Homocysteine promotes human endothelial cell dysfunction via site-specific epigenetic regulation of p66shc

Abstract

Aims: Hyperhomocysteinaemia is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial dysfunction. Homocysteine modulates cellular methylation reactions. P66shc is a protein that promotes oxidative stress whose expression is governed by promoter methylation. We asked if homocysteine induces endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter, and whether p66shc mediates homocysteine-stimulated endothelial cell dysfunction.

Methods and results: Homocysteine stimulates p66shc transcription in human endothelial cells and hypomethylates specific CpG dinucleotides in the human p66shc promoter. Knockdown of p66shc inhibits the increase in reactive oxygen species, and decrease in nitric oxide, elicited by homocysteine in endothelial cells and prevents homocysteine-induced up-regulation of endothelial intercellular adhesion molecule-1. In addition, knockdown of p66shc mitigates homocysteine-induced adhesion of monocytes to endothelial cells. Inhibition of DNA methyltransferase activity or knockdown of DNA methyltransferase 3b abrogates homocysteine-induced up-regulation of p66shc. Comparison of plasma homocysteine in humans with coronary artery disease shows a significant difference between those with highest and lowest p66shc promoter CpG methylation in peripheral blood leucocytes.

Conclusion: Homocysteine up-regulates human p66shc expression via hypomethylation of specific CpG dinucleotides in the p66shc promoter, and this mechanism is important in homocysteine-induced endothelial cell dysfunction.

Figure 1

Homocysteine increases endothelial p66shc expression. (A) Homocysteine (Hcy) dose-dependently increases p66shc protein expression in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged with Hcy (200 µM) for 8 h. *P< 0.05, **P< 0.01 compared with untreated cells (n= 3–4). (B) Hcy dose-dependently increases p66shc RNA expression in endothelial cells. *P< 0.05, **P< 0.01 compared with untreated cells. (n= 3). (C), Hcy stimulates p66shc promoter activity. Promoter activity was measured in HEK293 cells transfected with an 1141 bp p66shc (WT) promoter-reporter construct. *P< 0.05, **P< 0.01 (n= 3) compared with untreated cells.

Figure 2

Homocysteine increases p66shc promoter activity through site-specific CpG hypomethylation. (A) Human p66shc promoter showing CpG dinucleotides sequenced. Shaded dinucleotides were methylated in HUVECs. (B) Hcy hypomethylates CpG(6,7) in p66shc promoter. CpG(6,7) methylation was quantified by methylation-specific real-time PCR (*P< 0.05, n= 5). (C) CpG(6,7) mediates Hcy-stimulated p66shc promoter activity. Promoter activity was measured in HEK293 cells transfected with the WT or CpG(6,7) mutated promoter-reporter construct. *P< 0.05, **P< 0.01, and ***P< 0.001 (n= 3–8). (D) Sss1 methylase inhibits activation of p66shc promoter by Hcy. Promoter activity was measured in HEK293 cells transfected with unmethylated or Sss1-methylated promoter-reporter construct. *P< 0.05, **P< 0.01, and †P= NS (n= 3–4). (E) Methylation of CpG(6,7) inhibits p66shc promoter activity. Promoter activity was measured in HEK293 cells transfected with unmethylated or Sss1-methylated promoter-reporter construct. *P< 0.05 and †P= NS (n= 3) compared with corresponding unmethylated promoter. (F) Hcy leads to hyperacetylation of H3 on the p66shc promoter. HUVECs were treated with 50 µM Hcy, 300 nM of Trichostatin A (TSA) or 1 µM 5-azacytidine (5-AZA) for 24 h. Chromatin immunoprecipitation (ChIP) of HUVECs chromatin was performed using acetylated-H3 antibody. PCR was performed using primers specific for a 169 bp fragment of the human p66shc promoter encompassing the CpG dinucleotides. A fragment of human β-actin was amplified from input DNA. Result is representative of three independent experiments.

Figure 3

Homocysteine induces p66shc expression via DNMT3b. (A) Inhibition of DNMTs with 5-AZA abrogates Hcy-induced p66shc expression. HUVECs, with and without pre-treatment with 5-AZA (100 µM, 60 h), were challenged with Hcy (200 µM, 48 h). P66shc mRNA was quantified by QPCR. (*P< 0.05 and #P= NS, n= 3). (B and C): Knockout of DNMT1 and DNMT3b in HCT116 cells abrogates Hcy-induced p66shc expression. Wild-type HCT116 cells and HCT116 DNMT1,3b−/− (knockout) cells were challenged with Hcy for (200 µM, 48 h). P66shc protein (B) and mRNA (C) were quantified by immunoblotting and QPCR, respectively (*P< 0.05 and #P= NS, n= 3). (D) and (E) DNMT3b, but not DNMT1, mediates effect of Hcy on p66shc expression in HUVECs. DNMT1 or DNMT3b were knocked down in HUVECs with siRNA, and cells were treated with Hcy (200 µM, 48 h). Control cells were transfected with a scrambled siRNA. P66shc (D) mRNA and (E) protein was quantified by QPCR and immunoblotting and is expressed relative to untreated cells. (*P< 0.05, and #P= NS, n= 3). (F) siRNA-induced knockdown of DNMT1 and DNMT3b in HUVECs (*P< 0.05, n= 3).

Figure 4

Homocysteine stimulates endothelial cell ROS production, ICAM-1 expression, and adhesion of monocytes to endothelial cells, and decreases endothelial cell NO by a p66shc-mediated mechanism. (A) p66shc mediates Hcy-induced oxidative stress. HUVECs were infected with an adenovirus encoding a shRNA for p66shc (Adp66shcRNAi) or the inert E. coli LacZ gene (AdLacZ) for 24 h and hydrogen peroxide levels were measured in conditioned media (*P< 0.05 compared with AdLacZ, n= 3). (B) p66shc mediates Hcy-induced decrease in endothelium-derived NO. HUVECs were infected with Adp66shcRNAi or AdLacZ for 24 h. The NO metabolites nitrite and nitrate were measured in conditioned media of cells in the presence and absence of Hcy (50 µM for 24 h). Decrease in nitrate and nitrate with Hcy, normalized to HUVECs protein, is shown (**P< 0.01, n= 4). (C and D): Hcy increases endothelial ICAM-1. ICAM-1 (C) protein and (D) RNA was measured in endothelial HUVECs challenged with Hcy (8 h). RNA was normalized to GAPDH RNA (*P< 0.05, **P< 0.01 compared with untreated cells, n= 3). (E) P66shc mediates Hcy-induced ICAM-1. HUVECs infected with Adp66shcRNAi or AdLacZ were challenged with Hcy for 8 h. ICAM-1 protein expression was normalized to β-actin (*P< 0.05 and #P= NS compared with untreated cells, n= 3). All immunoblots shown are representative of three independent experiments. (F) Hcy and TNF-α induce U937 monocyte adhesion to HUVECs. HUVECs treated with Hcy and TNF-α for 8 h were incubated with U937 monocyte cells for 30 min. Representative photomicrographs of U937 cells adherent to HUVECs (top) and quantification of adherent cells (bottom) is shown. (*P< 0.05 compared with non-treated cells, n= 3). (G) P66shc mediates Hcy-induced adhesion of monocytes to HUVECs. HUVECs infected with Adp66shcRNAi or AdLacZ were challenged with Hcy for 8 h and incubated with U937 cells for 30 min. Representative photomicrographs of U937 cells adherent to HUVECs (left) and quantification of adherent cells (right) are shown. (*P< 0.05 compared with AdLacZ-infected cells, n= 3).

Figure 5

Plasma homocysteine is inversely associated with p66shc promoter methylation in peripheral blood leucocytes in patients with coronary artery disease. (A) Plasma homocysteine concentrations in subjects with the highest and lowest tertiles of p66shc promoter CpG(6,7) methylation in peripheral blood leucocytes (*P= 0.03, n= 40). (B) Plasma homocysteine concentrations in subjects with the highest and lowest tertiles of p66shc promoter CpG(5) methylation in peripheral blood leucocytes (#P= 0.52, n= 37). (C) Plasma homocysteine concentrations in subjects with the highest and lowest tertiles of total p66shc promoter CpG methylation in peripheral blood leucocytes (#P= 0.36, n= 38). Each box plot shows the distribution of plasma homocysteine from 25 to 75th percentile, and the lines inside the boxes denote the medians. Whiskers denote the intervals between the 5 and 95th percentiles, with dots representing data points outside these percentiles. (D) Baseline clinical and demographic characteristics, % CpG(6,7) methylation, and plasma homocysteine, of subjects with the highest and lowest tertiles of CpG(6,7) methylation in peripheral blood leucocytes. Values are mean ± SD.