Cherubism Mutation in SH3BP2 Enhances TNF-induced Osteoclast Differentiation and Function

Cherubism is a craniofacial disorder characterized by the destruction of jaw bones with extensive growth of fibrous lesions containing osteoclasts. We have previously discovered gain-of-function cheubism mutations in SH3 domain-binding protein 2 (SH3BP2) and reported that RANKL stimulation of bone marrow-derived macrophages (BMMs) from heterozygous knock-in (KI) mouse model of cherubism promotes osteoclast differentiation, compared to those from wild-type (+/+) BMMs. Since RANK, a receptor of RANKL, belongs to the TNF receptor superfamily, we hypothesized that TNF stimulation also potentiates osteoclast differentiation of BMMs from cherubism mutant mice. Objectives: To investigate the role of SH3BP2 in TNF-induced osteoclastogenesis. Methods: BMMs from heterozygous cherubism mice (KI/+) were used to examine the role of mutant SH3BP2 in TNF-induced osteoclastogenesis. Bone marrow cells were harvested and cultured with M-CSF (25ng/ml) for 2 days, followed by TNF stimulation (100ng/ml) for 5 days. Osteoclast differentiation and function were evaluated by TRAP staining, osteoclast-associated gene expressions, actin ring formation, and bone resorption assay. NF-κB, c-Fos, and NFATc1 expression levels were examined by qPCR and western blotting. Results: TNF-stimulated KI/+ BMMs differentiated into a significantly larger number of TRAP-positive multinucleated osteoclasts with actin ring formation and mineralized matrix resorption than wild-type (1143±58 vs 42±20 cells/well). Osteoclast-associated gene expressions including Cathepsin K, Oscar, Acp5, and Dc-stamp were higher in KI/+ osteoclasts. Consistent with these results, expression level of NFATc1, a master transcription factor of osteoclastogenesis, was higher in TNF-induced KI/+ osteoclasts than that in wild-type. Nuclear translocations of NF-κB, c-Fos, and NFATc1 in KI/+ osteoclasts were increased, suggested that mutant SH3BP2 enhances TNF-induced osteoclastogenesis by promoting nuclear localization of these transcription factors. Conclusion: Cherubism mutation in SH3BP2 enhanced TNF-induced osteoclastogenesis by promoting nuclear localization of NF-κB, c-Fos, and NFATc1. These results suggest a role of SH3BP2 in the pathogenesis of inflammatory bone loss and destruction, which involves TNF.