Abstract
Purpose :
To investigate the protective role of autophagy on retinal pigment epithelial (RPE) cells during endoplasmic reticulum (ER) stress-induced cell death and its functional mechanisms in vitro.
Methods :
In human RPE primary cells and ARPE-19 cells, we analyzed autophagy induction in the response to ER stress condition using real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting analysis for 48 and 72 hr. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was used to examine for the cell viability of ER stress-induced cell death in the presence or absence with chloroquine (CQ) as autophagy inhibitor. ER stress specific inhibitor such as 4-phenylbutyrate (4-PBA) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor as diphenyleneiodonium (DPI) and scavenger of reactive oxygen species (ROS) as N-acetyl-L-cysteine (NAC) and mito-TEMPO were evaluated for determining the transcriptional and translational autophagy level of ER stress in human RPE cells.
Results :
Human RPE cells after treatment with tunicamycin as ER stress inducers were markedly elevated with the transcriptional and translational level of autophagy genes as Atg5 and LC3B, but not beclin-1. The inhibition of ER stress by 4-PBA were significantly attenuated by the increased level of autophagy induction such as Atg5, LC3B and p62 by tunicamycin. The inhibitor of ROS production as DPI, NAC, and mito-TEMPO markedly decreased ER stress-induced cell death in primary RPE cells. A significant decrease in the levels of autophagy by chloroquine was evaluated for the increased cell mortality and the pro-survival role of ER stress-induced autophagy.
Conclusions :
These data indicate that the induction of autophagy may attenuate acquisition of ER stress-induced cell death through the ROS production by NADPH oxidase, suggesting that autophagy activation may be used as a potential therapeutic strategy for age-related macular degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.