Abstract
Purpose: :
Retinal pigment epithelium (RPE) dysfunction is a hallmark of age-related macular degeneration (AMD). Autophagy is a vital cellular housekeeping mechanism by eliminating malfunctional organelles and damaged proteins. X-box binding protein 1 (XBP1) is a key coordinator of unfolded protein response (UPR). In this study, we investigated the role of XBP1 in regulation of autophagic pathways in the RPE and its implication in AMD.
Methods: :
Albinotic RPE-specific XBP1 knockout mice were generated by using Cre/lox system. Light-induced retinal damage (LIRD) model was produced by exposure of mice to 3 klux of white fluorescent light for 6h. Human RPE (ARPE-19) cell line was used for in vitro study. Up- and down-regulation of XBP1 activity was achieved by adenovirus expressing spliced XBP1 and quinotrierixin, a specific inhibitor of XBP1 activation, or XBP1 siRNA, respectively. Expression of key autophagic genes was determined by quantitative RT-PCR, western blotting or immunohistochemistry. Autophagic flux was measured by conversion of LC3BII to LC3BI. RPE cell apoptosis was assessed TUNEL staining.
Results: :
We observed a decline in XBP1 activation in aged mouse RPE when exposed to ER stress stimulators. Decreased XBP1 activation was also observed in the RPE after light damage in a LIRD model. To study the role of XBP1 in the RPE and in AMD, we generated a RPE-specific XBP1 KO mouse line. Mice deficient of XBP1 in the RPE displayed reduced LC3BI/II expression with aging, accompanied by RPE apoptosis and reduced outer nuclear layer (ONL) thickness. XBP1 KO mice exposed to light stress exhibited exaggerated photoreceptor damage, suggesting a protective role of XBP1 in the RPE. In ARPE-19 cells, over-expression of XBP1 significantly increased ATG7, ATG12, ATG5 and LC3B I/II expression. Conversely, knockdown of XBP1 decreased Beclin-1 and LC3BI/II level. Moreover, incubation with 4-HNE, an oxidative stress inducer, or nutrient deprivation, stimulated autophagic flux in ARPE-19 cells or mouse RPE/Choroid complex, which was remarkably reduced in XBP1-deficient RPE. In addition, lack of XBP1 resulted in RPE apoptosis and potentiated cell death induced by 4-HNE, which was reversed by enhancing autophagy using rapamycin.
Conclusions: :
Taken together, our data indicate that XBP1 is a key protective gene that regulates UPR and autophagy in the RPE. Declined XBP1 activation in the RPE may contribute to the pathogenesis of AMD.
Keywords: age-related macular degeneration • retinal pigment epithelium • apoptosis/cell death