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

将Tsc2 (tuberous sclerosis 2 gene) cDNA表达载体导入Tsc2缺陷小鼠肾肿瘤（RT）细胞系mkoc1 -277，建立了表达tuberin （T2细胞）的稳定细胞系。与空载体对照细胞系相比，T2细胞中p70 S6激酶（S6K）的磷酸化被抑制。裸鼠RC细胞的致瘤潜能受tuberin表达的抑制。给药雷帕霉素（一种mTOR抑制剂）也阻断了RC细胞的肿瘤发生。这些结果提示mTOR ~ S6K通路在tsc2突变引起的肿瘤发生中具有一定的作用。有趣的是，Erc mRNA（间皮素基因同源物）的表达在T2细胞中也受到抑制。相比之下，雷帕霉素处理亲代MKOC1-277细胞没有改变Erc mRNA的表达水平。这些结果表明Erc受tuberin下游信号通路的调控，该信号通路不依赖于mTOR ~ S6K。这一新的调控途径可能成为结节性硬化症治疗分子靶点的新候选。为了寻找调节Erc表达的途径，我们用各种刺激或信号转导途径的化学抑制剂和激活剂处理Hela细胞，并通过northern和western blot分析。在血清饥饿的情况下，Erc蛋白氨基末端的一半增加，可能是通过蛋白酶介导的裂解。因此，Erc蛋白的表达和功能可能受到与细胞生长有关的翻译后水平的调控。本研究还进行了结核菌素结合蛋白候选物的寻找。以含Rapt-GAP同源结构域的tuberin羧基末端为诱饵，采用双杂交方法鉴定了γ -adaptin和myomegalin。

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.