title: Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.
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.
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Chunjuan
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Sayak K
authors.initials: SK
authors.lastname: Mitter
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Eleni
authors.initials: E
authors.lastname: Beli
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Haripriya V
authors.initials: HV
authors.lastname: Rao
authors.affiliation: Departments of Ophthalmology and Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Jindong
authors.initials: J
authors.lastname: Ding
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Colin S
authors.initials: CS
authors.lastname: Ip
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Gu
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Debra
authors.initials: D
authors.lastname: Akin
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: William A
authors.initials: WA
authors.lastname: Dunn
authors.affiliation: Departments of Ophthalmology and Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Catherine
authors.initials: C
authors.lastname: Bowes Rickman
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Maria B
authors.initials: MB
authors.lastname: Grant
authors.affiliation: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Boulton
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D008268:Macular Degeneration / Q000209:etiology / Q000503:physiopathology; D051379:Mice; D016328:NF-kappa B / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology*; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000502:physiology*; D000071456:Sequestosome-1 Protein / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D016328:NF-kappa B; C118585:P62 protein, human; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D013481:Superoxides