Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 7, 2020; 26(13): 1450-1462
Published online Apr 7, 2020. doi: 10.3748/wjg.v26.i13.1450
Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
Ru-Jia Xie, Xiao-Xia Hu, Lu Zheng, Shuang Cai, Yu-Si Chen, Yi Yang, Ting Yang, Bing Han, Qin Yang
Ru-Jia Xie, Lu Zheng, Shuang Cai, Yu-Si Chen, Yi Yang, Ting Yang, Bing Han, Qin Yang, Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases, College of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, Guizhou Province, China
Ru-Jia Xie, Lu Zheng, Shuang Cai, Yu-Si Chen, Yi Yang, Ting Yang, Bing Han, Qin Yang, Department of Pathophysiology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, Guizhou Province, China
Xiao-Xia Hu, Department of Physiology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, Guizhou Province, China
Author contributions: Xie RJ, Han B and Zheng L performed the experiments; Hu XX, Cai S, Chen YS, Yang Y and Yang T analyzed the data; Xie RJ and Yang Q designed the study; Xie RJ wrote the manuscript; all authors approved the final manuscript. Xie RJ and Hu XX contributed equally to this work.
Supported by the National Natural Science Foundation of China, No. 81560105; the Department of Science and Technology of Guizhou Province, No. LH (2014) 7074.
Institutional animal care and use committee statement: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals.
Conflict-of-interest statement: The authors declare no competing interests.
Data sharing statement: The datasets in the present study are available upon reasonable request.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Bing Han, MD, Academic Research, Department of Pathophysiology, College of Basic Medical Sciences, Guizhou Medical University, Dongqing Road, Guiyang 550025, Guizhou Province, China. 47569390@qq.com
Received: December 2, 2019
Peer-review started: December 2, 2019
First decision: December 12, 2019
Revised: February 20, 2020
Accepted: March 9, 2020
Article in press: March 9, 2020
Published online: April 7, 2020
Processing time: 127 Days and 11.6 Hours
Abstract
BACKGROUND

Calpain-2 is a Ca2+-dependent cysteine protease, and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.

AIM

To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum (ER) stress-related apoptosis in rat hepatocyte BRL-3A cells.

METHODS

BRL-3A cells were treated with varying doses of dithiothreitol (DTT), and their viability and apoptosis were quantified by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-H-tetrazolium bromide and flow cytometry. The expression of ER stress- and apoptosis-related proteins was detected by Western blot analysis. The protease activity of calpain-2 was determined using a fluorescent substrate, N-succinyl-Leu-Leu-Val-Tyr-AMC. Intracellular Ca2+ content, and ER and calpain-2 co-localization were characterized by fluorescent microscopy. The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.

RESULTS

DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis. DTT treatment significantly upregulated 78 kDa glucose-regulated protein, activating transcription factor 4, C/EBP-homologous protein expression by >2-fold, and enhanced PRKR-like ER kinase phosphorylation, caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence. DTT treatment also significantly increased intracellular Ca2+ content, calpain-2 expression, and activity by >2-fold in BRL-3A cells. Furthermore, immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2. Moreover, calpain-2 silencing to decrease calpain-2 expression by 85% significantly mitigated DTT-enhanced calpain-2 expression, caspase-12 cleavage, and apoptosis in BRL-3A cells.

CONCLUSION

The data indicated that Ca2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

Keywords: Calcium; Calpain-2; Caspase-12; Endoplasmic reticulum stress; Apoptosis; Hepatocyte

Core tip: Hepatocyte apoptosis is associated with many liver diseases. During the process of apoptosis, calpain-2 can cleave several apoptosis-related proteins. However, the regulatory mechanisms by which calpain-2 regulates the endoplasmic reticulum (ER) stress-mediated hepatocyte apoptosis remain unclear. In this study, the effect of calpain-2 on ER stress-mediated hepatocyte apoptosis and the underlying regulatory mechanisms was investigated. Our data indicate that calpain-2 is crucial for the aberrant ER stress-induced apoptosis of hepatocytes and may be a novel therapeutic target for liver diseases.