Effect of mutant IκB on cytokine-induced activation of NF-κB in cultured human RPE cells

Ping Yang, Brian S. McKay, Janice B. Allen, Wendy L. Roberts, Glenn J. Jaffe

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)1339-1347
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number3
DOIs
StatePublished - Mar 1 2003

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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