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.
Keywords: NF-E2-related factor 2; antioxidants; hypoxia inducible factor-1α; low oxygen; unfolded protein response.