Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells

Abstract

Neuronal cell death after traumatic brain injury, Alzheimer's disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D(341) detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.

Sorcin Induces a Drug-Resistant Phenotype in Human Colorectal Cancer by Modulating Ca2+ Homeostasis

“…Accordingly, the selective downregulation of 22 kDa sorcin in HCT-116 CRC cells treated with 10 mmol/L FU resulted in reduced levels of caspase-3 and the proteolytic cleavage of the caspase-12 precursor, a molecular event which triggers apoptotic signaling in response to ER stress (ref. 35; Fig. 3B).…”

“…A significant upregulation of 22 kDa sorcin was observed upon treatment of HCT-116 cells with 2 mmol/L Tg for 7 hours, a condition that induced ER stress (ref. 35; Fig. 3A).…”

Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels

“…In addition, increases in intracellular Ca 2+ levels may contribute to ROS accumulation and ER stress (17). Ca proteases, which have been reported to promote cysteinyl aspartate specific proteinase (caspase)-4 activation during ER stress-induced apoptosis (19)(20)(21). Furthermore, it has been reported that LCA induces apoptosis in HepG2 hepatocellular carcinoma cells through induction of ER stress via a phospholipase C gamma 1-, Ca 2+ -and ROS-dependent pathway (22).…”

Glucopsychosine increases cytosolic calcium to induce calpain-mediated apoptosis of acute myeloid leukemia cells

“…We tested protein levels of GRP78, a chaperone protein which is released as a precursor to the UPR to prevent the cells from committing to the ER stress pathway of apoptosis [37,95]. The ER is a center for protein folding and when there is insufficient protein folding, cytosolic GRP78 will shuttle unfolded proteins back into the ER to refold them [95]. We treated OCI-AML2 and U937 cells with 10 µM glucopsychosine for increasing time points and looked for changes in GRP78 protein expression (Fig.…”

“…Caspase-12 is normally bound to the ER as pro-caspase-12 until it becomes unbound and cleaved by calpains during ER stress [95]. Active caspase-12 can then initiate the caspase cascade and lead to apoptosis [38].…”