TRAF6 Mediates Suppression of Osteoclastogenesis and Prevention of Ovariectomy-Induced Bone Loss by a Novel Prenylflavonoid.
Given the limitations of current therapeutic options for postmenopausal osteoporosis, there is a need for alternatives with minimal adverse effects. In this study, we evaluated the effects of icaritin (ICT), a natural prenylflavonoid, on osteoclastogenesis both in vitro and in an ovariectomized (OVX) rat model and investigated its underlying molecular mechanism(s) of action. ICT inhibited osteoclast formation in two osteoclast precursor models, RAW 264.7 mouse monocyte cell line and human PBMC. ICT also inhibited sealing zone and resorption pit formation in a dose-dependent manner. Mechanistically, ICT inhibited RANKL-induced NF-κB and MAPK/AP-1 pathways to suppress gene expression of nuclear factor of activated T cells (NFAT)c1, the master transcription regulator of osteoclast differentiation. ICT, by inhibiting the TRAF6/c-Src/PI3K pathway, suppressed NADPH oxidase-1 activation to attenuate intracellular ROS production and downregulate calcineurin phosphatase activity. As a result, NFATc1 nuclear translocation and activity was suppressed. Crucially, ICT promoted proteasomal degradation of TRAF6, the critical adaptor protein that transduces RANKL/RANK signaling, and the inhibitory effect of ICT on osteoclastogenesis was reversed by the proteasomal inhibitor MG 132. ICT administration inhibited OVX-induced bone loss and resorption by suppressing osteoclast formation and activity. Consistent with cellular studies, ICT downregulated TRAF6 and NFATc1 protein expression in CD11b+/Gr-1-/lowosteoclast precursors isolated from OVX rats. Put together, we present novel findings that ICT, by downregulating TRAF6, coordinates inhibition of NF-κB, MAPK/AP-1, and ROS signaling pathways to reduce expression and activity of NFATc1. These results demonstrate the potential of ICT for treatment of postmenopausal osteoporosis and point to TRAF6 as a promising target for novel anti-osteoporotic drugs. © 2017 American Society for Bone and Mineral Research.
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Ee Min
EM
Tan
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Lei
L
Li
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Inthrani Raja
IR
Indran
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Division of Infectious Diseases, University Medicine Cluster, National University Hospital Singapore, Singapore.
Nicholas
N
Chew
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Eu-Leong
EL
Yong
D000818:Animals; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D010010:Osteoclasts / Q000378:metabolism* / Q000473:pathology; D015663:Osteoporosis, Postmenopausal / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D010052:Ovariectomy; D000067996:RAW 264.7 Cells; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*
D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D048029:TNF Receptor-Associated Factor 6; C000604754:Tifab protein, human; C499403:icaritin
ANIMAL MODELS; CELL/TISSUE SIGNALING-TRANSCRIPTION FACTORS; OSTEOCLASTS; OSTEOPOROSIS; THERAPEUTICS