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Previous Article | Next Article 
Molecular and Cellular Biology, February 2002, p. 992-1000, Vol. 22, No. 4
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.22.4.992-1000.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
B Ligand (RANKL) Activates TAK1 Mitogen-Activated Protein Kinase Kinase Kinase through a Signaling Complex Containing RANK, TAB2, and TRAF6
Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka 532-8505,1 Department of Molecular Biology, Graduate School of Science, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, Japan2
Received 16 July 2001/ Returned for modification 21 August 2001/ Accepted 20 November 2001
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ABSTRACT |
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B (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-B through IB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-B activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-B activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts. |
INTRODUCTION |
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B (RANK) is a member of the tumor necrosis factor (TNF) receptor family and is involved in osteoclastogenesis and lymph node development (1, 10). The ligand for RANK, RANKL (also called osteoclast differentiation factor [46], TNF-related activation induced cytokine [44], and osteoprotegerin ligand [21]), is a TNF receptor family ligand that regulates the functions of dendritic cells and osteoclasts. RANKL is expressed on osteoblasts and bone marrow stromal cells, while its receptor RANK is expressed on osteoclast progenitors or mature osteoclasts. RANKL interacts with RANK via direct cell-cell contact, thereby promoting the differentiation, survival, and bone-resorbing capability of osteoclasts (reviewed in references 13 and 35). RANK interacts with members of the family of TNF receptor-associated factors (TRAFs) that mediate activation of NF-B and c-Jun NH2-terminal kinase (JNK) (8, 11, 17, 43). Furthermore, the RANK cytoplasmic tail associates with c-Src kinase, which is responsible for the activation of Akt/PKB, a factor that has an antiapoptotic effect on osteoclasts (42). However, the proximal molecular components of RANK signal transduction and their interactions are not well understood. The TRAF family consists of six distinct proteins, each containing a ring and zinc finger motif in their N terminus and C-terminal TRAF domains that are responsible for self-association and protein interaction. The TRAF proteins serve as cytoplasmic adapters that can interact directly with the intracellular domains of cell surface receptors, such as the TNF receptor family, and mediate signaling (2). When overexpressed in cell lines, RANK can interact with TRAF1, -2, -3, -5, and -6. Among these TRAF molecules, TRAF6 has been shown to be a pivotal component in the RANK signaling pathway. TRAF6-deficient mice exhibit severe osteopetrosis and are defective in bone remodeling and tooth eruption caused by impaired osteoclast function (22, 25).
TRAF6 also mediates NF-B and JNK activation in the interleukin-1 (IL-1) signaling pathway (7). Recent studies have suggested a model by which the IL-1 signaling cascade is regulated. IL-1 signaling is initiated by the formation of a high-affinity complex composed of IL-1, the IL-1 receptor, and the IL-1 receptor accessory protein (12, 16, 20, 41). The intracellular adapter protein MyD88 is then recruited to the complex, where it mediates the association of IL-1 receptor-associated kinase (IRAK) with the receptor. (5, 6, 24, 40). IRAK then dissociates from the receptor complex and interacts with TRAF6, which transduces the IL-1 signal downstream, leading to NF-B and JNK activation. Thus, TRAF6 links several families of cytokine receptors to NF-B and JNK activation.
TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family and is activated by various cytokines, including the family of transforming growth factor-ß ligands (45). It was previously demonstrated that TAK1 is also involved in the IL-1 signaling pathway (26). Following exposure of cells to IL-1, endogenous TAK1 is recruited to the TRAF6 complex and activated, whereupon it stimulates both JNK and NF-B activation. Thus, TAK1 functions at the same point in the IL-1-activated signaling cascade as TRAF6. In previous studies, the yeast two-hybrid system was employed to isolate TAB2, a protein that interacts with TAK1. It was recently shown that TAB2 is also an intermediate in the IL-1 signaling pathway (36, 37). IL-1 stimulates the translocation of TAB2 from the membrane to the cytosol, where it interacts with TRAF6 and mediates its association with TAK1. These results suggest that TAB2 functions as an adapter that links TAK1 and TRAF6 in response to IL-1 and thereby mediates TAK1 activation. These results indicate that IL-1 activation of the NF-B and JNK cascades involves the formation of a TRAF6-TAB2-TAK1 complex.
In contrast to IL-1 signaling, the mechanism of RANKL-induced signal transduction is not well understood. In this work, we report that TAK1 and TAB2 are involved in the RANK signaling pathway. We show that TRAF6, TAB2, and TAK1 assemble into the RANK complex upon ligand engagement. Furthermore, RANKL stimulation activates endogenous TAK1 activity. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex with RANK is important for the RANK signaling.
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MATERIALS AND METHODS |
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TRAF6) were generously provided by H. Nakano (Juntendo University, Japan). Cell cultures and transfection. RAW 264.7 and 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37°C in 5% CO2. For the transfection studies, 293 cells were transfected with various expression vectors using Lipofectamine plus reagent (Gibco-BRL) according to the manufacturer's instructions. RAW 264.7 cells were transfected using FuGENE6 Transfection Reagent (Roche Diagnostics).
Generation of cell lines stably expressing RANKs.
To establish stable cell lines that express RANK, 293 cells were transfected with 1 µg of pcDNA3.1/Myc-His-RANK and selected in medium containing 800 µg of G418 (Geneticin; Gibco-BRL) per ml. Resistant colonies were isolated and characterized for RANKL-induced activation of NF-B by luciferase reporter assay.
Transient-transfection and reporter assay.
Cells were plated into 12-well plates and transfected with the luciferase reporter plasmid pNFB-Luc (Stratagene) or Ig--luciferase and pRSV-ß-gal (kindly provided by M. Tsuda at Toyama Medical and Pharmaceutical University). In some experiments, cells were cotransfected with multiple expression vectors and the total amount of DNA was adjusted with empty vector. After 24 h, cells were treated for 5 h with 1,000 ng of sRANKL (Pepro Tech) per ml or left untreated, and cell extracts were prepared and assayed for luciferase activity with a luciferase assay system (Wako). Relative luciferase activities were normalized to ß-galactosidase activity.
Antibodies.
Polyclonal rabbit antibody to RANK (anti-RANK) was produced against bacterially expressed RANK (amino acids 1 to 212). The expression vector for the RANK extracellular domain was generated by inserting human RANK cDNA encoding amino acids 1 to 212 into the SphI-HindIII site of pQE32 (Qiagen). Polyclonal rabbit antibodies to TRAF6 (anti-TRAF6C), TAK1 (anti-TAK1), TAB1 (anti-TAB1), and TAB2 (anti-TAB2) have been described previously (26, 36). Anti-c-Myc monoclonal antibody (9E10), anti-Flag M5 monoclonal antibody (Sigma), anti-T7 monoclonal antibody (Novagen), anti-HA (Y-11) polyclonal antibody, anti-JNK1 (FL) polyclonal antibody, anti-IKK
(H-744) polyclonal antibody, and anti-Xpress polyclonal antibody (Santa Cruz) were used for immunoprecipitation and immunoblotting. Polyclonal rabbit antibody to phospho-specific p38 MAP kinase (New England Biolabs) was used to detect the phosphorylated form of p38. Purified rabbit immunoglobulin G (IgG) (Sigma) were used as control antibodies.
Immunoprecipitation assay. 293-RANK cells plated in 10-cm-diameter dishes were treated with 1,000 ng of RANKL per ml for the indicated times or left untreated. Cells were washed once with ice-cold phosphate-buffered saline and lysed in 0.3 ml of lysis buffer (25 mM HEPES [pH 7.7], 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 10 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 10 µg of aprotinin per ml, and 10 µg of leupeptin per ml). Cell lysates were then diluted with an equal volume of dilution buffer (20 mM HEPES [pH 7.7], 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 10 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 10 µg of aprotinin per ml, and 10 µg of leupeptin per ml). After centrifugation, lysates were incubated with various antibodies on ice for 1.5 h and rotated with protein G-Sepharose (Amersham Pharmacia). The beads were washed three times with washing buffer (20 mM HEPES [pH 7.7], 0.15 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.05% Triton X-100). The immunoprecipitates or whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P membranes (Millipore). The membranes were immunoblotted with various antibodies, and the primary antibodies were detected with horseradish peroxidase-conjugated antibodies to rabbit IgG (Calbiochem) or mouse IgG (Sigma) and visualized by the ECL system (Amersham Pharmacia).
In vitro kinase assay.
Endogenous TAK1, JNK, or IKK was immunoprecipitated with anti-TAK1, anti-JNK, or anti-IKK antibody, respectively, as described above. Immunoprecipitates were incubated with 30 µl of kinase buffer (20 mM HEPES [pH 7.6], 20 mM MgCl2, 2 mM DTT, 20 µM ATP, 20 mM ß-glycerophosphate, 20 mM disodium 
-nitrophenylphosphate, 0.1 mM sodium orthovanadate, and 2 µCi of [
-32P]ATP). For the TAK1, JNK, or IKK kinase assay, 2.5 µg of bacterially expressed 6xHis-MKK6, glutathione S-transferase (GST)-c-Jun (1-79), or GST-IB (1-54) (29) was added in each kinase buffer as a substrate. The reaction mixtures were resolved by SDS-PAGE, followed by autoradiography.
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RESULTS |
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B activity was assayed with an NF-B-dependent luciferase reporter gene. As reported previously (11, 43), overexpression of RANK causes a 50- to 100-fold activation of NF-B in the absence of ligand. RANK-mediated NF-B activation was blocked by coexpression of a truncated version of TRAF6 (TRAF6) (Fig. 1A), which acts as a dominant negative inhibitor of NF-B activation in the IL-1 pathway (7). In the IL-1 signaling pathway, formation of the TRAF6-TAB2-TAK1 complex is essential for NF-B activation. Therefore, we investigated whether TAK1 and TAB2 participate in RANK signaling by examining the effects of dominant negative mutants on RANK-induced NF-B activation. Overexpression of a kinase-negative mutant of TAK1, TAK1(K63W), or a truncated form of TAB2, TAB2C, was previously shown to inhibit IL-1-induced activation of NF-B (26, 36). Similarly, we found that TAK1(K63W) and TAB2C blocked RANK-induced NF-B activation in a dose-dependent manner (Fig. 1A). Another member of the MAPKK family, MEKK1, has been shown to be involved in TNF--induced NF-B activation (14). Overexpression of a kinase-negative mutant of MEKK1 (MEKK1KN) had no effect on NF-B activation by RANK. Moreover, the inhibitory effect was not observed when the truncated form of MyD88 (MyD88C), an inhibitor of NF-B activation in the IL-1 pathway (40), was cotransfected (Fig. 1A). These results indicate that the dominant negative effects on RANK-induced NF-B activation are specific for TAK1(K63W) and TAB2C.
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B and JNK by IL-1, we next examined whether TAK1(K63W) and TAB2C also block RANK-induced JNK activation by an in vitro kinase assay. Coexpression of TAK1(K63W) and TAB2C reduced activation of JNK induced by RANK (Fig. 1B). However, MyD88C did not block RANK-induced JNK activation (Fig. 1B). These results suggest that TAK1 and TAB2 are involved in the RANK signaling pathway. TAB2 and TAK1 interact with RANK. When overexpressed, TRAF6 binds to RANK (8). We confirmed this interaction by coexpressing Flag-tagged TRAF6 (Flag-TRAF6) and Myc-tagged RANK (Myc-RANK) in 293 cells (Fig. 2A). We tested the potential of TAB2 and TAK1 to associate with RANK by coimmunoprecipitation experiments. Myc-RANK was coexpressed with T7-tagged TAB2 (T7-TAB2) or Flag-tagged TAK1 (Flag-TAK1) in 293 cells and immunoprecipitated with anti-Myc antibody. The immunoprecipitates were subjected to immunoblotting with anti-T7 or anti-Flag antibody, respectively. TAB2 and TAK1 were found to coprecipitate with RANK (Fig. 2B and C). These results indicate that TRAF6, TAB2, and TAK1 constitutively interact with RANK when overexpressed. In contrast to TAK1 and TAB2, Flag-tagged MyD88, which is known to be an adapter protein linking IRAK and IL-1 receptor in IL-1 signaling pathway, did not coprecipitate with RANK under the same conditions (Fig. 2D).
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B activation (11) (Fig. 3B). To elucidate the role of endogenous TAB2 and TAK1 in the RANK signaling pathway, we generated 293 cells stably expressing Myc epitope-tagged RANK (Myc-RANK). 293 cells were transfected with a Myc-RANK expression vector carrying the neomycin resistance gene and selected in medium containing G418. Twenty individual colonies were isolated and expanded. Each clone was transfected with an NF-B-dependent luciferase reporter gene, stimulated with RANKL, and assayed for luciferase activity. We chose the clone which showed maximum-fold activation of NF-B by RANKL. RANK expression in this clone, 293-RANK, was analyzed by immunoblotting. Whole-cell extracts were prepared from 293-RANK cells and immunoprecipitated with anti-RANK antibody. The immunoprecipitates were subjected to immunoblotting with a monoclonal antibody to c-Myc. Expression of Myc-RANK was detected in 293-RANK cells but not in parental 293 cells (Fig. 3A). 293-RANK cells also showed a dose-dependent response to RANKL in the NF-B reporter assay, while 293 cells showed a weak response (Fig. 3B).
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B, JNK, and p38 are activated in response to RANKL stimulation in 293-RANK cells and therefore can be used to analyze endogenous signal transduction mechanisms mediated by RANKL. RANKL induces the formation of a complex containing RANK, TRAF6, TAB2, and TAK1. When individually overexpressed in 293 cells, TRAF6, TAB2, and TAK1 interact with RANK, even in the absence of ligand stimulation (Fig. 2). To determine whether endogenous TRAF6, TAB2, and TAK1 form complexes with RANK, we investigated their association in 293-RANK cells stimulated with RANKL. After being stimulated for various lengths of time, endogenous TRAF6 was immunoprecipitated with anti-TRAF6 antibody. The immune complexes were subjected to immunoblotting with anti-Myc antibody to detect coimmunoprecipitated RANK. Association of endogenous TRAF6 with RANK was observed in cells stimulated with RANKL, but not in the unstimulated, control cells. The presence of RANK in TRAF6 immunoprecipitates was observed starting at 2 min after RANKL treatment (Fig. 4). This is consistent with the previous observation that TRAF6 and RANK associate in a RANKL-dependent manner in dendritic cells (42). The amounts of RANK found associated with TRAF6 peaked 2 min after RANKL induction and declined steeply thereafter. These results demonstrate that RANK forms a transient complex with TRAF6 in response to RANKL stimulation.
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We next examined the effect of RANKL stimulation on the interaction between endogenous TRAF6 and TAK1. Cell extracts from 293-RANK cells treated with RANKL were immunoprecipitated with anti-TAK1 antibody and analyzed by immunoblotting with anti-TRAF6 antibody. Association of endogenous TAK1 with TRAF6 was observed at 2 min after RANKL treatment and subsequently released from TRAF6 at 30 min (Fig. 6). The kinetics of RANKL-induced TRAF6-TAK1 association are similar to those for RANK-TRAF6 association (Fig. 4).
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TAK1 and TAB2 are involved in the RANK signaling pathway in RAW 264.7 cells.
To confirm the significance of TAK1 and TAB2 in the RANK signaling pathway, we tested the effects of TAK1(K63W) and TAB2C on RANKL-induced activation of NF-B under physiological conditions. The mouse macrophage cell line, RAW 264.7, was chosen because this cell line is known to express RANK and to differentiate into osteoclast-like cells in response to RANKL treatment (15). RANKL activated NF-B in RAW 264.7 cells in reporter gene assay (Fig. 8A), as reported previously (39). We found that this RANKL-induced NF-B activation was blocked by TAK1(K63W) and TAB2C (Fig. 8A), suggesting that TAK1 and TAB2 also play important roles in RANK signaling in osteoclasts. Moreover, endogenous TAK1 was activated within 3 min after addition of RANKL in RAW 264.7 cells (Fig. 8B). This rapid activation of TAK1 was comparable to that seen in 293-RANK cells. Thus, TAK1 activation is linked to RANKL-mediated signaling in RAW 264.7 cells as well as in 293-RANK cells.
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DISCUSSION |
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B and JNK pathways (8, 43). TRAF6 gene knockout mice exhibit severe osteopetrosis and are defective in osteoclast formation (22, 25). Thus, among TRAF family members TRAF6 is the most critical for RANK signaling. However, the molecular mechanisms by which TRAF6 exerts its biological effects in the RANK pathway have not previously been defined. In addition to its role in RANK signaling, TRAF6 has also been shown to be involved in the IL-1 signal transduction pathway linked to NF-B and JNK activation (7). Previous studies have demonstrated that TRAF6 interacts with the TAK1-associating protein TAB2 in an IL-1-dependent manner, resulting in the formation of a TRAF6-TAB2-TAK1 complex (36). Formation of this complex appears to be required for IL-1-mediated activation of NF-B and JNK. Thus, TAB2 acts as an adapter that links TRAF6 and TAK1 and thereby mediates the activation of TAK1 in the IL-1 signaling pathway. In this study we show that RANK employs a similar mechanism.
Our evidence suggesting that TAB2 and TAK1 function in the RANKL-RANK signal transduction pathway may be summarized as follows. First, dominant negative forms of TAB2 and TAK1 block activation of NF-B and JNK induced by overexpression of RANK. Second, in transient transfection experiments, overexpressed TAB2 and TAK1 constitutively associate with RANK. Third, in 293 cells stably expressing RANK (293-RANK), RANKL treatment induces the associations of RANK with TRAF6, RANK with TAB2, RANK with TAK1, TRAF6 with TAB2, and TRAF6 with TAK1, all with similar kinetics. This suggests that RANKL may induce the formation of a TRAF6-TAB2-TAK1 complex with RANK. Fourth, in 293-RANK cells, TAK1 activation occurs rapidly following RANKL stimulation, with kinetics that parallel the observed formation of complexes among RANK, TRAF6, TAB2, and TAK1. It therefore seems reasonable to assume that formation of the TRAF6-TAB2-TAK1 complex constitutes an early event in the activation of TAK1 by RANK. Fifth, dominant negative forms of TAB2 and TAK1 block RANKL-induced NF-B activation in RAW 264.7 cells, which are able to differentiate into osteoclast-like cells following this stimulation. Finally, RANKL stimulation also induces activation of endogenous TAK1 in RAW 264.7 cells, suggesting that TAK1 and TAB2 are involved in the RANK signaling pathway in osteoclasts. Taken together, these results suggest a model in which RANKL stimulation facilitates the formation of a RANK-TRAF6-TAB2-TAK1 complex, leading to activation of TAK1. In this model, TRAF6 acts as a mediator between RANK and TAK1 and TAB2 functions as an adapter that links TAK1 and TRAF6. However, the intramolecular mechanisms by which TAK1 is activated remain to be elucidated. Other studies have shown that TAK1 is activated by autophosphorylation (18, 28); thus, we speculate that complex formation may cause a conformational change in the catalytic domain of TAK1, which induces its kinase activity leading to autophosphorylation. Further study is required to clarify the role of each component of this large signaling complex in TAK1 activation.
In the case of IL-1 signaling, IRAK, not TRAF6, is recruited to the activated IL-1 receptors. IRAK then mediates the formation of a TRAF6-TAB2-TAK1 complex by interacting with TRAF6 (37). On the other hand, in RANK signaling, TRAF6 is directly recruited to the activated receptor, bypassing the dependence on IRAK seen in the IL-1 pathway. Consistent with this, overexpression of RANK was able to induce NF-B activation in IRAK-deficient cells (data not shown). Darnay et al. (9) have identified a novel TRAF6 binding motif (basic QXPXEX acidic) in RANK (340-RQMPTEDE-347) (homologous residues are underlined). Indeed, this TRAF6 binding region (positions 340 to 358) is necessary and sufficient for RANK-induced NF-B activation (9). Interestingly, this TRAF6 binding motif is also present in the C terminus of IRAK (701-RQGPEESD-708) (9). These results suggest that TRAF6 forms a complex with TAB2 and TAK1 on IRAK and RANK in the IL-1 and RANKL signaling pathways, respectively.
Wong et al. (42) have recently demonstrated that RANKL activates the antiapoptotic Akt/PKB pathway through TRAF6 and c-Src kinase. c-Src is constitutively bound to the cytoplasmic tail of RANK and further activated by recruitment of TRAF6 to the receptor complex following RANKL engagement (42). c-Src kinase is ubiquitously expressed and activated by many pathways, including tyrosine kinase growth factor receptors, G-protein-coupled receptors, and integrin cell surface adhesion molecules (4, 38). However, targeted disruption of the c-src gene in mice leads to only one predominant phenotype, osteopetrosis, resulting from an intrinsic functional defect in osteoclast development (33). Osteoclasts from Src-deficient mice failed to form ruffled borders or resorption lacunae (3). These observations, together with our present results, suggest that in osteoclasts, RANKL stimulates the formation of a signaling complex containing c-Src, TAK1, and other molecules at the cytoplasmic tail of RANK, and thereby activates downstream signaling events leading to osteoclast differentiation, cell survival, and cytoskeletal reorganization.
TRAF6-deficient (TRAF6-/-) mice also exhibit severe osteopetrosis and are defective in osteoclast formation due to blockade of the RANKL-RANK pathway (22). Recently, Kobayashi et al. (19) have delineated functional domains of TRAF6 by expressing mutant TRAF6 transgenes at endogenous wild-type levels in TRAF6-/- cells. These authors found that the finger domain of TRAF6 is responsible for the maturation of osteoclasts and activation of TAK1. Therefore, TAK1 may play a key role in osteoclastogenesis and may be an important pharmacological target in the treatment of bone-destructive diseases, such as osteoporosis and rheumatoid arthritis. Further development of a selective TAK1 inhibitor or generation of TAK1 or TAB2 gene knockout mice may provide more information on their roles in osteoclastogenesis.
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ACKNOWLEDGMENTS |
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This work was supported by special grants to Advanced Research on Cancer from the Ministry of Education, Culture and Science of Japan (K.M.).
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FOOTNOTES |
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