Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells

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 O₂ in lens epithelial cells (LECs). Excessive levels or a lack of O₂ are 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 O₂ levels 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 O₂ activated Nrf2/Keap1. The LECs shifted to 1% atmospheric O₂ from 0, 4 or 20% for 24 h showed decreased levels of Keap1. By contrast, hLECs cultured in 1% atmospheric O₂ for 24 h and then shifted to 0, 4 or 20% O₂ exhibited a significant upregulation of Nrf2. These results suggest that oxidative stress proteins were not expressed in a 1% O₂ environment. The O₂ levels 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 O₂ activated the UPR and the Nrf2/Keap1-mediated antioxidant system in LECs and chronic exposure to O₂ fluctuation led to ROS production and cell death. This study revealed that O₂ fluctuation-induced UPR/ER stress could be prevented by maintaining the cells in a 1% O₂ environment.