Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome C release and cell death, independent of vacuolation
Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome C release and cell death, independent of vacuolation
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DOI：
10.1074/jbc.m509404200
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发表时间：
2006-04-21
影响因子：
4.8
通讯作者：
Hirayama, T
中科院分区：
文献类型：
--
作者：
Yamasaki, E;Wada, A;Hirayama, T
幽门螺杆菌空泡化细胞毒素，VacA，可引起胃上皮细胞和其他类型的培养细胞空泡化，通过线粒体依赖途径刺激细胞凋亡。在本研究中，我们探讨了VacA诱导线粒体损伤的机制。免疫染色和共聚焦显微镜观察细胞内VacA的定位；在刺激细胞色素c释放的AZ-521细胞中，VacA主要定位于空泡，而不是线粒体。VacA降低线粒体膜电位而不诱导细胞色素c的释放，提示VacA不直接诱导线粒体释放细胞色素c，在完整细胞中，VacA诱导的细胞色素c释放与细胞凋亡相关因子(S)有关，如促凋亡的Bcl2家族蛋白。一致的是，使用针对激活的Bax的特异性抗体的流式细胞术分析表明，细胞内的Bax被VacA以浓度和时间依赖的方式激活。使用激活的形式特异性抗体，我们还观察到Bak-2家族蛋白被激活。在共聚焦显微镜下，在VacA诱导细胞色素c释放的AZ-521细胞中，Bax和Bak被激活。此外，小干扰RNA诱导的bax基因沉默导致VacA刺激的细胞色素c释放减少，这与VacA诱导的Bax激活对细胞色素c释放的贡献是一致的。NH4Cl促进了VacA诱导的空泡化和Bax的激活，而Bax的激活不被抑制VacA诱导的空泡化的bax激活所抑制。这些结果表明，VacA通过不同的信号通路通过Bax激活诱导细胞凋亡，而不依赖于空泡化。
Helicobacter pylori vacuolating cytotoxin, VacA, which causes vacuolation of gastric epithelial cells and other types of cultured cells, is known to stimulate apoptosis via a mitochondria-dependent pathway. In the present study, we examined the mechanisms of VacA-induced mitochondrial damage. Intracellular VacA localization was monitored by immunostaining and confocal microscopy; in AZ-521 cells in which cytochrome c release was stimulated, most of VacA was localized to vacuoles rather than mitochondria. VacA reduced the membrane potential of isolated mitochondria without inducing cytochrome c release, suggesting that it did not act directly to induce cytochrome c release from mitochondria and that in intact cells, VacA-induced cytochrome c release involved apoptosis-related factor(s), such as a proapoptotic Bcl-2 family protein. In agreement, flow cytometric analyses using antibodies specific for activated Bax revealed that intracellular Bax was activated by VacA in a concentration- and time-dependent manner. Using active form-specific antibodies, we also observed that the Bcl-2 family protein, Bak, was activated. By confocal microscopy, Bax and Bak were activated in AZ-521 cells in which cytochrome c release was induced by VacA. In addition, small interfering RNA-induced silencing of the bax gene resulted in reduction of VacA-stimulated cytochrome c release, consistent with a contribution of VacA-induced Bax activation to cytochrome c release. NH4Cl enhanced both VacA-induced vacuolation and Bax activation, whereas Bax activation was not inhibited by bafilomycin A1, which inhibited vacuolation caused by VacA. These results suggest that VacA acts through different signaling pathways to induce apoptosis via Bax activation, independent of vacuolation.