| Project/Area Number |
15209014
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | Nagoya University |
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Principal Investigator |
TAKAHASHI Masahide Nagoya University, Graduate School of Medicine, Professor, 大学院医学系研究科, 教授 (40183446)
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| Co-Investigator(Kenkyū-buntansha) |
ICHIHARA Masatoshi Chubu University, Life Health Sciences, Professor, 生命健康科学部, 教授 (00314013)
MURAKUMO Yoshiki Nagoya University, Graduate School of Medicine, Associate Professor, 大学院医学系研究科, 助教授 (40324438)
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| Project Period (FY) |
2003 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥50,310,000 (Direct Cost: ¥38,700,000、Indirect Cost: ¥11,610,000)
Fiscal Year 2006: ¥12,870,000 (Direct Cost: ¥9,900,000、Indirect Cost: ¥2,970,000)
Fiscal Year 2005: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
Fiscal Year 2004: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
Fiscal Year 2003: ¥13,780,000 (Direct Cost: ¥10,600,000、Indirect Cost: ¥3,180,000)
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| Keywords | GDNF / RET / tyrosine kinase / protein kinase A / JNK / cell movement / enteric nervous system / knock-in mice / RETチロシンキナーゼ / 遺伝子改変マウス / 形態形成 / 発癌 / トランスジェニックマウス / ノックアウトマウス / 発がん / 腎臓 |
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| Research Abstract |
The RET receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in RET represents a binding site for the PTB domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, PI3K/AKT, and JNK pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice were generated in which tyrosine 1062 in the Ret gene was replaced with phenylalanine. Although homozygous knock-in mice were born normally, they died by day 27 after birth with growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract as observed in Ret-deficient mice. The rest of the mutant mice exhibited the development of enteric neurons in the intestine to varying extent, although the size and number of ganglion cells were
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significantly reduced. Unlike Ret-deficient mice, the small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.
In addition, we demonstrated that the Rac1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of Ret in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the Ret signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not Erk, Akt and Src activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of Ret function regulates JNK signaling responsible for proper migration of the ENCCs in the developing gut. Less
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