Effect of mutant IkappaB on cytokine-induced activation of NF-kappaB in cultured human RPE cells.
OBJECTIVE
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.
Yang
Ping
P
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
McKay
Brian S
BS
Allen
Janice B
JB
Roberts
Wendy L
WL
Jaffe
Glenn J
GJ
D000260:Adenoviruses, Human / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D020672:I-kappa B Proteins / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000235:genetics
D018932:Chemokine CCL2; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D001616:beta-Galactosidase