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* Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Pharmaceutical Sciences and Upjohn Center for Clinical Pharmacology, University of Michigan, Ann Arbor, MI 48109; and
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602
Muramyl dipeptide (MDP), the NOD2 agonist, induces NF-
B and MAPK activation leading to the production of antimicrobial and proinflammatory molecules. MDP is internalized into acidified vesicles in macrophages. However, the endocytic mechanism of MDP uptake that induces NOD2 signaling is unknown. We now report the identification of an endocytosis pathway dependent on clathrin and dynamin that mediates MDP internalization and NOD2 activation. Intracellular MDP uptake was inhibited by chlorpromazine, a drug that disrupts clathrin-dependent endocytosis, but not by compounds that block pinocytosis or cellular entry via scavenger or mannose receptors. In contrast, MDP uptake and NOD2-dependent signaling were unimpaired in macrophages deficient in PepT1, a peptide transporter previously implicated in MDP internalization. Both chlorpromazine and knockdown of clathrin expression by RNA interference attenuated MDP-induced NF-B and MAPK activation. Furthermore, MDP uptake and NOD2-dependent signaling were impaired by inhibition of dynamin, a GTPase required for budding of clathrin-coated vesicles from the plasma membrane. Finally, bafilomycin A, a specific inhibitor of the vacuolar proton pump, blocked MDP accumulation in acidified vesicles and cytokine responses, suggesting that vacuolar maturation is important for MDP-induced NOD2 signaling. These studies provide evidence for a clathrin- and dynamin-dependent endocytosis pathway that mediates MDP uptake and NOD2 activation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants DK61707 (to G.N.) and GM035498 (to D.E.S.). L.F. was supported by a Fellowship from the Arthritis Foundation, and Y.-G.K. was supported by a Fellowship from the University of Michigan Comprehensive Cancer Center. MDP-derived compounds synthesis was supported by National Institutes of Health Grant GM065248 (to G.-J.B.).
2 N.M.-G. and L.F. contributed equally to the present work.
3 Current address: Molecular Immunopathology Unit, Pompeu Fabra University (DCEXS), Barcelona Biomedical Research Park, 08003 Barcelona, Spain.
4 Address correspondence and reprint requests to Dr. Gabriel Núñez, University of Michigan Medical School, Department of Pathology and Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, MI 48109. E-mail address: bclx{at}umich.edu
5 Abbreviations used in this paper: NOD, nucleotide-binding oligomerization domain; CPZ, chlorpromazine; DAPI, 4',6'-diamino-2-phenylindole; KO, knockout; MDP, muramyl dipeptide; NLR, NOD-like receptor; shRNA, small hairpin RNA; WT, wild type.
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  J. Lee, I. Tattoli, K. A. Wojtal, S. R. Vavricka, D. J. Philpott, and S. E. Girardin pH-dependent Internalization of Muramyl Peptides from Early Endosomes Enables Nod1 and Nod2 Signaling J. Biol. Chem., August 28, 2009; 284(35): 23818 - 23829. [Abstract] [Full Text] [PDF]
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