LGR4, A New Receptor For RANKL, Negatively Regulates Osteoclast Differentiation And Bone Resorption

RANKL, expressed or secretd by osteoblast, osteocyte and T cells, induces osteoclastogenesis and survival.However, mature osteoclasts still undergo apoptosis in a RANKL-containing environment, which indicates there exists a RANKL-induced signaling pathway limiting the survival of mature osteoclasts. Several members of the leucine-rich-repeat G protein-coupled receptor (LGR) family participate in bone remodeling, in which TSHR negatively regualtes osteoclast activation while FSHR promotes osteoclast differentiation. Therefore, we wonder whether other LGR memebers are involved in RANKL-inducing negative feedback loop regulation of osteoclast development. To indentify potential LGRs, we screened the expression of all Lgrs during osteoclast differentiation and found Lgr4 is the only gene that dramatically induced in bone marrow monocytes (BMMs) stimulated with RANKL in the presence of M-CSF in a time-dependent manner. Furthermore, our data revealed that Lgr4 is a direct transcriptional target of RANKL/NFATcl signaling during osteoclastogenesis. All of the data suggest that Lgr4 is a novel regulator during osteoclastogenesis.To clarify the function of Lgr4 in osteoclast, we examined the bone phenotypes of mice deficient in Lgr4 (Lgr4-/-/). These mice exhibit low bone mineral density (BMD). In addition, a large number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast cells were present in the femoral bones and calvariae of Lgr4-/- mice at all observed ages (8 weeks-old,16 weeks-old and 24 weeks-old).Furthermore, Lgr-/-promotes RANKL induced osteoclastogenesis and bone resorption in vitro as well. TUNNEL staining showed Lgr4//- also suppress osteoclast apoptosis. Therefore, Lgr4 is a negative regulateor induced by RANKL in osteoclast. Considering LGR4 regulates mouse bone formation and bone remodeling, human bone mass, immunity, mammary gland development, body temperature, cancer metastasis and energy expenditure, which Rankl-/- mice show several defects in similar functions, we hypothesized that LGR4 was another receptor for RANKL. Using Co-immunoprecipitation (Co-IP) and Surface Plasmon Resonance (SPR) analysis, we found LGR4 is able to associate with RANKL protein through its extracellular domain (ECD). Furthermore, RANKL-LGR4 signaling coupled with Gaq induces SRE-luc and transient calcium release. In osteoclast, RANKL-LGR4-Gaq signaling promotes GSK3β activation, leading to NFATc1 nuclear export that downregulate NFATc1 expression and ultimately impairing osteoclastogenesis. Besides, LGR4 can also compete with RANK for RANKL binding and attenuates RANK downstream signaling including NFκB pathway, calcium osscilation and AKT pathway. On the basis of our results we propose LGR4 negatively regulates osteoclast development, with two mechanisms of action, including Gaq-GSK3βpathway suppressing NFATcl expression and attenuating RANK downstream singling pathway.Since LGR4 associates with RANKL with its extracellular domain, we suppose LGR4ECD peptide could be a potential drug for treating osteoclast related disease. Via two mouse model, RANKL-inducing bone resorption mouse model and OPG-/-osteoporosis mouse model, we found LGR4ECD peptide could prevent and therapeutically block RANKL-induced bone loss via inhibition of osteoclast activity. Besides, LGR4ECD peptide dose-dependently suppressed osteoclast-like giant cell differentiation in giant cell tumor of the bone in vitro as well.In conclusion, our study demonstrates that Lgr4, a new receptor for RANKL, negatively regulates osteoclast differentiation and bone resorption and thus supports Lgr4 as a promising therapeutic target for osteoclast-related disease, including osteoporosis.