RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells
RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells
复制标题
RIP1依赖性Bid裂解介导L929成纤维细胞瘤细胞中TNFα诱导但不依赖Caspase-3的细胞死亡
DOI：
10.1007/s10495-014-1058-0
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发表时间：
2015-01-01
期刊：
影响因子：
7.2
通讯作者：
Yu, Xiaodan
中科院分区：
文献类型：
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作者：
Chen, Guozhu;Cheng, Xiang;Yu, Xiaodan
L929成纤维细胞瘤细胞（L929-A）和L929纤维肉瘤细胞（L929-N）是通常用于研究肿瘤坏死因子α（TNF α）的细胞毒性的不同细胞系。据报道，TNF α可诱导这两种细胞系的坏死。然而，比较这两种细胞系中TNF α诱导的细胞死亡，我们发现，与显示典型RIP 3依赖性坏死的L929-N细胞不同，L929-A细胞中TNF α诱导的细胞死亡是泛半胱天冬酶抑制剂Z-VAD-FMK（Z-VAD）敏感的，其不依赖于RIP 3。我们还证实了L929-A细胞中的细胞死亡信号是通过胞质溶胶预组装的ripoptosome启动的，并且Caspase-8或RIP 1蛋白的敲低阻断了细胞死亡。与L929-N细胞相比，L929-A细胞系具有较低水平的组成型和诱导型TNF α自分泌产生，并且泛半胱天冬酶抑制剂Z-VAD或Q-VD不杀死L929-A细胞，因为它们影响L929-N细胞。此外，L929-A细胞表达的RIP 3蛋白比L929-N细胞少;因此，TNF α未能诱导RIP 3依赖性坏死性凋亡。此外，ripoptosome介导的细胞死亡信号通过Caspase-8介导的和RIP 1激酶活性依赖的Bid切割转导到线粒体。RIP 1激酶抑制剂Necrostatin-1（Nec-1）或Caspase-8敲低可完全阻断Bid裂解，Bid或Bax/巴克敲低可阻止TNF α诱导的L929-A细胞死亡。虽然Bax/巴克的激活降低了线粒体膜电位，但线粒体膜间隙蛋白，包括细胞色素C（cyt-C）和Smac的水平下降，并且Western印迹和免疫荧光染色分析不能确定这些蛋白是否重新分布到胞质溶胶。此外，线粒体外膜蛋白Tom 20也减少，表明线粒体蛋白减少可能是由线粒体数量减少引起的。未观察到有效的cyt-C释放;因此，下游半胱天冬酶（包括半胱天冬酶-9、半胱天冬酶-7、半胱天冬酶-6和半胱天冬酶-3）的有限活化和裂解不足以杀死细胞。半胱天冬酶-9、半胱天冬酶-6和半胱天冬酶-3/7抑制剂或半胱天冬酶-9和半胱天冬酶-3敲低也不能阻断细胞死亡，并且Bcl-2的过表达也不能消除细胞死亡。透射电镜下死亡细胞呈坏死样而非凋亡样特征，与线粒体凋亡明显不同，提示效应型半胱氨酸天冬氨酸蛋白酶不是细胞死亡的执行者。这些新发现表明，TNF α诱导的L929-A细胞死亡不同于典型的RIP 3依赖性坏死和Caspase-8/Caspase-3介导的凋亡。这些结果强调了在使用不同的L929细胞作为研究TNF α诱导的细胞死亡的模型时需要谨慎。
L929 fibroblastoma cells (L929-A) and L929 fibrosarcoma cells (L929-N) are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNF alpha). TNF alpha has been reported to induce necrosis in both of these cell lines. However, comparing the TNF alpha-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNF alpha-induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sensitive, which does not depend on RIP3. We also confirmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8 or RIP1 protein blocked cell death. Compared with the L929-N cells, the L929-A cell line had lower levels of constitutive and inducible TNF alpha autocrine production, and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells. Moreover, the L929-A cells expressed less RIP3 protein than the L929-N cells; therefore, TNF alpha failed to induce RIP3-dependent necroptosis. In addition, the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-mediated and RIP1 kinase activity-dependent Bid cleavage. The RIP1 kinase inhibitor Necrostatin-1 (Nec-1) or Caspase-8 knockdown completely blocked Bid cleavage, and the knockdown of Bid or Bax/Bak prevented TNF alpha-induced cell death in the L929-A cells. Although the activation of Bax/Bak decreased the mitochondrial membrane potential, the levels of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-C) and Smac, declined, and western blotting and immunofluorescence staining analysis did not determine whether these proteins were redistributed to the cytosol. In addition, the mitochondrial outer membrane protein Tom20 was also reduced, indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers. No efficient cyt-C release was observed; therefore, the limited activation and cleavage of downstream caspases, including Caspase-9, Caspase-7, Caspase-6 and Caspase-3, was insufficient to kill the cells. The Caspase-9, Caspase-6 and Caspase-3/7 inhibitors or Caspase-9 and -3 knockdown also failed to block cell death, and the overexpression of Bcl-2 also did not abrogate cell death. Moreover, the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy, and these features were significantly different from mitochondrial apoptosis, indicating that the effector caspases were not the executioners of cell death. These new discoveries show that TNF alpha-induced cell death in L929-A cells is different than typical RIP3-dependent necrosis and Caspase-8/Caspase-3-mediated apoptosis. These results highlight that caution is necessary when using different L929 cells as a model to investigate TNF alpha-induced cell death.