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Effect of Mutant IB on Cytokine-Induced Activation of NF-B in Cultured Human RPE Cells

¹From the Departments of Ophthalmology and ²Cell Biology, Duke University Medical Center, Durham, North Carolina; and the ³Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina.

PURPOSE. The nuclear transcription factor (NF)-B is a central regulator of multiple inflammatory cytokines. The current study was conducted to determine whether infection of human retinal pigment epithelial (RPE) cells by adenovirus carrying a mutant inhibitory (I)-B (IB) transgene inhibits cytokine-induced activity of NF-B and expression of NF-B-dependent cytokines by preventing degradation of IB. The persistence of recombinant protein expression and function after the viral infection was also examined.

METHODS. Cultured human RPE cells were infected with adenovirus encoding either ß-galactosidase (LacZ) or mutant IB and were treated with interleukin (IL)-1ß or tumor necrosis factor (TNF)-. IB protein expression was determined by Western blot. NF-B nuclear translocation was evaluated by immunofluorescence, and functional NF-B activation was determined by luciferase reporter assay. NF-B-dependent cytokine gene expression was determined by reverse transcription-polymerase chain reaction. IL-1ß-induced monocyte chemoattractant protein (MCP)-1 protein secretion was measured by enzyme-linked immunosorbent assay.

RESULTS. Stimulation of RPE cells with IL-1ß or TNF- caused rapid degradation of the endogenous, but not mutant, IB protein. Expression of the mutant IB isoform inhibited cytokine-stimulated NF-B nuclear translocation, NF-B transcriptional activity, NF-B-dependent gene expression, and secretion of MCP-1. Significant levels of mutant IB protein were expressed for at least 7 weeks after infection.

CONCLUSIONS. Infection of human RPE by an adenoviral vector carrying a mutant IB transgene blocks NF-B activation and expression of multiple NF-B-dependent cytokine genes over an extended period. This technique will be useful to determine the role of NF-B in experimental proliferative vitreoretinopathy (PVR), and may offer a novel approach to treatment of PVR with a gene therapy approach.

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