Mechanisms of apoptosis in human retinal pigment epithelium induced by TNF-alpha in conditions of heavy metal ion deficiency.
OBJECTIVE
To investigate the mechanism underlying apoptosis in retinal pigment epithelium (RPE) induced by TNF-alpha 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 (Deltapsi(m)) and intracellular reactive oxygen species (rOx).


RESULTS
The apoptotic response of RPE cells was markedly enhanced when TNF-alpha 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 Deltapsi(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-alpha+act-D+TPEN, and rOx production did not require MPT or caspase activation. In addition, application of antioxidants significantly inhibited rOx production, Deltapsi(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-alpha in RPE.
Yang
Jun-Hai
JH
Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA. jhyang@acucela.com
Le
Wei-Dong
WD
Basinger
Scott F
SF
Wu
Samuel M
SM
Yang
Chao-yuh
CY
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D003609:Dactinomycin / Q000633:toxicity; D004338:Drug Combinations; D004789:Enzyme Activation; D005029:Ethylenediamines / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D019216:Metals, Heavy / Q000378:metabolism*; D008928:Mitochondria / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000473:pathology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
D002614:Chelating Agents; D004338:Drug Combinations; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D007473:Ion Channels; D019216:Metals, Heavy; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D016572:Cyclosporine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine