Epac1 deacetylates HMGB1 through increased IGFBP-3 and SIRT1 levels in the retinal vasculature

Abstract

Purpose: Inflammation is a key component of retinal disease. We previously reported that exchange protein for cAMP 1 (Epac1) reduced inflammatory mediators, including total levels of high mobility group box 1 (HMGB1) in retinal endothelial cells (RECs) and the mouse retina. The goal of this study was to determine intermediate pathways that allow Epac1 to reduce HMGB1, which could lead to novel targets for therapeutics.

Methods: We used endothelial cell-specific conditional knockout mice for Epac1 and RECs to investigate whether Epac1 requires activation of insulin like growth factor binding protein 3 (IGFBP-3) and sirtuin 1 (SIRT1) to reduce acetylated HMGB1 levels with immunoprecipitation, western blot, and enzyme-linked immunosorbent assay (ELISA).

Results: Data showed that high glucose reduced IGFBP-3 and SIRT1 levels, and increased acetylation of HMGB1 in RECs. An Epac1 agonist reduced acetylated HMGB1 levels in high glucose. The Epac1 agonist could not reduce HMGB1 or SIRT1 levels when IGFBP-3 siRNA was used. The agonist also could not reduce HMGB1 when SIRT1 siRNA was used. The mouse retina showed that loss of Epac1 increases acetylated HMGB1 levels and reduces IGFBP-3 and SIRT1 levels.

Conclusions: Taken together, the data suggest that Epac1 activates IGFBP-3 to increase SIRT1, leading to a significant reduction in acetylated HMGB1. These findings provide novel therapeutic targets for reducing key inflammatory cascades in the retina.

Figure 1

Protein data from Epac1 floxed and endothelial cell–specific Epac1 knockout mice. A: Acetylation of HMGB1. B: SIRT1 enzyme-linked immunosorbent assay (ELISA). C: IGFBP-3 levels. *p<0.05 versus floxed mice. n=5 mice (two eyes per mouse pooled). Panels A and C show representative blots from the mice. Data are mean ± standard error of the mean (SEM).

Figure 2

Retinal endothelial cells grown in NG, HG, or normal glucose plus mannitol. Cells in normal glucose (NG) and high glucose (HG) were treated with an Epac1 agonist. A: Enzyme-linked immunosorbent assay (ELISA) results for SIRT1. B: HMGB1 levels. *p<0.05 versus NG, #p<0.05 versus HG, n=4 for all groups. Data are mean ± standard error of the mean (SEM).

Figure 3

Retinal endothelial cells grown in NG or HG. Cells in normal glucose (NG) and high glucose (HG) were treated with an Epac1 agonist, and some cells received the Epac1 agonist after transfection with IGFBP-3 siRNA or scrambled siRNA. A: Acetylation of HMGB1. B: SIRT1 levels. C: IGFBP-3 to verify knockdown. *p<0.05 versus NG, #p<0.05 versus HG, $p<0.05 versus HG+Epac1 agonist. n=4 for all groups. Data are mean ± standard error of the mean (SEM).

Figure 4

Retinal endothelial cells grown in NG or HG. Cells in normal glucose (NG) and high glucose (HG) were treated with an Epac1 agonist, and some cells received the Epac1 agonist after transfection with SIRT1 siRNA. A: SIRT1 levels. B: HMGB1 acetylation. *p<0.05 versus NG, #p<0.05 versus HG, $p<0.05 versus HG+Epac1 agonist. n=4 for all groups. Data are mean ± standard error of the mean (SEM).