Formononetin Attenuates Osteoclastogenesis via Suppressing the RANKL-Induced Activation of NF-?B, c-Fos, and Nuclear Factor of Activated T-Cells Cytoplasmic 1 Signaling Pathway.
Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-?, interleukin (IL)-1?, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1? (MIP-1?) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-?B p65 subunit, degradation of inhibitor ?B? (I?B?), induction of NF-?B, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-?B siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.