| Project/Area Number |
09671896
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
UENO Akemichi The University of Tokushima, School of Dentistry, Associate Professor, 歯学部, 助教授 (80136267)
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| Co-Investigator(Kenkyū-buntansha) |
MIWA Yoshihiro University of Tsukuba, Institute of Basic Medical Science, Instructor, 基礎医学系, 講師 (70263845)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | thrombospondin / mineralization / extracellular matrix proteins / MC3T3-E1 cells / antisense oligonucleotides / von Kossa staining / stable transfectant / overexpression / Stable transformant / 欠損変異株 / プロテオグリカン / MC 3T3-E1細胞 / von Kossa 染色 / Lac Swich / IPTG / stable transformant |
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| Research Abstract |
TSP1 was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. Bovine TSP1 cDNA containing acomplete ORF of 1170 amino acids has high homologies to its human and mouse counteroarts. Immunohistochemical analyses of bovine anterior teeth with anti-TSP1 antibody, 5G11 revealed that TSP1 was localized at predentin between. Northern blot analysis of total RNA from bovine endodontal and periodontal tissues with bovine TSP1 cDNA showed that two sizes of TSP1 mRNAs were highly expressed in odontoblasts but not in dental pulp and gingiva. To make clear the role of TSP1 in predentin and osteoid, located at interface between mineralized and unmineralized tissues, mouse calvaria-derived osteoblastic cells, MC3T3-E 1 were treated with antisense phosphorothioate oligodeoxynucleotides complementary to TSP1 sequences. The 18mer antisense probe, TAS-2, caused a potent and concentration-dependent increase in the number of mineralized nodules and adecrease in the steady state expression of TSP1 in MC3T3-E1 cells even with submicromolar concentrations in the absence of β-glycerophosphate. The corresponding sense, TS-2 or scrambled oligonucleotide, STAS-2 did not show any effects. Then, MC3T3-E1 cells were transfected with a TSP1 expression vector, pBK-CMV-mTSP1, stably transfected cell lines overexpressing recombinant TSP1 were established, and assayed for TSP1 protein and the capacity to mineralize. Constitutive expression of TSP1 dose-dependently inhibited mineralization by these cells. Furthermore, exogenous purified TSP1 also suppressed the production of a mineralized matrix by MC3T3-E1 cells.
These results suggest that TSP1 is a negative regulator of biomineralization by bone and odontoblastic cells and might play a role in protection from mineralization of pulp as well as tissues adjacent to bone.
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