Formononetin Attenuates Osteoclastogenesis via Suppressing the RANKL-Induced Activation of NF-fκB, 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)-a, interleukin (IL)-1 beta, IL-6, monocyte chemoattractant protein-1 alpha (MCP-1 alpha), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1a (MIP-1 alpha) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-kappa B p65 subunit, degradation of inhibitor kappa B alpha (I kappa B alpha), induction of NF-kappa 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-kappa 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.