2001 Fiscal Year Final Research Report Summary
Regulation of the alkaline phosphatase gene expression by fat-soluble vitamins in human osteoblastic cells
| Project/Area Number |
12670123
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General medical chemistry
|
|---|
| Research Institution | Nippon Medical School |
|---|
Principal Investigator |
ORIMO Hideo Nippon Medical School, Faculty of Medicine, Associate Professor, 医学部, 助教授 (40177308)
|
|---|
| Project Period (FY) |
2000 – 2001
|
| Keywords | tissue-nonspecific alkaline phosphatase / retinoic acid / vitamin D / regulation of expression / retinoic acid response element / vitamin D response element / osteosarcoma cell lines |
|---|
| 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.
|
Research Products
(2 results)
