2002 Fiscal Year Final Research Report Summary
Isolation and analysis of molecules which associate with Csx
| Project/Area Number |
12470148
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | The University of Tokyo |
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Principal Investigator |
HIROI Yukio The University of Tokyo Hospital, Research Associate, 医学部附属病院, 助手 (30311624)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Dobun University of Tokyo Pharmacoepidemiology, Assistant Professor, 大学院・医学系研究科, 寄付講座教員 (80313104)
KOMURO Issei Chiba University, Faculty of Medicine, Professor, 医学部附属病院, 教授 (30260483)
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| Project Period (FY) |
2000 – 2002
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| Keywords | Csx / Nkx2-5 / Tbx5 / ANP / synergistic action / cardiac defect / Holt-Oram syndrome |
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| Research Abstract |
To elucidate how Csx/Nkx2-5 regulates cardiac development, we screened a human heart cDNA library by the yeast two-hybrid system using human Csx/Nkx2-5 as a bait. We isolated a T-box transcription factor, Tbx5 whose mutations have been reported to cause heart and upper limb malformations, Holt-Oram syndrome (HOS). Co-transfection of Tbx5 and Csx into COS-7 cells revealed that they associate with each other also in mammalian cells. GST pull-down assays indicated that the N-terminal domain and N-terminal part of T-box of Tbx5 and the homeodomain of Csx were necessary for their interaction. Tbx5 activated the cardiac specific atrial natriuretic peptide (ANP) promoter like as Csx. Both transcription factors directly bound to the ANP promoter in tandem and they showed a synergistic activation. Deletion analysis revealed that both the N-terminal domain and the T-box were important for this transactivation. Two misssense mutations of Tbx5, Gly80Arg and Arg237Gln, have been reported in HOS. The former causes marked cardiac defects with minor skeletal abnormalities, while the latter causes extreme upper limb malformations without significant cardiac abnormalities. While the Arg237Gln mutant activated the ANP promoter as strong as the wild type Tbx5, the Gly80Arg mutant activated it significantly less than the wild type Tbx5. P19CL6 cell lines overexpressing the wild type Tbx5 started to beat much earlier and stronger and expressed more abundant cardiac specific genes than the parental P19CL6. The cell lines overexpressing the Gly80Arg mutant did not differentiate into beating cardiomyocyte. These results suggest that Tbx5 plays a critical role in cardiomyocyte differentiation in concert with Csx/Nkx2-5.
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[Publications] Takimoto E, Mizuno T, Terasaki F, Shimoyama M, Honda H, Shiojima I, Hiroi Y, Oka T, Hayashi D, Hirai H, Kudoh S, Toko H, Kawamura K, Nagai R, Yazaki Y, Komuro I.: "Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice"Biochem Biophys Res Commun.. 270(3). 1074-1079 (2000)
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[Publications] Hiroi Y, Chen R, Sawa H, Hosoda T, Kudoh S, Kobayashi Y, Aburatani H, Nagashima K, Nagai R, Yazaki Y, Medof ME, Komuro I.: "Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1"Am J Physiol.. 279(1). C205-C212 (2000)
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[Publications] Monzen K, Hiroi Y, Kudoh S, Akazawa H, Oka T, Takimoto E, Hayashi D, Hosoda T, Kawabata M, Miyazono K, Ishii S, Yazaki Y, Nagai R, Komuro I.: "Smads, tak1, and their common target atf-2 play a critical role in cardiomyocyte differentiation"J Cell Biol.. 153(4). 687-698 (2001)
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[Publications] Zou Y, Hiroi Y, Uozumi H, Takimoto E, Toko H, Zhu W, Kudoh S, Mizukami M, Shimoyama M, Shibasaki F, Nagai R, Yazaki Y, Komuro I.: "Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy"Circulation.. 104(1). 97-101 (2001)
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[Publications] Harada T, Aoyagi T, Endo Y, Uno K, Takenaka K, Nakamura F, Sonoda M, Takahashi T, Yamazaki T, Hiroi Y, Ohno M, Hirata Y, Nagai R.: "Effusive constrictive pericarditis due to rheumatoid arthritis revealed by pericardiocentesis with simultaneous pressure recording--a case report"Angiology.. 53(1). 105-108 (2002)
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[Publications] Toko H, Zhu W, Takimoto E, Shiojima I, Hiroi Y, Zou Y, Oka T, Akazawa H, Mizukami M, Sakamoto M, Terasaki F, Kitaura Y, Takano H, Nagai T, Nagai R, Komuro I.: "Csx/Nkx2-5 is required for homeostasis and survival of cardiac myocytes in the adult heart"J Biol Chem.. in press. (2002)
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