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 contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster 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 activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the 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, the 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 aldosterone 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.
Coulombe
N
N
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, QC, Canada.
Lefebvre
A
A
Lehoux
J G
JG
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D006224:Cricetinae; D019405:Cytochrome P-450 CYP11B2 / Q000096:biosynthesis* / Q000235:genetics*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D015698:Genomic Library; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008647:Mesocricetus; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014162:Transfection; D015384:Zona Glomerulosa / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D019405:Cytochrome P-450 CYP11B2; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I