Regulation of RANKL-induced osteoclastogenesis by TGF-β through molecular interaction between Smad3 and Traf6

Abstract

Previous studies have shown that transforming growth factor β (TGF-β) promotes receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF-β signals were blocked either by a specific inhibitor of TGF-β type 1 receptor kinase activity, SB431542, or by introducing a dominant-negative mutant of TGF-β type 2 receptor, RANKL-induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c-Ski markedly suppressed RANKL-induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6-TAB1-TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6-TAB1-TAK1 complex formation was not observed when TGF-β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6-TAB1-TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL-induced osteoclast differentiation. In summary, TGF-β is indispensable in RANKL-induced osteoclastogenesis, and the binding of Smad3 to the TRAF6-TAB1-TAK1 complex is crucial for RANKL-induced osteoclastogenic signaling.