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. 2014 Oct 9;55(11):7256-65.
doi: 10.1167/iovs.14-15193.

Epigenetic modifications of Keap1 regulate its interaction with the protective factor Nrf2 in the development of diabetic retinopathy

Manish Mishra  1 , Qing Zhong  1 , Renu A Kowluru  1
Affiliations

Epigenetic modifications of Keap1 regulate its interaction with the protective factor Nrf2 in the development of diabetic retinopathy

Manish Mishra et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Diabetes induces oxidative imbalance in the retina and impairs Nrf2-mediated antioxidant response, and elevates Keap1, the cytoplasmic repressor of Nrf2. The goal of this study was to understand the role of epigenetic modifications at Keap1 promoter in regulation of Nrf2 function.

Methods: The effect of high glucose on the binding of transcriptional factor Sp1 at Keap1 promoter and histone methylation status of the promoter was investigated in retinal endothelial cells. Role of histone methylation was confirmed in cells transfected with siRNA of methyltransferase enzyme Set7/9 (SetD7). In vitro results were confirmed in the retina from streptozotocin-induced diabetic rats. The role of epigenetic modifications of Keap1 promoter in the metabolic memory was examined in rats maintained in poor control for 3 months followed by good control for 3 months.

Results: Hyperglycemia increased the binding of Sp1 at Keap1 promoter, and enriched H3K4me1 and activated SetD7. SetD7-siRNA prevented increase in Sp1 binding at Keap1 promoter and Keap1 expression, and ameliorated decrease in Nrf2-regulated antioxidant genes. Cessation of hyperglycemia failed to attenuate increased binding of Sp1 at Keap1, and the promoter continued to be methylated with increased expression of Keap1 and decreased expression of Nrf2-regulated genes.

Conclusions: Epigenetic modifications at Keap1 promoter by SetD7 facilitate its binding with Sp1, increasing its expression. Keap1 restrains Nrf2 in the cytosol, impairing its transcriptional activity. Reversal of hyperglycemia fails to provide any benefit to epigenetic modifications of Keap1 promoter, suggesting their role in both the development of diabetic retinopathy and the metabolic memory phenomenon.

Keywords: Keap1; Nrf2; diabetic retinopathy; epigenetic modifications; metabolic memory.

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Figures

Figure 1
Figure 1
Transfection of retinal endothelial cells with SetD7-siRNA. The transfection efficiency was determined in cells transfected with SetD7-siRNA or scramble RNA by quantifying the (a) protein (Western blot) and (b) gene (q-PCR) expressions of SetD7. Transfection experiments were repeated in three to four different cell preparations, and the mRNA values are represented as mean ± SD. UT, untransfected cells, Set-si and SC, cells transfected with SetD7-siRNA or scramble RNA, respectively. *P < 0.05 versus UT.
Figure 2
Figure 2
High glucose increases the binding of Sp1 at Keap1 promoter and elevates its expression. Untransfected cells, or cells transfected with SetD7-siRNA or scramble RNA, were incubated in 20 mM glucose for 4 days, or untransfected cells incubated in 20 mM glucose for 4 days followed by 5 mM glucose for 4 additional days (20-5) were analyzed for (a) Sp1 binding at Keap1 by ChIP technique. The q-PCR value in each immunoprecipitate was normalized to the Ct value from the input sample by using the delta-delta-Ct (ddCt) method. Rabbit IgG (^) was used as a negative antibody control. (b) Keap1 mRNA was quantified by q-PCR using β-actin as a housekeeping gene. Set-si and SC, cells transfected with SetD7-siRNA or scramble RNA, respectively, and incubated in 20 mM glucose for 4 days. Data are represented as mean ± SD from four to six preparations in each group. *P < 0.05 compared with 5 mM glucose and #P < 0.05 versus high glucose.
Figure 3
Figure 3
Enrichment of H3K4me1 at Keap1 promoter impairs Nrf2 signaling. (a) H3K4me1 at Keap1 was quantified by ChIP technique using rabbit IgG as negative antibody control (^), and (b) the products were confirmed by agarose gel electrophoresis. (c) Nrf2 binding at Gclc promoter was determined by ChIP technique, and mRNA levels of (d) Gclc and (e) HO1 were quantified by q-PCR. Data are mean ± SD from three to four preparations in each group. *P < 0.05 and #P < 0.05 compared with 5 mM glucose and 20 mM glucose, respectively.
Figure 4
Figure 4
High glucose increases SetD7 in retinal endothelial cells. (a) SetD7 mRNA was quantified by q-PCR using β-actin as housekeeping gene. (b) Activity was assayed by an ELISA-based methyltransferase activity assay. Values from the 5-mM glucose are considered as 1 for mRNA or 100% for activity. Values are mean ± SD from three to four experiments, and each experiment was performed in duplicate. *P < 0.05 vs. 5 mM glucose.
Figure 5
Figure 5
Diabetes increases the binding of Sp1 at Keap1 promoter. Retina from rats in poor glycemic control for 3 months followed by good glycemic control for 3 months (PC-Rev), or continuous PC or GC for 6 months was analyzed for (a) Sp1 binding at Keap1 using ChIP assay. Keap1 (b) mRNA was quantified by q-PCR using β-actin as a housekeeping gene, and (c) its protein expression by Western blotting technique and β-actin was used as a loading control. Results are represented as mean ± SD from five to six rats in each group, each analysis performed in duplicate. *P < 0.05 versus normal and #P < 0.05 versus PC rats.
Figure 6
Figure 6
H3K4me1 is increased at retinal Keap1 promoter in diabetes. (a) H3K4me1 at Keap1 was quantified by ChIP technique, and (b) confirmed by agarose gel electrophoresis. Norm, normal; IgG control, immunoprecipitation with IgG (^); Input, total genomic DNA without ChIP. Data are represented as mean ± SD from five to seven rats in each group. *P < 0.05 and #P < 0.05 versus normal and PC rats, respectively.
Figure 7
Figure 7
Diabetes increases retinal SetD7. (a) SetD7 mRNA was quantified by q-PCR using β-actin as housekeeping gene. Fold change was normalized to the values of normal control by ddCt method. (b) SetD7 enzyme activity was measured by an ELISA-based activity assay kit. Values obtained from normal rat are considered as 1 for mRNA and 100% for activity. Values are mean ± SD from five or more rats in each group. *P < 0.05 versus normal rats.
Figure 8
Figure 8
Retinal KEAP1 promoter is epigenetically modified in the human donors with diabetic retinopathy. (a) SP1 binding at KEAP1 was measured by ChIP technique by amplifying for the SP1 binding region at KEAP1 promoter in protein-DNA complex using normal rabbit IgG (^) as antibody control. (b) KEAP1 and (c) SETD7 mRNA levels were quantified by qPCR using β-actin as a housekeeping gene. The values obtained from nondiabetic donors were considered as 1. Diab, donors with documented diabetic retinopathy; Norm, age-matched nondiabetic donors. Data are presented as mean ± SD from four donors in each group. *P < 0.05 versus nondiabetic donors.
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