β-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death
β-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death
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DOI：
10.1242/jcs.01339
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发表时间：
2004-09-15
影响因子：
4
通讯作者：
Han, JH
中科院分区：
文献类型：
--
作者：
Li, JQ;Li, QX;Han, JH
肿瘤坏死因子（TNF）-α诱导纤维肉瘤细胞系L929中的半胱天冬酶非依赖性细胞死亡。这种细胞死亡具有坏死表型，并且依赖于线粒体中活性氧（ROS）的产生。为了鉴定参与这种TNF诱导的ROS依赖性细胞死亡途径的基因，我们利用逆转录病毒插入介导的随机诱变来产生TNF抗性L929细胞系，并且我们随后鉴定了其突变负责TNF抗性表型的基因。在一个这样的抗性系中，β-肌动蛋白被病毒插入破坏，随后在突变系中β-肌动蛋白表达水平的重建肌动蛋白（mut）恢复了其对TNF的敏感性肌动蛋白（mut）细胞中对TNF的抗性是信号特异性的，因为对其他死亡刺激的敏感性没有改变或甚至增加。在TNF处理的野生型和肌动蛋白（mut）细胞中，相当的NF-κ B活化和p38磷酸化也表明，肌动蛋白表达的减少仅选择性地阻断了一些TNF诱导的细胞变化。与HeLa细胞一样，在TNF处理的L929细胞死亡中不发生涉及凋亡的肌动蛋白裂解。caspase裂解产物，一个15 kDa的肌动蛋白片段，一致的过表达对TNF诱导的L929细胞坏死没有影响。相比之下，TNF诱导的线粒体聚集和ROS的产生显着减少在Actinmut细胞，表明肌动蛋白缺乏介导的TNF抗性是最有可能由于受损的线粒体对TNF刺激的反应。我们的研究结果表明，在TNF处理的L929细胞中，细胞死亡信号向线粒体的转导需要肌动蛋白的完全补充。
Tumor necrosis factor (TNF)-alpha induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, beta-actin was disrupted by viral insertion, and subsequent reconstitution of P-actin expression levels in the mutant line Actin(mut) restored its sensitivity to TNF Resistance to TNF in Actin(mut) cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-kappaB activation and p38 phosphorylation in TNF-treated wild-type and Actin(mut) cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actinmut cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.