[Cloning and expression of 25-hydroxyvitamin D3-1 alpha-hydroxylase gene]

Abstract

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1 alpha-hyroxylase, was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When 1 alpha-hydroxylase cDNA was transfected into COS-7 cells, they expressed 25-hydroxyvitamin D3-1 alpha-hydroxylase activity. The sequence analysis showed that 1 alpha-hydroxylase cDNA consisted of 2469 bp in length and contained an open reading frame encoding 501 amino acids. The expression of 1 alpha-hydroxylase mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3 (rats fed a low Ca diet), expression of 1 alpha-hydroxylase mRNA was greatly enhanced in the renal proximal convoluted tubules. These results clearly demonstrate that the expression of 1 alpha-hydroxylase is regulated at a transcriptional level. The DNA flanking the 5'-sequence of the mouse 1 alpha-hydroxylase gene has been cloned and sequenced. The promoter has 3 potential CRE sites, 2 perfect and 1 imperfect AP-1 sites, while no DR-3 was detected. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, while forskolin stimulates it by 3-fold. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1 alpha-hydroxylase by acting on the promoter of the 1 alpha-hydroxylase gene.