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Toshimichi Shinohara, Palsamy Periyasamy, Keshore Bidasee, Masahiko Ayaki, Elanchezhian Rajan; Epilepsy Cataract: Valproic Acid Suppresses the Nrf2 Dependent Stress/Antioxidant Protection. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5948.
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The incidence of cataracts is higher in epileptic patients but the basic mechanism of this cataract is unknown. Most epileptic patients take anti-epileptic drugs such as Valproic Acid (VPA). VPA is also known to be a DNA demethylation agent. We previously reported that DNA demethylation was found in the promoter of the Kelch-like ECH-associated protein 1 (Keap1) gene in the cataract with diabetes. Keap1 is a negative regulator for nuclear factor-erythroid-2-related factor 2 (Nrf2) and Nrf2 is a central transcriptional activator which regulates more than 200 stress/antioxidant genes. We hypothesize that VPA activates the transcription of the Keap1 gene to suppress the Nrf2 dependent stress/antioxidant protection and results in the epileptic cataract.
Human lens epithelial cells (LECs), Nrf2-/- and Nrf2+/+ mouse LECs were cultured with VPA. Nrf2-/- and Nrf2+/+ mice were treated with VPA. H2-DCFH-DA and EthD staining detects ROS and cell death, respectively. Live cell confocal imaging was used for Ca++ release. Protein blot analyses were used with specific Abs to detect unfolded protein response (UPR) specific proteins. Bisulfite converted DNA sequencing and qPCR analysis were used for studying DNA methylation status and to identify the mRNA levels, respectively.
HLECs treated with pharmacological- or higher-levels of VPA activated UPR stress-specific proteins, produced ROS, released Ca++ from the endoplasmic reticulum. The levels of mRNA of Nrf2 and DNA methyltransferases were increased but these protein levels were significantly decreased by the proteosomal degradation. Contrary, mRNA of the DNA demethylation enzyme (TET1) and its protein were significantly elevated. The DNA from human LECs treated with VPA lost methylated cytosine in the Keap1 promoter, which expressed higher levels of mRNA and Keap1 protein. Keap1 protein levels were inversely associated with Nrf2 levels and the Nrf2 dependent stress/antioxidant protection. VPA is much more toxic to Nrf2-/- than Nrf2+/+ mouse.
VPA activates chronic UPR to suppress the Nrf2 dependent stress/antioxidant protection by epigenetic modification of the Keap1 gene and by proteosomal degradation. Thus, VPA induces failure of the Nrf2 dependent stress/antioxidant protection which leads to the lens oxidation and cataract formation.
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