title: Loss of calpastatin leads to activation of calpain in human lens epithelial cells.
abstract: OBJECTIVE
  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 Ca(2+), 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.
authors: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors: Takeshi
authors: T
authors: Nakajima
authors: Department of Integrative Biosciences, Oregon Health & Science University, Portland, Oregon, United States.
authors: Thomas R
authors: TR
authors: Shearer
authors: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors: Mitsuyoshi
authors: M
authors: Azuma
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D002154:Calpain / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D059748:Proteolysis; D034741:RNA, Small Interfering; D013049:Spectrin / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D034741:RNA, Small Interfering; C039304:calpain inhibitors; D013049:Spectrin; C033668:calpastatin; D002154:Calpain; D002118:Calcium
keywords: SNJ-1945; calpain; calpastatin; human lens epithelial cells; proteolysis