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Bhavana Chhunchha, Eri Kubo, Dhirendra P Singh; Nrf2 Accelerates Oxidative Stress And Lens Epithelial Cell Death Through Induction of KLF9-Driven Repression of Prdx6 In Response To Excessive Oxidative Load. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5601.
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© ARVO (1962-2015); The Authors (2016-present)
Regulation of oxidative stress is critical to cell survival. An antioxidant protein, Peroxiredoxin (Prdx) 6, protects cells against oxidative stress. We earlier reported that Sumoylation of Prdx6 reduces its activity, increasing oxidative load and cell death. In this study, we demonstrated that in response to increased level of ROS over a threshold, Nrf2 activation of transcription Kruppel-like factor (Klf9) expression results in Klf9-dependent repression of Prdx6 transcription, leading to further increases in levels of ROS and cell death.
Cultured lens epithelial cells (LECs) treated with variable concentrations (conc.) of H2O2 (0 to 300μM) were used to examine expression of Prdx6, Nrf2, Klf9 and Sumo1 by real-time PCR and Western analyses. Actinomycin D, a transcription inhibitor, treatment determined modulation in expression of Nrf2 and Klf9 mRNA. Nrf2-mediated activation of Klf9 and Klf9-dependent Prdx6 repression was examined by transfecting LECs with shRNA (sh) Nrf2 and shKlf9, respectively. LECs overexpressed with GFP-linked Nrf2 or Klf9 and/or Sumo1, and underexpressed with shKlf9 were used to examine effects on cell viability and ROS by using MTS and H2DCFH-DA dye, respectively. Transactivation assay with Human Prdx6 promoter-CAT (-1589/+109nts) monitored Klf9 regulation of Prdx6.
LECs exposed to excessive oxidative stress (>100μM H2O2 conc.) had increased expression of Nrf2, Klf9, Sumo1 and ROS with decreased Prdx6 mRNA and protein, consistent with increased H2O2 conc. The process was amplified in cells overexpressing Sumo1. Actinomycin D treatment revealed that increased Nrf2 and Klf9 expression was linked to activation of their transcription. shRNA experiments showed Nrf2-dependent regulation of Klf9 and Klf9-mediated repression of Prdx6 mRNA. Promoter assay revealed that Klf9 repressed Prdx6 transcription.Cells overexpressing Klf9 showed increased ROS expression and reduced Prdx6 expression and were more susceptible to cell death. shKlf9 reversed the process, suggesting Klf9 independently caused the ROS-driven cell death.
Data revealed an Nrf2-mediated feed-forward regulation of ROS by stimulating Klf9-dependent suppression of Prdx6 and accumulation of ROS leading to increased LECs death. Since Klf9 depletion suppresses oxidative stress, this may be a promising target in oxidative disorders.
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