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Yang Shan, Renu A Kowluru; DNA Methylation of matrix metalloproteinase-9 promoter in diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5200.
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Diabetes increases retinal oxidative stress, and oxidative stress is shown to modify histones at the promoter of matrix metalloproteinase-9 (MMP-9), regulating its transcription. Activated MMP-9 damages mitochondria, and in the pathogenesis of diabetic retinopathy, it also plays a pro-apoptotic role. Gene expression is also regulated by DNA methylation, and this study aims to investigate its role in regulation of MMP9 in diabetic retinopathy.
Retina from streptozotocin-induced diabetic C57/BL6, or overexpressing Sod2 mice were analyzed for methylation status of MMP-9 promoter using methylated DNA immunoprecipitation method. The gene transcripts were determined by qPCR, and protein-DNA interaction were quantified by chromatin immunoprecipitation. The results were validated in bovine retinal endothelial cells incubated in normal glucose (5mM) or high glucose (20mM) with/without MnTBAP (250uM), a superoxide dismutase mimic.
Diabetes decreased methylation of MMP-9 promoter, and significantly increased transcripts of retinal MMP-9. This was accompanied by increased expressions of the major methylation and demethylation enzymes, Dnmt1 and Tet1/2/3 respectively. The binding of Dnmt1 at MMP-9 promoter was also significantly increased compared to the values obtained from age-matched normal mice. Sod2 overexpression prevented diabetes-induced demethylation of MMP-9 promoter and increase in its transcription, and also ameliorated increase in DNA methylating enzymes. Similar increases in Dnmt1, Tets were observed in retinal endothelial cells incubated in high glucose compared to those in normal glucose. Supplementation of high glucose with MnTBAP prevented elevations in Dnmt1 and Tet2 binding at MMP-9 promoter and increases in Dnmt1 and Tets transcripts.
Although diabetes increased expressions of Dnmt1 and Tets, the levels of 5mC and Dnmt1 binding at Mmp9 promoter remained subnormal, suggesting regulation of MMP-9 by a highly dynamic methylation-demethylation process. This methylation machinery appears to be under the control of oxidative stress as its regulation ameliorated decrease in methylation of MMP-9 promoter, and increase in Dnmt1 and Tets. Thus, regulation of oxidative stress, via modulating epigenetic changes in MMP-9 promoter, should prevent increase in its expression. This will safeguard the mitochondria function and prevent the development of diabetic retinopathy.
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