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

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