TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ
10.7644/j.issn.1674-9960.2017.05.005
- VernacularTitle:肿瘤坏死因子α通过抑制线粒体呼吸链复合体Ⅲ诱导L929-A细胞发生RIP1激酶依赖性细胞凋亡
- Author:
Shule WANG
;
Xiang CHENG
;
Guozhu CHEN
;
Ming ZHAO
;
Xiaodan YU
- Keywords:
tumor necrosis factor α;
RIP1 kinase;
Bid;
caspase-8;
membrane potential,mitochondrial;
mitochondrial respiratory chain complex
- From:
Military Medical Sciences
2017;41(5):346-351
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.
