Abstract
Purpose:
Dowregulation of DJ-1 and decreased nuclear localization of nuclear factor erythroid-derived 2-like 2 (Nrf2) has been detected in Fuchs endothelial corneal dystrophy endothelium. DJ-1 has been reported to stabilize Nrf2, which binds to antioxidant response element in the nucleus to protect cells from apoptosis. In this study, we investigated the effect of DJ-1 downregulation on Nrf2 nuclear translocation, Nrf2-associated protein regulation, and corneal endothelial cell susceptibility to oxidative stress.
Methods:
An immortalized normal human corneal endothelial cell line (HCECi) was transfected with 50 nM of siRNA specific to the human DJ-1 gene using Lipofectamine. Scrambled siRNA was used as control. Nrf2 and p53 levels in nuclear and cytosolic extracts were evaluated by western blotting. Nrf2-associated proteins such as Cul3 and Keap1 were detected by immunoprecipitation (IP) with anti-Nrf2 antibody, followed by western blotting with target antibodies. Oxidative stress was induced by exposing HCECi cells to a UVA broadband lamp with the fluence of 10 J/cm2. Cell pellets were harvested for western blotting with anti-caspase 3 and p53 antibodies, while supernatants were collected for a cell viability assay.
Results:
Despite similar levels of cytoplasmic Nrf2, nuclear Nrf2 protein levels decreased by 2.2-fold (p=0.04) in DJ-1 siRNA-treated HCECi cells as compared to scrambled siRNA-treated cells. IP studies detected Nrf2 association with Cul3 and Keap1 with an increase in Cul3-Nrf2 complex in DJ-1 siRNA-treated cells relative to controls. UVA irradiation led to an 8.5% increase in cell death in DJ-1 siRNA-treated cells as compared to controls. Moreover, downregulation of DJ-1 led to a 16.4% increase in active caspase 3 levels. This effect was augmented by UVA treatment, which led to a 28.6% increase in caspase 3 as compared to controls. Downregulation of DJ-1 resulted in an increase in cytoplasmic p53 levels, while UVA irradiation resulted in a 17.7% increase in total p53 levels in DJ-1 siRNA treated cells as compared to controls.
Conclusions:
Downregulation of DJ-1 impairs nuclear translocation of Nrf2 potentially by targeting Nrf2 for degradation through Cul3 and Keap1 pathways. Decline in DJ-1 levels leads to heightened cell susceptibility to UVA-induced apoptosis and activates p53-dependent pathway in corneal endothelium.
Keywords: 481 cornea: endothelium •
634 oxidation/oxidative or free radical damage •
739 transcription factors