Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene.
Mitochondrial cytochrome P450(24) expression in the vitamin D-degradation pathway is induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The molecular basis of this enzyme regulation was investigated by isolating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction process. Constructs containing different lengths of 5'-flanking region of the gene were linked to a luciferase reporter gene and transiently co-transfected with a human vitamin D receptor (hVDR) expression vector (pRSV-hVDR) into COS-1 cells. These experiments showed that the flanking region from -298 to -122 directed a 24-fold increase in luciferase activity in response to 1,25-(OH)2D3 provided that the cells were co-transfected with pRSV-hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)2D3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase promoter. Mutagenesis revealed that the sequence from position -150 to -136 is required for induction by 1,25-(OH)2D3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) reported for other genes. Gel shift mobility assays showed this region specifically bound a nuclear protein complex from 1,25-(OH)2D3 treated COS-1 cells that had been co-transfected with pRSV-hVDR. The retarded band was specifically competed with the well characterized VDRE from the mouse osteopontin gene. A VDRE at position -150 to -136 in the promoter of the rat P450(24) gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.
Department of Biochemistry, University Adelaide, Australia.
C N
CN
Hahn
D M
DM
Kerry
J L
JL
Omdahl
B K
BK
May
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics*; D014162:Transfection; D015854:Up-Regulation; D065668:Vitamin D3 24-Hydroxylase
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol