Recent studies have provided critical insight into the signaling components and regulatory steps that are induced upon NOD2 activation in response to MDP stimulation. However, the cellular events that act upstream of NOD2 to induce MDP-mediated signaling have remained poorly understood. Stimulation of phagocytic and epithelial cells with MDP can result in NF-κB and MAPK activation, although delivery of MDP to the cytosol by transfection is thought to enhance MDP-induced signaling (3
). These results suggest that both macrophages and epithelial cells possess the machinery to internalize MDP. In this study, we have identified an endocytic pathway that mediates MDP uptake and induces NOD2-dependent signaling. Studies with both pharmacological inhibitors and RNAi interference revealed that MDP internalization is mediated by a clathrin-dependent endocytosis pathway. This endocytosis pathway was also dependent on dynamin, a GTPase that is required for budding of the clathrin-coated pits from the plasma membrane (32
). Clathrin-dependent endocytosis constitutes a major route for the uptake of nutrients, pathogens, growth factors and transmembrane proteins in mammalian cells (37
). Similarly, LPS is endocytosed by an endocytic pathway that is dependent on clathrin and dynamin (42
). The mechanism by which MDP is targeted to clathrin-coated pits for endocytosis remains unclear. Because endocytosis is typically mediated through transmembrane proteins that are selectively recruited to coated pits by interaction to clathrin adaptors, the findings suggest that MDP might be recognized by a plasma membrane receptor that mediates its delivery to clathrin-coated pits. Our initial studies revealed that MDP uptake is unimpaired by treatment with an inhibitor of scavenger receptors or mannan, a molecule that interacts with the mannose receptor. These results appear to rule out the latter receptors, although other surface receptors might be involved in promoting MDP internalization. Many pathogens that express NOD2-stimulatory activity are internalized into host cells by clathrin-dependent endocytosis (41
). Thus, both MDP and bacteria appear to utilize the same route to trigger NOD2 activation and signaling. Further studies are needed to determine the mechanism whereby extracellular MDP istargeted to the clathrin-dependent endocytosis pathway.
Recent studies have shown that MDP induces caspase-1 activation through the Nlrp3 inflammasome, an event that requires exogenous ATP to deliver MDP from acidified vesicles into the cytosol via the pannexin-1 pore (22
). In contrast, other authors have concluded that MDP-mediated caspase-1 activation induced by an inflammasome complex containing Nlrp1 and NOD2 (46
). Regardless of the mechanism involved, our results indicate that MDP internalization via dynamin-mediated endocytosis is required for caspase-1 activation. Unlike the activation of caspase-1, NF-κB and MAPK activation induced by MDP is mediated solely via NOD2 and does not require exogenous ATP (3
). These results suggest that after internalization via clathrin-dependent endocytosis, MDP relies on different mechanisms to activate Nlrp3 and NOD2. One possibility is that NOD2 activation by MDP is mediated via a surface receptor also present in endocytic vesicles or by another mechanism independent of cytosolic recognition. Alternatively, after internalization in acidified vesicles, MDP may leak or be actively transported into the cytosol where is sensed by NOD2. Our results rule out the peptide transporter PepT1, although other transporter systems operating in endosomes/lysosomes may be involved. Furthermore, the studies with bafilomycin A suggest that vacuolar acidification and/or maturation is important for MDP-induced cytokine responses. These results are in line with recent findings showing that bafilomycin A blocks activation of NOD2 in macrophages infected with a Listeria
mutant that cannot escape the phago-lysosome (45
). Consistent with our findings, localization of the Listeria
mutant in the phago-lysosome, which leads to the degradation of the bacterial cell wall and release of peptidoglycan fragments, was required for NOD2-induced signaling (45
). Further studies are needed to understand the mechanism by which MDP and peptidoglycan fragments derived from bacteria activate NOD2.