title: Norisoboldine suppresses osteoclast differentiation through preventing the accumulation of TRAF6-TAK1 complexes and activation of MAPKs/NF-κB/c-Fos/NFATc1 Pathways.
abstract: Norisoboldine (NOR) is the main alkaloid constituent in the dry root of Lindera aggregata (Sims) Kosterm. (L. strychnifolia Vill.). As reported previously, orally administered NOR displayed a robust inhibition of joint bone destruction present in both mouse collagen-induced arthritis and rat adjuvant-induced arthritis with lower efficacious doses than that required for ameliorating systemic inflammation. This attracted us to assess the effects of NOR on differentiation and function of osteoclasts, primary effector cells for inflammatory bone destruction, to get insight into its anti-rheumatoid arthritis mechanisms. Both RAW264.7 cells and mouse bone marrow-derived macrophages (BMMs) were stimulated with RANKL (100 ng/mL) to establish osteoclast differentiation models. ELISA, RT-PCR, gelatin zymography, western blotting, immunoprecipitation and EMSA were used to reveal related signalling pathways. NOR (10 and 30 µM), without significant cytotoxicity, showed significant reduction of the number of osteoclasts and the resorption pit areas, and it targeted osteoclast differentiation at the early stage. In conjunction with the anti-resorption effect of NOR, mRNA levels of cathepsin K and MMP-9 were decreased, and the activity of MMP-9 was attenuated. Furthermore, our mechanistic studies indicated that NOR obviously suppressed the ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of ERK and p38 MAPK, and reduced the nuclear translocation of NF-κB-p65 and DNA-binding activity of NF-κB. However, NOR had little effect on expressions of TRAF6 or the phosphorylation and degradation of IκBα. Moreover, NOR markedly inhibited expressions of transcription factor NFATc1, but not c-Fos. Intriguingly, the subsequent nuclear translocations of c-Fos and NFATc1 were substantially down-regulated. Hence, we demonstrated for the first time that preventing the differentiation and function of osteoclasts at the early stage was an important anti-bone destruction mechanism of NOR, which might be attributed to inhibition of ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of MAPKs/NF-κB/c-Fos/NFATc1 pathways.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Zhi-Feng
authors.initials: ZF
authors.lastname: Wei
authors.forename: Bei
authors.initials: B
authors.lastname: Tong
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Xia
authors.forename: Qian
authors.initials: Q
authors.lastname: Lu
authors.forename: Gui-Xin
authors.initials: GX
authors.lastname: Chou
authors.forename: Zheng-Tao
authors.initials: ZT
authors.lastname: Wang
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D001862:Bone Resorption / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D053245:RANK Ligand / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D054875:Ubiquitination / Q000187:drug effects; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D000135:Acid Phosphatase; D000470:Alkaloids; D007527:Isoenzymes; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; D000071681:Tartrate-Resistant Acid Phosphatase; D000099151:MAP Kinase Kinase Kinase 7; C464745:norisoboldine
references.citation: Arthritis Res. 2002;4 Suppl 3:S265-72
references.pmid: 12110146
references.citation: Expert Opin Ther Targets. 2011 Feb;15(2):169-81
references.pmid: 21204734
references.citation: Nature. 1999 Jan 28;397(6717):315-23
references.pmid: 9950424
references.citation: Genes Dev. 1998 Sep 15;12(18):2821-30
references.pmid: 9744859
references.citation: J Cell Biol. 2007 Mar 12;176(6):877-88
references.pmid: 17353363
references.citation: Cancer Res. 2008 Jul 15;68(14):5803-11
references.pmid: 18632634
references.citation: J Ethnopharmacol. 2007 May 4;111(2):322-8
references.pmid: 17204385
references.citation: Aust Fam Physician. 2010 Sep;39(9):626-8
references.pmid: 20877764
references.citation: Arthritis Res Ther. 2007;9 Suppl 1:S2
references.pmid: 17634141
references.citation: Arthritis Res Ther. 2007;9(1):203
references.pmid: 17316459
references.citation: J Bone Miner Metab. 2007;25(4):219-25
references.pmid: 17593491
references.citation: Biochem Biophys Res Commun. 2006 Dec 8;351(1):99-105
references.pmid: 17052691
references.citation: Cell. 1998 Apr 17;93(2):165-76
references.pmid: 9568710
references.citation: Int Immunopharmacol. 2011 May;11(5):543-8
references.pmid: 21081190
references.citation: FEBS Lett. 2006 Sep 18;580(21):4984-90
references.pmid: 16949582
references.citation: Mol Cell Biol. 2002 Feb;22(4):992-1000
references.pmid: 11809792
references.citation: J Immunol. 2009 Sep 1;183(5):3390-9
references.pmid: 19641137
references.citation: Int J Biochem Cell Biol. 2010 May;42(5):585-9
references.pmid: 20060931
references.citation: Rheumatol Int. 2012 Nov;32(11):3397-403
references.pmid: 22057136
references.citation: Int J Biochem Cell Biol. 2010 May;42(5):576-9
references.pmid: 20035895
references.citation: J Cell Biochem. 2008 Oct 15;105(3):896-904
references.pmid: 18759249
references.citation: Cell Res. 2011 Jan;21(1):169-82
references.pmid: 21079651
references.citation: Arthritis Care Res (Hoboken). 2010 Apr;62(4):537-44
references.pmid: 20391509
references.citation: Nat Rev Drug Discov. 2004 Jan;3(1):17-26
references.pmid: 14708018
references.citation: J Cell Biochem. 2012 Aug;113(8):2785-95
references.pmid: 22473817
references.citation: Rev Endocr Metab Disord. 2006 Jun;7(1-2):23-32
references.pmid: 17115268
references.citation: Rheum Dis Clin North Am. 2010 May;36(2):385-404
references.pmid: 20510240
references.citation: Arthritis Res Ther. 2011 Jul 28;13(4):235
references.pmid: 21861862
references.citation: J Bone Miner Res. 2008 Jun;23(6):907-14
references.pmid: 18251700
references.citation: Curr Rheumatol Rep. 2009 Jul;11(3):196-201
references.pmid: 19604464
references.citation: Food Chem Toxicol. 2012 May;50(5):1610-5
references.pmid: 22386813
references.citation: Curr Opin Rheumatol. 2007 May;19(3):289-95
references.pmid: 17414958
references.citation: Rheumatology (Oxford). 2006 Sep;45(9):1068-76
references.pmid: 16490750
references.citation: Int J Biochem Cell Biol. 2007;39(6):1077-81
references.pmid: 17174136
references.citation: Phytomedicine. 2010 Aug;17(10):726-31
references.pmid: 20363113
references.citation: J Biol Chem. 2007 May 25;282(21):15921-9
references.pmid: 17403683
references.citation: Joint Bone Spine. 2010 Dec;77(6):537-41
references.pmid: 20646949
references.citation: PLoS One. 2008;3(12):e4064
references.pmid: 19112497
references.citation: Apoptosis. 2005 Aug;10(4):731-41
references.pmid: 16133865