Abstract
Purpose: :
Calpains are calcium-activated, intracellular, nonlysosomal, cysteine proteases. Activation of calpains (CAPN2 and Lp82) in rodent lenses readily causes proteolysis and cataract formation. In contrast, primate lenses are quite resistant to activation of calpain, presumably because of the high levels of endogenous calpain inhibitor, calpastatin (CS). Loss of CS activity during aging, such as in Alzheimer's disease, is under investigation. Thus, purpose of present study was to examine the consequences of knock down of CS in a human lens epithelial cell line, HLE-B3.
Methods: :
Transcripts for CS and calpains were measured by qPCR. Small interference RNAs (siRNAs) were used to reduce the expression of calpastatin in HLE-B3 cells, which were then cultured with the calcium ionophore ionomycin, with or without calpain inhibitor SNJ1945. Calpain activity in cells was detected by immunoblotting for the calpain-specific, α-spectrin breakdown product at 145 kDa and for activation-associated, fragments of calpain. Release of LDH into the culture medium was measured as a marker for cell death.
Results: :
Without CS knockdown, proteolysis of α-spectrin was observed in soluble proteins from αTN-4 cells (mouse comparator cell line) incubated with calcium, but not in the human HLE-B3 cells. Expression of CS in HLE-B3 cells was higher than αTN-4 cells. Transfection of CS siRNA into HLE-B3 cells reduced expression of CS mRNA and protein. When the CS siRNA-transfected-HLE-B3 cells were cultured with ionomycin; calpains were activated, specific proteolysis of α-spectrin was observed, and cell death ensued. Calpain inhibitor SNJ1945 inhibited these changes.
Conclusions: :
Our data demonstrate that the high level of endogenous CS normally fully inhibits calpains in human lens epithelial cells. Our hypothesis is that aging of human lens epithelial cells causes loss of CS activity; allowing activation of long-dormant calpains, proteolysis of critical cytoskeletal proteins, and cataract formation.
Keywords: calcium • apoptosis/cell death • cataract