Caspase-8-mediated apoptosis in human RPE cells.
OBJECTIVE
Tumor necrosis factor (TNF)-alpha is an important cytokine associated with age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). TNF-alpha activates the extrinsic apoptotic pathway. In many cells, nuclear transcription factor (NF)-kappaB upregulates antiapoptotic proteins and prevents TNF-alpha-mediated apoptosis. However, retinal pigment epithelial (RPE) cells are resistant to TNF-alpha-induced apoptosis, even after specific NF-kappaB blockade. Herein, the authors investigated the role of caspase-8 in RPE cell resistance to TNF-alpha-mediated cell death.


METHODS
Caspase-8 mRNA and protein expression were measured in human RPE cells, human lens epithelial cells, human trabecular meshwork (TM) cells, human choroidal endothelial cells, human uveal melanoma cells (OCM-1, 92.1 and MKT-BR), T-98G, OVCAR-3, HCT116, and Jurkat cancer cells by real-time reverse transcription-polymerase chain reaction and Western blot, respectively. RPE cells were coinfected with adenovirus encoding caspase-8 and Cre. RPE and T-98G cells were infected with adenovirus encoding mutant inhibitory (I)-kappaB and then were treated with media alone or with TNF-alpha. Cell viability was determined by WST-1 assay, and apoptosis was evaluated with DNA fragmentation assay and M30 assay. Caspase-3, -7, -9 expression and Bid protein expression after caspase-8 overexpression were examined by Western blot.


RESULTS
Human RPE cell caspase-8 mRNA and protein levels were low compared with levels in nonneoplastic ocular cells and cancer cells. Overexpression of caspase-8 significantly decreased cell number, caused caspase-8 and caspase-3 activation, decreased full-length Bid, caspase-9, and caspase-7, and significantly increased DNA fragmentation and M30-positive RPE cells. Without TNF-alpha treatment, NF-kappaB blockade had no effect on caspase-8-mediated RPE cell death. In the presence of TNF-alpha, NF-kappaB blockade slightly but significantly enhanced caspase-8-mediated RPE cell death.


CONCLUSIONS
RPE cell caspase-8 protein levels are low compared with levels for other cell types and may be regulated posttranscriptionally. Low caspase-8 levels may protect RPE cells from apoptosis normally and in diseases such as AMD and may promote the survival of abnormal cells in PVR. Introduction of caspase-8 into RPE cells may be a potential strategy to treat PVR.
Yang
Ping
P
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Peairs
James J
JJ
Tano
Ryotaro
R
Zhang
Nanfei
N
Tyrell
Jillian
J
Jaffe
Glenn J
GJ
D000256:Adenoviridae / Q000502:physiology; D000328:Adult; D017209:Apoptosis; D015153:Blotting, Western; D053181:Caspase 8 / Q000502:physiology*; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D053938:DNA Fragmentation; D006801:Humans; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C505530:CASP8 protein, human; D053181:Caspase 8