Apoptosis

, Volume 15, Issue 12, pp 1480–1493

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

  • Juan A. Martinez
  • Zhiqun Zhang
  • Stanislav I. Svetlov
  • Ronald L. Hayes
  • Kevin K. Wang
  • Stephen F. Larner
Original Paper

DOI: 10.1007/s10495-010-0526-4

Cite this article as:
Martinez, J.A., Zhang, Z., Svetlov, S.I. et al. Apoptosis (2010) 15: 1480. doi:10.1007/s10495-010-0526-4

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 D341 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.

Keywords

ER stress Thapsigargin Unfolded protein response Calpain Caspase PC12 cells 

Abbreviations

ER

Endoplasmic reticulum

UPR

Unfolded protein response

GRP-78

Glucose responsive protein of 78 kDa

TG

Thapsigargin

BDP

Breakdown product

TBI

Traumatic brain injury

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Juan A. Martinez
    • 1
  • Zhiqun Zhang
    • 1
  • Stanislav I. Svetlov
    • 1
  • Ronald L. Hayes
    • 1
  • Kevin K. Wang
    • 1
  • Stephen F. Larner
    • 1
  1. 1.Center of Innovative Research, Banyan Biomarkers IncAlachuaUSA

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