The dual roles of peptidoglycans: NOD1 and NOD2 inversely regulate bone metabolism

Gut microbiota and microbial components are known to regulate bone metabolism. Peptidoglycan, a key bacterial cell wall component, is recognized by NOD1 and NOD2. Muramyl dipeptide (MDP), a ligand for NOD2 found in most bacteria, increases bone mass by promoting bone formation via Runx2 and β-catenin. However, the effects of NOD1 ligands, such as L-Ala-γ-D-glu-meso-diaminopimelic acid (TriDAP) from Gram-negative bacteria, remain poorly understood. Here, we demonstrate that, unlike MDP, TriDAP elicits bone resorption by decreasing osteoblast and Runx2 levels and increasing osteoclasts in the distal femurs of mice intraperitoneally administered TriDAP. This treatment inhibited osteoblast differentiation by downregulating Runx2 expression and also decreased the protein stability of Runx2 by increasing its ubiquitination. TriDAP, but not MDP, reduced the expression of IκB and increased NF-κB transcriptional activity in osteoblasts. The inhibition of osteoblast differentiation by TriDAP occurred through NF-κB activation and NOD1 recognition. TriDAP exhibited a marginal increase in osteoclast differentiation in the presence of RANKL, but it enhanced osteoclast differentiation in an osteoblast-osteoclast co-culture system. This suggests that TriDAP directly affects osteoblasts and indirectly affects osteoclasts. TriDAP did not induce osteoclast differentiation in the presence of NOD1-deficient osteoblasts. Other NOD1 ligands, C12-iE-DAP and C14-Tri-LAN-Gly, also inhibited osteoblastogenesis and promoted osteoclastogenesis, similar to TriDAP. Bacillus cereus peptidoglycan preferentially stimulates NOD1 but not NOD2, leading to increased bone resorption. In conclusion, activation of NOD1 in osteoblasts plays a role in regulating bone homeostasis by enhancing bone resorption.