Characterization of a spontaneous osteopetrosis model using RANKL-dysfunctional mice.

Tumor necrosis factor superfamily member 11 (TNFSF11), or receptor activator of nuclear factor-κB ligand (RANKL), is a crucial osteoclast-stimulating factor binding to RANK on osteoclast membranes. Mouse models are powerful tools for understanding the genetic mechanisms of related diseases. Here, we examined the utility of Tnfsf11 mutation in mice for understanding the mechanisms of bone remodeling and dysmorphology. The Tnfsf11gum mouse, discovered in 2011 at Jackson Laboratory, was used to study the genetic landscape associated with TNFSF11 inactivation in bone marrow tissues. Tnfsf11gum/+ and Tnfsf11+/+ mice were subjected to Micro-CT observation, ELISA analysis, histological evaluation, and massively-parallel mRNA sequencing (RNA-Seq) analysis. Tnfsf11gum/+ mice exhibited severe osteopetrotic changes in the bone marrow cavity, along with significantly lower serum RANKL levels and a reduced number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the bone marrow compared to those in Tnfsf11+/+ mice. However, tooth eruption between Tnfsf11gum/+ and Tnfsf11+/+ mice did not differ. Furthermore, genes involved in osteoblast proliferation and differentiation, including Gli1, Slc35b2, Lrrc17, and Junb were differentially expressed. Heterozygous mutation of TNFSF11 was also associated with a slightly increased expression of genes involved in osteoclast proliferation and differentiation, including Tcirg1, Junb, Anxa2, and Atp6ap1. Overall, we demonstrate that single gene mutations in Tnfsf11 cause bone resorption instability without significantly altering the genes related to osteoblast and osteoclast activity in the bone marrow cavity, thus establishing an optimal resource as an experimental animal model for bone resorption in bone biology research.

An LGR4 agonist activates the GSK­3ß pathway to inhibit RANK­RANKL signaling during osteoclastogenesis in bone marrow­derived macrophages.

The binding between receptor­activated nuclear factor­κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine­rich repeat­containing G­protein­coupled receptor 4 (LGR4) acts as a negative regulator of RANK­RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow­derived macrophages and a mouse model of RANKL­induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild­type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT­glycogen synthase kinase­3ß (GSK­3ß)­nuclear factor of activated T cells, cytoplasmic, calcineurin­dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL­induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK­3ß phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL­induced osteoclastogenesis through an LGR4­dependent pathway and this could be exploited to develop new therapies for osteoporosis.

RANKL immunisation inhibits prostate cancer metastasis by modulating EMT through a RANKL-dependent pathway.

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 ß-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and ß-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.

A novel modified RANKL variant can prevent osteoporosis by acting as a vaccine and an inhibitor.

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-ß 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.

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL.

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-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-3ß 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.