11B2 gene to -167 bp resu lted in less than two-thirds of the maximal activity, indicating the p robability of putative inhibitory cis elements in this area of the gen e. Forskolin stimulated the expression of the reporter gene of deletio n plasmids excepting the construct containing only the TATA box, and t he highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, t he promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the a ldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.