Involvement of p38 Mitogen-activated Protein Kinase Signaling Pathway in Osteoclastogenesis Mediated by Receptor Activator of NF-κB Ligand (RANKL)
2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês
10.1074/jbc.m001229200
ISSN1083-351X
AutoresMasahito Matsumoto, T Sudo, Tamio Saito, Hiroyuki Osada, Masafumi Tsujimoto,
Tópico(s)NF-κB Signaling Pathways
ResumoThe receptor activator of NF-κB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38α MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells. The receptor activator of NF-κB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38α MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells. receptor activator of NF-κB ligand receptor activator of NF-κB macrophage colony-stimulating factor mitogen-activated protein MAP kinase kinase reverse transcription-polymerase chain reaction tumor necrosis factor receptor-associated factor extracellular signal-regulated kinase c-Jun N-terminal kinase osteoprotegerin tartrate-resistant acid phosphatase non-essential amino acid glutathione S-transferase Bone morphogenesis, remodeling, and resorption are controlled in part by osteoclasts. These cells differentiate from hematopoietic myeloid precursors of monocyte/macrophage lineage under control of osteotropic hormones and local factors produced by supporting cells such as osteoblasts and stromal cells (1Ash P. Loutit J.F. Townsend K.M. Nature. 1980; 283: 669-670Crossref PubMed Scopus (236) Google Scholar, 2Scheven B.A. Visser J.W. Nijweide P.J. Nature. 1986; 321: 79-81Crossref PubMed Scopus (218) Google Scholar, 3Kurihara N. Suda T. Miura Y. Nakauchi H. Kodama H. Hiura K. Hakeda Y. Kumegawa M. Blood. 1989; 74: 1295-1302Crossref PubMed Google Scholar, 4Roodman G.D. Endocr. Rev. 1996; 17: 308-332PubMed Google Scholar, 5Manolagas S.C. Jilka R.L.N. N. Engl. J. Med. 1995; 332: 305-311Crossref PubMed Scopus (1559) Google Scholar, 6Hayashi S. Yamane T. Miyamoto A. Hemmi H. Tagaya H. Tanio Y. Kanda H. Yamazaki H. Kunisada T. Biochem. Cell Biol. 1998; 76: 911-922Crossref PubMed Scopus (56) Google Scholar, 7Udagawa N. Takahashi N. Akatsu T. Tanaka H. Sasaki T. Nishihara T. 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The model includes the appearance of mononuclear osteoclasts, the fusion process prior to multinucleated osteoclast formation, and the osteoclast maturation process (6Hayashi S. Yamane T. Miyamoto A. Hemmi H. Tagaya H. Tanio Y. Kanda H. Yamazaki H. Kunisada T. Biochem. Cell Biol. 1998; 76: 911-922Crossref PubMed Scopus (56) Google Scholar). Moreover, it has been shown that mutant mice disrupted with either RANKL or its receptor RANK revealed severe osteopetrosis and osteoclast defects (16Kong 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 (2834) Google Scholar, 17Dougall W.C. Glaccum M. Charrier K. Rohrbach K. Brasel K. De Smedt T. Daro E. Smith J. Tometsko M.E. Maliszewski C.R. Armstrong A. Shen V. Bain S. Cosman D. Anderson D. Morrissey P.J. Peschon J.J. Schuh J. 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Aggarwal B.B. J. Biol. Chem. 1999; 274: 724-731Abstract Full Text Full Text PDF Scopus (355) Google Scholar). The intracellular domain of RANK contains two distinct TRAF-binding domains, each of which recognizes different TRAF proteins specifically (18Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1408) Google Scholar, 19Galibert L. Tometsko M.E. Anderson D.M. Cosman D. Dougall W.C. J. Biol. Chem. 1998; 273: 34120-34127Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). While the C-terminal region of RANK interacts with TRAF2 and TRAF5, the TRAF6-binding domain resides in the middle of the RANK intracellular region. Overexpression of RANK C-terminal deletion mutants has revealed that activation of the RANK-mediated signaling pathway results in the activation of NF-κB and c-Jun N-terminal kinase (JNK) which correlate with the TRAF6 interaction activity of mutants (18Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1408) Google Scholar, 19Galibert L. Tometsko M.E. Anderson D.M. Cosman D. Dougall W.C. J. Biol. Chem. 1998; 273: 34120-34127Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In addition, mice with disrupted TRAF6 gene exhibit an osteopetrotic phenotype due to a defect in bone resorption (22Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1068) Google Scholar, 23Naito 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 (538) Google Scholar). Therefore it is speculated that JNK might play an important role in osteoclast differentiation.Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that are important in cell growth, differentiation, and apoptosis (24Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 25Avruch J. Zhang X.F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 26Davis R.J. Trends Biochem. 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It was reported that the differentiation inducing activity of these reagents was mediated by RANKL, since the addition of OPG/OCIF, a decoy receptor for RANK, completely inhibited the generation of osteoclasts (39Simonet 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. De Rose 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. 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 (4299) Google Scholar, 40Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Goto M. Mochizuki S. Suda E. Morinaga T. Udagawa N. Takahashi N. Suda T. Higashino K. Bone. 1999; 25: 109-113Crossref PubMed Scopus (228) Google Scholar). These results suggest that in combination with M-CSF, RANKL plays an essential role in the induction of osteoclast differentiation.In this paper, we have confirmed that both M-CSF and sRANKL are required to induce terminal differentiation of bone marrow cells into multinucleated osteoclasts. Our results suggest that M-CSF and RANKL act on bone marrow cells sequentially to induce the terminal differentiation to osteoclasts and that RANKL is the differentiation inducing factor in our assay system since sRANKL alone could induce the TRAP-positive mononulear cells in the absence of the increase in cell number. Several reports indicate that M-CSF plays an essential role in osteoclastogenesis through RANK induction, the stimulation of cell survival and proliferation, and by acting as a competence factor for differentiation (34Arai F. Miyamoto T. Ohneda O. Inada T. Sudo T. Brasel K. Miyata T. Anderson D.M. Suda T. J. Exp. Med. 1999; 190: 1741-1754Crossref PubMed Scopus (552) Google Scholar, 48Lagasse E. Weissman I.L. Cell. 1997; 89: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 49Hayashi S. Miyamoto A. Yamane T. Kataoka H. Ogawa M. Sugawara S. Nishikawa S. Nishikawa S. Sudo T. Yamazaki H. Kunisada T. J. Cell. Physiol. 1997; 170: 41-47Crossref Scopus (48) Google Scholar).It was quite unexpected for us that SB203580 but not PD98059 inhibited the M-CSF/sRANKL-induced differentiation of bone marrow cells into multinucleated osteoclasts. Since an increase in the number of adherent cells, albeit no appearance of TRAP-positive cells, was observed even in the presence of SB203580, we can speculate that SB203580 inhibits the differentiation of bone marrow cells by interfering with the RANKL-induced p38 MAP kinase activity.To elucidate the causal link between the activation of the p38 MAP kinase pathway and RANKL-induced osteoclast differentiation, we employed RAW264 cells which expressed RANK and differentiated into TRAP-positive multinucleated cells through sRANKL treatment. As was the case with bone marrow cells, SB203580 but not PD98059 inhibited differentiation induced by sRANKL. In RAW264 cells, RANKL-induced activations of ERKs, JNKs, and p38 MAP kinase were clearly detected and SB203580 inhibited only the p38 MAP kinase activity. These results strongly suggest that at least in part p38 MAP kinase plays a critical role in RANKL-induced differentiation. However, we cannot rule out the possible involvement of ERK and JNK in osteoclastogenesis at present.Expression of the dominant negative form of p38α MAP kinase resulted in a significant decrease in the number of TRAP-positive cells induced by RANKL. Significant decrease in the RANKL-induced p38 MAP kinase activation was also observed. These results further support the notion that the activation of the p38 MAP kinase signaling pathway is necessary for sRANKL-induced differentiation of bone marrow cells and RAW264 cells into osteoclasts. However, complete inhibition was not observed in CX38DN cells expressing the dominant negative form of p38α MAP kinase. It is conceivable that other isoforms of p38 MAP kinase could compensate for a possible defect in the signaling pathway through p38α MAP kinase. In addition to the α-type, at least three isoforms (β-, γ-, and δ-types) of p38 MAP kinase are reported (50Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. 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Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). We detected the expression of the mRNA for β-type in RAW264 cells by RT-PCR (data not shown). Therefore it is possible that p38β MAP kinase functions in CX38DN cells. Because SB203580 inhibited osteoclastogenesis completely, it is likely that only p38α and p38β MAP kinases are involved in differentiation since these are the only isoforms inhibited by SB203580 at the low doses (38Kumar S. Jiang M.S. Adams J.L. Lee J.C. Biochem. Biophys. Res. Commun. 1999; 263: 825-831Crossref PubMed Scopus (219) Google Scholar).MKK6 is a direct and specific activator of all p38 MAP kinase isoforms so far identified (56Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 57Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1139) Google Scholar). Expression of the dominant negative form of MKK6 inhibited both sRANKL-induced differentiation of RAW264 cells and p38 MAP kinase activation. It is likely that the dominant negative form of MKK6 inhibits both α- and β-types of p38 MAP kinase, thus conferring a greater range of differentiation inhibition than the inhibition performed by the dominant negative form of p38α MAP kinase. The quantitative differences between the suppression of differentiation seen with dominant negative forms of p38α MAP kinase and MKK6 could be completely explained by the extent to which the transfected forms are able to suppress p38 activity in cells. Presumably if they were more efficient, they could both achieve complete suppression.In conclusion, we have demonstrated in this paper that the p38 MAP kinase signaling pathway plays a crucial role in RANKL-mediated differentiation of bone marrow cells into osteoclasts. However, experiments of the overexpression of wild type kinases revealed no correlation between p38 MAP kinase activity and RANKL-induced differentiation, suggesting the role of other factors in the RANKL-mediated osteoclastogenesis. Since the activation of other MAP kinase families (i.e. ERKs and JNKs) during differentiation was clearly observed, the contribution of these kinases to osteoclast differentiation should be elucidated in the near future. Bone morphogenesis, remodeling, and resorption are controlled in part by osteoclasts. These cells differentiate from hematopoietic myeloid precursors of monocyte/macrophage lineage under control of osteotropic hormones and local factors produced by supporting cells such as osteoblasts and stromal cells (1Ash P. Loutit J.F. Townsend K.M. Nature. 1980; 283: 669-670Crossref PubMed Scopus (236) Google Scholar, 2Scheven B.A. Visser J.W. Nijweide P.J. Nature. 1986; 321: 79-81Crossref PubMed Scopus (218) Google Scholar, 3Kurihara N. Suda T. 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The receptor activator of NF-κB ligand (RANKL)1 (10Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Du Bose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar), also refereed to as osteoclast differentiation factor (11Yasuda 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 (3526) Google Scholar), tumor necrosis factor-related activation-induced cytokine (12Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett 3rd, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar), or osteoprotegerin ligand (13Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. 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