The Effect of Adenovirus-mediated Overexpression of Mutant IB on IL-1ß-induced NF-B Activation in Cultured Human RPE Cells

Abstract

Abstract: : Purpose: There is accumulating evidence that cytokines initiate and perpetuate PVR. The transcription factor NF-ΚB is a central regulator of multiple inflammatory cytokines. Activation of NF-ΚB requires both phosphorylation and degradation of its natural cytoplasmic inhibitor, IΚB. Herein, we determined whether infection of human RPE by adenovirus carrying a mutant IΚB transgene inhibited expression of NF-ΚB-dependent cytokines, and if so, whether this effect persisted following viral infection. Methods: Cultured human RPE cells were infected with adenovirus encoding either LacZ gene or mutant IΚB at a multiplicity of infection (MOI) of 1-10, then treated with IL-1ß (5units/ml). Mutant and endogenous IΚB protein production was determined by Western blot. Functional NF-ΚB activation was determined by a NF-ΚB-luciferase reporter assay, and NF-ΚB-dependent cytokine gene expression was determined by RT-PCR. Results: Endogenous IΚB protein was rapidly degraded, within 30 minutes, after the cells were stimulated with IL-1ß. However, mutant IΚB protein was not degraded by IL-1ß treatment. IL-1ß-induced NF-ΚB activity was inhibited significantly in mutant IΚB-infected cells compared to un-infected cells (P<0.01) or LacZ-infected cells (P<0.01). Expression of the NF-ΚB-dependent genes MGSA-gro-α, MCSF-1 and IL-1ß was blocked in mutant IΚB-infected cells but not in un-infected or LacZ-infected cells. Significant levels of mutant IΚB protein were expressed in cells infected with adenovirus at an MOI of 1, and expression was detected up to 7 weeks following infection. This persistent transgene expression was associated with prolonged blockade of NF-ΚB-dependent cytokines. In contrast, ß-catenin, a protein phosphorylated and degraded by a different signaling pathway, was not affected by IΚB mutant transgene expression in either treated or untreated cells. Conclusion: 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 time period. This technique will be a useful method to determine the role of NF-ΚB in experimental PVR, and may offer a novel approach to PVR treatment with a gene therapy approach.

Keywords: 524 proliferative vitreoretinopathy • 567 retinal pigment epithelium • 307 adenovirus