| Co-Investigator(Kenkyū-buntansha) |
AKIYAMA Shuichi School of Dentistry, Showa University, Assistant, 歯学部, 助手 (70276591)
MIYAURA Chisato School of Dentistry, Showa University, Lecturer, 歯学部, 講師 (20138382)
SUDA Tatsuo School of Dentistry, Showa University, Professor, 歯学部, 教授 (90014034)
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| Research Abstract |
The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D_3 1alpha- and 24-hydroxylases, Previously, we reported that renal proximal convoluted tubules, the major site of 1alpha, 25-hydroxyvitamin D_3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1alpha, 25-dihydroxyvitamin D_3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D_3 1alpha- and 24-hydroxylase activities. In rat models of renal production of 1alpha, 25-hydroxyvitamin D_3, expression of vitamin D receptors and 25-hydroxyvitamin D_3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts.
By using the method of mixed oligonucleotide-primed amplification of cDNA,we have cloned a new sequence of the cytochrome P450 mixed function oxidase from the kid
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ney of vitamin D-deficient rats. A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D_3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1 (+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D_3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha consisted of 2469bp in length and contained an open reading frame encoding 501 amino acids. The deduced amino acid sequence showed less than 30% similarity to those of all other P450s reported to date. Thus, it is a new member of the P450 superfamily. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha, 25-dehydroxyvitamin D_3 (rats fed a low Ca diet), expression of P4501alpha mRNA was greatly enhanced in the renal proximal convoluted tubules. These results clearly demonstrate that the expression of P4501alpha is regulated at a transcriptional level. Less
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