A2E Triggers an Inflammatory Gene Expression Program in ARPE–19 Cells

Abstract

Purpose: : A2E is a major hydrophobic fluorophore formed during the visual cycle and becomes part of the RPE lipofuscins. During aging and in retinal degenerations such as Stargardt’s disease, A2E accumulates and promotes RPE cell injury and death. Here we have explored genome–wide gene expression patterns in retinal pigment epithelial (ARPE–19) cells exposed to the A2E as well as stress–inducing cytokines IL–1ß, TNFα, the oxidizing peroxide H_{202, the combination of TNFα+H202.}

Methods: : Three days old ARPE–19 were incubated with IL–1ß (10 ng/ml), TNFα (10 ng/ml), H_{202 (0.6 uM), TNFα+H202 (10 ng/ml and 0.6 uM) and A2E (10 uM) for 8 hours, after which total RNA was isolated. Gene expression profiles were examined using HG–U133 plus 2.0 GeneChips (Affymetrix). Specific changes in select messenger RNA levels were confirmed using Northern and Western immunoassay.}

Results: : The inflammatory cytokines IL–1ß and TNFα, hydrogen peroxide (H_{2O2), the combination of TNFα+H2O2 together, and A2E, a major cationic, ampiphilic pyridinium bisretinoid and molecular constitutent of human ocular lipofuscin, each induce specific inflammatory gene expression patterns in stressed ARPE–19 cells. We observe significant up–regulation of a pro–inflammatory and pro–oxidative gene family that includes beta amyloid precursor protein (ßAPP), cyclooxygenase–2 (COX–2) and the 70 kilodalton heat shock protein (HSP70) in IL–1ß–, TNFα–, H2O2– and A2E–stressed cells. Alterations in RNA and protein levels for COX–1 and COX–3 were not significant.}

Conclusions: : Up–regulation in the expression of ßAPP, COX–2, HSP70 and related genes in stressed ARPE–19 cells may initiate and/or propagate pathological inflammatory pathways in the retinal pigment epithelium. Understanding the mechanism of coordinate induction and regulation of these and related genes at the level of the transcriptome may lead to a clearer understanding of the significance of inflammatory signaling in retinal degeneration.

Keywords: age-related macular degeneration • signal transduction • gene/expression