| Project/Area Number |
07671124
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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 |
内分泌・代謝学
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| Research Institution | Nagoya University |
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Principal Investigator |
KAMBE Fukushi Nagoya Univ., Res.Inst.Environ.Med.Research Associate, 環境医学研究所, 助手 (00211871)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAYA Takashi Nagoya Univ., Res.Inst.Environ.Med.Research Associate, 環境医学研究所, 助手 (80262913)
MURATA Yoshiharu Nagoya Univ., Res.Inst.Environ.Med.Associate Professor, 環境医学研究所, 助教授 (80174308)
SEO Hisao Nagoya Univ., Res.Inst.Environ.Med.Professor, 環境医学研究所, 教授 (40135380)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | TTF-1 / Pax-8 / Thyrotropin(TSH) / Transcription factor / Thyroid / Thyroglobulin / Redox / Gene Expression / 甲状腺刺激ホルモン / インスリン / プロモーター |
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| Research Abstract |
Thyroid-transcription factors, Pax-8 and TEE-1, are involved in the thyroid-specific expression of thyroglobulin (TG) gene. We studied the redox regulation of both factors in vitro and in vivo. When analyzed by electrophoretic mobility shift assay (EMSA), oxidation with diamide abolished the DNA binding of Pax-8. Subsequent reduction with dithiothretiol (DTT) restored the binding. Thioredoxin (TRX), a cellular reducing catalyst, restored the binding more efficiently than DTT.When TTF-1 was oxidized with diamide, its binding was decreased and the TTF-1-DNA complex migrated faster on EMSA.DTT reversed these effects. These observations indicate that reduction is required for full DNA binding of Pax-8 and TTF-1 in vitro. We then examined whether TSH modulates their binding through redox regulation. Whole cell extracts were prepared from FRTL-5 cells at intervals after TSH treatment without reducing agents and subjected to EMSA.Pax-8 and TTF-1 binding activities were gradully increased during the initial 6 hours after TSH.This increase was due to reduction of the factors, since treatment of the extracts with DTT masked the increase by enhancing their binding activities. These results suggest that TSH up-regulates the binding of Pax-8 and TTF-1 at least in part by reducing the pre-existing, oxidized forms in FRTL-5 cells. Northern analysis showed that the increase in TRX mRNA level by TSH in FRTL-5 cells was associated with the increase in the binding activities. Cotransfection of luciferase-reporter plasmid driven by TG promoter with Pax-8-and TRX-expressing plasmids into heterologous cells revealed that TRX up-regulated the Pax8-mediated TG promoter activity. Taken together, the present study suggests that the redox regyulation of Pax-8 and TTF-1 by TSH probably through TRX modulates the TG gene expression.
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