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

Atoh 1/Hath 1是肠上皮细胞分化所必需的，但Hath 1功能的调控机制尚不清楚。因此，我们评估了Wnt信号对Hath 1的影响，并在Wnt信号中揭示了Hath 1的新信号通路。Hath-1蛋白在SW 480细胞中被主动降解，其中Wnt信号被组成性激活。相反，在Wnt信号保持失活的293 T细胞中，Hath-1的分解没有发生。蛋白酶体抑制剂MG 132处理SW 480细胞后，Hath-1的降解被抑制，Hath-1的积累被泛素化，表明泛素-蛋白酶体途径在此过程中发挥作用。Hath-1的突变分析表明，在SW 480细胞中调节蛋白水解的关键残基是S54和S58，其对应于GSK-3底物的共有序列。事实上，药理学GSK-3抑制剂阻断了SW 480细胞中的Hath-1降解。重要的是，共转染wt-APC也抑制了SW 480细胞中Hath-1的降解，这也促进了β-catenin的降解。相反，Wnt 1在293 T细胞中的共表达导致了Hath-1的蛋白水解，同时促进了β-catenin的积累。通过RNA微阵列分析和芯片分析筛选出多个基因，发现Wnt-GSK-3轴信号通路中存在一个新的分支，提示其通过反向调节Hath-1和β-catenin蛋白的稳定性，在平衡细胞分化和增殖中发挥重要作用。

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.