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Cailing Liu, Yuming Chen, Irene E Kochevar, Ula Jurkunas; UVA Irradiation Activates Nrf2 Antioxidant Defense in Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2060.
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© ARVO (1962-2015); The Authors (2016-present)
UVA light induces formation of reactive oxygen species which cause oxidative stress and cellular damage. Corneal endothelial cells are particularly susceptible to oxidative stress. Previously, we reported that downregulation of Nrf2-regulated antioxidant defense contributes to oxidant-antioxidant imbalance in Fuchs corneal endothelial dystrophy, which in turn leads to oxidative DNA damage and apoptosis. The aim of this project is to determine whether UVA activates Nrf2-regulated antioxidant defense in corneal endothelium.
An immortalized human corneal endothelial cell line (HCECi) grown in Chen’s complete medium or pretreated in OPTI-MEM base medium were exposed to UVA irradiation (2.5, 5, 10 or 25 J/cm2). Post UVA treatment, the cells were incubated in OPTI-MEM base medium for 0, 3, 5, 18 or 24 hrs. Controls were cells not receiving UVA irradiation. Activated caspase3 was detected by immunoblotting and by a rhodamine-based fluorescence assay. Cell death was evaluated by measuring the released lactate dehydrogenase in supernatants. The mRNA levels of Nrf2, HO-1 and NQO1 were quantified by real-time PCR. The protein levels of Nrf2, HO-1, NQO-1 and phospho-p53 were examined by immunoblotting.
Exposure of HCECi cells to UVA induced caspase3 activation. The caspase3 levels increased with higher UVA doses and longer recovery time. Elevated caspase3 activation led to increased cell death ranging from 5 to 58% at 3 to 24 hrs recovery. Initial UVA irradiation caused caspase3-independent cell death. At 18 and 24 hrs recovery, phospho p53 level increased with higher UVA doses. Moreover, 2.5 J/cm2 UVA irradiation led to a 1.5-fold increase in Nrf2 mRNA at 3 hrs recovery and a 2.0-fold increase in protein level at 6 hrs recovery as compared to controls. Similarly, HO-1 and NQO-1 mRNA levels were about 1.6-fold increased using 2.5 J/cm2 UVA treatment at 3 hrs recovery.
Irradiation of HCECi cell with UVA resulted in initial upregulation of phospho-p53 and subsequent activation of caspase3. While acute UVA exposure led to caspase3-independent cell death, recovery-induced UVA damage was phospho-53 and caspase3 dependent. Nrf2-regulated antioxidant defense was activated at lower doses of UVA and preceded activation of caspase3. Role of Nrf2 in mediating UVA-induced damage should be investigated further.
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