Loss of calpastatin leads to activation of calpain in human lens epithelial cells

Takeshi Nakajima, Thomas R. Shearer, Mitsuyoshi Azuma

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Activation of calpains (calpain 2 and Lp82) in rodent lenses readily causes proteolysis and cataract formation. In contrast, primate lenses are quite resistant to activation of calpains. The hypothesis is that high levels of human endogenous calpain inhibitor, calpastatin (CS), prevent calpain activation in human lenses. The purpose of the present study was to directly test if CS is a major inhibitory factor in a human lens epithelial cell line, HLE B-3. Methods. Small interfering RNAs (siRNAs) were used to knock down expression of CS in HLE B-3. The cells then were cultured with the calcium ionophore, ionomycin, with or without a calpain inhibitor SNJ-1945. Transcripts for calpain 2 and CS were measured by quantitative PCR (qPCR). Calpain 2 activity was detected by immunoblotting for the calpain-specific, α-spectrin breakdown product and for activation-associated, fragments of calpain 2. Results. Expression of CS in HLE B-3 was remarkably higher than in α-TN4 (mouse comparator cell line). Proteolysis of α-spectrin was observed in the soluble proteins from α-TN4 incubated with Ca2+, but not in the human HLE B-3. When CS-reduced HLE B-3 cells (transfected with CS siRNA) were cultured with ionomycin, calpain 2 was activated, specific proteolysis of α-spectrin occurred, and cell death ensued; SNJ-1945 inhibited these changes. Conclusions. Our data demonstrated that the high levels of endogenous CS do, indeed, inhibit calpain activity in normal human lens epithelial cells. We speculate that age-related oxidation might cause loss of CS activity in human lens epithelial cells, allowing activation of long-dormant calpain 2, proteolysis of critical cytoskeletal proteins, and cataract formation.

Original languageEnglish (US)
Pages (from-to)5278-5283
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Issue number8
StatePublished - Jul 22 2014
Externally publishedYes


  • Calpain
  • Calpastatin
  • Human lens epithelial cells
  • Proteolysis
  • SNJ-1945

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience


Dive into the research topics of 'Loss of calpastatin leads to activation of calpain in human lens epithelial cells'. Together they form a unique fingerprint.

Cite this