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RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells

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Abstract

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 (TNFα). TNFα has been reported to induce necrosis in both of these cell lines. However, comparing the TNFα-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNFα-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 TNFα 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, TNFα 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 TNFα-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 TNFα-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 TNFα-induced cell death.

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Abbreviations

TNFα:

Tumor necrosis factor alpha

Z-VAD:

Z-VAD-FMK

Nec-1:

Necrostatin-1

MMP:

Mitochondrial membrane potential

ROS:

Reactive oxygen species

cyt-C:

Cytochrome-c

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Acknowledgments

We are grateful to Prof. Yan Li and Yuqin Liu for kindly providing the L929 cell lines, and we thank Prof. Tao Zhou for sending the Bcl-xL plasmid. This work was supported in part by Grant 2012CB518200 from the “973” Program of the Ministry of Science and Technology of China (to X. Yu) and by Grants 31371434 (to X. Yu) and 31201041 (to G. Chen) from the National Natural Science Foundation of China.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Cognitive Science, Institute of Basic Medical Sciences, Cognitive and Mental Health Research Center, #27 Taiping Road, Beijing, 100850, China

    Guozhu Chen, Xiang Cheng, Ming Zhao, Song Lin & Xiaodan Yu

  2. Department of Pathology, The First Affiliate Hospital of PLA, Beijing, China

    Jiangyang Lu & Jiarui Kang

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  1. Guozhu Chen
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  2. Xiang Cheng
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  3. Ming Zhao
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  4. Song Lin
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  5. Jiangyang Lu
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  7. Xiaodan Yu
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Correspondence to Xiaodan Yu.

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Guozhu Chen and Xiang Cheng have contributed equally to this work.

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Chen, G., Cheng, X., Zhao, M. et al. RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells. Apoptosis 20, 92–109 (2015). https://doi.org/10.1007/s10495-014-1058-0

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