Disturbance of intracellular SHP-2 activity and cell growth regulation by Helicobacter pylori CagA

1.利用Tet-On系统建立了条件性表达CagA的AGS人胃上皮细胞系。表达CagA的细胞表现出与cagA阳性幽门螺杆菌感染的情况相同的生长因子样形态变化。利用该细胞，我们研究了CagA对Ras/MAPK通路的影响。结果表明，CagA诱导的细胞形态学改变依赖于MAPK活性，而不依赖于Ras活性。另一方面，我们观察到酪氨酸激酶FAK的酪氨酸磷酸化水平在CagA表达细胞中降低。SHP-2直接使与FAK活化相关的FAK磷酸化位点去磷酸化，从而使FAK失活。这些结果表明，CagA通过激活SHP-2和Ras非依赖性MAPK级联反应，抑制FAK的激活，从而干扰细胞内信号传导。2.利用基因芯片技术分析cagA基因在胃上皮细胞中异位表达引起的基因表达变化，发现多个cagA应答基因。分析推定的启动子序列的cagA反应基因，特定的转录因子的结合位点显着过度代表在启动子区域的cagA激活基因。此外，我们发现CagA能够激活基因转录，这依赖于细胞中的特定转录因子。提示CagA生物学活性导致细胞内基因表达谱失调。3.构建CagA转基因小鼠，研究CagA生物学活性及其介导的胃癌发生机制。我们已经建立了几个转cagA基因的转基因株系。

1.We established AGS human gastric epithelial cells that conditionally express CagA by employing Tet-On system. The cells expressing CagA exhibited the growth factor-like morphological change as in the case of cagA-positive H.pylori infection. Using the cells, we investigated the CagA effect on Ras/MAPK pathway. As results, we revealed that the morphological change induced by CagA was dependent on MAPK activity but not on Ras activity. On the other hand, we observed that tyrosine phosphorylation levels of tyrosine kinase FAK decreased in CagA-expressing cells. SHP-2 directly dephosphorylates FAK phosphorylation sites which associate with FAK activation, and thereby inactivates FAK. From these results, we clarified that CagA disrupt intracellular signaling by in activation of FAK through SHP-2 activation as well as activation of Ras-independent MAPK cascade.2.We analyzed changes in gene expression caused by ectopic expression of the cagA gene in gastric epithelial cells using a DNA microarray, and accordingly found multiple cagA-responsive genes. Analyzing putative promoter sequences of the cagA-responsive genes, binding sites for particular transcription factors were significantly over-represented in the promoter regions of CagA-activated genes. Furthermore, we found that CagA is capable of activating gene transcription which is dependent on a specific transcriptional factor in the cells. It is suggested that CagA biological activity causes dysregulation of intracellular gene-expression profile.3.We constructed cagA transgenic mouse to investigate CagA biological activity and the mechanism of CagA mediated-development of gastric cancer in vivo. We have established several transgenic lines which show expression of cagA gene.