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• W1998128158 abstract "To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-κB ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-κB, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-κB and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing IκB phosphorylation and thus NF-κB activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways. To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-κB ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-κB, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-κB and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing IκB phosphorylation and thus NF-κB activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways. Bone-resorbing osteoclasts, cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage lineage, play an essential role in regulating bone morphogenesis and remodeling. It is now clear that two molecules produced by bone marrow mensenchymal cells are required for osteoclastogenesis, macrophage colony-stimulating factor (M-CSF) 1M-CSFmacrophage colony-stimulating factorRANKreceptor for activation of nuclear factor κBNF-κBnuclear factor κBRANKLRANK ligandTNFtumor necrosis factorIL-4interleukin-4IL-1interleukin-1OCosteoclastSTATsignal transducer and activator of transcriptionJNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinaseTRAFTNF receptor-associated factorIKKIκB kinaseTRAPtartrate-resistant acid phosphataseICAMintracellular adhesion moleculeTLRToll-like receptorMAPmitogen-activated proteinMKKMAP kinase kinaseRTreverse transcriptionEMSAelectrophoretic mobility shift assaySHIPSH2-containing inositol phosphatasePPARγ1peroxisome proliferator-activated receptor γ1 1M-CSFmacrophage colony-stimulating factorRANKreceptor for activation of nuclear factor κBNF-κBnuclear factor κBRANKLRANK ligandTNFtumor necrosis factorIL-4interleukin-4IL-1interleukin-1OCosteoclastSTATsignal transducer and activator of transcriptionJNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinaseTRAFTNF receptor-associated factorIKKIκB kinaseTRAPtartrate-resistant acid phosphataseICAMintracellular adhesion moleculeTLRToll-like receptorMAPmitogen-activated proteinMKKMAP kinase kinaseRTreverse transcriptionEMSAelectrophoretic mobility shift assaySHIPSH2-containing inositol phosphatasePPARγ1peroxisome proliferator-activated receptor γ1 and receptor for activation of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL) (1Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (2967) Google Scholar, 2Ross F.P. J. Clin. Invest. 2000; 105: 555-558Crossref PubMed Scopus (61) Google Scholar). M-CSF binding to its receptor, c-Fms, on osteoclast precursors provides signals required for their survival and proliferation. RANKL, a member of the tumor necrosis factor (TNF) cytokine superfamily, exists in both soluble and membrane-bound forms, and its function is critical to the differentiation and fusion of precursors into mature osteoclasts (3Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4546) Google Scholar, 4Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3500) Google Scholar, 5Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar, 6Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1905) Google Scholar, 7Lam J. Takeshita S. Barker J.E. Kanagawa O. Ross F.P. Teitelbaum S.L. J. Clin. Invest. 2000; 106: 1481-1488Crossref PubMed Scopus (1081) Google Scholar). TNFα and IL-1 are two other cytokines that impact osteoclastogenesis while being incapable of replacing either of the its two key regulators, M-CSF and RANKL. Recent gene-targeting studies show that the commitment of mononuclear precursors to mature osteoclasts involves transcription factors such as AP-1, NF-κB, and p38α (8Grigoriadis A.E. Wang Z.-Q. Cecchini M.G. Hofstetter W. Felix R. Fleisch H.A. Wagner E.F. Science. 1994; 266: 443-448Crossref PubMed Scopus (1056) Google Scholar, 9Iotsova V. Caamano J. Loy J. Yang Y. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (862) Google Scholar, 10Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. 1997; 11: 3482-3496Crossref PubMed Scopus (853) Google Scholar, 11Matsumoto M. Sudo T. Maruyama M. Osada H. Tsujimoto M. FEBS Lett. 2000; 486: 23-28Crossref PubMed Scopus (74) Google Scholar). macrophage colony-stimulating factor receptor for activation of nuclear factor κB nuclear factor κB RANK ligand tumor necrosis factor interleukin-4 interleukin-1 osteoclast signal transducer and activator of transcription c-Jun N-terminal kinase extracellular signal-regulated kinase TNF receptor-associated factor IκB kinase tartrate-resistant acid phosphatase intracellular adhesion molecule Toll-like receptor mitogen-activated protein MAP kinase kinase reverse transcription electrophoretic mobility shift assay SH2-containing inositol phosphatase peroxisome proliferator-activated receptor γ1 macrophage colony-stimulating factor receptor for activation of nuclear factor κB nuclear factor κB RANK ligand tumor necrosis factor interleukin-4 interleukin-1 osteoclast signal transducer and activator of transcription c-Jun N-terminal kinase extracellular signal-regulated kinase TNF receptor-associated factor IκB kinase tartrate-resistant acid phosphatase intracellular adhesion molecule Toll-like receptor mitogen-activated protein MAP kinase kinase reverse transcription electrophoretic mobility shift assay SH2-containing inositol phosphatase peroxisome proliferator-activated receptor γ1 Interleukin-4 (IL-4) is an immunoregulatory protein produced mainly by activated T lymphocytes. This Th2 cytokine functions as a growth and/or differentiation factor for a wide variety of bone marrow-derived hematopoietic progenitor cells (12Nelms K. Keegan A.D. Zamorano J. Ryan J.J. Paul W.E. Annu. Rev. Immunol. 1999; 17: 701-738Crossref PubMed Scopus (1269) Google Scholar). The hematopoietic origin of osteoclasts has led to the hypothesis that IL-4 may play an important role in the regulation of bone metabolism. Indeed, IL-4 inhibits the in vitro bone resorption stimulated by various agents, and the inhibitory effect of IL-4 on bone resorption is abolished by a monoclonal anti-IL-4 antibody (13Watanabe K. Tanaka Y. Morimoto I. Yahata K. Zeki K. Fujihira T. Yamashita U. Eto S. Biochem. Biophys. Res. Commun. 1990; 172: 1035-1041Crossref PubMed Scopus (96) Google Scholar). IL-4 also inhibits spontaneous and parathyroid hormone-related protein-stimulated osteoclast formation and modulates development of humoral hypercalcemia malignancy in intact mice (14Nakano Y. Watanabe K. Morimoto I. Okada Y. Ura K. Sato K. Kasono K. Nakamura T. Eto S. J. Bone Miner. Res. 1994; 9: 1533-1539Crossref PubMed Scopus (47) Google Scholar). Furthermore, IL-4 inhibits in vitro osteoclast formation by affecting the commitment of its precursors to the osteoclast (15Lacey D.L. Erdmann J.M. Teitelbaum S.L. Tan H.-L. Ohara J. Shioi A. Endocrinology. 1995; 136: 2367-2376Crossref PubMed Scopus (67) Google Scholar). Although these observations establish IL-4 as a potent anti-bone resorption factor, the mechanism(s) by which IL-4 inhibits osteoclastogenesis remain poorly understood. The availability of RANKL permits generation of both human and murine osteoclasts from purified myeloid precursors without stromal cell/osteoblast co-culture (3Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4546) Google Scholar, 4Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3500) Google Scholar, 16Quinn J.M. Elliott J. Gillespie M.T. Martin T.J. Endocrinology. 1998; 139: 4424-4427Crossref PubMed Google Scholar). The present study was designed to investigate the molecular mechanism(s) mediating IL-4 inhibition of osteoclastogenesis. We find that IL-4 inhibits formation of osteoclasts (OCs) when murine OC precursors, namely bone marrow macrophage (BMMs), are treated with M-CSF and RANKL. The cytokine arrests multiple aspects of RANKL-induced signaling including the following: 1) preventing IκBα phosphorylation and thus NF-κB activation, and 2) blockade of the JNK, p38, and ERK MAP kinase pathways. In addition, IL-4 selectively inhibits TNF signaling and enhances that induced by IL-1 in BMMs. These inhibitory events of IL-4 on RANK signaling are reversible and require signal transducer and activator of transcription-6 (STAT6). In contrast, exposure of BMMs to IL-4 fails to impact those components of M-CSF-induced intracellular signaling relating to osteoclastogenesis, namely activation of the MAP kinase and Akt pathways. Polyclonal anti-IκBα, polyclonal anti-RANK, and polyclonal anti-TRAF6 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-IKKα and monoclonal anti-IKKβ antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). All other antibodies and MEK inhibitors were from Cell Signaling Technology (Beverly, MA). Protease inhibitor mixtures were from Calbiochem. The bicinchoninic acid kit for protein determination and enhanced chemiluminescence kits were obtained from Pierce Endogen (Rockford, IL). Recombinant murine M-CSF, IL-4, TNFα, and IL-1α were from R & D (Minneapolis, MN). Murine RANKL was expressed in our laboratory as described previously (17McHugh K.P. Hodivala-Dilke K. Zheng M.H. Namba N. Lam J. Novack D. Feng X. Ross F.P. Hynes R.O. Teitelbaum S.L. J. Clin. Invest. 2000; 105: 433-440Crossref PubMed Scopus (567) Google Scholar). All other chemicals were obtained from Sigma. Wild type C57BL/6 were purchased from Harlan Industries (Indianapolis, IN). STAT6- and SHIP1-deficient mice were kindly provided by Dr. James Ihle (St. Jude Children's Research Hospital, Memphis, TN) and Dr. Keith Humphries (British Columbia Cancer Agency, Vancouver, British Columbia, Canada), respectively. BMMs were isolated from whole bone marrow of 4–6-week-old mice and incubated in tissue culture dishes at 37 °C in 5% CO2. After 24 h in culture, the non-adherent cells were collected and layered on a Ficoll-Paque gradient, and the cells at the gradient interface were collected. The cells were replated at 65,000/cm2 in α-minimal essential medium and supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2in the presence of recombinant mouse M-CSF (10 ng/ml). Cells were stimulated with cytokines (M-CSF, RANKL, TNF, or IL-1) on day 4 or 5. In some experiments (for MEK1/2, MKK3/6, Akt activation, and M-CSF signaling), cells were cultured in serum and M-CSF-free medium for 24 h before stimulation. BMMs were cultured in 48- or 96-well cell culture dishes in the presence of M-CSF (10 ng/ml) and RANKL (40 ng/ml) and other cytokines as appropriate, and medium was changed on day 3. Osteoclast-like cells were characterized by staining for tartrate-resistant acid phosphatase (TRAP). The numbers of osteoclasts were assessed by TRAP solution assay as described previously (18Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Boyle W.J. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4261) Google Scholar). Cytokine-treated or control monolayers of BMMs were washed twice with ice-cold phosphate-buffered saline. Cells were lysed in the buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm Na3VO4, 1 mm NaF, and 1× protease inhibitor mixture. Fifty μg of cell lysates were boiled in the presence of SDS sample buffer (0.5m Tris-HCl, pH 6.8, 10% (w/v) SDS, 10% glycerol, 0.05% (w/v) bromphenol blue) for 5 min and subjected to electrophoresis on 8% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad, Hercules, CA) and incubated in blocking solution (5% non-fat dry milk in TBS containing 0.1% Tween 20) for 1 h to reduce nonspecific binding. Membranes were then exposed to primary antibodies overnight at 4 °C, washed three times, and incubated with secondary goat anti-mouse or rabbit IgG horseradish peroxidase-conjugated antibody for 1 h. Membranes were washed extensively, and enhanced chemiluminescence detection assay was performed following the manufacturer's directions. EMSA was performed as described previously (19Wei S. Wang M.W.-H. Teitelbaum S.L. Ross F.P. Endocrinology. 2001; 142: 1290-1295Crossref PubMed Scopus (95) Google Scholar). In brief, cells were lifted from the dish after treatment with 5 mm EDTA and 5 mm EGTA in phosphate-buffered saline, resuspended in hypotonic lysis buffer A (10 mm HEPES, pH 7.8, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 5 μg/ml leupeptin), and incubated on ice for 15 min, and then Nonidet P-40 was added to a final concentration of 6.4%. Nuclei were pelleted and resuspended in nuclear extraction buffer B (20 mm HEPES, pH 7.8, 420 mm NaCl, 1.2 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm dithiothreitol, 0.5 mm 4-(2-aminoethyl) benzenesulfonyl fluoride, 5 μg/ml pepstatin A, 5 μg/ml leupeptin) and rotated for 30 min at 4 °C. The samples were then centrifuged, and nuclear proteins in the supernatant were transferred to fresh tubes. Nuclear extracts (3 μg) were incubated with an end-labeled double-stranded oligonucleotide probe containing the sequence 5′-AAACAGGGGGCTTTCCCTCCTC-3′ derived from the κB3 site of the TNF promoter (20Drouet C. Shakhov A.N. Jongeneel C.V. J. Immunol. 1991; 147: 1694-1700PubMed Google Scholar). The reaction was performed in a total of 20 μl of binding buffer (20 mm HEPES, pH 7.8, 100 mm NaCl, 0.5 mm dithiothreitol, 1 μl of poly(dI-dC), and 10% glycerol) for 20 min at room temperature. The samples were fractionated on a 4–20% TBE gel (NOVEX, San Diego, CA) and visualized by exposing dried gel to film. RNA was isolated using RNeasy kits (Qiagen, Inc., Valencia, CA) and reverse-transcribed using 1 μg of total RNA, 0.5 μg of (dT)15 primer, 20 units of RNasin RNase inhibitor, 20 units of avian myeloblastosis virus-reverse transcriptase, 1× avian myeloblastosis virus-RT buffer (Promega Corp., Madison, WI), and 1 mm dNTP mix (Invitrogen) in a final volume of 20 μl. The reaction was carried out for 15 min at 42 °C, followed by 5 min at 99 °C, and then cooled to 4 °C. The RT reaction products were further diluted to 100 μl, and 1 μl of each was applied to a 49-μl volume PCR premix, containing 1 pmol of each primer, 0.2 mm dNTP mix, 5 units of Taqpolymerase, 2 mm MgCl2, and 1× PCR buffer (Invitrogen). PCR was carried out in a PCR Express thermal cycler (HYBAID, Franklin, MA) set for various cycles to monitor the linearity of the amplification, with annealing temperature at 60 °C. The oligonucleotide primers used are as follows: 1) IκBα, (5′) 5′-GCCTGGACTCCATGAAAGAC-3′ and (3′) 5′-CAAGTGGAGTGGAGTCTGCTGCAGGTTGTT-3′; 2) ICAM-1, (5′) 5′-CGGCTGGACGAGACGGACTG-3′ and (3′) 5′-GACGCTGCCATCACGAGGCC-3′; 3) TLR-2, (5′) 5′-GGAGACTCTGGAAGCAGGCG-3′ and (3′) 5′-GGCTTCCTCTTGGCCTGGAG-3′; and 4) α-actin, (5′) 5′-CATCGTGGGCCGCTCTAGGCACCA-3′ and (3′) 5′-CGGTTGGCCTTAGGGTTCAGGGGG-3′. We and others (15Lacey D.L. Erdmann J.M. Teitelbaum S.L. Tan H.-L. Ohara J. Shioi A. Endocrinology. 1995; 136: 2367-2376Crossref PubMed Scopus (67) Google Scholar, 21Shioi A. Teitelbaum S.L. Ross F.P. Welgus H.G. Ohara J. Suzuki H. Lacey D.L. J. Cell. Biochem. 1991; 47: 1-6Crossref PubMed Scopus (76) Google Scholar) have reported that IL-4 inhibits osteoclast formation when whole marrow is cultured with 1,25(OH)2D3 and dexamethasone. Thus, we first determined whether IL-4 inhibits osteoclastogenesis induced by M-CSF and RANKL. As seen in Fig. 1, IL-4 inhibits formation of TRAP-positive multinucleated OCs when BMMs are exposed to optimal concentrations of M-CSF and RANKL. The effect occurs at the lowest dose of the cytokine used in these studies, indicating the potent nature of the inhibition. To determine at which stage IL-4 inhibits osteoclastogenesis, IL-4 was added to OC-generating cultures on days 0–4, and TRAP staining was performed on day 5. We find that the cytokine is effective only when present during the first 2 days of culture, suggesting it targets early OC precursors (Fig.2). To determine whether IL-4 inhibition of osteoclastogenesis is reversible, the cytokine was added at the beginning of OC-generating culture and withdrawn on day 3 or day 5 by medium change. Withdrawal of IL-4, up to 5 days after its addition, restores OC differentiation. Thus, inhibition of OC formation by IL-4 is reversible (Fig.3). To determine whether IL-4 affects M-CSF signaling, we examined M-CSF-induced intracellular signaling. Although M-CSF does not activate JNK1, as reported previously (22Jaworowski A. Wilson N.J. Christy E. Byrne R. Hamilton J.A. J. Biol. Chem. 1999; 274: 15127-15133Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), it activates JNK2, ERK1/2, and Akt, but not p38 or NF-κB (Fig. 4). Exposure of cells to IL-4 fails to impact these aspects of M-CSF-induced signaling (Fig. 4). Since generation of osteoclasts from myeloid precursors requires the activity of two cytokines, M-CSF and RANKL (1Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (2967) Google Scholar, 2Ross F.P. J. Clin. Invest. 2000; 105: 555-558Crossref PubMed Scopus (61) Google Scholar), the mechanism by which the anti-inflammatory cytokine decreases osteoclastogenesis is by blunting RANKL-stimulated signaling. Activation of RANKL signaling involves binding of the cytokine to its unique receptor RANK, a member of the TNF receptor superfamily. To exclude the possibility that IL-4 impairs RANKL signaling by decreasing expression of RANK and/or TRAF6, a member of the TRAF superfamily that binds to RANK (23Wong B.R. Besser D. Kim N. Arron J.R. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) and is critical for osteoclastogenesis (24Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Crossref PubMed Scopus (533) Google Scholar, 25Kobayashi N. Kadono Y. Naito A. Matsumoto K. Yamamoto T. Tanaka S. Inoue J. EMBO J. 2001; 20: 1271-1280Crossref PubMed Scopus (367) Google Scholar), we performed Western blot analysis of BMMs exposed to IL-4 for 3 days. As can be seen in Fig.5, levels of both the receptor and its immediate effector are unchanged with IL-4 treatment. RANKL, the key osteoclastogenic cytokine upon binding its unique receptor, RANK, activates three major intracellular signals. These signals are as follows: 1) the NF-κB pathway; 2) all three MAPK members, namely ERKs (p42/p44), SAPK/JNK, and p38 and; and 3) the phosphatidylinositol 3-kinase/Akt axis (1Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (2967) Google Scholar, 2Ross F.P. J. Clin. Invest. 2000; 105: 555-558Crossref PubMed Scopus (61) Google Scholar). To define the molecular mechanism(s) by which IL-4 inhibits osteoclastogenesis, we examined each of the major RANKL-induced signaling pathways. Our findings confirm prior reports (19Wei S. Wang M.W.-H. Teitelbaum S.L. Ross F.P. Endocrinology. 2001; 142: 1290-1295Crossref PubMed Scopus (95) Google Scholar, 25Kobayashi N. Kadono Y. Naito A. Matsumoto K. Yamamoto T. Tanaka S. Inoue J. EMBO J. 2001; 20: 1271-1280Crossref PubMed Scopus (367) Google Scholar, 26Matsumoto M. Sudo T. Saito T. Osada A. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) that RANKL induces phosphorylation in BMMs of JNK, p38, ERK1/2, and IκBα, the latter prompting NF-κB translocation to the nucleus where it acts as a transcription factor. While pretreatment of IL-4 for 1 h does not significantly affect any RANKL-induced signals, exposure to the cytokine for 24 h dampens phosphorylation of IκBα, thus decreasing degradation of this protein (Fig. 6 A). Furthermore, the extent of inhibition is greater when cells are exposed for 3 days (Fig. 6 B). Documenting that IL-4 inhibition of osteoclast formation is reversible, 24 h after withdrawal of the cytokine, after 3 days of exposure, completely normalizes RANKL-induced IκBα phosphorylation. Cytokine-induced phosphorylation of IκBα is mediated by a large complex in which the active components are IKKα and IKKβ (27Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4024) Google Scholar). To exclude the possibility that IL-4 decreases levels of IKKα and/or IKKβ, lysates from RANKL-treated BMMs pre-exposed to vehicle or IL-4 were subjected to Western blot analysis with IKK-specific antibodies. As can be seen in Fig. 6 C, levels of neither IKKα nor and IKKβ are altered by IL-4. The fact that IL-4 pretreatment of BMMs decreases RANKL-stimulated phosphorylation of IκBα should dampen nuclear translocation of NF-κB (27Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4024) Google Scholar). To confirm this, we performed EMSA for NF-κB by using nuclear extracts from BMMs pretreated with IL-4 or vehicle and then exposed to RANKL. We find a 70% decrease in binding of nuclear levels NF-κB to a consensus oligonucleotide (Fig.7 A), which should diminish transcription of NF-κB-dependent genes. To test this hypothesis we used RT-PCR to measure mRNA levels of three NF-κB-dependent early response genes. Consistent with our prediction, IL-4 pretreatment decreases both basal and RANKL-stimulated mRNA levels of IκBα, ICAM-1, and TLR-2 (Fig. 7 B), all of which are transcriptionally activated by NF-κB (28Ito C.Y. Kazantsev A.G. Baldwin A.S., Jr. Nucleic Acids Res. 1994; 22: 3787-3792Crossref PubMed Scopus (208) Google Scholar, 29Roebuck K.A. Finnegan A. J. Leukocyte Biol. 1999; 66: 876-888Crossref PubMed Scopus (462) Google Scholar, 30Musikacharoen T. Matsuguchi T. Kikuchi T. Yoshikai Y. J. Immunol. 2001; 166: 4516-4524Crossref PubMed Scopus (108) Google Scholar). Pretreatment with IL-4 for 24 h also partially inhibits p38 activation, without significantly altering that of JNK or ERKs (Fig.8 A). Longer exposure to the cytokine (3 days) almost entirely blocks activation of JNK, p38, and ERK. Once again the alterations in MAP kinase activation, which mirror the effect of IL-4 on osteoclastogenesis, are reversed by withdrawal of the cytokine for 24 h (Fig. 8 B). Turning to effectors of the SAPK/JNK ERK and p38 pathways, IL-4 decreases RANKL-stimulated activation of SEK1/MKK4, which is upstream of JNK, MEK1/2, the kinases responsible for ERK activation, and MKK3/6, which enhance p38 activity (Fig. 8 C). RANKL blocks apoptosis by phosphorylating Akt/protein kinase B on serine 473 and threonine 308 through a signaling complex containing c-Src and TRAF6 (23Wong B.R. Besser D. Kim N. Arron J.R. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). To determine whether IL-4 alters RANKL-induced activation of the Akt pathway, we examined phosphorylation of Akt using antibodies specific for phosphothreonine 308. As seen in Fig.9, IL-4 alone stimulates Akt phosphorylation but does not alter subsequent RANKL-induced Akt activation in BMMs. We next asked whether the cytokine alters the signaling pathways induced by TNF, a molecule that synergizes with RANKL in stimulating osteoclastogenesis (7Lam J. Takeshita S. Barker J.E. Kanagawa O. Ross F.P. Teitelbaum S.L. J. Clin. Invest. 2000; 106: 1481-1488Crossref PubMed Scopus (1081) Google Scholar), and IL-1, an inflammatory cytokine that induces mature osteoclast activation and, like RANKL, also signals via TRAF6 (31Jimi E. Nakamura I. Duong L.T. Ikebe T. Takahashi N. Rodan G.A. Suda T. Exp. Cell Res. 1999; 247: 84-93Crossref PubMed Scopus (297) Google Scholar, 32Jimi E. Nakamura I. Ikebe T. Akiyama S. Takahashi N. Suda T. J. Biol. Chem. 1998; 273: 8799-8805Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 33Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1103) Google Scholar). We find that IL-4 selectively inhibits TNF-induced signaling, because it decreases JNK, p38, and Akt activation but fails to inhibit that of ERKs and NF-κB (Fig.10 A). Interestingly, exposure of BMMs to IL-4 enhances IL-1-induced NF-κB and MAP kinase activation (Fig. 10 B). IL-1-induced Akt activation in BMMs was not detectable (data not shown). Thus, the results in Fig. 10, combined with those in Fig. 4, reflect the specificity of IL-4 inhibition of RANKL-induced signaling in relation to osteoclast differentiation. As shown in Fig. 8 A and by others (26Matsumoto M. Sudo T. Saito T. Osada A. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), RANKL induces ERK activation, raising the possibility that activation of the ERK pathway plays a role in osteoclastogenesis. In contrast to a previous report (26Matsumoto M. Sudo T. Saito T. Osada A. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), and consistent with our observation that IL-4 inhibits RANKL-induced ERK activation, we find that both PD98059, a MEK1-specific inhibitor, and U0126, which blunts both MEK1 and MEK2, dose-dependently inhibit OC formation (Fig. 11,A and B). Withdrawal of the MEK inhibitors substantially rescues OC formation (Fig. 11 C), demonstrating that, in the doses used, the inhibitors are non-toxic and ERK activation is required for osteoclastogenesis. IL-4 signal transduction is initiated by binding of the cytokine to its heterodimeric membrane receptor, comprising IL-4Rα, which binds the cytokine with high affinity and the γC chain, a protein common to other members of the IL-4 receptor family (12Nelms K. Keegan A.D. Zamorano J. Ryan J.J. Paul W.E. Annu. Rev. Immunol. 1999; 17: 701-738Crossref PubMed Scopus (1269) Google Scholar). Receptor activation is followed by phosphorylation of both subunits, by Janus kinase, an event that initiates signaling by providing docking sites for adapter molecules or proteins capable of transducing signals. To date five adapters/transducers have been identified that bind the activated IL-4 r" @default.
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