cells were stained with TRAP. Bar, 100 m. B, TRAP activities were measured. C, TRAP-positive multinucleated osteoclasts. P < 0.05, P < 0.01. doi:10.1371/journal.pone.0130174.g007 rhinacanthin C. Therefore, suppression of TRAP activity and pit formation by rhinacanthin C are attributed to reduced levels of osteoclast specific/related gene expression governed by NFATc1. c-Fos induces NFATc1 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704093 expression and is an essential regulator of osteoclastogenesis. c-Fos knockout mice develop osteopetrosis due to a lack of osteoclast formation. Suppression of NFATc1 accumulation by rhinacanthin C may be caused by down-regulation of c-Fos. The NFATc1 promoter is initially activated by NF-B and AP-1 in the presence of RANKL. NFATc1 binds to its own promoter, followed by NFATc1-mediated auto-amplification of gene induction. c-Jun and c-Fos are phosphorylated and activated by JNK and ERK, respectively. Rhinacanthin C inhibited the RANKL-induced phosphorylation of ERK and JNK. Thus, rhinacanthin C may suppress AP-1 activation via inhibition of MAPKs. Rhinacanthin C also suppressed RANKL-induced phosphorylation of IB and NF-B/p-65, suggesting that the inhibitory effects of rhinacanthin C on osteoclast differentiation may also be caused by inhibition of NF-B activity. Thus, rhinacanthin C suppresses the early step of osteoclast differentiation via inhibition of RANKL-stimulated NF-B and MAPK activation. As a consequence of RANKL binding, RANK activates TRAF6 to initiate complex formation with TAK1, which facilitates NF-B and MAPK activation. TRAF6 siRNA and 12 / 17 Rhinacanthin C Suppresses Osteoclastogenesis Fig 8. Protective effects of rhinacanthin C on LPS-induced bone destruction in vivo. Vehicle or LPS with or without rhinacanthin C was daily injected into the subcutaneous tissue overlying the calvaria of 8-week-old wild type and OPG-/mice. The mice were sacrificed on day 5. TRAP staining of whole calvaria and 3D-images. Quantitative data of calvarial bone by CT analysis. BV/TV: Bone volume/total tissue volume ratio. Tb.Sp: Trabecular separation. TBPf: Trabecular bone pattern factor. Rhinacanthin C Suppresses Osteoclastogenesis TRAF6 decoy peptides inhibit the formation of TRAP-positive multinucleated cell and bone resorption. TRAF6 knockout mice exhibit severe osteopetrosis with defects in bone remodeling caused by impaired osteoclast function. Our immunoprecipitation assay revealed that association of TRAF6 and TAK1 was increased by RANKL, while rhinacanthin C suppressed this effect. Rhinacanthin C may disrupt intracellular signal PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705070 processing by targeting TRAF6TAK1 complex formation, thus impairing osteoclast differentiation. The direct targets of rhinacanthin C remain unknown. Recently, it was reported that -Vistin A and Norisoboldine inhibit osteoclast differentiation and prevent TRA6-TAK1 formation. Whether these natural 345627-80-7 price compounds and rhinacanthin C target the same molecule will be the topic of future research. Consistent with its anti-osteoclastogenic effects in vitro, rhinacanthin C also inhibits RANKL-induced osteoclast formation and bone destruction in vivo. Furthermore, caldecrin inhibits LPS-stimulated osteoclast formation from BMMS and calvarial bone resorption. LPS is recognized by Toll-like receptor, which triggers 
recruitment of TRAF6 and TAK1, leading to activation of the NF-B pathway. TRAF6 knockout cells fail to respond to LPS stimulation. Rhinacanthin C might inhibit LPS-induced osteoclastogenesis by suppressing TRAF6-TAK1-NF-B signal
