Cherubism (OMIM#118400) is a genetic disorder with excessive jawbone resorption caused by mutations in the signaling adaptor protein SH3BP2. Studies on the mouse model for cherubism carrying a P416R knock-in mutation have revealed that mutant SH3BP2 enhances TNF-α production and RANKL-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of TRAP-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2KI/KI). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2KI/+) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves SYK and PLCγ2 phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders.

Summary Cherubism is caused by mutations in SH3BP2. Studies of cherubism mice showed that TNF-α-dependent autoinflammation is a major cause for the disorder, but failed to explain why human cherubism lesions are restricted to jaws and regress after puberty. We demonstrate that the inflammation in cherubism mice is MYD88-dependent and is rescued in the absence of TLR2 and TLR4. However, germ-free cherubism mice also develop inflammation. Mutant macrophages are hyper-responsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns) that activate TLRs, resulting in TNF-α overproduction. Phosphorylation of SH3BP2 at Y183 is critical for the TNF-α production. Finally, SYK depletion in macrophages prevents the inflammation. These data suggest that the presence of a large amount of TLR ligands, presumably oral bacteria and DAMPs during jawbone remodeling, may cause the jaw-specific development of human cherubism lesions. Reduced levels of DAMPs after stabilization of jaw remodeling may contribute to the age-dependent regression.

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Tankyrase is a poly (ADP-ribose) polymerase that leads to ubiquitination and degradation of target proteins. Since tankyrase inhibitors suppress the degradation of AXIN protein, a negative regulator of the canonical Wnt pathway, they effectively act as Wnt inhibitors. Small molecule tankyrase inhibitors are being investigated as drug candidates for cancer and fibrotic diseases, in which the Wnt pathways are aberrantly activated. Tankyrase is also reported to degrade the adaptor protein SH3BP2 (SH3 domain-binding protein 2). We have previously shown that SH3BP2 gain-of-function mutation enhances receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in murine bone marrow-derived macrophages (BMMs). Although the interaction between tankyrase and SH3BP2 has been reported, it is not clear whether and how the inhibition of tankyrase affects bone cells and bone mass. Here, we have demonstrated that tankyrase inhibitors (IWR-1, XAV939, and G007-LK) enhanced RANKL-induced osteoclast formation and function in murine BMMs and human peripheral blood mononuclear cells through the accumulation of SH3BP2, subsequent phosphorylation of SYK, and nuclear translocation of NFATc1. Tankyrase inhibitors also enhanced osteoblast differentiation and maturation, represented by increased expression of osteoblast-associated genes accompanied by the accumulation of SH3BP2 protein and enhanced nuclear translocation of ABL, TAZ, and Runx2 in primary osteoblasts. Most importantly, pharmacological inhibition of tankyrase in mice significantly decreased tibia and lumbar vertebrae bone volumes in association with increased numbers of osteoclasts. Our findings uncover the role of tankyrase inhibition in bone cells and highlight the potential adverse effects of the inhibitor on bone.

Objective SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. The purpose of this study was to investigate the role of SH3BP2 in arthritis in human TNF-α transgenic (hTNFtg) and collagen-induced arthritis (CIA) models. Methods First, SH3BP2-deficient (Sh3bp2–/–) and wild-type (Sh3bp2+/+) mice were crossed with hTNFtg mice. Inflammation and bone loss were examined by clinical inspection and histological and micro-CT analyses. Osteoclastogenesis was evaluated with primary bone marrow-derived M-CSF-dependent macrophages (BMMs). Second, CIA was induced in Sh3bp2–/– and Sh3bp2+/+ mice, and the incidence and severity of arthritis were evaluated. Anti-mouse type II collagen (CII) antibody levels were measured by ELISA. Lymph node cell responses to CII were also determined. Results SH3BP2-deficiency did not alter the severity of joint swelling but suppressed bone erosion in the hTNFtg model. Bone loss of talus and tibia was prevented in Sh3bp2–/–/hTNFtg mice compared to Sh3bp2+/+/hTNFtg mice. RANKL- and TNF-α-induced osteoclastogenesis was suppressed in Sh3bp2–/– BMM cultures. NFATc1 nuclear localization in response to TNF-α was decreased in Sh3bp2–/– BMMs compared to Sh3bp2+/+ BMMs. In the CIA model, SH3BP2-deficiency suppressed the incidence of arthritis, which was associated with decreased anti-CII antibody production, while the antigen-specific T-cell responses in lymph nodes were not significantly different between Sh3bp2+/+ and Sh3bp2–/– mice. Conclusion SH3BP2-deficiency prevents bone loss via impaired osteoclastogenesis in the hTNFtg model and suppresses the induction of arthritis via decreased autoantibody production in the CIA model. Therefore, SH3BP2 could be a therapeutic target for rheumatoid arthritis.

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.

Cherubism is a genetic disorder of the craniofacial skeleton caused by gain-of-function mutations in the signaling adaptor protein, SH3-domain binding protein 2 (SH3BP2). In a knock-in mouse model for cherubism, we previously demonstrated that homozygous mutant mice develop T/B cell-independent systemic macrophage inflammation leading to bone erosion and joint destruction. Homozygous mice develop multiostotic bone lesions while cherubism lesions in humans are limited to jawbones. We identified a critical role of TNF-α in the development of autoinflammation by creating homozygous TNF-α-deficient cherubism mutants, where systemic inflammation and bone destruction were rescued. In the current study, we examined whether postnatal administration of an anti-TNF-α antagonist can prevent or ameliorate the disease progression in cherubism mice. Neonatal homozygous mutants, where active inflammation has not yet developed, were treated with a high dose of etanercept (25 mg/kg, twice/week) for 7 weeks. Etanercept-treated neonatal mice showed strong rescue of facial swelling and bone loss in jaws and calvariae. Destruction of joints was fully rescued in the high dose group. Moreover, the high dose treatment group showed a significant decrease in lung and liver inflammatory lesions. However, inflammation and bone loss, which were successfully treated by etanercept administration recurred after etanercept discontinuation. No significant effect was observed in low dose- (0.5 mg/kg, twice/week) and vehicle-treated groups. In contrast, when 10-week-old cherubism mice with fully active inflammation were treated with etanercept for 7 weeks, even the high dose administration did not decrease bone loss, lung or liver inflammation. Taken together, the results suggest that anti-TNF-α therapy may be effective in young cherubism patients, if treated before the inflammatory phase or bone resorption occurs. Therefore, early genetic diagnosis and early treatment with anti-TNF-α antagonists may be able to prevent or ameliorate cherubism, especially in patients with a mutation in SH3BP2.

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