NOD2/CARD15 Mediates Induction of the Antimicrobial Peptide Human Beta-defensin-2

Eske Voss,Jan Wehkamp,Kai Wehkamp,Eduard F Stange,Jens M Schröder,Jürgen Harder

doi:10.1074/jbc.m511044200

Eske Voss, Jan Wehkamp + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m511044200

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Abstract

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.

Highlights

Belongs to the beta-defensin family, a group of small (4 –5 kDa), cationic antibiotic peptides first discovered in cattle [6]. Human beta-defensin-2 (hBD-2) exhibits a broad spectrum of antimicrobial activity, and its capacity to kill bacteria in vivo has been demonstrated in a mouse gene therapy study with hBD-2transfected tumor cells
HEK293 cells transfected with the NOD2 gene carrying the 3020InsC mutation [36] revealed no NF-␬B activation upon muramyl dipeptide (MDP) treatment (Fig. 1b)
To verify that activation of NOD2 by its ligand MDP mediates the secretion of hBD-2, we used an Small Interfering RNA (siRNA) approach to selectively inhibit the expression of NOD2 in primary keratinocytes

Summary

Introduction

Belongs to the beta-defensin family, a group of small (4 –5 kDa), cationic antibiotic peptides first discovered in cattle [6]. hBD-2 exhibits a broad spectrum of antimicrobial activity, and its capacity to kill bacteria in vivo has been demonstrated in a mouse gene therapy study with hBD-2transfected tumor cells. To verify the efficiency of our NOD2 plasmid constructs we used these constructs to transiently transfect HEK293 cells and analyzed NF-␬B activation upon MDP treatment. HEK293 cells transfected with the NOD2 gene carrying the 3020InsC mutation (a frameshift mutation encoding a truncated MDP-nonresponsive NOD2 protein that is associated with Crohn disease) [36] revealed no NF-␬B activation upon MDP treatment (Fig. 1b).

Results

Conclusion