NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2.
Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.
Voss
Eske
E
Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany.
Wehkamp
Jan
J
Wehkamp
Kai
K
Stange
Eduard F
EF
Schröder
Jens M
JM
Harder
Jürgen
J
D000890:Anti-Infective Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D003424:Crohn Disease / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D016368:Frameshift Mutation; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D015603:Keratinocytes / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein; D010455:Peptides / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012717:Sesquiterpenes / Q000494:pharmacology; D045789:Sesquiterpenes, Guaiane; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D023083:beta-Defensins / Q000494:pharmacology*
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; C106641:DEFB4A protein, human; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D010455:Peptides; D010457:Peptidoglycan; D034741:RNA, Small Interfering; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; D018808:Transcription Factor AP-1; D023083:beta-Defensins; C001329:helenalin; D012313:RNA; D008156:Luciferases