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.