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Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.
PLoS One. 2017; 12(2):e0171940.Plos

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+Show Affiliations

Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.Departments of Ophthalmology and Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.Departments of Ophthalmology and Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28222108

Citation

Song, Chunjuan, et al. "Oxidative Stress-mediated NFκB Phosphorylation Upregulates p62/SQSTM1 and Promotes Retinal Pigmented Epithelial Cell Survival Through Increased Autophagy." PloS One, vol. 12, no. 2, 2017, pp. e0171940.
Song C, Mitter SK, Qi X, et al. Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy. PLoS ONE. 2017;12(2):e0171940.
Song, C., Mitter, S. K., Qi, X., Beli, E., Rao, H. V., Ding, J., Ip, C. S., Gu, H., Akin, D., Dunn, W. A., Bowes Rickman, C., Lewin, A. S., Grant, M. B., & Boulton, M. E. (2017). Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy. PloS One, 12(2), e0171940. https://doi.org/10.1371/journal.pone.0171940
Song C, et al. Oxidative Stress-mediated NFκB Phosphorylation Upregulates p62/SQSTM1 and Promotes Retinal Pigmented Epithelial Cell Survival Through Increased Autophagy. PLoS ONE. 2017;12(2):e0171940. PubMed PMID: 28222108.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy. AU - Song,Chunjuan, AU - Mitter,Sayak K, AU - Qi,Xiaoping, AU - Beli,Eleni, AU - Rao,Haripriya V, AU - Ding,Jindong, AU - Ip,Colin S, AU - Gu,Hongmei, AU - Akin,Debra, AU - Dunn,William A,Jr AU - Bowes Rickman,Catherine, AU - Lewin,Alfred S, AU - Grant,Maria B, AU - Boulton,Michael E, Y1 - 2017/02/21/ PY - 2016/07/13/received PY - 2017/01/27/accepted PY - 2017/2/22/entrez PY - 2017/2/22/pubmed PY - 2017/8/22/medline SP - e0171940 EP - e0171940 JF - PloS one JO - PLoS ONE VL - 12 IS - 2 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/28222108/Oxidative_stress_mediated_NFκB_phosphorylation_upregulates_p62/SQSTM1_and_promotes_retinal_pigmented_epithelial_cell_survival_through_increased_autophagy_ L2 - http://dx.plos.org/10.1371/journal.pone.0171940 DB - PRIME DP - Unbound Medicine ER -