Involvement of calpain 2 in ionomycin-induced cell death in cultured mouse lens epithelial cells

Takeshi Nakajima, Thomas R. Shearer, Mitsuyoshi Azuma

Research output: Contribution to journalArticle

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Abstract

Purpose: Calpains are calcium-activated, intracellular, non-lysosomal, cysteine proteases that hydrolyze lens crystallins and cytoskeletal proteins. Elevated calcium is a frequent finding in both rodent and human cataracts, and calpain 2 is present in lenses of both species. Lens epithelium forms a critical barrier to influx of calcium, but the role of calpain 2 in lens epithelium is poorly characterized. Thus, the purpose of the present experiment was to determine the role of calpain 2 in lens epithelial cell death. Methods: Mouse lens epithelial cells (α-TN4) were cultured with the calcium ionophore ionomycin to promote calcium influx. Release of LDH into the culture medium was measured as a general marker of cell death, while necrosis and apoptosis were detected by staining with ethidium homodimer III (EtD-III) or FITC-annexin V. Calpain activity was determined by zymography and immunoblotting for activation-associated, fragments of calpain. Breakdown products of calpain substrate α-spectrin were also detected by immunoblotting as additional markers of calpain activation. Results: Calpain 2 was found to be the major calpain isozyme in α-TN4 cells. Ionomycin caused leakage of LDH into the medium, activation of calpain 2, proteolysis of α-spectrin, and changes in α-TN4 cell morphology and staining characteristic of necrotic cell death. Calpain inhibitor SNJ-1945 significantly inhibited these changes. Conclusions: The ability of mouse lens epithelium to maintain lens transparency would be compromised by activation of calpain 2 and associated necrotic cell death. Since calpain 2 is ubiquitously present in all animal lenses so far observed, the current results may predict the pathological consequences of calpain 2 activation in animal lenses including those of man.

Original languageEnglish (US)
Pages (from-to)930-936
Number of pages7
JournalCurrent Eye Research
Volume36
Issue number10
DOIs
StatePublished - Oct 1 2011

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Keywords

  • Calpain
  • Lens epithelial cells
  • Necrosis
  • Proteolysis
  • SNJ-1945

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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