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.
Department of Physiology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, Guizhou Province, China.
World J Gastroenterol. 2020 Apr 7;26(13):1450-1462. doi: 10.3748/wjg.v26.i13.1450.
Calpain-2 is a Ca-dependent cysteine protease, and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.
To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum (ER) stress-related apoptosis in rat hepatocyte BRL-3A cells.
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, -succinyl-Leu-Leu-Val-Tyr-AMC. Intracellular Ca 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.
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 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.
The data indicated that Ca-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.
钙蛋白酶-2 是一种 Ca2+依赖性半胱氨酸蛋白酶，其高活性可增强多种触发因素介导的细胞凋亡。
探讨钙蛋白酶-2 是否可以调节大鼠肝细胞 BRL-3A 细胞中异常内质网（ER）应激相关的细胞凋亡。
用不同剂量的二硫苏糖醇（DTT）处理 BRL-3A 细胞，通过 3-[4,5-二甲基-2-噻唑基]-2,5-二苯基-2-H-四唑溴盐和流式细胞术检测细胞活力和凋亡。用 Western blot 分析检测 ER 应激和凋亡相关蛋白的表达。用荧光底物 -succinyl-Leu-Leu-Val-Tyr-AMC 测定钙蛋白酶-2 的蛋白酶活性。通过荧光显微镜检测细胞内 Ca 含量、ER 和钙蛋白酶-2 的共定位。用特异性小干扰 RNA 沉默钙蛋白酶-2 对 BRL-3A 细胞中 caspase-12 激活和凋亡的影响进行定量分析。
DTT 对 BRL-3A 细胞表现出剂量依赖性细胞毒性，2mmol/L DTT 处理可触发 BRL-3A 细胞凋亡。DTT 处理显著上调 78kDa 葡萄糖调节蛋白、激活转录因子 4、C/EBP 同源蛋白表达>2 倍，并增强 PRKR 样内质网激酶磷酸化、caspase-12 和 caspase-3 切割，呈时间依赖性。DTT 处理还使 BRL-3A 细胞内 Ca 含量、钙蛋白酶-2 表达和活性增加>2 倍。此外，免疫荧光显示 DTT 处理促进了钙蛋白酶-2 在 ER 中的积累。此外，钙蛋白酶-2 沉默使钙蛋白酶-2 表达减少 85%，显著减轻了 DTT 增强的钙蛋白酶-2 表达、caspase-12 切割和 BRL-3A 细胞凋亡。
数据表明，Ca2+依赖性钙蛋白酶-2 活性通过激活内质网中的 caspase-12 促进大鼠肝细胞异常的 ER 应激相关细胞凋亡。
