| Project/Area Number |
15591942
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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 |
Morphological basic dentistry
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| Research Institution | Health Sciences University of Hokkaido |
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Principal Investigator |
YAJIMA Toshihiko Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (10018749)
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| Co-Investigator(Kenkyū-buntansha) |
IRIE Kazuharu Health Sciences University of Hokkaido, School of Dentistry, Assistant Professor, 歯学部, 講師 (70223352)
TSURUGA Eichi Health Sciences University of Hokkaido, School of Dentistry, Instructor, 歯学部, 助手 (30295901)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | elastic system fibers / elastin / fibrillin / fibulin / MAGP-1 / fibroblasts / periodontal tissues / elaunin fibers / MAGP-2 / MMP-2 / TIMP-2 / フィブリン / オキシタラン線維 |
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| Research Abstract |
Elastic system fibers amsist of microflbrils and tropoelstin. There are three types-oxytalan, elaunin, and elastic fibers-which differ in their relative micmfibril and elastin eontens. During development, microfibrils act as a temolate on which tropoelastin is deposited.
1.We examined gene xepression and accumulation of tropoelastin in the Deli-matrix layer of human gingival fibroblasts (EHGF) and human periodontal ligament f fibroblasts (HPLF) in vitro. Northern blotting and RT-PCR analysis showed that only HGF expressed mRNA encoding tropoelastin. Western blotting analysis demonstrated ptopoelastin (77- and 68-kDa) only in the cell-matrix layer of HGF. These results suggest that the different tropoelastin expression patterns reflect the difference between HGF and HPLF Dhenotvoes.
2.Microfibril-associated glycoprotein-1 (MAGP-l) and fibrillin-2, the major components of microfibrils, provide the likely template far tropelastin deposition. We used the RNA interference (RNAi) technique to
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establish MAGP-1 and fibrillin-2 gene-specific knockdowns individually in elastin-producing cells (HGF) and examined the extr"- Ilular deposition of tropoelastin by western blotting. These two genes were specaifically suppressed to 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluoreseence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extraeellular deposition of tropoelastin during elastic fiber formation in HGF invitro.
3.Fibulin-5 (also knouwn as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (HGF) with non-producing cells (HPLP) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing e Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of tropoelastin gene, and the fibuifin-5 level was reduced upon RNAi-Mediated down-regulation of tmpoelastin. Fibulin.-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.
4.We examined possibility of involvement of matrix metalloprotenases (MMPs) in remodeling of fibrilins by HGF and HPLF. There was no difference in fibrillin-1 accumulation between HGF and HPLF, but fibrillin-2 accumulated to much smaller extent in the HPLF Pro MW-2 was detected in both cell types, while active MMP-2 was observed only HPLF. Addition of AMP-inhibitor or T1MP resulted to increase fibrillin-2 accummulation in HPLF significantly. These results suggested that MW-2 activated in the HPLF cell layer degrades fibrillin-2, and MMPs may play arole in remodeling of elastic system fibers in periodontal tissues, Less
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