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Lei Wang, Katayoon Ebrahimi, Marisol Cano, James Handa; p62, regulated by Nrf2 signaling, is a key factor in autophagic protection in retinal pigment epithelial (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3653. doi: https://doi.org/.
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Age-related macular degeneration (AMD) involves oxidative damage and impaired protein degradation in RPE cells. p62, a multifunctional protein, prevents the accumulation of cytoplasmic protein aggregates in age-related diseases. Its promoter contains Nrf2 binding sites, suggesting that p62 may link autophagy with the antioxidant response. The purpose of our study was to determine the role of p62 in autophagy in RPE cells, and its regulation by Nrf2 signaling.
ARPE19 cells were transfected with siRNA to p62, Nrf2, Keap1, or scrambled control, and treated with cigarette smoke (CS) extract for 24h. Total RNA and protein were extracted for quantitative RT-PCR and western blot analysis, respectively. Two mo old C57Bl6 mice were exposed to either air or CS for 1 or 6 months. Protein extracted from RPE/choroid was subjected to western blot analysis.
In APRE19 cells, p62 knockdown (KD) increased the amount of polyubiquitinated protein aggregates and reduced LC3 conversion following treatment with 125 ug/ml CS extract, indicating that p62 is essential for autophagic clearance. p62 KD also weakened Nrf2 signaling by decreasing the mRNA expression of Nrf2 dependent genes - Nqo1 to 63% (p=0.03) and Gclm to 72% (p=0.03). The expression of p62 itself was under the control of Nrf2 signaling - Nrf2 KD decreased the expression of p62 by 28% (p=0.02), while Keap1 KD increased p62 expression by 250% (p=0.02). Thus, p62 and Nrf2 form a positive feedback loop whereby low levels of p62 attenuate Nrf2 signaling and its own expression becomes reduced. Autophagy was studied in mouse RPE to examine the effect of aging and smoking. 8 mo old mice raised in air had reduced LC3 expression and higher levels of polyubiquitin protein aggregates, compared to 3 mo old mice. Mice exposed to 6 mo of CS had even higher polyubiquitin protein aggregates than age-matched air controls.
p62 is a pivotal factor in the autophagy of RPE cells, whose protection becomes deficient with aging and smoking. By forming a positive feedback loop with Nrf2, p62 senses and responds to oxidative damage.
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