Abstract
Purpose: :
We have shown previously that the Wnt signaling pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. The purpose of this study is to investigate the causative role of oxidative stress in the canonical Wnt pathway activation.
Methods: :
Cultured ARPE19 cells derived from human RPE and bovine retinal capillary endothelial cells (BRCEC) were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE) and an antioxidant, N-Acetyl-Cysteine (NAC). The activation of the canonical Wnt pathway was measured by TOPFLASH assay, an activity assay for luciferase reporter driven by a promoter with TCF/β-catenin-binding sites and by Western blot analysis of Wnt pathway components and target genes. In vivo, rats with streptozotocin-induced diabetes were treated by NAC in drinking water for 8 weeks. Levels of Wnt pathway components and target genes in the eyecups were measured by Western blot analysis. Oxidative stress in the retina was evaluated by immunostaining of 3-nitrotyrosine, a marker of oxidative stress.
Results: :
Levels of phosphorylated and total LRP6, a co-receptor of Wnts and totoal β-catenin, a Wnt effector, were significantly increased by HNE. Similarly, TOPFLASH activity and expression of connected tissue growth factor (CTGF), a Wnt target gene, were also up-regulated by HNE. NAC blocked the Wnt activation induced by HNE, suggesting the Wnt pathway activation was induced by oxidative stress. In diabetic rats, HNE and 3-nitrotyrosine immunosignal were more intensive the retina, compared to that of non-diabetic rats, suggesting an increase in oxidative stress. Retinal levels of total LRP6, β-catenin and CTGF were significantly increased in diabetic rats and reduced by the NAC treatment.
Conclusions: :
Lipid peroxidation products activated the canonical Wnt pathway through oxidative stress, which plays an important role in the development of retinal diseases.
Keywords: oxidation/oxidative or free radical damage • diabetic retinopathy • retinal pigment epithelium