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Functional analysis of tuberin by using animal models of tumor suppressor Tsc2-mutant.

利用抑癌基因 Tsc2 突变体动物模型对马铃薯蛋白进行功能分析。

基本信息

批准号：
14580804
负责人：
KOBAYASHI Toshiyuki
金额：
$ 2.24万
依托单位：
Japanese Foundation for Cancer Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580804/
关键词：
Tsc2 tuberin knockout mouse transgenic Eker rat renal tumor tuberous sclerosis S6 kinase Erc mesothelin

项目摘要

Expression vector for Tsc2 (tuberous sclerosis 2 gene) cDNA was introduced in Tsc2-deficient mouse renal tumor (RT) cell line (MKOC1.-277) and stable lines expressing tuberin (T2 cells) were established. Phosphorylation of p70 S6 kinase (S6K) was suppressed in T2 cells as compared with control cell lines with empty vector. Tumorigenic potential of RC cells in the nude mouse was suppressed by tuberin expression. Administration of rapamycin, a mTOR inhibitor, also blocked tumorigenesis of RC cells. These results suggest that mTOR〜S6K pathway has some role in tumorigenesis caused by Tsc2-mutation. Interestingly, expression of Erc mRNA (mesothelin gene homologue) was also suppressed in T2 cells. In contrast, rapamycin treatment of parental MKOC1-277 cells did not change expression level of Erc mRNA. These results suggest that Erc is regulated by downstream signaling pathway of tuberin, which is independent of mTOR〜S6K. This novel tuberin-regulated pathway may be a new candidate for therapeutic molecular target for tuberous sclerosis. To search for pathways regulating Erc expression, Hela cells were treated with various stimuli or chemical inhibitors and activators of signal transduction pathways and analyzed by northern and western blot analyses. In the case of serum starvation, amino-terminal half of Erc protein was increased, probably by protease-mediated cleavage. Thus, expression and function of Erc protein may be regulated by post-translational level in relation with cell growth. In this study, search for candidates of tuberin-binding protein was also performed. Gamma-adaptin and myomegalin were identified by two-hybrid analysis using carboxy-terminal region of tuberin containing Rapt-GAP homology domain as a bait.

将结节硬化症2(TSC2)基因的表达载体导入TSC2基因缺失的小鼠肾肿瘤(RT)细胞系(MKOC1.-277)，建立了稳定表达TSC2基因的T2细胞系。与空载体对照细胞系相比，T2细胞中p70S6激酶(S6K)的磷酸化受到抑制。Tuberin的表达抑制了RC细胞在裸鼠体内的致瘤潜能。给予mTOR抑制剂雷帕霉素也可以阻止RC细胞的肿瘤形成。提示mTOR~S6K通路在TSC2-突变所致肿瘤发生中起一定作用。有趣的是，T2细胞中ERC基因同源基因的表达也受到抑制。相反，雷帕霉素处理亲本MKOC1-277细胞并不改变ERCmRNA的表达水平。这些结果表明，ERC受tuberin下游信号通路的调控，而不受mTOR~S6K的影响。这一新的结节蛋白调控途径可能成为治疗结节性硬化症的新的分子靶点。为了寻找调控ERC表达的途径，用不同的信号转导通路的刺激物或化学抑制剂和激活剂处理Hela细胞，并进行Northern和Western印迹分析。在血清饥饿的情况下，ERC蛋白的氨基末端半部分增加，可能是通过蛋白酶介导的切割。因此，ERC蛋白的表达和功能可能受翻译后水平的调控，与细胞生长有关。在这项研究中，还搜索了结节蛋白结合蛋白的候选基因。以含有Rapt-GAP同源结构域的tuberin的羧基末端区域为诱饵，通过双杂交分析鉴定了Gamma-Adaptin和myomegalin。