Abstract
Purpose: :
Cigarette smoking is the most consistent risk factor for age-related macular degeneration (AMD). We investigated the role of an oxidant activated orphan nuclear receptor, aryl hydrocarbon receptor (AhR) and its binding partner ARNT, in retinal pigment epithelial (RPE) cell injury. AhRs are activated by environmental toxins and have been shown to increase oxidative stress and facilitate mitochondrial respiration-dependent increases in reactive oxygen species. We hypothesized that cigarette smoke pro-oxidants such as hydroquinone (HQ) may function as agonists for AhRs and mediate RPE mitochondrial dysfunction, leading to further cell damage and potentially sub-RPE deposit formation.
Methods: :
Post-confluent ARPE19 cells were treated acutely (24hrs) and chronically (two times a week for two weeks) with HQ. AhR activity was determined using a luciferase-based reporter assay and compared to AhR agonists [TCCD and beta-naphthoflavone (bNF)] and antagonist, [alpha-NF (aNF)]. Expression of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, CYP2S1, IL-8 and Nrf2, in oxidant treated cells and cells transfected with siRNA for AhR was determined by real-time PCR and Western blot analysis. Mitochondrial function was determined based on membrane potential, superoxide and reactive oxygen species production, and expression of metabolic and functional genes.
Results: :
AhR and ARNT are expressed in RPE cells. HQ treatment resulted in increased activity of AhR, comparable to TCCD and bNF, and significant up-regulation of AhR target genes including CYP1A1, CYP1B1 and IL8, supporting HQ as a relevant AhR ligand. Decreased target gene expression was seen following AhR knockdown, further suggesting HQ activity is mediated through the AhR pathway. Decrease in mitochondrial membrane potential and increased intracellular reactive oxygen species generation was seen in cells following AhR activation with oxidants.
Conclusions: :
Previous studies have shown that treatment of RPE cells with HQ results in disregulated extracellular matrix production in vitro and deposit formation in vivo. HQ is a known component of cigarette smoke and an activating ligand for AhR. AhR activation is associated with mitochondrial dysfunction. This study suggests that the AhR/ARNT pathway may regulate early phenotypic changes in RPE cells and AMD pathogenesis and that pharmacological manipulation of this pathway may have therapeutic potential.
Keywords: age-related macular degeneration • retinal pigment epithelium • signal transduction: pharmacology/physiology