title: Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells.
abstract: Cataractogenic stresses are associated with the induction of endoplasmic reticulum (ER) stress. However, little is known about oxygen (O2)-induced ER stress in the lens. Cataract research has focused on elevated levels of O2in lens epithelial cells (LECs). Excessive levels or a lack of O2are known to induce ER stress whereas chronic ER stress activates the unfolded protein response (UPR). The present study investigated the hypothesis that the fluctuation of O2levels induces a UPR, and may be controlled by maintaining human LECs (hLECs) in a specific concentration of O2. Human LECs were cultured in different atmospheric levels of O2. Hypoxic conditions were determined by the level of hypoxia-inducible factor (HIF)-1α. 2',7'-Dichlorodihydrofluorescein diacetate and ethidium homodimer-1 staining were conducted to detect reactive oxygen species (ROS) and cell death, respectively. Protein blot analyses were performed with antibodies specific to antioxidant and UPR-specific proteins. Reverse transcription-quantitatative polymerase chain reaction assays were performed to quantify the mRNA levels of activated NF-E2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1). The treatment of human LECs with 0 and 20% atmospheric O2activated Nrf2/Keap1. The LECs shifted to 1% atmospheric O2from 0, 4 or 20% for 24 h showed decreased levels of Keap1. By contrast, hLECs cultured in 1% atmospheric O2for 24 h and then shifted to 0, 4 or 20% O2exhibited a significant upregulation of Nrf2. These results suggest that oxidative stress proteins were not expressed in a 1% O2environment. The O2levels in the culture medium were equilibrated within 2 h in the cell culture plates. These results showed that an appropriate oxygen environment for the culture of LECs is ~1 % atmospheric O2. Either 0 or 20% of atmospheric O2activated the UPR and the Nrf2/Keap1-mediated antioxidant system in LECs and chronic exposure to O2fluctuation led to ROS production and cell death. This study revealed that O2fluctuation-induced UPR/ER stress could be prevented by maintaining the cells in a 1% O2environment.
authors.affiliation: Department of Ophthalmology, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Zheng
authors.affiliation: Department of Ophthalmology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Jia
authors.initials: J
authors.lastname: Xu
authors.affiliation: The Central Laboratory, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: X I
authors.initials: XI
authors.lastname: Chen
authors.affiliation: The Central Laboratory, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Ting-Yan
authors.initials: TY
authors.lastname: Wang
keywords: NF-E2-related factor 2; antioxidants; hypoxia inducible factor-1α; low oxygen; unfolded protein response