Previous studies have shown that transforming growth factor b (TGF-b) promotes receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF-b signals were blocked either by a specific inhibitor of TGF-b type 1 receptor kinase activity, SB431542, or by introducing a dominant-negative mutant of TGF-b 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-b 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 RANKLinduced osteoclast differentiation. In summary, TGF-b is indispensable in RANKL-induced osteoclastogenesis, and the binding of Smad3 to the TRAF6-TAB1-TAK1 complex is crucial for RANKL-induced osteoclastogenic signaling. ß

We have previously reported that transforming growth factor b (TGF-b) plays an essential role in receptor activator of nuclear factorkB ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-b, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF-b signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF-b regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos.

Class IA phosphatidylinositol 3-kinases (PI3Ks) are activated by growth factor receptors and regulate a wide range of cellular processes. In osteoclasts, they are activated downstream of a v b 3 integrin and colony-stimulating factor-1 receptor (c-Fms), which are involved in the regulation of bone-resorbing activity. The physiological relevance of the in vitro studies using PI3K inhibitors has been of limited value, because they inhibit all classes of PI3K. Here, we show that the osteoclast-specific deletion of the p85 genes encoding the regulatory subunit of the class IA PI3K results in an osteopetrotic phenotype caused by a defect in the bone-resorbing activity of osteoclasts. Class IA PI3K is required for the ruffled border formation and vesicular transport, but not for the formation of the sealing zone. p85a/b doubly deficient osteoclasts had a defect in macrophage colony-stimulating factor (M-CSF)-induced protein kinase B (Akt) activation and the introduction of constitutively active Akt recovered the bone-resorbing activity. Thus, the class IA PI3K-Akt pathway regulates the cellular machinery crucial for osteoclastic bone resorption, and may provide a molecular basis for therapeutic strategies against bone diseases. ß

Objective. Nitrogen-containing bisphosphonates are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the functional mechanism of one of the typical nitrogencontaining bisphosphonates, risedronate.Methods. Osteoclasts generated from murine bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and boneresorbing activity were examined. The mechanism of action of risedronate was examined by gene induction of constitutively active Akt-1 and constitutively active MEK-1, and by gene deletion of Bim. Bim ؊/؊ mice, in which osteoclasts were resistant to apoptosis, were treated with risedronate and analyzed radiographically, biochemically, and histologically.Results. Risedronate induced osteoclast apoptosis through the mitochondria-dependent pathway with an increased expression of Bim, and the proapoptotic effect of risedronate was suppressed by Bim deletion and constitutively active MEK-1 introduction. In contrast, the risedronate-induced suppression of bone resorption was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively active MEK-1 introduction. These results suggested that apoptosis and bone-resorbing activity of osteoclasts were regulated through the ERK/Bim axis and the Akt pathway, respectively, both of which were suppressed by risedronate. Although osteoclast apoptosis in response to risedronate administration was suppressed in the Bim ؊/؊ mice, risedronate treatment increased bone mineral density in Bim ؊/؊ mice at a level equivalent to that in wild-type mice.Conclusion. Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of osteoclasts and not by the induction of osteoclast apoptosis.

The transcription factor Stat5 inhibits the bone-resorbing function of osteoclasts, in part by inducing the expression of the phosphatases Dusp1 and Dusp2.