Clathrin- and Dynamin-Dependent Endocytic Pathway Regulates Muramyl Dipeptide Internalization and NOD2 Activation

Abstract

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.

pH-dependent Internalization of Muramyl Peptides from Early Endosomes Enables Nod1 and Nod2 Signaling

“…A recent report has demonstrated that MDP was internalized by macrophages through clathrin-dependent endocytosis (39), which supports our findings in HEK293T cells. However, MDP entry in macrophages appeared to require V-ATPase-dependent endosomal acidification, contrasting with our observations in HEK293T cells.…”

“…Indeed, a number of muramyl peptide transporters could exist and be differentially expressed in specific cell types and/or subcellular compartments. This hypothesis is supported by the fact that SLC15A1 was identified as an MDP transporter in Caco-2/bbe epithelial cells (40), but found to be dispensable for mediating MDP entry in macrophages (39). Similarly, SLC15A2 has been shown to trigger transport of the Nod1 ligand iE-DAP in human upper airway epithelial cells (41), but was found to be dispensable in our assays.…”

Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems

“…To reveal the primary physiological pathway of incorporation, two representative CPPs, colon cancer-homing CPP2 and myeloid leukaemia-homing CPP44, were chosen as examples. First, their internalization was examined using 5-(N-ethyl-N-isopropyl) amiloride (EIPA) 10 , Dynamin inhibitor I (DI; Dynasore) 11 , and chlorpromazine (CPZ) 12 . Pretreatment of the cells with EIPA, a macropinocytosis inhibitor, prevented the entry of polyarginine (r9) 13 in a dose-dependent manner; however, the entry of CPP44 into primary AML cells was not impeded.…”

The protein Nod2: An innate receptor more complex than previously assumed

“…3B). Finally, extracellular MDP can get access to the host cytosol through the intestinal apical di-/tripeptide transporter, hPepT1 [21] or by a clathrin-and dynamin-dependent endocytic pathway [22,23] (Fig. 3C).…”