Inflammation-induced up-regulation of TLR2 expression in human endothelial cells is independent of differential methylation in the TLR2 promoter CpG island

Toll-like receptors play an important role in endothelial inflammation; however, little is known on the mechanisms regulating their expression. Differential promoter DNA methylation is an increasingly recognized mechanism that determines a switch between gene silencing and gene transcription. We hypothesized that epigenetic mechanisms are involved in the regulation of endothelial TLR2 expression because of the localization of the TLR2 promoter on a CpG-island. Resting human umbilical vein endothelial cells (HUVECs) displayed rather low TLR2 mRNA expression, while a strong expression increase occurred under inflammatory conditions. We examined the TLR2 promoter methylation pattern in resting HUVECs and compared it to cells treated either with the inflammatory cytokine TNF-α or the DNA-demethylating agent 5-azacytidine. DNA bisulfite conversion was followed by either genomic sequencing or single nucleotide primer extension (SNuPE) HPLC. Results of both techniques showed a low- or non-methylated TLR2 promoter in resting HUVECs and no alteration of the methylation pattern under inflammatory conditions. Whereas 5-azacytidine significantly increased the mRNA expression of the epigenetically regulated gene H 19, TLR2 expression was not affected. Taken together, employing different methodological approaches, our data show no implication of methylation pattern changes in inflammatory induction of TLR2 expression in human endothelial cells.