| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Although tissue-nonspecific alkaline phosphatase (TNSALP) is a well-known marker of bone formation, its regulatory mechanism by fat-soluble vitamins affecting bone metabolism has not been understood. The aim of the present study was to clarify this mechanism. In a human osteoblastic osteosarcoma cell line, SaOS-2, mRNA levels and enzymatic activity of TNSALP were induced by 10^<-6> M all-trans-retinoic acid, and in another cell line, MG-63, they were induced by 10^<-7> M 1,25-dihydroxyvitamin D. In addition, 4.5 kb of the upstream region of the human TNSALP gene was cloned and sequenced, and inserted into luciferase expression vectors, and a set of deletion mutants were created. Luciferase assays using these vectors showed that the upstream region containing a retinoic acid response element-like motif possessed transcriptional activity. EMSA assays revealed specific binding of the nuclear extract of SaOS-2 cells to this motif, and the assays with the antibodies against retinoic acid receptors α,β, and retinoid X receptor β that forms a heterodimer with the retinoic acid receptors, showed supershift bands, indicating that retinoic acid regulates TNSALP via a retinoic acid response element in the upstream region (-1012 to -999). Expression of those receptors in the cells was also confirmed This study has been published in Bone 36 (2005) 866-876.
On the other hand, luciferase assays using MG-63 cells and vitamin D did not show activation of transcription and a vitamin D response element like-motif in the upstream region (-3444 to -3430) was not functional. Negative vitamin D response element-like motifs showed decreased transcription activity but no specific binding in EMSA assays. These results suggest that modulation of TNSALP by vitamin D may be indirect action. Currently, changes of mRNA stability by vitamin D are being analyzed, and the study on vitamin D with this data will be published.
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