An LGR4 agonist activates the GSK3β pathway to inhibit RANK-RANKL signaling during osteoclastogenesis in bone marrow-derived macrophages

Abstract

Abstract The binding between receptor-activated nuclear factor kappa B (RANK) and its specific ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. Recently, the leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was reported as a negative regulator of RANKL-RANK signaling that suppresses canonical RANK signaling during osteoclast differentiation. Hence, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In this study, we used bone marrow-derived macrophages (BMDM) and a mouse model of RANKL-induced bone loss to investigate the effect of a RANKL-derived mutant, (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild-type (WT) RANKL and MT RANKL for RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT-GSK-3β-NFATc1 signaling cascade was investigated using western blotting and confocal microscopy. In addition, the immunopositive expression of LGR4 and the colocalization of LGR4 and MT RANKL were analyzed in a mouse model of RANKL-induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4, decreased AKT phosphorylation, and increased GSK-3β phosphorylation, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL upregulated LGR4 expression, colocalized with LGR4, and inhibited bone resorption. These results indicate that MT RANKL, which is derived from RANKL and inhibits RANKL-induced osteoclastogenesis through an LGR4-dependent pathway, may be useful in the treatment of osteoporosis.