Muramyl dipeptide (MDP), the NOD2 agonist, induces NF-κB 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-κB 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.
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