March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Oxidative Stress-Induced p62/SQSTM1 Upregulation in the RPE
Author Affiliations & Notes
  • Chunjuan Song
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Sayak K. Mitter
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Haripriya V. Rao
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Jun Cai
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Xiaoping Qi
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Michael E. Boulton
    Anatomy and Cell Biology, University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  Chunjuan Song, None; Sayak K. Mitter, None; Haripriya V. Rao, None; Jun Cai, None; Xiaoping Qi, None; Michael E. Boulton, None
  • Footnotes
    Support  NIH grants EY019688 and EY021626
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4765. doi:
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      Chunjuan Song, Sayak K. Mitter, Haripriya V. Rao, Jun Cai, Xiaoping Qi, Michael E. Boulton; Oxidative Stress-Induced p62/SQSTM1 Upregulation in the RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4765.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : p62/SQSTM1 is a multifunctional protein implicated in oxidative stress responsive pathways, the ubiquitin-proteasome system (UPS) and selective macroautophagy. The aim of this study was to determine the mechanism and role of p62 upregulation under oxidative stress.

Methods: : Confluent ARPE19 cells in serum-free medium (Ham’s F-10) were treated with oxidative stressor (200, 400 or 800 µM H2O2) for 6, 24 or 48 hours. In some cases, to better mimic chronic oxidative stress, cells received sustained exposure to H2O2 (400 µM) for up to 14 days. Western blot was used to a) assess the LC3II:LC3I ratio as a marker of autophagy, b) to characterize the level of p62 protein and c) to determine polyubiquitinated protein accumulation and d) caspase-3 proteolytic cleavage. Caspase-3 activation was also examined by caspase-3 enzyme activity using its fluorogenic substrate Ac-DEVD-AFC. Apoptotic cell death was determined by an ELISA sandwich assay for DNA fragmentation. p62 mRNA expression was assessed by real time qRT-PCR. The proteasomal peptidase assay was performed to examine proteasome activity. 75nM bafilomycin A1, which blocked the fusion of autophagosome and lysosome, was used together with H2O2.

Results: : Exposure of cells to H2O2 resulted in an increase in autophagy. p62 expression increased at both the protein and mRNA level after H2O2 exposure compared to control. Co-treatment with bafilomycin A1 further enhanced the protein level of p62 in ARPE19 cells. In addition, we also found H2O2 inhibit proteasomal activity and caused the aggregation of polyubiquitinated proteins. As expected, proteasome inhibitor also increased p62 expression in ARPE19 cells. Sustained treatment with H2O2 dysregulated p62, decreased autophagy and activated caspases leading to apoptotic cell death.

Conclusions: : Oxidative stress inhibits proteasomal activity and aggregates polyubiquitinated proteins, which in turn upregulates p62 and triggers the autophagy-lysosome pathway to remove the protein aggregates. Furthermore, acute and chronic oxidative stress activate different pathways.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • apoptosis/cell death 
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