Abstract
Purpose :
Failure of redox equilibrium of lens epithelial cells (LECs) due to weakened antioxidant defense is linked to onset of cataract. Sulforaphane (SFN), an Nrf2 activator, affects cell fate by regulating antioxidant genes like peroxiredoxin (Prdx) 6. Here we showed that the mechanism of hormetic effects of SFN; higher doses of SFN (similar to H2O2) activated death signaling by overstimulation of Nrf2-mediated Kruppel-like factor (Klf9) repression of Prdx6.
Methods :
Cultured human LECs were treated with SFN (0 to 24μM) or H2O2 (0 to 300μM) for 12 to 24 h to induce biphasic responses. Cell viability, reactive oxygen species (ROS), Prdx6, Nrf2, and Klf9 levels were examined by MTS, H2DCF dye, qPCR and Western blot, respectively. In silico analysis identified Klf9 sites in Prdx6 (-1559/+30) and Nrf2 sites in Klf9 (-5925/+65) promoters. Effect of SFN or H2O2 was done by transfecting LECs with Prdx6-or Klf9-CAT promoter or its mutants with Nrf2-siRNA or Klf9 plasmids. ChIP assay with treated LEC revealed DNA-Nrf2 or DNA-Klf9 binding. LECs overexpressing Nrf2/Klf9 plasmid were used to examine cell viability. A two-tailed Student’s t-test was applied for statistical analysis.
Results :
Increased doses of SFN (>12μM) or H2O2 (>100μM) caused accumulation of Nrf2 and Klf9 upregulation with a decrease of Prdx6 mRNA and protein, and increased ROS and cell death (p<0.001). Like nontoxic doses (<50μM) of H2O2, lower doses of SFN (<6μM) did not upregulate Klf9 and increased Prdx6 level and cell survival. SiRNA transfection showed Nrf2-dependent regulation of Klf9 and Klf9-mediated Prdx6 repression in cells treated with higher amounts of SFN or H2O2 (p<0.001). Promoter and ChIP assays showed that all four antioxidant response elements participated in upregulating Klf9, and three Klf9 repressive sites (CA/GCCC) were responsible to repress Prdx6(p<0.001).Cells overexpressing Klf9 showed increased ROS (~2fold) and were vulnerable to death, validating that overstimulation of Nrf2 caused the ROS-driven cell death by repressing Prdx6 via Klf9.
Conclusions :
Given the hormetic shifting of SFN--low doses of SFN on cell survival and high doses in promotion of cell death, the phenomenon which occurred by unique modalities of Nrf2 action in cells-we propose the delivery of low doses of SFN to stimulate antioxidant defense to protect cells facing oxidative stress-induced disorders.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.