Effect of mutant IkappaB on cytokine-induced activation of NF-kappaB in cultured human RPE cells.

Journal Article (Journal Article)

PURPOSE: The nuclear transcription factor (NF)-kappaB 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)-kappaB (IkappaB) transgene inhibits cytokine-induced activity of NF-kappaB and expression of NF-kappaB-dependent cytokines by preventing degradation of IkappaB. 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 beta-galactosidase (LacZ) or mutant IkappaB and were treated with interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha. IkappaB protein expression was determined by Western blot. NF-kappaB nuclear translocation was evaluated by immunofluorescence, and functional NF-kappaB activation was determined by luciferase reporter assay. NF-kappaB-dependent cytokine gene expression was determined by reverse transcription-polymerase chain reaction. IL-1beta-induced monocyte chemoattractant protein (MCP)-1 protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: Stimulation of RPE cells with IL-1beta or TNF-alpha caused rapid degradation of the endogenous, but not mutant, IkappaB protein. Expression of the mutant IkappaB isoform inhibited cytokine-stimulated NF-kappaB nuclear translocation, NF-kappaB transcriptional activity, NF-kappaB-dependent gene expression, and secretion of MCP-1. Significant levels of mutant IkappaB protein were expressed for at least 7 weeks after infection. CONCLUSIONS: Infection of human RPE by an adenoviral vector carrying a mutant IkappaB transgene blocks NF-kappaB activation and expression of multiple NF-kappaB-dependent cytokine genes over an extended period. This technique will be useful to determine the role of NF-kappaB in experimental proliferative vitreoretinopathy (PVR), and may offer a novel approach to treatment of PVR with a gene therapy approach.

Full Text

Duke Authors

Cited Authors

  • Yang, P; McKay, BS; Allen, JB; Roberts, WL; Jaffe, GJ

Published Date

  • March 2003

Published In

Volume / Issue

  • 44 / 3

Start / End Page

  • 1339 - 1347

PubMed ID

  • 12601067

International Standard Serial Number (ISSN)

  • 0146-0404

Digital Object Identifier (DOI)

  • 10.1167/iovs.02-0878


  • eng

Conference Location

  • United States