Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells

Abstract

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H2O2-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H2O2 induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H2O2-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.

Selenoprotein S Is Highly Expressed in the Blood Vessels and Prevents Vascular Smooth Muscle Cells From Apoptosis

“…CHOP is a transcriptional factor that mediates ER‐initiated apoptotic cell death [Tabas, ]. Because ER stress is involved in H 2 O 2 ‐induced apoptosis in many other types of cells [Ariyama et al, ; Min et al, ; Katsoulieris et al, ; Wei et al, ; Weber et al, ], Western blot of selected ER stress markers (GRP78, phosphorylated PERK, and CHOP) was performed to examine whether their protein expression levels were affected by SelS silence with or without H 2 O 2 . Analysis of SelS expression confirmed its suppression in SelS silence cells.…”

“…ER stress is involved in H 2 O 2 -induced apoptosis in many other types of cells, such as pancreatic AR42J cells [Weber et al, 2013], pancreatic b-cells [Ariyama et al, 2008], mesenchymal stem cells [Wei et al, 2010], renal proximal tubular cells [Katsoulieris et al, 2010], human oral keratinocytes [Min et al, 2008]. In this paper, ER stress was activated by H 2 O 2 in VSMCs, as shown by the increased protein levels of three ER stress markers, GRP78, phosphorylated PERK, and CHOP (Fig.…”

Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown

“…With knowledge of specific fragments generated under specific conditions, more precise biomarker detection methods, such as those using fragment-specific antibodies (Zhang et al, 2009) to detect specific fragments, can be developed to replace the use of an antibody directed against αII-spectrin in Western blot analysis ( e.g. Weber et al, 2013). Additional specific fragments, such as the N-terminal segment (134,563.7 Da) and/or the “middle” segment (33,672.3 Da; SBDP37) mentioned above, may also be tested and developed as biomarkers.…”

Roles of the miR-137-3p/CAPN-2 gene pair in ischemia-reperfusion-induced neuronal apoptosis through modulation of p35 cleavage and subsequent caspase-8 overactivation

“…Acting as an upstream activator of caspase-3, caspase-8 is implicated in various models of CNS diseases and is critical for neuronal apoptosis [24,25]. Thus, preventing caspase-8 proteolysis is especially important for controlling a series of broad caspase activations [24,25]. Consistently, the p35 protein from baculovirus effectively blocked the apoptosis cascade by forming a p35-caspase-8 complex via a thioester bond [24,26].…”

“…In those studies, overexpressed CAPN-2 precisely cleaved the normally membrane-bound p35 into the more stable p25 form, which finally led to inappropriate increases in p25/Cdk5 activation and protein levels of caspase-3, a final executioner of neuronal apoptosis [19,22,23]. Acting as an upstream activator of caspase-3, caspase-8 is implicated in various models of CNS diseases and is critical for neuronal apoptosis [24,25]. Thus, preventing caspase-8 proteolysis is especially important for controlling a series of broad caspase activations [24,25].…”