Activation of the Nucleotide Oligomerization Domain Signaling Pathway by the Non-bacterially Derived Xanthone Drug 5′6-Dimethylxanthenone-4-acetic Acid (Vadimezan)

Guanjun Cheng,Jing Sun,Zvi G Fridlender,Liang-Chuan S Wang,Lai-Ming Ching,Steven M Albelda

doi:10.1074/jbc.m109.065631

Abstract

The cytosolic nucleotide-binding oligomerization domain 1 (NOD1)/CARD4 and NOD2/CARD15 proteins are members of NOD-like receptors recognizing specific motifs within peptidoglycans of both Gram-negative and Gram-positive bacteria. NOD1 and NOD2 signal via the downstream adaptor serine/threonine kinase RIP2/CARDIAK/RICK to initiate NF-kappaB activation and the release of inflammatory cytokines/chemokines. In this report, we show that 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a cell-permeable, small molecule that has anti-tumor activity, can also activate NOD1 and NOD2. This was demonstrated: 1) by using human embryonic kidney epithelial (HEK) 293 cells transfected with a NF-kappaB reporter plasmid in combination with NOD1 or NOD2 expression plasmids; 2) by inhibiting DMXAA-induced chemokine (CXCL10) mRNA and protein production in the AB12 mesothelioma cell line using a pharmacological inhibitor of RICK kinase, SB20358; and 3) by using small interfering RNA to knock down NOD2 and lentiviral short hairpin RNA to knock down RICK. These findings expand the potential ligands for the NOD-like receptors, suggesting that other xanthone compounds may act similarly and could be developed as anti-tumor agents. This information also expands our knowledge on the mechanisms of action of the anti-tumor agent DMXAA (currently in clinical trials) and may be important for its biological activity.

Highlights

The NOD1 and NOD2 proteins are members of the NOD-like receptor family, defined by a tripartite structure consisting of a variable N-terminal caspase-recruitment domain (CARD), a central NOD domain, and a C-terminal leucine-rich repeat that detects pathogen-associated molecular patterns [1, 2]
This similarity suggested the hypothesis that DMXAA might be a potential ligand for NOD2 and/or NOD1 that would signal via the downstream adaptor serine/threonine kinase RIP2/RICK to activate NF-␬B and induce the release of chemokines and cytokines
DMXAA Stimulates NF-␬B Activation in 293 Cells Only in the Presence of NOD2 or NOD1—To determine if DMXAA could engage NOD1 or NOD2, we employed the same HEK293 cell reporter system originally described to identify the peptidoglycan ligands of the NODs [2, 6]

Summary

Introduction

The NOD1 ( known as CARD4) and NOD2 ( known as CARD15) proteins are members of the NOD-like receptor family, defined by a tripartite structure consisting of a variable N-terminal caspase-recruitment domain (CARD), a central NOD domain, and a C-terminal leucine-rich repeat that detects pathogen-associated molecular patterns [1, 2]. DMXAA can activate NF-␬B, and this activation is markedly augmented by co-administration or priming with lipopolysaccharide [12] This appears to occur through a pathway that does not require the Toll-like receptor adaptor molecule MyD88 [11, 13]. The ability of DMXAA to activate NF-␬B in macrophages in a MyD88-independent fashion through a cytoplasmic process that is primed by lipopolysaccharide is reminiscent of the activities of MDP acting through NOD2. This similarity suggested the hypothesis that DMXAA might be a potential ligand for NOD2 and/or NOD1 that would signal via the downstream adaptor serine/threonine kinase RIP2/RICK to activate NF-␬B and induce the release of chemokines and cytokines. The purpose of this report was to test this hypothesis using transfected cell lines, pharmacologic inhibitors, and siRNA

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