Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

Abstract

Diabetes elevates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 damages mitochondria, accelerating retinal capillary cell apoptosis, a phenomenon which precedes the development of retinopathy. Diabetes also favors epigenetic modifications regulating expression of many genes. DNA methylation is maintained by methylating-hydroxymethylating enzymes, and retinal DNA methyltransferase (Dnmt) is activated in diabetes. Our aim is to investigate the role of DNA methylation in MMP-9 regulation. Effect of high glucose on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and binding of Dnmt1 and hydroxymethylating enzyme (Tet2) on MMP-9 promoter were quantified in retinal endothelial cells. Specific role of Tet2 in MMP-9 activation was validated using Tet2-siRNA. The results were confirmed in the retina from streptozotocin-induced diabetic mouse. Although glucose increased Dnmt1 binding at MMP-9 promoter, it decreased 5mC levels. At the same promoter site, Tet2 binding and 5hmC levels were elevated. Tet2-siRNA ameliorated increase in 5hmC and MMP-9 transcription, and protected mitochondrial damage. Diabetic mice also presented similar dynamic DNA methylation changes in the retinal MMP-9 promoter. Thus, in diabetes transcription of retinal MMP-9 is maintained, in part, by an active DNA methylation-hydroxymethylation process, and regulation of this machinery should help maintain mitochondrial homeostasis and inhibit the development/ progression of diabetic retinopathy.

The Role of DNA Methylation in the Metabolic Memory Phenomenon Associated With the Continued Progression of Diabetic Retinopathy

“…32,33 Experimental models have shown that the alterations in histone modifications that maintain mitochondrial homeostasis in diabetic retinopathy, including histone modifications at the glutamate-cysteine ligase, catalytic subunit-antioxidant response element 4, Sod2 promoter/enhancer, and MMP-9 promoter continue even after termination of hyperglycemic insult. 17,29 Altered methylation of both mtDNA ( D-loop region) and nDNA ( MMP-9 ) are associated with mitochondrial homeostasis, 21,22 and here we present novel results showing that reversal of hyperglycemic insult by normal glycemia does not benefit the machinery responsible for maintaining DNA methylation; enzymes responsible for DNA methylation and hydroxymethylation remain active for some time after removal of high glucose, and DNA methylation (mtDNA and nDNA) continue to be altered. The results demonstrate that the duration of normal glycemia that follows hyperglycemia (post poor glycemic control), affects the outcome of the good control; extension of this period to 8 days from 4 days has beneficial effects on DNA methylation.…”

“…Due to increased binding of Tet2 at the MMP-9 promoter, the levels of 5hmC are increased and hypomethylation of the promoter results in its transcriptional activation. 22 Novel results presented here show that once the retinal Tet is activated in diabetes, it does not benefit from the good glycemic control for a duration that follows the hyperglycemia, and the MMP-9 promoter continues to be hypomethylated with MMP-9 activation, further compromising mitochondrial homeostasis.…”

“…Our recent work has implicated Tet2 in transcriptional activation of MMP-9 in diabetic retinopathy 22 ; to confirm the role of Tet2, the transcripts of MMP-9 were quantified. Consistent with Tet2 , MMP-9 transcripts remain elevated in the cells in the 4d-4d group, but when the normal glucose phase was extended from 4 days to 8 days (4d-8d group), MMP-9 transcripts were significantly lower compared with the values from cells in the continuous high-glucose or in 4d-4d group (Fig.…”

“…DNA methylation is a dynamic process and methylated cytosine can be oxidized to 5hmC by Tet 23 ; to understand the role of Tet in the metabolic memory phenomenon, gene transcription of Tet2 (the only member of the Tet family upregulated in the retina in diabetes 22 ), was quantified. Consistent with Dnmt1 , Tet2 transcripts also remained elevated even after 4 days of normal glucose that had followed 4 days of high glucose; but extending the normal glucose phase to 8 days (4d-8d), had significant beneficial effects on Tet2 transcripts (Fig.…”

“…19 Our recent work has shown that Tet2 is also activated in diabetes, and its binding at the MMP-9 promoter is increased, allowing the promoter to be hypomethylated. 22 However, the role of DNA methylation machinery in the metabolic memory associated with the continued progression of diabetic retinopathy remains to be investigated.…”