EGCG prevents PCB-126-induced endothelial cell inflammation via epigenetic modifications of NF-κB target genes in human endothelial cells

Abstract

Anti-inflammatory polyphenols, such as epigallocatechin-3-gallate (EGCG), have been shown to protect against the toxicity of environmental pollutants. It is well known that bioactive food compounds such as polyphenols may exert their protection by modulating inflammatory pathways regulated through nuclear factor-kappa B (NF-κB) signaling. EGCG has been reported to inhibit NF-κB activation. We hypothesize that EGCG can protect against polychlorinated biphenyl (PCB)-induced endothelial inflammation in part through epigenetic regulation of NF-κB-regulated inflammatory genes. In order to test this hypothesis, human endothelial cells (EA.hy926) were exposed to physiologically relevant levels of coplanar PCB 126 and/or 15 or 30 μM of EGCG, followed by quantification of NF-κB subunit p65, histone acetyltransferase p300 and histone deacetylases (HDACs) accumulation through chromatin immunoprecipitation assay in the promoter region of inflammatory genes. In addition, the enrichment of the acetylated H3 was also quantified. PCB 126 exposure increased the expression of vascular inflammatory mediators, including interleukin (IL)-6, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and IL-1α/β, which were prevented by pretreatment with EGCG. This inhibitory effect by EGCG correlated with abolished nuclear import of p65, decreased chromatin binding of p65 and p300, as well as increased chromatin binding of HDAC 1/2. Furthermore, EGCG induced hypoacetylation of H3, which accounts for deactivation of downstream genes. These data suggest that EGCG-induced epigenetic modifications can decrease PCB-induced vascular toxicity.

Keywords: EGCG; HDACs; PCB 126; Vascular inflammation; p300; p65.

Figure 1. EGCG decreases PCB-induced NF-κB activation

(A) EGCG decreased PCB 126-induced p65 expression. Cellular p65 mRNA level was measured in endothelial cells which were pre-treated with 15 or 30 μM of EGCG (E15, E30) for 8 h, and then treated in fresh medium with 0.03 nM of PCB 126 for 16 h. Values are means ± SD with n=3. ^*Significantly increased compared to the DMSO control. ^#Significantly decreased compare to the PCB treatment group. (B) Protein levels of p65, IKK-α/β, IκB-α and -β were measured by immunoblotting. Actin was used as the loading control. (C) EGCG decreased PCB 126-mediated p65 nuclear translocation. The intracellular localization of endogenous p65 was analyzed by immunocytochemical staining using anti-p65 (green) and the nucleus was visualized by DAPI staining (blue).

Figure 2. EGCG attenuates PCB 126-induced inflammatory gene expression

The mRNA level of inflammatory genes was analyzed using qRT-PCR. Endothelial cells were pretreated with 15 or 30 μM of EGCG for 8 h, and followed by treatment in fresh medium with 0.03 nM of PCB 126 for 16 h. Values are the means ± SD with n=3. ^*Significantly increased compared to the DMSO control. ^{^}Significantly decreased compared to the DMSO control. ^#Significantly decreased compare to the PCB treatment group.

Figure 3. EGCG reverses PCB 126-induced chromatin binding of acetylation enzymes

The recruitment of p65, p300, and HDAC1-3 was modulated by EGCG in the promoters of IL-6 (A), CRP (B), ICAM-1 (C), VCAM-1 (D), IL-1α (E), IL-1β (F) and GAPDH (G). Sheared chromatin from whole-cell lysates was immunoprecipitated with antibodies against the indicated proteins. IgG was used as a negative control. The GAPDH promoter was used as control locus. Values are means ± SD with n=3. ^*Significantly increased compared to the DMSO control. ^{^}Significantly decreased compared to the DMSO control. ^#Significantly decreased compared to the PCB treatment group.

Figure 4. EGCG induces H3 hypo-acetylation as a result of HAT/HDAC binding exchange

The H3 acetylation was enhanced by PCB 126 and decreased by the pre-treatment with EGCG in the NF-κB-regulated genes. H3 is the control for nucleosomal occupancy. Values are means ± SD with n=3. ^*Significantly increased compared to the DMSO control. ^{^}Significantly decreased compared to the DMSO control. ^#Significantly decreased compared to the PCB treatment group.