Anti-DEF6 monoclonal antibody, clone 4C3

Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions. Def6 deficiency in Def6(-/-) mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer receptor activator for NF-kappa B ligand, and Def6(-/-) osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-alpha induced osteoclastogenesis in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-alpha serum levels correlated negatively with Def6 expression levels in osteoclast precursors obtained from RA patients, and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, B lymphocyte-induced maturation protein-1, and c-Fos by regulating an endogenous IFN-beta-mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction.

Rho-family GTPases have been implicated in actin remodeling and subsequent morphologic changes in various cells. DEF6, a pleckstrin homology domain-containing protein, has been reported to regulate Rho- family GTPases as a guanine nucleotide exchange factor. Here, we demonstrate that DEF6 also has the property of cooperating with activated Rac1. DEF6 bound selectively to Rac1 loaded with GTP. The interaction is mediated by the effector domain of Rac1. Overexpression of GFP-DEF6 together with constitutively active Rac1 in COS-7 cells significantly changed their cell shape; this was not seen in the absence of activated Rac1. This effect of DEF6 on cellular morphology was shown to be independent of its guanine nucleotide exchange activity. Because DEF6 does not contain any sequences previously known to interact with Rac, we explored the domain necessary for the binding. The amino-terminal portion and central parts of DEF6 were required for the binding. Finally, we succeeded in creating mutants of DEF6 with point mutations in the amino-terminal portion, which abrogate the binding to activated Rac1. These mutants did not exhibit the morphologic change in COS-7 cells when they were co-expressed with activated Rac1. These results suggest that DEF6 not only activates Rho- family GTPases but also cooperates with activated Rac1 to exert its cellular function.