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

Abstract

Abstract. Cataractogenic stresses are associated with the induction of endoplasmic reticulum (ER) stress. However, little is known about oxygen (O 2 )-induced ER stress in the lens. Cataract research has focused on elevated levels of O 2 in lens epithelial cells (LECs). Excessive levels or a lack of O 2 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 O 2 levels induces a UPR, and may be controlled by maintaining human LECs (hLECs) in a specific concentration of O 2 . Human LECs were cultured in different atmospheric levels of O 2 . 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 O 2 activated Nrf2/Keap1. The LECs shifted to 1% atmospheric O 2 from 0, 4 or 20% for 24 h showed decreased levels of Keap1. By contrast, hLECs cultured in 1% atmospheric O 2 for 24 h and then shifted to 0, 4 or 20% O 2 exhibited a significant upregulation of Nrf2. These results suggest that oxidative stress proteins were not expressed in a 1% O 2 environment. The O 2 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 O 2 . Either 0 or 20% of atmospheric O 2 activated the UPR and the Nrf2/Keap1-mediated antioxidant system in LECs and chronic exposure to O 2 fluctuation led to ROS production and cell death. This study revealed that O 2 fluctuation-induced UPR/ER stress could be prevented by maintaining the cells in a 1% O 2 environment.

Nrf2 as a target for prevention of age‐related and diabetic cataracts by against oxidative stress

“…O 2 level with the range of 0.1–1.0% can be defined as severe hypoxia, and the O 2 level in lens can effortlessly fall into the severe hypoxia range even in a normal (13–15%) atmospheric O 2 supply (Shui & Beebe, ), especially for diabetic patients (Holekamp et al ., ). Either 0 or 20% atmospheric O 2 induced significant ROS production and cell death in cultured HLECs, accompanied by increased UPR protein and inhibition of the Nrf2/Keap1‐dependent antioxidant protection in LECs, and the results showed that 1% O 2 was appropriate for culturing LECs (Zheng et al ., ).…”

The Nrf2 Signaling in Retinal Ganglion Cells under Oxidative Stress in Ocular Neurodegenerative Diseases

“…Then, Nrf2 initiates the transcription of ARE controlled anti-oxidative enzymes, such as superoxide dismutase (SOD), catalase, glutathione S-transferase (GSTP) 5 , NAD (P)H: quinone oxidoreductase 1 (NQO1) 6 , heme oxygenase-1 (HO-1) 7 , thioredoxin reductase (TrxR), glutathione reductase (GR) and glutathione-S-transferase (GST) 8 , 9 , which detoxify ROS through glutathione (GSH) regulation 10 . Nrf2 is known as the molecular switch of the Nrf2 signaling (also known as Nrf2/Keap1 or Nrf2/Keap1/ARE signaling) and serves as a foremost component of ROS signaling pathway that can be activated by oxidative stress inducers, such as sulforaphane (SF) 11 , hyperoside 12 , hyperglycemia 3 and inhibited by oxidative stress factors such as homocysteine (Hcy) 13 , O 2 fluctuation 14 and hypoglycemia under hypoxia 15 . The protective role of Nrf2 against oxidative stress in ocular diseases has been explained in the following researches: DR 3 , retinal I/R injury 4 , etc.…”

Age-related cataracts: Role of unfolded protein response, Ca 2+ mobilization, epigenetic DNA modifications, and loss of Nrf2/Keap1 dependent cytoprotection

“…Lens-specific cataract formation might be induced by a predominant interaction between lens specific protein(s) and the ER stressor(s). It has been demonstrated that human lens epithelial cells exposed to a certain oxygen environment activated a protective UPR while chronic hypoxic conditions induced ROS production and apoptotic UPR resulting in lens epithelial cell death and lens oxidation (Elanchezhian et al, 2012b; Zheng et al, 2015). Thus, cataract is one of the protein misfolding diseases, and misfolded protein conformation in the ER initiates pathogenic processes and results in misfolded crystallin aggregations.…”