DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription

The present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation.This report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells.The innate immune system recognizes conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), that are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs) including toll-like receptors (TLRs) [1-3]. After the recognition of microbial PAMPs by innate immune systems, TLRs activate signaling pathways that induce inflammatory cytokines and antimicrobial peptides to eliminate invading pathogens. Eleven members of the mammalian TLR family have been identified and cloned thus far. TLR2 and TLR4 have been studied in the greatest depth [4,5], and TLR4 appears to be primarily involved in the recognition of lipopolysaccharide (LPS) from Gram-negative bacteria [5]. In contrast, TLR2 responds to a variety of both Gram-negative and Gram-positive bacterial products, including peptidoglycan, lipoprotein, lipoteichoic acid, and lipoarabinomannan. This suggests that TLR2 plays a critical role in the host defence system [4].TLR2 is primarily expressed in monocytes, macrophages, dendritic cells, a