Oxidative stress-mediated NFeκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy

Chunjuan Song, Sayak K. Mitter, Xiaoping Qi, Eleni Beli, Haripriya V. Rao, Jindong Ding, Colin S. Ip, Hongmei Gu, Debra Akin, William A. Dunn, Catherine Bowes Rickman, Alfred S. Lewin, Maria B. Grant, Michael E. Boulton

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

P62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFeκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2 O2 )-mediated oxidative stress and NFeκB 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 H2 O2 -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, NFeκB 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 Mito- SOX 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 NFeκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2 O2 -induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFeκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFeκB 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. NFeκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Original languageEnglish (US)
Article numbere0171940
JournalPLoS One
Volume12
Issue number2
DOIs
StatePublished - Feb 1 2017

Fingerprint

Phosphorylation
Oxidative stress
Retinal Pigments
autophagy
Retinal Pigment Epithelium
Autophagy
cell viability
Cell Survival
phosphorylation
Oxidative Stress
epithelial cells
oxidative stress
Up-Regulation
epithelium
Epithelial Cells
pigments
apolipoprotein E4
Apolipoprotein E4
reactive oxygen species
Reactive Oxygen Species

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Oxidative stress-mediated NFeκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy. / Song, Chunjuan; Mitter, Sayak K.; Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V.; Ding, Jindong; Ip, Colin S.; Gu, Hongmei; Akin, Debra; Dunn, William A.; Rickman, Catherine Bowes; Lewin, Alfred S.; Grant, Maria B.; Boulton, Michael E.

In: PLoS One, Vol. 12, No. 2, e0171940, 01.02.2017.

Research output: Contribution to journalArticle

Song, C, Mitter, SK, Qi, X, Beli, E, Rao, HV, Ding, J, Ip, CS, Gu, H, Akin, D, Dunn, WA, Rickman, CB, Lewin, AS, Grant, MB & Boulton, ME 2017, 'Oxidative stress-mediated NFeκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy', PLoS One, vol. 12, no. 2, e0171940. https://doi.org/10.1371/journal.pone.0171940
Song, Chunjuan ; Mitter, Sayak K. ; Qi, Xiaoping ; Beli, Eleni ; Rao, Haripriya V. ; Ding, Jindong ; Ip, Colin S. ; Gu, Hongmei ; Akin, Debra ; Dunn, William A. ; Rickman, Catherine Bowes ; Lewin, Alfred S. ; Grant, Maria B. ; Boulton, Michael E. / Oxidative stress-mediated NFeκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy. In: PLoS One. 2017 ; Vol. 12, No. 2.
@article{8a929fc3209f4adb8b1a282648409212,
title = "Oxidative stress-mediated NFeκB 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 NFeκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2 O2 )-mediated oxidative stress and NFeκB 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 H2 O2 -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, NFeκB 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 Mito- SOX 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 NFeκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2 O2 -induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFeκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFeκB 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. NFeκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.",
author = "Chunjuan Song and Mitter, {Sayak K.} and Xiaoping Qi and Eleni Beli and Rao, {Haripriya V.} and Jindong Ding and Ip, {Colin S.} and Hongmei Gu and Debra Akin and Dunn, {William A.} and Rickman, {Catherine Bowes} and Lewin, {Alfred S.} and Grant, {Maria B.} and Boulton, {Michael E.}",
year = "2017",
month = "2",
day = "1",
doi = "10.1371/journal.pone.0171940",
language = "English (US)",
volume = "12",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "2",

}

TY - JOUR

T1 - Oxidative stress-mediated NFeκB 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.

AU - Rickman, Catherine Bowes

AU - Lewin, Alfred S.

AU - Grant, Maria B.

AU - Boulton, Michael E.

PY - 2017/2/1

Y1 - 2017/2/1

N2 - P62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFeκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2 O2 )-mediated oxidative stress and NFeκB 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 H2 O2 -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, NFeκB 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 Mito- SOX 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 NFeκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2 O2 -induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFeκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFeκB 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. NFeκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

AB - P62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFeκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2 O2 )-mediated oxidative stress and NFeκB 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 H2 O2 -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, NFeκB 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 Mito- SOX 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 NFeκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2 O2 -induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFeκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFeκB 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. NFeκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

UR - http://www.scopus.com/inward/record.url?scp=85013647228&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85013647228&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0171940

DO - 10.1371/journal.pone.0171940

M3 - Article

VL - 12

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 2

M1 - e0171940

ER -