Abstract
Abstract: :
Purpose: TNF–α is an important cytokine associated with proliferative vitreoretinopathy (PVR) development. The nuclear transcription factor (NF)–ΚB is a pivotal regulator of multiple inflammatory cytokine– and apoptosis–related genes. NF–ΚB blockade enhances tumor necrosis factor (TNF)–α–induced apoptosis in a variety of cell types otherwise resistant to TNF–α–induced cell death. However, RPE cells are resistant to TNF–α–induced apoptosis, even after specific NF–ΚB blockade. Uncontrolled RPE cell proliferation contributes to membrane formation in PVR. To further investigate the mechanisms underlying the survival of RPE cells in PVR, we determined whether TNF–α regulated survival factor expression, and whether regulation was dependent on NF–ΚB. Methods: Cultured human RPE cells were un–infected or infected with adenovirus encoding either mutant IΚB, to block NF–ΚB activation, or ß–galactosidase (used as a control) and then treated with or without TNF–α for varying times. Real–time PCR and Western Blot were used to determine survival factor gene and protein expression. ß–catenin, a protein that is degraded by the proteasome in response to a NF–ΚB–independent signal pathway, was used as a control for cytoplasmic degradation and protein loading. Results: TNF–α upregulated c–FLIP and TRAF1 gene and protein expression in a time–dependent manner. c–IAP1, c–IAP2, survivin, Bcl–xl and TRAF2 protein were expressed constitutively, and levels were not influenced by TNF–α treatment at any time point tested. Overexpression of mutant IΚB blocked TNF–α–induced c–FLIP and TRAF1 protein expression. NF–ΚB blockade failed to inhibit c–IAP1, and TRAF2 expression in the presence or absence of TNF–α. ß–catenin was not affected by TNF–α treatment. Conclusions: Multiple survival factors are expressed constitutively by human RPE cells, while others are upregulated by TNF–α in a NF–ΚB–dependent manner. These results help to explain RPE cell resistance to TNF–α–mediated apoptosis, and pathological cell survival in PVR.
Keywords: proliferative vitreoretinopathy • cell death/apoptosis • retinal pigment epithelium