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Cailing Liu, Dijana Vojnovic, Irene E Kochevar, Ula V Jurkunas; UVA Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3 Dependent Apoptosis in Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4897.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we reported that Nrf2 deficiency contributes to oxidant-antioxidant imbalance, causing apoptosis in Fuchs corneal endothelial dystrophy (FECD). To mimic the oxidative stress induced by sunlight, we utilized ultraviolet-A (UVA) irradiation to challenge human corneal endothelial cells (CEC). The aim of this project is to examine whether Nrf2-regulated antioxidant defense is activated in corneal endothelium by UVA light.
An immortalized human CEC (HCECi) was exposed to a UVA lamp to induce oxidative stress. Cells grown in complete medium were pretreated in OPTI-MEM medium 24 hrs prior to UVA treatment. Post UVA treatment with a dose of 2.5, 5, 10 or 25 J/cm2, the cells were recovered in OPTI-MEM medium for 0, 3, 6, 18 or 24 hrs at 37 oC. Reactive Oxygen Species (ROS) were detected with carboxy-H2DCFDA. Cell viability was evaluated by Trypan Blue staining or by measuring the released lactate dehydrogenase in supernatants. The mRNA levels of Nrf2 target genes HO-1 and NQO1 were quantified by real-time PCR. The protein levels of phospho-p53 were examined by immunoblotting. Activated Caspase3 was examined by immunoblotting and by a fluorescence assay. At each recovery time, the non-UVA treatment was used as a control.
Exposure of HCECi cells to 5 J/cm2, 10 J/cm2 and 25 J/cm2 UVA irradiation generated elevated levels of ROS production as compared to no UVA irradiation, but did not affect cell viability. At 6 hrs post irradiation, 5 J/cm2, 10 J/cm2 and 25 J/cm2 UVA irradiation led to a 1.5 to 1.6-fold increase in HO-1 mRNA as compared to controls. Similarly, 2.5 J/cm2 and 5 J/cm2 UVA irradiation caused a 1.3 to 1.5-fold increase in NQO-1 mRNA as compared to controls. Interestingly, 25 J/cm2 UVA treatment decreased NQO-1 mRNA levels at 6 hrs post irradiation. At 24 hrs post treatment, 5 J/cm2, 10 J/cm2 and 25 J/cm2 UVA exposure yielded a 1.3 to 1.8-fold increase in phospho- p53 and a 2.0 to 6.0-fold increase in activated Caspase3 levels as compared to no UVA treatment, resulting in 20-48% cell death.
Irradiation of HCECi cell with UVA light resulted in ROS elevation and induction of Nrf2-regulated anti-oxidant defense followed by phospho-p53 and Caspase3 activation. The initial activation of antioxidant defense exerted a cytoprotective effect which was lost with increasing recovery times and doses of UVA-induced stress.
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