Nucleic Acids Research, 1994, Vol. 22, No. 12 2410-2416
© 1994
MOLECULAR BIOLOGY |
Identification of a vitamin D responsive element in the promoter of the rat cytochrome P45024 gene
Department of Biochemistry, The University Adelaide South Australia 5005, Australia 1Department of Biochemistry, University of New Mexico Albuquerque, NM 87131, USA
*To whom correspondence should be addressed
Received December 10, 1993. Revised June 12, 1994. Accepted June 12, 1994.
Mitochondrial cytochrome P45024 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 P45024 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 P45024 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 P45024 gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.
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