2007 Fiscal Year Final Research Report Summary
Hypoxia inducible gene manipulation and clinical application.
| Project/Area Number |
18591040
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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 |
Hematology
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| Research Institution | University of Tsukuba |
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Principal Investigator |
IMAGAWA Shigehiko University of Tsukuba, GSCHS, Professor (60231164)
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| Co-Investigator(Kenkyū-buntansha) |
OHNEDA Osamu University of Tsukuba, 大学院・人間総合科学研究科, GSCHS, Professor (30311872)
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| Project Period (FY) |
2006 – 2007
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| Keywords | erythropoietin / HIF / GATA / VEGF / transcription factor / gene expression / promotor / enhancer |
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| Research Abstract |
Erythropoietin (Epo) gene expression is under the control of HIF-1 and is positively regulated through an HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. In response to anemia, Epo gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ecto
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pic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene. In oxygenated cells, hypoxia inducible factor-1 (HIF-1) α subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1 α protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1 α protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis. Less
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[Journal Article] c-Maf expression in angioimmunoblastic T-cell lymphoma2007
Author(s)
Idate Murakami Y, Yatabe Y, Sakaguchi Y, Saki E, Yamashita Y, Morito N, Yoh K, Fujioka Y, Matsuno F, Hata H, Mitsuya H, Imagawa S, Suzuki A, Esumi H, Sakai M, Takahasbi S, Mori N.
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Journal Title
Am J Surg Pathol 31
Pages: 1695-1702
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Detection of hypoxia-inducible gene manipulation2007
Author(s)
Imagawa S, Horie M, Matsumoto K, Hirano I, Suzuki N, Yamamoto M.
Organizer
The 2nd JST-ERATO Yamamoto Environmental Response Project International Symposium : The Environmental Response
Place of Presentation
Tsukuba, Japan
Year and Date
2007-12-22
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Detection of hypoxia inducible gene manipulation.2007
Author(s)
Imagawa S, Horie M, Matsumoto K, Hirano I, Suzuki N, Yamamoto M.
Organizer
The 2nd JST-ERATO Yamamoto Environmental Response Project Internationl Symposium: The Environmetntal Response.
Place of Presentation
Tsukuba, Japan
Year and Date
2007-12-21
Description
「研究成果報告書概要(和文)」より
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