Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-κB signaling pathways

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oo-un, Bra561561f KoReceived 4 July 2009Received in revised form 22 February 2010Accepted 14 March 2010European Journal of Pharmacology 636 (2010) 2835Contents lists available at ScienceDirectEuropean Journal oe l1. IntroductionBone remodeling is a physiological process that involves theresorption of bone by osteoclasts and the synthesis of bone matrix byosteoblasts (Karsenty and Wagner, 2002). Osteoclasts are known tobe formed by the fusion of hematopoietic cells of the monocyte-macrophage lineage during the early stage of the differentiationprocess (Mohamed et al., 2007). Terminal differentiation in thislineage is characterized by acquisition of mature phenotypic markerssuch as expression of tartrate-resistant acid phosphatase (TRAP),calcitonin receptor, matrix metalloproteinase 9 (MMP9), and cathep-sin K, as well as morphological conversion into large multinucleatedcells and the capability to form resorption lacunae on bone(Anusaksathien et al., 2001; Motyckova et al., 2001; Reddy et al.,(macrophage colony-stimulating factor) in bone marrow-derivedmacrophage precursor cells (Takayanagi et al., 2002). RANKL inducesthe signaling essential for precursor cells to differentiate into osteoclasts(Theill et al., 2002),whereasM-CSF, secretedbyosteoblasts, provides thesurvival signal to these cells (Yoshida et al., 1990). Binding of RANKL toits receptor RANK activates TNF receptor-associated factor 6, which islinked to nuclear factor B (NF-B) and mitogen-activated proteinkinases (Kobayashi et al., 2001; Lee et al., 2002; Chang et al., 2007).Active extracellular signal-regulated kinase (ERK) can directly phos-phorylate c-Fos and active c-Jun-N-terminal kinase (JNK) phosphor-ylates c-Jun (Miyazaki et al., 2000). Thus, AP-1 transcription factor, aheterodimer composed of a Fos family member and a Jun familymember, can be a target of ERK and JNK in response to RANKLstimulation of osteoclast precursor cells. In addition, RANKL induces the1995).The essential signaling molecules for oinclude RANKL (receptor activator of NF- Corresponding author. Tel.: +82 63 270 4038; fax:E-mail address: (Y. Soh).0014-2999/$ see front matter 2010 Elsevier B.V. Adoi:10.1016/j.ejphar.2010.03.023Available online 29 March 2010Keywords:Ikarisoside AOsteoclastOsteoclastogenesisBoneRAW 264.7TRAPRANKLplay a crucial homeostatic role in skeletal modeling and remodeling and destroy bone in many pathologicconditions. Receptor activator of NF-B ligand (RANKL) is essential to osteoclastogenesis. In this study, weinvestigated the effects of Ikarisoside A, isolated from Epimedium koreanum (Berberidaceae), onosteoclastogenesis in RANKL-treated murine monocyte/macrophage RAW 264.7 cells. The results indicatethat Ikarisoside A is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW 264.7 cells as well asin bone marrow-derived macrophages. The inhibitory effect of Ikarisoside A resulted in decrease ofosteoclast-specic genes like matrix metalloproteinase 9 (MMP9), tartrate-resistant acid phosphatase(TRAP), receptor activator of NF-B (RANK), and cathepsin K. Moreover, Ikarisoside A blocked the resorbingcapacity of RAW 264.7 cells on calcium phosphate-coated plates. Ikarisoside A also has inhibitory effects onthe RANKL-mediated activation of NF-B, JNK, and Akt. Finally, Ikarisoside A clearly decreased the expressionof c-Fos and nuclear factor of activated T cells c1 (NFATc1) as well as the transcriptional activity of NFATc1,the master regulator of osteoclast differentiation. The data indicate that Ikarisoside A has potential for use intreatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, andperiodontal bone erosion. 2010 Elsevier B.V. All rights reserved.steoclast differentiationB ligand) and M-CSFkey transcriptionT cells c1 (NFATcet al., 2002).As the bone aall factors that reeffect on bone anbone disease fo+82 63 270 4037.ll rights reserved.derived from multipotent myeloid progenitor cells. TheyArticle history: Osteoclasts are specialized bone-resorbing cellsa b s t r a c ta r t i c l e i n f oMolecular and Cellular PharmacologyInhibition of osteoclastogenic differentiatiJNK and NF-B signaling pathwaysHwa Jung Choi a, Young Ran Park a, Manoj Nepal a, BSoo Rye Heo c, Hyung Sup Kim c, Moon-Sik Yang d, Ya Department of Dental Pharmacology, School of Dentistry and Institute of Oral Bioscienceb Department of Oral Pathology, School of Dentistry, Chonbuk National University, Jeon-Juc Department of Periodontology, School of Dentistry, Chonbuk National University, Jeon-Jud Division of Biological Sciences, Chonbuk National University, Jeonju, 561-756, Republic oj ourna l homepage: www.n by Ikarisoside A in RAW 264.7 cells viaYun Choi a, Nam-Pyo Cho b, Seoung Hwan Choi c,jo Soh a,in Korea 21 project, Chonbuk National University, Jeon-Ju 561-756, Republic of Korea-756, Republic of Korea-756, Republic of Koreareaf Pharmacologysev ie locate /e jpharfactor for osteoclastogenesis, nuclear factor of activated1) (Takayanagi et al., 2002; Yamashita et al., 2007; Zhound the immune system are so closely intermingled,gulate immune cells should be investigated for theird vice versa. For this reason, treatment strategies forcus on the suppression of bone destruction andinammation-associated bone loss. Bone-resorbing osteoclasts areimportant effector cells in inammation-induced bone loss such asrheumatoid arthritis or periodontitis (Jimi et al., 2004; Mino et al.,1998). Inammatory cytokines and prostaglandins up-regulateRANKL in osteoblasts, synovial broblasts, and activated T cells(Coon et al., 2007; Kotake et al., 1999). RANKRANKL signaling wasshown to be essential for osteoclast differentiation in inammatorybone destruction (Anandarajah and Schwarz, 2006). In addition,many cytokines affected by inammation, including the proinam-matory cytokines TNF- and interleukin-1 (IL-1), may contribute toosteoclast differentiation and activation (Han et al., 2007).Flavonoids among novel therapeutic agents are well known toespecially suppress inammation. Ikarisoside A is a natural avonoidof the Ikarisoside family (Kuroda et al., 2000; Li et al., 1998, 1996).Data from a previous study of ours showed that Ikarisoside A isolatedfrom Epimedium koreanum (Berberidaceae) exerted antioxidantpotential and anti-inammatory effects in LPS-stimulated bonemarrow-derived macrophage precursor cells and RAW 264.7 cells(Choi et al., 2008a). Therefore, we examined the anti-osteoclastogeniceffects and signaling pathways of Ikarisoside A with RANKL-stimulated macrophages. We demonstrate here for the rst timethat Ikarisoside A signicantly suppresses RANKL-induced osteoclastdifferentiation by modulating osteoclast-specic genes, transcriptionfactors, and signaling molecules.2. Materials and methods2.1. MaterialsCell culture medium, fetal bovine serum (FBS), and horse serumwere obtained from Invitrogen (Gaithersburg, MD, USA). RANKL wasavailable OAAS kit for osteoclastic bone resorption assay was obtainedfrom Oscotec (Choongnam, Korea). All other chemicals werepurchased from Sigma and/or the same as described previously(Soh et al., 2000, 2003, 1998), unless otherwise indicated.2.2. Isolation of Ikarisoside AIkarisoside A (Fig. 1A) from E. koreanum methanol extract wasisolated by successive fractionation with ethyl acetate, n-butanol,chloroform, and hexane as Icariside II was puried (Choi et al., 2008b).2.3. Isolation of bone marrow-derived macrophages and co-cultureSix-week-old ICR (Institute of Cancer Research) mice werepurchased from Damool Science (Daejeon, Korea), bred and main-tained in accordance with the guidelines of the Chonbuk NationalUniversity Animal Ethics Commiittee. Cells were obtained from tibiaand femur bone marrow and were cultured in -MEM with 10% FBScontaining 20 ng/ml macrophage colony-stimulating factor (M-CSF).After 3 days, the nonadherent cells were removed by washing andadherent cells were used as bone marrow-derived macrophages. Forco-culture experiment, primary osteoblasts were prepared from thecalvaria of 1 day-old mouse and seeded on 96-well plates (1104cells per well). After 1 day, bone marrow cells were added at 1105cells perwell to the osteoblasts and cultured for 5 days in the presenceof vitamin D3 (108 M) and prostaglandin E2 (106 M).2.4. Cell culture and treatmentThe murine monocyte/macrophage cell line RAW 264.7 waspurchased from American Type Culture Collection (Manasas, VA, USA)cellsays,ay. T29H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 2835obtained from PeproTech (Rocky Hill, NJ, USA). M-CSF was from R&DSystems (Minneapolis, MN, USA). Leukocyte Acid Phosphatase AssayKit was obtained from Sigma (St. Louis, MO, USA). CommerciallyFig. 1. Inhibitory effects on osteoclast differentiation in RANKL-stimulated RAW 264.7indicated concentration of Ikarisoside A in the presence of RANKL (50 ng/ml). After 6 dwere counted. (D) The effect of Ikarisoside A on cell viability wasmeasuredwithMTT ass(#).and grown in DMEM supplemented with 10% heat-inactivated FBS,penicillin (100 U/ml), and streptomycin (100 g/ml). All cells weregrown in a humidied atmosphere containing 5% CO2 at 37 C. For. (A) Chemical structure of Ikarisoside A. (B) RAW 264.7 cells were cultured with thecells were xed and stained for TRAP. (C) TRAP-positive multinucleated cells (TRAP+)he results are expressed asmeanS.E.M. **Pb0.01, *Pb0.05 versus vehicle-treated cellsosteoclastic differentiation, RAW264.7 cells were suspended in-MEMcontaining10% FBS, 2 mM L-glutamate, 100 U/ml penicillin, and 100 g/ml streptomycin and then seeded at 3103 cells/well in 96-well cultureplates and cultured with 50 ng/ml soluble RANKL for 6 days. The bonemarrow-derived macrophages were further cultured for 3 days in anosteoclastogenic medium (-MEM containing 10% FBS, 30 ng/ml M-CSF, and 200 ng/ml RANKL). Cells were cytochemically stained fortartrate-resistant acid phosphatase (TRAP), an osteoclast markerprotein.2.5. Tartrate-resistant acid phosphatase (TRAP) stainingTRAP staining was performed according to Han et al. (2007). Briey,cells were washed with PBS and xed with 3.7% formaldehyde for10 min. After washing with PBS, cells were incubated with 0.1% (v/v)Triton X-100 for 1 min. Cells were washed, and then incubated for30 H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 283540 min at 37 C in the dark with a mixture of solutions of Fast GarnetGBC, sodium nitrite, naphthol AS-BI phosphoric acid, acetate, andtartrate of the Leukocyte Acid Phosphatase Assay kit (Sigma) accordingto the manufacturer's instructions. Cells were washed with distilledwater and TRAP-positive multinucleated cells containing three or morenuclei were counted under a light microscope.2.6. RT-PCR analysisTotal RNAs were isolated from cultured cells using TRIzol(Invitrogen) and cDNA synthesis was performed with SuperScript IIreverse transcriptase (Invitrogen) according to the manufacturer'sprotocol. PCR primers were purchased from Bioneer (Daejeon, Korea).Primer sequences and PCR conditions used in this study are listed inTable 1. After initial denaturation at 94 C for 1 min, PCR wasperformed for various cycles (30 s at 94 C, 1 min at annealingtemperature and 2 min at 72 C) using Taq polymerase (Promega,Madison, WI, USA). Reaction products were separated on a 1% agarosegel, stained with ethidium bromide, and analyzed densitometricallyusing a Phosphoimager and Quantity One software (Version 4.3.1)(Bio-Rad, Hercules, CA, USA).2.7. Immunoblot analysisCells were harvested and resolved in lysis buffer [20 mM TrisHCl(pH 7.5), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4,1 mMphenylmethylsulfonyluoride (PMSF), and 1 X protease inhibitorcocktail]. After centrifugation at 16,000g for 15 min, supernatantswere used as cell extracts. Cell extracts were separated by SDS-PAGE on810% gels and then transferred to polyvinylidene diuoride (PVDF)membranes (Bio-Rad). Membranes were blocked with 5% nonfat skimmilk in Tris-buffered saline (TBS) containing 0.25% Tween-20 (TTBS) atroom temperature for 1 h and then incubated for 16 h at 4 Cwith rabbitTable 1Primer sequences and conditions for RT-PCR.Target genes Primers AnnealingTm (C)PCRcyclesProductsize (bp)(Accession number) (Forward, Reverse)TRAP(NM_007388)5-ctgctgggcctacaaatcat-35-ggtagtaagggctggggaag-354 21 400RANK(NM_009399)5-aaaccttggaccaactgcac-35-accatcttctcctcccgagt-353.7 30 377MMP9(NM_013599)5-cgtcgtgatccccacttact-35-agagtactgcttgcccagga-357.5 30 433Cathepsin K(NM_007802)5-aggcggctatatgaccactg-35-ccgagccaagagagcatatc-357.5 17 403c-Fos(NM_010234)5-atgggctctcctgtcaacac-35-ggctgccaaaataaactcca-357.5 30 480-actin(NM_007393)5-ttctacaatgagctgcgtgt-35-ctcatagctcttctccaggg-350 25 456anti-phospho-ERK (Cell Signaling Technology Inc., Beverly, MA, USA),anti-phospho-p38 (Cell Signaling Technology), anti-phospho-JNK (CellSignaling Technology), anti-ERK (Cell Signaling Technology), anti-JNK(Cell Signaling Technology), anti-p38 (Cell Signaling Technology), anti-MMP9 (Santa Cruz Biotechnology), anti-NFATc1 (Santa Cruz Biotech-nology), anti-c-Fos (Cell Signaling Technology), or anti--actin (SantaCruz Biotechnology) antibodies diluted 1:1000 in 5% nonfat skimmilk inTTBS. Horseradish peroxidase-conjugated anti-rabbit or anti-mouseantibodies (Santa Cruz Biotechnology) were used as secondaryantibodies (1:50001:10,000 dilution in 5% nonfat skim milk in TTBS,1 h incubation at room temperature) and the antigenantibodycomplexeswere visualizedwith an ECL Plus kit (AmershamBiosciences,Piscataway, NJ, USA).2.8. Cell transfection and luciferase reporter assayRAW 264.7 cells were seeded at 5104/well in a 24-well dish andgrown to 9095% conuence in complete growth media. For each well,1.0 g of luciferase reporter plasmid construct harboring the NFATc1and NF-B binding sites and 0.5 g of pCMV--galactosidase controlvector were co-transfected into cells with Lipofectamine 2000 (Invitro-gen) according to themanufacturer's instructions. After transfection for24 h, the medium was changed and the cells were treated with RANKLand Ikarisoside A. The cells were thenwashedwith PBS, and lysed in 1reporter lysis buffer (Promega). The activities of rey luciferase in thecellular extracts were measured using the luciferase reporter assaysystem according to the manufacturer's instructions (Promega).Relative luciferase activity was obtained by normalizing the reyluciferase activity against the -galactosidase activity.2.9. Pit formation assayRAW 264.7 cells were suspended in phenol -MEM containing 10%FBS and plated at a concentration of 1104 cells/well on an OsteoclastActivity Assay Substrate (OAAS) in thepresence or absence of 50 ng/mlRANKL and Ikarisoside A. The medium was replaced every 2 days. After6 days of culture, the plates were washed in 6% sodium hypochloritesolution to remove the cells. The resorbed areas on the plates werecaptured with a digital camera attached to the microscope and analyzedby Soft Imaging System (Olympus Soft Imaging Solutions GmbH,Munster, Germany).2.10. Statistics analysisAll the experimental data shown are expressed as meansS.E.M.and all experiments were repeated at least three times, unlessotherwise indicated. Statistical analyses were performed by Dunnett'smultiple comparison test using SPSS ver. 12.0 software and P-valuesless than 0.05 were considered signicant.3. Results3.1. Inhibitory effects on osteoclast differentiationWe tested the effect of Ikarisoside A on osteoclast formation usingthe murine monocyte/macrophage cell line RAW 264.7 and bonemarrow-derived macrophages. Fifty ng/ml of RANKL induced TRAP-positive multinucleated osteoclast differentiation in RAW 264.7 cells.However, Ikarisoside A inhibited osteoclast differentiation in aconcentration-dependent manner (Fig. 1B). Ikarisoside A reducedthe number of TRAP-positive multinucleated cells generated with46.55.8% and 78.88.7% inhibition at 5 M and 10 M concentra-tions, respectively (Fig. 1C). To examine the effect of Ikarisoside A oncell growth, we treated cells with various concentrations of Ikariso-side A for 24 h and measured cell growth by an MTT assay. IkarisosideA did not affect the cell growth rate of RAW 264.7 cells (Fig. 1D),sustaining substantial viability even when used at concentrations of20 M Ikarisoside A (N93%). To investigate the effect of Ikarisoside Aon osteoclastogenesis, we used a co-culture systemwhere osteoblastsand bone marrow cells were cultured together in the presence ofvitamin D3 and prostaglandin E2. Ikarisiside A signicantly inhibitedosteoclast differentiation in a concentration-dependent manner(Fig. 2A and B). To examine whether Ikarisoside A directly affectosteoclast precursor cells, we used bone marrow-derived macro-phages which are known to differentiate to osteoclasts when inducedby M-CSF and RANKL. Ikarisoside A reduced the formation of TRAP-positive multinucleated cells (MNC) in a concentration-dependentmanner (Fig. 2C). Almost complete inhibition of osteoclastogenesiswas observed at 20 M concentration. Ikarisoside A reduced thenumber of TRAP-positive multinucleated cells generated with70.75.2% and 85.88.3% inhibition at concentrations of 10 Mand 20 M, respectively (Fig. 2D). Ikarisoside A did not affect cellgrowth of bone marrow-derived macrophages at the concentrationsused in this study (data not shown).3.2. Effects on regulation of osteoclastic genes in RANKL-stimulated RAW264.7 cellsOsteoclast differentiation is associated with up-regulation ofspecic genes in response to RANKL. To determine if the inhibitoryeffect of Ikarisoside A corresponds with the expression of osteoclast-specic genes, total RNA was prepared and analyzed by RT-PCR.RANKL (50 ng/ml) signicantly induced the expression of MMP9,TRAP, RANK, and cathepsin K in RAW264.7 cells. However, IkarisosideA reduced the expression of these osteoclast-specic genes in aconcentration-dependent manner (Fig. 3A and B). Ikarisoside A did3.3. Effects on pit formation of RANKL-stimulated RAW 264.7 cellsWe further examined if Ikarisoside A has an effect on the ability ofmature osteoclasts to resorb bone. RAW 264.7 cells were stimulatedwith RANKL to induce the differentiation of mature osteoclasts withbone-resorbing capacity and plated on calcium phosphate-coatedculture plates. RANKL-stimulated cells formed a number of pits on thebottom of the plate, suggesting that the bone resorption activity ofRANKL-treated RAW 264.7 cells made them functionally active asosteoclasts (Fig. 3C). Treatment with Ikarisoside A signicantlyreduced the formation of resorption pits in numbers and in overallarea in a concentration-dependent manner as compared to treatmentwith RANKL alone (Fig. 3D): 45.311.8%, 75.911.3%, and 91.04.0% inhibition at 5 M, 10 M, and 20 M, respectively.3.4. Effect on NF-B activation in RANKL-stimulated RAW 264.7 cellsActivation of the NF-B transcription factor is an essential step forosteoclast differentiation (Franzoso et al., 1997;Vaira et al., 2008). Recentstudies have suggested that NF-B is another upstream transcriptionfactor modulating NFATc1 expression (Yamashita et al., 2007). Toinvestigate the molecular mechanism of Ikarisoside A-mediated inhibi-tion of osteoclast differentiation, phosphorylation of inhibitory B (IB),which is a marker of ubiquitination, and subsequent proteasome-mediated degradation, was investigated. IB degradation unmasks thenuclear localization signalmotif of NF-B, which allows the transcriptionfactor to move into the nucleus. In time-dependant experimentalconditions, 20 M Ikarisoside A reduced RANKL-induced IB phosphor-ylation markedly within 10 min compared to total IB (Fig. 4A upper).Furthermore, phosphorylation of p65, which enhances its own tran-scriptional potential (Viatour et al., 2005), was investigated. Ikarisoside AerivE2.ys, c31H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 2835Fig. 2. Inhibitory effects on osteoclast differentiation in co-culture and bone marrow-dindicated concentrations of Ikarisoside A in the presence of vitamin D3 and prostaglandinof Ikarisoside A in the presence of M-CSF (30 ng/ml) and RANKL (200 ng/ml). After 6 danot affect the expression of the housekeeping gene -actin. These datashow that Ikarisoside A has a specic effect on the regulation of somegenes induced during osteoclast differentiation.were counted. The results are expressed as meanS.E.M. **Pb0.01, *Pb0.05 versus RANKLalso reduced the RANKL-induced phosphorylation of p65 markedlycompared to total p65 (Fig. 4A lower). We also examined NF-Btranscriptional activity by using a luciferase reporter plasmid constructed macrophages. (A) Bone marrow cells and calvarial osteoblasts were cultured with(C) Bonemarrow-derivedmacrophages were cultured with the indicated concentrationells were xed and stained for TRAP. (B, D) TRAP-positive multinucleated cells (TRAP+)-treated cells (#).32 H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 2835containing the NF-B binding site. RANKL caused a 1.3 fold-increase NF-B transcriptional activity within 15 min, but did not increase its activityat later time points. However, Ikarisoside A signicantly suppressedRANKL-induced NF-B transcriptional activity for 15 min (data notshown).3.5. Effects on activation of MAPKs in RANKL-stimulated RAW 264.7 cellsThe three families of MAPKs, ERK, JNK, and p38, have all beenshown to be activated in response to RANKL in osteoclast precursorcells (Lee et al., 2002) and RAW 264.7 cells (Mozar et al., 2008). Todetermine the intracellular mechanism of Ikarisoside A, we investi-gated the activation of MAPKs involved in the RANKL-signalingpathway using the RAW 264.7 cell line. The JNK, ERK, p38, and Aktactivation states were determined by immunoblot analysis usingantibodies specically directed against the phosphorylated forms ofthe enzymes, compared to data obtained with antibodies directedagainst the unphosphorylated states of the kinases. RANKL (50 ng/ml)Fig. 3. Suppression of RANKL-induced gene expression and RANKL-stimulated pit formationIkarisoside A in the presence of RANKL (50 ng/ml). After 6 days, the mRNA expression levelswith that of -actin. (B) The histogram represents the levels of the mRNA expression (%) cActivity Assay Substrate (OAAS) with the indicated concentration of Ikarisoside A in thesodium hypochlorite solution and resorbed areas were measured. (D) The histogram represeexpressed as meanS.E.M. **Pb0.01, *Pb0.05 versus RANKL-treated cells (#).markedly induced the activation of all three MAPKs within 10 min oftreatment in RAW 264.7 cells. However, Ikarisoside A inhibited thephosphorylation of JNK but not the phosphorylation of ERK or p38(Fig. 4B). RANKL (50 ng/ml) also induced the activation of Akt within30 min of treatment and a further increase in activation was observedat 60 min of treatment. Furthermore, this RANKL-mediated activationof Akt appeared to be inhibited by treatment with Ikarisoside A(Fig. 4B).3.6. Effects on expression of c-Fos in RANKL-stimulated RAW 264.7 cellsc-Fos plays an important role in RANKL-induced NFATc1 expres-sion by forming AP-1 complexes with c-Jun (Karsenty and Wagner,2002; Yamashita et al., 2007). RANKL has been shown to elevate thelevels of c-Fos in osteoclast precursor cells (Han et al., 2007; Park et al.,2007). As shown in Fig. 5, RANKL also induced the expression of c-Fosin RAW 264.7 cells. The RANKL-induced increase in c-Fos mRNA levelswas signicantly attenuated by Ikarisoside A (Fig. 5A). The proteinby Ikarisoside A. (A) RAW 264.7 cells were cultured with the indicated concentration ofofMMP9, TRAP, RANK, and cathepsin K genes were determined by RT-PCR and comparedompared with that of the control. (C) RAW 264.7 cells were cultured on an Osteoclastpresence of RANKL (50 ng/ml). After 6 days, cells were removed by treating with 6%nts the relative resorbed area (%) compared with that of the control (#). The results arefactor. These results suggest that Ikarisoside A might have suppressedNFATc1 by inhibiting the up-regulation of c-Fos in RANKL-stimulatedRAW 264.7 cells.4. DiscussionIn this study, we found that Ikarisoside A is a potent inhibitor ofosteoclastogenesis in RANKL-stimulated RAW 264.7 cells as well as inand bone marrow-derived macrophages. The inhibitory effect ofIkarisoside A also led to the decrease of osteoclast-specic genes likeMMP9, TRAP, RANK, and cathepsin K. Moreover, Ikarisoside Ainhibited the resorbing capacity of RAW 264.7 cells on calciumphosphate-coated plates. Ikarisoside A has inhibitory effects on theactivation of NF-B, JNK, and Akt. Finally, Ikarisoside A clearlydecreased the expression of c-Fos and NFATc1 and reduced thetranscriptional activity of NFATc1, the master regulator of osteoclastdifferentiation.The signaling mechanism of RANKL has been extensively studied.Osteoblast lineage cells express a membrane-bound form of RANKL, a33H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 2835level of c-Fos was also markedly increased by 3 h of RANKL exposure.However, pretreatment of RAW 264.7 cells with Ikarisoside A reducedthe RANKL-induced up-regulation of this transcription factor ascompared to an untreated control (Fig. 5B).3.7. Effects on activation of NFATc1 in RANKL-stimulated RAW 264.7cellsImportantly, RANKL specically and strongly induces nuclearfactor of activated T cells, cytoplasmic 1 (NFATc1), the masterregulator of osteoclast differentiation, and this induction is dependenton both the NF-B and c-Fos pathways (Takayanagi et al., 2002). Wenext investigated whether Ikarisoside A reduced the activation ofNFATc1 in osteoclastogenesis of RAW 264.7 cells. RANKL induced thetranscriptional activity of NFATc1 in RAW 264.7 cells with 3 hexposure, as shown in the luciferase reporter assay (Fig. 6A), andincreased the expression of NFATc1 as determined by Western blot(Fig. 6B). However, 20 M Ikarisoside A reduced the transcriptionalactivity of NFATc1 as well as the protein level of the transcriptionmember of the TNF cytokine family. Like other members of the TNFreceptor superfamily, RANK strongly activates the NF-B pathway. Inthe canonical NF-B pathway, ligation of RANK activates the inhibitorof IB kinase complex, which phosphorylates NF-B-associated IB,leading to its ubiquitination and proteosomal degradation. Theseevents release NF-B dimers containing RelA and c-Rel in the cytosol,allowing them to translocate into the nucleus where they enhancetranscription of target genes (Luo et al., 2005). Ikarisoside A reducedFig. 4. Effect on NF-B (A), MAPKs, and Akt (B) activation in RANKL-stimulated RAW264.7 cells. RAW 264.7 cells were serum-starved for 16 h, pretreated with 20 MIkarisoside A for 30 min, and stimulated with RANKL (50 ng/ml) for the indicated time.Cell extracts were analyzed by Immunoblot analysis using antibodies specicallydirected against the phosphorylated forms of the enzymes, compared to data obtainedwith antibodies directed against the unphosphorylated states of the kinases. Equalamounts of protein were loaded in each lane as demonstrated by the level of -actin.Fig. 5. Suppression of RANKL-induced c-Fos expression by Ikarisoside A. (A) RAW 264.7cells were cultured with the indicated concentration of Ikarisoside A in the presence ofRANKL (50 ng/ml). After 6 h, the mRNA expression levels of c-fos genes weredetermined by RT-PCR and compared with that of -actin. The lower histogramrepresents the levels of themRNA expression (%) comparedwith that of the control (#).The results are expressed as meanS.E.M. *Pb0.05 versus RANKL-treated cells (#).(B) RAW 264.7 cells were serum-starved for 16 h, pretreated with 20 M Ikarisoside Afor 30 min, and stimulated with RANKL (50 ng/ml) for the indicated time. Cell extractswere analyzed by Immunoblot analysis using antibodies specically directed against c-Fos protein. Equal amounts of protein were loaded in each lane as demonstrated by thelevel of -actin.34 H.J. Choi et al. / European Journal of Pharmacology 636 (2010) 2835Fig. 6. Inhibition of NFATc1 activity by Ikarisoside A. (A) RAW 264.7 cells transfectedwith a NFATc1-dependent luciferase reporter gene construct were treated with RANKL(50 ng/ml) in the absence or presence of 20 M Ikarisoside A for the indicated time.Cells were lysed and luciferase activity was measured. *Pb0.05 versus RANKL-treatedcells (#). (B) RAW 264.7 cells were serum-starved for 16 h, pretreated with 20 MIkarisoside A for 30 min, and stimulated with RANKL (50 ng/ml) for 24 h. Cell extractsRANKL-induced NF-B transcriptional activity as well as phosphory-lation of IB and p65. Akt is activated by both RANKL and M-CSF andserves as a central player in the regulation of osteoclast survival(Stern, 2007). The RANKL-mediated activation of Akt is dependent onTRAF6-Src-PI 3-kinase interaction (Wong et al., 1999). Ikarisoside Adisplayed a potent inhibitory effect on osteoclast differentiation,which may have resulted from reduced survival of osteoclastprecursor cells during differentiation due to Akt inhibition. In ourstudy, Ikarisoside A did not affect the activation of either ERK or p38 inresponse to RANKL. Therefore, it is unlikely that Ikarisoside A directlymodulates the activation of p38 or ERK under the conditions used inthis study. Taken together, Ikarisoside A may specically inhibit theactivation of RANKL signaling proximal to both Akt and NF-B, and itis unlikely that Ikarisoside A inhibits RANKLRANK interaction, as isthe case of the RANKL decoy receptor osteoprotegerin.In addition to AP-1, NFATc1 is also a crucial transcription factor inRANKL-induced osteoclastogenesis (Takayanagi et al., 2002). Genes suchas TRAP, cathepsin K, andMMP-9, which are specically induced duringthe terminal differentiation of osteoclasts, contain multiple NFAT andAP-1 binding sites (Anusaksathien et al., 2001; Motyckova et al., 2001;Reddy et al., 1995). BothAP-1 andNF-Bbinding sites are presentwithinthepromoter regionof theNFATc1gene (Zhou et al., 2002). It is thereforepossible that the initial induction of NFATc1 is mediated by these twotranscription factors, and is then followed by NFATc1-mediatedautoamplication of gene induction. In our study, Ikarisoside Asignicantly suppressed not only c-Fos induction but also NFATc1 up-regulation by RANKL. We suggest that the down-regulation of c-Fos byIkarisoside Amay be, at least in part, the cause of suppression of RANKL-induced NFATc1 expression.In summary, we have demonstrated, for the rst time, thatIkarisoside A has an inhibitory effect on osteoclastogenesis fromMed. 66, 575577.a new approach to cancer therapy. J. Clin. Invest. 115, 26252632.were analyzed by Immunoblot analysis using antibodies specically directed againstNFATc1 protein. Equal amounts of protein were loaded in each lane as demonstrated bythe level of -actin.Mino, T., Sugiyama, E., Taki, H., Kuroda, A., Yamashita, N., Maruyama, M., Kobayashi, M.,1998. Interleukin-1alpha and tumor necrosis factor alpha synergistically stimulateprostaglandin E2-dependent production of interleukin-11 in rheumatoid synovialbroblasts. 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Choi et al. / European Journal of Pharmacology 636 (2010) 2835Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and .....IntroductionMaterials and methodsMaterialsIsolation of Ikarisoside AIsolation of bone marrow-derived macrophages and co-cultureCell culture and treatmentTartrate-resistant acid phosphatase (TRAP) stainingRT-PCR analysisImmunoblot analysisCell transfection and luciferase reporter assayPit formation assayStatistics analysisResultsInhibitory effects on osteoclast differentiationEffects on regulation of osteoclastic genes in RANKL-stimulated RAW 264.7 cellsEffects on pit formation of RANKL-stimulated RAW 264.7 cellsEffect on NF-B activation in RANKL-stimulated RAW 264.7 cellsEffects on activation of MAPKs in RANKL-stimulated RAW 264.7 cellsEffects on expression of c-Fos in RANKL-stimulated RAW 264.7 cellsEffects on activation of NFATc1 in RANKL-stimulated RAW 264.7 cellsDiscussionAcknowledgementsReferences


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