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Elanchezhian Rajan, Palsamy Periyasamy, Madson J. Christian, Lynch W. David, Toshimichi Shinohara; Influence on the Nrf2/Keap1 Gene Expression by Different Atmospheric Oxygen Levels in Human and Nrf2 Knockout Mice Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4149.
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© ARVO (1962-2015); The Authors (2016-present)
Although, many cataractogenic stresses induce the endoplasmic reticulum (ER) stress, little is known for the ER stress regulated antioxidant protection genes in the lens. The sequential reduction of molecular oxygen through the addition of electrons leads to the formation of a number of Reactive Oxygen Species (ROS). Recent reports showed that cataracts are associated with the elevated levels of molecular oxygen (O2). Excess levels or lack of O2 are well known to induce ER stress, and the chronic ER stress activates the unfolded protein response (UPR). The UPR and/or oxidants regulate a major transcription factor, NF-E2-related factor 2 (Nrf2) for many antioxidant enzyme genes to preserve cellular redox homeostasis. Nrf2 is negatively regulated by a Kelch-like ECH-associated protein 1 (Keap1). We hypothesized that the either excess or lack of O2 stress generates the production of ROS and up-regulate Nrf2 associated antioxidant protection.
Human lens epithelial cells (LECs) and mouse Nrf2 -/- and Nrf2+/+ LECs were cultured in different atmospheric O2. H2-DCFH-DA and EthD staining detects ROS and cell death, respectively. Protein blot analyses were performed with Abs specific to antioxidant specific UPR proteins. RT-PCR were used to identify the mRNA levels.
Human LECs treated with 0 and 20% of atmospheric O2 activated Nrf2/Keap1. The LECs were transferred into 1% atmospheric O2 for 1, 2, 4 and 8 h, the Nrf2/Keap1 was decreased to basal level. In contrast, human LECs were cultured for 24 h in 1% atmospheric O2 and then transfer to the 0, 1, 4, and 20% O2. Significant up-regulation of Nrf2/Keap1 in LECs treated with 0, and 20% oxygen but not in 1 and not much in 4% O2. These results suggest that oxidative stress proteins were not expressed in 1% O2 environment. The O2 levels in the culture medium were equilibrated within 2 h in the cell culture plates. The LECs isolated from the Nrf2 -/- LECs were cultured in either 0. 1, 4, and 20% of O2 and 0 and 20% O2 induce significant cell death but not that from Nrf2+/+ LECs. These results showed that an appropriate oxygen environment for cultured LECs is about 1% of atmospheric O2.
Either 0 or 20% of atmospheric O2 activated Nrf2/Keap1 in LECs. Minimum levels of oxidative stress for LECs culture is found to be 1% of atmospheric O2 and decreased or increased levels of O2 from the 1% induces the oxidative stress to the LECs.
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