Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells
Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells
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作者：
Mitsuno, Y;Yoshida, H;Omata, M
幽门螺杆菌感染可诱导胃粘膜表达促炎细胞因子如白细胞介素（IL）-8和肿瘤坏死因子α（TNF-α），它们的基因在启动子区具有AP-1结合位点。c-Fos对启动子区具有SRE的AP-1的反式激活是重要的。我们进行了这项研究，以确认幽门螺杆菌诱导的反式激活这些binding sites. Methods-SRE和AP-1的反式激活在人胃癌细胞TMK 1和MKN 45通过荧光素酶报告基因检测瞬时转染进行了评估。我们比较了与四种幽门螺杆菌菌株共培养的效果，cag致病岛（派）阳性菌株TN 2，其同基因vacA阴性（TN 2-Delta vacA）或cagE阴性（TN 2-Delta cagE）突变体，以及cag派阴性临床分离株T68。用免疫印迹法检测ERK 1/2、JNK和c-Jun的磷酸化，用ELISA法检测IL-8的分泌，用抑制剂U 0126检测MEK的作用。尽管TN 2-Delta vacA诱导了相当的反式激活，但TN 2-Delta cagE和T68显示SRE（65%和51%）和AP-1（分别为TN 2的71%和54%）的反式激活降低。热杀死TN 2或间接接触使用渗透膜抑制转录。磷酸化ERK 1/2、JNK和c-Jun的水平通过与TN 2共培养而增加。MEK抑制剂U 0126可使TN 2诱导的SRE和AP 1的反式激活以及IL-8的分泌分别减少TN 2的83%、87%和53%。结论在与Pi pylori共培养的胃癌细胞中，SRE和AP-1的反式激活分别通过ERK/MAPK和JNK/SAPK级联反应进行。与具有完整cag派的活细菌直接接触是导致AP-1反式激活的细胞内信号传导开始的先决条件。
Background and aims-Helicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor a (TNF-alpha) in gastric mucosa, and their genes have AP-1 binding sites in the promoter region. c-Fos is important for transactivation of AP-1 which has SRE in the promoter region. We conducted this study to confirm H pylori induced transactivation of these binding sites.Methods-Transactivation of SRE and AP-1 was evaluated in human gastric cancer cells TMK1 and MKN45 by luciferase reporter assay in transient transfection. We compared the effects of coculture with four H pylori strains, a cag pathogenicity island (PAI) positive strain TN2, its isogenic vacA negative (TN2-Delta vacA) or cagE negative (TN2-Delta cagE) mutants, and a cag PAI negative clinical isolate T68. Phosphorylation of ERK1/2, JNK, and c-Jun was measured by immunoblot, induction of IL-8 secretion by ELISA, and the effects of MEK by inhibitor U0126.Results-Both SRE and AP-1 were transactivated by coculture with TN2. Although TN2-Delta vacA induced comparable transactivation, TN2-Delta cagE and T68 showed decreased transactivation of SRE (65% and 51%) and AP-1 (71% and 54%, respectively, of TN2). Heat killed TN2 or indirect contact using a permeable membrane inhibited transactivation. Levels of phosphorylated ERK1/2, JNK, and c-Jun were increased by coculture with TN2. MEK inhibitor U0126 reduced TN2 induced transactivation of SRE and AP1, as well as secretion of IL-8, by 83%, 87%, and 53%, respectively, of TN2.Conclusions-Transactivation of SRE and AP-1, through ERK/MAPK and JNK/SAPK cascades, respectively, was found in gastric cancer cells cocultured with Pi pylori. Direct contact with viable bacteria possessing intact cag PAI is a prerequisite for the onset of intracellular signalling leading to AP-1 transactivation.