Abstract
p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.
Highlights
The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) comprise two major pathways for the removal and clearance of proteins and damaged organelles in eukaryotic cells
We have previously reported that acute sublethal dose of H2O2 increases autophagy in retinal pigment epithelial (RPE) cells by measuring the lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3) as well as autophagosome numbers by light-chain 3 (LC3) immunostaining (9)
We confirmed that p62 gene expression was increased in a dose-dependent fashion following exposure of RPE cells to oxidative stress compared to untreated control (Fig 1D)
Summary
The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) comprise two major pathways for the removal and clearance of proteins and damaged organelles in eukaryotic cells. The pathogenesis of AMD remains unclear there is strong evidence that oxidative stress and impaired protein degradation and clearance pathways in retinal pigment epithelial (RPE) cells may lead to RPE damage and dysfunction [6,7,8,9,10]. It has been reported that proteasome activity is reduced in RPE cells during aging or under oxidative stress [13, 14] It remains unclear how autophagy is activated under oxidative stress in RPE cells and which signaling factors and pathways are involved
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