Role of endosomal RANKL–LGR4 signaling during osteoclast differentiation

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also known as GPR48) is a membrane receptor that negatively regulates the RANK signaling cascade during osteoclastogenesis. Traditionally, cell signaling and endocytic membrane trafficking via membrane receptors have been considered distinct processes; however, they are now recognized to be closely and bidirectionally linked. The present study investigated the difference between membrane-bound and endosomal LGR4 signaling and whether the LGR4 signaling pathway influences RANK–RANKL signaling during RANKL-induced osteoclastogenesis. We used CRISPR-Cas9 to create LGR4 conditional knock-out (CKO) in RAW 264.7 cells and Drg2 knockout (KO) in mice to study the impacts of LGR4 and DRG2 on osteoclastogenesis. LGR4 was endocytosed into endosomes after binding to RANKL in RAW 264.7 s osteoclast precursor cells. Within the early endosomes, internalized LGR4 activates LGR4–RANKL signaling. When bound to RANKL, LGR4 is endocytosed and localized in the RAB5-positive endosomes. In Lgr4 CKO RAW 264.7 cells, early endosome signaling was increased and the inhibitory phosphorylation of GSK-3β was decreased, both in the whole lysate and endosome fraction. RANKL treatment increased nuclear translocation of NFATC1 in Lgr4 CKO RAW 264.7 cells and Drg2 KO mice. Overall, our results suggested that RANKL–LGR4 signaling is regulated by membrane-to-endosomal trafficking during osteoclastogenesis.