Nrf2による新しい酸化ストレス応答機構の解析

• Project/Area Number: 11877025
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Pathological medical chemistry
• Research Institution: University of Tsukuba
• Principal Investigator: 山本 雅之 筑波大学, 基礎医学系, 教授 (50166823)
• Project Period (FY): 1999 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
• Keywords: 異物代謝 / 環境異物 / 転写制御 / モデル動物 / bZip型転写因子 / 転写因子 / Nrf2 / 酸化ストレス / 親電子性物質 / Keap1 / 蛋白質分解 / プロテオソーム

Research Abstract

今年度は以下の成果を得た.
1)親電子性物質・活性酸素種によるNrf2蛋白質蓄積の分子機構の解析
Nrf2が親電子性物質により蓄積するメカニズムを腹腔マクロファージを用いて調べた。親電子性物質とプロテオソーム阻害剤にはNrf2蓄積に対する協調効果が認められなかったことより、新電子性物質は、プロテオソーム阻害剤と同じ経路でNrf2を蓄積することが示唆された。さらに、Nrf2の半減期が親電子性物質により延長することより、新電子性物質がNrf2を安定化することが明らかになった。Keaplとの相互作用に重要であるNeh2ドメインを用いたNeh2-GFP融合蛋白質の半減期は,GFP蛋白質やNeh5-GFP蛋白質に比較して著明に短縮していることより,Neh2ドメインが,Nrf2蛋白質の分解に寄与することが示唆された。
2)Keapl遺伝子破壊実験によるKeaplの生体機能の解析
Keapl遺伝子破壊マウスを作製して,肝臓におけるNrf2の蓄積を調べたところ,同因子が著明に蓄積していることが明らかになった.また,Nrf2の標的遺伝子である異物代謝系第2相の酵素群の発現が上昇していることも明らかになった.さらに,Keapl :: Nrf22重欠損マウスでは,これらの酵素の誘導が認められず,Keapl遺伝子欠損マウスの表現型は,Nrf2の過剰状態を反映していることが示された.以上により,Nrf2の活性は,Keaplにより負に制御されていることが個体レベルで証明された.

Research Products

• [Publications] Nishimura,S.: "A GATA box in the GATA-1 gene hematopoietic enhancer is critical element in the network of GATA factors and sites that regulate this gene."Mol.Cell.Biol.. 20. 713-723 (2000)
• [Publications] Pan,X..: "Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions."J.Biochem.. 127. 105-112 (2000)
• [Publications] Kobayashi,A.: "A combinatorial code for gene expression generated by transcription factor B ach2 and MAZR (MAZ-related factor) through BTB/POZ domain."Mol.Cell.Biol.. 20. 1733-1746 (2000)
• [Publications] Onodera,K.: "Perinatal synthetic lethality and hematopoietic defects in compound mafG :: mafK mutant mice."EMBO J. 19. 1335-1345 (2000)
• [Publications] Katsuoka,F.: "One enhancer mediates mafK transcriptional activation in both hematopoietic and cardiac muscle cells."EMBO J.. 19. 2980-2991 (2000)
• [Publications] Ishii,T.: "Induction of murine intestinal and hepatic peroxiredoxin MSP23 by dietary butylated hydroxyanisole."Carcinogenesis. 21. 1013-1016 (2000)
• [Publications] Hoshino,H.: "Oxidative stress abolishes leptomycine B-sensitive nuclear export of transcription repressor B ach2 that counteracts activation of Maf recognition element."J.Biol.Chem.. 275. 15370-15376 (2000)
• [Publications] Ishii,T.: "Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages."J.Biol.Chem.. 275. 16023-16029 (2000)
• [Publications] Takahashi,S.: "GATA factor transgenes under GATA-1 locus control rescue germ line GATA-1 mutant deficiencies."Blood. 96. 910-916 (2000)
• [Publications] Takahashi,S-L: "Cloning of a coproporphyrinogen oxidase promoter regulatory element binding protein."Biochem.Biophys.Res.Commun.. 2732. 596-602 (2000)
• [Publications] Hayes,J.D.: "The Nrf2 transcription factor contributes both to the basal expression of glutathion S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin."Biochem.Soc.Trans.. 28. 33-41 (2000)
• [Publications] Zhou,Y.: "GATA2 is required for the generation of V2 intemeurons."Development. 127. 3829-3838 (2000)
• [Publications] Toki,T.: "Functional characterization of the two alternative promoters of human p45 NF-E2 gene."Exp.Hematol.. 28. 1113-1119 (2000)
• [Publications] Wakabayashi,N.: "A novel cis-acting element regulates HES-1 gene expression in P19 embryonal carcinoma cells treated with retinoic acid."J.Biochem.. 128. 1087-1095 (2000)
• [Publications] Motohashi,H.,: "Positive or negative MARE-dependent transcriptional regulation is determined by the abundance of small Maf proteins."Cell. 103. 865-875 (2000)
• [Publications] Itoh,K.: "Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the N-terminal Neh2 domain."Genes Dev.. 13. 76-86 (1999)
• [Publications] Kobayashi,A.: "Molecular cloning and functional characterization of a new CNC family transcription factor Nrf3."J.Biol.Chem.. 274. 6443-6452 (1999)
• [Publications] Minegishi,N.: "The mouse GATA-2 gene is expressed in the aorta-gonads-mesonephros region."Blood. 93. 4196-4207 (1999)
• [Publications] Kawauchi,S.: "Regulation of lens fiber cell differentiation by transcription factor c-Maf."J.Biol Chem.. 274. 19254-19260 (1999)
• [Publications] Onodera,K.: "Characterization of the murine mafF gene."J.Biol.Chem.. 274. 21162-21169 (1999)
• [Publications] Yoshida,C.: "Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1."Genes Cells. 4. 643-655 (1999)
• [Publications] Nakajima,O.: "Heme deficiency in erythroid Iineage causes differentiation arrest and cytoplasmic iron overload."EMBO J.. 18. 6282-6289 (1999)
• [Publications] Nishimura,S.: "A GATA box in the GATA-1 gene hematopoietic enhancer is critical element in the network of GATA factors and sites that regulate this gene."Mol.Cell.Biol.. 20. 713-723 (2000)
• [Publications] Kobayashi,A.: "Acombinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through BTB/ POZ domain."Mol.Cell.Biol.. 20. 1733-1746 (2000)
• [Publications] Pan,X.: "Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions."J.Biochem.. 127. 105-112 (2000)
• [Publications] Onodera,K.: "Perinatal Synthetic Lethality and Hematopoietic Defects in Compound mafG:: mafK Mutant Mice."EMBO.J.. (印刷中). (2000)