2-Methoxystypandrone represses RANKL-mediated osteoclastogenesis by down-regulating formation of TRAF6-TAK1 signalling complexes.
OBJECTIVE
2-Methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum, a Chinese herb used to treat bone diseases. Here we have determined whether 2-MS antagonised osteoclast development and bone resorption.


METHODS
RAW264.7 cells were treated with receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to induce differentiation into osteoclasts. RT-PCR and Western blot were used to analyse osteoclast-associated gene expression and signalling pathways.


RESULTS
The number of multinuclear osteoclasts, actin rings and resorption pit formation were markedly inhibited by 2-MS, targeting osteoclast differentiation at an early stage and without significant cytotoxicity. The anti-resorption effect of 2-MS was accompanied by decreasing dendritic cell-specific transmembrane protein and matrix metalloproteinase-9 (MMP-9) mRNA expression. RANKL-increased MMP-9 gelatinolytic activity was also attenuated by concurrent, but not by subsequent addition of 2-MS. 2-MS markedly inhibited not only the RANKL-triggered nuclear translocations of NF-kappaB, c-Fos and nuclear factor of activated T cells c1 (NFATc1), but also the subsequent NFATc1 induction. Degradation of IkappaB and phosphorylation of mitogen-activated protein kinases were also suppressed. RANKL facilitated the formation of signaling complexes of tumour necrosis factor receptor-associated factor 6 and transforming growth factor beta-activated kinase 1 (TRAF6-TAK1), important for osteoclastogenesis and formation of such signalling complexes was prevented by 2-MS.


CONCLUSIONS
The anti-osteoclastogenic effects of 2-MS could reflect the block of RANKL-induced association of TRAF6-TAK1 complexes with consequent decrease of IkappaB-mediated NF-kappaB and mitogen-activated protein kinases-mediated c-Fos activation pathways and suppression of NFATc1 and other gene expression, essential for bone resorption.
Chiou
W F
WF
National Research Institute of Chinese Medicine, Taipei, Taiwan. ccchen@sunrise.hk.edu.tw
Liao
J F
JF
Huang
C Y
CY
Chen
C C
CC
D000818:Animals; D050071:Bone Density Conservation Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D015536:Down-Regulation; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification / Q000494:pharmacology*; D045468:Fallopia japonica / Q000737:chemistry; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009285:Naphthoquinones / Q000302:isolation & purification / Q000494:pharmacology*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D053245:RANK Ligand / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
D050071:Bone Density Conservation Agents; D004365:Drugs, Chinese Herbal; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009285:Naphthoquinones; D011480:Protease Inhibitors; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; D000099151:MAP Kinase Kinase Kinase 7; C447630:2-Methoxystypandrone