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• W1963556204 abstract "Long-term administration of glucocorticoids (GCs) causes osteoporosis with a rapid and severe bone loss and with a slow and prolonged bone disruption. Although the involvement of GCs in osteoblastic proliferation and differentiation has been studied extensively, their direct action on osteoclasts is still controversial and not conclusive. In this study, we investigated the direct participation of GCs in osteoclastogenesis. Dexamethasone (Dex) at <10–8m stimulated, but at >10–7m depressed, receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation synergistically with transforming growth factor-β. The stimulatory action of Dex was restricted to the early phase of osteoclast differentiation and enhanced the priming of osteoclast progenitors (bone marrow-derived monocytes/macrophages) toward differentiation into cells of the osteoclast lineage. The osteoclast differentiation depending on RANKL requires the activation of NF-κB and AP-1, and the DNA binding of these transcription factors to their respective consensus cis-elements was enhanced by Dex, consistent with the stimulation of osteoclastogenesis. However, Dex did not affect the RANKL-induced signaling pathways such as the activation of IκB kinase followed by NF-κB nuclear translocation or the activation of JNK. On the other hand, Dex significantly decreased the endogenous production of interferon-β, and this cytokine depressed the RANKL-elicited DNA binding of NF-κB and AP-1, as well as osteoclast formation. Thus, the down-regulation of inhibitory cytokines such as interferon-β by Dex may allow the osteoclast progenitors to be freed from the suppression of osteoclastogenesis, resulting in an increased number of osteoclasts, as is observed in the early phase of GC-induced osteoporosis. Long-term administration of glucocorticoids (GCs) causes osteoporosis with a rapid and severe bone loss and with a slow and prolonged bone disruption. Although the involvement of GCs in osteoblastic proliferation and differentiation has been studied extensively, their direct action on osteoclasts is still controversial and not conclusive. In this study, we investigated the direct participation of GCs in osteoclastogenesis. Dexamethasone (Dex) at <10–8m stimulated, but at >10–7m depressed, receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation synergistically with transforming growth factor-β. The stimulatory action of Dex was restricted to the early phase of osteoclast differentiation and enhanced the priming of osteoclast progenitors (bone marrow-derived monocytes/macrophages) toward differentiation into cells of the osteoclast lineage. The osteoclast differentiation depending on RANKL requires the activation of NF-κB and AP-1, and the DNA binding of these transcription factors to their respective consensus cis-elements was enhanced by Dex, consistent with the stimulation of osteoclastogenesis. However, Dex did not affect the RANKL-induced signaling pathways such as the activation of IκB kinase followed by NF-κB nuclear translocation or the activation of JNK. On the other hand, Dex significantly decreased the endogenous production of interferon-β, and this cytokine depressed the RANKL-elicited DNA binding of NF-κB and AP-1, as well as osteoclast formation. Thus, the down-regulation of inhibitory cytokines such as interferon-β by Dex may allow the osteoclast progenitors to be freed from the suppression of osteoclastogenesis, resulting in an increased number of osteoclasts, as is observed in the early phase of GC-induced osteoporosis. Osteoclasts, the cells primarily responsible for bone resorption, are of hemopoietic stem cell origin. Precursors of osteoclasts have been demonstrated to share common properties with those of the monocyte/macrophage (M/MØ) 1The abbreviations used are: M/MØ, monocyte/macrophage; RANK, receptor activator of NF-κB; RANKL, RANK ligand; M-CSF, macrophage colony-stimulating factor; TGF-β, transforming growth factor-β; IFN, interferon; GC, glucocorticoid; Dex, dexamethasone; TRAP, tartrate-resistant acid phosphatase; MNC, multinucleate cell; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription; ISGF-3, IFN-stimulated gene factor-3; PBS, phosphate-buffered saline; JNK, c-Jun NH2-terminal kinase; TRAF, tumor necrosis factor receptor-associated factor; sRANKL, soluble RANKL; GST, glutathione S-transferase; GR, glucocorticoid receptor; MEM, minimum Eagle's medium; FBS, fetal bovine serum; RT, reverse transcription; p-ABSF, p-aminoethyl-benzenesulfonyl fluoride; EMSA, electrophoretic mobility shift assay.1The abbreviations used are: M/MØ, monocyte/macrophage; RANK, receptor activator of NF-κB; RANKL, RANK ligand; M-CSF, macrophage colony-stimulating factor; TGF-β, transforming growth factor-β; IFN, interferon; GC, glucocorticoid; Dex, dexamethasone; TRAP, tartrate-resistant acid phosphatase; MNC, multinucleate cell; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription; ISGF-3, IFN-stimulated gene factor-3; PBS, phosphate-buffered saline; JNK, c-Jun NH2-terminal kinase; TRAF, tumor necrosis factor receptor-associated factor; sRANKL, soluble RANKL; GST, glutathione S-transferase; GR, glucocorticoid receptor; MEM, minimum Eagle's medium; FBS, fetal bovine serum; RT, reverse transcription; p-ABSF, p-aminoethyl-benzenesulfonyl fluoride; EMSA, electrophoretic mobility shift assay. cell lineage (1Felix R. Cecchini M.G. Hofstetter W. Elford P.R. Stutzer A. Fleisch H. J. Bone Miner. Res. 1990; 5: 781-789Crossref PubMed Scopus (248) Google Scholar, 2Kodama H. Yamasaki A. Nose M. Niida S. Ohgame Y. Abe M. Kumegawa M. Suda T. J. Exp. Med. 1991; 173: 269-272Crossref PubMed Scopus (331) Google Scholar). Although many systemic hormones and local cytokines participate in regulating osteoclast differentiation (3Chambers T.J. J. Clin. Pathol. 1985; 38: 241-252Crossref PubMed Scopus (219) Google Scholar, 4Suda T. Udagawa N. Nakamura I. Miyaura C. Takahashi N. Bone. 1995; 17: 87S-91SCrossref PubMed Scopus (281) Google Scholar), the receptor activator of NF-κB (RANK) ligand (RANKL) is the most critical molecule for osteoclastogenesis in cooperation with macrophage colony-stimulating factor (M-CSF) in the interaction between stromal cells and cells of the osteoclast lineage (5Lacey 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, 6Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3500) Google Scholar, 7Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2799) Google Scholar). Extensive studies indicate that the induction of osteoclast differentiation by RANKL requires the activation of NF-κB and JNK pathways via tumor necrosis factor receptorassociated factor (TRAF) family proteins from RANK, the RANKL receptor (8Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 9Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (519) Google Scholar, 10Armstrong A.P. Tometsko M.E. Glaccum M. Sutherland C.L. Cosman D. Dougall W.C. J. Biol. Chem. 2002; 277: 44347-44356Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). We demonstrated recently (11Kaneda T. Nojima T. Nakagawa M. Ogasawara A. Kaneko H. Sato T. Mano H. Kumegawa M. Hakeda Y. J. Immunol. 2000; 165: 4254-4263Crossref PubMed Scopus (96) Google Scholar) that the endogenous production of transforming growth factor (TGF-β) was also essential for osteoclastogenesis as a cofactor with RANKL and M-CSF. Besides these proteins, a variety of local cytokines participate in regulating osteoclast differentiation (3Chambers T.J. J. Clin. Pathol. 1985; 38: 241-252Crossref PubMed Scopus (219) Google Scholar, 4Suda T. Udagawa N. Nakamura I. Miyaura C. Takahashi N. Bone. 1995; 17: 87S-91SCrossref PubMed Scopus (281) Google Scholar), Most of the osteotropic factors regulating osteoclast differentiation also affect the immune system, suggesting an intimate relationship between these two systems. These immune mediators such as interleukins, tumor necrosis factor-α, and interferons (IFNs) can be categorized into different groups based on their stimulatory or inhibitory effects and their direct or indirect action on osteoclast differentiation (12Palmqvist P. Persson E. Conaway H.H. Lerner U.H. J. Immunol. 2002; 169: 3353-3362Crossref PubMed Scopus (400) Google Scholar, 13Martin T.J. Romas E. Gillespie M.T. Crit. Rev. Eukaryot. Gene Expr. 1998; 8: 107-123Crossref PubMed Scopus (85) Google Scholar, 14Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. Martin T.J. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar, 15Kitazawa R. Kimble R.B. Vannice J.L. Kung V.T. Pacifici R. J. Clin. Invest. 1994; 94: 2397-2406Crossref PubMed Scopus (277) Google Scholar, 16Horowitz M.C. Xi Y. Wilson K. Kacena M.A. Cytokine Growth Factor Rev. 2001; 12: 9-18Crossref PubMed Scopus (182) Google Scholar). We and others reported more recently (17Hayashi T. Kaneda T. Toyama Y. Kumegawa M. Hakeda Y. J. Biol. Chem. 2002; 277: 27880-27886Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Takayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. Wagner E.F. Taniguchi T. Nature. 2002; 416: 744-749Crossref PubMed Scopus (577) Google Scholar) that the endogenous production of type-I IFNs such as IFN-β induced by RANKL in osteoclast progenitors intrinsically inhibited the differentiation of osteoclasts. Thus, osteoclast differentiation is governed by a delicate balance between the above stimulatory and inhibitory cytokines. Glucocorticoids (GCs) have a multitude of effects on the immune response at several sites and are both anti-inflammatory and immunosuppressive when administered therapeutically (19Pitzalis C. Pipitone N. Perretti M. Ann. N. Y. Acad. Sci. 2002; 966: 108-118Crossref PubMed Scopus (124) Google Scholar, 20Langenegger T. Michel B.A. Clin. Ortho. 1999; 366: 22-30Crossref Scopus (13) Google Scholar, 21Riccardi C. Zollo O. Nocentini G. Bruscoli S. Bartoli A. D'Adamio F. Cannarile L. Delfino D. Ayroldi E. Migliorati G. Therapie. 2000; 55: 165-169PubMed Google Scholar). Although GCs are effective for the treatment of a wide variety of disorders ranging from autoimmune diseases to acute situations such as spinal cord injury, long-term therapy with GCs causes osteoporosis, resulting in severe bone loss that, at present, has become a big clinical problem (22Canalis E. Delany A.M. Ann. N. Y. Acad. Sci. 2002; 966: 73-81Crossref PubMed Scopus (288) Google Scholar, 23Weinstein R.S. Rev. Endocr. Metab. Disord. 2001; 2: 65-73Crossref PubMed Scopus (156) Google Scholar, 24Patschan D. Loddenkemper K. Buttgereit F. Bone. 2001; 29: 498-505Crossref PubMed Scopus (165) Google Scholar, 25Manolagas S.C. Weinstein R.S. J. Bone Miner. Res. 1999; 14: 1061-1066Crossref PubMed Scopus (332) Google Scholar). GCs decrease calcium absorption in the gastrointestinal system and increase calcium excretion in the renal system, resulting in a high level of parathyroid hormone (26Bikle D.D. Halloran B. Fong L. Steinbach L. Shellito J. J. Clin. Endocrinol. Metab. 1993; 76: 456-461Crossref PubMed Scopus (36) Google Scholar, 27Klein R.G. Arnaud S.B. Gallagher J.C. Deluca H.F. Riggs B.L. J. Clin. Invest. 1977; 60: 253-259Crossref PubMed Scopus (286) Google Scholar). Therefore, GC-induced osteoporosis has been accepted for a long time to be caused by the secondary hyperparathyroidism (26Bikle D.D. Halloran B. Fong L. Steinbach L. Shellito J. J. Clin. Endocrinol. Metab. 1993; 76: 456-461Crossref PubMed Scopus (36) Google Scholar). In addition, another possible mechanism for GC-induced osteoporosis is that GCs decrease gonadotropin production, which may result in increased bone resorption because of estrogen deficiency (28Michael A.E. Cooke B.A. Mol. Cell. Endocrinol. 1994; 100: 55-63Crossref PubMed Scopus (65) Google Scholar, 29Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1267) Google Scholar). Considerable evidence, however, indicates that elevated levels of parathyroid hormone or subnormal vitamin D metabolite concentrations are not typical of patients receiving GC therapy, and there is no direct evidence indicating that GC-induced hypogonadism is responsible for the enhanced bone resorption, thus suggesting the existence of some other mechanism for GC-induced bone loss (30Paz-Pacheco E. Fuleihan G.E. LeBoff M.S. J. Bone Miner. Res. 1995; 10: 1713-1718Crossref PubMed Scopus (63) Google Scholar). On the other hand, many in vitro studies have indicated the direct action of GCs on osteoblasts (31Chyun Y.S. Kream B.E. Raisz L.G. Endocrinology. 1984; 114: 477-480Crossref PubMed Scopus (124) Google Scholar, 32Smith E. Coetzee G.A. Frenkel B. J. Biol. Chem. 2002; 277: 18191-18197Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 33Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1998; 102: 274-282Crossref PubMed Scopus (1371) Google Scholar). Recent studies (34Hofbauer L.C. Gori F. Riggs B.L. Lacey D.L. Dunstan C.R. Spelsberg T.C. Khosla S. Endocrinology. 1999; 140: 4382-4389Crossref PubMed Google Scholar, 35Rubin J. Biskobing D.M. Jadhav L. Fan D. Nanes M.S. Perkins S. Fan X. Endocrinology. 1998; 139: 1006-1012Crossref PubMed Scopus (76) Google Scholar) show that GC acts directly on osteoblasts to up-regulate the expression of RANKL and M-CSF and that the steroid oppositely down-regulates osteoprotegerin, a decoy receptor of RANKL that prevents the transmission of the RANKL signal into cells of the osteoclast lineage. This regulation is likely to be a mechanism for induction of bone resorption by GCs. However, direct effects of GCs on osteoclasts are controversial and are not conclusive (36Shuto T. Kukita T. Hirata M. Jimi E. Koga T. Endocrinology. 1994; 134: 1121-1126Crossref PubMed Scopus (63) Google Scholar, 37Kaji H. Sugimoto T. Kanatani M. Nishiyama K. Chihara K. J. Bone Miner. Res. 1997; 12: 734-741Crossref PubMed Scopus (77) Google Scholar, 38Tobias J. Chambers T.J. Endocrinology. 1989; 125: 1290-1295Crossref PubMed Scopus (74) Google Scholar). The aim of this study was to evaluate precisely the direct action of GCs on osteoclast differentiation and to determine the point of GC action in the process of osteoclastogenesis. In this study, we found that dexamethasone (Dex), at low concentrations (<10–8m), enhanced RANKL-induced osteoclast formation synergistically with TGF-β by stimulating the priming of bone marrow-derived M/MØ as osteoclast progenitors for differentiation toward osteoclasts. Although numerous studies (39Adcock I.M. Caramori G. Immunol. Cell Biol. 2001; 79: 376-384Crossref PubMed Scopus (285) Google Scholar, 40Jenkins B.D. Pullen C.B. Darimont B.D. Trends Endocrinol. Metab. 2001; 12: 122-126Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 41Wissink S. van Heerde E.C. vand der Burg B. van der Saag P.T. Mol. Endocrinol. 1998; 12: 355-363Crossref PubMed Google Scholar, 42McKay L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar) have indicated the negative regulation of NF-κB and AP-1 activation by GCs as anti-inflammatory and immunosuppressive agents, the enhancement of osteoclastogenesis was accompanied by the additional activation by Dex of these transcription factors evoked by RANKL. However, Dex did not influence the signaling pathways from RANK by which these transcription factors are translocated into nucleus. On the other hand, Dex significantly depressed the endogenous production of a type-I IFN, IFN-β. IFN-β potently inhibited osteoclastogenesis, as demonstrated previously (17Hayashi T. Kaneda T. Toyama Y. Kumegawa M. Hakeda Y. J. Biol. Chem. 2002; 277: 27880-27886Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Takayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. Wagner E.F. Taniguchi T. Nature. 2002; 416: 744-749Crossref PubMed Scopus (577) Google Scholar), and the cytokine depressed the activation of NF-κB and AP-1 in osteoclast progenitors. Thus, down-regulation of the inhibitory cytokines such as IFN-β by Dex may cause release from the suppression of osteoclastogenesis, thus resulting in an increase in osteoclast number, as is observed in the early phase of GC-induced bone loss. Reagents—Recombinant human M-CSF and recombinant mouse soluble RANKL (sRANKL) were kindly provided by Morinaga Milk Industry Co. (Tokyo, Japan) and Snow Brand Milk Industry Co. (Tochigi, Japan), respectively. Recombinant human TGF-β1 and recombinant mouse IFN-β were obtained from Genzyme/Techne (Cambridge, MA) and PBL Biomedical Laboratories (New Brunswick, NJ), respectively. Recombinant GST-conjugated c-Jun was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Anti-glucocorticoid receptor (GR), anti-NF-κB p50, anti-NF-κB p65, anti-IκB-α, anti-phospho-IκB-α, anti-JNK, anti-c-Jun/AP-1, anti-c-Fos, anti-STAT-1, anti-STAT-2, and anti-ISGF-3γ antibodies were purchased from Santa Cruz Biotechnology, Inc. (San Diego, CA). Anti-phospho-NF-κB p65 was from Cell Signaling Technology, Inc. (Beverly, MA). Osteoclastogenesis from M/MØ-like Hemopoietic Cells—Femora and tibiae were obtained from 4–5-week-old ICR mice (Shizuoka Laboratories Animal Center, Shizuoka, Japan), and the connective soft tissues were removed from the bones. Bone marrow cells were flushed out from the bone marrow cavity, suspended in α-MEM (ICN Biomedicals, Aurora, OH) supplemented with 10% FBS (Intergen, Purchase, NY), M-CSF (100 ng/ml), and 100 units/ml of penicillin, and cultured in Petri dishes in a humidified atmosphere of 5% CO2. During 3 days in culture, the cells remaining on the bottom of the dishes consisted of a large population of adherent M/MØ-like cells, expressing MØ-specific antigens such as Mac-1 and F4/80 and exhibiting phagocytotic activity (11Kaneda T. Nojima T. Nakagawa M. Ogasawara A. Kaneko H. Sato T. Mano H. Kumegawa M. Hakeda Y. J. Immunol. 2000; 165: 4254-4263Crossref PubMed Scopus (96) Google Scholar), and only a small population of nonadherent cells and adherent stromal cells. After removal of these nonadherent cells and stromal cells by washing the dishes with PBS and by subsequent incubation for 5 min in 0.25% trypsin/0.05% EDTA, respectively, the M/MØ-like hemopoietic cells were harvested in α-MEM/10% FBS by vigorous pipetting. The isolated M/MØ-like hemopoietic cells were seeded at an initial density of 2.5×104/cm2 and cultured in α-MEM/10% FBS/M-CSF (20 ng/ml) with or without sRANKL (40 ng/ml) and/or other cytokines or agents. The culture medium was exchanged for fresh medium every 2 days. After a culture period of the desired length, the cells were fixed in 10% formalin and stained for tartrate-resistant acid phosphatase (TRAP) activity with a leukocyte acid phosphatase kit (Sigma). The number of TRAP-positive multinucleate cells (MNCs) with more than three nuclei, which were considered to be osteoclastic cells, was counted under a microscope. Quantification of TRAP by Fluorescence Spectroscopy—Cellular TRAP activity was measured by fluorescence spectroscopy as described by Gallwitz et al. (43Gallwitz W.E. Mundy G.R. Lee C.H. Qiao M. Roodman G.D. Raftery M. Gaskell S.J. Bonewald L.F. J. Biol. Chem. 1993; 268: 10087-10094Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Isolated M/MØ-like hemopoietic cells were pre-cultured in α-MEM/10% FBS/M-CSF (20 ng/ml) and/or other cytokines or agents for 2 days and then further cultured in the presence of sRANKL (40 ng/ml) under various conditions for 4 days. After the culture period, the cells were washed with PBS and lysed by two cycles of freezing and thawing in 0.05% Triton X-100. After centrifugation, the supernatant was used for determination of TRAP activity. The cell lysate was incubated for 30 min at 37 °C in a reaction mixture consisting of 0.48 m sodium acetate/0.48 m acetic acid (pH 5.0), 20 mm tartaric acid, and 2 mm methylumbelliferyl phosphate as a substrate and then the reaction was terminated by adding glycine and EDTA (pH 10.5), each for a final concentration of 50 mm. The concentration of methylumbelliferone produced in the reaction mixture was determined by fluorometry with excitation at 366 nm and emission at 456 nm. Enzyme activity was expressed as nanomoles of methylumbelliferyl phosphate hydrolyzed/min/mg protein. The concentration of proteins in the cell lysate was measured with a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc.). RT-PCR—Total RNA (1 μg) extracted from cultured cells was used as a template for cDNA synthesis. cDNA was prepared by use of a Superscript II preamplification system (Invitrogen). Primers were synthesized on the basis of the reported mouse cDNA sequences for TRAP, cathepsin K, calcitonin receptor, integrin αv, integrin β3, CD14, GR, RANK, TRAF2, TRAF6, c-fos, fra-1, c-jun, IFN-β, suppressor of cytokine signaling-1 (SOCS-1), SOCS-3, STAT-1, STAT-2, ISGF-3γ, and β-actin. Sequences of the primers used for PCR were as follows: TRAP forward, 5′-CACGATGCCAGCGACAAGAG-3′; TRAP reverse, 5′-TGACCCCGTATGTGGCTAAC-3′; cathepsin K forward, 5′-GGAAGAAGACTCACCAGAAGC-3′; cathepsin K reverse, 5′-GTCATATAGCCGCCTCCACAG-3′; calcitonin receptor forward, 5′-ACCGACGAGCAACGCCTACGC-3′; calcitonin receptor reverse, 5′-GCCTTCACAGCCTTCAGGTAC-3′; integrin αv forward, 5′-GCCAGCCCATTGAGTTTGATT-3′; integrin αv reverse, 5′-GCTACCAGGACCACCGAGAAG-3′; integrin β3 forward, 5′-TTACCCCGTGGACATCTACTA-3′; integrin β3 reverse, 5′-AGTCTTCCATCCAGGGCAATA-3′; CD14 forward, 5′-AAGTTCCCGACCCTCCAAGTT-3′; CD14 reverse, 5′-CTGCCTTTCTTTCCTTACATC-3′; GR forward, 5′-CGCTCAGTGTTTTCTAATGG-3′; GR reverse, 5′-ATCAGGAGCAAAGCATAGCA-3′; RANK forward, 5′-CTCTGCGTGCTGCTCGTTCC-3′; RANK reverse, 5′-TTGTCCCCTGGTGTGCTTCT-3′; TRAF2 forward, 5′-CCGTGAAGTAGAGAGGGTAGC-3′; TRAF2 reverse, 5′-TTGGACACAGGGCAGAAGAGG-3′; TRAF6 forward, 5′-AGCCCACGAAAGCCAGAAGAA-3′; TRAF6 reverse, 5′-CCCTTATGGATTTGATGATGC-3′; c-fos forward, 5′-CATCGGCAGAAGGGGCAAAG-3′; c-fos reverse, 5′-GAGAAGGGGCAGGGTGAAGG-3′; fra-1 forward, 5′-AACCTTGCTCCTCCGCTCACC-3′; fra-1 reverse, 5′-GCTGCTGGCTGTTGATGCTGT-3′; c-jun forward, 5′-CTGGCGGCGGTGGTGGCTAC-3′; c-jun reverse, 5′-CGGTCTGCGGCTCTTCCTTC-3′; IFN-β forward, 5′-CTTCTCCACCACAGCCCTCTC-3′; IFN-β reverse, 5′-CCCACGTCAATCTTTCCTCTT-3′; SOCS-1 forward, 5′-AGGATGGTAGCACGCAACCAGGT-3′; SOCS-1 reverse, 5′-GATCTGGAAGGGGAAGGAACTCAGGTA-3′; SOCS-3 forward, 5′-GCCATGGTCACCCACAGCAAGTT-3′; SOCS-3 reverse, 5′-AAGTGGAGCATCATACTGATCCAGGA-3′; STAT-1 forward, 5′-CGAAGAGCGACCAAAAACAG-3′; STAT-1 reverse, 5′-TGCTGGAAGAGGAGGAAGGT-3′; STAT-2 forward, 5′-TTTGCTGGGACCTGTGCTCA-3′; STAT-2 reverse, 5′-TGGGTTCTCGGGGATGTTCT-3′; ISGF-3γ forward, 5′-GAACCCTCCCTAACCAACCA-3′; ISGF-3γ reverse, 5′-AGGTGAGCAGCAGCGAGTAG-3′; and β-actin forward, 5′-TCACCCACACTGTGCCCATCTAC-3′; β-actin reverse, 5′-GAGTACTTGCGCTCAGGAGGAGC-3′. Amplification was conducted for 22–32 cycles, each of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min in a 25-μl reaction mixture containing 0.5 μl of each cDNA, 25 pmol of each primer, 0.2 mm dNTP, and 1 unit of Taq DNA polymerase (Qiagen, Valencia, CA). After amplification, 15 μl of each reaction mixture was analyzed by 1.5% agarose gel electrophoresis, and the bands were then visualized by ethidium bromide staining. Western Blot Analysis—After various treatments, the cells were washed with PBS, scraped into a solution consisting of 10 mm sodium phosphate (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm EDTA, 1 mm p-aminoethyl-benzenesulfonyl fluoride (p-ABSF), 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin and sonicated for 15 s. The samples, containing equal amounts of protein, were subjected to 10% SDS-PAGE, and the proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane. After having been blocked with 5% skim milk, the membrane was incubated with anti-GR, anti-NF-κB p50, anti-NF-κB p65, anti-phospho-NF-κB p65, anti-IκB-α, or anti-phospho-IκB-α antibodies and subsequently with peroxidase-conjugated antimouse or anti-rabbit IgG antibody. Immunoreactive proteins were visualized with Western blot chemiluminescence reagents (Amersham Biosciences) following the manufacturer's instructions. Assay of JNK Activity—M/MØ-like hemopoietic cells were pretreated or not with Dex and/or TGF-β in the presence of M-CSF for 2 days prior to treatment with RANKL for 1 h. After the treatment, the cells were extracted in a lysis buffer consisting of 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin, and 1 mm p-ABSF. The cell lysis was then incubated with anti-JNK antibody (2 μg) at 4 °C overnight and further incubated with protein A-beads for an additional 3 h. After several washes, the immunoreacted beads were suspended in a kinase reaction mixture (25 mm Tris-HCl (pH 7.5), 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate, 10 mm MgCl2) supplemented with 100 μm [γ-32P]ATP (5 μCi) and 1 μg of recombinant GST-c-Jun and incubated at 30 °C for 30 min. The reaction was terminated by adding SDS sample buffer, and the mixture was then boiled. After the samples had been subjected to SDS-PAGE (12% gel), the phosphorylated GST-c-Jun in the gel was visualized by autoradiography at –80 °C. Electrophoretic Mobility Shift Assay (EMSA)—After pretreatment or not with Dex and/or TGF-β in the presence of M-CSF for 2 days, the cells were treated with sRANKL for 1 h and then washed twice with ice-cold PBS, incubated for 10 min on ice in 1 ml of ice-cold Buffer A (10 mm Hepes (pH 7.4), 10 mm KCl, 0.1 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 1 mm p-ABSF, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin), and scraped into the buffer. The cell lysates were incubated further for 10 min on ice and then transferred to tubes. The nuclei obtained by centrifugation for 1 min at 5000 × g were extracted by a 30-min incubation in ice-cold Buffer C, consisting of 50 mm Hepes (pH 7.5), 420 mm KCl, 0.1 mm EDTA, 5 mm MgCl2, 20% glycerol, 1 mm dithiothreitol, 1 mm p-ABSF, 2 μg/ml aprotinin, 2 μg/ml pepstatin, and 2 μg/ml leupeptin. Then, the extracts were centrifuged at 14,000 × g for 30 min, and the supernatants were used for EMSA. Double-stranded oligonucleotides containing an NF-κB binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′), an AP-1 binding site (5′-CGCTTGATGAGTCAGGAA-3′), or an IFN-stimulated response element (5′-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3′, element underlined (44Petricoin III, E. David M. Igarashi K. Benjamin C. Ling L. Goelz S. Finbloom D.S. Larner A.C. Mol. Cell Biol. 1996; 16: 1419-1424Crossref PubMed Scopus (34) Google Scholar)) were end-radiolabeled with [γ-32P]ATP by using T4 polynucleotide kinase (Promega, Madison, WI) according to the manufacturer's instruction and combined and incubated for 30 min at room temperature with 1 μg of nuclear extract in binding buffer (10 mm Tris-HCl (pH 7.5), 4% glycerol, 50 mm NaCl, 1 mm MgCl2, 0.5 mm EDTA, and 0.5 mm dithiothreitol). The specificity of the reaction was confirmed by competition with a 50-fold molar excess of nonlabeled oligonucleotides. The protein-DNA complexes were resolved by PAGE in 0.5× TBE buffer and visualized by autoradiography. In addition, other nuclear extracts were incubated with anti-NF-κB p50, anti-NF-κB p65, anti-c-Jun/AP-1, anti-c-Fos, anti-STAT-1, anti-STAT-2, or anti-ISFG-3γ antibodies for 30 min on ice after binding to the oligonucleotides and then were subjected to PAGE. Densitometry—Intensity of bands obtained from RT-PCR, JNK activity, and EMSA analyses was quantified by densitometry with an image analyzer (B. I. Systems Corp., Ann Arbor, MI). Statistical Analysis—Means of groups were compared by analysis of variance, and significance of differences was determined by post-hoc testing using Bonferroni's method. Dexamethasone Stimulates Osteoclastogenesis from Bone Marrow M/MØ Cells—When bone marrow-derived M/MØ were incubated with M-CSF and sRANKL, and particularly when TGF-β was also present, most of the cells differentiated into cells of the osteoclast lineage and became mature osteoclasts, as described previously (11Kaneda T. Nojima T. Nakagawa M. Ogasawara A. Kaneko H. Sato T. Mano H. Kumegawa M. Hakeda Y. J. Immunol. 2000; 165: 4254-4263Crossref PubMed Scopus (96) Google Scholar). The addition of exogenous Dex increased the number of osteoclastic TRAP-positive MNCs formed in these cultures in the presence of TGF-β in a Dex dose-related manner, with the maximal increase at 10–9-10–8m (Fig. 1A); whereas the total number of nuclei was not changed (data not shown). However, at a higher concentration of 10–7m, Dex completely depressed the osteoclast formation. Thus, Dex revealed biphasic effects on osteoclastogenesis, one being stimulatory at lower concentrations (≤10–8m) and the o" @default.
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• W1963556204 title "Dexamethasone Enhances Osteoclast Formation Synergistically with Transforming Growth Factor-β by Stimulating the Priming of Osteoclast Progenitors for Differentiation into Osteoclasts" @default.
• W1963556204 cites W1519118083 @default.
• W1963556204 cites W1534645474 @default.
• W1963556204 cites W1557762633 @default.
• W1963556204 cites W1771759116 @default.
• W1963556204 cites W1886623575 @default.
• W1963556204 cites W1902098806 @default.
• W1963556204 cites W1948304169 @default.
• W1963556204 cites W1963700437 @default.
• W1963556204 cites W1968858083 @default.
• W1963556204 cites W1972001482 @default.
• W1963556204 cites W1972692051 @default.
• W1963556204 cites W1975801129 @default.
• W1963556204 cites W1980418071 @default.
• W1963556204 cites W1982078028 @default.
• W1963556204 cites W1988620146 @default.
• W1963556204 cites W1996028579 @default.
• W1963556204 cites W1999032828 @default.
• W1963556204 cites W2001682983 @default.
• W1963556204 cites W2002315967 @default.
• W1963556204 cites W2005326563 @default.
• W1963556204 cites W2008737860 @default.
• W1963556204 cites W2010277922 @default.
• W1963556204 cites W2014237818 @default.
• W1963556204 cites W2018602999 @default.
• W1963556204 cites W2031157573 @default.
• W1963556204 cites W2032972084 @default.
• W1963556204 cites W2033864326 @default.
• W1963556204 cites W2042623077 @default.
• W1963556204 cites W2043651158 @default.
• W1963556204 cites W2045779571 @default.
• W1963556204 cites W2048299560 @default.
• W1963556204 cites W2054306576 @default.
• W1963556204 cites W2065317510 @default.
• W1963556204 cites W2067397506 @default.
• W1963556204 cites W2071883746 @default.
• W1963556204 cites W2072697232 @default.
• W1963556204 cites W2080570046 @default.
• W1963556204 cites W2081889483 @default.
• W1963556204 cites W2084102864 @default.
• W1963556204 cites W2085222566 @default.
• W1963556204 cites W2094402463 @default.
• W1963556204 cites W2108700225 @default.
• W1963556204 cites W2113898373 @default.
• W1963556204 cites W2126446849 @default.
• W1963556204 cites W2134055375 @default.
• W1963556204 cites W2139996507 @default.
• W1963556204 cites W2147277158 @default.
• W1963556204 cites W2148930555 @default.
• W1963556204 cites W2153314004 @default.
• W1963556204 cites W2154393492 @default.
• W1963556204 cites W2155283231 @default.
• W1963556204 cites W2157528306 @default.
• W1963556204 cites W2157744411 @default.
• W1963556204 cites W2163247322 @default.
• W1963556204 cites W2163584089 @default.
• W1963556204 cites W2165787609 @default.
• W1963556204 cites W2168569369 @default.
• W1963556204 cites W4240799970 @default.
• W1963556204 cites W82097804 @default.
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