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-degradat ion pathway is induced by 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3]. The molecular basis of this enzyme regulation was investigated by iso lating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction proc ess. 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 flankin g region from - 298 to - 122 directed a 24-fold increase in luciferase activity in response to 1,25(OH)(2)D-3 provided that the cells were c o-transfected with pRSV - hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)(2)D-3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase pr omoter. Mutagenesis revealed that the sequence from position -150 to - 136 is required for induction by 1,25(OH)(2)D-3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) repo rted for other genes. Gel shift mobility assays showed this region spe cifically bound a nuclear protein complex from 1,25-(OH)(2)D-3 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 pro moter of the rat P450(24) gene is identified in this study and found t o be important in mediating the enhanced expression of the gene by 1,2 5-(OH)(2)D-3.