| Project/Area Number |
16592081
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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 |
Periodontal dentistry
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| Research Institution | Nihon University |
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Principal Investigator |
OGATA Yorimasa Nihon University, School of Dentistry at Matsudo, Professor, 松戸歯学部, 教授 (90204065)
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| Co-Investigator(Kenkyū-buntansha) |
SAMOTO Hiroshi Nihon University, School of Dentistry at Matsudo, Research Assistant (Full-Time), 松戸歯学部, 助手 (50366613)
NAKAO Sumi Nihon University, School of Dentistry at Matsudo, Research Assistant (Full-Time), 松戸歯学部, 助手 (20102577)
清水 映美 日本大学, 松戸歯学部, 講師 (80349969)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Bone sialoprotein / Extracellular matrix / Osteoblasts / Transcription factor / Transcriptional regulation / Gene promoter / Enamel matrix derivative / LPS / メカニカルストレス / インシュリン様成長因子 / リポポリサッカライド / FGF2 / cAMP / 歯周組織再生 / 石灰化 / 腫瘍壊死因子 / プロスタグランジンE2 / 静磁場 |
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| Research Abstract |
BSP sialoprotein (BSP) is a glycoprotein that essentially unique to mineralize tissues. Therefore, BSP may function in the initial nucleation of hydroxyapatite crystal in de novo bone formation.
Enamel matrix derivative (EMD) has been developed as a periodontal regenerative treatment. While EMD is believed to induce regeneration of periodontal tissue, the precise mechanism is not known. Using the osteoblast like ROS 17/2.8 cells, we revealed that BSP mRNA levels increased 〜2.8-fold by EMD. In transient transfection analyses, EMD (50 μg/ml, 12 h) increased luciferase activities of pLUC4 (nt -425 to +60) and pLUC5 (nt -801 to +60), transfected into ROS 17/2.8 cells. Within the pLUC4 and 5, a homeodomain binding element (HOX) and a TGF-beta activation element (TAE) are present. Gel mobility shift assays with radiolabeled HOX and TAE ds-oligonucleotides revealed increased binding of nuclear proteins from EMD stimulated ROS 17/2.8 cells.
Lipopolysaccharide (LPS) is mediator of inflammatory responses in periodontal disease. LPS (1 μg/ml, 12 h) caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC ; 20mM) 30 min prior to stimulation with LPS attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, revealed that LPS (1 μg/ml) suppressed expression of luciferase construct (-108 to +60), transfected into ROS17/2.8 cells. The effects of LPS were inhibited by PKA inhibitor H89 and the tyrosine kinase inhibitor, herbimycin A. Introduction of 2bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 showed that the LPS effects were mediated by the CRE and FRE. Whereas the FRE and 3'-FRE DNA-protein complexes were decreased by LPS, CRE-protein complex did not change after LPS treatment. These studies show that LPS suppresses BSP gene transcription through PKA and tyrosine kinase-dependent pathways and that the LPS effects are mediated through CRE and FRE elements in the proximal BSP gene promoter.
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