Functional characterization of the NF-kB binding site in the human NOD2 promoter

Nucleotide-binding and oligomerization domain 2(NOD2),a member of the NOD protein family,plays an important role in innate immunity.In response to pathogen attack,NOD2 stimulates cytokine and defensin production by activating nuclear factor(NF)-kB,a key transcription factor responsible for mediating downstream reactions.However,the mechanism linking NOD2 regulation and NF-kB activation is poorly understood.Using bioinformatics,we found a completely preserved canonical NF-kB binding site in the NOD2 core promoter(216 to 225 bp)in both humans and chimpanzees.The functional role of this NF-kB binding site was investigated using the enhanced green fluorescent protein(EGFP)reporter system,site-directed mutagenesis,the NF-kB activation inhibitor(JSH-23)and the chromatin immunoprecipitation(ChIP)assay.The results show that the NF-kB binding site is critical for regulation of the NOD2 gene.Either deletion of the NF-kB binding elements within the NOD2 promoter or treatment with an NF-kB activation inhibitor could lead to a significant loss of NOD2 promoter activity as detected by reporter gene assay.The canonical NF-kB binding site was bound by NF-kB as determined by the ChIP method.Based on these results,we suggest a positive feedback regulation between NF-kB and NOD2,which may represent an efficient mechanism in response to pathogen invasion.