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

Apoptosis. 2010 Dec;15(12):1480-93. doi: 10.1007/s10495-010-0526-4.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calpain* / metabolism
  • Caspase 12 / metabolism*
  • Caspases* / metabolism
  • Cell Death / drug effects
  • Cell Death / physiology*
  • Cell Differentiation
  • Cell Line, Tumor
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / enzymology*
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology*
  • Enzyme Inhibitors / pharmacology
  • Heat-Shock Proteins / metabolism*
  • Humans
  • Microfilament Proteins / analysis*
  • Microfilament Proteins / chemistry
  • Molecular Sequence Data
  • Neurons / chemistry
  • Neurons / physiology
  • Oxidative Stress / drug effects
  • Oxidative Stress / physiology*
  • Peptide Fragments / analysis*
  • Peptide Fragments / chemistry
  • Rats
  • Substrate Specificity
  • Thapsigargin / pharmacology
  • Time Factors
  • Unfolded Protein Response / drug effects
  • Unfolded Protein Response / physiology*
  • Vesicular Transport Proteins / analysis*
  • Vesicular Transport Proteins / chemistry


  • Enzyme Inhibitors
  • Heat-Shock Proteins
  • Microfilament Proteins
  • Peptide Fragments
  • Sptan1 protein, rat
  • Vesicular Transport Proteins
  • Thapsigargin
  • CASP12 protein, human
  • Calpain
  • Caspase 12
  • Caspases
  • molecular chaperone GRP78