Mechanisms of apoptosis in human retinal pigment epithelium induced by TNF-α in conditions of heavy metal ion deficiency

PURPOSE. To investigate the mechanism underlying apoptosis in retinal pigment epithelium (RPE) induced by TNF-α in conditions of heavy metal ion deficiency. METHODS. Apoptotic morphology was assessed with Hoechst 33342. FITC-VAD-fmk or antibody specific to cleaved caspase 3 was used to detect in situ activated caspases or cleaved caspase 3, respectively. JC-1 and carboxylated dichlorodihydrofluorescein diacetate were used as probes to measure mitochondrial membrane potential (Δψ_m) and intracellular reactive oxygen species (rOx). RESULTS. The apoptotic response of RPE cells was markedly enhanced when TNF-α plus actinomycin D (act-D) was coapplied with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a heavy metal ion chelator. The apoptosis was caspase dependent, and a blockade with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT), but not FK506, a calcineurin inhibitor, abolished caspase activation and subsequent apoptosis, suggesting that apoptosis requires the MPT, and that caspase activation is downstream to the MPT. MPT, observed in situ as Δψ_m loss, was prevented when cells were pretreated with either CsA or the pan-caspase inhibitor z-VAD-fmk. This result suggests that apoptotic signaling is initiated by the MPT and further amplified by downstream caspases, probably through a feedback loop. An increase in rOx was observed in cells exposed to TNF-α+act-D+TPEN, and rOx production did not require MPT or caspase activation. In addition, application of antioxidants significantly inhibited rOx production, Δψ_m loss, and apoptosis. These data suggest that the rOx may play a role as a proapoptotic signal. CONCLUSIONS. The data suggest that intracellular heavy metal ions play a role in determining the apoptosis induction threshold of the inflammatory response to TNF-α in RPE.