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Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes

BACKGROUND: Calpain-2 is a Ca(2+)-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...

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Autores principales: Xie, Ru-Jia, Hu, Xiao-Xia, Zheng, Lu, Cai, Shuang, Chen, Yu-Si, Yang, Yi, Yang, Ting, Han, Bing, Yang, Qin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Baishideng Publishing Group Inc 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152521/
https://www.ncbi.nlm.nih.gov/pubmed/32308346
http://dx.doi.org/10.3748/wjg.v26.i13.1450
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author Xie, Ru-Jia
Hu, Xiao-Xia
Zheng, Lu
Cai, Shuang
Chen, Yu-Si
Yang, Yi
Yang, Ting
Han, Bing
Yang, Qin
author_facet Xie, Ru-Jia
Hu, Xiao-Xia
Zheng, Lu
Cai, Shuang
Chen, Yu-Si
Yang, Yi
Yang, Ting
Han, Bing
Yang, Qin
author_sort Xie, Ru-Jia
collection PubMed
description BACKGROUND: Calpain-2 is a Ca(2+)-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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.
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spelling pubmed-71525212020-04-19 Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes Xie, Ru-Jia Hu, Xiao-Xia Zheng, Lu Cai, Shuang Chen, Yu-Si Yang, Yi Yang, Ting Han, Bing Yang, Qin World J Gastroenterol Basic Study BACKGROUND: Calpain-2 is a Ca(2+)-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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER. Baishideng Publishing Group Inc 2020-04-07 2020-04-07 /pmc/articles/PMC7152521/ /pubmed/32308346 http://dx.doi.org/10.3748/wjg.v26.i13.1450 Text en ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved. http://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (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.
spellingShingle Basic Study
Xie, Ru-Jia
Hu, Xiao-Xia
Zheng, Lu
Cai, Shuang
Chen, Yu-Si
Yang, Yi
Yang, Ting
Han, Bing
Yang, Qin
Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title_full Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title_fullStr Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title_full_unstemmed Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title_short Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
title_sort calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes
topic Basic Study
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152521/
https://www.ncbi.nlm.nih.gov/pubmed/32308346
http://dx.doi.org/10.3748/wjg.v26.i13.1450
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