Abstract
Purpose: :
Macrophage–released TNF–α is an important cytokine associated with age–related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). TNF–α activates the extrinsic apoptotic pathway via TNF receptors. In many cells, the transcription factor NF–ΚB upregulates anti–apoptotic proteins and thereby prevents TNF–α–mediated apoptosis. However, we have shown that RPE cells are resistant to TNF–α–induced apoptosis, even after specific NF–ΚB blockade. Herein, we investigated the role of caspase–8, an upstream extrinsic apoptosis pathway regulator, in RPE cell resistance to TNF–α–mediated RPE cell death.
Methods: :
Cultured human RPE, T–98G glioma, HCT116 colon carcinoma, and OVCAR–3 ovarian carcinoma cell endogenous caspase–8 mRNA and protein expression were measured by real–time PCR, and Western blot, respectively. RPE cells were co–infected with adenovirus encoding caspase–8 and site–specific recombinase Cre (used as an on/off switching unit of caspase–8) (Ad8) to express caspase–8 to a level similar to endogenous caspase–8 in T–98G cells. RPE and T–98G cells were then infected with adenovirus encoding either mutant IΚB (AdIΚB) to block NF–ΚB activation or ß–galactosidase (lacZ, used as a control) and then treated with or without TNF–α for 24h. Cell viability was determined by trypan blue and WST–1 assays.
Results: :
Primary cultured human RPE cells from 11 different donors expressed moderate caspase–8 mRNA levels, but very low levels of caspase–8 protein compared to T–98G cells, HCT116, and OVCAR–3 cells. Activated caspase–8 protein was detected in untreated Ad8–infected RPE cells and TNF–α–treated–AdIΚB–infected T–98G cells. Cell number was significantly decreased in Ad8–infected RPE cells compared to that in lacZ–infected RPE cells. Addition of TNF–α to Ad8–infected cells slightly enhanced the effect of caspase–8 on RPE cell death. NF–ΚB blockade had no effect on RPE cell death, with or without TNF–α treatment. In contrast, TNF–α markedly enhanced T–98G cell death following NF–ΚB blockade.
Conclusions: :
RPE cell caspase–8 protein levels are very low compared to other cell types and may be regulated post–transcriptionally. Low caspase–8 levels may protect RPE cells from TNF–α–mediated cell death in diseases such as AMD, and may allow pathological cell survival in PVR.
Keywords: retinal pigment epithelium • apoptosis/cell death • adenovirus