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Clarification of the interaction between the differentiation and immune regulation of goblet cells by Math1.

Math1阐明杯状细胞分化与免疫调节之间的相互作用。

基本信息

批准号：
17590621
负责人：
TSUCHIYA Kiichiro
金额：
$ 2.24万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590621/
关键词：
Wnt signal Hath1 Transcription Intestine 腸管分化杯細胞 Wntシグナル蛋白分解

项目摘要

Atoh1/Hath1 is indispensable to the differentiation of the intestinal epithelial cells, however what regulates Hath1 function has unknown. So we assessed the effect of Wnt signal for Hath1 and then, we revealed new signaling pathway for Hath1 in Wnt signals. The Hath-1 protein was actively degraded in SW480 cells where the Wnt signal is constitutively activated. In contrast, the breakdown of Hath-1 did not occur in 293T cells in which the Wnt signal remains inactivated. The degradation of Hath-1 in SW480 cells was inhibited by the treatment with MG132, an inhibitor for the proteasome, and the accumulated Hath-1 was shown to be ubiquitinated, indicating a role of ubiquitin-proteasome pathway in this process. Mutational analysis of Hath-1 showed that the critical residues regulating the proteolysis in SW480 cells are S54 and S58 that correspond to the consensus sequence for GSK-3 substrates. Indeed, pharmacological GSK-3 inhibitors blocked the Hath-1 degradation in SW480 cells. Importantly, the degradation of Hath-1 in SW480 cells was also inhibited by cotransfection of wt-APC, which reciprocally enhanced the degradation of β-catenin. Conversely, coexpression of Wnt1 in 293T cells resulted in the proteolysis of Hath-1, while it facilitated the accumulation of β-catenin.Next we demonstrated that the identification of genes directly targeted by Hath1. Several genes were selected by RNA micro array analysis or CHIP-on-chip analysis.We showed a novel branching of the Wnt-GSK-3 axis of signaling pathway, suggesting its important role in balancing the cellular differentiation and proliferation via the inverse regulation for the stability of Hath-1 and p-catenin proteins.

Atoh1/Hath1对肠上皮细胞的分化是不可或缺的，但Hath1功能的调节机制尚不清楚。因此，我们评估了Wnt信号对Hath1的影响，进而揭示了Hath1在Wnt信号中的新的信号通路。在Wnt信号被结构性激活的SW480细胞中，Hath-1蛋白被活跃地降解。相反，在Wnt信号仍然失活的293T细胞中，Hath-1的分解没有发生。蛋白酶体抑制剂MG132可抑制SW480细胞中Hath-1的降解，且Hath-1的积聚被泛素化，这表明泛素-蛋白酶体途径在此过程中起作用。Hath-1的突变分析表明，调控SW480细胞蛋白降解的关键残基是S54和S58，它们与GSK-3底物的共识序列相对应。事实上，药物GSK-3抑制剂阻止了SW480细胞中Hath-1的降解。重要的是，共转染wt-APC也抑制了Hath-1在SW480细胞中的降解，从而促进了β-catenin的降解。相反，WNT1在293T细胞中的共表达导致了Hath-1的蛋白分解，同时促进了β-Catenin的积累。通过RNA微阵列分析或芯片分析选择了几个基因，我们发现了Wnt-GSK-3信号通路轴的一个新的分支，表明它通过反向调节Hath-1和p-catenin蛋白的稳定性而在平衡细胞分化和增殖中发挥重要作用。