Involvement of Calpain 2 in Ionomycin-induced Cell Death in Cultured Mouse Lens Epithelial Cells

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.

Keywords::

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

ACKNOWLEDGMENTS

Declaration of interest: Dr Shearer is a paid consultant for Senju Pharmaceutical Co., Ltd., a company that may have a commercial interest in the results of this research and technology. Dr Azuma and Dr Nakajima are employees of Senju Pharmaceutical Co., Ltd. These potential conflicts of interest were reviewed, and management plans approved by the OHSU Conflict of Interest in Research Committee were implemented.