Fujii Jun, Matsui Takashi, Heatherly Daniel P, Schlegel Kailo H, Lobo Peter I, Yutsudo Takashi, Ciraolo Georgianne M, Morris Randal E, Obrig Tom
Department of Internal Medicine/Nephrology, University of Virginia, Charlottesville, Virginia 22908, USA.
Infect Immun. 2003 May;71(5):2724-35. doi: 10.1128/IAI.71.5.2724-2735.2003.
Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.
将HeLa细胞暴露于细菌志贺毒素（Stx1和Stx2；10 ng/ml）后，细胞凋亡迅速被诱导。通过DNA片段化、末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记（TUNEL）分析和电子显微镜检测发现，约60%的HeLa细胞在4小时内发生凋亡。Stx1诱导的凋亡需要Stx1A亚基的酶活性，单独的Stx2B亚基或抗球三糖基神经酰胺抗体均不能诱导凋亡。布雷菲德菌素A也能抑制这种活性，表明毒素需要通过高尔基体进行加工。导致凋亡的细胞内途径得到了进一步明确。用合成底物进行酶活性分析以及通过蛋白质免疫印迹法检测这些半胱天冬酶的蛋白水解激活形式，结果显示，将HeLa细胞暴露于Stx1会激活半胱天冬酶3、6、8和9。用半胱天冬酶3、6和8的底物抑制剂对HeLa细胞进行预孵育，可保护细胞免受Stx1依赖性凋亡。这些结果促使人们对凋亡的线粒体途径进行更详细的研究。Stx1诱导的凋亡伴随着线粒体膜损伤，表现为线粒体膜电位降低，且在3至4小时时线粒体细胞色素c释放增加。Bid是一种已知可使线粒体膜通透的内源性蛋白质，它以Stx1依赖性方式被激活。已知半胱天冬酶-8可激活Bid，一种半胱天冬酶-8特异性抑制剂可防止线粒体损伤。尽管这些数据表明半胱天冬酶-8介导的Bid裂解、线粒体细胞色素c释放以及半胱天冬酶-9激活是凋亡的原因，但用半胱天冬酶-9特异性抑制剂对HeLa细胞进行预孵育并不能防止凋亡。内源性XIAP（一种半胱天冬酶-9的直接抑制剂）同时出现Stx1依赖性增加，这一发现解释了这些结果。我们得出结论，HeLa细胞中Stx1诱导的凋亡和DNA片段化的主要途径是独特的，包括半胱天冬酶8、6和3，但与线粒体途径中的事件无关。
