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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.

Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. RANKL was associated with epithelial–mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Therefore, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa.

Background The discovery of receptor activator of nuclear factor‐ĸB ligand (RANKL) as the final effector in the pathogenesis of osteoporosis has led to a better understanding of bone remodeling. When RANKL binds to its receptor (RANK), osteoclastic differentiation and activation are initiated. Herein, we propose a strategy using a novel RANKL variant as a competitive inhibitor for RANKL. The RANKL variant activates LGR4 signaling, which competitively regulates RANK and acts as an immunogen that induces anti‐RANKL antibody production. Methods We modified the RANK‐binding site on RANKL using minimal amino acid changes in the RANKL complex and its counterpart receptor RANK and tried to evaluate the inhibitory effects on osteoclastogenesis. Results The novel RANKL variant did not bind RANK in osteoclast progenitor cells, but activated LGR4 through the GSK3‐β signaling pathway, thereby suppressing activated T cell cytoplasmic nuclear factor calcineurin‐dependent 1 (NFATc1) expression and activity during osteoclastogenesis. Our RANKL variant generated high levels of RANKL‐specific antibodies, blocked osteoclastogenesis, and inhibited osteoporosis in ovariectomized mouse models. Generated anti‐RANKL antibodies showed a high inhibitory effect on osteoclastogenesis in vivo and in vitro. Conclusions We observed that the novel RANKL indeed blocks RANKL via LGR4 signaling and generates anti‐RANKL antibodies, demonstrating an innovative strategy in the development of general immunotherapy., Schematic diagram of mRANKL‐MT3 in dual inhibitory effect against RANKL during osteoclastogenesis. The first effect of mRANKL‐MT3 is induced to RANKL‐LGR4 modulation of the RANKL–NFATc1 signaling cascade by a negative‐feedback mechanism, which controls osteoclast activity. The second effect is induced to anti‐RANKL generation by mutant RANKL, which inhibits osteoclastogenesis and bone erosion.

Background: Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Method: In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa.Results: RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Conclusion: Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCaTrial registration: Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/ site=hospital&mn=151&type=view&catename=IRB

Here, we proposed the use of mutated RANKL as an immunogen for active immunization and to induce anti-cytokine antibodies for osteoporosis treatment. Osteoclasts are responsible for bone resorption in bone-related disorders. Anti-cytokine therapeutic antibodies such as denosumab are effective for the treatment of osteoporosis. However, problems with antibody manufacturing and the immunogenicity caused by multiple antibody doses have led to the use of auto-cytokines as immunogens to induce anti-cytokine antibodies. RANKL was point-mutated based on the crystal structure of the complex of RANKL and its receptor RANK. As a proof of concept, immunization with RANKL produced high levels of specific antibodies and blocked osteoclast development in vitro and inhibited osteoporosis in RANKL-treated or ovariectomized mouse models. The results demonstrate the successful use of mutated RANKL as an immunogen for the induction of anti-RANKL immune response. This strategy is useful in general anti-cytokine immunotherapy to avoid toxic side effects of osteoporosis treatment.

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK&ndash, RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis., Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3&beta, phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models, and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.