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Palsamy Periyasamy, Elanchezhian Rajan, Masahiko Ayaki, Toshimichi Shinohara; Promoter Demethylation of the Keap1 Gene in Human Aging Lens: A Possible Epigenetic Mechanism for Lens Oxidation by Failure of Keap1/Nrf2 Dependent Antioxidant Protection. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5122.
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Aging is a major risk factor associated with age-related cataracts (ARCs). So lens oxidation increases eventually as an individual is aged. However, limited information is available on the master transcriptional regulators, especially the Kelch like-ECH-associated protein 1 (Keap1)/Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), for lens antioxidant genes. It is known that Nrf2, upon oxidative stress, activates expression of glutathione reductase, catalase, thioredoxin, etc. to preserve cellular redox homeostasis. Also, the Nrf2 level is regulated by its negative regulator, Keap1 by proteasomal degradation. Epigenetic mechanisms may also play a pivotal role in the regulation of gene expressions. We hypothesized that Keap1/Nrf2 dependent antioxidant protection might be failed with aging. Here, we investigated the Keap1 promoter DNA methylation status in human aged and cataract lenses.
The genomic DNA from human cataract capsulotomy specimens and clear lenses (NDRI, Philadelphia, PA) were treated with sodium bisulfite. The bisulfite-modified DNA was amplified by bisulphite sequencing PCR with specific primers. The PCR products were purified, then cloned into pCR®4-TOPO vector using TOPO TA Cloning® kit (Invitrogen, CA). About 10 independent clones of each amplicon were sequenced to identify CpG methylation status.
A significant change in the DNA methylation status was found in the CpG islands of Keap1. The CpG island (-433 to -96) of the Keap1 promoter was hypermethylated at age 17. But, these methylated CpGs were significantly demethylated at age 37 and further at age 63. In addition, the Keap1 promoter DNA methylation was highly heterogeneous in each lens epithelial cells of single lens, suggested the heterogeneity in lens oxidation status. Based on the sequencing data, we found transcriptional element binding sites for AP2, CREB, E2F, Sp1, and NF-ΚB, which further activates transcription, in the demethylated CpG island of Keap1 promoter. This suggested the increased level of Keap1 during aging; thereby Nrf2 is reduced by proteasomal degradation. Although changes in methylation pattern were found in the ARC lenses, demethylation status during aging was significantly greater than that of ARC lenses.
Thus, promoter DNA demethylation of Keap1 with aging leads to lower levels of Nrf2 which results in the reduced antioxidant protection and increased lens oxidation.
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