Abstract
Purpose: :
Low levels of macular zinc have been reported in age-related macular degeneration (AMD), and oral zinc supplementation ameliorated some symptoms. We previously reported that the membrane-permeable zinc chelator TPEN caused cell death in cultured, primary, human retinal pigment epithelium (RPE) cells due to activation of calpain protease. However, we did not determine if the other major executor of cell death, caspase, was also involved in TPEN-dependent RPE cell death. Thus, the purpose of the present study was to investigate caspase and calpain activation during cell death, using more readily available monkey RPE cells.
Methods: :
Monkey primary RPE cells were cultured with TPEN, and cell viability was assessed by the MTS assay. Activation of calpains and caspases and proteolysis of their substrate, α-spectrin, were detected by immunoblotting for activation-specific fragments. Calpain inhibitor, SNJ-1945, and caspase inhibitor, z-VAD-fmk, were also used to confirm activation of the proteases.
Results: :
TPEN caused a time dependent decrease in viable RPE cells. Activation of calpain 1 (presence of 78/76 kDa active fragments), mitochondria-dependent caspase-9 (37/34 kDa fragments) and executer caspase-3 (17 kDa fragments) occurred during TPEN-induced cell death. Immunoblots showed the calpain-specific spectrin breakdown product (SBDP) at 145 kDa and the caspase-3 SBDP at 120 kDa. Calpain inhibitor, SNJ-1945, inhibited calpain activation and slightly inhibited caspase-9 activation. Pan-caspase inhibitor, z-VAD-fmk, inhibited caspase and calpain 1 activation. TPEN did not cause endoplasmic reticulum (ER) stress or caspase-12 activation, while the positive control thapsigargin did.
Conclusions: :
Relative zinc deficiency in monkey RPE cells causes activation of the cytosolic calpain and mitochondrial caspase pathways, but ER stress appears not to be involved. Caspase activation generally indicates apoptotic cell death. Further investigation is necessary to determine whether the observed calpain activation also induces necrosis/apoptosis in the zinc deficient macula.Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co. Ltd.
Keywords: retinal pigment epithelium • apoptosis/cell death • age-related macular degeneration