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

幽门螺杆菌 CagA 对细胞内 SHP-2 活性和细胞生长调节的干扰

• 批准号: 15590264
• 负责人: HIGASHI Hideaki
• 金额: $ 2.3万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590264/
• 关键词: Helicobacter pylori; CagA; SHP-2; FAK; Ras; DNA chip; protein phosphorylation; 癌; シグナル伝達; 感染症

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.

1.利用Tet-On系统建立了有条件表达CagA的人胃上皮细胞株AGS。表达CagA的细胞表现出与cagA阳性幽门螺杆菌感染相同的生长因子样形态改变。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，我们观察到在表达CagA的细胞中，酪氨酸激酶FAK的酪氨酸磷酸化水平降低。SHP-2直接使与FAK激活相关的FAK磷酸化位点去磷酸化，从而使FAK失活。根据这些结果，我们阐明了CagA通过激活SHP-2激活FAK以及激活RAS非依赖的MAPK级联来干扰细胞内信号转导。2.我们利用DNA芯片分析了cagA基因在胃上皮细胞中异位表达引起的基因表达的变化，从而发现了多个cagA反应基因。分析cagA反应基因的启动子序列，发现特定转录因子的结合位点在CagA激活基因的启动子区域显著过度表达。此外，我们还发现CagA能够激活依赖于细胞内特定转录因子的基因转录。3.构建cagA转基因小鼠，研究CagA的生物学活性及CagA介导的胃癌发生机制。我们已经建立了几个表达cagA基因的转基因株系。

项目成果

Takebayashi, T.: "NF-kB-dependent induction of cyclin D1 by pRB family proteins and tumor-derived pRB mutants."J.Biol.Chem.. 278. 14897-14905 (2003)

Takebayashi, T.：“pRB 家族蛋白和肿瘤衍生的 pRB 突变体对细胞周期蛋白 D1 的 NF-kB 依赖性诱导。”J.Biol.Chem.. 278. 14897-14905 (2003)

The effects of cure of Helicobacter pylori infection on the signal transduction of gastric epithelial cells.

幽门螺杆菌感染治愈对胃上皮细胞信号转导的影响

• 发表时间: 2003
• 期刊: Aliment.Pharmacol.Ther. 18
• 作者: Zhou W.;Yamazaki S.;Yamakawa A.;Ohtani M.;Ito Y.;Keida Y.;Higashi H.;Hatakeyama M.;Si J.;Azuma T.;Higashi H.;Azuma T.;Higuchi M.;Azuma T.;Ozawa H.;Zhou T.;東 秀明;Azuma T.
• 通讯作者: Azuma T.

生化学75

生物化学75

• 发表时间: 2003
• 作者: Yamazaki S.;Yamakawa A.;Ito Y.;Ohtani M.;Higashi H.;Hatakeyama M.;Azuma T.;東 秀明;東 秀明;東 秀明
• 通讯作者: 東 秀明

Higashi, H.: "Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitment and activation."J.Biol.Chem.. (In press.).

Higashi, H.：“幽门螺杆菌 CagA 通过 SHP-2 招募和激活诱导 Ras 独立的形态发生反应。”J.Biol.Chem..（正在出版）。

The CagA protein of Helicobacter pylori is translocated into epithelial cells and binds to SHP-2 in human gastric mucosa.

• DOI: 10.1086/367807
• 发表时间: 2003-01
• 期刊: The Journal of infectious diseases
• 作者: S. Yamazaki;A. Yamakawa;Yoshiyuki Ito;M. Ohtani;H. Higashi;M. Hatakeyama;T. Azuma