March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
The Role Of DJ-1 In Nrf2-regulated Antioxidant Defense In Human Corneal Endothelial Cells
Author Affiliations & Notes
  • Cailing Liu
    Schepens / Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Thore Schmedt
    Schepens / Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Ula Jurkunas
    Schepens / Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  Cailing Liu, None; Thore Schmedt, None; Ula Jurkunas, None
  • Footnotes
    Support  RO1 EY020581 (UVJ);New England Corneal Transplant Fund (UVJ); Research to Prevent Blindness Award (UVJ)
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6011. doi:
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    • Get Citation

      Cailing Liu, Thore Schmedt, Ula Jurkunas; The Role Of DJ-1 In Nrf2-regulated Antioxidant Defense In Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6011.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Corneal endothelial cell loss related to aging and dystrophies has been shown to be associated with oxidative stress. Specifically, a decline in nuclear factor erythroid-derived 2-like 2 (Nrf2) has been detected in Fuchs endothelial corneal dystrophy. In some cell types, DJ-1 is reported as a stabilizer of Nrf2, which binds to antioxidant response element and activates antioxidant activity. In this study, we investigated the role of DJ-1 in antioxidant defense and, specifically, regulation of Nrf2 transcriptional function in corneal endothelium.

Methods: : An immortalized normal human corneal endothelial cell line (HCECi) was transfected with 25, 50 or 100 nM of siRNA specific to the human DJ-1 gene (referred to as siDJ-1) using LipofectamineTM 2000. Scrambled siRNA was used as a control. Cellular toxicity was evaluated by phase contrast microscopy. Knockdown efficiency and the effect of DJ-1 downregulation on Nrf2 and its major transcriptional target, heme oxygenase (HO-1), were evaluated by TaqMan® real-time PCR and Western blot analyses. Protein oxidative modification was evaluated by measuring levels of protein carbonyls via ELISA assay.

Results: : HCECi produced DJ-1 protein in a monomeric form at a molecular weight of 20 kD. siRNA transfection did not cause any cellular toxicity as observed by normal hexagonal endothelial cell morphology. The most efficient knockdown was achieved with 50 nM siDJ-1 that led to 90% reduction of DJ-1 protein level and 99% of mRNA expression as compared to controls. Downregulation of DJ-1 led to a 4.5-fold decrease in Nrf2 mRNA (p<0.01) and a 1.7-fold decrease in protein levels. Similarly, real-time PCR showed that HO-1 expression was 9.0-fold decreased by siDJ-1 in HCECi as compared to controls (p<0.01). Moreover, the amount of protein carbonyls was increased by 29% in siDJ-1-treated HCECi as compared to controls.

Conclusions: : DJ-1 downregulation decreases Nrf2 at the transcriptional and translational levels, and impairs antioxidant cytoprotection, as seen by heightened protein oxidation. Our studies show that DJ-1 plays a potentially important role in Nrf2-regulated defense against oxidative damage in human corneal endothelium.

Keywords: cornea: endothelium • oxidation/oxidative or free radical damage • transcription factors 
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