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Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis

2001; Springer Nature; Volume: 20; Issue: 6 Linguagem: Inglês

10.1093/emboj/20.6.1271

1460-2075

Norihiko Kobayashi, Yuho Kadono, Asuka Naito, Kunihiro Matsumoto, Tadashi Yamamoto, Sakae Tanaka, Jun‐ichiro Inoue,

Immune Response and Inflammation

Article15 March 2001free access Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis Norihiko Kobayashi Norihiko Kobayashi Department of Hematology, Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo, 162-8655 Japan Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Yuho Kadono Yuho Kadono Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Asuka Naito Asuka Naito Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Kunihiro Matsumoto Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Sakae Tanaka Sakae Tanaka Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Jun-ichiro Inoue Corresponding Author Jun-ichiro Inoue Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Norihiko Kobayashi Norihiko Kobayashi Department of Hematology, Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo, 162-8655 Japan Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Yuho Kadono Yuho Kadono Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Asuka Naito Asuka Naito Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Kunihiro Matsumoto Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Sakae Tanaka Sakae Tanaka Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Jun-ichiro Inoue Corresponding Author Jun-ichiro Inoue Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan Search for more papers by this author Author Information Norihiko Kobayashi1,5, Yuho Kadono2,5, Asuka Naito3, Kunihiro Matsumoto4, Tadashi Yamamoto3, Sakae Tanaka2 and Jun-ichiro Inoue 5 1Department of Hematology, Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo, 162-8655 Japan 2Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan 3Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan 4Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan 5Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, 223-8522 Japan ‡N.Kobayashi and Y.Kadono contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1271-1280https://doi.org/10.1093/emboj/20.6.1271 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signals emanating from the receptor for interleukin-1 (IL-1), lipopolysaccharide (LPS) or osteoclast differentiation factor/receptor activator of NFκB ligand (ODF/RANKL) stimulate transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation and NFκB through IκB kinase (IKK) activation. These kinases are thought to be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). However, molecular mechanisms by which TRAF6 activates various downstream kinases remain to be elucidated. We identified functional domains of TRAF6 under physiological conditions established by appropriate expression of TRAF6 mutants in TRAF6-deficient cells. In IL-1 and LPS signaling pathways, the RING finger and first zinc finger domains are not required for NFκB activation but are required for full activation of MAPK. However, IL-1 and LPS signals utilize distinct regions within the zinc finger domains of TRAF6 to activate NFκB. Furthermore, the RING finger domain is not required for differentiation of splenocytes to multinuclear osteoclasts, but is essential for osteoclast maturation. Thus, TRAF6 plays essential roles in both the differentiation and maturation of osteoclasts by activating various kinases via its multiple domains. Introduction The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family proteins are cytoplasmic adapter proteins that mediate cytokine signaling (Arch et al., 1998). Among the receptors that recruit TRAF proteins are members of the TNFR superfamily and the Toll/interleukin-1 receptor (IL-1R) family. To date, six members of this family have been described. The TRAF domain is located in the C-terminal region of all members of the TRAF family and is composed of two subregions (Cheng et al., 1995): TRAF-C, which is the C-terminal half of the TRAF domain, is highly conserved among all members, and TRAF-N, which is the N-terminal half of the TRAF domain, is predicted to form a coiled-coil structure. Crystallographic studies of the TRAF domain of TRAF2 suggest that this domain forms a mushroom-shaped trimer, which is likely to interact with the trimer form of CD40 or TNFRII in response to ligand stimulation (McWhirter et al., 1999; Park et al., 1999). With the exception of TRAF1, all TRAFs contain an N-terminal RING finger domain and a stretch of predicted zinc fingers. Whereas TRAF2, TRAF5 and TRAF6 activate transcription factors NFκB through IκB kinase (IKK) activation and AP-1 through activation of mitogen-activated protein kinases (MAPKs) including Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), neither TRAF3 nor TRAF4 activates these pathways (Arch et al., 1998). Recently, signal-dependent interaction of MAP kinase kinase kinase (MAP3K) with TRAFs was demonstrated. TRAF6 interacts with transforming growth factor β (TGFβ)-activated kinase 1 (TAK1) via TAK1 binding protein 2 (TAB2) to activate NFκB-inducing kinase (NIK) and JNK (Ninomiya et al., 1999; Takaesu et al., 2000). TRAF2 interacts with apoptosis signal-regulating kinase-1 (ASK1) (Nishitoh et al., 1998) and MEKK1 (Baud et al., 1999). Thus, TRAF2, TRAF6 and probably TRAF5 exert their function by activating downstream MAP3Ks or MAP4Ks. TRAF6 has the most divergent TRAF-C and is the only TRAF that is involved in the signal from members of the Toll/IL-1R family by interacting with the IL-1R-associated kinase (IRAK) (Cao et al., 1996; Ishida et al., 1996a) and that from p75 neutrophin receptor (Khursigara et al., 1999). Furthermore, TRAF2, TRAF3 and TRAF5 bind to the membrane-distal domain in the cytoplasmic tail of CD40 and receptor activator of NFκB (RANK), whereas TRAF6 interacts with the membrane-proximal domain (Galibert et al., 1998; Wong et al., 1998; Tsukamoto et al., 1999). We have previously shown that TRAF6-deficient (TRAF6−/−) mice exhibit severe osteopetrosis and are defective in osteoclast formation due to defective signaling from RANK upon binding of osteoclast differentiation factor/RANK ligand (ODF/RANKL) (also known as OPGL and TRANCE) (Naito et al., 1999). Furthermore, TRAF6−/− mice are defective in normal B-cell differentiation, lymph node organogenesis and IL-1 signaling. Thus, TRAF6 plays pivotal roles in immune and inflammatory systems in vivo. Although TRAF6-mediated signal transduction is indispensable, the molecular mechanism by which TRAF6 exerts its biological effects remains unknown. Moreover, results of earlier studies designed to investigate the role of TRAF zinc-binding regions, which are required for TRAF signal transduction, were often contradictory, most likely because TRAF proteins were grossly overexpressed in cell lines that endogenously express TRAF (Cao et al., 1996; Takeuchi et al., 1996; Dadgostar and Cheng, 1998; Baud et al., 1999). In this study, we sought to identify critical regions within TRAF6 that are involved in activation of NFκB and MAPK pathways, and to define the essential roles of TRAF6 in osteoclastogenesis by expressing mutant TRAF6 transgenes at endogenous wild-type levels in TRAF6−/− cells. Results TRAF6 is essential for activation of NFκB, JNK and p38 by IL-1 and lipopolysaccharide (LPS) signaling TRAF6−/− mice display osteopetrosis with defects in bone remodeling due to impaired ODF/RANKL–RANK signaling (Lomaga et al., 1999; Naito et al., 1999). However, we scarcely observed tartrate-resistant acid phosphatase-positive (TRAP+) osteoclast-like cells (OCLs) in bone tissue, whereas normal numbers of TRAP+ OCLs that lacked contact with bone surfaces were observed by Lomaga et al. (1999). Furthermore, our in vitro culture experiments revealed that osteoclast progenitors derived from TRAF6−/− mice are unable to differentiate into OCLs in response to ODF/RANKL. This discrepancy could be due to differences in targeting constructs. However, other unexpected genetic defects besides TRAF6 deficiency are possible. Thus, whether TRAF6 is essential for NFκB and MAPK activation via IL-1 and LPS signaling was first tested. Two independent mouse embryonic fibroblast (MEF) cell lines were generated from wild-type and TRAF6−/− mice (Figure 1B). MEF cells were stimulated with either IL-1 or LPS, and activation of NFκB, JNK and p38 was analyzed. Activation of NFκB, JNK and p38 in response to IL-1 and LPS was abrogated in TRAF6−/− MEF cells (Figures 2 and 3). In contrast, activation of NFκB and MAPK pathways by tumor necrosis factor α (TNFα) or sorbitol in TRAF6−/− MEF cells was similar to that in wild-type MEF cells (data not shown). To confirm that failure of MEF cells to respond to IL-1 and LPS was due solely to lack of TRAF6 expression, TRAF6 cDNA was introduced into TRAF6−/− MEF cells by a retrovirus vector carrying a puromycin resistance gene. The amount of TRAF6 expressed from the introduced cDNA was comparable to that of endogenous TRAF6 in wild-type MEF cells by incubating TRAF6−/− MEF cells with diluted virus solution to avoid multiple infection of virus (Figure 1B). Complementation of TRAF6−/− MEF cells with TRAF6 restored activation of NFκB, JNK and p38 in response to IL-1 and LPS treatment (Figures 2 and 3). These results indicate that TRAF6 is essential for IL-1 and LPS signaling linked to NFκB and MAPK activation. Figure 1.Establishment of TRAF6−/− embryo-derived fibroblasts stably expressing physiological levels of exogenous wild-type or mutant TRAF6. (A) Structure of wild-type and N-terminally truncated TRAF6 mutants. Numbers in parentheses indicate amino acids that comprise wild-type and mutant TRAF6. (B) Expression level of wild-type and mutant TRAF6 in MEF cells. Cells were metabolically labeled with [35S]methionine, and wild-type and mutant TRAF6 were immunoprecipitated with anti-TRAF6 antibody. The number of methionine residues in each protein: T6 (18), T6ΔR (15), T6ΔZ1 (14), T6ΔZ3 (13) and T6ΔZ5 (10). Dots denote the positions of protein bands that correspond to the indicated form of TRAF6 (shown above each lane). Download figure Download PowerPoint Figure 2.The RING finger and first zinc finger of TRAF6 are not required for NFκB activation in response to IL-1 and LPS in MEF cells. MEF cells were derived from wild-type (+/+) or TRAF6−/− (−/−) mice and infected with recombinant retrovirus harboring either wild-type or mutant TRAF6 transgenes. (A) DNA binding activity of nuclear NFκB. Cells were either unstimulated (–) or stimulated (+) with IL-1 (20 ng/ml) for 30 min or LPS (200 ng/ml) for 60 min. Nuclear extracts were prepared and EMSAs were performed. The quantity of nuclear extracts used was confirmed by the DNA binding activity of Sp1. (B) NFκB-induced expression of endogenous IκBα gene. Cells were either unstimulated (−) or stimulated (+) with IL-1 (20 ng/ml) for 60 min or LPS (200 ng/ml) for 90 min. Expression of endogenous IκBα gene was determined by northern blotting. The fold increase in IκBα mRNA in stimulated cells compared with unstimulated cells was determined by phosphoimaging and normalized to the level of total RNA loaded into each lane as determined by ethidium bromide (EtBr) staining. Download figure Download PowerPoint Figure 3.Both the RING finger and the zinc finger domain are required for full activation of JNK and p38 in MEF cells. MEF cells were derived from wild-type (+/+) or TRAF6−/− (−/−) mice. (A) Activation of JNK. Cells were either unstimulated (−) or stimulated (+) with IL-1 (20 ng/ml) for 15 min or LPS (500 ng/ml) for 90 min. After stimulation, cells were assayed for endogenous JNK activity by immunocomplex kinase assay with GST–c-Jun (1–89) as a substrate (GST–c-Jun). The amount of JNK in the immunocomplex is also shown (JNK). The fold increase in endogenous JNK activity in stimulated cells compared with untreated cells was determined by phosphoimaging and normalized to the level of JNK in the immunocomplex, which was determined by immunoblotting using ImageQuant (Molecular Dynamics). (B) Activation of p38 MAPK. After stimulation as described in (A), cells were lysed with sample buffer. p38 MAPK (p38) and phosphorylated p38 (p-p38) were visualized by immunoblotting with anti-p38 and anti-p-p38 antibodies. The fold increase in the amount of p-p38 in stimulated cells compared with unstimulated cells was determined and normalized to the amount of p38 as measured by ImageQuant. Download figure Download PowerPoint TRAF6 does not associate with TRAF2 or TRAF5 in response to IL-1 Since both TRAF2 and TRAF5 activate NFκB and MAPK (Rothe et al., 1994; Ishida et al., 1996b; Nakano et al., 1996), and since the TRAF family of proteins can associate to form homomeric or heteromeric complexes (Arch et al., 1998), TRAF6 is also likely to form complexes with TRAF2 or TRAF5. Thus, complex formation was analyzed under two different conditions where TRAF6 activates NFκB and MAPK pathways. TRAF6 did not associate with TRAF2 or TRAF5 when they were overexpressed in 293T cells, although TRAF6 forms a homomeric complex (Figure 4A). Furthermore, endogenous TRAF6 did not co-precipitate with TRAF2 or TRAF5 irrespective of IL-1 stimulation of MEF cells (Figure 4B). In contrast, stimulation-dependent association of TRAF6 with IRAK was reproduced (Cao et al., 1996). Thus, wild-type and mutant TRAF6 described below are not likely to associate with TRAF2 or TRAF5 in MEF cells, even upon stimulation. Figure 4.TRAF6 does not associate with TRAF2 or TRAF5 in MEF cells. (A) TRAF6 does not heterodimerize with TRAF2 or TRAF5 when overexpressed in 293T cells. Myc-TRAF6 expression plasmid was co-transfected with control expression plasmid or expression plasmid encoding Flag-TRAF2, Flag-TRAF5 or Flag-TRAF6 into 293T cells. After 36 h, cell lysates were prepared and incubated with anti-FLAG monoclonal antibody. Co-precipitating Myc-TRAF6 was detected by immunoblot analysis with anti-Myc monoclonal antibody (top). Amounts of Myc-TRAF6 in total lysates (middle) and FLAG-TRAFs in immunocomplexes (bottom) are shown. (B) Endogenous TRAF6 does not associate with TRAF2 or TRAF5 upon IL-1 stimulation. MEF cells derived from wild-type mice were either unstimulated (−) or stimulated with IL-1 (20 ng/ml) for 15 min (+). Cell lysates were prepared and incubated with anti-TRAF6 antibody. Amounts of TRAF2, TRAF5, TRAF6 and IRAK in the immunoprecipitates were determined by immunoblot analysis with antibodies recognizing each protein (upper three panels). Amounts of endogenous TRAF2, TRAF5 and IRAK in the lysates were determined by immunoblotting (lower two panels). Download figure Download PowerPoint IL-1 and LPS signals utilize distinct regions within the zinc finger domain of TRAF6 to activate NFκB pathways Because IL-1- and LPS-dependent activation of NFκB and MAPK were restored by complementation of TRAF6−/− MEF cells with TRAF6, we attempted to delineate the domains in TRAF6 required for NFκB and MAPK activation. Previous studies designed to determine functional domains of TRAF6 were performed by overexpression of TRAF6 mutants programmed by transient transfection in cell lines having endogenous TRAF6 (Cao et al., 1996; Baud et al., 1999). Thus, the ability of TRAF6 mutants to transmit a physiological signal has never been appropriately addressed. Since the TRAF domain is required for binding to upstream molecules such as CD40 or IRAK (Cao et al., 1996; Ishida et al., 1996a), a series of N-terminal deletion mutants (Figure 1A) was generated, and their cDNAs were inserted into retrovirus vector. To avoid multiple integration of the mutant TRAF6 transgene, TRAF6−/− MEF cells were incubated with adequately diluted solutions of recombinant virus and subsequently selected for puromycin resistance. Expression levels of exogenously introduced TRAF6 mutants were comparable to that of endogenous TRAF6 in wild-type MEF cells (Figure 1B). The ability of TRAF6 mutants to activate NFκB in response to IL-1 was first determined by electrophoretic mobility shift assays (EMSAs) (Figure 2A). T6ΔR, T6ΔZ1 and T6ΔZ3 were capable of activating the NFκB pathway to a level almost comparable to that observed with wild-type TRAF6. Thus, these results indicate that the RING finger and first three zinc fingers were not required for NFκB activation in response to IL-1. T6ΔZ5, which does not have any zinc-binding region, still had residual yet detectable activity. These results were supported by the results of another set of experiments in which IL-1-induced expression of the IκBα gene was analyzed (Figure 2B). The IκBα gene is under the transcriptional control of NFκB (Sun et al., 1993). Consistent with the EMSA results, full induction of the IκBα gene was observed in MEF cells expressing wild-type TRAF6, T6ΔR, T6ΔZ1 and T6ΔZ3. However, expression of T6ΔZ5 did not result in IκBα gene induction. LPS signal transduction was also analyzed (Figure 2A and B). Both EMSAs and IκBα northern blot analysis revealed that NFκB was fully activated in cells expressing T6ΔZ1. Thus, the RING finger and the first zinc finger are not required for LPS-induced NFκB activation. Interestingly, unlike IL-1 signaling, further deletion of the second and third zinc fingers completely abolished the ability of TRAF6 to mediate the NFκB signaling pathway. Taken together, results indicate that the RING finger and first zinc finger of TRAF6 are not required for full activation of the NFκB pathway by both IL-1 and LPS. Interestingly, the second and third zinc fingers were essential for NFκB activation by LPS but not required for that by IL-1. In contrast, NFκB activation by IL-1 required the fourth and fifth zinc fingers. RING finger and zinc finger domains of TRAF6 are required for full activation of MAPKs Domains of TRAF6 required for JNK or p38 activation were mapped. Wild-type MEF cells or TRAF6−/− MEF cells expressing full-length TRAF6 or one of the TRAF6 mutants described above were stimulated by either IL-1 or LPS. JNK activity was determined by in vitro kinase assay (Figure 3A), and p38 activation was estimated by immunoblotting phosphorylated p38 (Figure 3B). Deletion of the RING finger resulted in ∼40% reduction of IL-1-induced JNK and p38 activation. Further deletion of zinc fingers led to the gradual reduction of JNK and p38 activation, and the entire deletion of all zinc-binding domains abolished the ability of TRAF6 to activate JNK and p38 almost completely. Almost identical results were obtained when JNK and p38 activation were induced by LPS. Taken together, these results indicate that the zinc-binding motifs including the RING finger as well as all of the zinc fingers are required for full activation of JNK and p38. Thus, the RING finger and first zinc finger of TRAF6 are solely required for full activation of MAPK but not essential for activation of NFκB. The RING finger domain of TRAF6 is essential for IL-1-induced activation of TAK1 TAK1, a member of MAP3K, is activated by association with TRAF6 via TAB2 in an IL-1 signal-dependent manner, and TAK1 in turn activates NIK, which is likely to activate the IKK complex (Ninomiya et al., 1999; Takaesu et al., 2000). TAK1 is capable of activating NFκB and JNK in the presence of TAB1. Thus, experiments designed to identify the domain of TRAF6 required for TAK1 activation by IL-1 were carried out. Activation of TAK1 coincides with phosphorylation of TAK1 and TAB1, which results in the mobility shift of these two proteins on SDS–polyacrylamide gels (Ninomiya et al., 1999). MEF cells were treated with IL-1, and lysates were prepared and assayed for TAB1 mobility shift (Figure 5). No activation of TAK1 was observed in TRAF6−/− MEF cells treated with IL-1, whereas TAK1 activation was observed in IL-1-treated TRAF6−/− MEF cells complemented with TRAF6. Thus, TRAF6 is essential for IL-1-induced activation of TAK1. Interestingly, in TRAF6−/− MEF cells expressing T6ΔR, no activation of TAK1 by IL-1 was detected, whereas IL-1 activated JNK by 60% in these cells (Figure 3A). These results indicate that TRAF6 activates other JNK-activating kinases via its zinc finger domain. Furthermore, since the RING finger is not required for NFκB activation, the contribution of TAK1 to NFκB activation via an IL-1 pathway is negligible in MEF cells. Therefore, involvement of TAK1 in NFκB activation is likely to be cell-type specific. Figure 5.The RING finger of TRAF6 is essential for TAK1 activation in response to IL-1. Various MEF cells were either unstimulated (−) or stimulated (+) with IL-1 (20 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting with anti-TAB1 antibody. Phosphorylated TAB1 (p-TAB1) shows less mobility than TAB1. Download figure Download PowerPoint The RING finger domain of TRAF6 is essential for osteoclast maturation but is not required for osteoclast differentiation Formation of functional osteoclasts requires differentiation of monocyte–macrophage lineage precursor cells (colony-forming unit monocyte–macrophage, CFU-M) to TRAP+ multinuclear cells and subsequent maturation, where cells polarize and form actin rings and ruffled border membranes for bone resorption (Teitelbaum, 2000). We have previously shown that TRAF6−/− mice exhibit severe osteopetrosis due to the lack of osteoclast formation (Naito et al., 1999). Furthermore, in vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6−/− mice are unable to differentiate into osteoclasts in response to ODF/RANKL and macrophage colony-stimulating factor (M-CSF). However, Lomaga et al. (1999) demonstrated that the TRAF6−/− mice are osteopetrotic and have abundant dysfunctional osteoclasts. These observations prompted us to think that the RANK–TRAF6 pathway could be required for both differentiation and maturation of osteoclasts. Since the numbers of osteoclast progenitor cells in the spleens of wild-type and TRAF6−/− mice are comparable (Naito et al., 1999), we first checked various cytokine-signaling pathways in splenocytes pre-cultured for 3 days in the presence of M-CSF. Degradation of IκBα and phosphorylation of JNK and p38 in response to IL-1 or soluble ODF/RANKL (sODF/RANKL) were observed only in wild-type progenitor cells but not in TRAF6−/− progenitors (Figure 6). These results indicate that ODF/RANKL-induced RANK signaling is impaired in TRAF6−/− progenitor cells. Figure 6.TRAF6 is essential for ODF/RANKL-induced activation of NFκB and MAPK in osteoclast progenitors. Spleen cells derived from wild-type (+/+) or TRAF6−/− (−/−) mice were cultured for 3 days in the presence of M-CSF. After serum starvation for 30 min, cells were either unstimulated (None) or stimulated with IL-1 (10 ng/ml) or sODF/RANKL (100 ng/ml) for 20 min. Cell lysates were prepared and immunoblot analysis was performed with antibodies reactive with IκBα, JNK, p38, phosphorylated JNK (p-JNK) or phosphorylated p38 (p-p38). Download figure Download PowerPoint To address the role of TRAF6 in the differentiation and maturation of osteoclasts, splenocytes from wild-type or TRAF6−/− mice were either mock infected or infected with retrovirus carrying wild-type TRAF6 or TRAF6 mutant in the presence of M-CSF. The infected cells were further cultured in the presence of both M-CSF and sODF/RANKL, and the number of OCLs identified as multinuclear TRAP+ cells was counted (Figure 7). Under this condition, splenocytes from wild-type mice efficiently differentiated into OCLs, ∼90% of which formed apparent actin rings. Non-infected or mock-infected splenocytes did not differentiate into OCLs. Expression of wild-type TRAF6 in TRAF6−/− splenocytes resulted in the appearance of OCLs, ∼50% of which formed actin rings (Figure 7A, c and d). Complementation of TRAF6−/− splenocytes with T6ΔR or T6ΔZ1 resulted in the appearance of significant numbers of OCLs, whereas complementation of these cells with T6ΔZ3 or T6ΔZ5 did not lead to formation of OCLs (Figure 7C). Interestingly, none of the OCLs generated by the expression of either T6ΔR or T6ΔZ1 was able to form the actin ring (Figure 7A, e and f and data not shown), although these OCLs expressed typical marker genes required for osteoclasts (Figure 7B). These results indicate that the RING finger domain is not required for differentiation of progenitor cells to TRAP+ OCLs, but is required for formation of the actin ring. Figure 7.The RING finger and first zinc finger of TRAF6 are not required for the formation of multinuclear TRAP+ cells. (A) Microscopic observation of TRAP+ cells and actin ring formation. Spleen cells from wild-type mice (a) and TRAF6−/− mice (b–f). TRAF6−/− cells were mock-infected (b) or infected with retrovirus encoding wild-type TRAF6 (c and d), or T6ΔR (e and f). After infection, cells were cultured in the presence of M-CSF (10 ng/ml) and sODF/RANKL (100 ng/ml). Then, cells were fixed and stained for TRAP (a, b, c and e) and F-actin (d and f) to visualize actin rings. (B) Expression of osteoclast marker genes. MMP-9 (nucleotides 111–864), cathepsin K (CATK) (nucleotides 1–505), calcitonin receptor (CTR) (nucleotides 1311–1765), TRAP (nucleotides 586–1082) and GAPDH (nucleotides 319–1047) were amplified by RT–PCR. (C) Quantitative analysis of the formation of OCLs. TRAP+ cells containing more than three nuclei were counted as OCLs. Download figure Download PowerPoint We next analyzed the bone-resorbing activity of these OCLs. Splenocytes from TRAF6−/− mice infected with retrovirus harboring wild-type or mutant TRAF6 transgene were co-cultured with osteoblasts from wild-type mice in the presence of 1α,25-dihydroxyvitamin D3 and prostaglandin E2 for 7 days. The number of OCLs was counted, and the area of resorption pits was measured. Formation of OCLs was comparable to the results shown in Figure 7C, where recombinant M-CSF and sODF/RANKL were used instead of osteoblasts (data not shown). Complementation of TRAF6−/− splenocytes with wild-type TRAF6 restored resorption activity, as was shown by the generation of OCLs, which formed a number of resorption pits (Figure 8A). However, complementation of TRAF6−/− splenocytes with T6ΔR did not restore OCL resorption activity. Quantitation of resorption, expressed as the resorbed area per single OCL, revealed that OCLs derived from TRAF6−/− splenocytes expressing T6ΔR or T6ΔZ1 have impaired bone resorbing ability (Figure 8B). Therefore, the RING finger of TRAF6 is essential for the maturation of osteoclasts, but is not required for generating multinuclear TRAP+ OCLs. Because OCLs were absent from cultures of ODF/RANKL-treated TRAF6−/− splenocytes (Naito et al., 1999), it is clear that TRAF6 is required for both differentiation and maturation of osteoclasts. Figure 8.The RING finger of TRAF6 is essential for osteoclast maturation. (A) Microscopic view of resorption pits. Cells were cultured with normal osteoblasts on dentine slices in the presence of prostaglandin E2 (1 μM) and 1α,25-dihydroxyvitamin D3 (10 nM). Spleen cells from TRAF6−/− mice were mock infected (left) or infected with retrovirus encoding wild-type TRAF6 (center) and T6ΔR (right). (B) Quantitative analysis of the resorbed area. Resorption pits on dentine slices were visualized by staining with 0.5% toluidine blue, and the total pit area on dentine slices was measured. OCLs formed under identical conditions were counted, and the resorption area per single OCL was calculated. Download figure Download PowerPoint Discussion Identification of functional domains of TRAF6 under physiologic