Atorvastatin inhibits osteoclast differentiation by suppressing NF-κB and MAPK signaling during IL-1β-induced osteoclastogenesis

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1 ORIGINAL ARTICLE Koren J Intern Med 218;33: Atorvsttin inhiits osteoclst differentition y suppressing NF-κB nd MAPK signling during IL-1β-induced osteoclstogenesis Won-Seok Lee, Eun-Gyeong Lee, Myung-Soon Sung, Yun-Jung Choi, nd Wn-Hee Yoo Division of Rheumtology, Deprtment of Internl Medicine, Chonuk Ntionl University Medicl School, Reserch Institute of Clinicl Medicine of Chonuk Ntionl University- Biomedicl Reserch Institute of Chonuk Ntionl University Hospitl, Jeonju, Kore Received : July 28, 215 Revised : Octoer 12, 215 Accepted : Mrch 11, 216 Correspondence to Wn-Hee Yoo, M.D. Division of Rheumtology, Deprtment of Internl Medicine, Chonuk Ntionl University Medicl School, Reserch Institute of Clinicl Medicine of Chonuk Ntionl University-Biomedicl Reserch Institute of Chonuk Ntionl University Hospitl, 2 Geonji-ro, Deokjin-gu, Jeonju 5497, Kore Tel: Fx: E-mil: ywhim@jnu.c.kr Bckground/Aims: To define the effect of sttins on interleukin 1β (IL-1β)-induced osteoclstogenesis nd elucidte the underlying mechnisms. Methods: Bone mrrow cells were otined from 5-week-old mle ICR (Institute for Cncer Reserch) mice, nd they were cultured to differentite them into osteoclsts with mcrophge colony-stimulting fctor nd the receptor ctivtor of nucler fctor (NF)-κB lignd in the presence or sence of IL-1β or torvsttin. The formtion of osteoclsts ws evluted y trtrte-resistnt cid phosphtse (TRAP) stining nd resorption pit ssy with dentine slice. The moleculr mechnisms of the effects of torvsttin on osteoclstogenesis were investigted using reverse trnscription polymerse chin rection nd immunolotting for osteoclst specific molecules. Results: Atorvsttin significntly reduced the numer of TRAP-positive multinucleted cells s well s the one resorption re. Atorvsttin lso downregulted the expression of the NF of ctivted T-cell c1 messenger RNA nd inhiited the expression of osteoclst-specific genes. A possile underlying mechnism my e tht torvsttin suppresses the degrdtion of the inhiitors of NF-κB nd locks the ctivtion of the c-jun N-terminl kinse, extrcellulr signl-regulted kinse, nd p38; thus, implicting the NF-κB nd mitogen-ctivted protein kinses pthwy in this process. Conclusions: Atorvsttin is strong inhiitor of inflmmtion-induced osteoclstogenesis in inflmmtory joint diseses. Keywords: Atorvsttin; Joint diseses; Osteoprotegerin; Mice INTRODUCTION Bone erosion is centrl feture of rheumtoid rthritis (RA), nd it is ssocited with disese severity nd poor functionl outcome. Bone loss results from n imlnce in which one resorption y osteoclsts is fvored over one formtion y osteolsts [1]. A role for osteoclsts in one erosion in RA ws identified in multinucleted cells t sites of erosion in suchondrl one nd t the pnnus/one interfce in tissue smples from ptients with RA [2]. The pthogenesis of one erosion nd the role of osteoclsts in RA were gretly ugmented y the discovery of cytokine system of osteoclst differentition nd ctivtion. Osteotropic gents such s interleukin 1 (IL-1), IL-6, IL-11, IL-15, IL-17, tumor necrosis fctor α (TNF-α), prostglndin E2, nd the prthyroid hormone induce one loss y incresing osteoclst form- Copyright 218 The Koren Assocition of Internl Medicine This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( y-nc/4./) which permits unrestricted noncommercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. pissn eissn

2 The Koren Journl of Internl Medicine Vol. 33, No. 2, Mrch 218 tion [3,4]. A cell-surfce molecule, the receptor ctivtor of nucler fctor (NF)-κB lignd (RANKL), recruits TNF receptor-ssocited fctor (TRAF) proteins y inding to its receptor, the receptor ctivtor of NF-κB (RANK), nd it hs een found to e key fctor in the stimultion of osteoclst formtion [5,6]. TRAFs ctivte vrious signling pthwys, including c-src, PI3-kinse/Akr, nd mitogen-ctivted protein kinse (MAPK) [7]. RANKL ctivtes numerous trnscription fctors, including c-fos, NF of ctivted T-cell (NFAT) c1, PU1, nd microphthlmi-ssocited trnscription fctors, s these re essentil to osteoclst differentition [8]. c-fos is responsile for the expression of NF of ctivted T-cells (NFATc1) during RANKL-medited osteoclst differentition [9,1]. Atorvsttin is 3-hydroxy-3-methylglutryl coenzyme A (HMG-CoA) reductse inhiitor widely used s lipid-lowering gent. Inhiition of the enzyme results in reduced conversion of HMG-CoA to mevlonte nd susequent isoprenoid precursors, nd it minly reduces the cholesterol level in liver cells [11]. Reduced cholesterol vilility leds to incresed liver low density lipoprotein (LDL) receptor synthesis nd incresed removl of LDL from the loodstrem [12]. Severl studies hve shown tht sttins such s torvsttin my lso exert n immunomodultory effect y the inhiition of proinflmmtory cytokine production [13], s well s n inhiitory effect on T-cell recruitment nd T-cell stimultion [14,15]. Furthermore, torvsttin my potentilly hve eneficil effects on one metolism in ptients with hypercholesterolemi, mostly y reducing one resorption rther thn y stimulting one formtion [16]. Herein, we showed tht the inhiitory mechnism of torvsttin on IL-1β-induced, RANKL-medited osteoclst differentition vi the MAPK/NF-κB pthwy. METHODS Regents nd ntiodies Recominnt humn IL-1β ws purchsed from R&D System (Minnepolis, MN, USA). Atorvsttin ws otined from Axon MedChem (Groningen, the Netherlnd). Humn solule RANKL nd the mcrophge-colony stimulting fctor (M-CSF) were otined from PeproTech (Rocky Hill, NJ, USA). Monoclonl ntiodies (As) ginst extrcellulr signl-regulted kinse (ERK) 1/2, phosphte (p)-erk, c-jun N-terminl kinses (JNK), p-jnk, p38, p-p38, NF-κB (p65), inhiitor of NF-κBα (IκBα), nd β-ctin were purchsed from Cell Signling Technology (Dnvers, MA, USA). As ginst c-fos, NFATc1, nd trtrte-resistnt cid phosphtse (TRAP) were purchsed from Snt Cruz Biotechnology (Dlls, TX, USA). The got nti-rit immunogloulin G (IgG, ENZO Life Sciences, Frmingdle, NY, USA) nd rit nti-got horserdish peroxidse (HRP)-conjugte were purchsed from Bethyl Lortories (Montgomery, TX, USA). Osteoclst differentition Bone mrrow cells (BMCs) were collected from the tiis nd femurs with α-minimum essentil medi (α-mem, WelGENE, Gyeongsn, Kore) contining ntiiotics (Sigm-Aldrich, St. Louis, MO, USA) of 5-week-old mle Institute for Cncer Reserch (ICR) mice. The niml experiments were pproved y the Institutionl Animl Cre nd Use Committee of Chonuk Ntionl University (CNU), nd they were conducted in ccordnce with the guidelines of CNU. The BMCs were cultured in α-mem contining 1% fetl ovine serum (FBS), ntiiotics, nd M-CSF (1 ng/ml), nd then they were cultured for 1 dy. Nondherent cells were plted on 1- mm petri dishes nd were then cultured for 3 dys in the presence of M-CSF (3 ng/ml). Adherent cells were used s one mrrow-derived mcrophges (BMMs). The BMMs were cultured for 3 dys with M-CSF (3 ng/ ml) nd RANKL (1 ng/ml) to induce pre-fusion osteoclsts. To differentite osteoclsts from the BMMs, they were cultured for 4 dys with M-CSF (3 ng/ml) nd RANKL (5 ng/ml) in the presence or sence of IL-1β (1 ng/ml) or torvsttin (.5 µm). RNA isoltion nd reverse trnscription-polymerse chin rection Totl RNA ws isolted with TRIzol regent (Invitrogen, Crlsd, CA, USA) ccording to the mnufcturer s recommended protocol. The complement (c)-dna ws synthesized using Mxime RT Premix Kit (intron Biotechnology, Seongnm, Kore), nd then cdna ws mplified using Mxime polymerse chin rection (PCR) Premix Kit (intron Biotechnology). The primers used in the PCR were s follows: NFATc1 forwrd, 5 -tgtgcgccttccctg-3, reverse, 5 -tccccccgc

3 Lee WS, et l. Sttins nd osteoclstogenesis cgctc-3 ; c-fos forwrd, 5 -tcggggggggctgc-3, reverse, 5 -ggccggcggtccct-3 ; TRAP forwrd, 5 -gtgctggctggcctg-3, reverse, 5 -cccctcgcctgccc-3 ; E-cdherin forwrd, 5 -tgtgtggctcccccc-3, reverse, 5 -ctctccctggtgcccc-3 ; cthepsin K forwrd, 5 -cggtgggggcgggtc-3, reverse, 5 -gctttctcgttcccccgg-3 ; osteoclst-ssocited receptor (OSCAR) forwrd, 5 -ctttttctttctggccgcgt-3, reverse, 5 -ctggtggctccccctc-3 ; dendritic cell-specific trnsmemrne protein (DC-STAMP) forwrd, 5 -gcggcccgggtcg-3, reverse, 5 -cgttggcccggggg-3 ; c-src forwrd, 5 -ctggttcccggcgtc-3, reverse, 5 -ttctgttcgtgcccgcc-3 ; nd glycerldehyde 3-phosphte dehydrogense (GAP- DH) forwrd, 5 -tctctctccgccccttct-3, reverse, 5 -gcttcccgttcgctctgg-3. PCR products were electrophoresed on 1% grose gel stined with ethidium romide. Densitometric nlysis ws performed using FusionCpt Advnce softwre (Viler Lourmt, Eerhrdzell, Germny). Cell viility nlysis Cell viility ws determined using CCK-8 kit (Dojindo Lortories, Kummoto, Jpn) ccording to the mnufcturer s instructions. Briefly, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrzolium (CCK-8) ws reduced y dehydrogenses in cells to yield n ornge-colored product (formzn) [17]. The mount of formzn dye generted y dehydrogenses in the cells ws directly proportionl to the numer of living cells. The BMMs were plted on 96-well pltes with α-mem contining 1% FBS nd ntiiotics with M-CSF (3 ng/ml) nd RANKL (1 ng/ml) for 3 dys. The cells were wshed with complete α-mem medi nd were then incuted for 24 hours with IL-1β (1 ng/ml) or torvsttin (.5 µm) in the presence of M-CSF (3 ng/ml) nd RANKL (5 ng/ml). CCK-8 (1 µl/well) ws dded to ech well of the pltes, nd the cells were incuted for 3 hours. The sornce ws mesured t 45 nm using microplte reder. Western lot nlysis Cultured cells were lysed in the lysis uffer (5 mm Tris- HCl, 15 mm NACl, 5 mm ethylenediminetetrcetic cid, 1% Triton X-1, 1 mm sodium fluoride, 1 mm sodium vndte, nd 1% deoxycholte) contining protese inhiitors. The protein concentrtion ws determined using the Bio-Rd protein ssy regent (Bio-Rd Lortories, Hercules, CA, USA). Smples contining 3 μg of protein were oiled in sodium dodecyl sulfte (SDS) smple uffer t 95ºC for 5 minutes, seprted y 1% SDS-polycrylmide gel electrophoresis, nd trnsferred to polyvinylidene difluoride memrnes. Primry As (1 μg/ml) ginst NFATc1, c-fos, TRAP, ERK 1/2, p-erk, JNK, p-jnk, p38, p-p38 NF-κB (p65), IκBα, nd β-ctin were incuted overnight t 4ºC. Then the secondry HRP-conjugted A, which ws got nti-rit IgG or rit nti-got IgG, ws wshed three times for 3 minutes. Rective proteins were detected y enhnced chemiluminescence (Amershm Life Sciences, Arlington, IL, USA) using Fusion FX7 (Viler Lourmt). Cytochemicl ssessment of osteoclst formtion (TRAP stining) The BMMs were seeded t density of cells/ well in 96-well plte in triplicte for 3 dys. The cells were cultured for 4 dys with IL-1β (1 ng/ml) or torvsttin (.5 µm) in the presence of M-CSF (3 ng/ml) nd RANKL (5 ng/ml). After 4 dys, the cells were fixed with 3.7% formlin nd permeilized in.1% Triton X-1 for 1 minutes. The cells were stined with TRAP stin solution using leukocyte cid phosphtse kit (Sigm-Aldrich) ccording to the mnufcturer s instructions. TRAP positive cells contining three or more nuclei were counted s multinucler osteoclsts. Photogrphs were tken under light microscope (Crl Zeiss, Thornwood, NY, USA). Resorption pit ssy The formtion of osteoclsts ws evluted y the resorption pit re s follows. The BMMs (2 1 4 cells/well) were seeded in 96-well plte contining dentine slice 5 mm in dimeter (Immunodignostic Systems, Githersurg, MD, USA) with IL-1β (1 ng/ml) or torvsttin (.5 µm) in the presence of M-CSF (3 ng/ml) nd RANKL (5 ng/ml) for 3 weeks. Cells on the dentine slices were rued with cotton ud nd stined with hemtoxylin (Sigm-Aldrich) or TRAP. Quntifiction of the re of resorption ws conducted using the IMT i-solution softwre (Smwoo Scientific Co., Seoul, Kore). Sttisticl nlysis All dt re expressed s men ± stndrd devition

4 The Koren Journl of Internl Medicine Vol. 33, No. 2, Mrch 218 Atorvsttin.5 µm Atorvsttin IL-1β IL-1β Atorvsttin IL-1β + Atorvsttin Resorption pit TRAP(+) MNCs 15 A Atorvsttin.5 µm IL-1β + Atorvsttin Atorvsttin IL-1β + Atorvsttin Resorption pit re (%) TRAP(+) MNCs per well 2 IL-1β B IL-1β Atorvsttin IL-1β + Atorvsttin Figure 1. Inhiition of receptor ctivtor of nucler fctor κb lignd (RANKL)-medited, interleukin 1β (IL-1β)-induced trtrte-resistnt cid phosphtse (TRAP) (+) cell formtion y torvsttin. (A) Osteoclst precursors were cultured with mcrophge colony-stimulting fctor (3 ng/ml) nd RANKL (5 ng/ml) for 3 dys in the presence or sence of IL-1β (1 ng/ml) or torvsttin (.5 μm). The cells were stined with TRAP solution. Atorvsttin significntly decresed RANKL-medited or IL-1β stimulted formtion of TRAP (+) cells compred to the sence of torvsttin. (B) Atorvsttin (.5 μm) lso significntly decresed the RANKL-medited or IL-1β-stimulted formtion of resorption pits compred to the sence of torvsttin. The results re presented s men ± stndrd devition (n = 3). MNC, multinucleted cell. p <.5 vs. no IL-1β nd torvsttin, p <.5 vs. IL-1β without torvsttin. of triplicte mesurements. The men vlues of ech group were compred using Student t test or nlysis of vrince s pproprite, nd p <.5 ws considered sttisticlly significnce. Sttisticl nlyses were performed using SPSS version 16. (SPSS Inc., Chicgo, IL, USA). dictor of osteoclst formtion. IL-1β significntly incresed the resorption re compred to the sence of IL-1β (48.3 ± 4.5 vs. 8.8 ± 3.2, respectively; p <.5). Atorvsttin lso significntly decresed the RANKL-medited or IL-1β stimulted formtion of resorption pits compred to the sence of torvsttin (4.9 ± 3.5 vs ± 2.5, respectively; p <.5) (Fig. 1B). RESULTS Atorvsttin inhiits RANKL-medited or IL-β-stimulted survivl of osteoclst precursors Atorvsttin inhiits RANKL-medited or IL-β-stimulted osteoclst differentition To evlute the effects of torvsttin on the growth properties of the BMCs from the 5-week-old mle ICR mice, we mesured cell survivl with M-CSF (3 ng/ml) nd RANKL (1 ng/ml) in the presence or sence of torvsttin (.5 μm) or IL-1 (1 ng/ml) for 3 dys s previously descried. As shown in Fig. 2A, IL-1β significntly incresed the survivl of osteoclst precursor cells compred to the control without IL-1β (p <.5). Atorvsttin significntly inhiited the survivl of osteoclst precursor cells compred to IL-1β (p <.5). However, there ws no difference in the survivl of osteoclst precursor cells etween the control nd cultures with torvsttin. To determine the dose-dependent effects of torvsttin on IL-1β-induced survivl of osteoclst precursor cells, we dded vrious doses of torvsttin To determine the effects of torvsttin on RANKL-medited or IL-1β-stimulted osteoclstogenesis, we initilly exmined the formtion of TRAP (+) multinucler cells s n indictor of osteoclst differentition, which stisfied most of the morphologicl criteri of osteoclsts. IL-β significntly incresed the formtion of TRAP (+) cells compred to the sence of this cytokine (48.2 ± 4.3 vs ± 7.5, respectively; p <.5). Atorvsttin significntly decresed RANKL-medited or IL-1β-stimulted formtion of TRAP (+) cells compred to the sence of torvsttin (34.3 ± 4.5 vs ± 5.2, respectively; p <.5) (Fig. 1A). We lso mesured the resorption re s nother in

5 Lee WS, et l. Sttins nd osteoclstogenesis Survived cell quntity y CCK-8 Asornce t 45 nm A IL-1β Atorvsttin IL-1β + Atorvsttin IL-1 (1 ng/ml) B Atorvsttin Survived cell quntity y CCK-8 Asornce t 45 nm µm + 1 µm + 5 µm Figure 2. (A, B) Interleukin 1β (IL-1β) increses the trtrte-resistnt cid phosphtse (TRAP) (+) cell formtion nd torvsttin inhiits IL-1β-induced survivl of osteoclst precursors dose-dependently. The survivl of one mrrow cells is evluted with CCK-8 kit fter culture for 3 dys with/without IL-1β (1 ng/ml) nd torvsttin (.5 μm). Atorvsttin significntly inhiited IL-1β stimulted prolifertion of TRAP (+) multinucleted cells. The results re presented s men ± stndrd devition (n = 3). Atorvsttin lso inhiited IL-1β stimulted prolifertion of TRAP (+) cell dose-dependently. p <.5 vs. no IL-1β nd torvsttin, p <.5 vs. IL-1β without torvsttin. (.5, 1, nd 5 μm) to the osteoclst precursor cell cultures with IL-1β (1 ng/ml) for 3 dys nd performed CCK-8 ssy. The inhiitory effects of torvsttin were significntly enhnced s the concentrtion of torvsttin incresed (Fig. 2B). These effects were lso time dependent (dt not shown). Atorvsttin suppresses c-fos nd NFATc1 expression induced y RANKL or IL-1β Osteoclst differentition is regulted y the induction of vrious genes in response to RANKL nd other osteotropic gents, including IL-1β nd TNF-α. IL-1β is very importnt inflmmtory cytokine in RA nd cuses one loss y incresing osteoclst formtion. Both c-fos nd NFATc1 ply essentil roles in the differentition of osteoclst precursors [1]. Therefore, we exmined whether torvsttin regulted the expression of c-fos, NFATc1 messenger RNAs (m-rnas), nd proteins in response to RANKL or IL-1β. The densitometric vlue of reverse trnscription (RT)-PCR normlized y the GAPDH intensity showed tht torvsttin significntly decresed the RANKL-medited or IL-1β-stimulted expression of c-fos nd NFATc1 mrna (Fig. 3A). Consistent with the results of the RT-PCR nlyses, expression of c-fos nd NFATc1 protein levels incresed in response to RANKL nd IL-1β, nd the expression of c-fos nd NFATc1 ws significntly inhiited y torvsttin (Fig. 3B). Thus, torvsttin my inhiit osteoclst differentition y inhiiting c-fos nd NFATc1 expressions in response to RANKL nd IL-1β. Mny molecules re implicted in cell-cell fusion, including E-cdherin [18], DC-STAMP [19], nd Src fmily kinses [2]. Therefore, we exmined whether torvsttin regulted the expression of E-cdherin, DC-STAMP, nd c-src. The densitometric vlue of RT-PCR normlized y the GAPDH intensity showed tht torvsttin significntly decresed RANKL or IL-1β, s well s the expression of E-cdherin, DC-STAMP, nd c-src mrna (Fig. 4). Atorvsttin suppresses MAPK signl pthwys induced y RANKL or IL-1β To determine the involvement of signl trnsduction nd the mechnisms underlying the effect of torvsttin on IL-1β-stimulted, RANKL-medited osteoclst differentition, we evluted the ctivtion of MAPKs in the BMCs. The densitometric vlues on immunolots normlized ccording to β-ctin intensity showed tht IL-1β ctivted intrcellulr MAPKs, including ERK, p38, JNK (p <.5), nd torvsttin significntly inhiited RANKL-medited or IL-1β-stimulted ctivtion of intrcellulr MAPK, including ERK nd JNK, except p38 (p <.5) (Fig. 5). These results indicted tht torvsttin inhiits RANKL-medited or IL-1β-stimulted osteoclst differentition nd survivl vi intrcellulr MAPKs pthwys. DISCUSSION The modultion of one formtion nd inflmmtion re considered mong the vrious functions of sttins [21-23]. Numerous clinicl dt hve shown tht sttins

6 The Koren Journl of Internl Medicine Vol. 33, No. 2, Mrch 218 NFATc1 NFATc1 c-fos c-fos TRAP TRAP β-actin GAPDH IL-1β Atorvsttin IL-1β + IL-1β Atorvsttin IL-1β + Atorvsttin Atorvsttin Reltive expression of trget/β-ctin NFATc1 c-fos TRAP Reltive expression of trget/gapdh NFATc1 c-fos TRAP A IL-1β Atorvsttin IL-1β + Atorvsttin B IL-1β Atorvsttin IL-1β + Atorvsttin Figure 3. Effects of torvsttin on interleukin 1β (IL-1β)-stimulted, receptor ctivtor of nucler fctor-κb lignd-medited nucler fctor of ctivted T-cells (NFATc1), c-fos, nd trtrte-resistnt cid phosphtse (TRAP) ctivtion. To evlute the expression of NFATc1, c-fos, nd TRAP messenger RNA (mrna) nd protein, we performed immunolotting nd reverse trnscription polymerse chin rection. IL-1β enhnced the expression of NFATc1, c-fos, nd TRAP compred to the results without IL-1β. Atorvsttin (.5 μm) inhiited the IL-1β stimulted expression of NFATc1, c-fos, TRAP protein (A), nd mrna (B). The results re presented s men ± stndrd devition (n = 3). GAPDH, glycerldehyde 3-phosphte dehydrogense. p <.5 vs. no IL-1β nd torvsttin, p <.5 vs. IL-1β without torvsttin. E-cdherin Cthepsin K OSCAR DC-STAMP c-src Reltive expression of trget/gapdh E-cdherin Cthepsin K OSCAR DC-STAMP c-src GAPDH A IL-1β Atorvsttin IL-1β + Atorvsttin IL-1β Atorvsttin IL-1β + Atorvsttin Figure 4. (A, B) Effects of torvsttin on interleukin 1β (IL-1β)-stimulted, receptor ctivtor of nucler fctor κb lignd-medited dendritic cell-specific trnsmemrne protein (DC-STAMP), cthepsin K, osteoclst-ssocited receptor (OSCAR), DC-STAMP, nd c-src expression. IL-1β enhnces the expression of E-cdherin, cthepsin K, OSCAR, DC-STAMP, nd c-src compred to the results without IL-1β. Atorvsttin (.5 μm) inhiits the IL-1β-stimulted expression of E-cdherin, cthepsin K, OSCAR, DC-STAMP, nd c-src. The results re presented s men ± stndrd devition (n = 3). GAPDH, glycerldehyde 3-phosphte dehydrogense. p <.5 vs. no IL-1β nd torvsttin, p <.5 vs. IL-1β without torvsttin. B modulted utoimmune disese ctivity or indeed modified vsculr risk fctors in rheumtic disese [24-26]. McCrey et l. [26] showed mrked suppression with torvsttin of cute-phse vriles nd significnt reduction in the swollen joint count in ptients with RA presenting with ctive disese, despite existing dis

7 Lee WS, et l. Sttins nd osteoclstogenesis p-erk NF-κB ERK IκB p-p38 β-actin p38 IL-1β Atorvsttin IL-1β + Atorvsttin p-jnk JNK IL-1β Atorvsttin IL-1β + Atorvsttin Reltive expression of trget/unrective signl p-erk/erk p-p38/p38 p-jnk/jnk Reltive expression of trget/β-ctin NF-κB IκB A IL-1β Atorvsttin IL-1β + Atorvsttin IL-1β Atorvsttin IL-1β + Atorvsttin Figure 5. Effects of torvsttin on interleukin 1β (IL-1β)-stimulted, receptor ctivtor of nucler fctor-κb (NF-κB) lignd-medited mitogen-ctivted protein kinse (MAPK) signl pthwys nd NF-κB ctivtion. To evlute the mechnism of torvsttin on IL-1β-induced osteoclst differentition, we evluted the MAPK signling pthwy nd NF-κB ctivtion y immunolotting. (A) IL-1β enhnced the phosphoryltion of extrcellulr signl-regulted kinse (ERK), p38, nd c-jun N-terminl kinses (JNK), nd torvsttin inhiited the IL-1β-simulted ctivtion of ERK, p38, nd JNK. (B) IL-1β ctivted NF-κB nd decresed cytoplsmic inhiitor of NF-κBα (IκB), nd torvsttin inhiited the IL-1β-stimulted ctivtion of NFκB. The results re presented s men ± stndrd devition (n = 3). p <.5 vs. no IL-1β nd torvsttin, p <.5 vs. IL-1β without torvsttin. B ese-modifying ntirheumtic drug therpy. There re mny experimentl studies on the effect of torvsttin on osteolsts, wheres there re only few out the effect of torvsttin on osteoclsts. In recent study, torvsttin mrkedly reduced RANKL expression in the firolst-like synoviocytes (FLSs) of ptients with RA, nd torvsttin inhiited osteoclstogenesis in FLSs. Atorvsttin inhiits osteoclstogenesis nd one destruction in ptients with RA [27]. A recent study suggested n inhitnt effect of torvsttin on osteoclsts. Susequently, no studies hve reported on this effect. Therefore, our reserch tem ttempted to directly differentite osteoclsts from mouse nd oserve the effect. The present study findings showed tht torvsttin inhiits IL-1β-stimulted, RANKL-medited osteoclst differentition in dose-dependent mnner y inhiiting ctivtion of the ERK nd p-jnk kinse s well s inhiiting c-fos nd NFATc1 expression. These findings suggest tht torvsttin hs n importnt inhiitory role in one loss y preventing osteoclst formtion, nd it my e useful s new preventive gent for mnging one destruction in inflmmtory diseses such s RA. IL-1 is produced y mny cell types relevnt in rheumtoid synovitis nd is redily detected in the rheumtoid joint, oth in the synovil fluid (IL-1β) nd within cells (IL-1α > IL-1β) [28]. IL-1β is criticl in the pthogenesis of inflmmtory synovitis nd joint destruction in RA, s it induces the differentition nd ctivtion of osteoclsts [29]. IL-1 cn induce nd enhnce TRAF ctivtion vi heterodimeric IL-1 receptors. Thus, IL-1 differentites nd ctivtes osteoclsts y engging nd ctivting TRAF6 vi MAPKs, c-fos, nd NFATc1 sig

8 The Koren Journl of Internl Medicine Vol. 33, No. 2, Mrch 218 nling pthwys [3,7]. However, IL-1 lone is unle to stimulte NFATc1 expression, ut it cn do so in the presence of permissive level of RANKL [3]. RANKL plys crucil role in IL-1 in terms of inducing the expression of NFATc1, which in turn coopertes with other trnscriptionl fctors to simulte gene expression nd thus osteoclstogenesis in osteoclst precursors. We found tht torvsttin inhiits osteoclst differentition in dose-dependent mnner in IL-1β-stimulted, RANKL-medited conditions. This suggests tht torvsttin my e potent therpeutic compound for RA. However, further studies re required to investigte the overll effects nd underlying mechnisms of torvsttin on the pthophysiology of osteoclstogenesis in in vivo systems such s niml models of RA, including collgen-induced rthritis. RANKL-RANK interction ctivtes mny trnscription fctors, including NFATc1, clcineurin nd clcium-regulted trnscription fctor. RANKL induced the expression of c-fos during erly osteoclstogenesis. The inding of c-fos to the NFATc1 promoter region is incresed y RANKL, which induces NFATc1 gene expression [1,31]. The presence of NFATc1 in precursor cells prompts them to undergo osteoclstogenesis in the sence of RANKL [1]. In this study, to elucidte the mechnisms ehind the effects of torvsttin on IL-1βinduced, RANKL-medited osteoclst differentition, the expression of c-fos nd NFATc1 were exmined. We found tht the inhiition of osteoclst differentition y torvsttin coincided with the regultion of c-fos nd NFATc1. Furthermore, NFATc1 induced the expression of osteoclst-specific genes such s TRAP, cthepsin K, nd OSCAR [9,32]. However, we found tht torvsttin synergisticlly reduced the expression of TRAP, cthepsin K, nd OSCAR y inhiiting NFATc1. The inding of RANKL to RANK lso recruits TRAF- 6 to ctivte ll MAPK pthwys, nmely ERK, JNK, nd p38 s well s phosphtidylinositol 3-kinse [33]. TRAF- 6 / mice re severely osteopetrotic either with undnt, dysfunctionl osteoclsts [34] or without osteoclsts [35]. In the present study, to elucidte the mechnisms ehind the effects of torvsttin on IL-1β-induced, RANKL-medited osteoclst differentition, the expression of ERK, p38 MAPKs, nd JNK were exmined. We found tht the inhiition of osteoclst differentition y torvsttin coincided with the regultion of ERK nd p-jnk. However, further studies re required to define the precise mechnisms of these effects on osteoclst differentition in the sence or presence of stimulting fctors. Further investigtions re lso required to elucidte the mechnisms y which intrcellulr signling fctors re more specificlly or directly involved in the effects of torvsttin on osteoclst differentition. Much progress hs een mde in recent yers for understnding osteoclst fusion nd its regultion. DC-STAMP is significnt fusion regultor [19], nd its expression is regulted y direct nd indirect mechnisms [36]. Regrding the direct mechnism, RANKL/ RANK downstrem trnscription fctors, which re importnt for RANKL-medited osteoclst formtion, re criticl for osteoclst fusion, nd this includes c-fos nd NFATc1. The fct tht torvsttin down-regulted the fusion of osteoclst precursors led us to hypothesize tht torvsttin my control the expression of DC- STAMP. However, further investigtions re required to define severl importnt fusogenic genes, including the osteoclst stimultory trnsmemrne protein [37], CD9 [38], ATP6vd2 [39], nd dose-dependent effect of torvsttin on osteoclst fusion. In conclusion, the findings presented herein demonstrted tht torvsttin inhiits IL-1β-stimulted, RANKLmedited osteoclst differentition. This study lso showed tht torvsttin inhiits the phosphoryltion of MAPK pthwys nd the ctivtion of c-fos nd NFATc1. These findings need to e vlidted in in vivo models prior to their extrpoltion to the pthophysiologicl condition, nd further evlutions re needed to define the exct mechnisms of torvsttin s intrcellulr signls on these effects. However, etter insight into the mechnisms of torvsttin regrding osteoclst differentition my provide new therpeutic trget for preventing rticulr one nd crtilge destruction in inflmmtory joint diseses, including RA. KEY MESSAGE 1. Atorvsttin hs n importnt inhiitory role in one loss y preventing osteoclst formtion. 2. Atorvsttin my e useful s new preventive gent for mnging one destruction in inflmmtory diseses such s rheumtoid rthritis

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