Hyperglycemia-induced reactive oxygen species increase expression of the receptor for advanced glycation end products (RAGE) and RAGE ligands

Abstract

Objective: RAGE interacts with the endogenous ligands S100 calgranulins and high mobility group box 1 (HMGB1) to induce inflammation. Since hyperglycemia-induced reactive oxygen species (ROS) activate many pathways of diabetic tissue damage, the effect of these ROS on RAGE and RAGE ligand expression was evaluated.

Research design and methods: Expression of RAGE, S100A8, S100A12, and HMGB1 was evaluated in human aortic endothelial cells (HAECs) incubated in normal glucose, high glucose, and high glucose after overexpression of either uncoupling protein 1 (UCP1), superoxide dismutase 2 (SOD2), or glyoxalase 1 (GLO1). Expression was also evaluated in normal glucose after knockdown of GLO1. Expression was next evaluated in high glucose after knockdown of nuclear factor (NF)-kappaB p65 (RAGE) and after knockdown of activated protein-1 (AP-1) (S100A8, S100A12, and HMGB1), and chromatin immunoprecipitation (ChIP) was performed +/- GLO1 overexpression for NFkappaB p65 (RAGE promoter) and AP-1 (S100A8, S100A12, and HMGB1 promoters). Finally, endothelial cells from nondiabetic mice, STZ diabetic mice, and STZ diabetic mice treated with the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) were evaluated.

Results: High glucose increased RAGE, S100A8, S100A12, and HMGB1 expression, which was normalized by overexpression of UCP1, SOD2, or GLO1. GLO1 knockdown mimicked the effect of high glucose, and in high glucose, overexpression of GLO1 normalized increased binding of NFkappaB p65 and AP-1. Diabetes increased RAGE, S100A8, and HMGB1 expression, and MnTBAP treatment normalized this.

Conclusions: These results show that hyperglycemia-induced ROS production increases expression of RAGE and RAGE ligands. This effect is mediated by ROS-induced methylglyoxal, the major substrate of glyoxalase 1.

FIG. 1.

Hyperglycemia-induced ROS increase expression of RAGE, S100 calgranulins, and HMGB1. A: Primary HAECs were infected with UCP1, SOD2, GLO1, or empty control adenovirus (CTL). After incubation with either low glucose (LG) (5 mmol/l) or high glucose (HG) (30 mmol/l) for 5 days, mRNA levels were determined by real-time PCR (n = 3). B: Intracellular protein levels of RAGE, S100A8, and HMGB1 were determined by Western blotting (n = 4). Protein sizes were evaluated by standard protein markers, and their sizes were as follows: RAGE (46 kDa), S100A8 (11 kDa), and HMGB1 (25 kDa). C: Levels of secreted HMGB1 were determined in culture medium by enzyme-linked immunosorbent assay (ELISA) (n = 5). Values are shown as means + SD, *P < 0.05 vs. LG/CTL group. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 2.

Overexpression of UCP1, SOD2, and GLO1 prevents hyperglycemia-induced ROS generation and methylglyoxal formation. Primary HAECs were infected with UCP1, SOD2, GLO1, or empty control adenovirus (CTL). After incubation with either low glucose (LG) (5 mmol/l) or high glucose (HG) (30 mmol/l) for 1 or 5 days, the cells were used for analysis. A: ROS formation on day 5. B: Methylglyoxal-modified protein on day 5. C: Representative blots for B. D: GLO1 activity on day 1. E: GLO1 protein on day 1. F: Representative blots for E. G: GLO1 activity on day 5. H: GLO1 protein level on day 5. I: Representative blots for H. n = 4. *P < 0.05 vs. LG/CTL group. Values are shown as means + SD. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 3.

GLO1 knockdown duplicates the effect of hyperglycemia on expression of RAGE, S100 calgranulins, and HMGB1. Conditionally immortalized HAECs were infected with either shGLO1 or nontargeting control (CTL) lentivirus and then incubated in either low glucose (LG) or high glucose (HG) for 5 days. A: mRNA levels were determined by real-time PCR (n = 3). B: Protein levels were determine by Western blotting (n = 4). Protein sizes were evaluated and confirmed by standard protein markers, and their sizes were as follows: RAGE (46 kDa), S100A8 (11 kDa), and HMGB1 (25 kDa). C: Intracellular methylglyoxal-modified proteins were quantitated by Western blotting (n = 4). D: Representative blots for C. Values are shown as means + SD. *P < 0.05 vs. CTL group. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 4.

Hyperglycemia-induced methylglyoxal increases binding of NFκB to the RAGE promoter and binding of AP-1 to the S100A8, S10012, and HMGB1 promoters. A: Conditionally immortalized HAECs were treated with low glucose (LG), high glucose (HG), or HG after transfection with either scrambled oligonucleotides (LG and HG) or siRNA for p65 (HG/siP65) for 5 days, and the RAGE mRNA level was determined by qPCR (n = 3). B: Primary HAECs were treated with LG, HG, or HG after infections with either nontarget control or GLO1 adenovirus (HG/GLO1) for 5 days. Chromatin immunoprecipitation was performed using the p65 antibody, and the RAGE promoter was amplified by qPCR (n = 4). C: Conditionally immortalized HAECs were treated with LG, HG, or HG after transfection with either scrambled oligonucleotides or siRNA for AP-1 (c-Jun) (HG/siAP-1) for 5 days, and mRNA levels of S100A8, S10012, and HMGB1 were determined by qPCR (n = 3). D: Primary HAECs were treated with LG, HG, or HG after infections with either nontarget control or GLO1 adenovirus (HG/GLO1) for 5 days. Chromatin immunoprecipitation was performed using c-Jun antibody, and the S100A8, 100A12, and HMGB1 promoters were amplified by qPCR (n = 4). Values are shown as means + SD. *P < 0.05 vs. LG group. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 5.

Treatment of diabetic mice with MnTBAP normalizes increased expression of RAGE, S100 calgranulins, and HMGB1. A: Aortic endothelial cells (AEC) or smooth muscle cells (SMC) were isolated from either wild-type (WT) mice, STZ-induced diabetic mice (STZ), or STZ-diabetic mice treated with MnTBAP (STZ/MnTBAP), using laser capture microdissection. mRNA levels for vWF (A) and α-actin (B) were determined by qPCR (n = 3, *P < 0.05 vs. AEC group). C: 3-Nitrotyrosine content of aortic cells from the above mice was determined by Western blot (n = 4, *P < 0.05 vs. CTL group). D: Representative blots for C. E: Methylglyoxal-modified protein content of aortic cells from the above mice was determined by Western blot (n = 4, *P < 0.05 vs. CTL group). F: Representative blots for E. G: Aortic endothelial cells were used for analysis of gene expression by qPCR (n = 3, *P < 0.05 vs. WT group). Values are shown as means + SD. (A high-quality color digital representation of this figure is available in the online issue.)