Calpain-2 activity promotes aberrant endoplasmic reticulum stressrelated apoptosis in hepatocytes

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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, Nsuccinyl-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.

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Xie, R. J., Zheng, L., Cai, S., Chen, Y. S., Yang, Y., Yang, T., … Hu, X. X. (2020). Calpain-2 activity promotes aberrant endoplasmic reticulum stressrelated apoptosis in hepatocytes. World Journal of Gastroenterology, 26(13), 1450–1462. https://doi.org/10.3748/WJG.V26.I13.1450

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