Purpose: 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.