CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis.

Jingtian Zhang,Linlin Zeng,Xu Zhang,Han Niu,Fuqiang Zhang,Yuxiang Wang,Xueqi Fu,Zhizhuang Joe Zhao

doi:10.1155/2019/9071297

Abstract

TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax—members of the Bcl-2 family—are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK−/− cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak−/− cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.

Highlights

Tumor necrosis factor-α (TNFα)—a member of the family of death receptor ligands—induces cell death through the extrinsic death receptor apoptosis pathway
Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and proapoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHXinduced PARP cleavage and caspase activation were blocked in BAK−/− cells or using speci c siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using speci c siRNA
Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak signi cantly decreased this translocation. ese results indicate that TNFα/CHX-induced apoptosis does not occur in Bak−/− cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent

Summary

Introduction

Tumor necrosis factor-α (TNFα)—a member of the family of death receptor ligands—induces cell death through the extrinsic death receptor apoptosis pathway. TNFα induced apoptosis in human gastric cancer cells [2]. It promoted hepatocyte death [3]. TNFα is critical for the survival of dendritic cells, and TOM-independent complex formation of Bax and Bak in mitochondrial fraction in TNFα-induced apoptosis in HeLa cells [4, 5]. FASL and TRAIL are other members of the family of death receptor ligands. FASL is the ligand of FAS (or CD95), which triggers cell death that depends upon caspase-8 activation. The molecular events spanning from receptor engagement until cell death have already been investigated in details, precise mechanisms underlying TNFαinduced apoptosis remain unclear

Methods

Results

Conclusion